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The present invention relates to appetite-suppressing peptides or an appetite-suppressing peptide-containing fraction for the treatment of obesity or type II diabetes.
1. A method of treating diseases or disorders associated with impaired appetite regulation, comprising administering to an individual in need of such treatment a pharmaceutical composition comprising a pharmaceutically acceptable excipient or vehicle and an effective amount of a peptide having the following amino acid sequence (SEQ ID NO: 1)
X1 is NH2, DFPEEVAIVEELGRR (SEQ ID NO:2), DFPEEVTIVEELGRR (SEQ ID NO:3), DFPEEVNIVEELRRR (SEQ ID NO:4), or a fragment thereof,
X2 is Ala or Gly,
X3 is Ile or Val,
X4 is Asn, Ser or His,
X5 is Ala or Thr,
X6 is Arg or Lys,
X7 is Ile or Leu,
X8 is Gln or His, and
X9 is OH, Lys, Arg, Arg-Lys, Lys-Arg, Arg-Arg or Lys-Lys.
2. The method of claim 1, wherein the disease or disorder is obesity or type II diabetes.
3. The method of claim 1, wherein the amount of the peptide is in the range of from about 10 μg/kg body weight to about 5 mg/kg body weight.
4. The method of claim 1, wherein the pharmaceutical composition further comprises another appetite-suppressing or satiety-inducing agent.
5. The method of claim 4, wherein the other appetite-suppressing or satiety-inducing agent is glucagon-like peptide-1.
6. The method of claim 1, wherein X1 is NH2.
7. The method of claim 1, wherein X2 is Ala.
8. The method of claim 1, wherein X3 is Ile.
9. The method of claim 1, wherein X4 is Asn.
10. The method of claim 1, wherein X5 is Ala.
11. The method of claim 1, wherein X6 is Arg.
12. The method of claim 1, wherein X7 is Ile.
13. The method of claim 1, wherein X8 is Gln.
14. The method of claim 1, wherein X9 is OH.
15. The method of claim 1, wherein the peptide has an amino acid sequence of
16. The method of claim 1, wherein the peptide has an amino acid sequence of
17. The method of claim 1, wherein the peptide has an amino acid sequence of
This application claims priority of U.S. provisional applications 60/015,403 and 60/018,865 both filed Mar. 15, 1996 and of Danish applications 0230/96 filed Mar. 1, 1996 and 0231/96 filed Mar. 1, 1996, the contents of which applications are fully incorporated herein by reference.
The present invention relates to use of a pharmaceutical composition comprising an appetite-suppressing peptide or an appetite-suppressing peptide-containing fraction, as well as a method of obtaining appetite regulation by means of said peptide.
Glucagon is produced by the pancreatic A-cell and released in response to low blood glucose levels. Its major site of action is the liver where it stimulates glucose production. It is thus the major hormone counteracting insulin in blood glucose homeostasis (Unger, R. H. and L. Orci (1990). Glucagon, in: Diabetes Mellitus, 4th ed. New York, Elsevier. pp 104-120).
Glucagon is processed from a larger precursor by limited proteolysis. Molecular cloning of the glucagon gene revealed that the proglucagon precursor contained not only glucagon but also two additional glucagon-like peptides named GLP-1 and GLP-2. GLP-1 and GLP-2 are encoded by separate exons suggesting distinct biological activities. It was later demonstrated that the proglucagon precursor was subjected to differential processing in the three different tissues known to produce proglucagon: the pancreatic A-cell, the intestinal L-cell, and in the central nervous system (CNS). Glucagon is thus selectively excised from the precursor in the islet A-cell, while GLP-1 and GLP-2 are selectively liberated from the intestinal L-cell and the CNS reviewed in (Unger, R. H. and L. Orci (1990). Glucagon. in: Diabetes Mellitus, 4th ed. New York, Elsevier. pp 104-120)!.
Specific GLP-1 receptors have been identified (Thorens, B. (1992) Proc. Natl. Acad. Sci. USA 89: 8641-8645) which are clearly distinct from the glucagon receptor (L. J Jelinek, et al. (1993) Science 259: 1614-1616) and they have different tissue distributions (R. V Campos, et al. (1994) Endocrinology 134: 2156-2164). GLP-1 is released from the L-cell after a meal and functions as an incretin hormone (i.e. it potentiates glucose induced insulin release from the pancreatic B-cell). The GLP-1 receptor is thus expressed at high levels on the surface of islet B-cells (K. Moens, et al. (1996) Diabetes 45: 257-261).
Induction of intestinal epithelial proliferation by GLP-2 was demonstrated (Drucker, D. J. et al (1996) Proc. Natl. Acad. Sci. USA 93: 7911-7916) and treatment of gastrointestinal deseases by cells grown in GLP-2 medium was disclosed (Drucker, D. J and Keneford, J. R., WO 96/32414).
No GLP-2 receptor has so far been reported.
Proglucagon derived peptides and feeding behaviour
We have previously reported the derivation and establishment of transplantable anorectic glucagonomas (O. D. Madsen et al. (1993) Endocrinology 133: 2022-2030) as well as of hypoglycemic insulinomas in the rat (O. D Madsen, et al. (1988) Proc. NatI. Acad. Sci. USA 85: 6652-6656). Such tumors can be derived from common clonal origin of pluripotent MSL-cells (O. D. Madsen, et al. (1986) J. Cell Biol. 103: 2025-2034) and reflects a maturation process towards islet A-cell and B-cells, respectively (O. D. Madsen, et al. (1993) Endocrinology 133: 2022-2030).
The glucagonoma associated anorexia is very severe: it has an acute onset and leads after few days to a complete stop in food intake. This severity of anorexia is hardly matched by other experimental tumors in rodents and suggests the production by the glucagonoma of a very powerful satiety factor which acts by a peripheral route of administration. It has previously been demonstrated that the anorectic glucagonomas displayed an unphysiological processing resulting in the formation of both glucagon and GLP-1 (O. D. Madsen, et al. (1993) Endocrinology 133: 2022-2030). Moreover, a non-anorectic glucagonoma variant was unable to process the precursor (O. D. Madsen, et al. (1995) Scand. J. Clin. Lab. Invest. 55, suppl 220: 27-36. Weight loss is mentioned as a component also of the glucagonoma syndrome in man (J. J. Holst (1985) Glucagon-producing tumors, in: Hormone-producing tumors of the gastrointestinal tract. New York, Churchill Livingstone. pp 57-84) although with a high degree of variability among different patients (S. J. Bhathena, et al. (1981). Glucagonoma and glucagonoma syndrome, in: Glucagon. Physiology, pathophysiology and morphology of the pancreatic A-cells. New York, Elsevier. 413-438).
Glucagon has been shown to be involved in the regulation of spontaneous meal size in rats but the overall effect is minimal and is exerted via the vagal connections to the liver (N. Geary, et al. (1993) Am. J. Physiol. 264: R116-R122). This effect is observed only by hepatic portal infusion of glucagon while intraperitoneal administration of pharmacological doses show no effect on food intake in fasted rats (O. D. Madsen, et al. (1993) Endocrinology 133: 2022-2030).
A central role for GLP-1 in the regulation of feeding was recently reported (M. D. Turton, et al. (1996) Nature 379: 69-72). Intracerebroventricular (ICV) administration of GLP-1 inhibited feeding in fasted rats. Again peripheral administration of GLP-1 had no effect on feeding behaviour (M. D. Turton, et al. (1996) Nature 379: 69-72; O. D. Madsen, et al. (1993) Endocrinology 133: 2022-2030) suggesting that tumor produced GLP-1 may not contribute significantly to the observed anorexia.
It has been found that GLP-2 has a powerful effect on inhibiting food intake when administered peripherally.
It is proposed that GLP-2 normally released together with GLP-1 from the intestinal L-cell serves its own distinct role as a peripheral satiety factor.
Accordingly, the present invention relates to use of a pharmaceutical composition comprising, together with a pharmaceutically acceptable excipient or vehicle, an HPLC fraction of a glucagonoma tumour extract prepared by acid ethanol extraction, gel filtration and preparative HPLC, said fraction being shown as fraction G4H9 in FIG. 2 and containing glucagon-like peptide 2 (GLP-2) as a major component (i.e. more than 40%) or comprising any single component of said fraction or a combination of two or more of the components of said fraction.
In another aspect, the invention relates to use of a pharmaceutical composition comprising glucagon-like peptide-2 (GLP-2) or a variant or homologue thereof for the prohylaxis or treatment of diseases or disorders associated with impaired appetite regulation.
In a further aspect, the invention relates to use of a pharmaceutical composition comprising a peptide with the following amino acid sequence
X1 is NH2, DFPEEVAIVEELGRR (SEQ ID NO:2), DFPEEVTIVEELGRR (SEQ ID NO:3), DFPEEVNIVEELRRR (SEQ ID NO:4), or a fragment thereof,
X2 is Ala or Gly,
X3 is Ile or Val,
X4 is Asn, Ser or His,
X5 is Ala or Thr,
X6 is Arg or Lys,
X7 is Ile or Leu,
X8 is Gln or His, or
X9 is OH, Lys, Arg, Arg-Lys, Lys-Arg, Arg-Arg or Lys-Lys for the prophylaxis or treatment of diseases or disorders associated with impaired appetite regulation.
In a still further aspect, the invention relates to method of treating diseases or disorders associated with impaired appetite regulation, the method comprising administering to an individual in need of such treatment an amount of a peptide as specified herein sufficient to suppress appetite or induce satiety in said individual.
In a still further aspect, the invention relates to the use of a peptide as specified herein for the manufacture of a medicament for the prophylaxis or treatment of diseases or disorders associated with impaired appetite regulation.
FIG. 1 A gel filtration chromatogram of fractions G1, G2, G3, G4, and G5.
FIG. 2 A HPLC chromatogram of G4 material.
In the present description, the term "peptide" is understood to include the mature GLP-2 peptide or a precursor form thereof as well a functional fragment thereof which essentially has the activity of the full-length peptide. Furthermore, the term "peptide" is intended to include homologues of said peptide. Such homologues comprise an amino acid sequence exhibiting a degree of identity of at least 50%, such as at least 75%, and more particularly at least 90% identity with the amino acid sequence of human GLP-2. The degree of identity may be determined by conventional methods, see for instance, Altshul et al., i Bull. Math. Bio. 48: 603-616, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915-10919, 1992.
Homologues of the present peptide may have one or more amino acid substitutions, deletions or additions. These changes may be of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding or activity of the peptide, small deletions, typically of one to about five amino acids, small amino- or carboyxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 15 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain. See in general Ford et al., Protein Expression and Purification 2: 95-107, 1991. Examples of conservative substitutions are within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine).
The homologue may be an allelic variant, i.e. an alternative form of a gene that arises through mutation, or an altered peptide encoded by the mutated gene, but having substantially the same activity as the native GLP-2 peptide. Hence mutations can be silent (no change in the encoded peptide) or may encode peptides having altered amino acid sequence.
The homologue of the present peptide may also be a species homologue, i.e. a peptide with a similar activity derived from another species. Examples of species homologues of the GLP-2 peptide are human, bovine, rat, hamster, guinea pig and porcine GLP-2.
In a preferred embodiment of the present invention, the GLP-2 peptide is one in which X1 is NH2, X2 is Ala, X3 is Ile, X4 is Asn, X5 is Ala, X6 is Arg, X7 is Ile, X8 is Gln, or X9 is OH. In particular, the peptide has the following amino acid sequence
H A D G S F S D E M N T I L D N L A A R D F I N W L I Q T K I T D (human GLP-2) (SEQ ID NO:5) or
H A D G S F S D E M N T I L D N L A T R D F I N W L I Q T K I T D (rat GLP-2) (SEQ ID NO:6) or
H A D G S F S D E M N T V L D N L A T R D F I N W L L H T K I T D (porcine GLP-2) (SEQ ID NO:7).
A homologue of the peptide may be isolated by preparing a genomic or cDNA library of a cell of the species in question, and screening for DNA sequences coding for all or part of the homologue by using synthetic oligonucleotide probes in accordance with standard techniques, e.g. as described by Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd. Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, or by means of polymerase chain reaction (PCR) using specific primers as described by Sambrook et al., supra.
The present invention also relates to a composition comprising a variant of the GLP-2 peptide. The variant is one in which one or more amino acid residues have been substituted by other amino acid residues. In a particularly preferred embodiment Ala has been substituted by Gly in position 2 of the mature peptide. It is expected that this variant will exhibit a longer plasma half-life than the native peptide, which is an advantage because the dosage required to obtain an adequate appetite-suppressing or satiety-inducing effect will generally be smaller.
The GLP-2 peptide or homologue or variant thereof as specified above may be made by recombinant DNA techniques in accordance with procedures well established in the art.
More specifically, a DNA sequence encoding the GLP-2 peptide may be isolated or synthesized on the basis of the published human preproglucagon DNA sequence (cf. J. W. White et al., Nucleic Acids Res. 14, 1986, pp. 4719-4730; G. I. Bell et al., Nature 304, 1983, pp. 368-371), for instance obtained by preparing a genomic or cDNA library from an appropriate tissue and screening for DNA sequences coding for all or part of the GLP-2 peptide by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., supra). For the present purpose, the DNA sequence encoding the GLP-2 peptide is preferably of human origin.
The DNA construct encoding the GLP-2 peptide may also be prepared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. According to the phosphoamidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.
Furthermore, the DNA construct may be of mixed synthetic and genomic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate), the fragments corresponding to various parts of the entire DNA construct, in accordance with standard techniques.
The DNA construct may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491, or Sambrook et al., supra.
In a currently preferred embodiment, the DNA construct comprises the DNA sequence shown in FIG. 3 of G. I. Bell et al., Nature 304, 1983, pp. 368-371, as well as nucleic acid sequences encoding human GLP-2, but which differ from the DNA sequence shown in FIG. 3 of Bell et al., supra, by virtue of the degeneracy of the genetic code. The DNA construct further includes nucleic acid sequences which hybridize to a nucleic acid molecule (either genomic, synthetic or cDNA or RNA) encoding human GLP-2 under the conditions of high stringency (i.e. presoaking in 5X SSC and prehybridizing for 1 hr at about 40° C. in a solution of 20% formamide, 5X Denhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 μg denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 μM ATP for 18 hrs. at about 40° C., followed by a wash in 0.4X SSC at a temperature of about 45° C.). This could, for instance, be DNA sequences encoding GLP-2 from other species, e.g. rat, bovine, hamster, guinea pig or porcine GLP-2.
To express GLP-2, the DNA construct encoding the GLP-2 peptide is inserted into an appropriate recombinant vector. This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
The vector is preferably an expression vector in which the DNA sequence encoding the GLP-2 peptide is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g. transcription initiates in a promoter and proceeds through the DNA sequence coding for the peptide.
The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of the DNA encoding the GLP-2 peptide in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814) or the adenovirus 2 major late promoter.
An example of a suitable promoter for use in insect cells is the polyhedrin promoter (U.S. Pat. No. 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), the P10 promoter (J. M. Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosis virus basic protein promoter (EP 397 485), the baculovirus immediate early gene 1 promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222), or the baculovirus 39K delayed-early gene promoter (U.S. Pat. No. 5,155,037; U.S. Pat. No. 5,162,222).
Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No. 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654) promoters.
Examples of suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter. Examples of other useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase. Preferred are the TAKA-amylase and gluA promoters.
Examples of suitable promoters for use in bacterial host cells include the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the Bacillus lichenifonnis alpha-amylase gene, the Bacillus amyloliquefaciens BAN amylase gene, the Bacillus subtilis alkaline protease gen, or the Bacillus pumilus xylosidase gene, or by the phage Lambda PR or PL promoters or the E. coli lac, trp or tac promoters.
The DNA sequence encoding the GLP-2 peptide may also, if necessary, be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) terminators. The vector may further comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).
The recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of replication.
When the host cell is a yeast cell, suitable sequences enabling the vector to replicate are the yeast plasmid 2μ replication genes REP 1-3 and origin of replication.
The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130), or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate. For filamentous fungi, selectable markers include amdS, pyrG, argB, niaD, sC.
To direct the GLP-2 peptide into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, preprosequence or presequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the peptide. The secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.
For secretion from yeast cells, the secretory signal sequence may encode any signal peptide which ensures efficient direction of the expressed peptide into the secretory pathway of the cell. The signal peptide may be naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides have been found to be the α-factor signal peptide (cf. U.S. Pat. No. 4,870,008), the signal peptide of mouse salivary amylase (cf. O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modified carboxypeptidase signal peptide (cf. L. A. Valls et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (cf. WO 87/02670), or the yeast aspartic protease 3 (YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).
For efficient secretion in yeast, a sequence encoding a leader peptide may also be inserted downstream of the signal sequence and uptream of the DNA sequence encoding the GLP-2 peptide. The function of the leader peptide is to allow the expressed peptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to a secretory vesicle for secretion into the culture medium (i.e. export of the peptide across the cell wall or at least through the cellular membrane into the periplasmic space of the yeast cell). The leader peptide may be the yeast α-factor leader (the use of which is described in e.g. U.S. Pat. No. 4,546,082, EP 16 201, EP 123 294, EP 123 544 and EP 163 529). Alternatively, the leader peptide may be a synthetic leader peptide, which is to say a leader peptide not found in nature. Synthetic leader peptides may, for instance, be constructed as described in WO 89/02463 or WO 92/11378.
For use in filamentous fungi, the signal peptide may conveniently be derived from a gene encoding an Aspergillus sp. amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, a Humicola lanuginosa lipase. The signal peptide is preferably derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral α-amylase, A. niger acid-stable amylase, or A. niger glucoamylase.
For use in insect cells, the signal peptide may conveniently be derived from an insect gene (cf. WO 90/05783), such as the lepidopteran Manduca sexta adipokinetic hormone precursor signal peptide (cf. U.S. Pat. No. 5,023,328).
The procedures used to ligate the DNA sequences coding for the GLP-2 peptide, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op.cit.).
The DNA sequence encoding the GLP-2 peptide introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e. produced by the host cell in nature, it will typically be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a cDNA sequence encoding a polypeptide native to the host organism in question. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.
The host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells.
Examples of bacterial host cells which, on cultivation, are capable of producing the GLP-2 peptide are grampositive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothemophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or B. thuringiensis, or strains of Streptomyces, such as S. lividans or S. murinus, or gramnegative bacteria such as Echerichia coli. The transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra).
When expressing the peptide in bacteria such as E. coli, the peptide may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the peptide is refolded by diluting the denaturing agent. In the latter case, the peptide may be recovered from the periplasmic space by disrupting the cells, e.g. by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the peptide.
Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appi. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.
Examples of suitable yeasts cells include cells of Saccharomyces spp. or Schizosaccharomyces spp., in particular strains of Saccharomyces cerevisiae or Saccharomyces kluyveri. Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides therefrom are described, e.g. in U.S. Pat. Nos. 4,599,311, 4,931,373, 4,870,008, 5,037,743, and U.S. Pat. No. 4,845,075, all of which are hereby incorporated by reference. Transformed cells are selected by a phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient, e.g. leucine. A preferred vector for use in yeast is the POT1 vector disclosed in U.S. Pat. No. 4,931,373. The DNA sequence encoding the GLP-2 peptide may be preceded by a signal sequence and optionally a leader sequence, e.g. as described above. Further examples of suitable yeast cells are strains of Kluyveromyces, such as K. lactis, Hansenula, e.g. H. polymorpha, or Pichia, e.g. P. pastoris (cf. Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; U.S. Pat. No. 4,882,279).
Examples of other fungal cells are cells of filamentous fungi, e.g. Aspergillus spp., Neurospora spp., Fusarium spp. or Trichodenna spp., in particular strains of A. oryzae, A. nidulans or A. niger. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277 and EP 230 023. The transformation of F. oxysporum may, for instance, be carried out as described by Malardier et al., 1989, Gene 78: 147-156.
When a filamentous fungus is used as the host cell, it may be transformed with the DNA construct encoding the GLP-2 peptide, conveniently by integrating the DNA construct in the host chromosome to obtain a recombinant host cell. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g. by homologous or heterologous recombination.
Transformation of insect cells and production of heterologous polypeptides therein may be performed as described in U.S. Pat. Nos. 4,745,051; 4,879,236; 5,155,037; 5,162,222; EP 397,485) all of which are incorporated herein by reference. The insect cell line used as the host may suitably be a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (cf. U.S. Pat. No. 5,077,214). Culture conditions may suitably be as described in, for instance, WO 89/01029 or WO 89/01028, or any of the aforementioned references.
The transformed or transfected host cell described above is then cultured in a suitable nutrient medium under conditions permitting the expression of the GLP-2 peptide, after which the resulting GLP-2 peptide is recovered from the culture.
The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The GLP-2 peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e. g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like.
In the pharmaceutical composition of the invention, the GLP-2 peptide may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences, 1985. The composition may be in a form suited for systemic injection or infusion and may, as such, be formulated with a suitable liquid vehicle such as sterile water or an isotonic saline or glucose solution. The compositions may be sterilized by conventional sterilization techniques which are well known in the art. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
The pharmaceutical composition of the present invention may also be adapted for nasal, transdermal, pulmonal or rectal administration. The pharmaceutically acceptable carrier or diluent employed in the composition may be any conventional solid carrier. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
It may be of particular advantage to provide the composition of the invention in the form of a sustained release formulation. As such, the composition may be formulated as microcapsules or microparticles containing the GLP-2 peptide encapsulated by or dispersed in a suitable pharmaceutically acceptable biodegradable polymer such as polylactic acid, polyglycolic acid or a lactic acid/glycolic acid copolymer.
For nasal administration, the preparation may contain GLP-2 peptide dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilizing agents, e.g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.
Generally, the compounds of the present invention are dispensed in unit dosage form comprising 0.5-500 mg of the peptide together with a pharmaceutically acceptable carrier per unit dosage.
The GLP-2 peptide is contemplated to be advantageous to use in appetite suppression or satiety induction, such as for the prophylaxis or treatment of diseases or disorders associated with impaired appetite regulation. Examples of such diseases or disorders are obesity and type II diabetes. The dosage of the GLP-2 peptide administered to a patient will vary with the type and severity of the condition to be treated, but is generally in the range of from about 10 μg/kg to about 5 mg/kg body weight.
In the pharmaceutical composition of the invention, the GLP-2 peptide may be combined with another appetite-suppressing or satiety-inducing agent. An example of such an agent is GLP-1 which has been shown to have some effect on appetite suppression (cf. M. D. Turton et al., Nature 379, Jan. 4, 1996, pp. 69-72).
It is further contemplated that the GLP-2 peptide in suitably labelled form, e.g. radiolabelled GLP-2, may be used to identify a receptor for GLP-2 in binding studies using tissue(s) expected to express the GLP-2 receptor, e.g. hypothalamus tissue. Once localized by GLP-2 binding, the receptor may be cloned by expression cloning, i.e. by preparing a cDNA library of the tissue in question, cloning the cDNA into suitable vectors and introducing the vectors into an appropriate cell to effect expression of the cDNA, after which a clone expressing the receptor is identified by binding to GLP-2. A cell line stably expressing the receptor may then be used in a screening assay for GLP-2 agonists (i.e. compounds acting on the receptor to induce satiety or suppress appetite) or GLP-2 antagonists (i.e. compounds which antagonize the action of GLP-2 on the receptor, e.g. for use in the treatment of cancer anorexia or anorexia nervosa).
The invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed.
Anorectic tumors were produced in rats as previously described (Madsen, O.D. et al. (1993) Endocrinology 133, 2022-2030). Fifty anorectic 12C3AN (MSL-G-AN) tumours (at -80° C.) corresponding to 50.07 g of wet tissue were homogenised at 4° C. with 700 ml of acid ethanol (96% ethanol/0.7M HCl, 3/1, vol/vol). The homogenisation was carried out for 5 min in a precooled (4° C.) 2 liter Waring Commercial Blender at maximum speed. After homogenisation the mixture was stirred at 4° C. for 16 hours. The mixture was centrifuged at 9000 RPM at 4° C. for 1 hour. The volume of the supernatant was reduced to 20% by vacuum rotation. During this process, in which the main part of the ethanol is removed, some precipitate is formed. This precipitate was removed by centrifugation at 4° C. for one hour at 20.000 RPM. The supernatant, which still contained some lipid-like material, was filtered and applied to a LiChroprep RP-18 (Merck) column (2.5×10 cm) equilibrated with 0.1% TFA at a flow rate of 2 ml/min. The column was washed with 100 ml of 0.1% of TFA at a flow rate of 4 ml/min. Bound material was eluted with 400 ml of 0.1% TFA containing 70% (vol/vol) of acetonitrile. The acetonitrile was removed by vacuum rotation and the resulting mixture was lyophilised. After lyophilization the material was dissolved in 50 ml of water and the pH was adjusted to 5.3 with 425 μl of 1N NaOH. Further titration of the mixture to pH 6.0 resulted in the formation of a precipitate. Upon back titration to pH 5.3 this precipitate was dissolved again. Therefore the pH was left at 5.3 and the mixture was lyophilised. The total yield of lyophilised material from 50 tumours was 359 mg of dry powder.
Lyophilized material (278 mg) from the acid ethanol extract corresponding to 38 individual tumors was redissolved in 20 ml of 1 M HAc and applied to Sephadex G75 column (5×50 cm). The column was equilibrated and eluted with 1 M HAc at a flow rate of 55 ml/h, and fractions corresponding to 10 ml were collected. The absorption at 280 nm was recorded for each fraction. The gel filtration chromatogram is shown in FIG. 1. Individual fractions were pooled in the following 5 main fractions: G1 (Fr. 30-39), G2 (Fr. 40-45), G3 (Fr. 46-66), G4 (Fr. 67-91) and G5 (Fr. 92-118) and subjected to bioassay after lyophilization.
Some of the appetite suppression activity of gel filtration pools showed the activity to be present in the G4 pool, and this pool was further fractionated by preparative HPLC. Lyophilized G4 material (corresponding to 80 tumors) was redissolved in 15 ml 0.1% TFA and pumped onto a Vydac 214TP1022 C4 column (2.2×25 cm) equilibrated in 0.1% TFA. The column was washed with 20 ml of 0.1% TFA, followed by 100 ml of MeCN/H2 O/TFA (10.0:89.9:0.1, v/v/v). The material was eluted at 25° C. at a flow rate of 4 ml/min with a linear gradient formed from MeCN/H2 O/TFA (10:79.9:0.1, v/v/v) and MeCN/H2 O/TFA (65.0:34.9:0.1, v/v/v) over 110 min. UV absorption was monitored at 214 nm and 280 nm. The HPLC chromatogram (monitored at 280 nm) is shown in FIG. 2. Fractions corresponding to 10 main pools were generated as indicated in FIG. 2. The volume was reduced to approx. 25% by vacuum rotation and the fractions were lyophilized and tested in the bioassay.
The appetite suppression activity was found in fraction G4H9 (Example 6) and the peptides of this fraction were analysed by amino acid sequence analysis and mass spectrometry analysis (Example 4) .
Amino acid sequence analysis was carried out by automated Edman degradation using an Applied Biosystems Model 477 gas-phase sequencer essentially as described by the manufacturer. Mass spectrometry analysis was performed using an API III LC/MS/MS system (Sciex, Thornhill, Ont., Canada). The triple quadrupole instrument has a mass-to-charge (m/z) range of 2400 and is fitted with a pneumatically assisted electrospray (also referred to as ion-spray) interface (Bruins, A. P., Covey, T. R., & Henion, J. D. (1987) Anal. Chem. 59, 2642-2646 and Covey, T. R., Bonner, R. F., Shushan, B. I., & Henion, J. D. (1988) Rapid Commun. Mass Spectrom. 2, 249-256). Sample introduction was done by a syringe infusion pump (Sage Instruments, Cambridge, Mass.) through a fused capillary /75 mm i.d.) with a liquid flow rate set at 0.5-1 ml/min. The instrument m/z scale was calibrated with the singly-charged ammonium adduct ions of poly(propylene glycols) (PPGs) under unit resolution. The accuracy of mass measurements is generally better than 0.02%.
The dominating peptide in this fraction was found to have the following amino acid sequence:
The molecular weight found by mass spectrometry was: 3796.
This peptide is identical to rat GLP-2 (1-33). Minor amounts of the following two peptides were also found:
D F P E E V A I A E E L G R R H A D G S F S D E M N T I L D N L A T R D F I N W L I Q T K I T D (SEQ ID NO:8) and
These peptides are identical to rat GLP-2 which is N-terminally extended with spacer peptide 2 and rat GLP-1 (1-36amide), respectively.
Mice were deprived of their normal feed for two days and given free access to a 20% sucrose solution on the first day of feed deprivation. After the 2-day feed deprivation period, mice were injected intraperitoneally with 0.5 ml of a solution containing the test substance. Thirty minutes after injection, individual mice were placed in one of eight 15 cm2 test box with a stainless steel grid floor and a glass drinking tube which projected into the box. The drinking tube was connected to a reservoir containing a 20% sucrose solution, and the interior of the drinking tube contained an electrode enabling the detection of drinking contacts with the solution by measuring the flow of a weak (unnoticeable) electric current through mice by means of an electronic apparatus connected to the drinking tube electrode and the stainless steel grid floor. Consumption of the sucrose solution was measured over a 10 minute period by electronically recording the total amount of contact with the sucrose solution during the test session. The degree of appetite suppression produced by a given test substance was determined by statistical comparison of the duration of sucrose consumption by control (vehicle treated) mice with that of mice treated with a test substance. The degree of appetite suppression in a treated group of mice was expressed as percent of the control groups response.
Mice were tested for appetite suppression (see Example 5) after treatment with a test substance. The test substance consisted of extracts of the anorectic glucagonoma tumor prepared according to Example 3 (Gelfiltration fraction G4) or according to Example 4 (HPLC fraction G4H9) dissolved in phosphate buffered saline. The test solution containing lyophilized material from the Gelfiltration fraction G4 corresponding to 3.3 tumors suppressed sucrose consumption by 72%. Of the 10 HPLC sub-fractions of the G4 Gelfiltration fraction (see Example 4 and FIG. 2), only the GLP-2 containing fraction, G4H9, gave a statistically significant suppression of appetite, suppressing sucrose consumption by 49%, when lyophilized material corresponding to 5.3 tumors was given.
Mice were tested for appetite suppression as described in Example 5 after treatment with a test substance consisting of synthetic porcine GLP-2 dissolved in phosphate buffered saline. Porcine GLP-2 has the following amino acid sequence:
An intraperitoneal injection of test solution containing 50 micrograms of synthetic porcine GLP-2 suppressed sucrose consumption by 38%.
The method is as in Example 5, but instead of 20% sucrose, a solution of infant formula milk (Complan®) is used. The test substance is dissolved in a vehicle consisting of phosphate buffered saline with 1% albumin. Test substances dissolved in vehicle are injected either intravenously (IV) in a volume of 100 microliters, or intra-cerebroventricularly (ICV) in a volume of 10 microliters.
Mice were tested for appetite suppression as described in Example 8 after treatment with a test substance consisting of synthetic human GLP-2. Human GLP-2 has the following amino acid sequence:
An IV injection of test solution containing 3 micrograms of synthetic human GLP-2 suppressed milk consumption by 24%, while ICV injections of 3 micrograms and 10 micrograms of synthetic human GLP-2 suppressed milk consumption by 32% and 35%, respectively.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 9- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 35 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: None- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:#Met Asn Thr Xaa Leu Asper Phe Ser Asp Glu# 15#Leu Xaa Xaa Thr Lys Ilesp Phe Ile Asn Trp# 30- Thr Asp Xaa 35- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: None- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:#Glu Leu Gly Arg Arglu Val Ala Ile Val Glu# 15- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: None- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:#Glu Leu Gly Arg Arglu Val Thr Ile Val Glu# 15- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 15 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: None- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:#Glu Leu Arg Arg Arglu Val Asn Ile Val Glu# 15- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 33 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: None- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:#Asn Thr Ile Leu Asp Asnhe Ser Asp Glu Met# 15#Ile Gln Thr Lys Ile Thrhe Ile Asn Trp Leu# 30- Asp- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 33 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: None- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:#Asn Thr Ile Leu Asp Asnhe Ser Asp Glu Met# 15#Ile Gln Thr Lys Ile Thrhe Ile Asn Trp Leu# 30- Asp- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 33 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: None- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:#Asn Thr Val Leu Asp Asnhe Ser Asp Glu Met# 15#Leu His Thr Lys Ile Thrhe Ile Asn Trp Leu# 30- Asp- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 48 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: None- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:#Glu Leu Gly Arg Arg Hisal Ala Ile Ala Glu# 15#Thr Ile Leu Asp Asn Leuer Asp Glu Met Asn# 30#Gln Thr Lys Ile Thr Asple Asn Trp Leu Ile# 45- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 36 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear- (ii) MOLECULE TYPE: None- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:#Thr Phe Thr Ser Asp Valrg His Ala Glu Gly# 15#Glu Phe Ile Ala Trp Leuly Gln Ala Ala Lys# 30- Val Lys Gly Arg 35__________________________________________________________________________
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Michael E. DeBakey VA Medical Center - Houston, Texas
Houston VA Work Program Offers Veterans A "Way Back"
July 28, 2003
Houston VA Work Program Offers Veterans A "Way Back"
Veterans Industries has provided vocational rehabilitation services to veterans for decades and yet, little is known outside of the VA about the treatment successes of these programs.
Released: 2003/07/28
Transitional Work Experience program participant, Lawrence Sanders (left), stops and talks with Peggy Moore, assistant manager of the HVAMC Environmental Management Section. Five days a week, eight hours a day, you will recognize Sanders as the smiling and affable housekeeping aid on the HVAMC's 4th floor, taking care of the auditorium, business offices, and the Oncology Clinic. "I think this job will give me the break I need to get back on my feet and into the workforce, plus I enjoy talking with everyone around here," said Sanders.
PHOTO: Mike Spratt, HVAMC Media Section
HOUSTON, TX - Lawrence Sanders, an Army veteran, has always dreamed of opening his own vehicle detail shop. He has encountered a few bumps along the way, but now believes he is back on track. "Everybody has dreams and goals, and I'm trying to find my way back," he said. Sanders admits he got mixed up with the wrong crowd a few years ago and made some mistakes in judgment. "I've paid my dues for that and now I want a better life. It's all about attitude."
This past March, Sanders started in the Incentive Therapy (IT)/Veterans Industries (VI) program with the Houston VA Medical Center (HVAMC), then advanced to the Transitional Work Experience program (TWE). Five days a week, eight hours a day, you will recognize him as the smiling and affable housekeeping aid on the hospital's 4th floor, taking care of the auditorium, business offices, and the Oncology Clinic. "I think this job will give me the break I need to get back on my feet and into the workforce, plus I enjoy talking with everyone around here," said Sanders.
The Veterans Health Administration (VHA) maintains over a hundred Veterans Industries Work Therapy programs nationwide. Approximately 7,000 veterans complete these programs each year. VI is a therapeutic work-for-pay program in which the Department of Veterans Affairs (VA) contracts with private industry and federal agencies for work performed by veterans. These Compensated Work Therapy (CWT) programs were established in the 1950s. The majority of veterans involved participate in outpatient mental health programs, and live in VA domiciliaries or supportive housing in the community.
At the HVAMC, the comprehensive treatment plan for patients with substance use or misuse, or mental health problems includes assessment and referral for vocational rehabilitation services. This program began at the HVAMC in 1994, and each veteran is offered an opportunity to participate in TWE.
Last April, TWE significantly expanded at the HVAMC to include two certificate-training programs in the areas of housekeeping and patient escort. During these 90-day on-the-job training programs, some veterans receive a certificate that documents their proficiency in hospital linen processing duties while other veterans may receive a general housekeeping certificate. Veterans with the desire to develop more advanced skills receive classroom and on-the-job training in hospital safety, body mechanics, patient room cleaning, equipment operation, proper handling of biohazard trash, customer service standards, infection control, special procedures for transporting wheelchair and stretcher patients, and techniques to clean critical care areas such as surgical suites, spinal cord units, and nursing home areas.
In addition to the housekeeping and patient escort training programs, veterans receive valuable work experience in shipping and receiving, medical supply, grounds keeping, food service, and wheelchair repair. HVAMC management has already expanded the TWE program to other job sites. In May, eleven veterans began work as grounds keepers at the Houston National Cemetery.
Veterans Industries has provided vocational rehabilitation services to veterans for decades and yet, little is known outside of the VA about the treatment successes of these programs. Recently, a segment of the video "VI/CWT Partnering with America's Veterans" was filmed at the HVAMC. The goal of this video is to challenge corporations searching for quality workers to consider an invaluable and greatly underutilized resource, a veteran. The HVAMC Veterans Industries program has been accredited by the Council for the Accreditation of Rehabilitation Facilities since 1999, and recently received reaccreditation until 2005.
Of the TWE program at the HVAMC, Sanders said, "I didn't want to go through another drug program. Too many just don't work. Here at the VA, it was different. I started listening to the counselors and the possibilities. It's about the best program I've come across. It's motivation. The counselors give us motivation. They don't treat us like criminals or addicts. They don't classify us as that was what you were, that's what you will be. The program here gives you a better outlook, keeps your motivation high, builds up your self-esteem, keeps your head clear."
Sanders is also pursing his dreams by starting a vehicle detail business out of his home. "The VA offered me a second chance. You have to stick with it. If you give up, it's over," said Sanders.
If you would like more information about the HVAMC TWE program, call the HVAMC Specialized Evaluation and Rehabilitation Program Director at (713) 794-7116/7044.
# # #
Point of Contact: VHAHOU Public Affairs04/21/04 08:25

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text:
Hillary Clinton, pro-abortion triangulator
The leading Democratic candidate for president is being criticized by a pro-life figure for her reaction to Planned Parenthood officials caught in undercover videos.
A legal group representing a California first grader says it stands by the youngster's censorship allegations against a public school teacher.
The teacher, Tammy Williams, allegedly told Brynn Williams, "Jesus is not allowed in school," when the child presented the Star of Bethlehem at a Christmas-themed show-and-tell last December.
The teacher now claims she never censored the child.
The child's family enlisted the help of California-based Advocates for Faith and Freedom.
"We stand behind the facts as provided by our clients," says Advocates general counsel Robert Tyler.
The school district, Temecula Valley Unified School District, released a statement from Williams in which she claimed she cut the student short because she was having trouble completing the speech. In addition, the teacher said she stopped the first-grader after she shared about Mary and Joseph and asked her about her Christmas tradition.
Advocates reported that the student was told to stop her presentation and take her seat, the only child not allowed to finish the one-minute presentation.
After the school responded to the allegation, Tyler says the law group submitted a 20-page response to the school district addressing the claims of the teacher and principal.
Advocates alleges correspondence between the student's mother and the school district show that the principal is trying to cover up.
Williams was allegedly prevented from reciting John 3:16 to complete her speech, and Tyler says the student was only allowed to recite the verse in the principal's office to make her feel better.
Tylers says the law group has a "valid basis to believe our clients and what we were told by them."
The leading Democratic candidate for president is being criticized by a pro-life figure for her reaction to Planned Parenthood officials caught in undercover videos.


text:
$4.50 NNN? You Must Be Joking
October 23, 2009
We've generally refrained from turning this blog into a Burbed for commercial real estate, but occasionally we see a listing that makes us reconsider. So in the Burbed spirit ladies and gentlemen, we would like to present you with 350 Winchester Blvd! Only $4.50 NNN (that's $54 for those of you in SF).
At these rates, we hope it's got entitlements for a grow house, because without those entitlements it's maybe $2.00 space.
And in case you are wondering, this is the same property that was subject of an ongoing lawsuit that last we read put FRIT on the hook for a pretty penny.
We'll have to come up with a special tag for these kind of listings – feel free to comment with any ideas. Here's a couple to start with: "dreaming" and "out of touch".
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Tags: 350 Winchester, Commercial Real Estate, Federal Realty, Office Space, Santana Row
I love these type of posts! They expose the fact that many people are just not living in reality. One thing I noticed in the brochure is that the smallest space they can provide is 6,700 square feet. Needless to say, $4.50 NNN for 6,700 square feet is VERY STEEP. It would be one thing if it was a 750 – 1,200 square foot suite in an A+ location, but this seems to miss that mark by a wide margin.
I like the grow house comment. Appropriate for NorCal and the numbers would still work at $4.50 NNN.
just because it is next to mantana row doesn't mean it's a great retail location. i bet if you look at the non restaurant retailers at santana row, they actually don't do that well, and to expect people to walk down the alley to get to this building is a stretch too.
Didn't the owners of Santana Row try to buy this property back before they started construction and they refused to sell? I wonder if they wished they sold now…


text:
…the Preferred Choice for Preferred Care!
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Surgical Abortion
Abortion Pill and other No Surgery options
Pregnancy Test
We offer you experience you can trust…
A Preferred Women's Health Centers have offered no surgery abortion options for our patients for over 10 years! In 2000, we became one of the first medical facilities in the United States to provide abortion services with the use of medications only. Given the convenience and privacy of this treatment, medical abortions for terminating early pregnancies continues to be a preferred option for many of our patients.
If you choose to pursue a medical abortion, A Preferred Women's Health Center can meet your unique needs. We have successfully treated thousands of women over the past 10 years with these medications to enable them to have an abortion without surgery.
We also offer expedited care…
We try to minimize the time you have to spend in the office. And most recently, we have modified staff assignments so our medical abortion patients have their own patient care provider! There is someone assigned just to you to facilitate your care and eliminate any unnecessary delays while you are at our office!
Unlike other providers, A Preferred Women's Health Centers offer this expedited care service at no additional charge. Convenience without additional cost is what we provide!
We have expanded services to meet your needs…
In order to offer a no surgery abortion option to more of our patients, we have implemented a revised treatment plan that allows us to use the Abortion Pill with patients up to 9 weeks in gestation (63 days).
Given our many years of successful experience, we continue to offer the methotrexate treatment regimen as an option. RU-486, Mifeprex, Abortion Pill and Methotrexate all require the use of Misoprostal, also called Cytotec, as the second medication in these treatments.
In consultation with your physician at A Preferred Women's Health Center, you can choose which no surgery abortion method will be best for you!
What to expect when you arrive…
Upon arrival, you will be asked to fill out a medical history, complete routine laboratory tests (Hemoglobin, Rh testing, urine pregnancy test), and have an ultrasound evaluation to verify the length of your pregnancy. If you are less than 9 weeks, you will proceed with counseling and consultation from the APWHC staff and your physician to discuss your abortion care options. They will be glad to answer any questions you have about the abortion pill.
How does it work…
Whether the choice for treatment is Mifepristone, also known as RU-486, or Methotrexate, both medications produce the same outcome. They interrupt growth and inhibit further development of the early pregnancy. More specifically, Methotrexate stops cell growth of the pregnancy; mifepristone inhibits continued implantation of the pregnancy. The effect of both is to stop the gestation from further growth or development.
After the initial medication has been given, Misoprostal is administered to induce uterine cramping. These cramps cause the uterus to bleed and shed its' contents in much the same way cramps promote a monthly menstrual period. With a medical abortion, the induced bleeding may be heavier than a normal period and contain some clot formations, but usually nothing more significant.
The length of bleeding varies and sometimes lasts longer than a normal mentrual cycle. After a patient returns for follow up care, periods very quickly return to normal. There are no usual residual effects of the abortion pill treatment and they do not known to interfere with future fertility or pregnancies.
More questions? Need an appointment?
A Preferred Women's Health Center has phone counselors available to answer your questions and schedule an appointment 7 days a week:
1 (888) 665-4126.
Or, if you prefer, confidential appointments can be made on this secured website. It is your choice!
Thank you for choosing A Preferred Women's Health Center.
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…the Preferred Choice for Preferred Care!
Abortion Counseling...Learn More


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First Look: Gringo Offers Taco Options Like Al Pastor, Octopus and Grasshopper in the Central West End [Photos]
From the formidable forces behind the successful pizza empire that is St. Louis chain Pi Pizzeria comes a new concept called Gringo (398 North Euclid; 314-449-1212), which opened its doors in the Central West End last week with a full menu of tacos, margaritas and more.
Mabel Suen El Jibarito, a fried plantain "sandwich" with pork belly, tomato-chipotle jam, avocado and pickled red onion.
See also:
- Pi Pizzeria Team Opening Gringo
- First Look: New Moon Room at the Moonrise Hotel
- First Look: Central Table Food Hall
Co-owner Chris Sommers describes Gringo as, "a mid-century Baja beach party," and the brightly lit 3,300-square-foot dining room complete with a hanging swordfish matches his vision accordingly. Finished wood, a recurring bright blue and yellow logo, and black-and-white portraits of Gringos in Latin American garb accentuate the space.
There's room for 24 at the bar, 84 in the dining room and 68 on an outdoor patio that wraps around the building at the corner of Euclid and McPherson Avenues.
Pi executive chef Steve Caravelli heads up the menu with sous-vide cooked meats. Each meal begins with chile dusted tortilla chips and house roasted tomato-chipotle salsa -- the first one's on them and then it's about a buck for a refill.
Mabel Suen
Tacos come in options such as al pastor ($3.50), grilled octopus ($3.50) and Sommers' personal favorite, pescado ($3.50), with grilled or fried snapper, Napa cabbage slaw, chipotle crema and toasted pepitas. Brave and bold bug-loving folks can go for chapulines ($4.50) made with sauteed grasshoppers, guajillo pepper and avocado.
See a photo of the grasshopper taco on the next page!
Location Info
Sponsor Content


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Robert Newman & daughter Kendal (Christine Fix, Soaps.com)
It seems that proud fathers love showing off their daughters at the glitzy events! The 2008 Daytime Emmys was an example of dads bringing their girls with them Emmy night for a taste of the red carpet and the awards show.
Robert Newman (Josh, Guiding Light) and his Daughter Kendal (his date for the evening) stopped by to chat on the red carpet before the show began. Robert was wearing a suit by Armani for the big night. When I asked if there are any big celebrating plans if GL takes home best show, Robert replied: "You know, I work with some incredibly gifted people on both sides of the camera and it would be well deserved. We would totally enjoy it and have fun with it!" Chrissi and I then took the opportunity to chat with Kendal for a moment. We asked her "What's it like watching your father on TV everyday?" Kendal chuckled and said "Oh, you get used to it when it happens for your entire life. It does get a little weird though watching him on TV and in my living room…at the same time!" Kendal then discussed her thoughts on the new look of Guiding Light. "I love it", she exclaimed! "I do a lot of video as a hobby, and I think it's great that that style is kind of making it into television." I then inquired "Do you feel that the show is now more modern and trendy?" Kendal informed us, "Yes, I do. I like it a lot!" Robert took the opportunity to wrap it up by saying "That is such a great thing to hear, cause this is sort of the age group (16 to 18 to 20 in that area) that's what we're hoping to capture with this." Robert is correct; that is the age group of the viewers of the future, so hopefully they like what they are seeing!
Doug Davidson & daughter Calyssa (Christine Fix, Soaps.com)
Kristoff St. John & daughter Lola (Christine Fix, Soaps.com)
Soaps.com was as impressed with the lovely, talented and down to earth daughters of these soap vets as we were with these soap vets. Only one of the father's was nominated this year for an award and Kristoff took home the coveted Outstanding Lead Actor Award, with his family at his side. Congratulations to Kristoff, especially since it's his first win! We wonder if he let Lola hold it for him on the way home?


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Earlier this week, a bunch of people gathered at North Charleston City Hall to yell at a cardboard cut-out of Sen. Lindsey Graham.
Not too long ago, a Myrtle Beach tea party group threw our senior senator a "retirement party." Another group wanted to put Graham's face on a milk carton.
And Thursday, the Tea Party Express — which bills itself as the largest tea party political action committee — came to Charleston to complain about Graham's unwillingness to join their fight to defund Obamacare.
"He needs to be in the state, holding town halls and listening to his constituents," said Amy Kremer, chairwoman of the Tea Party Express. "He thinks he's better, thinks he knows better. He's one of the elites."
You know, a guy could get the feeling some people out there just don't like him — especially the 10 or so people who showed up Thursday for the Tea Party Express press conference (not including two of the people running against Graham).
Make no mistake, these folks are passionate. And they believe they are the only ones fighting to "restore liberty" and "honor the Constitution."
Problem is, they are a little fuzzy on the details. And honestly, Graham does know more than they do.
And that's why he's going to eat their lunch.
Do the math
These tea party people are right about one thing: Obamacare is unpopular.
Most recent polls show that a plurality, if not majority, of Americans have an unfavorable view of the Affordable Care Act.
But polls also show a clear majority oppose Republican efforts to defund the program, which is what the Tea Party Express is pushing.
Although the group claimed it's not getting into individual races at this time, it was all over Graham for not signing Utah Sen. Mike Lee's pledge to vote against any budget that includes money for Obamacare.
Tellingly, they said little about Sen. Tim Scott, who has not signed on either.
But Graham has a target on his back. He's a Republican in Name Only, they say. You can hear the disdain dripping from their voices when they mention his willingness, on occasion, to compromise.
That's a four-letter word in the tea party's world.
Instead, these groups adore Sens. Lee, Ted Cruz, Marco Rubio and Rand Paul. You have to wonder if the mostly retired folks in the crowd Thursday realized that those guys, if in power, would soon turn their attention to Medicaid and, yes, Medicare.
Richard Cash, a corporate computer analyst, small business owner and missionary, is almost without question the most formidable opposition Graham faces. He told the Tea Party Express crowd that his hero is Jim DeMint.
And what does DeMint have in common with Cruz, Rubio and the others? Yeah, he helped them get elected.
What they don't point out is that DeMint's endorsements of numbskulls in Delaware, Indiana and Missouri cost the Republican Party the U.S. Senate.
That's why Graham isn't wasting time on this defund Obamacare thing. He can count.
And the conservatives don't have the votes in the Senate.
Thanks to DeMint.
Dreams of sheep
Of course, Ronald Reagan's hallowed name was invoked Thursday at the Tea Party Express meeting.
Funny, these folks don't remember that Reagan compromised often with Democratic House Speaker Tip O'Neill, and even — gasp — raised taxes a few times. A lot of mainstream Republicans like to grouse that Reagan couldn't even make it through a primary these days if he faced tea party litmus tests.
And it's possible Graham could lose to a DeMint disciple — this is, after all, South Carolina.
Trouble is, every time the tea party knocks out one of the Republican Party's elder statesmen, they open the door for Democrats to take a seat that would otherwise go to the GOP (see Missouri, Indiana).
And that's the thing. Clearly, the tea partiers can read polls. They should look at the ones that show they are in the clear minority. Most people don't believe in their brand of extremism.
They know that politics is the art of compromise. At least it used to be, back in the days of Reagan.
That's why this idea of a Senate filled with Lees, Cruzes and Pauls is a pipe dream.
And the goal of defeating Graham is, at best, about as likely as defunding Obamacare.
Reach Brian Hicks at firstname.lastname@example.com
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Narrow your search
The Somfy myLink and motorized window shades combine with mobile app for complete smartphone control.
Three GMC trucks and two Buick models will feature Apple's smartphone integration system in the 2016 model year.
At a press preview event in San Francisco, Chevrolet demonstrated both Android Auto and Apple CarPlay, and promised implementations in 10 of its models.
The MyLink software from Chevy is more user-friendly than before.
Chevrolet showed off a 2014 Impala equipped with the next generation of its MyLink infotainment system at CES 2013.
Chevrolet showed off a 2014 Impala at CES 2013, featuring its next-generation MyLink system, with smartphone-like icons and flexibility.
Even without piling on the options, the 2013 Chevrolet Malibu LTZ offers a pretty good list of standard cabin tech features from its MyLink infotainment system.
Looking for tips on safely using your Android phone with your car's stereo? CNET has you covered.
The 2013 Chevrolet Spark LT's doesn't come with a CD player, but it makes up for this absence with a full suite of digital-audio connections and smartphone app connectivity. You probably don't listen to CDs anymore anyway, right?
Chevrolet's Spark is the least expensive vehicle in GM's lineup, but that doesn't mean that it has to have the lowest tech.


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Join us in celebrating 21 years of business. We'll have live music by Echo Park, special wine flights and sangria available for additional purchase + a trio of sliders for ($16 / $14 for Wine Club members) while supplies last. Sliders include: Cabernet Onion & Sharp Cheddar Slider, Coffee-Rubbed Slider with Swiss & Stampede BBQ Sauce, Bombay Slider with Garlic-Curry Sauce.
Anniversary Barbecue at Duck Pond Cellars
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The Olympic torch is set to arrive in Hull and North East Lincolnshire in June 2012 as part of a 70-day tour of the UK.
The torch will arrive in Hull on Monday, 18 June and in Cleethorpes on Tuesday, 26 June, en route to London.
BBC News - Olympic 2012 torch relay to visit Hull and Cleethorpes


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Physical activity can help promote brain health
Physical activity not only benefits your body, it's a great work-out for your brain too. As we mentioned last week, engaging in physical activity on a regular basis has been proven to reduce levels of stress and anxiety, as well as improve learning and memory.
There is also some recent medical research that suggests physical activity may even go as far as helping to reduce the risk of being diagnosed with dementia or significantly lessen its damaging effects.
One of those studies published in the American Heart Association's Journal, "Stroke," showed that older adults who exercised regularly reduced their risk of vascular-related dementia by 40 percent.
Physical activity is great for elevating one's mood, helping to increase self-esteem and improving the ability to react calmly in given situations.
From a physiological standpoint, physical activity allows for more oxygen and blood flow to the brain, thereby fostering an environment for the production of brain cell growth as well as working to potentially improve brain cells that may be damaged.
It can also stimulate certain neurotransmitters to release signals allowing for pain reduction. Additionally, there are also numerous cognitive benefits from physical activity that include enhancing the ability to store learned information, assisting to focus one's attention on certain tasks and aiding in memory improvement.
Before beginning any physical activity routine, it is always best to consult a physician to determine what level of physical activity is safest and most appropriate for you, especially for those already in a declining mental state.
Source: American Heart Association
Healthy Habits 2013 is a partnership among Cabell Huntington Hospital, Marshall University Joan C. Edwards School of Medicine and St. Mary's Medical Center. We are a community working together to improve our health. Our goal is a simple one: to inform and encourage area residents on ways to improve their health. Join our conversation and "like" us on Facebook at www.facebook.com/healthyhabits2013.


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|Product #: EMC3377016_TQ
What's in This Book? High-Frequency Words: Stories and Activities, Level B (R eBookKindergarten|Grade 1
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This vocabulary development unit from 'High-Frequency Words: Stories and Activities, Level B' provides teacher directions for the materials and strategies available in each unit. Includes the award form entitled 'You Are a Star!'
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The summer project is completed and I am finally home. It feels good to be in my own surroundings. It feels good to not be surrounded by people all day. It feels good not to have exhausting hours. It feels good not to have to rethink what I am saying in Spanish or to struggle […]
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Service Learning
My midwifery program has a strong social justice bent. Because of this, service-learning is built into the curriculum in many ways. This summer I am working with migrant farm workers through a farm worker health clinic. We do general well-child checks with the children at a summer school program during the day an in the […]
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Helping Babies Breathe
At the ACNM Annual Conference I attended a workshop entitled Helping Babies Breathe. It turned out to be a trainer training for a program from the American Academy of Pediatrics on a basic neonatal resuscitation program. This is not the Neonatal Resuscitation used in developed countries. This is an evidence based program designed to be […]
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Xavier opens the exhibition schedule against Marquette on Friday.
Aug. 16, 2012
CINCINNATI - The Xavier men's soccer team gets its first taste of preseason action against Marquette at the Shindigz National Soccer Festival on Friday in Fort Wayne, Ind.
It will be the first exhibition match of the 2012 season for the two-time defending Atlantic 10 Champion Musketeers that have had record-breaking success under third-year head coach Andy Fleming.
Xavier went 12-5-4 last season and was bumped from its second-straight NCAA Tournament by West Virginia. Returning from that squad are all 11 starters that posted 26 of 31 goals and 35 of 37 assists. Leading the offensive returners are senior Luke Spencer (7g, 6a) and junior Gino De Paoli (6g, 7a), who account for half of the returning goals.
Junior Nick Hagglund, a candidate for the Hermann Trophy, anchors one of the top defenses in the conference. Opponents scored only 19 times against the Musketeers last season, the lowest mark in program history. Joining Hagglund in the back are seniors James Queree and Sean Ryan, and junior Adar Cohen.
Starting again in goal for the Musketeers is senior Justin Marshall. The four-year starter is Xavier's all-time goals against average leader (1.11) and has had two-consecutive record-breaking years in net, which earned him NSCAA region honors.
Friday's match begins at 5 p.m. on field two.


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People & Places
Campus Dance Groups Create Community
SU Dance Corps, SU Belly Dance Circle and SU Swings give students the ability to dance at college without being a dance major.
Class and Lecture Examine the Impact of Arab Spring
Marking the first anniversary of the Arab Spring, Susquehannans explored the implications of this series of revolutions and protests that toppled three totalitarian regimes in the Middle East.
Initiative Explores Intersection of Liberal Arts and Medicine
The medical humanities Initiative (MHI), founded in 2005 under the direction of Associate Professors of History Ed Slavishak and Karol K. Weaver, explores how humanities disciplines illuminate the nature and practice of medicine.
Exhibit Explores Travel, Foreignness
Award-winning artist Asya Reznikov was behind the Up-Routed For exhibit at the Lore Degenstein Gallery.
Residence Halls Earn "Green" Certification
Two residence halls in Susquehanna University's West Village complex recently earned silver-level LEED.


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Ener-G Foods, Wheat-Free Crackers, 7 oz (200 g) (Discontinued Item)
(Save Image)
Ener-G Foods, Wheat-Free Crackers, 7 oz (200 g) (Discontinued Item)
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Shipping Weight
The Shipping Weight includes the product, protective packaging material and the actual shipping box. In addition, the Shipping Weight may be adjusted for the Dimensional Weight (e.g. length, width & height) of a package. It is important to note that certain types of products (e.g. glass containers, liquids, fragile, refrigerated or ice packed) will often require protective packaging material. As such, these products will reflect a higher Shipping Weight compared to the unprotected product.
Product Overview
Gluten-free crackers are suitable for gluten sensitive enteropathies.
Corn starch, palm oil, soy flour, salt, sugar, leavening agents (ammonium bicarbonate, sodium bicarbonate, sodium acid pyrophosphate), sodium carboxymethylcellulose, yeast, soy lecithin (emulsifier).


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Top Celebrity Engagements and Weddings from 2013
Watch Miley Cyrus Fall Down on Stage Wearing a Fake Butt
Beyonce Isn't as Flawless as We Thought
Kylie Jenner Says She Has To Act Fake For The Kardashian Image
Michael Jackson's Daughter Paris Reveals Her Boyfriend
Is Jon Hamm a Single Man?
Scott Disick's Heart Breaking Message To His Daughter
Mel Gibson Is Head Over Heels For A Very Young Woman
One "Hidden" Nest Egg Most Americans Don't Realize They Have
What Does Kylie Jenner Look Like Without Makeup?
Jake Gyllenhaal Breaks His Silence on Taylor Swift
Shannon Beador Drops Another Affair Truth Bomb
Twitter Reacts To Bobbi Kristina Brown's Death
Scott Disick Talks Baby Number 4 With Kourtney Kardashian
Caitlyn Jenner Shares Magnificent Father's Day Photo
The Truth About Tourism in Southeast Asia
Is Kaitlyn Bristowe's Engagement Already Over?
Yolanda Foster's Health Takes A Turn For The Worse?
Celebrities with Diseases: You'll Be Surprised by Who Has Been Diagnosed
Why Can't John and Katy Make it Work?
Brad Pitt & Angelina Jolie: When is it Time to Seek Help?
Chris Hemsworth is One Hot Dad
32 Ridiculously Cute Queer Women Couples
Did You know These Stars Were Born Rich?
Cindy Crawford's 13-Year-Old Daughter Looks Just Like Her
Celeb Kids Who Look Just Like Dad
Watch Ellen Shut Down Bethenny Frankel's Ignorant Comments
Woman Removes Makeup, Reveals Stunning Transformation
Kendall and Kylie Jenner Celebrate High School Graduation with Star-Studded Party
Caitlyn Jenner Shows Off Her Tomboy Side


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Welcome to Wolf & Pravato, P.A. Florida Personal Injury Lawyer
Hiring a lawyer is an important decision that requires careful consideration. At Wolf & Pravato, P.A., we focus our legal practice on personal injury cases, such as: car accidents; injured at work; medical malpractice; faulty products like Stryker hip, Chinese drywall, and spray foam insulation. Since 1996 we've been helping injured people get the just and equitable compensation they need to get back on their feet.
Why Hire Wolf & Pravato?
Wolf & Pravato, P.A. has served Southern Florida since 1996 and is dedicated to providing an unwavering commitment to clients. With a team of six very highly accomplished personal injury lawyers and a staff of fourteen client relations experts, we provide each client with personal, one-on-one attention with regular communication and updates about the progress of your case.
We are proud of the prestigious awards we have earned, including:
- Board Certified Civil Trial Attorney – The highest level of evaluation by the Florida Bar, it identifies the attorney as a "specialist" or "expert" in their field. – Only about 1% of all Florida Attorneys are Board Certified Civil Trial Attorneys.
- Lifelong Member of the Million Dollar Advocates Forum, reserved for trial attorneys who have won multi-million dollar trial verdicts.
Named one of 2013 Southern California Super Lawyers.
Personal injury claims settled with the help of an attorney often result in higher values than those settled alone. Our lawyers' extensive experience includes skill in the following areas:
- Personal Injury Experience: Wolf & Pravato's attorneys have handled complicated high-stakes car accidents, including wrongful death litigation, and cases other injury lawyers have turned to us for assistance. Whether you've been injured at work, involved in a car accident or are a victim of medical malpractice, we can help.
- Trial Experience: Our lawyers prepare your case as if it were going to trial – even if an out-of-court settlement is expected. Our willingness to take your case to court alone is often the key factor that lets us negotiate you a higher settlement.
- Win Large Class Action Law Suits: Not every law firm is equipped with the resources and the know-how to handle a large scale class action law suit. Wolf & Pravato have handled many large class action lawsuits, such as Stryker hip, Chinese drywall, and spray foam insulation.
Contact us immediately and we'll answer all of your questions, explain your rights, and if we take on your case, we will help you achieve justice. Justice is not automatic – let us fight for you.
Call 1-844-334-1783 to schedule a consultation in South Florida, including Fort Lauderdale, Miami, Fort Myers, West Palm Beach, Boynton Beach and Tampa. For internet based inquiries, please fill out our online consultation form to schedule a private consultation with one of our attorneys.


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|Business and Economics home | About us | Future students | Current students | Alumni | Contact us
|Intranet | Staff directory | A-Z index | Site map
International Human Resource Management: China and North Asia
Scope of the Research:
Key Researchers:
Project 1: The Changing Role of HRM in Transitional Economies: Evidence from China
Project researchers: Cherrie Zhu, Helen De Cieri, Brian Cooper,
Project 2: Enhancing Knowledge Sharing in Multinational Corporations in China
Michailova, S. and Hutchings, K. 2006. "National Cultural Influences on Knowledge Sharing in China and Russia", Journal of Management Studies, 43(3): 383-405.
Hutchings, K. and Michailova, S. 2006. "Impacts of Culture and Institutions on Knowledge Sharing in Russia and China", International Journal of Emerging Markets, 1(1): 21-34.
Hutchings, K. and Michailova, S. 2004. "Facilitating knowledge sharing in Russian and Chinese subsidiaries: The role of personal networks", Journal of Knowledge Management, 8(2):84-94.
Project researcher: Kate Hutchings
Project 3: Retrospective and prospective analysis of HRM research in China: Implications for further study
Project researchers: Cherrie Zhu, S. Bruce Thomson and Helen De Cieri
Project 4: Differences between Planned, Implemented and Perceived HR Practices and their Implications for SHRM: Empirical Test in MNCs Operating in China
Project researchers: Cherrie Zhu and Helen De Cieri
Project 5: Outward FDI by Chinese Multinational Enterprises: Implications for International Human Resource Management
Zhu, C.J., Thomson, B., De Cieri, H. & Dowling, P.J. 2006. Outward FDI by Chinese MNEs: Developments, progress and emerging HRM issues. Paper presented in Panel session at the Academy of International Business Annual Conference, Beijing China, June 23-26.
Project researchers:
Project 6: International HRM in the Asia Pacific Region
Hutchings, K. 2005. Koalas in the land of the pandas: Reviewing Australian expatriates' China preparation. International Journal of Human Resource Management, 16(4): 553-566.
De Cieri, H., Fenwick, M., & Hutchings, K. 2005. The challenge of international human resource management: Reconciling strategic academic approaches with practical complexity. International Journal of Human Resource Management, 16(4): 584-598.
Project 7: Social Security Reform and HRM: Empirical Evidence from China
Project researchers: Chris Nyland, Cherrie Zhu, and Brian Cooper
Project 8: Intercultural Networking in China and the Arab World
Hutchings, K. & Weir, D. 2006. Guanxi and Wasta: A comparative examination of the impact of internationalisation and modernisation on traditional ways of networking in China and the Arab World. Thunderbird International Business Review, 48(1): 141-156.
Project researchers:
Project 9: Expatriate Managers vs. Chinese Managers vs. Local-hire Foreign Managers: A Comparison of Australian and German Organisations in China
Project researchers:


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Many types of engines are available in the market, what sets them apart are the models, types of power supply, what risks each of them will have been in contact with the water and further, each electric motor, has your number record like a car that has its chassis. The electric motor can not be sold without proper registration and also have all the specifications noted in it.
Motor rollertable2 with inscription IR55 baldor ebm3554t
This type of motor is presented with high strength and robustness to extreme operating conditions, it has radial fins to prevent the accumulation of waste in housing, and also count on the advanced sealing system, and a protection against corrosion and mechanical rigidity.
Water IR2
The engines are cooled to water and were developed to meet the growing market demand for compact and quieter engines and at the same time has a power relationship as defined by the engine outer shell size.


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source: http://www.writersweekly.com/news_from_the_home_office/006468_12292010.html
December 29, 2010
The Christmas Bug!
Caleb was sick on the 23rd with a stomach bug. I obsessively santized the house and, over and over again, but to no avail. Max woke up with it on Christmas morning. I took pictures of him opening his presents in bed. By mid-afternoon, Frank was sick and Rita and I succumbed by bedtime. On Sunday night, Zach was down for the count and Kimia (Caleb's girlfriend) sent me a text that she had it as well. Nobody has gotten sick since then so we are hoping everybody else in the house is immune. We had to postpone our Boxing Day party, of course.
As I'm typing this on Monday, we are getting the big blizzard that's all over the news (14 inches by noon) and Ali is cooking a gourment meal for us in the kitchen while the adults are watching comedies in the living room, next to the Christmas tree.
The Boxing Day party will be tomorrow and we're looking forward to a rip-roarin' good time!
This week's Maxism:
"Does Santa put hidden cameras in kids' rooms to see if they're naughty or nice?"
Hugs to all!
source: http://www.writersweekly.com/news_from_the_home_office/006468_12292010.html
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George Ou's networking and security insights keep enterprise managers in the know and vendors up at night.
George Ou, a former ZDNet blogger, is an IT consultant specializing in Servers, Microsoft, Cisco, Switches, Routers, Firewalls, IDS, VPN, Wireless LAN, Security, and IT infrastructure and architecture.</p>
How higher RPM hard drives rip you off
Is encryption really crackable?
Why VPN can't replace Wi-Fi security
Don't believe the low bit-rate 'HD' lie
Why Satellite Internet service is so slow


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Posted at: 02/14/2013 11:31 AM
Updated at: 02/14/2013 11:35 AM
Elizabeth Reil of Scraps Galore offers tips for making homemade Valentine's Day cards.
Scraps Galore
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Few things feel sexier than applying red lipstick, puckering up, and batting those long lashes. Let your accessories in on the fun with this pouty little clutch, designed in Manhattan's fashion-savvy SoHo neighborhood. A natural beauty of woven straw, this handbag is all done up for a night on the town…which is exactly where you should be taking it.
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