Pyrazole-based cathepsin S inhibitors with arylalkynes as P1 binding elements

A crystal structure of 1 bound to a Cys25Ser mutant of cathepsin S helped to elucidate the binding mode of a previously disclosed series of pyrazole-based CatS inhibitors and facilitated the design of a new class of arylalkyne analogs. Optimization of the alkyne and tetrahydropyridine portions of the pharmacophore provided potent CatS inhibitors (IC₅₀ = 40–300 nM), and an X-ray structure of 32 revealed that the arylalkyne moiety binds in the S1 pocket of the enzyme.     

Munc18c is not rate-limiting for glucose and long-chain fatty acid uptake in the heart

The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)- and SNARE-associated proteins have not yet been assessed in regulation of cardiac glucose uptake, nor in the regulation of long-chain fatty acid (LCFA) uptake in any tissue. Munc18c is a SNARE-associated protein that regulates GLUT4 translocation in skeletal muscle and adipose tissue. Using cardiomyocytes from Munc18c^?/+ mice (with 56% reduction of Munc18c protein expression), we investigated whether this syntaxin4-associated protein is involved in regulation of cardiac substrate uptake. Basal, insulin- and oligomycin (a 5′ AMP-activated protein kinase-activating agent)-stimulated glucose and LCFA uptake were not altered significantly in Munc18c^?/+ cardiomyocytes compared to wild-type cells. We conclude, therefore, that Munc18c is not rate-limiting for cardiac substrate uptake, neither under basal conditions nor when maximally stimulated metabolically.     

Molecular areas of phospholipids as determined by 2H NMR spectroscopy. Comparison of phosphatidylethanolamines and phosphatidylcholines.

The role of lipid diversity in biomembranes is one of the major unsolved problems in biochemistry. One parameter of possible importance is the mean cross-sectional area occupied per lipid molecule, which may be related to formation of nonbilayer structures and membrane protein function. We have used 2H NMR spectroscopy to compare the properties of 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-d62) and 1,2-diperdeuteriopalmitoyl-sn-glycero-3-phosphocholine (DPPC-d62) in the L alpha phase. We find that DPPE has greater segmental order than DPPC, and that this increase in order is related to the smaller area per acyl chain found for DPPE. Values of the mean cross-sectional chain area are calculated using a simple diamond lattice model for the acyl chain configurational statistics, together with dilatometry data. The results obtained for the mean area per molecule are comparable with those from low angle x-ray diffraction studies.     

Amplification of the rbcL gene from dissolved and particulate DNA from aquatic environments.

The carboxylation of ribulose biphosphate by the enzyme ribulosebisphosphate carboxylase/oxygenase is the mechanism for CO2 fixation and primary production in nearly all ecosystems on this planet. Although certain algal isolates and higher plants contain conserved nucleotide sequences in the large subunit of the gene (rbcL) for this enzyme, such genes from natural microbial assemblages have not been heretofore examined. Using oligonucleotide primers designed for conserved regions of the rbcL gene of a Synechococcus sp. (Anacystis nidulans), we have amplified rbcL from DNA preparations from planktonic samples from a Florida reservoir and from algal isolates by the polymerase chain reaction. We have also detected rbcL by gene amplification in the extracellular DNA fraction of this reservoir, indicating that phytoplankton can be a source of dissolved DNA. These results suggest that gene amplification can be applied for the detection of conserved genes encoding enzymes involved in important ecological functions in aquatic environments.     

Hormonally regulated programmed cell death in barley aleurone cells

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.     

The use of loglinear model to evaluate factors associated with sero-prevalence of Campylobacter fetus in dairy cattle

Campylobacter fetus is one of the leading infections causing bovine infertility and abortion. In a cross-sectional study, the associations of parity, herd, serological status against Leptospira hardjo and Haemophilus somnus with the sero-prevalence of Campylobacter fetus in dairy cattle were evaluated. Four hundred dairy cows from three herds were included in the study. Blood samples from the cows were collected in December 1986. An enzyme-linked immunosorbant assay (ELISA) was used to determine positive or negative status of cows against Campylobacter fetus , Haemophilus somnus and Leptospira hardjo antigens. Loglinear analysis was used to describe the relationship of four categorical variables with the sero-prevalence of Campylobacter fetus . The final loglinear model was interpreted as a logit model. The final loglinear model included a three-way interaction among Campylobacter fetus , Leptospira hardjo and herd and a two-way interaction between Campylobacter fetus and parity. The adjusted odds of being a positive Campylobacter fetus reading were highest for cows with parity >6 (e.g. 27.8, 95% confidence interval (CI) = 12.9, 60.1); it was next highest for cows in parity <4 (e.g. 18.0, 95% CI = 13.5, 24.2); and it was lowest for cows in parity 4 to 6 (e.g. 13.3, 95% CI 7.3, 24.4). The herd management practice that was associated with the carrier state in early and late parities is identified in the study, and the biological implication of a three-way interaction among Campylobacter fetus , Leptospira hardjo and herd are discussed.     

Munc18c regulates insulin-stimulated glut4 translocation to the transverse tubules in skeletal muscle

To examine the intracellular trafficking and translocation of GLUT4 in skeletal muscle, we have generated transgenic mouse lines that specifically express a GLUT4-EGFP (enhanced green fluorescent protein) fusion protein under the control of the human skeletal muscle actin promoter. These transgenic mice displayed EGFP fluorescence restricted to skeletal muscle and increased glucose tolerance characteristic of enhanced insulin sensitivity. The GLUT4-EGFP protein localized to the same intracellular compartment as the endogenous GLUT4 protein and underwent insulin- and exercise-stimulated translocation to both the sarcolemma and transverse-tubule membranes. Consistent with previous studies in adipocytes, overexpression of the syntaxin 4-binding Munc18c isoform, but not the related Munc18b isoform, in vivo specifically inhibited insulin-stimulated GLUT4-EGFP translocation. Surprisingly, however, Munc18c inhibited GLUT4 translocation to the transverse-tubule membrane without affecting translocation to the sarcolemma membrane. The ability of Munc18c to block GLUT4-EGFP translocation to the transverse-tubule membrane but not the sarcolemma membrane was consistent with substantially reduced levels of syntaxin 4 in the transverse-tubule membrane. Together, these data demonstrate that Munc18c specifically functions in the compartmentalized translocation of GLUT4 to the transverse-tubules in skeletal muscle. In addition, these results underscore the utility of this transgenic model to directly visualize GLUT4 translocation in skeletal muscle.     

Comparison of Ketosis,Clinical Mastitis,Somatic Cell Count,and Reproductive Performance between Free Stall and Tie Stall Barns in Norwegian Dairy Herds with Automatic Feeding

Differences in ketosis and clinical mastitis rates, mean somatic cell counts (SCC) and reproductive performance scores (fertility status index = FS-index) between herds housed in free stall (n = 533) and tie stall (n = 59) barns were measured in relation to management and environmental factors in a retrospective cohort study of Norwegian dairy farms with automatic grain feeding systems. Herds with tie stalls were found to have a higher clinical mastitis rate, (61.6 cases per 100 cow-years versus 46.4 cases per 100 cow-years (p<0.01)); a significantly higher proportion of herds with ketosis, (relative risk = 1.59, (p<0.01)); and a lower mean fertility status index, (67.3 versus 82.8 (p<0.01)). No significant difference in mean SCC was found between the 2 groups of dairies (p = 0.32). Large herds had higher Loge geometric mean SCC than small herds (p<0.01) and herds with high SCC had lower milk production than herds with low SCC (p<0.01). Herds that scored “very good” on cow cleanliness had significantly lower adjusted mean SCC than herds with scores of “average” or “good” (p<0.05). Herds in barns with a concrete floor had an adjusted mean number of cases of clinical mastitis of 51.4 per 100 cow-years, 14 per cent higher than herds in barns with rubber mats, litter bed or wood (45.2 cases per 100 cow-years) (p<0.05). Herds with high milk production level had higher FS-index than herds with low milk production (p<0.01). Lower disease rates and the higher fertility status measured in the present study favor free stall herds over tie stall herds.     

Associations among prostaglandin F2alpha, plasma zinc, copper and iron concentrations and fetal loss in cows and mares

The objective of this study was to test the hypothesis that PGF2alpha is associated with abortion and changes in plasma Zn, Cu, and Fe concentrations in cows and mares in their first trimester of pregnancy. Eleven pregnant cows were infused with endotoxin (n = 5) or endotoxin plus an inhibitor of cycloxygenase, flunixin meglumine (n = 6). Blood was collected over a 5-d period. Additionally, 4 mares were treated every 24 h with cloprostenol sodium and blood was collected hourly until abortion. Plasma Zn, Cu, and Fe were determined. Three of five cows treated with endotoxin aborted, but none of the six cows treated with endotoxin and flunixin meglumine aborted. Aborting cows had lower plasma Zn (P = 0.048) over the 5-d study period compared with the nonaborting cows. The changes in Zn corresponded to release of PGF2alpha. All 4 mares aborted and plasma Zn concentrations were lower (P = 0.008) and Cu/Zn was higher (P = 0.02) 12 h after cloprostenol treatment. Plasma Zn may be a useful biomarker for risk of spontaneous abortion, and the decline in plasma Zn may be caused by PGF2alpha.     

Munc18c function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles

To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulin-responsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4- and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane. Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.