Significance of hydrogen bonding at the S(1)' subsite of calpain I.

alpha-Ketohydroxamates were synthesized as bioisosteres of alpha-ketoamides. The alpha-ketohydroxamates were generally more potent than the corresponding alpha-ketoamides. The potency of the compounds suggests that hydrogen bonding and steric bulk of substituents on the nitrogen atom of the ketoamide moiety influence calpain inhibition.     

EFFECTIVENESS OF SELECTION IN REDUCING THE GENETIC LOAD IN POPULATIONS OF PEROMYSCUS POLIONOTUS DURING GENERATIONS OF INBREEDING

It has been hypothesized that natural selection reduces the “genetic load” of deleterious alleles from populations that inbreed during bottlenecks, thereby ameliorating impacts of future inbreeding. We tested the efficiency with which natural selection purges deleterious alleles from three subspecies of Peromyscus polionotus during 10 generations of laboratory inbreeding by monitoring pairing success, litter size, viability, and growth in 3604 litters produced from 3058 pairs. In P. p. subgriseus, there was no reduction across generations in inbreeding depression in any of the fitness components. Strongly deleterious recessive alleles may have been removed previously during episodes of local inbreeding in the wild, and the residual genetic load in this population was not further reduced by selection in the lab. In P. p. rhoadsi, four of seven fitness components did show a reduction of the genetic load with continued inbreeding. The average reduction in the genetic load was as expected if inbreeding depression in this population is caused by highly deleterious recessive alleles that are efficiently removed by selection. For P. p. leucocephalus a population that experiences periodic bottlenecks in the wild, the effect of further inbreeding in the laboratory was to exacerbate rather than reduce the genetic load. Recessive deleterious alleles may have been removed from this population during repeated bottlenecks in the wild; the population may be close to a threshold level of heterozygosity below which fitness declines rapidly. Thus, the effects of selection on inbreeding depression varied substantially among populations, perhaps due to different histories of inbreeding and selection.     

Specific neuronal expression of human NGF receptors in the basal forebrain and cerebellum of transgenic mice

Transgenic mice carrying multiple copies of the human NGF receptor gene have been generated. Using a monoclonal antibody specific for the human receptor, we have detected specific expression in cholinergic neurons in the basal forebrain and Purkinje cells in the cerebellum during the postnatal period. Expression in the PNS was exemplified by immunostaining of sympathetic and sensory neurons during an early embryonic age. Transection of the sciatic nerve in transgenic animals resulted in induction of human NGF receptors, indicating that the inserted gene can be appropriately regulated. These transgenic mice will provide an opportunity to study the elements regulating the NGF receptor. Furthermore, the ability to obtain specific expression in transgenic mice will permit directed expression of heterologous genes in discrete cells important in the cholinergic septal-hippocampal pathway and the PNS.     

Mapping the anthrax protective antigen binding site on the lethal and edema factors.

Entry of anthrax edema factor (EF) and lethal factor (LF) into the cytosol of eukaryotic cells depends on their ability to translocate across the endosomal membrane in the presence of anthrax protective antigen (PA). Here we report attributes of the N-terminal domains of EF and LF (EF(N) and LF(N), respectively) that are critical for their initial interaction with PA. We found that deletion of the first 36 residues of LF(N) had no effect on its binding to PA or its ability to be translocated. To map the binding site for PA, we used the three-dimensional structure of LF and sequence similarity between EF and LF to select positions for mutagenesis. We identified seven sites in LF(N) (Asp-182, Asp-187, Leu-188, Tyr-223, His-229, Leu-235, and Tyr-236) where mutation to Ala produced significant binding defects, with H229A and Y236A almost completely eliminating binding. Homologous mutants of EF(N) displayed nearly identical defects. Cytotoxicity assays confirmed that the LF(N) mutations impact intoxication. The seven mutation-sensitive amino acids are clustered on the surface of LF and form a small convoluted patch with both hydrophobic and hydrophilic character. We propose that this patch constitutes the recognition site for PA.     

Improved Culture Flask for Obligate Anaerobes

An improved flask system for the growth of extremely oxygen-sensitive bacteria in liquid culture is described. The improvement described utilizes an all-glass, neoprene-stoppered flask designed for growth of 50- to 1,000-ml cultures of bacteria with continuous gassing.     

Maintenance of genetic variation in quantitative traits of a woodland rodent during generations of captive breeding

Breeding programs to conserve diversity are predicated on the assumption that genetic variation in adaptively important traits will be lost in parallel to the loss of variation at neutral loci. To test this assumption, we monitored quantitative traits across 18 generations of Peromyscus leucopus mice propagated with protocols that mirror breeding programs for threatened species. Ears, hind feet, and tails became shorter, but changes were reversible by outcrossing and therefore were due to accumulated inbreeding. Heritability of ear length decreased, because of an increase in phenotypic variance rather than the expected decrease in additive genetic variance. Additive genetic variance in hind foot length increased. This trait initially had low heritability but large dominance or common environmental variance contributing to resemblance among full-sibs. The increase in the additive component indicates that there was conversion of interaction variances to additive variance. For no trait did additive genetic variation decrease significantly across generations. These findings indicate that the restructuring of genetic variance that occurs with genetic drift and novel selection in captivity can prevent or delay the loss of phenotypic and heritable variation, providing variation on which selection can act to adapt populations to captivity and perhaps later to readapt to more natural habitats after release. Therefore, the importance of minimizing loss of gene diversity from conservation breeding programs for threatened wildlife species might lie in preventing immediate reduction in individual fitness due to inbreeding and protecting allelic diversity for long-term evolutionary change, more so than in protecting variation in quantitative traits for rapid re-adaptation to wild environments.     

Molecular dynamics simulations of the aggregation behaviour of overlapped graphene sheets in linear aliphatic hydrocarbons

Molecular dynamics simulations were used to investigate the aggregation of two partially overlapped graphene sheets in hexane, dodecane and eicosane. When partially overlapped graphene sheets are adjacent to one another, they will expel the adsorbed layers of the solvent molecules on the graphene surface, and the amount of overlap will increase. When the overlapped regions of the graphene sheets are separated by solvent molecules, they cannot expel the adsorption layers between them, and so the sheets remain separated. The driving force for aggregation is the van der Waals interaction between the two graphene sheets, while the van der Waals interaction between the graphene sheets and the solvent molecules inhibits graphene aggregation. The diffusion rate of the hydrocarbon molecules with shorter chain lengths is higher. Thus, they diffuse faster during graphene aggregation, which leads to a higher rate of graphene overlapping in the shorter hydrocarbons. This work provides useful insights into graphene aggregation in linear hydrocarbon solvents of varying lengths at the nanoscale.     

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.