Proteomic profile changes in membranes of ethanol-tolerant <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.     

Discovery of selective imidazole-based inhibitors of mammalian 15-lipoxygenase: highly potent against human enzyme within a cellular environment

A series of 2,4,5-tri-substituted imidazoles has proven to be highly potent in inhibiting mammalian 15-lipoxygenase (15-LO) with excellent selectivity over human isozymes 5- and P-12-LO. Non-symmetrical sulfamides (e.g., 21a-n) were found to be suitable replacements for the earlier arylsulfonamide-containing members of this series (e.g., 2, 14a-p). Several members of these series also demonstrated potent inhibition of human 15-LO in a cell-based assay.     

Morphometric Differentiation among Experimental Lines of the Housefly in Relation to a Bottleneck

Differentiation in morphometric traits among experimental populations of the housefly subjected to an experimental bottleneck was examined for replicate lines founded with one, four or 16 pairs of flies. Differentiation among lines within a bottleneck size was significantly greater than predicted by drift in relation to the additive genetic variation for these traits within the founding population. Two models of nonadditive genetic variance were investigated to interpret these results, one involving dominance of allelic effects within loci and another incorporating multiplicative epistasis. Both models generated more variation among lines as a direct result of sampling during the bottleneck than predicted by a model with additive gene action. The pattern of differentiation among our experimental lines in relation to these models conformed more to the model incorporating epistasis. Nevertheless, it may be difficult to distinguish differentiation among lines occurring during a bottleneck as a result of nonadditive gene action from that caused by diversifying selection among lines after the bottleneck.     

Effect of muscle action and metabolic strain on oxidative metabolic responses in human skeletal muscle.

A recent report suggests that differences in aerobic capacity exist between concentric and eccentric muscle action in human muscle (T. W. Ryschon, M. D. Fowler, R. E. Wysong, A. R. Anthony, and R. S. Balaban. J. Appl. Physiol. 83: 867-874, 1997). This study compared oxidative response, in the form of phosphocreatine (PCr) resynthesis rates, with matched levels of metabolic strain (i.e., changes in ADP concentration or the free energy of ATP hydrolysis) in tibialis anterior muscle exercised with either muscle action in vivo (n = 7 subjects). Exercise was controlled and metabolic strain measured by a dynamometer and (31)P-magnetic resonance spectroscopy, respectively. Metabolic strain was varied to bring cytosolic ADP concentration up to 55 microM or decrease the free energy of ATP hydrolysis to -55 kJ/mol with no change in cytoplasmic pH. PCr resynthesis rates after exercise ranged from 31.9 to 462.5 and from 21.4 to 405.4 micromol PCr/s for concentric and eccentric action, respectively. PCr resynthesis rates as a function of metabolic strain were not significantly different between muscle actions (P > 0.40), suggesting that oxidative capacity is dependent on metabolic strain, not muscle action. Pooled data were found to more closely conform to previous biochemical measurements when a term for increasing oxidative capacity with metabolic strain was added to models of respiratory control.     

Low utilization of circulating glucose after food withdrawal in Snell dwarf mice

Glucose metabolism is altered in long-lived people and mice. Although it is clear that there is an association between altered glucose metabolism and longevity, it is not known whether this link is causal or not. Our current hypothesis is that decreased fasting glucose utilization may increase longevity by reducing oxygen radical production, a potential cause of aging. We observed that whole body fasting glucose utilization was lower in the Snell dwarf, a long-lived mutant mouse. Whole body fasting glucose utilization may be reduced by a decrease in the production of circulating glucose. Our isotope labeling analysis indicated both gluconeogenesis and glycogenolysis were suppressed in Snell dwarfs. Elevated circulating adiponectin may contribute to the reduction of glucose production in Snell dwarfs. Adiponectin lowered the appearance of glucose in the media over hepatoma cells by suppressing gluconeogenesis and glycogenolysis. The suppression of glucose production by adiponectin in vitro depended on AMP-activated protein kinase, a cell mediator of fatty acid oxidation. Elevated fatty acid oxidation was indicated in Snell dwarfs by increased utilization of circulating oleic acid, reduced intracellular triglyceride content, and increased phosphorylation of acetyl-CoA carboxylase. Finally, protein carbonyl content, a marker of oxygen radical damage, was decreased in Snell dwarfs. The correlation between high glucose utilization and elevated oxygen radical production was also observed in vitro by altering the concentrations of glucose and fatty acids in the media or pharmacologic inhibition of glucose and fatty acid oxidation with 4-hydroxycyanocinnamic acid and etomoxir, respectively.     

Relationship between estrone sulfate in plasma and litter size at farrowing for sows and gilts

A positive association (P < 0.01) was detected between estrone sulfate (ES) concentrations in maternal plasma at Day 30 of pregnancy and litter size at parturition in swine. This relationship was best described by a fifth order regression equation (R(2) = 0.5) which indicated that as ES increased from 1 to 7.5 ng/ml on Day 30, litter size increased from 0 (nonpregnant) to 18 piglets farrowed. Day of sampling (P < 0.02), month (P < 0.04) and parity (P < 0.08) were major sources of variation in the model. This indicated that effects of environmental factors such as heat stress, which influence conception rate and embryonic survival, are reflected in changes in maternal ES. Also, larger litter size associated with parous sows is reflected in increased ES in maternal plasma. We conclude that measurement of ES early in gestation may be useful in reproductive management to identify nonpregnant gilts and sows as well as those with small litters.     

Isothiazolidinone inhibitors of PTP1B containing imidazoles and imidazolines

The structure-based design and synthesis of isothiazolidinone (IZD) inhibitors of PTP1B containing imidazoles and imidazolines and their modification to interact with the B site of PTP1B are described here. The X-ray crystal structures of 3I and 4I complexed with PTP1B were solved and revealed the inhibitors are interacting extensively with the B site of the enzyme.     

Simulation of LV pacemaker lead in marginal vein: potential risk factors for acute dislodgement

Although left ventricular (LV) coronary sinus lead dislodgement remains a problem, the risk factors for dislodgement have not been clearly defined. In order to identify potential risk factors for acute lead dislodgement, we conducted dynamic finite element simulations of pacemaker lead dislodgement in marginal LV vein. We considered factors such as mismatch in lead and vein diameters, velocity of myocardial motion, branch angle between the insertion vein and the coronary sinus, degree of slack, and depth of insertion. The results show that large lead-to-vein diameter mismatch, rapid myocardial motion, and superficial insertion are potential risk factors for lead dislodgement. In addition, the degree of slack presents either a positive or negative effect on dislodgement risk depending on the branch angle. The prevention of acute lead dislodgment can be enforced by inducing as much static friction force as possible at the lead-vein interface, while reducing the external force. If the latter exceeds the former, dislodgement will occur. The present findings underscore the major risk factors for lead dislodgment, which may improve implantation criterion and future lead design.     

Secondary nucleating sequences affect kinetics and thermodynamics of tau aggregation

Tau protein was scanned for highly amyloidogenic sequences in amphiphilic motifs (X)(n)Z, Z(X)(n)Z (n ≥ 2), or (XZ)(n) (n ≥ 2), where X is a hydrophobic residue and Z is a charged or polar residue. N-Acetyl peptides homologous to these sequences were used to study aggregation. Transmission electron microscopy (TEM) showed seven peptides, in addition to well-known primary nucleating sequences Ac(275)VQIINK (AcPHF6*) and Ac(306)VQIVYK (AcPHF6), formed fibers, tubes, ribbons, or rolled sheets. Of the peptides shown by TEM to form amyloid, Ac(10)VME, AcPHF6*, Ac(375)KLTFR, and Ac(393)VYK were found to enhance the fraction of β-structure of AcPHF6 formed at equilibrium, and Ac(375)KLTFR was found to inhibit AcPHF6 and AcPHF6* aggregation kinetics in a dose-dependent manner, consistent with its participation in a hybrid steric zipper model. Single site mutants were generated which transformed predicted amyloidogenic sequences in tau into non-amyloidogenic ones. A M11K mutant had fewer filaments and showed a decrease in aggregation kinetics and an increased lag time compared to wild-type tau, while a F378K mutant showed significantly more filaments. Our results infer that sequences throughout tau, in addition to PHF6 and PHF6*, can seed amyloid formation or affect aggregation kinetics or thermodynamics.