Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man₆₍₇₎GlcNAc₂-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 M, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man₉GlcNAc₂-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man₅GlcNAc₂-R and Man₆GlcNAc₂-R. Man₇GlcNAc₂-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).     

Differential inhibition of indirect immunofluorescence in rat liver sections incubated with antibodies against microsomal cytochromes P-450a, b, and c and epoxide hydrolase

Rat liver sections were incubated with antibodies (100-1000 micrograms IgG/ml) against microsomal cytochromes P-450a, P-450b, and P-450c, and epoxide hydrolase. Inhibition of indirect immunofluorescence, which progressed with higher concentrations of primary antibody, corresponded with antigen-enriched tissue in frozen liver sections from male and female rats. It was found in liver sections from phenobarbital-treated rats incubated with anti-P-450b and anti-epoxide hydrolase and from 3-methylcholanthrene-treated rats incubated with anti-P-450c. No inhibition was found in sections from untreated rats or rats receiving treatments that did not induce the specific antigen. No inhibition was found in sections incubated with anti-P-450a. Inhibition of immunofluorescence was abolished in frozen sections subjected to dehydration-rehydration protocols known to extract antigens, and was prevented by certain solvents and detergent-wash. Inhibition of immunofluorescence provides a unique method for confirming the antigen-rich regions of the liver lobules specific for microsomal expoxide hydrolase and the cytochrome P-450s.     

Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.     

Influence of Seismic Stress on Photosynthetic Productivity, Gas Exchange, and Leaf Diffusive Resistance of Glycine max (L.) Merrill cv Wells II

Relative growth rate (RGR), leaf water potential (Ψw), transpiration rate (Tr), photosynthetic rate (Pn), and stomatal and mesophyll resistances to CO₂ exchange were measured or calculated to determine how periodic seismic (shaking) stress decreased dry weight accumulation by soybean (Glycine max [L.] Merrill cv Wells II). Seismic stress was applied with a gyratory shaker at 240 to 280 revolutions per minute for 5 minutes three times daily at 0930, 1430, and 1930 hours. Fifteen days of treatment decreased stem length 21%, leaf area 17%, and plant dry weight 18% relative to undisturbed plants. Seismic stress also decreased RGR 4%, which was due entirely to decreased net assimilation rate. Transpiration decreased 17% and leaf Ψw increased 39% 30 minutes after treatment. A reduction in Pn began within seconds after the onset of treatment and had declined 16% after 20 minutes, at which time gradual recovery began. Assimilation rate recovered fully before the next seismic treatment 5 hours later. Resistance analysis and calculation of leaf internal CO₂ levels indicated that the transitory decrease in Pn caused by periodic seismic stress was due to increased stomatal resistance on the abaxial leaf surface.     

Ferric iron reductase of Rhodopseudomonas sphaeroides.

Partially digested chromosomal DNA of Bacillus brevis ATCC 9999, a producer of the cyclic peptide antibiotic gramicidin S, was ligated into the BamHI site of the Escherichia coli expression vector pUR2-Bam. The ligated molecules were used to transfer E. coli to ampicillin resistance. Of 5 X 10(3) colonies tested by in situ immunoassay for a cross-reaction with antibodies against the gramicidin S synthetase 2, 6 colonies were found to be immunoreactive. A clone designated MK2, which had a 3.9-kilobase insert of B. brevis DNA, directed in E. coli under the lac promoter control the synthesis of polypeptides that were cross-reactive with the antibody to the gramicidin S synthetase 2. Partial purification of the gene products by gel filtration revealed a major fraction with an approximate molecular weight of 140,000 and with specific ornithine-dependent ATP-32PPi and 2'-dATP-32PPi exchange activities. These unique activities of the gramicidin S synthetase 2 were not detected in the E. coli strain harboring the vector.     

Prevalence of helminths in a cyclic snowshoe hare population

Five species of helminths were monitored in a population of snowshoe hares (Lepus americanus) near Rochester, Alberta, during 1961-1977. Prevalence of both Obeliscoides cuniculi and Protostrongylus boughtoni among young hares averaged about 50% by age 2 mo, then tended to level off. Prevalence of Taenia pisiformis (cysticerci) and Dirofilaria scapiceps rose more slowly, but continued to increase steadily beyond their mean levels of 8% and 1% at age 2 mo. There were well defined seasonal (within-year) cycles in prevalence of O. cuniculi and P. boughtoni that were generated evidently to a major degree by arrested development of larvae in fall and renewed development in late winter. It was hypothesized that renewed larval development was triggered (in February) in O. cuniculi by the seasonal rise of circulating pituitary gonadotrophins, and (in April) in P. boughtoni by the seasonal rise of gonadal androgens and estrogens. Indices to gonadal hormone levels in hares indicated that these increased most rapidly among males, and may have accounted for the higher prevalences of P. boughtoni in males during April-May. Neither T. pisiformis nor D. scapiceps exhibited conspicuous seasonal changes in prevalence. Maximum prevalence of T. pisiformis was attained at about 1 yr of age, whereas D. scapiceps increased among adult snowshoes through age 2 yr before stabilizing. Long-term (between-year) changes in prevalence of O. cuniculi, T. pisiformis, and D. scapiceps were correlated significantly with the cyclic hare population which declined from a peak in fall 1961 to a low in 1965-1966, rose to another peak by fall 1970, and declined again to a low in 1975. There was no detectable time lage between this "10-yr" cycle in hare density and the cycles of parasite prevalence among juveniles (less than 1 yr of age). Among adult hares, the cycle of O. cuniculi prevalence was likewise synchronous with that of the hare population, but the cycles of D. scapiceps and T. pisiformis lagged by approximately 1 and 2 yr, respectively. This lag in T. pisiformis prevalence was largely inexplicable to us. Our data on P. boughtoni were not suitable for analyses of between-year trends; nor were those for the fifth helminth, Taenia serialis (coenuri), because mean prevalence was less than 1% among both juveniles and adults. An apparent decline in T. serialis after the early 1950's, and its continued scarcity thereafter, paralleled a major change in numbers of one important definitive host--the red fox (Vulpes vulpes).(ABSTRACT TRUNCATED AT 400 WORDS)     

Caldesmon: A calmodulin — Binding actin — Regulatory protein

The protein caldesmon, originally isolated from smooth muscle tissue where it is the most abundant calmodulin-binding protein, has since been shown to have a wide distribution in actin- and myosin- containing cells where it is localized in sub-cellular structures concerned with motility, shape changes and exo- or endo-cytosis. Caldesmon is believed to be an actin- regulatory protein, and binds with high affinity to actin or actin-tropomyosin. Caldesmon inhibits the activation by actin-tropomyosin of myosin MgATPase activity, and the inhibition can be reversed by Ca2+.calmodulin. The binding of caldesmon to smooth muscle proteins has been studied in detail, enabling a model to be constructed which could account for the observed Ca2+ regulation of smooth muscle thin filaments. The abundance of caldesmon, and the Ca2+-regulation of its activity via calmodulin, mean that it is potentially an important intracellular regulator of processes such as smooth muscle contraction, cell motility and secretion.     

Aerobic ferrisiderophore reductase assay and activity stain for native polyacrylamide gels

The reduction of ferric iron from microbial iron-binding compounds (siderophores) releases the iron from the siderophore so that it may be utilized by the microorganism. A method to detect aerobic ferrisiderophore reductase activity using ferrozine as a ferrous iron trap is shown to be applicable to cytoplasmic fractions from Rhodopseudomonas sphaeroides and four other different species of bacteria. The ferrisiderophore reductase uses reduced nicotinamide cofactors as reducing agents, and activity is stimulated by flavins. This assay has been adapted as a staining method to locate ferrisiderophore reductase activity in native polyacrylamide gels.