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We describe the identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with tub4‐1, encoding a mutated yeast γ‐tubulin. The cytoplasmic Gim proteins encoded by these GIM genes are present in common complexes as judged by co‐immunoprecipitation and gel filtration experiments. The disruption of any of these genes results in similar phenotypes: the gim null mutants are synthetically lethal with tub4‐1 and super‐sensitive towards the microtubule‐depolymerizing drug benomyl. All except Δgim4 are cold‐sensitive and their microtubules disassemble at 14°C. The Gim proteins have one function related to α‐tubulin and another to Tub4p, supported by the finding that the benomyl super‐sensitivity is caused by a reduced level of α‐tubulin while the synthetic lethality with tub4‐1 is not. In addition, GIM1/YKE2 genetically interacts with two distinct classes of genes, one of which is involved in tubulin folding and the other in microtubule nucleation. We show that the Gim proteins are important for Tub4p function and bind to overproduced Tub4p. The mammalian homologues of GIM1/YKE2 and GIM2/PAC10 rescue the synthetically lethal phenotype with tub4‐1 as well as the cold‐sensitivity and benomyl super‐sensitivity of the yeast deletion mutants. We suggest that the Gim proteins form a protein complex that promotes formation of functional α‐ and γ‐tubulin.
Microtubules are hollow cylinders with a diameter of 25 nm. They are part of the cytoskeleton of eukaryotic cells and play an important role in numerous cellular processes, including chromosome segregation in mitosis and meiosis, organelle positioning and intracellular transport. The wall of the microtubule cylinder consists of 13 protofilaments which are strings of alternating α‐ and β‐tubulin subunits pointing in the same direction (reviewed by Mandelkow and Mandelkow, 1993). Therefore, microtubules are polar structures with biochemically distinct ends. Microtubule formation is a complex process that includes the post‐translational folding of α‐ and β‐tubulin, their assembly into the heterodimer tubulin and finally the formation of microtubules from tubulin subunits (Solomon, 1991). All of these steps are assisted by proteins in vivo.
The first post‐translational step in the pathway leading to the formation of the tubulin heterodimer is the folding of α‐ and β‐tubulin. Genetic (Ursic and Culbertson, 1991; Chen et al., 1994; Vinh and Drubin, 1994; Archer et al., 1995) and biochemical (Frydman et al., 1992; Yaffe et al., 1992; Melki et al., 1993; Sternlicht et al., 1993; Tian et al., 1996) evidence suggests that tubulins undergo facilitated folding via their interaction with cytoplasmic chaperonins. Conditional lethal mutations in TCP1, BIN2, BIN3 and ANC2, coding for subunits of cytoplasmic chaperonin (TRiC), affect microtubule as well as actin functions (Ursic and Culbertson, 1991; Chen et al., 1994; Li et al., 1994; Miklos et al., 1994; Vinh and Drubin, 1994). For example, tcp1‐1 cells are cold‐sensitive for growth, and accumulate multi‐nucleated and anucleated cells with abnormal microtubule structures. This mutant is also sensitive towards the microtubule‐depolymerizing drug benomyl (Ursic and Culbertson, 1991). Remarkably, bin3‐1 cells display a normal distribution of unbudded, small‐budded and large‐budded cells, although the microtubules of bin3‐1 cells disassemble at 14°C (Chen et al., 1994).
While the cytoplasmic chaperonin is sufficient for the production of native actin in an ATP‐dependent manner (Gao et al., 1992), folding of α‐ and β‐tubulin requires the participation of additional cofactors. Such proteins, named cofactors A, B, C, D and E, have been identified by biochemical approaches (Tian et al., 1996, 1997). Potential yeast homologues of cofactors A, B, D and E are Rbl2p, Alf1p, Cin1p and Pac2p (Archer et al., 1995; Tian et al., 1996; Geiser et al., 1997). Surprisingly, ALF1, RBL2, CIN1 and PAC2 are not essential for growth of yeast cells (Hoyt et al., 1990; Stearns et al., 1990; Archer et al., 1995). However, their deletion causes sensitivity towards benomyl and they show multiple genetic interactions with genes coding for components of the microtubule system. In addition, Rbl2p interacts directly with yeast β‐tubulin (Archer et al., 1995).
In many cell types, microtubule assembly is initiated at centrosomes, basal bodies, spindle pole bodies (SPBs) or nucleus‐associated bodies, structures for which Pickett‐Heaps (1969) coined the generic term microtubule‐organizing centre (MTOC). MTOCs assist the assembly of the first tubulin monomers into oligomers, a step known as microtubule nucleation (Mitchison and Kirschner, 1984). A universal component of MTOCs involved in microtubule nucleation is γ‐tubulin (Oakley et al., 1990; Horio et al., 1991; Stearns et al., 1991). γ‐Tubulin forms a complex with additional proteins (Stearns and Kirschner, 1994; Moudjou et al., 1996; Akashi et al., 1997), and purification of such a complex from Xenopus laevis eggs identified α‐, β‐ and γ‐tubulin and proteins with mol. wts of 195, 133, 109 and 75 kDa (Zheng et al., 1995).
In the yeast Saccharomyces cerevisiae, the MTOC is known as the SPB. It is a multi‐layered structure which is embedded in the nuclear envelope (Byers and Goetsch, 1975; Byers, 1981). The yeast γ‐tubulin is encoded by the essential TUB4 gene (Sobel and Snyder, 1995; Marschall et al., 1996; Spang et al., 1996). Tub4p forms a 6S complex with the SPB components Spc98p and Spc97p (Geissler et al., 1996; Spang et al., 1996; Knop et al., 1997). Purification of this complex suggests that it contains only one molecule of Spc98p and Spc97p, but two or more molecules of Tub4p (Knop and Schiebel, 1997). Fractionation experiments suggest that the Tub4p complex assembles in the cytoplasm followed by its nuclear import via an essential nuclear localization sequence in Spc98p (G. Pereira et al., 1998). Finally, Spc98p and Spc97p of the Tub4p complex interact in the nucleus with the amino‐terminal domain of the SPB component Spc110p (Knop and Schiebel, 1997). The binding site for the Tub4p complex at the outer plaque is still unknown.
To identify genes that are involved in yeast γ‐tubulin functions, we performed a genetic screen for mutants that are synthetically lethal with tub4‐1. We identified SPC98 and SPC97 coding for components of the yeast γ‐tubulin complex and five additional genes, which we named GIM1–GIM5. Their products are not associated with the SPB, instead they form cytoplasmic multi‐protein complexes which promote formation of functional α‐tubulin and Tub4p. Most interestingly, the Gim proteins are phylogenetically conserved proteins, and mouse and human homologues function in yeast, indicating that the mammalian proteins fulfil a very similar role.
Identification of GIM1/YKE2, GIM2/PAC10, GIM3, GIM4 and GIM5 in a screen for mutants that are synthetically lethal with a mutated yeast γ‐tubulin
In the yeast S.cerevisiae, the γ‐tubulin Tub4p forms a complex with the SPB components Spc98p and Spc97p. Physical interaction between Tub4p, Spc98p and Spc97p is reflected by multiple genetic interactions, including synthetic lethality (Geissler et al., 1996; Knop et al., 1997). The latter is an indication of a functional relationship of two gene products. To identify further components that functionally interact with TUB4, we performed a genetic screen for mutants that are synthetically lethal with tub4‐1, yielding 12 mutants with this phenotype. Subsequent analysis showed that two of these mutants were defective in SPC98 and two in SPC97. The other eight belong to five complementation groups which we named GIM1–GIM5 (genes involved in microtubule biogenesis). Plasmids containing GIM1–GIM5 were isolated by transforming the mutants with a yeast genomic library. Subcloning of DNA fragments and complementation analysis showed that GIM1 is identical to YKE2 (Shang et al., 1994), coding for a protein of 114 amino acids (Table I). GIM2 had already been identified as PAC10, a gene that becomes essential in the absence of the CIN8‐encoded kinesin motor (Geiser et al., 1997). GIM3 corresponds to the open reading frame (ORF) YNL153c, encoding a protein of 129 amino acids. The DNA fragment complementing gim4 cells contained three overlapping ORFs, one of which was interrupted by an intron. The various coding regions were cloned directly behind the ADH promoter of plasmid p415‐ADH. Only the ADH promoter fusion with the intron‐containing ORF YEL003w complemented gim4 cells (data not shown). Gim4p is a protein of 132 amino acids. GIM5 (ORF YML094w) also contains a small intron and encodes a protein of 163 amino acids.
The Gim proteins are phylogenetically conserved coiled‐coil proteins
As reported for Gim2p/Pac10p (Geiser et al., 1997), all Gim proteins have a high probability of forming coiled‐coils (Lupas, 1996). Sequence comparisons have shown that Gim1p/Yke2p, Gim2p/Pac10p, Gim3p, Gim4p and Gim5p are related proteins. They also have relatives in mammals, Caenorhabditis elegans and Schizosaccharomyces pombe, revealing that they are phylogenetically conserved (Figure 1). Remarkably, Gim1p/Yke2p and Gim5p are homologous to two proteins of the archaebacterium Methanococcus jannaschii, while no relatives were found in eubacteria. We also noticed that the Gim proteins are more closely related to their homologues from other species than to each other (Figure 1).
Deletion of the GIM genes causes microtubule defects
To understand the function of the GIM gene products, we investigated whether they are essential for growth of yeast cells. We disrupted the entire coding regions of the GIM genes in the diploid yeast strain YPH501. As shown for Δgim5 (Figure 2A), spore analysis revealed that GIM1/YKE2, GIM3, GIM4 and GIM5 are not essential for growth. In agreement with a previous report (Geiser et al., 1997), we observed that Δpac10 cells were defective in spore germination. Such a defect was not apparent for the other GIM mutants, raising the possibility that GIM2/PAC10 has an additional specialized function in spore germination. The disruption of any of the GIM genes resulted in a slow growth phenotype at 30°C (Figure 2A and B). A more detailed analysis indicated that the doubling time of Δgim1/yke2, Δgim2/pac10, Δgim3 and Δgim5 cells grown in liquid medium at 30°C was increased by a factor of 1.4 compared with the wild‐type, while the doubling time of Δgim4 cells was only affected 1.1‐fold. It has been reported previously that a deletion mutant of GIM1/YKE2 shows normal growth over a wide range of temperatures (Shang et al., 1994). This discrepancy with our study is explained either by strain differences or by the fact that Shang et al. (1994) did not disrupt the entire coding region of YKE2.
Common phenotypes of mutants affecting the microtubule cytoskeleton are a cold‐sensitive growth defect and an increased sensitivity towards the microtubule‐depolymerizing drug benomyl (Neff et al., 1983; Huffaker et al., 1988). Therefore, we investigated whether the growth defects of the haploid gim null strains were intensified at lower temperatures. The gim null strains showed an enhanced growth defect at reduced temperatures in comparison with the wild‐type (Figure 2B; compare 30°C with 23°C and 14°C plates). An exception was Δgim4 cells, which did not show an enhanced growth defect at lower temperatures (Table I). In contrast to the wild‐type, all gim null strains were super‐sensitive to 2.5 μg/ml benomyl (Figure 2C). Such a strong benomyl sensitivity has only been observed for mutants defective in microtubule biogenesis (Stearns et al., 1990; Chen et al., 1994; Archer et al., 1995; Tian et al., 1997).
We investigated whether the Gim proteins function in parallel pathways. In this case double mutants should have more pronounced defects compared with the single mutants. However, the double deletion mutants of Δgim1/yke2 together with any one of Δgim2/pac10, Δgim3, Δgim4 or Δgim5 were viable and as benomyl‐sensitive as the single mutants. We also tested whether overexpression of any of the GIM genes rescued the synthetically lethal phenotype of gim tub4‐1 cells. This was the case for GIM1 which weakly suppressed the defect of gim4 tub4‐1 cells (data not shown).
The strong benomyl super‐sensitivity of the haploid gim null strains suggests that the gene products are needed for either tubulin formation or microtubule stability. To analyse these possibilities, we investigated, by indirect immunofluorescence, the microtubule arrays of Δgim1/yke2, Δgim2/pac10 and Δgim3 cells incubated at 14°C for 20 h. The phenotype is shown for Δgim1/yke2 cells as a representative, since it is very similar to that of the other mutants. Δgim cells lost most of their cytoplasmic and nuclear microtubules, leaving only a small spot near the nucleus, most likely at the SPB (Figure 3A). This observation suggests that microtubule attachment to the SPB via the Tub4p complex is still taking place in Δgim cells. Instead microtubule stability seems to be impaired. Furthermore, cells with a large bud (Figure 3A, arrow) had only one 4′,6'‐diamidino‐2‐phenylindole (DAPI) staining region in one of the two cell bodies, a phenotype that is consistent with the observed defect in microtubule organization. Despite these defects, gim null cells incubated for 20 h at 14°C were still as viable as wild‐type cells when then grown at 30°C (data not shown). Based on the distributions of the DNA content (Figure 3B) and of the cell morphologies (Figure 3A), Δgim cells did not arrest at a defined stage of the cell cycle. This observation was surprising, since many mutants with a defect in spindle formation arrest in mitosis due to a mitotic check point (Hoyt et al., 1990; Li and Murray, 1991). The non‐arrest phenotype could point to a role for the Gim proteins in mitotic check point control. However, all GIM deletion mutants arrested in the cell cycle like wild‐type cells in response to the microtubule‐depolymerizing drug nocodazole, while the mitotic check point control mutant bub2 did not (Hoyt et al., 1990; Li and Murray, 1991; Geiser et al., 1997; data not shown). This led us to conclude that the GIM gene products are not part of a mitotic check point. Taken together, the strong benomyl sensitivity and the microtubule defects of the gim null mutants at reduced temperature are most consistent with a role for the Gim proteins in tubulin biogenesis.
gim null mutants are sensitive towards the actin inhibitor latrunculin‐A and are osmotically sensitive
We noticed that the volume of cells of the GIM deletion mutants incubated at 14°C was ∼2.2‐ to 2.4‐fold increased compared with the wild‐type (Table I). Such a phenotype has been reported, among others, for mutants affecting the actin cytoskeleton (reviewed by Drubin, 1990). Many mutants with actin defects are sensitive towards the actin‐binding drug latrunculin‐A (Ayscough et al., 1997) and are osmotically sensitive (Drubin, 1990). We found that the gim null strains were more sensitive towards latrunculin‐A than the wild‐type, which indicates an influence of the Gim proteins on the actin cytoskeleton (Figure 3C). This sensitivity was stronger compared with that of the cold‐sensitive mutant tcp1‐1, but was, however, clearly weaker compared with the actin mutant act1‐2. TCP1 encodes a component of TRiC which is required for actin folding (Ursic and Culbertson, 1991). Furthermore, similarly to actin mutants, Δgim1/yke2, Δgim2/pac10 and Δgim5 cells did not grow on plates containing 1.4 M sorbitol. In contrast, Δgim3 cells grew slowly, while Δgim4 grew as wild‐type cells (Figure 3D). At 1.8 M sorbitol, Δgim3 cells failed to grow and a weak growth defect of Δgim4 cells was observed (Figure 3D). We sought additional evidence for a role for the Gim proteins in actin function. However, deletion of GIM1/YKE2 was synthetically lethal with neither act1‐2 nor act1‐3. In addition, Δgim1/yke2, Δgim2/pac10, Δgim3 and Δgim4 cells did not show an obvious defect in actin organization as judged by indirect immunofluorescence using anti‐actin antibodies (data not shown), suggesting that the gim null mutants have only subtle actin defects. In conclusion, the GIM genes may have multiple functions, since their deletion causes microtubule defects as well as an increased sensitivity towards the actin‐specific drug latrunculin‐A and towards high osmolarity.
The GIM genes interact genetically with genes involved in microtubule biogenesis
To gain further insight into the role of the GIM genes, we investigated their genetic interactions with other genes involved in microtubule function. We first addressed the question of whether total loss of GIM gene function is synthetically lethal with mutants of the Tub4p complex. Using the approach described in Figure 4A, we could show that this is indeed the case for tub4‐1. This result was confirmed by tetrad analysis using a GIM4/Δgim4::HIS3MX6 TUB4/tub4‐1 strain. No viable temperature‐sensitive HIS3 spores were obtained, confirming that Δgim4::HIS3MX6 combined with tub4‐1 is lethal. Microscopic inspection of the non‐colony‐forming spores identified several hundred cells. Therefore, Δgim4 tub4‐1 cells died only after multiple duplications. A similar result was obtained with GIM3 (data not shown).
In a similar manner, we tested whether GIM1/YKE2, GIM2/PAC10 and GIM4 showed interactions with SPC98 and SPC97, coding for components of the yeast γ‐tubulin complex. Δgim1/yke2 was synthetically lethal with spc98‐2 and synthetically toxic when combined with spc98‐1 (Figure 4B). Allele‐specific genetic interactions of GIM1/YKE2 were also observed with SPC97: deletion of GIM1/YKE2 was synthetically toxic in spc97‐20 cells, while it did not affect growth of the spc97‐14 mutant (Figure 4C). In contrast to Δgim1/yke2, Δgim2/pac10 or Δgim4 combined with spc98‐1, spc98‐2 or spc97‐20 hardly affected growth (Table II). It is important to note that the spc98 and spc97 alleles have distinct phenotypes and are suppressed differently by TUB4 (Geissler et al., 1996; Knop et al., 1997). In particular, spc97‐20 has an SPB duplication defect, while spc97‐14 fails to form a mitotic spindle (Knop et al., 1997).
Finally, GIM1/YKE2, GIM2/PAC10 and GIM4 were in part tested for their genetic interactions with TUB1, TUB2, BIN2, BIN3 and RBL2. TUB1 and TUB2 code for α‐ and β‐tubulin in yeast (Neff et al., 1983; Schatz et al., 1986a), while RBL2 encodes a β‐tubulin‐binding protein, the homologue of mammalian cofactor A (Archer et al., 1995; Tian et al., 1996). Bin2p and Bin3p are subunits of TRiC (Chen et al., 1994). Δgim1 showed synthetic lethality with tub1‐4, tub2‐403, tub2‐405, bin2‐1, bin3‐1 and Δrbl2 (Table II). In contrast, Δgim4 was only synthetically lethal in combination with bin2‐1, bin3‐1 and Δrbl2, but not with tub2‐403 or tub2‐405. In summary, the GIM genes show a broad range of genetic interactions with genes involved in tubulin biogenesis and microtubule nucleation.
Deletion of the GIM genes results in reduced levels of α‐tubulin, which explains the benomyl super‐sensitivity, but not the synthetic lethality with tub4‐1
A defect of the gim null mutants in tubulin biogenesis may result in reduced levels of α‐, β‐ or γ‐tubulin. The amount of these proteins was determined by immunoblotting using specific antibodies. While the α‐tubulin content was about half to a third compared with wild‐type, β‐tubulin and Tub4p levels were approximately the same (Figure 5A and B). It is noteworthy that deletion of GIM4 affected α‐tubulin less severely than deletion of the other GIM genes. We also noticed that the reduction of α‐tubulin levels was independent of the incubation temperature of the cultures (data not shown).
An increase of β‐tubulin over α‐tubulin levels, which is the case in the gim null mutants, is toxic for yeast cells (Burke et al., 1989; Katz et al., 1990; Schatz et al., 1986b). Therefore, increasing the α‐tubulin content by overexpression of TUB1 or reducing the pool of free β‐tubulin by overproduction of the β‐tubulin‐binding protein Rbl2p may suppress the tubulin defects. Indeed, the benomyl super‐sensitivity of the five Δgim mutants was fully rescued when TUB1 or RBL2 were overexpressed using either elevated gene dosage (2μ plasmids) or the strong ADH promoter (Table I), while overexpression of TUB4 showed no effect (Figure 5C; shown for Δgim1/yke2). We then investigated whether the reduced level of α‐tubulin is also the cause for the synthetically lethal phenotype with tub4‐1. However, overexpression of either TUB1 or RBL2 did not suppress the synthetic lethality, while plasmid‐encoded TUB4 and the GIM genes complemented the mutants (Figure 5D; shown for Δgim1/yke2). In summary, the benomyl super‐sensitivity of the GIM mutants is the result of a reduced level of α‐tubulin; however, this is not the reason for the synthetically lethal phenotype with tub4‐1. This suggests that the Gim proteins have at least two functions, one related to α‐tubulin and the other to Tub4p.
Gim1p/Yke2p, Gim2p/Pac10p, Gim3p and Gim5p bind to overproduced Tub4p
The failure of overexpressed TUB1 and RBL2 to suppress the synthetically lethal phenotype with tub4‐1 and the allele‐specific interaction of GIM1/YKE2 with SPC98 and SPC97 point to a more direct role for the Gim proteins in Tub4p complex function. This notion was supported further by the finding that overexpression of TUB4 was toxic in the Δgim1/yke2 null strain (Figure 6A, sector 1), while it hardly affected growth of the wild‐type (Figure 6A, sector 3) (Spang et al., 1996; Knop et al., 1997). Δgim1/yke2 cells overexpressing TUB4 frequently were large‐budded and showed abnormal bud morphologies (Figure 6A), phenotypes which were observed neither in Δgim1/yke2 cells nor in wild‐type cells overexpressing TUB4 (data not shown). Similarly, simultaneous overexpression of chromosomally integrated TUB4, SPC98 and SPC97 was not toxic for wild‐type cells; however, it strongly affected growth of the gim1/yke2 null mutant (Figure 6B). As expected, this defect was rescued by plasmid‐encoded Gim1p.
This result, together with the observed synthetically lethal phenotype with tub4‐1, suggests that the GIM genes become essential either after overexpression of TUB4 or in the presence of a mutated TUB4. This consideration led us to test whether the Gim proteins bind to overproduced Tub4p. A gene fusion consisting of TUB4 and three repeats of the haemagglutinin (HA) epitope‐encoding sequence was used for this experiment. TUB4‐3HA was overexpressed in wild‐type cells (Figure 6C), followed by the immunoprecipitation of Tub4p‐3HA using anti‐HA antibodies. Interestingly, the precipitate not only contained Tub4p‐3HA (lane 3), but also Gim1p/Yke2p (lane 5), Gim2p/Pac10p (lane 7), Gim3p (lane 9) and Gim5p (lane 11). This co‐immunoprecipitation was specific as no, or hardly any, Gim protein was detected when the anti‐HA precipitation was performed with an extract containing overproduced Tub4p (lanes 6, 8, 10 and 12) instead of Tub4p‐3HA. Gim4p was not tested due to the lack of an antibody. In summary, our results show that GIM1/YKE2 becomes essential after TUB4 overexpression and that the Gim proteins bind to overproduced Tub4p.
The Gim proteins interact with each other and are present in common complexes
The interactions between the Gim proteins were investigated by immunoprecipitation experiments, using functional, epitope‐tagged versions of the Gim proteins (Figure 7A). For example, co‐immunoprecipitation of Gim2p/Pac10p‐MYC with Gim1p/Yke2p‐3HA was observed. Similarly, Gim3p‐MYC co‐precipitated with Gim2p/Pac10p‐3HA and Gim4p‐3HA precipitated Gim2p/Pac10p‐MYC. The result of the immunoprecipitation was confirmed using a functional gene fusion between GIM5 and protein A (GIM5‐ProA). Gim5p‐ProA was enriched from cell lysates with IgG–Sepharose. We detected not only Gim5p‐ProA in the precipitate, but also Gim1p/Yke2p, Gim2p/Pac10p, Gim3p and Gim4p‐3HA (Figure 7B). Similar results were obtained with Gim2p/Pac10p‐ProA (data not shown). Multiple interactions between the Gim proteins were also observed using the yeast two‐hybrid system (Fields and Song, 1989) (data not shown).
The results of the immunoprecipitations do not allow a decision to be made as to whether the Gim proteins form only one or several complexes. To address this point, yeast extract from wild‐type cells was fractionated by gel filtration (Figure 7C). Immunodetection of the Gim proteins revealed that Gim1p/Yke2p, Gim2p/Pac10p, Gim3p and Gim5p co‐fractionated over a wide range, suggesting that the complexes were heterogeneous in their composition. However, all four proteins peaked in the same fraction corresponding to an apparent mol. wt of ∼170–230 kDa. It is noteworthy that Gim2p/Pac10p was also detected in earlier fractions corresponding to mol. wts up to 2 MDa. In conclusion, the immunoprecipitation experiments demonstrate that the Gim proteins interact with each other and that they are present in common complexes.
The Gim complexes are localized mainly in the cytoplasm of yeast cells
In order to obtain a better understanding of the Gim complexes, we investigated their cellular localization. Localization studies by indirect or direct immunofluorescence using epitope‐tagged or green fluorescent protein (GFP) fusion proteins excluded a localization of the Gim proteins at the SPB. Instead, a cytoplasmic staining was observed (data not shown). This result was confirmed by fractionation experiments (Figure 8). Yeast cell lysates (lane 1) were separated by differential centrifugation into a soluble cytoplasmic fraction (lane 2) and a sediment (lane 3) containing nuclei, mitochondria and other organelles. The distribution of the α‐ and β‐subunits of the cytoplasmic fatty acid synthase (Fas) and the nuclear protein nucleolin (Nop1p) established that the two fractions were hardly contaminated by each other. Gim1p/Yke2p, Gim2p/Pac10p, Gim3p, Gim4p‐3HA and Gim5p were detected predominantly in the cytoplasmic fraction (lane 2). Taken together, the localization studies suggest that the Gim complexes are localized predominantly in the cytoplasm of yeast cells.
The mammalian homologues of Gim1p/Yke2p and Gim2p/Pac10p are fully functional in yeast
Gim1p/Yke2p is 47% identical to the mouse KE2 gene product (Shang et al., 1994) and Gim2p/Pac10p is 43% identical to the human VBP‐1 protein (Geiser et al., 1997). We were interested to see whether the KE2 and VBP‐1 gene products can substitute for Gim1p/Yke2p and Gim2p/Pac10p in yeast. The KE2 and VBP‐1 genes were amplified by PCR from a mouse and a human cDNA library, respectively. Both genes were cloned behind the yeast ADH promoter. Like plasmid‐encoded GIM1/YKE2, the ADH–KE2 promoter fusion complemented the synthetically lethal phenotype of Δgim1/yke2 tub4‐1 (data not shown) as well as the cold‐ and benomyl‐sensitive growth defects of Δgim1/yke2 cells (Figure 9A). Similar results were observed with the human VBP‐1 gene using the Δgim2/pac10 mutant (Figure 9B). Our results indicate that the Gim proteins are not only structurally conserved, but that they also fulfil very similar functions in mammalian and in yeast cells.
Gim1p/Yke2p, Gim2p/Pac10p, Gim3p, Gim4p and Gim5p interact with each other and form complexes
In this study, we performed a genetic screen for mutants that are not viable in combination with a mutated TUB4. We expected to find genes coding either for components of the Tub4p complex or for proteins that are involved in the folding of Tub4p or the assembly of the Tub4p complex. Since the Tub4p complex is associated with SPB substructures that are located in the nucleus (inner plaque) and in the cytoplasm (outer plaque) (Rout and Kilmartin, 1990; Spang et al., 1996; Knop et al., 1997), we could also expect to identify factors that play a role in the nuclear import of the Tub4p complex (G.Pereira et al., 1998). Finally, we expected to find either α‐ or β‐tubulin, since it has been proposed that these proteins interact with γ‐tubulin (Oakley, 1992).
Our genetic screen identified SPC98 and SPC97, which was not surprising, since previous results indicated that they interact genetically with TUB4 (Geissler et al., 1996; Knop et al., 1997). In addition, we identified five novel genes that we named GIM1–GIM5. Two of the GIM genes have been described previously: YKE2 was analysed due to its homology to the mouse KE2 gene, located on the mouse MHC locus (Shang et al., 1994), and PAC10 was found in a screen for genes which become essential in the absence of CIN8, encoding a kinesin‐related protein (Geiser et al., 1997). The co‐immunoprecipitation and the fractionation studies indicate that all five Gim proteins function together and that they are part of either one complex or several complexes containing different Gim proteins and possibly as yet unidentified proteins. In retrospect, our screen was quite selective, as it only identified components of the Tub4p or the Gim complexes.
Gim proteins have multiple functions related to the yeast cytoskeleton
All GIM deletion mutants had a reduced level of α‐tubulin, while the amounts of β‐tubulin and Tub4p were normal. The imbalance of α‐ and β‐tubulin results in an elevated pool of free β‐tubulin which is probably the cause of the microtubule defects. Our conclusion is supported by the observation that the deletion of TUB3, coding for the minor α‐tubulin in yeast, also results in benomyl super‐sensitivity (Schatz et al., 1986b) and that overexpression of TUB2, coding for β‐tubulin, is toxic (Katz et al., 1990). We reasoned, that any manipulation that increases the amount of α‐tubulin or decreases the pool of free β‐tubulin should suppress the tubulin defects. This was indeed the case, as overexpression of TUB1 or RBL2, both of which reduce the level of free β‐tubulin (Archer et al., 1995), cured the benomyl super‐sensitivity of the gim null strains. We also believe that the synthetically lethal defects of GIM deletions with Δrbl2 are the result of too little α‐tubulin, as loss of Rbl2p will increase the pool of free β‐tubulin further, which is then lethal for the cells (Katz et al., 1990; Archer et al., 1995). Furthermore, the reduced level of α‐tubulin in Δpac10 cells may explain why this gene becomes essential in the absence of the microtubule‐binding protein Cin8p (Geiser et al., 1997). Alternatively, Pac10p may have additional, motor protein‐related functions.
GIM deletion mutants are slightly more sensitive towards the actin inhibitor latrunculin‐A, than tcp1‐1 which codes for a mutated subunit of TRiC. Besides having other functions, the latter plays an essential role in actin folding (Ursic and Culbertson, 1991; Chen et al., 1994; Miklos et al., 1994; Vinh and Drubin, 1994). An additional hint of a function for the Gim proteins in actin‐related processes comes from the sensitivity of GIM deletion mutants towards high osmolarity, which is also a common phenotype of actin mutants (Drubin, 1990). Since we did not observe direct defects of the actin cytoskeleton in gim null mutants and since latrunculin‐A is highly specific towards actin (Ayscough et al., 1997), the actin defects in the gim strains are probably only subtle.
A number of results point to specific functions of the Gim proteins with respect to Tub4p. First, all GIM genes genetically interact with TUB4. Second, GIM1/YKE2 shows allele‐specific genetic interactions with SPC98 and SPC97, coding for components of the yeast γ‐tubulin complex. Third, overexpression of TUB4 or co‐overexpression of TUB4, SPC98 and SPC97 is toxic in a gim1/yke2 null strain, while it does not affect growth of wild‐type cells (Spang et al., 1996; Knop and Schiebel, 1997). This suggests that the GIM genes become essential in the presence of a mutated Tub4p protein or after overproduction of Tub4p. Under the latter condition, we could show that the tested Gim1p/Yke2p, Gim2p/Pac10p, Gim3p and Gim5p proteins were associated with Tub4p.
Do all of the Gim proteins have the same function? The very similar phenotypes of the gim null strains as well as complex formation suggest that the Gim proteins have at least overlapping functions. However, we also obtained evidence for specialized functions of the Gim proteins. For example, only Δpac10 cells but not the other Δgim mutants have a spore germination defect (Geiser et al., 1997). In addition, Δgim4 cells have a less severe growth defect and are not as osmotically sensitive as the other GIM deletion mutants. Finally, GIM1/YKE2, GIM2/PAC10 and GIM4 interact differently with SPC98 and SPC97.
The GIM genes encode phylogenetically conserved proteins which may function in folding or assembly processes
How do the Gim proteins function? The cellular localization, the effects of gim null mutants on the actin and tubulin systems as well as the phylogenetic conservation of the Gim proteins are reminiscent of the cytoplasmic chaperonin TRiC. Our localization studies suggests that the Gim proteins, like the TRiC proteins, form cytoplasmic multiprotein complexes. While TRiC contains eight subunits (Kubota et al., 1995; Willison and Horwich, 1996; Nitsch et al., 1997), it is still unclear whether additional Gim proteins exist. The tubulin defects of TRiC mutants and gim null strains are remarkably similar. TRiC mutants are also frequently super‐sensitive towards benomyl, and their microtubules depolymerize at reduced temperatures. In addition, some of the TRiC mutants also do not arrest in the cell cycle, although microtubule structures are impaired (Ursic and Culbertson, 1991; Chen et al., 1994; Miklos et al., 1994). tcp1‐1 was similarly sensitive towards latrunculin‐A as the gim null strains. Finally, a similar phylogenetic conservation as for the Gim proteins has been observed for the subunits of TRiC (Kubota et al., 1995; Nitsch et al., 1997): like TRiC, the Gim proteins have relatives in human, mouse, C.elegans and S.pombe (Figure 1). As for the Gim proteins, only two homologues of TRiC subunits were identified in archaebacteria and none in eubacteria. In addition to this, the Gim proteins as well as the subunits of TRiC bear a stronger homology to their relatives from other species than to each other (Kubota et al., 1995; Waldmann et al., 1995; Nitsch et al., 1997).
The Gim complexes may play a role in the biogenesis of α‐ and γ‐tubulin and probably actin, possibly as a molecular chaperone. A number of our results are consistent with this notion. First, the GIM genes show genetic interactions with components of TRiC, which is expected if both complexes function in related processes. Secondly, the Gim proteins bind to overproduced Tub4p. Thirdly, the reduced level of α‐tubulin may be the consequence of a folding defect. Fourthly, the phenotypes of TRiC and GIM mutants are very similar. The Gim complexes may assist protein folding either before or after TRiC function. They could also bind to folded monomeric Tub4p or actin, thereby reducing potential toxic effects of the monomers. In any case, the Gim proteins only become essential for growth under certain circumstances, such as in the presence of a mutated Tub4p protein or overexpressed Tub4p.
Our results demonstrate that the mammalian homologues of Gim1p/Yke2p and Gim2p/Pac10p, the KE2 and VBP‐1 gene products, are functional in yeast. Their expression complemented the synthetically lethal defects of Δgim1/yke2 or Δgim2/pac10 with tub4‐1 as well as the benomyl super‐sensitivity and cold sensitivity. Interestingly, KE2 mRNA is most abundant in brain and testis (Abe et al., 1988), tissues which have a high content of tubulin. It is expected that these tissues have a high level of proteins that are required for tubulin biogenesis, as is the case for TRiC (Silver et al., 1979, 1987). The homologue of Gim2p/Pac10p, the VBP‐1 protein, has been identified in a two‐hybrid screen using the von Hippel–Lindau tumor suppressor gene as a bait (Tsuchiya et al., 1996). Whether this interaction has any physiological significance remains to be determined. Homologues of Gim3p and Gim5p were also found; however, we have not yet tested whether they function in yeast. The fact that the KE2 and VBP‐1 proteins can substitute for Gim1p/Yke2p and Gim2p/Pac10p in yeast raises the possibility that the homologues of the Gim proteins also form complexes that promote the formation of functional α‐ and γ‐tubulin in mammals and other species.
Materials and methods
Media and general methods
Basic yeast methods and growth media were as described (Guthrie and Fink, 1991). For Gal1‐controlled gene expression, yeast strains were grown in synthetic complete medium (SC) containing raffinose (2%) as carbon source. Glucose (2%) or galactose (2%) was added for the repression or induction of the Gal1 promoter, respectively. Yeast strains were transformed by the lithium acetate method (Schiestl and Gietz, 1989). Escherichia coli strains were transformed by electroporation (Dower et al., 1988). PCR was performed with Vent polymerase (New England Biolabs). Recombinant DNA methodology was performed as reported (Sambrook et al., 1989).
Plasmids and yeast strains
Plasmids and yeast strains used and constructed during the course of this study are listed in Table III. The mouse KE2 gene and the human VBP‐1 gene were amplified by PCR from a mouse and human cDNA library, respectively and the PCR products obtained were cloned into p415‐ADH. Epitope‐tagged versions of GIM genes were generated by recombinant PCR, cloned into pRS315 (Sikorski and Hieter, 1989) and tested for functionality in gim mutants. The coding regions of the intron containing the GIM4 ORF were amplified by PCR from chromosomal yeast DNA and cloned into p415‐ADH (Mumberg et al., 1995). The intron of GIM5 was removed by recombinant PCR. The coding region of GIM5 was cloned into expression vector p415‐ADH. Double deletion mutants of Δgim1/yke2 together with any one of Δgim2/pac10, Δgim3, Δgim4 or Δgim5 were generated by subsequent deletion of the respective ORFs using either kanMX4 (Wach et al., 1994) or HIS3MX6 (Wach et al., 1997) as selectable markers. Chromosomally integrated C‐terminal fusions of the GIM genes with GFP, protein A and 3HA were generated by homologous recombination with PCR‐amplified GIM‐GFP‐kanMX4 (Wach et al., 1997), GIM‐ProA‐kanMX4 and GIM‐3HA‐kanMX4 cassettes (E.Schiebel, unpublished). TUB1 and RBL2 were amplified by PCR and cloned into p415‐ADH and pRS425 (Christianson et al., 1992), respectively.
Isolation of mutants which are synthetically lethal with tub4‐1
In order to identify mutants which are synthetically lethal with tub4‐1, we used the ade2/ade3 red/white colony sectoring system (Koshland et al., 1985; Huffaker et al., 1987). The haploid strain K2348 (ade2 ade3) was transformed with an ADE3/URA3 plasmid containing TUB4 (pSG24). This plasmid had a mutated centromere rendering it unstable under non‐selective growth conditions. The chromosomal TUB4 gene was disrupted by insertion of the HIS3 marker using the Δtub4::HIS3 cassette of plasmid pSM219 (Spang et al., 1996). This strain was transformed with a TRP1‐based plasmid carrying the temperature‐sensitive tub4‐1 allele (pSG23), yielding strain SGY33. The tub4‐1 allele harbours a single amino acid substitution which causes cells to die at 37°C while they stay alive at 30°C (Spang et al., 1996). Strain SGY33 grows with red colony colour, since it is genotypic ade2, resulting in the accumulation of a red intermediate of the adenine biosynthetic pathway. Loss of the ADE3 plasmid pSG24 under non‐selective conditions changes the genotype from ade2 to ade2 ade3, resulting in white or red/white sectoring colonies. Strain SGY33 was mutagenized with methane sulfonic acid ethyl ester (EMS, Sigma) to ∼30% survival. Mutations that conferred synthetic lethality with tub4‐1 were identified on plates without selection for the plasmid pSG24 as red, non‐sectoring colonies, since these cells depend on TUB4 of the ADE3‐containing plasmid. Among 60 000 screened colonies, 12 colonies with this phenotype were obtained. These were unable to grow on plates containing 5‐fluoro‐orotic acid (5‐FOA), a drug that kills cells harbouring a URA3 gene. They all regained the red/white sectoring phenotype and their ability to grow on 5‐FOA when transformed with a LEU2‐based plasmid containing the TUB4 gene but not when transformed with a plasmid carrying tub4‐1. Although all 12 mutants showed a red/white sectoring phenotype when transformed with an SPC98‐containing plasmid, it was shown by crossings to strain SGY88 (SPC98‐LEU2) and subsequent linkage analysis (see below) that the mutation causing synthetic lethality resided only in two cases in SPC98. In contrast, the red/white sectoring phenotype of the other mutants was due to suppression of the tub4‐1 phenotype by SPC98 (Geissler et al., 1996). The mutants were back‐crossed twice to strain SGY24, which revealed that the phenotypes were caused by a single, recessive mutation. Crosses between the mutants identified seven complementation groups.
Identification and disruption of the GIM genes
Two of the mutants from our screen were complemented by SPC97, recently identified as the third component of the γ‐tubulin complex in yeast (Knop et al., 1997). The other eight mutants were transformed with DNA from a LEU2/CEN‐based yeast genomic library (kindly provided by Dr K.Nasmyth), and the transformants were screened for regaining of the red/white sectoring phenotype. The genomic inserts of plasmids allowing cells to lose the TUB4‐containing ADE3/URA3 plasmid after re‐isolation and re‐transformation were sequenced. Subcloning and sequencing identified five genes which we named GIM1–GIM5.
The GIM, SPC98 and SPC97 genes on pRS305 (Sikorski and Hieter, 1989) were targeted to their chromosomal locations in strain SGY24. The resulting strains were then crossed with the corresponding mutants. Random spore analysis of the diploid strains revealed that, for example, GIM1 was linked to the mutation conferring the synthetically lethal phenotype with tub4‐1, since all sectoring haploid progenies did not contain the labelled GIM1‐LEU2. The GIM genes were disrupted by insertion of the heterologous markers kanMX4 (Wach et al., 1994) or HIS3MX6 (Wach et al., 1997), resulting in the deletion of the entire ORFs. The kanMX4 and HIS3MX6 disruption cassettes were generated by PCR and transformed into diploid YPH501 or haploid YPH499 (in the case of GIM2/PAC10). The deletion of the GIM genes in the resulting strains was confirmed by PCR.
Anti‐Gim1p/Yke2p, anti‐Gim2p/Pac10p, anti‐Gim3p, anti‐Gim5p antibodies, immunoblots, immunoprecipitation experiments and immunofluorescence microscopy
The entire coding regions of GIM1/YKE2, GIM2/PAC10, GIM3 and GIM5 were cloned into pGEX expression plasmids (Pharmacia). The GST fusion proteins were expressed and purified by affinity purification using glutathione–Sepharose according to the manufacturer's recommendations. Antibodies against the purified proteins were raised in rabbits as described (Harlow and Lane, 1988). Antibodies against Gim1p/Yke2p and Gim2p/Pac10p were affinity purified as described (Spang et al., 1995). The specificity of the antibodies was demonstrated by performing immunoblots with extracts from GIM and Δgim strains.
For immunoprecipitations of the Gim proteins, cells of strains carrying 3HA‐ or MYC‐tagged derivatives were grown in selective media at 30°C to a density of 2×107 cells/ml. Washed cells were resuspended in H‐buffer [50 mM HEPES, pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 1 mM EGTA, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM benzamidine, 10 μM leupeptin, 20 μg/ml chymostatin and 20 μg/ml E64] and lysed by vortexing with glass beads on ice until >95% of the cells were lysed. Cell debris were removed by centrifugation (5 min, 20 000 g). Cell lysates were incubated with anti‐HA antibodies (12CA5) covalently bound to protein A–Sepharose for 3 h at 4°C. The anti‐HA antibodies were cross‐linked to protein A–Sepharose as reported (Harlow and Lane, 1988). The precipitates were washed twice with H‐buffer, resuspended in HU‐buffer (Knop et al., 1996) and heated for 10 min at 65°C prior to SDS–PAGE (Laemmli, 1970). Gim5p‐ProA was precipitated identically using IgG–Sepharose. As molecular weight standards for SDS–PAGE, we used myosin (200 kDa), β‐galactosidase (116 kDa), phosphorylase B (97.4 kDa), serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), lysozyme (14.4 kDa) and aprotinin (6.5 kDa).
For the overexpression of TUB4‐3HA and TUB4 from the Gal1 promoter, yeast cells were grown in SC medium with 2% raffinose as sole carbon source at 30°C to a cell density of 5×106 cells/ml. The Gal1 promoter was induced by the addition of 2% galactose for 6 h. Harvested cells were resuspended in L‐buffer (20 mM Tris–HCl, pH 7.5, 100 mM NaCl, 10 mM EDTA, 1 mM EGTA, 5% glycerol, 1 mM GTP and protease inhibitors) and lysed with glass beads as described above. Cell lysates were incubated with anti‐HA antibodies covalently bound to protein A–Sepharose for 1 h at 4°C.
For the quantifications of α‐tubulin, β‐tubulin and Tub4p, cells were lysed as described by Knop et al. (1996). The primary antibodies used for immunoblots were mouse monoclonal anti‐HA (12CA5, Hiss Diagnostics), mouse monoclonal anti‐MYC (9E10; Boehringer Ingelheim), rabbit anti‐Gim1p/Yke2p, rabbit anti‐Gim2p/Pac10p, rabbit anti‐Gim3p, rabbit anti‐Gim5p, rabbit anti‐Tub4p (Geissler et al., 1996), rabbit anti‐α‐tubulin, rabbit anti‐β‐tubulin (α‐ and β‐tubulin antibodies were a gift of Dr F.Solomon), rabbit anti‐Nop1p (Dr E.Hurt) and rabbit anti‐Fas antibodies (Dr R.Egner). As secondary antibodies, rabbit anti‐mouse or goat anti‐rabbit IgGs coupled to horseradish peroxidase (Jackson Immuno Research Laboratories) were used. The immunoreaction was visualized by an ECL Kit from Amersham.
Immunofluorescence of formaldehyde‐fixed yeast cells was performed as described (Knop et al., 1996) with 1 h fixation time. The primary antibodies were mouse monoclonal anti‐β‐tubulin WA3 (kindly provided by U.Euteneuer‐Schliwa) or mouse monoclonal anti‐actin (Amersham). Secondary antibodies were goat anti‐mouse IgGs coupled to CY3 (Jackson Immuno Research Laboratories). DNA was stained with DAPI (Boehringer Mannheim).
Nocodazole arrest, flow cytometry, determination of cell size and protein determination
For nocodazole arrest, cells were treated with 15 μg/ml nocodazole (Sigma) in liquid medium at 30°C for 3 h. For flow cytometry, cells were prepared as reported (Hutter and Eipel, 1979). The DNA content of 20 000 cells was determined using a flow cytometer (Facscalibur, Becton‐Dickinson). Mean cell volumes were determined using a CASY1 Cell Counter and Analyzer System (Schärfe System GmbH). Protein concentrations were determined according to Bradford (1976).
Size exclusion chromatography
Cell lysates of YPH499 were prepared in H‐buffer as described above. Proteins were separated according to their apparent size using a Smart System from Pharmacia with a Superose 6 column (PC 3.2/30). The column was equilibrated with H‐buffer, and molecular weight determination was carried out according to a calibration curve obtained with standard proteins (thyroglobulin, apoferritin, β‐amylase, alcohol dehydrogenase, bovine serum albumin, ovalbumin, carbonic anhydrase, RNase A, blue dextran; Sigma) separated in H‐buffer. Separation was performed with a flow rate of 15 μl/min. The eluate was collected in fractions of 50 μl each. Each three consecutive fractions were pooled and analysed by immunoblotting.
We are very grateful to Dr A.Lupas for sequence analysis of the Gim proteins and to Dr D.Jenne for the PCR amplification of VBP‐1. Katrin Grein, Elvira Glatt and Tanja Hilsendegen are acknowledged for excellent technical assistance. We would like to thank Dr F.Solomon for the gift of anti‐α‐tubulin and anti‐β‐tubulin antibodies and Drs D.Botstein, K.Nasmyth and T.Stearns for providing us with strains. We thank S.Elliott and Dr M.Knop for carefully reading the manuscript and Dr F.‐U.Hartl for helpful discussions. This work was supported by a grant of the BMBF.
- Copyright © 1998 European Molecular Biology Organization


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One of Many Odd Script Ideas
July 10th, 2010 19:05 pm | by Ed |
An exploration ship encounters a relentlessly hostile alien. After a battle in which they take damage to most ship systems they finally manage to out maneuver the enemy ship long enough to deal it a crippling blow, they escape.
They're only able to get about two days head start on the alien ship when they are forced to stop and make repairs before their warp engines fail completely. The only habitable planet within range is inhabited by a civilization that appears to be in the early stages of deep space exploration in spite of the fact that they have fairly advanced technology.
The natives of this planet are unwilling to allow them to approach the planet at all and warn them that automatic defenses will destroy them if they attempt it. Soon after this the planetary government stops responding to attempts to communicate.
The damaged ship dares not approach the inner planets of the system because they detect automated defense systems that are routinely targeting and destroying even small asteroids and space debris that moves within range and the ships defenses are unable to withstand such an attack in their damage condition.
Monitoring communications within the system, it's discovered that the locals have very good reason for the extremely heavy defense system. The race that lives here gains weight at a steady pace until, once every two years, they all go on the most severe and debilitating quick weight loss diets to return to a healthy weight and during this time they are physically incapacitated and incapable of any kind of defense for nearly a full month at a time.
The automated defense network was built and deployed as soon as they discovered the hostile race that the exploration ship encountered when they learned from neighboring races that this hostile would do anything it could to interrupt their lifecycle and halt their weight loss efforts, turning them into a planet of overweight, sluggish beings that would be used as food by the voracious hostiles.
Technorati Tags: science fiction, short story, script idea

subset: commoncrawl


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A big ambulance replacement drive is nearing its conclusion across the East of England.
The programme which began in early 2014 sees 267 emergency ambulances being replaced across six counties – including here in Hertfordshire - with 27 additional ambulances for the fleet.
By the end of next month there will be no emergency ambulance older than five years.
The huge undertaking is part of the East of the England Ambulance Service NHS Trust (EEAST) six-point plan to improve patient care.
The ambulances come with a full set of new medical devices and consumables which altogether means better, more reliable transport for staff and patients with a full stock of equipment fit for purpose.
Earlier last year, 147 ambulances were introduced around the region - 120 were replacements and 27 were new ones.
Since September, another 120 replacement ambulances were purchased and the remaining 37 will be delivered into operational service by the end of next month.
Trust chief executive Anthony Marsh, who introduced the plan said: "I am delighted that the fleet modernisation programme has remained ahead of schedule.
"Our ageing fleet was a real concern amongst staff when I joined EEAST last year, but to see a new fleet under five years old with new equipment on board by the end of March is an excellent achievement.
"These new emergency ambulances will be more reliable and provide a better experience for patients and crews.
"I would like to thank our fleet and medical devices teams for their hard work getting these new vehicles on the road."
The fleet was also well prepared for winter as they kept 16 ambulances scheduled for disposal.
Snow socks for all operational vehicles enabled them to be used in more treacherous conditions, as did windscreen protectors, snow shovels and long-handled scrapers.
Paul Henry, head of operations support said: "I am pleased to see that we have transformed our fleet over the last busy 13 months and we are moving ahead with the next ambulance and response car replacement requirements for 2015/16 and 2016/17.
"We will ensure that ambulance and car replacement is delivered in time with the trust's agreed life cycle for ambulances and cars which is now five years and three years respectively.
"Again, all vehicles will be equipped with new medical devices to maintain the standard of devices available to patients and staff."


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Gold Mining Museum
& Family History Research
World War I
Waihi's association with World War I.
1912 Waihi Strike Commemorations
These events are being held in Waihi during the commemoration of the 1912 Miners' Strike. More events and event details will be posted as we have them.
WACMA 50th Jubilee - 2012
Fifty years ago, Waihi Arts Centre and Museum Association came into being.
How it all began...
The transformation from the Technical School building to the Waihi Arts Centre and Museum.
Family History Research
The Waihi Arts Centre and Museum Association (WACMA) have a volunteer research team.
Waihi History
From gold mining to tunnelling and electronics, explore Waihi's rich history.
Photo Gallery
A taste of some of the many historic photographs that have now been digitally archived by the Waihi Museum.
Videos on the history of Waihi and the surrounding district.
Books For Sale
Explore the colourful history of Waihi in our range of books about the region's past.
DVDs For Sale
Purchase DVD's on gold mining and the railway in the early days of Waihi.
Do you know?
Help us to identify and provide some history for some of the items in our collection...
Here's a sample of some digitized maps from our archive.
Waihi Arts Centre and Museum is a community asset which is managed by volunteers. New members are always welcome.


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Status: Closed
Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Drive in for anything from oil changes or a tune-up to new tires and brakes for foreign or domestic vehicles.
We have 3 Locations to help with all your car care needs.-please visit our Towing Center, Car Care/ Mech.Repair Center & Collision Center Pages for More Details.
Collision Center www.facebook.com/WestlandCarCareAutomotiveGroup-Collision Center
Towing Center
Westland Car Care Center is your best source for quality maintenance and repair for your car or truck. Our team of professional technicians are ASE-certified and provide expert care for your vehicle. Our business takes quality and customer service seriously. As a NAPA AutoCare Center, we follow a strict Code of Ethics so customers will know up front what to expect.
Be the first MLive.com user to add photos or videos of Westland Car Care Center
Drive in for anything from oil changes or a tune-up to new tires and brakes for foreign or domestic vehicles. Read More
Drive in for anything from oil changes or a tune-up to new tires and brakes for foreign or domestic vehicles.
I needed a jump after my little one must have left a light on in the car. The interaction with the driver was awful. He was ridiculously rude as if I wanted to be there and it wasn't his job to be there. I contacted the company and AAA. He'll get a slap on the wrist at best but if AAA pulls business they'll get a clue.
I have Progressive ins.road service,my sons car broke down today andtthey sent these Bozo`s to get it and tow it from M-14 and Beck rd.to my house in Southgat. AAccording to progressive they pay for 15 miles,thats find so the guy said it 4.00 a mile after that when they got to my house they charged my wife 80.00.and Google maps said from pick up point was 27.1 miles,by my math she iwed them...
Westland Car Care Center can be found at N Wayne Rd 7666. The following is offered: Auto Repairs. The entry is present with us since Sep 8, 2010 and was last updated on Nov 14, 2013. In Livonia there are 30 other Auto Repairs. An overview can be found here.
Learn about ACDelco's quality auto parts & vehicle service at ACDelco.com. Find replacement brakes, filters, spark plugs, shocks & struts for your car


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NEW 2016 TRAILER http://youtu.be/ki572gdKG6o
QUOTES & REVIEWS from audience members at MALCO Premiere Memphis screening:
"What a phenomenal story and a masterpiece of a movie!" "Beautiful music"
More at www.facebook.com/ChristmasRideReviews
SCORE Music by award-winning singer songwriters Danny Jones and other composers, many prize-winning.
RATINGS This film Rated PG by MPAA. Learn more on IMDB.com
AWARDS The script by Karen Francis (aka Leah Kia) earned a semi-finalist award from Charleston International Film Festival, 2013.
BOOKINGS Seeking additional theatrical distribution w/o NDA before PPV, VOD or CABLE, TV or DVD release.
CHRISTMAS RIDE is a dramatic 108 minute narrative feature film, a qualified Academy Award eligible film for Best Picture, 2015, and ready for the big screens of cinemas in a DCP [digital cinema print] format. See article in in The Wrap http://www.thewrap.com/oscars-announce-323-films-eligible-…/
WHAT'S IT ABOUT? After hearing a frightening supernatural warning, a woman takes an urgent road trip south at Christmastime with her two kids and a mysterious veteran haunted by ghosts from his past. She faces dangerous situations and ultimately learns a heartbreaking truth which tests the limits of forgiveness.
PRODUCED BY Karen E. Francis of Art Promotions in cooperation with Yarbrough Media Productions in TN, Christmas Ride had its first theatrical run in DCP 2K format at the Music Hall, one of the Laemmle theaters on Wilshire Blvd in Beverly HIlls, CA. This was a week long qualifying run with four screenings a day at a commercial theater.
THE DOVE FOUNDATION AWARDS SEAL OF APPROVAL, AGES 12+ (Click here to see Photo Blog for details)
BLOGS In addition to the Photo Blog on this site (see tab above), see christmasride.wordpress.com at http://www.facebook.com/ChristmasRide
Warning! Audience Reactions include smiles, laughs and tears.


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Disability Tax Credit
Do you qualify?
The Disability Tax Credit helps Canadians who are living with a severe or prolonged impairment in physical or mental functions, or who are caring for an individual with an impairment.
In order to qualify, physical or mental impairments must affect an individual's ability to perform daily activities and must last, or be expected to last, for a continuous period of at least 12 months. There are many impairments that qualify for this non-refundable tax credit, so it is best to check with our office if you have any questions.
To qualify for the Disability Tax Credit, individuals living with impairments, or their caregivers, must:
- Complete Form T2201, the Disability Tax Credit Certificate;
- Have the Disability Tax Credit Certificate certified by a qualified medical practitioner; and
- Have the Disability Tax Credit Certificate form approved by the Canada Revenue Agency
Please note that individuals are personally responsible for all expenses and fees that may be charged by a medical practitioner to fill out the Disability Tax Credit Certificate. Additionally, if you are currently receiving Canada Pension Plan disability benefits, workers' compensation benefits, or any other disability or insurance benefits, this DOES NOT necessarily mean you are automatically eligible for the Disability Tax Credit.
If you have any questions about the Disability Tax Credit, please feel free to contact our constituency office.


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Cemetery staff continue to assess and clear damage from yesterday's storm. Visitors are advised to avoid areas with large branches or fallen trees.
Effective October 26, 2016:
Bicycling will no longer be allowed on cemetery grounds without a family pass.
The primary purpose of ANC's roads are to facilitate funeral services, vehicles operating in the daily care of the cemetery, and families visiting the final resting place of loved ones. While we know that riders intend no disrespect, bicyclists traversing the cemetery grounds do impact funeral services and the experience that families expect and deserve as they visit their loved one's grave.
Additionally, there are legitimate safety concerns regarding bicyclists mixing with the volume of pedestrians who visit Arlington National Cemetery each day. As there are no bike paths on the cemetery grounds, mixing cyclists with pedestrians and vehicles creates a safety hazard. This new policy has been deemed necessary to alleviate these concerns.
Family members and guests desiring to visit a loved one's grave can still show their family pass and, with that pass, ride their bicycles to and from the grave site.
We thank you for your understanding and adherence to this new policy.
For more information call 1-877-907-8585.


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What are Amazon MTurk Scripts?
If you've ever worked on HITs on Amazon's MTurk, then it's likely you've experienced the same little inconveniences that many others have. All those extra clicks required on some pages can slow you down and waste your valuable time.
MTurk scripts can shave seconds (even minutes) off your in-between time, ultimately increasing your hourly wage.
Spripts are each designed by individual people as a solution to a problem–a problem that you've probably experienced yourself within MTurk. And they have even made a plugin to install and operate these scripts (for non-techies like us) so we don't have to learn how to write code!
We won't be getting into all of that technical stuff here.. just what you need to know:
- Which extension you'll need to download..
- Where to find your extension..
- Where to find your scripts..
- What each script can do for you..
Install your extension.. GreaseMonkey or TamperMonkey?
For Firefox, install: GreaseMonkey.
For Chrome, install: TamperMonkey.
Install Your Amazon MTurk Scripts..
Once you find your script and click install, it is automatically added to your GreaseMonkey or TamperMonkey Extension. You don't have to do anything.
Be sure to be working in the browser where you want the script uploaded. You can install the scripts to both Firefox and Chrome, but you'll need to do it twice.. once while you're in Firefox, and again while you're in Chrome.
Once they're installed, you won't notice it anywhere until you are actually on the MTurk website. Then they will show up on your MTurk pages and automatically start doing their job.
You can search for MTurk scripts (or Turking scripts) at GreasyFork, but here are some of the best ones that should fix you right up..
A very valuable tool that shows you what other Turkers think about the requester you're working for. It also allows you to rate the requester yourself, according to your own experience.
Block Requesters
You can use this script to hide all HITS from any specific requester that you do not want to work for. If the requester's work is not worth the pay, or if they have bad reviews and you choose to stay away from them, this is the script for you.
Block Individual Requests
Displays an X at the end of the line where it says: "View a HIT in this group". Click the X to hide any HIT you don't want in your search results, and it will disappear the next time your page is refreshed. You have to look carefully for the X though, it is very tiny.
Show Captcha and Accept Button
Hate having to wait for the entire page to load before the captcha or the Accept Hit button shows up? This script makes it load first, so you don't have to waste anymore time.
MTurk HIT DataBase Testing
The HIT Database Testing analyzes past HITs completed, as well as pending and projected earnings with a progress bar, Requester notes and specific Requester blocking.
MTurk Expanded Header
This script displays your account info on MTurk pages, including Worker ID and transferable earnings..
Requester ID and Auto-approval Time
This script cleverly displays the Requester's ID in case they decide to change their name and the HIT's auto approval time. I guess it happens!
This is a very cool tool that really comes in handy, once you learn how to use it. It adds a list of filters to the left of the account page, and it alerts you for each filter you activate. If you want to know when your favorite Requester lists new HITs, tell the TurkMaster and it will alert you right away. You can be notified when new high-paying HITs are listed, or of a certain qualification level. You can even change settings to alert you while you're not working on MTurk. In order to receive your notifications, however, you must always have a separate tab running your account page.
HIT Scraper WITH EXPORT
This is a pretty awesome script that does a great job of sorting through the HITs for you so you can get right to those that are worth your time. It might take you a little bit of time and patience to learn how to work it though. Here's a great step-by-step tutorial that will get you started with HIT Scraper
Pending Earnings
This script always displays the amount you are owed for the work you've done, and what you can expect to be coming in (as long as they're marked as successful by the Requester).
Today's Projected Earnings
Similar to pending earnings. It shows you your total day's earnings that you've worked for–what you should expect to be paid for.
MTurk Dashboard Change Notifier
Many people find this a useful addition to the MTurk account page. The Dashboard Change Notifier tracks changes of 12 different values from the last time you refreshed your page. This includes your earnings such as approved, bonuses, transferable, and your HITs status, the number of approved, pending, rejected, and so on. This can help you keep track of changes that aren't immediately noticeable without doing the math.
MTurk Time Tracker
This is a helpful script that shows you the number of HITS, times each HIT, and offers estimated reward and hourly rate for your latest
activity, as well as the projected earnings and hourly rate for the day. This works within the following Status Page Chart script..
Status Page Chart
This scripts adds a lot of substance to the Status section of "Your Account" page. It gives more detail to the table of earnings, including those that the MTurk Time Tracker (from above) keeps track of. It also gives a nice big line chart at the bottom of the page. Very helpful..
ID Copy/Paste
Don't you hate having to open a new tab every time a Requester asks for your Worker ID? This script places a simple little button on the page for you to easily copy your Worker ID, so you can place it within the HIT as requested. Can't believe Amazon didn't think to do this themselves!
Once you've installed all the scripts you want, you can see them in your GreaseMonkey or TamperMonkey plugin in the top right corner.
When you click on the icon, it will appear that no scripts are running, unless you are in the MTurk site. Then only certain scripts will be running on any given MTurk page, depending on what each is designed for.
TamperMonkey MTurk scripts running.
GreaseMonkey MTurk scripts running..
Are the MTurk scripts safe?
All of the scripts on this post are perfectly safe. They have been tested by many users over, and each one is running on my own computer right now.
However, some scripts can be harmful. Scripts are designed and created by real people, and some people like to be malicious for various reasons. Each script is really just a bunch of code telling your computer how to manipulate a web page.
Firefox and Chrome somewhat protect you by limiting the capabilities of the script. They do not have as much power as an actual extension or add-on does when it comes to your computer. But it is possible for a script to access any website, capture information you enter into web pages, and impersonate you. So, you should really do your research when you're looking to add a new script. Check out the number of installs by other users and any reviews or ratings you can find. If people are still installing it on a daily basis, it can probably be trusted.
Check out this great ebook.. It shows you how you can zero in on MTurk's BIG money..
Let me know with a comment if there are any other valuable scripts I'm missing..


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|Mayor's Office image of Rahm Emanuel jogging with police recruits in March 2017.
On September 21 last year, Emanuel promised that the city will replace every cop who retires and will further increase the department's manpower by 1,000 cops overall before the end of 2018.
Since Emanuel made his promise, Chicagoans have been awash in a seemingly endless river of footage showing the mayor at CPD graduation ceremonies and jogging with legions of new recruits. But, as of July 21, all of the hype had resulted in a net gain of just 37 cops.
|Emanuel inspects new police graduates in August 2017 | City of Chicago
Police units that saw the greatest increases in strength were the Area North and Area Central detective units; the recruit training division; and the Area Central Bureau of Patrol.
Ignoring "The Backbone"But the units that lost the most manpower are largely in what's called the "backbone of policing"—Chicago's 22 police districts. The district units are on the front line, handling day-to-day patrols and responding to 911 calls.
You can support CWB's newsgathering efforts with a donation to our Tip Jar.
In the violence-ripped Englewood District, there are 55 fewer cops than when Rahm promised to add 1,000 officers citywide. Englewood lost more cops than any other ongoing unit in the entire department, according to the city's data.
And the 10th (Ogden) District lost 38 officers; the 4th (South Chicago) lost 27 and the 2nd (Wentworth) is off by 26.
The 18th District, which protects the heart of Chicago's tourism district between the Chicago River, Lake Michigan, and Fullerton lost 21 cops. The 19th District, spanning from Fullerton to Lawrence and the river to the lake, lost 15.
Fewer cops translate directly to slower emergency response times and greater frequency of so-called "RAP" events. RAPS, or "Radio Assignment Pending" status means that a district does not have any police officers available to handle incoming calls for emergency service.
Shuddup About ItBut if you're frustrated with the fact that your local police district is losing cops while politicians keep talking about how many "new cops" there are, don't bring it up.
Across the city, police commanders—nearly all working with fewer officers than a year ago—have been instructed to shut up about their manpower needs.
At a recent 14th District Community Policing meeting, officers were forbidden to speak about district manpower numbers. Instead, they bounced the question to an alderman who was in attendance. The 14th District serves Logan Square, Avondale, Bucktown, and Wicker Park.
CWBChicago's FOIA letter sought the department's total manpower by unit on or about September 21, 2016, and on July 21, 2017, for comparison. Our request, filed on July 21, was answered on September 1, two days after CPD says it swore in 182 new cops.


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Wedding Photos
Real Weddings at Langrish House
Just to show you how lovely Langrish House can look in your Wedding photos, we have collected a few of our favourites.
Choosing a wedding photographer to capture your big day is probably one of the most important decisions you'll have to make when planning your wedding. The photographer's role is an important one, as he or she is responsible for creating lasting keepsakes for you and your family to cherish for years to come.
Three things to consider when choosing your photographer:
- Personality: Remember this person is going to be with you throughout your wedding day – it's important that you get along. When you meet him or her for the first time ask yourself: Do I like this person? Do I trust them to handle such an important day in my life? Do I feel comfortable with this person? Will my friends and family like him? Would I invite him or her to my wedding? If the answer to any of those questions is 'no' then you may need to find someone else.
- Style: Take a look at photographers' work. It's important that you like their style. Every photographer's work is different. Do their images move you? Do they tell the complete story? Are they artistic and creative? Are they real, and do the people look comfortable?
- Cost: After the wedding is over the most important reminder of the day will be your wedding photos. Your pictures are your memories. Try not to choose your photographer on price alone as you may regret it later. According to a survey run buy an online wedding website, the number one regret couples had is that they wished they had spent more money on their wedding photography. Get the best you can afford.
We have worked with many photographers at Langrish House who we can recommend. We have shown you a few examples above. However it is very important that you meet with one or more of them before you make your final decision. There are links to a few of our regular photographers on our Wedding Suppliers page.
wedding photos at langrish house


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Two clowns won, who will laugh? The crisis is not returning and also is. Britain vs. Greece.
Italian election results are known to all of you. Technocrat Monti leaves and he will be most probably replaced with a coalition hastily cobbled together under the slogan saying: virtue of necessity. And maybe there will be even early elections. Anyway, Italy is finally where it belongs – in the spotlights.
Economically the third largest member of the euro zone, it has a high potential to sink both ESM and EFSF just like stones. It already had its head in the loop twice, when first in the summer of 2011 and then a year later the yields on its bonds skyrocketed and the ECB had to intervene in order to calm down the situation by buying bonds, or by a promise to buy them. Although Italy has a primary surplus in the budget, it does little help, as the debt reaches nearly 130% of GDP, or two trillion euro in absolute terms, (after the U. S. and Japan, the Italian bond market is the largest in the world and is the largest in Europe).
What is even worse, the Italian economy has been stagnating for the last three decades. Since 1991, GDP grew at an average of 0.79% per year, which is slower figure than that in Japan and a negative record for a western economy at the same time. To make matters worse, the population of Italy is one of the oldest in the world, making the demographic pressure on the budget coming a little earlier than in the rest of Europe.
Italy cannot afford any stronger economic cough. Therefore, European leaders monitor anxiously what will happen in Italy after the election. Peer Steinbrück, a potential future chancellor of Germany, directly stated that two clowns won. President Napolitano offended by this, cancelled the meeting with him, but Berlusconi and Grillo caused concerns that Italy will cease to continue with reforms and will rather ask the German wallets for help.
One of the latest moves by the Italian Government was the introduction of the transaction tax that begins to be effective in Italy already on Friday. Let's see how the investors will respond. After a brief enthusiasm in December, when we saw a reverse of the deposit flow and money started coming to banks in the periphery again, the zeal now quickly cooled down. In January, money again fled from the banks.
The most afraid are Germans. Merkel's adviser said that the crisis has returned with a vengeance. Finance Minister Schauble said something similar, according to him, there is a real threat of contagion of the problems from Italy to other countries. Quite contrasting statements compared with reassurances from Barroso, Monti, Draghi, Lagarde, and latest also from Hollande, who do not forget to point out that the crisis is over even when they are ordering hamburgers.
Let the numbers to show us the situation rather than politicians talks. According to the latest forecasts from the Commission, six members of the euro zone will have debt above 100% of GDP in 2014. Of the seventeen countries only one will have a budget surplus (Estonia) and nine of the seventeen members will violate the 3% deficit rule (remember that this is a violation of the Fiscal compact?).
France also belongs to the sinners. They already requested a postponement, and Dutch decided to proceed similarly. They no longer intend to save, the reason is, as always, an "exceptional circumstances." French unemployment rate reached a record since 1997 in the meantime, making the whole government nervous. Industry Minister, Arnaud Montebourg, thinks that ECB should solve this by money printing. Although ECB has already thrown up one trillion euros to the system over the LTRO loans and bought bonds worth more than 200 billion, it seems too little to him.
Cyprus still ranks as a potential candidate for future assistance. In the election there, the president (from a communist party) was replaced by a conservative party candidate who is in favour of the EU rescue. This could be the ultimate break-point on the way for the request for bailout. Cyprus allegedly has enough money only until May. Also with another candidate for help, there was an exchange of power, but with an opposite effect. Slovenian Prime Minister stepped down due to a tax scandal; he is supposed to be replaced with a coalition not very keen on assistance from Brussels, wishing instead to spend on jobs and growth. Let's see whether they will have enough capacity to spend from, as in the middle of this year a € 2 billion of their debt matures, which is quite a large amount for Slovenia.
Thanks to two bankruptcies, Greece managed to reduce its debt by 62 billion in the year-to-year comparison. Still, debt reaches almost 160% of GDP and the social situation there continues to deteriorate. Foreign manufacturers will significantly reduce the supply of drugs, because the traffickers were buying these at regulated very low prices and solling them back again abroad with profits, not to mention the fact that health care facilities often did not pay for the drugs at all. People are angry and politicians cannot feel as safe as they once could. For example, the former mayor of the second largest Greek city Thessaloniki was convicted for life just a few days ago due to embezzlement of city's money.
Bleak weather is not only in the euro zone, Britain has had its rating downgraded by Moody's. There is no wonder as their public finances look worse than the Greek one.
The only hint of positive news came from Ireland. Eligible Liabilities Guarantee Scheme, a special guarantee mechanism introduced in 2008, ends now definitely. Like to the fund for deposits protection, the banks were paying there some little change and the state guaranteed their obligations in exchange. It was a simple mechanism for transferring the costs of incorrect decisions from shoulders of bank owners to those of taxpayers, which cost them 62 billion euros. To recap, Ireland population is about 4. 6 million. Unlike Spaniards, Iris paid the costs for the restructuring of their banking system by themselves via ELGS (like Slovaks and Czechs did at the beginning of the millennium, when it cost them about 10% of GDP), but they are doing everything possible to ensure that this created ultradebt would be passed through the ECB and possibly ESM to the whole Euro zone.
Please choose from these two provocations for the farewell: our campaign Richer Slovakia or review of European civil servants pensions.
A rich week wishes
Martin Vlachynský
(Translated by Jakub Pivoluska)
Fiat Euro! is English version of Euro Crisis weekly newsletter. It intends to inform about the latest development in eurozone from economic point of view.
What is Euro Crisis and what is INESS?
How can I support these activities?
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text: Pine Marten Small 320×259 Medium 640×517 Large 1024×827 Download TIFF Original 2400×1939 | (1.5 MB) Description The pine marten lives most of its life in trees, but does hunt on the ground. It feeds primarily on mice, squirrel, birds, frogs and fish. Keywords Mammalia, mammals, Martes americana, Pine Marten, sable Galleries Mammals: M-N Source: Chandler B. Beach The New Student's Reference Work for Teachers Students and Families (Chicago, IL: F. E. Compton and Company, 1909)


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HI ya ladies!! Just a quick hello to give you an update on my workouts. Yesterday was Leg day. I made it a bit more fun by counting and taking pics of each piece of "equipment" I used for my workout~ how many pieces of "equipment" did you use in your workout? I put my pics on my facebook– please follow me there if you don't already~
My tally was 19, can u beat me?
- Iphone app for interval timer
- hair band
- bra
- gloves
- water bottle
- visor
- I pod shuffle
- mirror
- bench
- MMA gloves
- mat
- curl bar
- dead lift rack
- Squat rack
- Knee extension
- Bicep curl (preacher curl)
- bike
- treadmill
I did a warm up on the bike then boxing intervals of running then hitting Bobx10's
then I did dead lifts, leg extensions, bicep curls, squats, knee ups, cats/dogs, sit ups and handstands
It was a good workout for yesterday~
Do you think that more equipment = a better workout or do you like keeping things more simple?
Be sure to post your tally!! and pics too if you can!!
Thanks, Michelle


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A good reminder on why pregnant and breast-feeding mothers need their vitamins
You know you're supposed to take your prenatal vitamin before and during pregnancy as well as when you are nursing, but you might not know exactly why and what your prenatal vitamin should have in it. Here's a great info graphic from LabDoor, which helps consumers research supplements.
In case you wondered where LabDoor got its information, here are the sources:
- Infographic designed by Jessie Orvidas (jessieorvidas.com)
- Co-author: Shoua Kue
- Effects of Prenatal Micronutrient and Early Food Supplementation on Maternal Hemoglobin, Birth Weight, and Infant Mortality among Children in Bangladesh: the MINIMat Randomized Trial – Journal of the American Medical Association
- Maternal Nutrition and Infant Mortality in the Context of Relationality – Joint Center for Political and Economic Studies Health Policy Institute
- The Role of Nutrition in Infant Mortality: a Public Health Perspective – Association of State Public Health Nutritionists
- Recommended Intakes – Institutes of Medicine
- Vitamins and Minerals During Pregnancy – March of Dimes
- Birth Defects – Child Health USA 2014
- Folate and Neural Tube Defects – The American Journal of Clinical Nutrition
- Folic Acid, Data, and Statistics – Centers for Disease Control and Prevention
- Prevention of Neural-Tube Defects with Folic Acid in China. China-U.S. Collaborative Project for Neural Tube Defect Prevention – New England Journal of Medicine
- Association Between Maternal Use of Folic Acid Supplements and Risk of Autism Spectrum Disorders in Children – The Journal of the American Medical Association
- Prenatal Vitamins: a Review of the Literature on Benefits and Risks of Various Nutrient Supplements – Anna K. Sfakianaki, MD, MPH, ModernMedicineNetwork
- New Recommendations Call for Iodine in All Prenatal Vitamins – Massachusetts General Hospital
- Iodine in Prenatal Vitamins – Medscape
- Vitamin D and Pregnancy – American Pregnancy Association
- Iodine Deficiency – American Thyroid Association


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Frenetic game where you're going to need a lot of skill to dodge all the obstacles and traps until you reach the door leading to the next level. You have to reach all the platforms by jumping really fast and throwing yourself at them to dodge the enemy robots' bullets.
Copyright (c) 2005-2019 | www.minigamers.com | Privacy policy, conditions of use and legal matters


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[Caml-list] Why systhreads?
Lauri Alanko
Sven Luther
Chris Hecker
- Basile STARYNKEVITCH
- Chris Hecker
- Vitaly Lugovsky
- Christopher Quinn
- Xavier Leroy
- Sven Luther
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|2002-11-24 (18:13)
|Basile STARYNKEVITCH <basile@s...>
|Re: [Caml-list] Why systhreads?
>>>>> "Chris" == Chris Hecker <firstname.lastname@example.com> writes: Chris> On a related note, now that the first CPUs with Chris> HyperThreading are shipping, is there any plan to Chris> multithread the GC so caml programs can take advantage of Chris> HT? I can understand why it was not a high priority to Chris> support real threads for multiprocessor machines when that Chris> was the only way to get parallelism with threads, but once Chris> HT is ubiquitous, it has the potential to make it worth the Chris> trouble to thread a regular application to increase Chris> performance. I don't think this is a high priority now, Chris> because there's 0% penetration of HT right now, but Chris> hopefully there's some plan for the future. I would suppose that HyperThreading chips can already successfully being used on Linux (I was told, IIRC, that such chips gives 2 cpu when asked thru /proc/cpuinfo). So, I would suppose that they can take advantage of native threads on Ocaml already. Chris> I guess the question is, is a multithreaded GC an open Chris> research problem, or is there a known good solution and it Chris> just hasn't gotten to the top of the priority list yet? I have a question to the Ocaml team : Could they explain what is the current (and perhaps near future) status of multithreading in Ocaml, notably with respect to garbage collection? I thought that the current GC in Ocaml (3.06) is already multithread-capable, at least because each thread has his own birth region and can do minor garbage collections independently of other threads, so threads have to synchronize only on major (ie full) garbage collections. Is my assumption correct? I just had a glance into ocaml/byterun/minor_gc.c and did not found any thread-local variables there... Also, ocaml/otherlibs/systhreads/posix.c mentions /* The global mutex used to ensure that at most one thread is running Caml code */ What is (with ocamlopt -thread on x86/linux) the use of multithreading in Ocaml? I really thought that it was really (in practical terms) biprocessor support, not only some limited kind of (throwable once only) continuations? Regards. -- Basile STARYNKEVITCH http://starynkevitch.net/Basile/ email: basile<at>starynkevitch<dot>net alias: basile<at>tunes<dot>org 8, rue de la Faïencerie, 92340 Bourg La Reine, France ------------------- To unsubscribe, mail firstname.lastname@example.com Archives: http://caml.inria.fr Bug reports: http://caml.inria.fr/bin/caml-bugs FAQ: http://caml.inria.fr/FAQ/ Beginner's list: http://groups.yahoo.com/group/ocaml_beginners


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Servings are based on 1.5"x2" pieces
*Cakes that have pieces pre sectioned (such as the rose bud and baby shower cake seen in the gallery) are 2"x2" pieces which means less servings
Flavors:Basic white, chocolate, strawberry, marble, or yellow
Double layer 8" round 20 servings
Double layer 9" round 24 servings
9x13" sheet cake 39 servings
11x15" sheet cake 55 servings
12x18" sheet cake 72 servings
Smash cakes for babies
Cupcakes 24 cupcakes
11 1/2" diameter decorated cookie
Chocolate chip or sugar cookie
The cupcake tower shown below can be rented for your next event. It holds up to 72 cupcakes. The ribbon around the edges can be changed out to match your event colors.
Rental will be for 48 hours unless other arrangements are made.
A $50 deposit will be held and returned to you upon the return of the undamaged tower.
The actual rental cost with cupcake order is $5.


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Do you like zombies? If you do, you've come to the right place! If you don't, well...you should read this anyway. Very soon - next week in fact! - the second annual Blogger Zombie Walk will take place, and this year we're taking part too, so you know it's going to be amazing ;P
Bloggers that want to participate can click the picture above to see full details on the Ghost Hunting Theories blog. Once you've registered, you'll need to make a zombie-themed post on Friday, 30th September, with a list (and link to) all the other participating blogs along with it. The list will be in the same order at every blog, so anyone who reads each post can easily follow the "zombie walk" to the next blog.


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Digital Marketing
Customer Experience
Chief Digital Officer
NRMA Insurance opens Crashed Car Showroom in Sydney
NRMA Insurance has combined Oculus Rift virtual reality with a physical hydraulics system for a simulation that's both fun and frightening.
The author of this article (pictured) marvels at his virtual-reality arms.
We swear that the car used in the simulation was damaged before we arrived.
The Crashed Car Showroom is located on York St in the Sydney CBD. You won't find new cars sold here.
On the show floor.
In addition to crash test simulation, the showroom features a distracted driving test that uses eye-tracking software to test whether users can hold their attention on the road while driving 60km/h – even when an SMS suddenly comes in.
The test attempts to fool the user into answering their text message. Fail to keep your eyes on the road and the test comes to a crashing halt.
Another area of the exhibit projected images on a car showing damage caused by a crash.
This Peugot was sawed into three pieces to show various safety features.
Each part of the car is embedded with Near Field Communications (NFC) chips that can load video on interested users' smartphones.
Car lovers may wish to look away.
A hail gun lets the driver switch places and see weather damage from the angry storm cloud's perspective.
Turns out shooting ice at a car door is a blast.
Besides safety technology, the showroom boasts some pretty brutal crash damage.
Airbags: before, during and after.
A video demonstrated autonomous car technology.
NRMA head of research, Robert McDonald, says interactivity is key to teaching people about car safety.


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Thank you for a great Turn out!
NOW AN ADULT CO-ED Division Too!!!!!
(maximum of 6 players per team)
8 teams max per division
4 Games Guaranteed
Trophies awarded to all division winners!!!
Tournament will be played at the
Preston Community Park, 13 Route 117 Preston CT 06365.
Please make checks payable to: Preston Parks and Recreation.
**This is a Qualifying Event For the 3v3 Challenge Tour**
3v3Challenge National CHAMPIONSHIP at ESPN Wide World of Sports Complex at Walt Disney World® Resort
Challenge 3v3 Nationals is the culminating tournament of the Challenge Sports 3v3 tour, which takes place year
round throughout the country. Teams participating in Nationals can expect top notch 3v3 competition and a professionally run tournament experience.
1st-4th place individual and team awards are provided for all age divisions, and all U10 and younger players receive individual awards. For more information
about Challenge Sports visit www.challengesports.com.
Visitor #


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Photos from Prometheus, Dark Shadows, Men in Black 3, Wrath of the Titans and Jack and Diane.
A poster featuring "Hellboy" and "Sons of Anarchy" star Ron Perlman as a transgender computer hacker in Frankie Goes Boom.
"Producer Megan Ellison has hinted that we might see an October release for Paul Thomas Anderson's much anticipated "The Master"…" (full details)
"Both Keanu Reeves and Alex Winter have confirmed a script for a third "Bill and Ted" movie is done and they're both happy with it, now comes the time to sort out making it…" (full details)
"Sony Pictures has jumped onboard MGM's "Robocop" remake as a distributor, akin to their arrangement for "The Girl With The Dragon Tattoo" and the upcoming Bond film "Skyfall"…" (full details)
"Warner Bros has scheduled a one-night showing of classic "Casablanca" on March 21st in nearly 500 cinemas across the country, preceding the March 27th release of the latest (the third) restoration of the film to hit Blu-ray and DVD…" (full details)
"Classic 80's comedy "Animal House" is being turned into a stage musical with Canadian rockers The Barenaked Ladies hired to pen the music…" (full details)
"IMAX Corporation and The Walt Disney Studios today announced that Tim Burton's stop-motion, animated film Frankenweenie will be digitally re-mastered into the immersive IMAX® 3D format and released in IMAX® digital theatres worldwide day-and-date on October 5th 2012…" (full details)
"Security technician Joshua Thompson has filed a class action in Wayne County Circuit Court against his local AMC theater in Michigan in the hopes of forcing cinemas statewide to dial down snack prices…" (full details)
"Mass Hysteria Entertainment has acquired rights to the upcoming action thriller "The Boiling Point" with Warren P. Sonoda attached to direct. The story follows a gangster assigned to take out a mob informer who's his ex-girlfriend and disappeared years ago into the witness protection program…" (full details)


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Why I feel so pissed off at the lack of help and support.
I am not the kind of person who sits around and waits to be asked or to be told when I comes to helping out. I use this thing - I think they even have a word for it - initiative
For those still not understanding me
Come on people! Its not that hard.
I am the person who:
- cooks dinner when you are babysitting my children.
- folds the washing that was sitting on the floor while we had coffee at your house.
- washes the dishes after a meal at your place.
- puts away the toys my kids have pulled out while visiting.
- turns up just to drop of a meal or baking when you need a hand (baby/sickness/stress).
- takes your child so you can go to the hairdresser/clothes shopping/groceries without the stress of little Tommy.
- Has your kids for sleep overs because I know you then get some time out.
- Cleans your toilet if I arrive and your are cleaning the shower.
- I will make things I know you like, even when I costs me more than I can afford and ask nothing in return.
- I will say yes to helping even when it means rearranging my plans/day/week/family because you are my friends and I want to help where I can.
- I go about researching and finding out information that might help when you need it.
- I will ask how you are and keep asking when I know you aren't telling me the truth.
- I will do my best to know I have done at least something in my power to help!


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Farmax Products and Services
Farmax has changed how its products (and services) are named, delivered and priced, to make them simpler and easier to understand.
To find out more about the Farmax sheep, beef and deer tools click here
To find out more about the Farmax dairy tools click here
FAQ's from Rural Professionals
Farmax for Modelling
To explain the FARMAX pricing, firstly we need to look at the two main uses of the FARMAX tools and who utilises them.
Farmax is widely used across the pastoral industry to 'model' a farm system and then investigate and analyse different scenarios. While FARMAX is used in this way by farmers, researchers, students, and others, agricultural consultants are the main user of FARMAX in this manner.
Instead of forcing consultants to pay an upfront annual subscription to use FARMAX, the pricing will be the equivalent of a "pay as you go" system. This pay as you go system applies to the product: FARMAX Analysis. FARMAX Analysis allows you to create a Farmax model of a farm entity and do unlimited scenario analysis. Each Farm entity either real or hypothetical that is modelled using Farmax Analysis will incur an annual subscription of $200. Our strong recommendation is that Farmax models should only be created after you have been trained to use Farmax, or by accredited Farmax consultants.
Farmax for Monitoring
The second predominate use of Farmax is monitoring, and this is largely done by farmers. Monitoring functionality or the entering of actual information into a Farmax farm file cannot be done using FARMAX Analysis. To unlock that functionality the user needs to step up to the monitoring range of products. This is where Farmax really comes to the fore in providing the information for proactive tactical and operational decision making on farm within a season. The unique value Farmax provides is the constant 'reforecasting' of the likely outcome for the season as actual data is entered throughout the year.
Moving forward, anyone will be able to view (read only) Farmax models, as the Farmax application will be available for download at no charge. The ability to open and view a Farmax model will be granted by the owner of the Farmax farm file. This opens exciting opportunities and possibilities to share the information in Farmax files with other trusted parties. One area we are currently investigating is making Farmax demo farm files available online for public access.
International Pricing
Please click here to view our international prices.
Take me to the Farmax sheep, beef and deer tools information page
Take me to the Farmax dairy tools information page


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up sectional
Using a simple expedient similar to Lego's snap-on system, the height of the Up sofa's backrest can be adjusted according to personal choice. Place the back cushions on top of the low backrest to create a higher backrest and a deeper seat. The particular shape of the cushions ensures maximum stability. The goose feather-padded cushions and armrests create a particularly soft, snug sofa. Also available with raw-edged leather upholstery.
Several Configurations are available. Dimensions vary based on components.
Fabric or Leather Upholstery.
Solid wood frame.
Feather and Down encased high resilient foam core cushions.
Black PVC legs.
Custom (4-5 months)
starting at
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With a focus on resource management, emissions reduction and innovative cost management, we can help improve your plant's business and environmental performance.
Ecolab Greater China
18G, Lane 168 Waterfront Place
Da Du He Road
Shanghai 200062, China
To request additional information on Ecolab Greater China, please send us an email.
Email: firstname.lastname@example.com
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