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Activity of the Kinesin Spindle Protein Inhibitor Ispinesib (SB-715992) in Models of Breast Cancer
Purcell, James W.
Davis, Jefferson
Reddy, Mamatha
Martin, Shamra
Samayoa, Kimberly
Vo, Hung
Thomsen, Karen
Bean, Peter
Kuo, Wen Lin
Ziyad, Safiyyah
Billig, Jessica
Feiler, Heidi S.
Gray, Joe W.
Wood, Kenneth W.
Cases, Sylvaine
Clinical cancer research : an official journal of the American Association for Cancer Research
Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein, a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have shown a 9% response rate in patients with locally advanced or metastatic breast cancer and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities, or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer, we explored the activity of ispinesib alone and in combination with several therapies approved for the treatment of breast cancer.
Experimental Design
We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo and tested the ability of ispinesib to enhance the antitumor activity of approved therapies.
In vitro, ispinesib displayed broad antiproliferative activity against a panel of 53 breast cell lines. In vivo, ispinesib produced regressions in each of five breast cancer models and tumor-free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the antitumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine and exhibited activity comparable with paclitaxel and ixabepilone.
These findings support further clinical exploration of kinesin spindle protein inhibitors for the treatment of breast cancer.
Related Articles
Initial Testing (Stage 1) of the Kinesin Spindle Protein Inhibitor Ispinesib by the Pediatric Preclinical Testing Program
Carol, Hernan
Lock, Richard
Houghton, Peter J.
Morton, Christopher L.
Kolb, E. Anders
Gorlick, Richard
Reynolds, C. Patrick
Maris, John M.
Keir, Stephen T.
Billups, Catherine A.
Smith, Malcolm A.
Pediatric blood & cancer
Ispinesib is a highly specific inhibitor of kinesin spindle protein (KSP, HsEg5), a mitotic kinesin required for separation of the spindle poles. Here we report the activity of ispinesib against the in vitro and in vivo panels of the Pediatric Preclinical Testing Program (PPTP).
Ispinesib was tested against the PPTP in vitro panel cell lines at concentrations from 0.1 nM to 1 μM and against the in vivo tumor panel xenografts by intraperitoneal administration (5 or 10 mg/kg) every 4 days for 3 doses repeated at day 21.
Ispinesib was highly potent against the PPTP's in vitro cell lines with a median IC50 of 4.1 nM. Ispinesib (10 mg/kg) induced unexplained toxicity in mice bearing osteosarcoma xenografts and exceeded the MTD in 12 of 40 non-osteosarcoma xenografts. Ispinesib induced significant tumor growth delay in 88% (23/26) of evaluable xenografts. Using a time to event measure of efficacy, ispinesib had intermediate and high levels of activity against 4 (21%) and 5 (26%) of the 19 evaluable solid tumor xenografts, respectively. Ispinesib induced maintained complete responses (CR) in a rhabdoid tumor, a Wilms tumor and a Ewing sarcoma xenograft. Ispinesib (5 mg/kg) produced 2 complete and 2 partial responses among 6 evaluable xenografts in the ALL panel. The in vivo pattern of activity was distinctive from that previously reported for vincristine.
Ispinesib demonstrated broad in vivo antitumor activity, including maintained complete responses for several xenografts, although with high toxicity rates at the doses studied.
Preclinical Testing; Developmental Therapeutics; Ispinesib
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KIF11 inhibition for glioblastoma treatment: reason to hope or a struggle with the brain?
Valensin, Silvia
Ghiron, Chiara
Lamanna, Claudia
Kremer, Andreas
Rossi, Marco
Ferruzzi, Pietro
Nievo, Marco
Bakker, Annette
BMC Cancer
Glioblastomas (GBM) are typically comprised of morphologically diverse cells. Despite current advances in therapy, including surgical resection followed by radiation and chemotherapy, the prognosis for patients with GBM remains poor. Unfortunately, most patients die within 2 years of diagnosis of their disease. Molecular abnormalities vary among individual patients and also within each tumor. Indeed, one of the distinguishing features of GBM is its marked genetic heterogeneity. Due to the brain location of the tumor, the potential target inhibition for anticancer therapy must exhibit a manageable neurotoxicity profile in the concentration range in which the compounds show anti-proliferative activity.
Kinesin KIF11 inhibition by small molecules such as Monastrol or Ispinesib is currently under investigation in the field of malignant tumors. In the current study we have assessed the relevance of the anti-mitotic Kinesin-like protein KIF11 in human GBM cell-lines.
In this study the target was validated using a set of well characterised and potentially specific small molecule inhibitors of KIF11: an ispinesib analog, Monastrol, a Merck compound and 3 simplified derivatives of the Merck compound. Following an in silico selection, those compounds predicted to bear a favorable BBB permeation profile were assessed for their phenotypic effect on cell lines derived both from primary (U87MG) as well as treated (DBTRG-05-MG) glioblastomas. For some compounds, these data could be compared to their effect on normal human astrocytes, as well as their neurotoxicity on primary rat cortical neurons. The ispinesib analogue 1 showed an anti-proliferative effect on GBM cell lines by blocking them in the G2/M phase in a concentration range which was shown to be harmless to primary rat cortical neurons. Furthermore, ispinesib analog increased caspase 3/7-induced apoptosis in U87MG cells.
In the area of cell cycle inhibition, KIF11 is critical for proper spindle assembly and represents an attractive anticancer target. Our results suggest that KIF11 inhibitors, when able to permeate the blood-brain-barrier, could represent an interesting class of anticancer drugs with low neurotoxic effects in the treatment of brain tumors.
Related Articles
Triphenylbutanamines: Kinesin Spindle Protein Inhibitors with in Vivo Antitumor Activity†
Wang, Fang
Good, James A. D.
Rath, Oliver
Kaan, Hung Yi Kristal
Sutcliffe, Oliver B.
Mackay, Simon P.
Kozielski, Frank
Journal of Medicinal Chemistry
The human mitotic kinesin Eg5 represents a novel mitotic spindle target for cancer chemotherapy. We previously identified S-trityl-l-cysteine (STLC) and related analogues as selective potent inhibitors of Eg5. We herein report on the development of a series of 4,4,4-triphenylbutan-1-amine inhibitors derived from the STLC scaffold. This new generation systematically improves on potency: the most potent C-trityl analogues exhibit Kiapp ≤ 10 nM and GI50 ≈ 50 nM, comparable to results from the phase II clinical benchmark ispinesib. Crystallographic studies reveal that they adopt the same overall binding configuration as S-trityl analogues at an allosteric site formed by loop L5 of Eg5. Evaluation of their druglike properties reveals favorable profiles for future development and, in the clinical candidate ispinesib, moderate hERG and CYP inhibition. One triphenylbutanamine analogue and ispinesib possess very good bioavailability (51% and 45%, respectively), with the former showing in vivo antitumor growth activity in nude mice xenograft studies.
Related Articles
A Pediatric Phase 1 Trial and Pharmacokinetic Study of Ispinesib: A Children's Oncology Group Phase I Consortium Study
Souid, Abdul-Kader
Dubowy, Ronald L.
Ingle, Ashish M.
Conlan, Maureen G.
Sun, Junfeng
Blaney, Susan M.
Adamson, Peter C.
Pediatric blood & cancer
To determine the maximum-tolerated dose, dose-limiting toxicities and pharmacokinetics of the kinesin spindle protein inhibitor ispinesib in pediatric patients with recurrent or refractory solid tumors.
Subjects and Methods
Ispinesib was administered as 1-hour intravenous infusion weekly × 3, every 28 days. Cohorts of 3-6 patients were enrolled at 5, 7, 9 and 12 mg/m2/dose. Serial plasma samples for pharmacokinetic analyses were obtained after the first dose.
Twenty-four (13 females) patients with a median (range) age of 10 years (1-19) were enrolled on the study. At the 12 mg/m2 dose level dose-limiting neutropenia occurred in 2/6 patients and hyperbilirubinemia in 1/6 patients, while at the 9 mg/m2 dose level 1/6 patients had dose-limiting neutropenia. There were no objective responses, but 3 patients (diagnoses of anaplastic astrocytoma, alveolar soft part sarcoma and ependymoblastoma) had stable disease for 4 to 7 courses. There was substantial inter-patient variation in drug disposition. The median (range) terminal elimination half-life was 16 hours (8-44) and the plasma drug clearance was 5 L/hr/m2 (1-14).
The maximum tolerated and recommended phase II dose for ispinesib administered weekly × 3 every 28 days for children with solid tumors is 9 mg/m2/dose. Plans for a phase II trial in select pediatric solid tumors are in development.
Ispinesib; pediatric cancer; solid tumors; Children Oncology Group; phase 1 trials
Related Articles
A phase I trial of ispinesib, a kinesin spindle protein inhibitor, with docetaxel in patients with advanced solid tumours
Blagden, S P
Molife, L R
Seebaran, A
Payne, M
Reid, A H M
Protheroe, A S
Vasist, L S
Williams, D D
Bowen, C
Kathman, S J
Hodge, J P
Dar, M M
de Bono, J S
Middleton, M R
British Journal of Cancer
The aim of this study is to define the maximum tolerated dose (MTD), safety, pharmacokinetics (PKs) and efficacy of ispinesib (SB-715992) in combination with docetaxel. Patients with advanced solid tumours were treated with ispinesib (6–12 mg m−2) and docetaxel (50–75 mg m−2). Docetaxel was administered over 1 h followed by a 1-h infusion of ispinesib on day 1 of a 21-day schedule. At least three patients were treated at each dose level. Blood samples were collected during cycle 1 for PK analysis. Clinical response assessments were performed every two cycles using RECIST guidelines. Twenty-four patients were treated at four dose levels. Prolonged neutropaenia and febrile neutropaenia were dose limiting in six and two patients, respectively. The MTD was ispinesib 10 mg m−2 with docetaxel 60 mg m−2. Pharmacokinetic assessment demonstrated concentrations of ispinesib and docetaxel, consistent with published data from single agent studies of the drugs. Seven patients (six hormone refractory prostate cancer (HRPC), one renal cancer) had a best response of stable disease (⩾18 weeks). One patient with HRPC had a confirmed >50% prostatic-specific antigen decrease. The MTD for ispinesib and docetaxel was defined and the combination demonstrated an acceptable toxicity profile. Preliminary PK data suggest no interaction between ispinesib and docetaxel.
docetaxel; kinesin spindle protein; mitotic kinesin; phase I; SB-715992
Related Articles
Optimized S-Trityl-l-cysteine-Based Inhibitors of Kinesin Spindle Protein with Potent in Vivo Antitumor Activity in Lung Cancer Xenograft Models
Good, James A. D.
Wang, Fang
Rath, Oliver
Kaan, Hung Yi Kristal
Talapatra, Sandeep K.
Podgórski, Dawid
MacKay, Simon P.
Kozielski, Frank
Journal of Medicinal Chemistry
The mitotic kinesin Eg5 is critical for the assembly of the mitotic spindle and is a promising chemotherapy target. Previously, we identified S-trityl-l-cysteine as a selective inhibitor of Eg5 and developed triphenylbutanamine analogues with improved potency, favorable drug-like properties, but moderate in vivo activity. We report here their further optimization to produce extremely potent inhibitors of Eg5 (Kiapp < 10 nM) with broad-spectrum activity against cancer cell lines comparable to the Phase II drug candidates ispinesib and SB-743921. They have good oral bioavailability and pharmacokinetics and induced complete tumor regression in nude mice explanted with lung cancer patient xenografts. Furthermore, they display fewer liabilities with CYP-metabolizing enzymes and hERG compared with ispinesib and SB-743921, which is important given the likely application of Eg5 inhibitors in combination therapies. We present the case for this preclinical series to be investigated in single and combination chemotherapies, especially targeting hematological malignancies.
Related Articles
Screening Compounds with a Novel High-Throughput ABCB1-Mediated Efflux Assay Identifies Drugs with Known Therapeutic Targets at Risk for Multidrug Resistance Interference
Ansbro, Megan R.
Shukla, Suneet
Ambudkar, Suresh V.
Yuspa, Stuart H.
Li, Luowei
PLoS ONE
ABCB1, also known as P-glycoprotein (P-gp) or multidrug resistance protein 1 (MDR1), is a membrane-associated multidrug transporter of the ATP-binding cassette (ABC) transporter family. It is one of the most widely studied transporters that enable cancer cells to develop drug resistance. Reliable high-throughput assays that can identify compounds that interact with ABCB1 are crucial for developing new therapeutic drugs. A high-throughput assay for measuring ABCB1-mediated calcein AM efflux was developed using a fluorescent and phase-contrast live cell imaging system. This assay demonstrated the time- and dose-dependent accumulation of fluorescent calcein in ABCB1-overexpressing KB-V1 cells. Validation of the assay was performed with known ABCB1 inhibitors, XR9576, verapamil, and cyclosporin A, all of which displayed dose-dependent inhibition of ABCB1-mediated calcein AM efflux in this assay. Phase-contrast and fluorescent images taken by the imaging system provided additional opportunities for evaluating compounds that are cytotoxic or produce false positive signals. Compounds with known therapeutic targets and a kinase inhibitor library were screened. The assay identified multiple agents as inhibitors of ABCB1-mediated efflux and is highly reproducible. Among compounds identified as ABCB1 inhibitors, BEZ235, BI 2536, IKK 16, and ispinesib were further evaluated. The four compounds inhibited calcein AM efflux in a dose-dependent manner and were also active in the flow cytometry-based calcein AM efflux assay. BEZ235, BI 2536, and IKK 16 also successfully inhibited the labeling of ABCB1 with radiolabeled photoaffinity substrate [125I]iodoarylazidoprazosin. Inhibition of ABCB1 with XR9576 and cyclosporin A enhanced the cytotoxicity of BI 2536 to ABCB1-overexpressing cancer cells, HCT-15-Pgp, and decreased the IC50 value of BI 2536 by several orders of magnitude. This efficient, reliable, and simple high-throughput assay has identified ABCB1 substrates/inhibitors that may influence drug potency or drug-drug interactions and predict multidrug resistance in clinical treatment.
Related Articles
Pseudo-merohedral twinning and noncrystallographic symmetry in orthorhombic crystals of SIVmac239 Nef core domain bound to different-length TCRζ fragments
Kim, Walter M.
Sigalov, Alexander B.
Stern, Lawrence J.
Acta Crystallographica Section D: Biological Crystallography
2010;66(Pt 2):163-175.
P212121 crystals of SIV Nef core domain bound to a peptide fragment of the T-cell receptor ζ subunit exhibited noncrystallographic symmetry and nearly perfect pseudo-merohedral twinning simulating tetragonal symmetry. For a different peptide fragment, nontwinned tetragonal crystals were observed but diffracted to lower resolution. The structure was determined after assignment of the top molecular-replacement solutions to various twin or NCS domains followed by refinement under the appropriate twin law.
HIV/SIV Nef mediates many cellular processes through interactions with various cytoplasmic and membrane-associated host proteins, including the signalling ζ subunit of the T-cell receptor (TCRζ). Here, the crystallization strategy, methods and refinement procedures used to solve the structures of the core domain of the SIVmac239 isolate of Nef (Nefcore) in complex with two different TCRζ fragments are described. The structure of SIVmac239 Nefcore bound to the longer TCRζ polypeptide (Leu51–Asp93) was determined to 3.7 Å resolution (R work = 28.7%) in the tetragonal space group P43212. The structure of SIVmac239 Nefcore in complex with the shorter TCRζ polypeptide (Ala63–Arg80) was determined to 2.05 Å resolution (R work = 17.0%), but only after the detection of nearly perfect pseudo-merohedral crystal twinning and proper assignment of the orthorhombic space group P212121. The reduction in crystal space-group symmetry induced by the truncated TCRζ polypeptide appears to be caused by the rearrangement of crystal-contact hydrogen-bonding networks and the substitution of crystallographic symmetry operations by similar noncrystallographic symmetry (NCS) operations. The combination of NCS rotations that were nearly parallel to the twin operation (k, h, −l) and a and b unit-cell parameters that were nearly identical predisposed the P212121 crystal form to pseudo-merohedral twinning.
pseudo-merohedral twinning; noncrystallographic symmetry; pseudosymmetry; human immunodeficiency virus; Nef; T-cell receptor
Related Articles
DrugTargetSeqR: a genomics- and CRISPR/Cas9-based method to analyze drug targets
Kasap, Corynn
Elemento, Olivier
Kapoor, Tarun M.
Nature chemical biology
To identify the physiological targets of drugs and bioactive small molecules we have developed an approach, named DrugTargetSeqR, which combines high-throughput sequencing, computational mutation discovery and CRISPR/Cas9-based genome editing. We apply this approach to ispinesib and YM155, drugs that have undergone clinical trials as anti-cancer agents, and demonstrate target identification and uncover genetic and epigenetic mechanisms likely to cause drug resistance in human cancer cells.
Related Articles
The Kinesin-12 Kif15 is a processive track-switching tetramer
Drechsler, Hauke
McHugh, Toni
Singleton, Martin R
Carter, Nicholas J
McAinsh, Andrew D
Kinesin-12 motors are a little studied branch of the kinesin superfamily with the human protein (Kif15) implicated in spindle mechanics and chromosome movement. In this study, we reconstitute full-length hKif15 and its microtubule-targeting factor hTpx2 in vitro to gain insight into the motors mode of operation. We reveal that hKif15 is a plus-end-directed processive homotetramer that can step against loads of up to 3.5 pN. We further show that hKif15 is the first kinesin that effectively switches microtubule tracks at intersections, enabling it to navigate microtubule networks, such as the spindle. hKif15 tetramers are also capable of cross-linking microtubules, but unexpectedly, this does not depend on hTpx2. Instead, we find that hTpx2 inhibits hKif15 stepping when microtubule-bound. Our data reveal that hKif15 is a second tetrameric spindle motor in addition to the kinesin-5 Eg5 and provides insight into the mechanisms by which hKif15 and its inhibitor hTpx2 modulate spindle microtubule architecture.
DOI: http://dx.doi.org/10.7554/eLife.01724.001
eLife digest
Before a cell can divide, it produces an extra copy of all its chromosomes, and it must then ensure that each daughter cell ends up with one copy of each chromosome. During the division process, a structure called the spindle forms in the cell. This spindle is made of thread-like extensions called microtubules that grow from two poles at opposite ends of the cell. These microtubules are responsible for getting the chromosomes to line up in the middle of the cell, and then pulling half of the chromosomes to one end of the cell, and half to the other end. The cell then divides into two daughter cells.
Two motor proteins—so-called because they consume chemical energy to 'walk' along the microtubules—have important roles in this process: Kif11 motor proteins mainly drive the formation of the spindle and thus division of the chromosomes. A cell that does not contain Kif11 can only divide if it contains extra copies of a second motor protein called Kif15: this suggests that Kif15 can serve as some sort of back up for Kif11.
Normal cells only divide when new cells are needed for growth or to replace old cells that have died. Cancer cells, on the other hand, divide in a way that is not controlled. Drugs that interfere with Kif11 have been developed in the hope that they will stop cancer cells dividing, but these drugs have not been very effective in clinical tests, possibly due to the Kif15 back up. Scientists hope, therefore, that a better understanding of the role of Kif15 may lead to improved cancer treatments.
Drechsler et al. have isolated individual Kif15 motor proteins and used advanced microscopy techniques to study them in action. These experiments showed that Kif15 motor proteins can travel long distances along a single microtubule, and can also switch to a different microtubule at intersections. This movement of Kif15 is stopped when they bump into Tpx2 proteins, which are sitting on the microtubules. Together, these proteins can also form links between microtubules that can withstand high forces. These properties provide a starting point to understand how Kif15 can act as a back up for Kif11 in cells. In the future, it will be important to work out how Kif11 and Kif15 motor proteins work together in teams to build the spindle.
DOI: http://dx.doi.org/10.7554/eLife.01724.002
kinesin-12; mitotic spindle; mitosis; human
Related Articles
Mapping the Structural and Dynamical Features of Kinesin Motor Domains
Scarabelli, Guido
Grant, Barry J.
PLoS Computational Biology
Kinesin motor proteins drive intracellular transport by coupling ATP hydrolysis to conformational changes that mediate directed movement along microtubules. Characterizing these distinct conformations and their interconversion mechanism is essential to determining an atomic-level model of kinesin action. Here we report a comprehensive principal component analysis of 114 experimental structures along with the results of conventional and accelerated molecular dynamics simulations that together map the structural dynamics of the kinesin motor domain. All experimental structures were found to reside in one of three distinct conformational clusters (ATP-like, ADP-like and Eg5 inhibitor-bound). These groups differ in the orientation of key functional elements, most notably the microtubule binding α4–α5, loop8 subdomain and α2b-β4-β6-β7 motor domain tip. Group membership was found not to correlate with the nature of the bound nucleotide in a given structure. However, groupings were coincident with distinct neck-linker orientations. Accelerated molecular dynamics simulations of ATP, ADP and nucleotide free Eg5 indicate that all three nucleotide states could sample the major crystallographically observed conformations. Differences in the dynamic coupling of distal sites were also evident. In multiple ATP bound simulations, the neck-linker, loop8 and the α4–α5 subdomain display correlated motions that are absent in ADP bound simulations. Further dissection of these couplings provides evidence for a network of dynamic communication between the active site, microtubule-binding interface and neck-linker via loop7 and loop13. Additional simulations indicate that the mutations G325A and G326A in loop13 reduce the flexibility of these regions and disrupt their couplings. Our combined results indicate that the reported ATP and ADP-like conformations of kinesin are intrinsically accessible regardless of nucleotide state and support a model where neck-linker docking leads to a tighter coupling of the microtubule and nucleotide binding regions. Furthermore, simulations highlight sites critical for large-scale conformational changes and the allosteric coupling between distal functional sites.
Author Summary
Kinesin motor proteins transport cargo along microtubule tracks to support essential cellular functions including cell growth, axonal signaling and the separation of chromosomes during cell division. All kinesins contain one or more conserved motor domains that modulate binding and movement along microtubules via cycles of ATP hydrolysis. Important conformational transitions occurring during this cycle have been characterized with extensive crystallographic studies. However, the link between the observed conformations and the mechanisms involved in conformational change and microtubule interaction modulation remain unclear. Here we describe a comprehensive principal component analysis of available motor domain crystallographic structures supplemented with extensive unbiased conventional and accelerated molecular dynamics simulations that together characterize the response of kinesin motor domains to ATP binding and hydrolysis. Our studies reveal atomic details of conformational transitions, as well as novel nucleotide-dependent dynamical couplings, of distal regions and residues potentially important for the allosteric link between nucleotide and microtubule binding sites.
Related Articles
Probing Spindle Assembly Mechanisms with Monastrol, a Small Molecule Inhibitor of the Mitotic Kinesin, Eg5
Kapoor, Tarun M.
Mayer, Thomas U.
Coughlin, Margaret L.
Mitchison, Timothy J.
The Journal of Cell Biology
Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest–deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.
monastrol; Eg5; kinesin; MAD2; kinetochore
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The Origin of Minus-end Directionality and Mechanochemistry of Ncd Motors
Jana, Biman
Hyeon, Changbong
Onuchic, José N.
PLoS Computational Biology
Adaptation of molecular structure to the ligand chemistry and interaction with the cytoskeletal filament are key to understanding the mechanochemistry of molecular motors. Despite the striking structural similarity with kinesin-1, which moves towards plus-end, Ncd motors exhibit minus-end directionality on microtubules (MTs). Here, by employing a structure-based model of protein folding, we show that a simple repositioning of the neck-helix makes the dynamics of Ncd non-processive and minus-end directed as opposed to kinesin-1. Our computational model shows that Ncd in solution can have both symmetric and asymmetric conformations with disparate ADP binding affinity, also revealing that there is a strong correlation between distortion of motor head and decrease in ADP binding affinity in the asymmetric state. The nucleotide (NT) free-ADP (φ-ADP) state bound to MTs favors the symmetric conformation whose coiled-coil stalk points to the plus-end. Upon ATP binding, an enhanced flexibility near the head-neck junction region, which we have identified as the important structural element for directional motility, leads to reorienting the coiled-coil stalk towards the minus-end by stabilizing the asymmetric conformation. The minus-end directionality of the Ncd motor is a remarkable example that demonstrates how motor proteins in the kinesin superfamily diversify their functions by simply rearranging the structural elements peripheral to the catalytic motor head domain.
Author Summary
Proteins belonging to the kinesin superfamily are responsible for vesicle or organelle transport, spindle morphogenesis, and chromosome sorting during cell division. Interestingly, while most proteins in kinesin superfamily that share the common catalytic motor head domain have plus-end directionality along microtubules, kinesin-14 (Ncd) exhibits minus-end directionality. Despite the several circumstantial evidences on the determining factors for the motor directionality in the last decade, a comprehensive understanding of the mechanism governing the Ncd minus-end directionality is still missing. Our studies provide a clear explanation for this minus-end directionality and the associated mechanochemical cycle. Here, we modeled an Ncd motor by employing structural details available in the literature to simulate its conformational dynamics. Simulations using our structure-based model of Ncd assert that the dynamics due to a simple rearrangement of structural elements, peripheral to the catalytic motor domain, is the key player in determining both the directionality and mechanochemistry unique to Ncd motors. Although Ncd has a different directionality, it uses a similar strategy to kinesin-1 of structural adaptation of the catalytic motor domain. Therefore using the same physical principle of protein folding and very similar structural elements, motors in the kinesin superfamily are able to achieve a variety of biological function.
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The crystal structure and biochemical characterization of Kif15: a bifunctional molecular motor involved in bipolar spindle formation and neuronal development
Klejnot, Marta
Falnikar, Aditi
Ulaganathan, Venkatasubramanian
Cross, Robert A.
Baas, Peter W.
Kozielski, Frank
Acta Crystallographica Section D: Biological Crystallography
2013;70(Pt 1):123-133.
The structural and biochemical study of Kif15 provides insight into this potential drug target and allows comparison with Eg5, a kinesin that partially shares the functions of Kif15.
Kinesins constitute a superfamily of microtubule-based motor proteins with important cellular functions ranging from intracellular transport to cell division. Some kinesin family members function during the mitotic phase of the eukaryotic cell cycle and are crucial for the successful progression of cell division. In the early stages of mitosis, during prometaphase, certain kinesins are required for the formation of the bipolar spindle, such as Eg5 and Kif15, which seem to possess partially overlapping functions. Because kinesins transform the chemical energy from ATP hydrolysis into mechanical work, inhibition of their function is a tractable approach for drug development. Drugs targeting Eg5 have shown promise as anticancer agents. Kif15 has recently come to the fore because it can substitute the functions of Eg5, and may itself have potential as a prospective drug target. Here, the initial biochemical, kinetic and structural characterization of Kif15 is reported and it is compared with the functionally related motor Eg5. Although Kif15 contains ADP in the catalytic site, its motor-domain structure was captured in the 'ATP-like' configuration, with the neck linker docked to the catalytic core. The interaction of Kif15 with microtubules was also investigated and structural differences between these two motors were elucidated which indicate profound differences in their mode of action, in agreement with current models of microtubule cross-linking and sliding.
human kinesins; mitosis; bipolar spindle formation; Kif15; Eg5
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Mitotic Spindle Assembly around RCC1-Coated Beads in Xenopus Egg Extracts
Halpin, David
Kalab, Petr
Wang, Jay
Weis, Karsten
Heald, Rebecca
PLoS Biology
Beads coated with the guanine nucleotide exchange factor RCC1 and a kinesin motor protein are sufficient to induce mitotic spindle assembly in Xenopus egg cytoplasm.
During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.
Author Summary
The mitotic spindle is a bipolar structure that is responsible for separating the two sets of duplicated chromosomes in a dividing cell, thereby delivering one set to each of the two daughter cells. It is built from dynamic filaments called microtubules, as well as hundreds of other components that contribute to the organization and dynamics of the microtubules and to chromosome movement. To understand which proteins are essential for spindle formation and function, we would like to be able to build it from purified components. As a step towards this goal, we coupled individual proteins to inert glass beads (as a substitute for chromosomes), to determine what combination of proteins can induce spindle assembly in a complex cytoplasm derived from frog eggs. We found that a single enzyme called RCC1 is sufficient to activate a pathway that stabilizes and organizes microtubules into a bipolar structure around the bead, but that this bead then oscillated back and forth between the poles of the spindle. By coupling a microtubule-based motor protein together with RCC1 on the bead, we were able to balance the bead in the center of the spindle. Thus, two proteins immobilized on a bead can substitute for a chromosome and induce stable spindle formation.
Related Articles
B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast
Chee, Mark K.
Haase, Steven B.
PLoS Genetics
Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.
Author Summary
The assembly of a bipolar mitotic spindle is essential for the accurate segregation of sister chromatids during mitosis and, hence, for successful cell division. Spindle assembly depends on the successful duplication of the spindle poles, followed by their separation to opposing ends of the cell. Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle, the relevant CDK targets have not been identified. Motor proteins of the kinesin-5 family generate movement on the microtubules that make up the spindle and are believed to power spindle pole separation. By employing the budding yeast Saccharomyces cerevisiae as a model, we have found evidence that cyclin/CDKs control spindle assembly by phosphorylating the kinesins-5 Kip1 and Cin8. When phosphorylation at a conserved CDK site in the motor domain of Kip1 is blocked, spindle pole separation is greatly diminished but neither protein abundance nor localization is affected. We have also obtained direct evidence by mass spectrometry that Kip1 and Cin8 are phosphorylated in vivo at consensus CDK sites in their tail domains. Our findings suggest that B-cyclin/CDKs regulate spindle assembly by regulating kinesin-5 motor activity.
Related Articles
A seesaw model for intermolecular gating in the kinesin motor protein
Sindelar, Charles V.
Biophysical Reviews
Recent structural observations of kinesin-1, the founding member of the kinesin group of motor proteins, have led to substantial gains in our understanding of this molecular machine. Kinesin-1, similar to many kinesin family members, assembles to form homodimers that use alternating ATPase cycles of the catalytic motor domains, or "heads", to proceed unidirectionally along its partner filament (the microtubule) via a hand-over-hand mechanism. Cryo-electron microscopy has now revealed 8-Å resolution, 3D reconstructions of kinesin-1•microtubule complexes for all three of this motor's principal nucleotide-state intermediates (ADP-bound, no-nucleotide, and ATP analog), the first time filament co-complexes of any cytoskeletal motor have been visualized at this level of detail. These reconstructions comprehensively describe nucleotide-dependent changes in a monomeric head domain at the secondary structure level, and this information has been combined with atomic-resolution crystallography data to synthesize an atomic-level "seesaw" mechanism describing how microtubules activate kinesin's ATP-sensing machinery. The new structural information revises or replaces key details of earlier models of kinesin's ATPase cycle that were based principally on crystal structures of free kinesin, and demonstrates that high-resolution characterization of the kinesin–microtubule complex is essential for understanding the structural basis of the cycle. I discuss the broader implications of the seesaw mechanism within the cycle of a fully functional kinesin dimer and show how the seesaw can account for two types of "gating" that keep the ATPase cycles of the two heads out of sync during processive movement.
Kinesin; Microtubule; Motor protein; ATPase; Cryo-electron microscopy; Structure
Related Articles
Harrington, Timothy D.
Naber, Nariman
Larson, Adam G.
Cooke, Roger
Rice, Sarah
Pate, Edward
Journal of theoretical biology
Loop 5 (L5) is a conserved loop that projects from the α2-helix adjacent to the nucleotide site of all kinesin-family motors. L5 is critical to the function of the mitotic kinesin-5 family motors and is the binding site for several kinesin-5 inhibitors that are currently in clinical trials. Its conformational dynamics and its role in motor function are not fully understood. Our previous work using EPR spectroscopy suggested that L5 alters the nucleotide pocket conformation of the kinesin-5 motor Eg5 [1]. EPR spectra of a spin-labeled nucleotide analog bound at the nucleotide site of Eg5 display a highly immobilized component that is absent if L5 is shortened or if the inhibitor STLC is added [1], which X-ray structures suggest stabilizes a L5 conformation pointing away from the nucleotide site. These data, coupled with the proximity of L5 to the nucleotide site suggest L5 could interact with a bound nucleotide, modulating function. Here we use molecular dynamics (MD) simulations of Eg5 to explore the interaction of L5 with the nucleotide site in greater detail. We performed MD simulations in which the L5-domain of the Eg5•ADP X-ray structure was manually deformed via backbone bond rotations. The L5-domain of Eg5 was sufficiently lengthy that portions of L5 could be located in proximity to bound ADP. The MD simulations evolved to thermodynamically stable structures at 300K showing that L5 can interact directly with bound nucleotide with significant impingement on the ribose hydroxyls, consistent with the EPR spectroscopy results. Taken together, these data provide support for the hypothesis that L5 modulates Eg5 function via interaction with the nucleotide-binding site.
kinesin-5; nucleotide-analog spin probes; EPR spectroscopy; molecular dynamics simulation
Related Articles
Structure of a monoclinic polymorph of human carbonic anhydrase II with a doubled a axis
Robbins, Arthur H.
Domsic, John F.
Agbandje-McKenna, Mavis
McKenna, Robert
Acta Crystallographica Section D: Biological Crystallography
2010;66(Pt 5):628-634.
The crystal structure of human carbonic anhydrase II with a doubled a axis from that of the usually observed monoclinic cell has been determined and refined to 1.4 Å resolution. The two molecules comprising the asymmetric unit are related by a noncrystallographic translation of ½ along a, but one of the molecules has two alternate orientations related by a rotation of approximately 2°.
The crystal structure of human carbonic anhydrase II with a doubled a axis from that of the usually observed monoclinic unit cell has been determined and refined to 1.4 Å resolution. The diffraction data with h = 2n + 1 were systematically weaker than those with h = 2n. Consequently, the scaling of the data, structure solution and refinement were challenging. The two molecules comprising the asymmetric unit are related by a noncrystallographic translation of ½ along a, but one of the molecules has two alternate positions related by a rotation of approximately 2°. This rotation axis is located near the edge of the central β-sheet, causing a maximum distance disparity of 1.7 Å between equivalent atoms on the diametrically opposite side of the molecule. The crystal-packing contacts are similar to two sequential combined unit cells along a of the previously determined monoclinic unit cell. Abnormally high final R cryst and R free values (20.2% and 23.7%, respectively) are not unusual for structures containing pseudo-translational symmetry and probably result from poor signal to noise in the weak h-odd data.
doubled axis; systematically weak data; crystal contacts; translational symmetry; rotational disorder
Related Articles
Common Mechanistic Themes for the Powerstroke of Kinesin-14 motors
Gonzalez, Miguel A.
Cope, Julia
Rank, Katherine C.
Chen, Chun Ju
Tittmann, Peter
Rayment, Ivan
Gilbert, Susan P.
Hoenger, Andreas
Journal of structural biology
Kar3Cik1 is a heterodimeric kinesin-14 from Saccharomyces cerevisiae involved in spindle formation during mitosis and karyogamy in mating cells. Kar3 represents a canonical kinesin motor domain that interacts with microtubules under the control of ATP-hydrolysis. In vivo, the localization and function of Kar3 is differentially regulated by its interacting stoichiometrically with either Cik1 or Vik1, two closely related motor homology domains that lack the nucleotide-binding site. Indeed, Vik1 structurally resembles the core of a kinesin head. Despite being closely related, Kar3Cik1 and Kar3Vik1 are each responsible for a distinct set of functions in vivo and also display different biochemical behavior in vitro. To determine a structural basis for their distinct functional abilities, we used cryo-electron microscopy and helical reconstruction to investigate the 3-D structure of Kar3Cik1 complexed to microtubules in various nucleotide states and compared our 3-D data of Kar3Cik1 with that of Kar3Vik1 and the homodimeric kinesin-14 Ncd from Drosophila melanogaster. Due to the lack of an X-ray crystal structure of the Cik1 motor homology domain, we predicted the structure of this Cik1 domain based on sequence similarity to its relatives Vik1, Kar3 and Ncd. By molecular docking into our 3-D maps, we produced a detailed near-atomic model of Kar3Cik1 complexed to microtubules in two distinct nucleotide states, a nucleotide-free state and an ATP-bound state. Our data show that despite their functional differences, heterodimeric Kar3Cik1 and Kar3Vik1 and homodimeric Ncd, all share striking structural similarities at distinct nucleotide states indicating a common mechanistic theme within the kinesin-14 family.
Kinesin-14; Kar3Cik1; Microtubules; Helical 3-D analysis; Molecular docking; Cryo-electron microscopy
Related Articles
Localization and function of Kinesin-5-like proteins during assembly and maintenance of mitotic spindles in Silvetia compressa
Peters, Nick T
Miller, Anne Catherine
Kropf, Darryl L
BMC Research Notes
Kinesin-5 (Eg-5) motor proteins are essential for maintenance of spindle bipolarity in animals. The roles of Kinesin-5 proteins in other systems, such as Arabidopsis, Dictyostelium, and sea urchin are more varied. We are studying Kinesin-5-like proteins during early development in the brown alga Silvetia compressa. Previously, this motor was shown to be needed to assemble a bipolar spindle, similar to animals. This report builds on those findings by investigating the localization of the motor and probing its function in spindle maintenance.
Anti-Eg5 antibodies were used to investigate localization of Kinesin-5-like proteins in brown algal zygotes. In interphase zygotes, localization was predominantly within the nucleus. As zygotes entered mitosis, these motor proteins strongly associated with spindle poles and, to a lesser degree, with the polar microtubule arrays and the spindle midzone. In order to address whether Kinesin-5-like proteins are required to maintain spindle bipolarity, we applied monastrol to synchronized zygotes containing bipolar spindles. Monastrol is a cell-permeable chemical inhibitor of the Kinesin-5 class of molecular motors. We found that inhibition of motor function in pre-formed spindles induced the formation of multipolar spindles and short bipolar spindles.
Based upon these localization and inhibitor studies, we conclude that Kinesin-5-like motors in brown algae are more similar to the motors of animals than those of plants or protists. However, Kinesin-5-like proteins in S. compressa serve novel roles in spindle formation and maintenance not observed in animals.
Related Articles
NSC 622124 Inhibits Human Eg5 and Other Kinesins Via Interaction with the Conserved Microtubule-Binding Site
Learman, Sarah S.
Kim, Catherine D.
Stevens, Nathaniel S.
Kim, Sunyoung
Wojcik, Edward J.
Walker, Richard A.
Kinesin-5 proteins are essential for formation of a bipolar mitotic spindle in most, and perhaps all, eukaryotic cells. Several Kinesin-5 proteins, notably the human version, HsEg5, are targets of a constantly expanding group of small-molecule inhibitors, which hold promise both as tools to probe mechanochemical transduction and as anti-cancer agents. Although most such compounds are selective for HsEg5 and closely related Kinesin-5 proteins, some, such as NSC 622124, exhibit activity against at least one kinesin from outside the Kinesin-5 family. Here we show NSC 622124, despite identification in a screen that yielded inhibitors now known to target the HsEg5 monastrol-binding site, does not compete with 14C-monastrol for binding to HsEg5, and is able to inhibit the basal and microtubule-stimulated ATPase activity of the monastrol-insensitive Kinesin-5, KLP61F. NSC 622124 competes with microtubules, but not ATP, for interaction with HsEg5, and disrupts the microtubule binding of HsEg5, KLP61F and Kinesin-1. Proteolytic degradation of an HsEg5•NSC622124 complex revealed that segments of the α3 and α5 helices map to the inhibitor-binding site. Overall, our results demonstrate that NSC 622124 targets the conserved microtubule-binding site of kinesin proteins. Further, unlike compounds previously reported to target the kinesin microtubule-binding site, NSC 622124 does not produce any enhancement of basal ATPase activity, and thus acts solely as a negative regulator through interaction with a site traditionally viewed as a binding region for positive regulators (i.e., microtubules). Our work emphasizes the concept that microtubule-dependent motor proteins may be controlled at multiple sites by both positive and negative effectors.
Related Articles
Computational Studies of the Farnesyltransferase Ternary Complex Part I: Substrate Binding†
Cui, Guanglei
Wang, Bing
Merz, Kenneth M.
Farnesyltransferase (FTase) catalyzes the transfer of farnesyl from farnesyldiphosphate (FPP) to cysteine residues at or near the C-terminal of protein acceptors with a CaaX motif (a, aliphatic; X, Met). Farnesylation is a critical modification to many switch proteins involved in extracellular signal transduction pathway, which facilitates their fixation on the cell membrane where the extracellular signal is processed. The malfunction caused by mutations in these proteins often causes uncontrolled cell reproduction and leads to tumor formation. FTase inhibitors have been extensively studied as potential anticancer agents in recent years, several of which have advanced to different phases of clinical trials. However, the mechanism of this biologically important enzyme has not been firmly established. Understanding how FTase recruits the FPP substrate is the first and foremost step towards further mechanistic investigations and the design of effective FTase inhibitors. Molecular dynamic simulations were carried out on the ternary structure of FTase complexed with the FPP substrate and an acetyl-capped tetrapeptide (acetyl-CVIM), which revealed that the FPP substrate maintains an inactive conformation and the binding of the diphosphate group can be largely attributed to residues R291β, K164α, K294β, and H248β. The FPP substrate assumes an extended conformation in the binding site with restricted rotation of the backbone dihedral angles; however, it does not have a well defined conformation when unbound in solution. This is evident from multinanosecond MD simulations of the FPP substrate in vacuum and solution. Our conclusion is further supported by theoretical j-coupling calculations. Our results on the FPP binding are in good agreement with previous experimental kinetic studies on FTase mutants. The hypothetical conformational activation of the FPP substrate is currently under investigation.
Related Articles
Effective killing of the human pathogen Candida albicans by a specific inhibitor of non-essential mitotic kinesin Kip1p
Chua, Penelope R
Roof, David M
Lee, Yan
Sakowicz, Roman
Clarke, David
Pierce, Dan
Stephens, Thoryn
Hamilton, Matthew
Morgan, Brad
Morgans, David
Nakai, Takashi
Tomasi, Adam
Maxon, Mary E
Molecular Microbiology
Kinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in 'rigor-like', tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a non-essential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery.
Related Articles
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Nuckols Farm named Blue Ribbon School
Henrico's Nuckols Farm Elementary School was one of nine Virginia schools named a 2012 National Blue Ribbon School today.
U.S. Secretary of Education Arne Duncan visited Arlington Traditional Elementary School in Arlington County earlier today to announce the schools that earned the designation.
The Blue Ribbon Schools Program honors public and private K-12 schools that are either academically superior or that demonstrate dramatic gains in student achievement.
Nuckols Farm was the only school in the Richmond region honored today.
"Each of these schools is at the center of a community where students, parents, teachers, the principal and everyone else at the school — from the office staff to the bus drivers — share high expectations for learning and achievement," Virginia Superintendent of Public Instruction Patricia I. Wright said.
Nuckols Farm and six of the other schools earned awards earlier this year through the Virginia Index of Performance program. Nuckols Farm earned the Governor's Award for Educational Excellence.
The U.S. Department of Education will honor all of the nation's 2012 National Blue Ribbon Schools during a conference and awards ceremony Nov. 12-13 in Washington, DC.
Richmond Solar Co-op sign ups end April 30
Citizen Staff Reports 04/25/2016
The sign up period for the Richmond Community Solar Co-op will close on April 30. Nearly 150 homeowners and businesses have joined the group to save money and make going solar easier. The group has started installations and is working with VA SUN to learn about solar technology and the process of going solar.
"If you've ever thought about going solar, this is a great opportunity to do so," said Sekar Veerappan Co-op member and the group's first installation. "Working with the group helps members learn about going solar and make an informed decision." > Read more.
YMCA branches to host 'Healthy Kids Day' Apr. 29-30
Citizen Staff Reports 04/22/2016
The YMCA of Greater Richmond will host its "Healthy Kids Day" April 29-30 at all local branches.
The event is part of the Y's national initiative to improve families' health and well-being.
Each event features games, healthy snacks, arts and crafts and physical activities, among other events. Its goal is to motivate and teach families how to develop healthy routines at home. > Read more.
Weekend Top 10
For our Top 10 calendar events this weekend, click here! > Read more.
Henrico Public Schools to offer parenting workshops
Glen Allen teens' project fights cyberbullying
Challa to receive 'Outstanding Women Award'
Highland Springs HS teacher earns national award
Citizen wins 2 Va. Press Association awards
- More News
Print Edition
Apr. 21, 2016Click here to read the print edition.
Weekend Top 10
Henrico students star in 'The Enchanted Toy Shop'
Weekend Top 10
Weekend Top 10
Reynolds CC hosting student art show
- More Entertainment
Obituary – Albert H. Buccini
Obituary – Rebecca Hagadorn Clarke
Obituary – David Allen Luebke
- More Obituaries
Richmond Solar Co-op sign ups end April 30
YMCA branches to host 'Healthy Kids Day' Apr. 29-30
Henrico PAL honors supporters
- More Community
Of character and parenting
Opinion: Rename school for late Henrico officer
Of car chats and grocery games
- More Opinions
Strawberry visits Tuckahoe Little League
Athlete of the Week – July 3: Kayla Sell
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CalendarLavender Fields Herb Farm, 11300 Winfrey Rd. in Glen Allen, will offer a free farm tour from 12 p.m. to 12:30 p.m. This walking tour includes farm history, a stop… Full text

subset: commoncrawl


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This side of a medical emergency there are four words that can definitely strike fear in the heart of a parent.
"Spend-the-night party."
Unless, of course, the big event, also known as a sleepover, is at someone else's house, and then sweeter words were never spoken.
For those new to this parental rite of passage, it's probably best to resist the temptation to leave town. Especially since the most independent child can suddenly become fearful once the lights are out and all is quiet. Or not quiet enough.
Such was the case with our older daughter. Her closest friend had planned a much-anticipated sleepover. It was to be the best party of all time. The girls were going to watch movies, eat lots of junk food and drink all the Mellow Yellow they wanted.
About 2 a.m. our phone rang.
"Mama," said our daughter. "I'm sick."
Prince Charming couldn't get out of the door fast enough to rescue his damsel in distress. Once she and her dad got home, the truth was revealed.
"I couldn't sleep," she admitted. "They wouldn't be quiet."
I'm not sure what she had expected.
It's common knowledge that boys wouldn't dare be caught enjoying anything as silly as a spend-the-night party. Oh no, they prefer the term campout, which according to my experience, is exactly like a sleepover only with a tent.
Those of us who are veteran parents would probably agree that any trip through parenthood wouldn't be complete without at least one spend-the-night party, I mean campout, set up within 15 feet of the backdoor.
When our older son was 9 years old he invited over a couple of buddies. We set up a tent, built a small fire and roasted hotdogs and marshmallows.
Eventually everyone went inside leaving the campers to their late evening plans, which at their age was pretty much limited to flashlight shadow puppets, scary stories and burping contests.
Surprisingly, it wasn't long before the campground was silent, at least for a while. Sometime just before dawn, I was awakened by an array of loud whoops and hollers.
Before the campers had a chance to awaken the entire neighborhood, my husband opened a window and vainly tried to get their attention. Then I heard the backdoor slam. Thinking I should go check things out, as well, I found my half-asleep husband standing outside chastising the boys. To this day, I'm not sure my husband is aware that at the time he was wearing nothing more than his fruit-of-the-looms.
Needless to say, he had their attention.
This weekend our 8-year-old daughter had her first sleepover. When Mary Kate, Ashli and Ciara arrived with our Anna right after school, I had a snack ready for them.
They devoured the chips and dip, eager to get upstairs to see what Anna's big sister had planned for them.
Before long the four little girls were dressed up in hand-me-down prom dresses having their makeup and hair done by the big sister.
After supper, the girls ate birthday cake and got ready for bed by pumping up a queen sized air mattress for the den floor.
"We're going to stay up all night," I heard Anna excitedly tell her friends.
I knew better.
Sometime in the night, I went upstairs to check on the girls and found four sleeping beauties all curled up on the air mattress, sweetly snoring away.
The next morning when the first mama arrived she expressed surprised disappointment that her little one had made it through the night without her.
"She didn't cry or anything?" inquired the mama.
"No, she was just fine. They had a blast," I said, and then I gave the mama a quick review of the past evening's activities.
"I don't think I'm ready for this," said the mama sadly.
"I know," I said. "No one ever is."


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Listen: Wale feat. Rick Ross & Common "Sunshine" Remix
Wale has revisited last year's The Gifted with his new release, the long-awaited remix to "Sunshine." For the reworking, the D.C. rapper grabbed Rick Ross and Common to bless Tone P and Stokley Williams' percussion-heavy production with uplifting words. Gruff voice and all, Rick Ross eschews his persona to drop honest bars about his unlikely rise: "I struggled at math, therefore perfected my craft/In the back of my mind I said establish a staff." Common offers similar wisdom, characterizing adversity as a maze: "In this maze they call us the black rats/'Cause we trapped from the projects we stacked at."
Though Wale recycles a verse from the original, his assertion that music is therapeutic ("Fuck a therapist, I put everything in this cone") is relatable to anyone who uses their passion as a distraction from their frustrations. Ironically, Wale got into a confrontation at a WWE event with a fan who called him out on Twitter. Video of the incident shows pushing between him and the heckler, but in the wake of last year's confrontation with Raptors analyst Matt Devlin and the now-infamous Complex magazine (disclosure: I write for them) phone call, people are eager to blow anything the rapper does out of proportion.
Now that his inner wrestler is out of his system, Wale can get back to working on what people really want to hear: The Album About Nothing.


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- Engage in Your Community
- Build Lasting Friendships
- Develop the Leader in You
Membership in the JLPA•MP is open to all women over the age of 21 regardless of race, religion or national origin, who are interested in community service.
Sign up for our email list to receive updates on our Summer open house schedule.
Or visit our Join page for more information on membership.
Done in a Day Projects
Done in a Day projects focus on short, intensive community activities that are typically completed in a single day or weekend. We hope you'll join us for an upcoming volunteer shift.
Latest Piper
Check out the most recent issue of Piper featuring League events, member news and The Shop's renovation and Sapphire Celebration.
Shopping Event
Luisa Spagnoli
Join us at the grand opening of the first Luisa Spagnoli store in the U.S. A portion of the sales from the evening will benefit the League.
Prosecco and hors d'oeuvres will be served. Silicon Valley Magazine will be taking photos at the event. This event is open to the public and you are welcome to bring guests. Please RSVP here.
Fundraising Quicklinks
Support our Annual Fund, Endowment Fund, FLASHDASH and The Shop by clicking here or using the green Donate button at the top of this page.
Thank You FlashDash Sponsors
The League appreciates the support of the following corporate partners who help us develop the potential of women, promote voluntarism and improve communities by supporting our FlashDash fundraiser:
Empowering girls to be STEM leaders of tomorrow
SPARK an idea•INSPIRE the next generation•CONNECT with leaders
The Junior League of Palo Alto•Mid Peninsula is investing in our next generation of female STEM leaders through strategic grant-making, coalition building and by partnering with local and national nonprofits on projects for direct service, capacity building and advocacy. We are excited to announce new multi-year partnerships with The Tech Museum of Innovation and Technovation to engage girls in our community with innovative STEM education and programs. This includes a $100,000 grant and volunteer support to launch Girls Day at The Tech and The Tech Challenge mentorship program.
In the news: Technovation & Innovation: Project READ Girls take on Tech! Sponsored by Technovation, the Junior League of Palo Alto•Mid Peninsula and BNY Mellon. (March 29, 2016)
Read more about our partnership with The Tech Museum of Innovation and their Tech Challenge in the San Jose Mercury News (March 2, 2016).
"Helping Girls See Themselves in STEM Careers," co-authored by our President Jan Hickman and Gretchen Walker, Vice President of Education at The Tech. Museum of Innovation
The first Girls Day @ The Tech, sponsored by the JLPA•MP, was covered here:


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Funny thing, the first trade is for 13k shares at .005. While the ask had someone willing to sell his shares at .0048. Wonder what today holds for our great investment?
Sentiment: Buy
BTW anyone wanting out, here's the time. I figure we have a few owning over 1M shares, there's someone willing to take them off your hands for a decent profit. I suggest you dangle them a little higher than the bid and make him/her chase. It will take one of us longs letting go, then you know they announce 5 minutes later the funding.
Is that your Bid?
Sentiment: Hold


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Stomach Cancer Topic Guide
Stomach Cancer: The stomach is a muscular bag with a capacity of about 1 liter or quart. It lies along the digestive tract between the esophagus and the small intestine. The stomach ...
Must Read Articles:
Gastrointestinal Bleeding (GI) Gastrointestinal (GI) bleeding either comes from the upper GI or lower GI tract. Upper GI bleeding can be caused by ulcers, gastritis, varices, cancer, or inflammation. Lower GI bleeding is caused by diverticulitis, cancers, IBD, infection, polyps, hemorrhoids, and anal fissures. Symptoms of GI bleeding include fatigue, weakness, shortness of breath, abdominal pain, vomiting blood, rectal bleeding, anemia, or a pale appearance. Treatment of GI bleeding depends on the cause.
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N.Y. sees small gains on medical pot
Associated Press
NEW YORK — Gov. Andrew Cuomo made a political splash by introducing a medical marijuana plan in the State of the State speech, but his cautious approach has been met with skepticism from pot advocates who question whether the proposal is mostly for show.
While nearly two dozen states have OK'd marijuana for medical purposes and Colorado and Washington have legalized its use for pleasure, Cuomo is tapping a 1980 state law to allow as many as 20 hospitals to dispense the drug to people with certain severe illnesses as an experimental research project.
"I'm absolutely thrilled that he's actually verbalized the words 'medical marijuana,' but he's just got to go further," said Susan Rusinko, a 52-year-old central New York resident who said a hit of pot is a "wonder drug" that relaxes immobilizing leg spasms from her multiple sclerosis. It's unclear whether she would even qualify for Cuomo's initiative or whether there would be a participating hospital near her.
The governor's office has yet to detail how the program would overcome key hurdles, including the lack of a legal, unadulterated supply of marijuana in the state and a federal law that still makes it illegal for doctors to write a prescription.
While advocates are frustrated, Cuomo's limited embrace of medical marijuana may be both politically astute and scientifically sensitive.
Some medical experts say that while the marijuana plant holds tantalizing possibilities for treating problems ranging from chemotherapy-related nausea to chronic pain, popular enthusiasm for the drug has outpaced a weak body of medical research.
Cuomo's initiative is styled as a test of whether pot can be effectively used as medicine without being abused.
"This does not start with a premise: 'Oh, this is a slam dunk. … We can do it without any ancillary problems,'" he said Monday. "It's the exact opposite."
Under his plan, people with cancer, glaucoma and possibly some other "life-threatening or sense-threatening" conditions could seek to get marijuana through studies based at hospitals yet to be named, with "stringent research protocols and eligibility requirements."
Cuomo's initiative bypasses a state Legislature that has declined to pass more ambitious medical marijuana laws. He's relying instead on his administrative powers to carry out a 1980 law allowing medical-marijuana research. A number of states passed such measures in that era.
Then California took a broader step, voting in 1996 to let doctors recommend cannabis for various conditions. Nineteen other states have since enacted medical marijuana laws. While the drug remains illegal under federal law, U.S. prosecutors were told in 2009 not to focus on people using it medically under state laws.
Critics feel medical marijuana is an entree to more recreational use of a drug that was widely outlawed in the U.S. in the 1930s. "I think it sends the wrong signal to our young people," said Michael Long, chairman of the New York Conservative Party.
Federal regulators have approved a few prescription drugs containing a synthetic version of the marijuana ingredient THC. But few clinical trials have been done to test whether the plant in its raw form is better than conventional therapies, partly because of federal restrictions on such research, notes Aron Lichtman, a pharmacology professor at Virginia Commonwealth University and the president of the International Cannabinoid Research Society.
"There are all sorts of claims being made, without any proper testing. So it's sort of a conundrum," he said.
Dr. J. Michael Bostwick, a Mayo Clinic psychiatrist who studies medical marijuana issues, sees therapeutic potential in cannabis. But he also has concerns that it is being dispensed in some states by questionable providers who make unsubstantiated claims about the drug's powers and provide little or no actual medical care or evaluation.
He said Cuomo's proposal seems to address some of those concerns by putting dispensaries within hospitals, but he said some New York hospitals might be wary of participating for fear of running afoul of federal authorities.
If hospitals do get on board, it isn't clear where they would get the marijuana. New York's 1980 law envisioned a possibility that the drug could be obtained from law enforcement agencies, but experts say pot seized from drug dealers could pose a safety hazard. The drug can't be imported from states where it is grown legally. Obtaining marijuana grown by federal authorities for research purposes is notoriously difficult.
Still, pharmaceutical pot is popular with New Yorkers. A Quinnipiac University poll in June found 70 percent of voters support it.
And for Cuomo, the issue represents an opportunity to plant a flag on the left — but not too far — as he faces re-election amid talk of presidential ambitions in 2016. Cuomo enjoys favorability ratings around 55 and 60 percent in recent polls, but some of the state's Democratic limelight has been shining lately on newly installed, staunchly liberal New York City Mayor Bill de Blasio.
Cuomo had said as recently as April that he opposed medical marijuana but that his thinking was evolving. With his new plan, "he can, at least, say, 'I'm pushing forward this conversation,'" said Christina Greer, a political science professor at Fordham University.
Advocates, who want state lawmakers to pass a broader medical marijuana law, question how workable Cuomo's program would be and how many people it would help.
"It moves New York forward, all the way to 1980," said Gabriel Sayegh, the New York state director of the Drug Policy Alliance, a group critical of war-on-drugs governing.
Carly Tangney-Decker isn't waiting for answers. She and her husband believe a particular strain of marijuana available through a Colorado dispensary could help their 8-month-old daughter, Mabel, who suffers from a genetic seizure disorder.
While doctors didn't recommend the marijuana treatment, the mother said, Mabel's neurologist supported the family's quest for alternatives to medications that aren't approved for regular use in infants and could cause permanent vision damage.
"People say that marijuana is a gateway drug," said Tangney-Decker, of Kingston. "Well, people in my situation consider it an exit drug to take us away from all the other drugs."
So she and baby Mabel are moving to Colorado next week.
Associated Press writers Michael Hill and Michael Virtanen in Albany contributed to this report.
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Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation
Article first published online: 17 JAN 2006
DOI: 10.1111/j.1574-6968.1998.tb13175.x
FEMS Microbiology Letters
Volume 166, Issue 1, pages 1–7, September 1998
Additional Information
How to Cite
Toyama, H., Anthony, C. and Lidstrom, M. E. (1998), Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation. FEMS Microbiology Letters, 166: 1–7. doi: 10.1111/j.1574-6968.1998.tb13175.x
Publication History
- Issue published online: 17 JAN 2006
- Article first published online: 17 JAN 2006
- Received 25 May 1998, Revised 18 June 1998, Accepted 23 June 1998
- Abstract
- Article
- References
- Cited By
- Methylobacterium extorquens AM1;
- Methylotroph;
- Methanol oxidation;
- Electroporation
Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which is widely used for analyzing pathways of C1 metabolism with biochemical and molecular biological techniques. To facilitate this approach, we have applied a new method to construct insertion or disruption mutants with drug resistance genes by electroporation. By using this method, mutants were obtained in four genes present in the mxa methylotrophy gene cluster for which the functions were unknown, mxaR, mxaS, mxaC and mxaD. These mutants were unable to grow on methanol except the mutant of mxaD, which showed reduced growth on methanol.


text:
Oklahoma City's Devon Energy goes for 'green' gold on former brownfield site
Devon Energy Center, when complete, won't be "green" just because of what went into it, but also because of what came out of the ground under it.
Part of the parcel of land now with the address 333 W Sheridan Ave. has a dirty history, one that reflects the changes in the city around it. But most recently, since Devon Energy Corp. acquired it, it's a story of colors: brown, green and gold.
Larry Nichols, executive chairman, told some of the tale as the closing speaker at the Oklahoma Brownfields Conference Wednesday at the Skirvin Hilton Hotel. Part of the city block was "brown" — in need of environmental remediation — because of former uses as a trolley barn, then a kind of "motor hotel," both of which left contaminated soil and water from fuel leakage.
'Downtown, period'
Selecting a site needing work was worth it for Devon, Nichols said, because the energy company wanted to do its part for Oklahoma City's renaissance. He said more than a few landowners and developers tried to woo Devon to the suburbs.
"I have yet to find a vibrant city that did not have a vibrant downtown. ... If the downtown core goes, the entire city seems to go right with it," he said. So, for Devon, even with suitors calling, it was "downtown, period," he said.
Around the turn of the 20th century, the "brown" property was the site of the terminal building, the hub, of the city's trolley system, a time when mass transit was the norm in the urban core.
In the late 1920s or early '30s, the building was converted to a "motor hotel," not the kind of roadside hotels that popped up to cater to motorists ("motels"), but a hotel for automobiles, where they could be stored during the workday — and serviced. The automobile was coming on strong.
That building came down in the 1950s, and a four-story parking garage went up, with a parking deck also providing fueling and maintenance, and remained until the early 1970s.
Pei's parking legacy
Then came I.M. Pei, architect and urban planner, Urban Renewal and plans for the Galleria, a downtown mall that never materialized — but that cost what is now Devon property some of the city's most resonant retail names: John A. Brown's Department Store, Kerr's Department Store, Katz Drugstore, Warner Theater.
But the "brown" part still remained: an odd, ugly parking deck with would-be support beams, intended to be basement parking for the Pei project.
Nichols said he took special joy in seeing it come down because it was "not only an ugly parking garage, but a reminder of failure." Under it were three abandoned fuel tanks, an abandoned water well and contaminated soil and water, all of which was removed.
Nichols ticked off a list of accomplishments contributing to Devon Energy Center's effort to gain gold LEED certification — for Leadership in Energy & Environmental Design — from the U.S. Green Building Council.
Demolition waste
All the material from the parking deck demolition was kept out of landfills.
• 11,200 tons of concrete was recycled and used for erosion control on a lakefront property.
• More than 15,200 tons of concrete went to the Oklahoma Department of Transportation as coarse rock.
• Nearly 500 tons of metal went to recycling centers for miscellaneous use.
• Some 500 light fixtures went to Oklahoma Electrical Supply Co. for reuse.
Construction waste
So far, 93 percent of construction waste has been recycled, a total of 69,026 tons.
• 2,565 tons of concrete.
• 2,485 tons of wood.
• 2,484 tons of metal.
• 1,486 tons of gypsum.
• 6 tons of plastics.
Building performance
"Green" features are built into building operations.
• District cooling and state-of-the-art energy management technologies maximize efficiency.
• The geometry of the tower and exterior glass fins reduce solar load.
• Energy-efficient light fixtures use minimal energy to operate.
• Light fixtures dim automatically depending on available natural light.
• Sensors dim lights when space is not occupied.
• Raised-floor air distribution increases efficiency and contributes to indoor air quality.
• The tower's geometry and glass walls allow abundant natural light in all areas.


text:
Beautyrest Black Alexia 12 Inch Extra Firm Mattress
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text:
by mpurvis11 8 years, 10 months ago
I am looking into Mishakoff/ Vasilof from alaska, also in the tree Denesha's which has already been researched pretty good in our family. I am also adding our generation into the tree.
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by moresmall 8 years, 10 months ago
I have a Gilbert Ross Doran b 1887 in St Andrews, Dundee and died about 1968. His occupations were Shifter in a Jute Mill, and Boil maker. He married Elizabeth (Betsy) Ritchie, (x Rosehearty) and they had 3 children, Gilbert, May &...
1 comment, latest 7 years, 5 months ago
by momster1 8 years, 10 months ago
grandmother - Anita Hernandez Medina dob 12,dec 1901 died 26,mar. 1978 her siblings are angelita (?) jesus (?) belen 1910 irene 1910 thomas 1914- Dionicio M Hernandez abo 1881 chihuahua,Mexico-father ????
1 comment, latest 8 years, 10 months ago
by ianmacmen 8 years, 10 months ago
My Great Grandfather was John MacMenigall b1864 in Redrow, Currie, Edinburgh, Scotland; parents John MacMenigall & Helen Leishman (family info reqd); m Jane Alice Hamilton from Manchester, England, (family info reqd); family went to Holyoake,...
0 comments
by barbc 8 years, 10 months ago
My ancestor left Copenhagen for Australia where she married Jacob Strandgard. Are there any Hansens related still in Copenhagen?
3 comments, latest 1 month, 1 week ago
by BILLYBOB 8 years, 10 months ago
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by JenPirok 8 years, 10 months ago
I have been searching with no luck on my family for almost three years now. The leads that I have run across have just managed to reunite other families with the same last name (which I have no problem doing), but I would really love to find...
0 comments
by jewelmoons 8 years, 10 months ago
I beleive that only the oldest left on the mountains know the the what fors and the real reasons why.And there is only a handful that God has given the talent to know this.. I have been lucky enough to get a few hints to why's and how's...
0 comments
by amber101 8 years, 10 months ago
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by Jamunjie 8 years, 10 months ago
I am trying to trace birth records of Robert John Clark presumably born in England in 1831. Any information would be gratefully accepted as records here in Tasmania are sketchy. His mother or father was an Indigenous Australian. Does anyone out...
6 comments, latest 8 years, 10 months ago
The Bammerlin or Bommerlin family from Baden Germany. Some of this family is located in Stark County, Ohio. I am very interested in the background of this family and am looking for any other Bammerlin researcher. My ancestor is Christina Catharina...
0 comments
We have compiled a great deal of information on this family from Baden. The family arrived in New York in 1847 as the Eglisdorfer family and moved from there to Pennsylvania, Ohio, Indiana and finally Michigan. My ancestor Ernest F. Eglisdorfer...
0 comments
The Neisser or Nizer family of Kent County, Michigan. Some of the names I am interested in are Carl, Charles, Mathilda (Stockman), William Reinhold, Sam or Samuel and Henrietta or Harriet. These Nizers or Neissers lived in Kent County between 1840...
0 comments
by wanda54 8 years, 10 months ago
i'm looking for information on joseph vickroy my g-grandfather.
0 comments
by Merrilyn 8 years, 10 months ago
Robert Cochrane was born in Glasgow Scotland in 1825 he Emigrated to Australia with his parents John and Mary in the 1830ies. The family first settled in Sydney and then Emu Plains, eventualy living in Dubbo NSW. Robert Married and went to the...
1 comment, latest 8 years, 10 months ago
by sunnyscunny 8 years, 10 months ago
My Grt grandfather was Robert Benjamin born (c) 1864 London He Married Alice ??? who was born (c) 1864 London There was a son my granfather Arthur Robert born 1890 Tottenham Who married Margaret Alice Richardson My father Arthur Richard was...
0 comments
by sabrecat00 8 years, 10 months ago
I received an e-mail from Dora Coffman futher confirming the John Holley line from which I am a descendant.
0 comments
by stdomsgirl 8 years, 10 months ago
As Mick Jagger sang: Please allow me to intoduce myself, I'm Judy {named after Judy Garland} - Judith Frances Corbin. I was raised Irish Catholic. I went to school at St. Dominic's School on Gray Street {where I now live 55 years later}. I've come...
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Creating a culture of cash and cost awareness
In 2008, Lutz Bertling, Eurocopter President & CEO, shifted the company's attention from EBIT and profitability, to instead increasing the weight of cash as a key performance indicator.
He also decided to launch a change towards a real cash culture. But how did Eurocopter manage to get all employees to understand how they have a direct impact on the company's cash position, how they proactively contribute to and drive cash flow, and how generating cash is well within their scope of activities?
The answer was Celemi Apples & Oranges™ - a hands on business simulation to demystify finance. "We have discovered that everybody understands what cash is, but when it comes to the real drivers - working capital, accounts receivable, inventories and the like - people don't share the same understanding," Mr. Prunet says.
"They don't see where their individual actions have an impact on the cash they have."
The solution was to train one third of the employees. In the first year of implementation, more than 2 000 employees had gone through Celemi Apples & Oranges in France, Germany, Spain and Australia, and the roll out continues.
"With sessions of 40 to 80 people, from all Directorates and from all levels, mixed by tables of four, each participant can see that he or she has hand in the company's cash position."
Bernard Prunet
Vice President ESPRIT Project


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Sondra Roberts
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Faceted glass beads glitter on the upper edge of a lavish satin clutch furnished with a drop-in chain strap.
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- By Sondra Roberts; imported.
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Ā I bought some flour sack cloths (next to cloth napkins) Āat Targets for a buck each and I jsut origami fold it, then tie around my lo waist. ĀThen I tuck the end under her bum and tuck into the front. ĀIt's kind of tarzanny but easy to pull out and tuck behind her on the potty and put back in. ĀAlso EASY to wash! ĀMight not hold crazy poops but at least some. Ā
Easy cheap EC wear- Flats or Flour slack towels...
Posted 12/10/2011
Comments (1)
Posted 06/01/2012
Sounds cool! Any chance you could post a pic? Thinner cloths would be better for me to notice her pees, than.our prefolds, plus she's gotten too big for me to snappi them anyway.
Would definitely try the napkins.
Would definitely try the napkins.


text:
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On The Box
The Olympics are over, which is just as well because we were in danger of being over them.
But then, the wonderful performances by those men and women in black made it worthwhile.
Then just when you thought it was safe to go back to the telly to watch non-Olympic stuff, along came the sensational news that Belarusian shot putter Nadzeya Ostapchuk was a drug cheat who had been stripped of her gold medal.
Mercifully, that was all she was stripped of, but it did mean that Valerie Adams was promoted to gold. That dominated the news for a couple of days and deservedly so. After all, our six golds were just one shy of the total of the West Islanders.
But then, we always knew we were good, but in a quiet way. The Aussies cover their self-doubt with brassy behaviour that borders on being boorish.
Simple maths tells us that if they have five times our population, they should have won five times as many golds. But they didn't and with Val's gold, our media enjoyed a second round of rubbing it in all over again. It felt good and, best of all, that will continue for another four years, at least.
While we wait for TVNZ, Prime and TV3 to launch a host of new series now the Olympics is over, I have to report that I am increasingly enjoying some of the reality TV series.
I've never been a fan, but they do grow on you; not the over-the-top American ones, but the home-grown variety.
This week on TV One on Tuesday were two of the better ones, Coastwatch at 7.30pm, followed by Highway Cops. They are really fly-on-the-wall documentaries, and give an insight into life in New Zealand - to some extent.
In Coastwatch any overseas viewer would think we are a bunch of lying cheats and thieves who rape and pillage our precious fish stocks.
It included those who tried to ignore the allowable catches for fish then made up a lot of fibs until the truth finally came out. What a bunch of naughty people we came across as.
At that point the ever-reliable Mrs Brown corrected me. "You mean naughty men, there were no women trying to steal fish and then lie about it."
She had a point. Not that I thought it was one that needed to be made, but as I've often said, she's a hard woman.
Anyway, back to Coastwatch. Right from the start the narrator makes it clear that this is a matter of some gravitas and we must all watch, or else risk being unpatriotic.
"480 million hectares of ocean, 15,000 kilometres of coastline, $4 billion of fish stocks at stake, poaching, overfishing and marine disasters. On the front line are officers from the Ministry for Primary Industries, police, air force, navy and coastguard."
You can't help but be a bit stirred by this stentorian start to the programme, especially so soon after the Olympics.
There are several items in the 30-minute programme.
There's a crippled yacht in big seas off the coast of Northland; naughty fishermen at sea trying to get away with more than their fair (and legal) share; naughty fishermen on the beaches trying to do the same thing, and Southland men who can't count when it comes to taking Bluff oysters. They are my favourite naughty men; they tell fibs when they get busted and reckon that 50 oysters per person, which is the allowable limit, isn't enough for a decent feed.
The narrator helps things to move along at a good pace and adds his own bit of humour. "Officers suspect these oystermen are bluffing."
It's obvious, but it keeps it light.
Before the end of the programme, all the loose ends are tied up. Some of the naughty ones get the benefit of the doubt, the others are fined, and the men and the yacht are towed to safety.
Then it's time for Highway Cops, which starts off in similar vein.
"New Zealand's highways, 11,000 kilometres of some of the most spectacular and unforgiving roads on the planet. Less (they mean fewer) than 250 men and women are tasked with keeping our highways and byways safe."
What a lucky country we are.
In the first segment we are told that there are only 40 officers on the highways of the lower North Island. Mrs B is surprised by that figure. "That means there are still some who haven't given you tickets for speeding then," she acidly observes.
"Yes dear," I mutter, wondering if there should be an Olympic event in sarcasm. If there was, there wouldn't be a Belarusian, drugged or otherwise, who'd get near to my wife.
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Read Across Oklahoma visits the Oklahoma City Zoo
Reading took center stage at the Oklahoma City Zoo on Tuesday.
Read Across Oklahoma gives kids the opportunity to receive a free book and encourages them to read in their spare time. Well-known local residents including OETA public television employees were among those who read to the kids.
The event was focused on the book "Oh Nuts," by Oklahoma author Tammi Sauer.