Successful field capture techniques for free‐ranging argali sheep (Ovis ammon) in Mongolia

Argali sheep (Ovis ammon) are the world's largest wild sheep and are threatened throughout their range in Mongolia. Little is known about the ecology of this highly cursorial species. This project was initiated to develop safe capture techniques in order to learn more about the ecology of argali in the Ikh Nartiin Chuluun Nature Reserve in southeastern Mongolia by using radiotelemetry. To our knowledge no one had ever successfully live‐caught a free‐ranging argali. We developed three techniques for capturing argali. From 2003 to 2007 we hand captured 65 argali lambs, losing one (our first capture) to maternal neglect because we stayed too long in the area and a second that was euthanized after being accidentally injured by a horse. Between 2000 and 2003 we captured six argali by darting, losing one ewe to an abdominal infection after a misplaced dart. Finally, between 2002 and 2007 we captured 32 argali by drive‐netting. We lost one ewe to over‐exertion after a long drive. The remaining 31 animals captured by drive‐netting did not appear to suffer any immediate effects (still alive in 30 days). This report will discuss the strengths and weaknesses for each of these capture techniques. Zoo Biol 27:137–144, 2008. © 2008 Wiley‐Liss, Inc.     

Comparison of Overall Obesity and Body Fat Distribution in Predicting Risk of Mortality

Results of studies comparing overall obesity and abdominal adiposity or body fat distribution with risk of mortality have varied considerably. We compared the relative importance and joint association of overall obesity and body fat distribution in predicting risk of mortality. Participants included 5,799 men and 6,429 women aged 30–102 years enrolled in the third National Health and Nutrition Examination Survey who completed a baseline health examination during 1988–1994. During a 12‐year follow‐up (102,172 person‐years), 1,188 men and 925 women died. In multivariable‐adjusted analyses, waist‐to‐thigh ratio (WTR) in both sexes (P_trend <0.01 for both) and waist‐to‐hip ratio (WHR) in women (P_trend 0.001) were positively associated with mortality in middle‐aged adults (30–64 years), while BMI and waist circumference (WC) exhibited U‐ or J‐shaped associations. Risk of mortality increased with a higher WHR and WTR among normal weight (BMI 18.5–24.9 kg/m²) and obese (BMI ≥30.0 kg/m²) adults. In older adults (65–102 years), a higher BMI in both sexes (P_trend <0.05) and WC in men (P_trend 0.001) were associated with increased survival, while remaining measures of body fat distribution exhibited either no association or an inverse relation with mortality. In conclusion, ratio measures of body fat distribution are strongly and positively associated with mortality and offer additional prognostic information beyond BMI and WC in middle‐aged adults. A higher BMI in both sexes and WC in men were associated with increased survival in older adults, while a higher WHR or WTR either decreased or did not influence risk of death.     

Valproic acid enhances fludarabine-induced apoptosis mediated by ROS and involving decreased AKT and ATM activation in B-cell-lymphoid neoplastic cells

Histone deacetylase (HDAC) inhibitors have been shown synergize with a number of cytotoxic drugs in leukemic cells. In chronic lymphocytic leukemia (CLL), the first line therapy is based on the combination of fludarabine, a nucleoside analogue, and rituximab, an anti-CD20 monoclonal antibody, and there are presently no HDAC inhibitors are used to manage CLL. In the present study, we found that the addition of valproic acid (VPA), a HDAC inhibitor, increases cell death in B-cell-neoplasm-derived cell lines, BJAB, NALM-6 and I-83. This increased apoptosis caused release of mitochondrial cytochrome c, activation of caspases, and increased reactive oxygen species (ROS). The addition of a ROS scavenger inhibited cell death induced by the VPA–fludarabine combination. In contrast, blocking the death receptor pathway failed to inhibit VPA increased fludarabine induced apoptosis. Combination of VPA and fludarabine treatment decreased both total and phosphorylated levels of AKT, an important anti-apoptotic protein, and ATM, a pivotal protein in DNA damage response. Chemical inhibition of AKT or ATM was sufficient to enhance fludarabine-induced apoptosis. We next examined patient samples from a local clinical trial where relapsed CLL patients were treated with VPA and examined the effects of VPA on AKT and ATM in vivo. After 30 days, there was a reduction in ATM levels in three out of the four patients treated, while AKT phosphorylation was reduced only in one patient. Taken together, VPA reduces ATM levels, thereby increasing ROS-dependent cell death via the mitochondrial apoptotic pathway when combined with fludarabine.     

Genetic analysis of the susceptibility of mouse cytomegalovirus to acyclovir.

Eight independently derived mouse cytomegalovirus (MCMV) mutants resistant to acyclovir (ACV) were obtained by the sequential plating of wild-type virus in increasing concentrations of ACV. Results of complementation studies among these eight mutants suggest that all had mutations within the same or closely associated genes. A ninth MCMV mutant resistant to phosphonoacetate (PAA) derived by plating wild-type virus in the presence of 100 micrograms of PAA per ml displayed coresistance to ACV and was unable to complement any of the ACV-derived mutants. Recombination experiments among all combinations of the nine MCMV mutants were performed and supported the complementation data in that no recombination could be detected. Seven of the eight ACV-resistant mutants demonstrated cross-resistance to PAA and hypersensitivity to aphidicolin. The one mutant not coresistant to PAA was more susceptible to PAA than was the parent virus. Only a few mutants demonstrated coresistance when the mutants were tested against 9-beta-D-arabinofuranosyladenine (ara-A). The ACV mutant that demonstrated increased susceptibility to PAA was 30-fold more susceptible to ara-A but remained unchanged in susceptibility to aphidicolin. Two of the parent-mutant combinations were selected for DNA synthesis analysis in the presence of ACV (5 microM). A significant decrease in DNA synthesis was demonstrated for both parent viruses, and there was little effect on mutant virus DNA synthesis at the same drug concentration. These results suggest that susceptibility of MCMV to ACV is confined to a product of a single gene and that a mutation of this gene can lead to an altered phenotype when compared with parent virus in susceptibility of DNA synthesis to PAA, ara-A, and aphidicolin, drugs that are known to inhibit DNA polymerase activity.     

Immobilized adriamycin: a tool for separating cell surface from intracellular mechanisms

Pharmacologic agents may exert their biological activity at the level of the cell membrane. Of particular interest is the anticancer agent adriamycin. This drug has previously been considered to act by intercalation with nuclear DNA, but recent evidence suggests the possibility that the cell surface membrane may represent an alternative target. To test this hypothesis, adriamycin was attached to insoluble supports, and conditions suggesting that the drug was actively cytotoxic without entering cells were demonstrated.     

p-Benzoquinone activation of metal oxide electrodes for attachment of enzymes

The mechanisms by which p-benzoquinone brings about the activation of a metal oxide surface and subsequent coupling of an enzyme has been investigated. Indium-tin oxide (ITO)coated glass plates were derivatized with an aminosilane and then activated with p-benzoquinone. Differential pulse voltammetry was used to distinguish between mono- and disubstitution of the benzoquinone, both for a model system in homogeneous solution and for the aminosilane derivatized plates. Glucose oxidase was attached covalently to the benzoquinone-aminosilane activated ITO plates to give an enzymatically active electrode.     

Structural basis for calcium-induced inhibition of rhodopsin kinase by recoverin

Recoverin, a member of the neuronal calcium sensor branch of the EF-hand superfamily, serves as a calcium sensor that regulates rhodopsin kinase (RK) activity in retinal rod cells. We report here the NMR structure of Ca(2+)-bound recoverin bound to a functional N-terminal fragment of rhodopsin kinase (residues 1-25, called RK25). The overall main-chain structure of recoverin in the complex is similar to structures of Ca(2+)-bound recoverin in the absence of target (<1.8A root-mean-square deviation). The first eight residues of recoverin at the N terminus are solvent-exposed, enabling the N-terminal myristoyl group to interact with target membranes, and Ca(2+) is bound at the second and third EF-hands of the protein. RK25 in the complex forms an amphipathic helix (residues 4-16). The hydrophobic face of the RK25 helix (Val-9, Val-10, Ala-11, Ala-14, and Phe-15) interacts with an exposed hydrophobic groove on the surface of recoverin lined by side-chain atoms of Trp-31, Phe-35, Phe-49, Ile-52, Tyr-53, Phe-56, Phe-57, Tyr-86, and Leu-90. Residues of recoverin that contact RK25 are highly conserved, suggesting a similar target binding site structure in all neuronal calcium sensor proteins. Site-specific mutagenesis and deletion analysis confirm that the hydrophobic residues at the interface are necessary and sufficient for binding. The recoverin-RK25 complex exhibits Ca(2+)-induced binding to rhodopsin immobilized on concanavalin-A resin. We propose that Ca(2+)-bound recoverin is bound between rhodopsin and RK in a ternary complex on rod outer segment disk membranes, thereby blocking RK interaction with rhodopsin at high Ca(2+).     

Cooperative attachment of cross bridges predicts regulation of smooth muscle force by myosin phosphorylation

Serine 19 phosphorylation of the myosin regulatory light chain (MRLC) appears to be the primary determinant of smooth muscle force development. The relationship between MRLC phosphorylation and force is nonlinear, showing that phosphorylation is not a simple switch regulating the number of cycling cross bridges. We reexamined the MRLC phosphorylation-force relationship in slow, tonic swine carotid media; fast, phasic rabbit urinary bladder detrusor; and very fast, tonic rat anococcygeus. We found a sigmoidal dependence of force on MRLC phosphorylation in all three tissues with a threshold for force development of approximately 0.15 mol P(i)/mol MRLC. This behavior suggests that force is regulated in a highly cooperative manner. We then determined whether a model that employs both the latch-bridge hypothesis and cooperative activation could reproduce the relationship between Ser(19)-MRLC phosphorylation and force without the need for a second regulatory system. We based this model on skeletal muscle in which attached cross bridges cooperatively activate thin filaments to facilitate cross-bridge attachment. We found that such a model describes both the steady-state and time-course relationship between Ser(19)-MRLC phosphorylation and force. The model required both cooperative activation and latch-bridge formation to predict force. The best fit of the model occurred when binding of a cross bridge cooperatively activated seven myosin binding sites on the thin filament. This result suggests cooperative mechanisms analogous to skeletal muscle that will require testing.     

Cyanophycin Production in a Phycoerythrin-Containing Marine Synechococcus Strain of Unusual Phylogenetic Affinity

Thirty-two strains of phycoerythrin-containing marine picocyanobacteria were screened for the capacity to produce cyanophycin, a nitrogen storage compound synthesized by some, but not all, cyanobacteria. We found that one of these strains, Synechococcus sp. strain G2.1 from the Arabian Sea, was able to synthesize cyanophycin. The cyanophycin extracted from the cells was composed of roughly equimolar amounts of arginine and aspartate (29 and 35 mol%, respectively), as well as a small amount of glutamate (15 mol%). Phylogenetic analysis, based on partial 16S ribosomal DNA (rDNA) sequence data, showed that Synechococcus sp. strain G2.1 formed a well-supported clade with several strains of filamentous cyanobacteria. It was not closely related to several other well-studied marine picocyanobacteria, including Synechococcus strains PCC7002, WH7805, and WH8018 and Prochlorococcus sp. strain MIT9312. This is the first report of cyanophycin production in a phycoerythrin-containing strain of marine or halotolerant Synechococcus, and its discovery highlights the diversity of this ecologically important functional group.