NMR spectroscopy of 113Cd(II)-substituted gene 32 protein

Gene 32 protein (g32P), the single-stranded DNA binding protein from bacteriophage T4, contains 1 mol of Zn(II)/mol of protein. This intrinsic zinc is retained within the DNA-binding core fragment, g32P-(A+B) (residues 22-253), obtained by limited proteolysis of the intact protein. Ultraviolet circular dichroism provides evidence that Zn(II) binding causes significant changes in the conformation of the peptide chain coupled with alterations in the microenvironments of tryptophan and tyrosine side chains. NMR spectroscopy of the 113Cd(II) derivative of g32P-(A+B) at both 44.4 and 110.9 MHz shows a single 113Cd resonance, delta 637, a chemical shift consistent with coordination to three of the four sulfhydryl groups in the protein. In vitro mutagenesis of Cys166 to Ser166 creates a mutant g32P that still contains 1 Zn(II)/molecule. This mutant protein when substituted with 113Cd(II) shows a 113Cd signal with a delta and a line width the same as those observed for the wild-type protein. Thus, the S-ligands to the metal ion appear to be contributed by Cys77, Cys87, and Cys90. Relaxation data suggest that chemical shift anisotropy is the dominant, but not exclusive, mechanism of relaxation of the 113Cd nucleus in g32P, since a dipolar modulation from ligand protons is observed at 44.4 MHz but not at 110.9 MHz. Complexation of core 113Cd g32P with d(pA)6 or Co(II) g32P with poly(dT) shows only minor perturbation of the NMR signal or d-d electronic transitions, respectively, suggesting that the metal ion in g32P does not add a ligand from the bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mucosal epithelial cells and Langerhans cells are targets for infection by the immunosuppressive type D retrovirus simian AIDS retrovirus serotype 1

Simian acquired immune deficiency syndrome (SAIDS) caused by the type D retrovirus SRV-1 results in opportunistic infections and a spectrum of oral lesions similar to those seen in humans with AIDS. To better understand the pathogenesis of these oral lesions we have retrospectively examined the oral mucosa from ten rhesus monkeys that died with SAIDS and prospectively examined the oral mucosa of ten additional animals inoculated with SRV-1 to determine at what time, and in what cells SRV-1 infection of the oral mucosa occurs. Using single and double label immunohistologic techniques, and electron microscopy we detected SRV-1 in clusters of oral epithelial cells and rare Langerhans cells as early as 1 month postinoculation.     

Motility response of Rhodobacter sphaeroides to chemotactic stimulation.

Tethered rotating cells of Rhodobacter sphaeroides varied widely in their stopping frequency; 45% of cells showed no stops of longer than 1 s, whereas others showed stops of up to several seconds. Individual cells alternated between stops and rotation at a fairly constant rate, without continuous variation. Addition of the chemoattractant propionate to free-swimming cells of R. sphaeroides increased the mean population swimming speed from 15 to 23 microns s-1. After correction for nonmotile cells, the percentage swimming at less than 5 microns s-1 dropped from approximately 22 to 8, whereas the percentage swimming at greater than 50 microns s-1 increased from 6 to 15. However, cells already swimming did not swim faster after propionate addition; the increase in the mean population speed after propionate addition was caused by an increase in the mean run length between stops from 25 to 101 microns. The increased run length was the result of a drop in both the stopping frequency and the length of a stop. Addition of propionate over the range of 10 microM to 1 mM decreased the stopping frequency; this decrease was almost entirely blocked by benzoate, a competitive inhibitor of propionate transport. The chemoattractants acetate and potassium had the same effect as propionate on the distribution of stopping frequency, which demonstrated that this is a general behavioral response to chemotactic stimulation. Adaptation to propionate stimulation was slow and very variable, cultures frequently showing little adaptation over 30 min. This characteristic may be the result of the lack of a highly specific chemosensory system in R. sphaeroides.     

Seleno-DL-methionine and the protection of human platelets against freezing injury

The effect of seleno-DL-methionine, which has antioxidative properties, on the recovery of human platelets after freezing with 0.5 mol/liter glycerol or 0.7 mol/liter (5% v/v) dimethyl sulfoxide was investigated. Incubation of platelets with 2 mumol/liter seleno-DL-methionine for 30 min at room temperature before equilibration with the protective additives improved the post-thaw uptake of 5-hydroxytryptamine and the percentage reversal in the hypotonic stress test. The effect was small, but in view of the ability of seleno-DL-methionine to inhibit lipid peroxidation in membranes, the results suggest that oxidative damage is implicated in freezing injury. The dimethyl sulfoxide protocol apparently afforded greater protection to the platelets than the glycerol protocol. But, if the platelets were incubated for 24 hr at 37 degrees C after thawing, there was a marked improvement in the response of cells in the hypotonic stress test, particularly in the samples frozen with glycerol, and there was no longer any difference between the two additives. There was, however, a concomitant loss of almost half the number of cells in the thawed suspensions during the prolonged incubation at 37 degrees C.     

m-Fluorotyrosine substitution in beta-galactosidase; evidence for the existence of a catalytically active tyrosine

The pH profiles of beta-galactosidase, having tyr replaced by m-fluorotyrosine, were compared to those of normal enzyme. The inflection point on the alkaline side was lowered about 1.5 pH units in the fluoro-enzyme, corresponding to the difference in the phenolic pKa values of m-fluorotyrosine and tyr. When glycosidic bond breakage was rate-limiting, the Vm at pH 7.0 was higher for the fluoro-enzyme. When hydrolysis was rate-limiting or when acceptors which made transgalactosylis rate-limiting were used, the Vm was lower for the fluoro-enzyme. This shows that a tyr in beta-galactosidase is a general-acid catalyst in the glycosidic bond breaking reaction and a tyr (probably the same one) is a general-base catalyst in the hydrolytic reaction.     

Location of polar substituents and fatty acyl chains on lipid A precursors from a 3-deoxy-D-manno-octulosonic acid-deficient mutant of Salmonella typhimurium. Studies by 1H, 13C, and 31P nuclear magnetic resonance

Eight anionic disaccharide precursors of lipid A accumulate at 42 degrees C in 3-deoxy-D-manno-octulosonic acid-deficient temperature-sensitive mutants of Salmonella typhimurium. These compounds comprise a series of lipids based on the minimal structure, O-[2-amino-2-deoxy-N2,O3-bis(3-hydroxytetradecanoyl)-beta-D-glucopyranos yl] -(1----6)-2-amino-2-deoxy-N2, O3-bis(3-hydroxytetradecanoyl)-alpha-D-glucopyranose 1,4'- bisphosphate (designated lipid IVA) that differ from each other by the presence of an additional phosphoethanolamine moiety (IIIA), or an aminodeoxypentose moiety (IIA), or both (IA). A homologous set of metabolites is further derivatized with a palmitoyl function; these are designated IVB, IIIB, IIB, and IB (Raetz, C. R. H., Purcell, S., Meyer, M. V., Qureshi, N., and Takayama, K. (1985) J. Biol. Chem. 260, 16080-16088). The attachment of the palmitoyl moiety, known to be on the reducing terminal GlcN residue by mass spectrometry, was determined to be O-beta of the N2-linked beta-hydroxymyristoyl group of that residue of IVB by 13C NMR and two-dimensional 1H chemical shift correlation spectroscopy experiments. 31P NMR indicated the presence of diphosphodiester moieties in IIIA, IIIB, and IA and monophosphodiester moieties in IIA and IA. Selective 1H decoupling of the 31P spectrum of IIIA demonstrated that the O-diphosphoethanolamine moiety is attached to the O4' position in IIIA. On the basis of the observed 31P chemical shifts it was concluded that the aminodeoxypentose is located at position 1 in IIA and IA, while diphosphoethanolamine is most likely located at O-4' in IA and IIIB, as in IIIA.     

The effect of three species of mites upon fungal growth on wheat

Every week, for 20 weeks, the growth of naturally occurring grain storage fungi on wheat infested with the three commonest British grain storage mites,Acarus siro, Glycyphagus destructor andTyrophagus longior, was compared with that on uninfested wheat.The number of colonies of theAspergillus glaucus group per gram were always less on grain infested with mites than on uninfested grain.Penicillium spp. were also less numerous on grain which was infested withA. siro but did not appear to be affected by the other mites. In contrast, two fungi which are pathogenic to mites,Aspergillus restrictus andWallemia sebi, were more abundant in the presence of certain mites. The former was associated withG. destructor, the latter withG. destructor andA. siro.The three species of mites either feed on theA. glaucus group andPenicillium spp., or inhibit them by an unknown secretion. Pathogenic fungi are probably avoided. Mites are therefore an important variable in studies on fungal growth during grain drying and storage.     

Population structure of some pond Microcrustacea

Summary The aestival species composition of microcrustacea of eight ponds in southwestern Kansas is described. Two benthic Ostracoda and three planktonic Copepoda and eight planktonic Cladocera were collected.On the whole, the Copepoda were more abundant than the Cladocera. Patterns of relative abundance varied from week to week and were more varied than the patterns described for the limnetic zone of lakes. There were two or three copepods in each pond. Except for one pond which had no Cladocera, there were two to five species of Cladocera per pond.There were fourteen significant affinities among the plankters. Two species showed no affinity. The relationships among four recurrent groups were established. Group I contained one cyclopoid, one calanoid and two cladocerans, a pattern not unlike the typical limnetic community.The population structure of each species that occurred in more that one pond was analyzed by analysis of variance. Significant differences between the ponds occurred from various species in per cent copepodids in the population, per cent females with eggs, and the number of eggs per egg sac. There was no significant difference among the ponds regarding the mean number of eggs per ovigerous female for any cladoceran.Interactions among the species and faunal characteristics of the ponds are discussed in relation to the environmental characteristics of the ponds.

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Zusammenfassung Die Zusammensetzung der estivalen Arten von Mikrocrustacean von acht Teichen im Südwesten von Kansas wird beschrieben. Zwei benthische Ostracoden, drei planktonische Copepoden und acht planktonische Cladoceren wurden gefunden.Die Copepoden waren im allgemeinen häufiger als die Cladoceren. Die Schemen der relativen Anzahl wechselten von Woche zu Woche und waren unterschiedlicher als die Schemen für die limnische Seezone. Zwei oder drei Copepoden, sowie zwei bis fünf Arten von Cladoceren kamen in jedem Teich vor, ausser in einem Teich, in dem keine Cladoceren gefunden wurden.Vierzehn wichtige Übereinstimmungen wurden unter den Planktonern gefunden. Zwei Arten zeigten keine Affinität. Der Zusammenhang zwischen vier wiederkehrenden Gruppen wurden nachgewiesen. Gruppe I enthielt einen Cyclopoiden, einen Calanoiden und zwei Cladoceren, ein für typisch limnische Gemeinschaften übliches Schema.Die Bevölkerungsstruktur jeder Art, die in mehr als einem Teich vorkam, wurde durch Streuungsanalyse untersucht. Es wurden signifikante Unterschiede im Prozentsatz der Copepoden: Bevölkerung, der Weibchen mit Eiern, der Anzahl Eier im Brutraum für die verschiedenen Arten zwischen den Teichen gefunden. Keine signifikanten Unterschiede wurden in der Anzahl Eier pro eitragendem Weibchen der Cladoceren zwischen den Teichen gefunden.Auch der Einfluss der Umweltfaktoren der Teiche auf die Arten und auf die Fauna des Teiches wurde untersucht.

     

Oxygen consumption and body temperature in yellow-bellied marmot populations from montane-mesic and lowland-xeric environments

Summary Yellow-bellied marmots characteristically live in montane-mesic environments, but in several areas in western North America, this species extended its range into lowland-xeric habitats. Body mass was significantly smaller in the lowland-xeric population from eastern Washington at 393 m than in the montane-mesic population from western Colorado at 2900 m. Oxygen consumption of marmots from montane-mesic and lowland-xeric environments was signiflcantly affected by ambient temperature (T_A) water regimen, population, and a population x water regimen x temperature interaction. Lowland-xeric animals had a higher metabolic rate at low T_As, but a lower metabolic rate at higher T_As than the montane-mesic aminals. Oxygen consumption was lower on a restricted-water regimen than on ad libitum water in both populations. Coefficients relating oxygen consumption to body mass were affected by T_A, water regimen, and population. These intraspecific coefficients are larger than the interspecific coefficients for all mammals. Body temperature (T_B) was affected significantly by T_A, water regimen, and population. T_A body mass, and a population x water regimen interaction significantly affected conductance. Conductance generally was higher in the lowland-xeric than in the montane-mesic marmots. Both populations increased conductance at high T_A, but the lowland-xeric population dissipated a much higher proportion of the heat by evaporative water loss (EWL) than did the montane-mesic population. Metabolic water production exceeded or equaled EWL at 5–20°C. Smaller body size, reduced metabolism at high T_A, and increased EWL at high T_A characterized the lowland-xeric population.Metabolic rates of yellow-bellied marmots were higher than predicted from body size during the reproductive season but decreased to 67% of that predicted from the Kleiber curve by late summer. Marmots minimize thermoregulatory costs by concentrating activity at times when the microclimate is favorable, by tolerating hyperthermia at high T_A in the field, and by having a conductance lower than that predicted from body size.Abbreviations DHC dry-heat conductance - EHL evaporative heat loss - EWL evaporative water loss - HP heat produced - T _A ambient temperature - T _n body temperature - M body mass