Molecular dynamics simulation of oligosaccharides containing N-acetyl neuraminic acid

αD -N-acetyl neuraminic acid (Neu5Ac, sialic acid) is a commonly occurring carbohydrate residue in various cell surface glycolipids and glycoproteins. This residue is linked terminally or internally to Gal residues via an α(2 → 3) or α(2 → 6) linkage. In the cell surface receptor, sialyl-Lewis^X, a terminal α(2 → 3) linkage is present. Previous studies from our laboratory have shown that in solution Lewis^X adopts a relatively rigid structure. In order to model the Neu5Ac residue, vacuum molecular dynamics of this monosaccharide were compared with simulations that explicitly include solvent water. The dynamical average of the monosaccharide conformation obtained from the two simulations was similar. Vacuum calculations for the disaccharide Neu5Ac α(2 → 3) Gal β-O-methyl show that a number of low energy minima are accessible to this disaccharide. Molecular dynamics simulations starting from the low energy minima show conformational transitions with a time scale of 10–50 ps among several of the minima while large barriers between other minima prevent transitions on the time scale studied. Simulations of this disaccharide in the presence of solvent show fewer conformational transitions, illustrating a dampening effect of the solvent that has been observed in some other studies. Our results are most consistent with an equilibrium among multiple conformations for the Neu5Ac α(2 → 3) Gal β linkage. © 1994 John Wiley & Sons, Inc.     

Reproductive status of cheetahs (Acinonyx jubatus) in North American Zoos: The benefits of physiological surveys for strategic planning

Under the mandate of a Species Survival Plan (SSP), reproductive status was assessed in 128 cheetahs maintained in 18 different institutions in North America. A mobile laboratory research team evaluated cheetahs using anesthesia, serial blood sampling, electroejaculation (males), and laparoscopy (females). Biomaterials were also collected for parallel studies of genetics, nutrition, and health. There was no mortality, and cheetahs were capable of reproducing naturally after these intense manipulatory examinations. No marked differences were observed in reproductive or endocrine characteristics between proven and unproven breeders. However, males consistently produced teratospermic ejaculates, and cheetah sperm were compromised in conspecific or heterologous in vitro fertilization systems. Structurally abnormal sperm were found to be filtered by the oocyte's zona pellucida. More than 80% of the females were anatomically sound, but morphological and endocrine evidence suggested that ～50% or more of the population may have had inactive ovaries at the time of the examination. Males ranging in age from 15 to 182 months produced spermic ejaculates, but motile sperm numbers/ejaculate and circulating testosterone concentrations were highest in males 60 to 120 months old. Parovarian cysts were observed in 51.5% of female cheetahs, but comparisons between proven and unproven subpopulations revealed that this abnormality likely had no influence on fertility. Fresh luteal tissue was not observed in any nonpregnant or nonlactating female, strongly suggesting that the cheetah is an induced ovulator. Overall survey results were discussed in the context of the etiology of reproductive inefficiency, especially with respect to the potential importance of biological versus management factors. Four high priority research areas in cheetah reproductive biology were identified: 1) continuous monitoring of ejaculate quality in the extant population, while studying the impact of pleiomorphisms on fertility; 2) determining the potential relationship between libido and androgen production (excretion) in males; 3) confirming the extent of cyclic, or acyclic, ovarian activity in females; and 4) continued development of assisted reproductive techniques for enhancing man-agement. In summary, a multidisciplinary, multi-institutional survey coordinated through the SSP is both possible and useful for generating a physiological and health database beneficial to driving further research and management initiatives. © 1993 Wiley-Liss, Inc.     

Lectin recognition of host-like saccharide motifs in streptococcal cell wall polysaccharides

Viridans streptococci that participate in the microbial colonizationof teeth have cell wall polysaccharides composed of linear phosphodiester-linkedhexa- or heptasaccharide repeating units, each containing ahost-like disaccharide motif, either Galß1     

Recovery of intestinal membrane binding sites for K88 E. coli from pig mucosal organ cultures

Summary Putative receptors for K88+ E. coli from piglet intestinal epithelium were released into the organ culture medium and were demonstrated by direct binding with K88+ E. coli through the utilization of an in vitro binding procedure or by immunoprecipitation with K88 antigen.Incorporation of ¹⁴C-glucosamine by newborn to day old and 3-week to 6-week old piglet jejunal and ileal mucosa, in organ culture, occurred throughout the 24 hr culture period. Uptake in both age groups and both areas of the intestine was similar with a somewhat greater incorporation by the older age group.Secretion of ¹⁴C-glucosamine-labeled components into the culture medium was demonstrated by gel filtration of the concentrated medium. Some large molecular weight components eluted in the void volume in excess of 2 x 10⁶ daltons. A second peak of activity was spread from approximately 690K to 25K daltons. All eluted fractions demonstrated binding to K88+ E. coli.Antibodies to purified brush borders from susceptible pigs produced prominent precipitation bands following double diffusion with concentrated organ culture media which confirmed that the organ culture media contained labeled proteins of brush border origin.Immunoprecipitation of the intestinal mucosal organ culture media with K88+ pili and pilus antisera, followed by electrophoresis with SDS and reduced conditions, demonstrated a subunit of approximately 35K daltons.     

Regeneration of masseter muscle following lidocaine-induced degeneration. A histochemical study.

The histoenzymatic characteristics of regenerating myofibers of rat masseter muscle following injection of 1% lidocaine, as well as morphometric and histochemical characteristics of the typical myofibers, were investigated. Myoblasts appeared initially by day 1 among numerous macrophages within the confines of degenerating myofibers. Myotubes predominated by the 3rd day. Complete regeneration of the muscle occurred by at least 45 days. Phosphorylase activity was absent at day 1 and reappeared by the 5th day when the regenerating myofibers showed slight activity. By the 15th day the myofiber types had partly differentiated; red myofibers were smaller and stained less intensely than the white myofibers. Myotubes stained uniformly for succinic dehydrogenase activity from 3 until 5 days. After 5 days this staining increased gradually. Myofiber types began differentiation by 15 days and were fully differentiated by 45 days. ATPase activity was barely evident by 1-3 days. This activity appeared uniformly low up to 5 days and increased to an intensity comparable with that of the typical myofiber by 15 days. Slight leucine aminopeptidase activity occurred in macrophages 1 day following injection. By 3 days this activity appeared in the remaining myoblasts and in the myotubes. Some activity was found in the fibroblasts. This staining intensity at 5 days was equal to that of earlier lesions. A trace of this activity was found at 7 days, and none at 15 days. Glucose-6-phosphate dehydrogenase activity was present in the macrophages by day 1. It increased by 3 days and occurred mainly in myoblasts and myotubes. This activity decreased by 5 days, and none was found by 7 days.     

The use of agar for gel electrophoresis of DNA

Agar can be used instead of agarose for electrophoresis of DNA. DNA restriction fragments migrate in proportion to the log of their molecular weights in the ranges studied. Bands of both restriction fragments and discrete small low molecular weight DNAs such as plasmids are sharp and clearly visible. The DNA can be Southern blotted with very low nonspecific background binding of radioactivity. Fragments can be removed from the gel and can be further restricted and ligated. Plasmid DNA retains its capacity to transform host bacterial cells. Agar is about $\text{1}\text{10}$ the cost of electrophoresis-grade agarose.     

Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.