Simple sequence repeat DNA markers in alfalfa and perennial and annual Medicago species.

Simple sequence repeat (SSR) or microsatellite DNA markers have been shown to function well in plant and mammalian species for genetic map construction and genotype identification. The objectives of the work reported here were to search GenBank for the presence of SSR-containing sequences from the genus Medicago, to assess the presence and frequency of SSR DNA in the alfalfa (Medicago sativa (L.) L. &L.) genome, and to examine the function of selected markers in a spectrum of perennial and annual Medicago species. The screening of an alfalfa genomic DNA library and sequencing of clones putatively containing SSRs indicated approximately 19 000 (AT)n + (CT)n + (CA)n + (ATT)n SSRs in the tetraploid genome. Inheritance was consistent with Mendelian expectations at four selected SSR loci with different core motifs. Additionally, genotypes of a range of Medicago species, including 10 perennial subspecies of the M. sativa complex and other perennial and annual Medicago species, were analyzed at each of the loci to ascertain the presence, number, and size of SSR alleles at each locus in each genotype. These studies indicate that SSR markers can function in alfalfa for the construction of genetic maps and will also be useful in a range of Medicago species for purposes of assessing genetic relatedness and taxonomic relationships, and for genotype identification.     

Analysis of the Structure of Surgical Activity for a Suturing and Knot-Tying Task

Background

Surgical tasks are performed in a sequence of steps, and technical skill evaluation includes assessing task flow efficiency. Our objective was to describe differences in task flow for expert and novice surgeons for a basic surgical task.

Methods

We used a hierarchical semantic vocabulary to decompose and annotate maneuvers and gestures for 135 instances of a surgeon’s knot performed by 18 surgeons. We compared counts of maneuvers and gestures, and analyzed task flow by skill level.

Results

Experts used fewer gestures to perform the task (26.29; 95% CI = 25.21 to 27.38 for experts vs. 31.30; 95% CI = 29.05 to 33.55 for novices) and made fewer errors in gestures than novices (1.00; 95% CI = 0.61 to 1.39 vs. 2.84; 95% CI = 2.3 to 3.37). Transitions among maneuvers, and among gestures within each maneuver for expert trials were more predictable than novice trials.

Conclusions

Activity segments and state flow transitions within a basic surgical task differ by surgical skill level, and can be used to provide targeted feedback to surgical trainees.     

Legume-Rhizobium interactions: host induced alterations in capsular polysaccharides and infectivity of cowpea Rhizobia

Klebsiella aerogenes NCTC418 was cultured anaerobically under glucose-limited conditions in chemostat cultures at various growth rates, ranging from 0.13 h^-1 to 0.82 h^-1. It was found that the specific uptake rate of glucose varied linearly with the growth rate and that under these conditions glucose was fermented solely to acetate and ethanol plus CO₂+H₂ and formate.When steady-state cultures were pulsed with cell saturating concentrations of glucose, the specific glucose aptake rate increased immediately and substantially. However, at steady-state growth rates lower than 0.5 h^-1, this increase was not accompanied by a change in the growth rate, in contrast to cultures growing at higher rates. It was found that relief of the glucose limitation resulted in a shift in fermentation pattern: at the lower growth rates 50% or more of the extra glucose taken up was fermented to D-lactate.Incubation experiments with sonified cells revcaled that K. aerogenes possessed all the enzymes needed to convert dihydroxyacetone phosphate to methylglyoxal and subsequently to D-lactate, and that the rate at which this overall conversion occurred in vitro was in close agreement with the production rate of D-lactate in vivo. Moreover, it was found that the activities of the enzymes of the methylglyoxal bypass were dependent on the imposed growth rate. At higher growth rates, where cells possessed the potential to increase their growth rate immediately, the activity of methylglyoxal synthase was relatively low.it could be shown that, under low growth rate conditions, the uncoupling effect of the methylglyoxal bypass was highly effective and that, as a consequence thereof, a significant increase in the uptake rate of the energy source was accompanied by only a marginal increase in the rate at which ATP was synthesized.     

Genetic analysis of attractin homologs

Attractin (ATRN) and Attractin-like 1 (ATRNL1) are highly similar type I transmembrane proteins. Atrn null mutant mice have a pleiotropic phenotype including dark fur, juvenile-onset spongiform neurodegeneration, hypomyelination, tremor, and reduced body weight and adiposity, implicating ATRN in numerous biological processes. Bioinformatic analysis indicated that Atrn and Atrnl1 arose from a common ancestral gene early in vertebrate evolution. To investigate the genetics of the ATRN system and explore potential redundancy between Atrn and Atrnl1, we generated and characterized Atrnl1 loss- and gain-of-function mutations in mice. Atrnl1 mutant mice were grossly normal with no alterations of pigmentation, central nervous system pathology or body weight. Atrn null mutant mice carrying a beta-actin promoter-driven Atrnl1 transgene had normal, agouti-banded hairs and significantly delayed onset of spongiform neurodegeneration, indicating that over-expression of ATRNL1 compensates for loss of ATRN. Thus, the two genes are redundant from the perspective of gain-of-function but not loss-of-function mutations.     

δ-Tocopherol Reduces Lipid Accumulation in Niemann-Pick Type C1 and Wolman Cholesterol Storage Disorders

Niemann-Pick disease type C (NPC) and Wolman disease are two members of a family of storage disorders caused by mutations of genes encoding lysosomal proteins. Deficiency in function of either the NPC1 or NPC2 protein in NPC disease or lysosomal acid lipase in Wolman disease results in defective cellular cholesterol trafficking. Lysosomal accumulation of cholesterol and enlarged lysosomes are shared phenotypic characteristics of both NPC and Wolman cells. Utilizing a phenotypic screen of an approved drug collection, we found that δ-tocopherol effectively reduced lysosomal cholesterol accumulation, decreased lysosomal volume, increased cholesterol efflux, and alleviated pathological phenotypes in both NPC1 and Wolman fibroblasts. Reduction of these abnormalities may be mediated by a δ-tocopherol-induced intracellular Ca²⁺ response and subsequent enhancement of lysosomal exocytosis. Consistent with a general mechanism for reduction of lysosomal lipid accumulation, we also found that δ-tocopherol reduces pathological phenotypes in patient fibroblasts from other lysosomal storage diseases, including NPC2, Batten (ceroid lipofuscinosis, neuronal 2, CLN2), Fabry, Farber, Niemann-Pick disease type A, Sanfilippo type B (mucopolysaccharidosis type IIIB, MPSIIIB), and Tay-Sachs. Our data suggest that regulated exocytosis may represent a potential therapeutic target for reduction of lysosomal storage in this class of diseases.