Improved procedure for the isolation of a double-strand-specific ribonuclease and its application to structural analysis of various 5S rRNAs and tRNAs

An improved method for the isolation of a double-strand-specific RNase from snake venom is presented. This RNase, called CSV, was used to cleave yeast tRNAPhe and tRNA2Glu and tRNAfMet from Escherichia coli. In addition these RNAs and E. coli tRNAPhe were examined with the single-strand-specific nuclease S1. The results are discussed in terms of the specificity of CSV RNase and the structure of tRNAs. S1 nuclease digestions at increasing temperatures allowed the melting of tertiary and secondary structure to be monitored. 5S rRNA from E. coli, Thermoplasma acidophilum and the chloroplasts of Spinacia oleracea were digested with CSV and S1. The information these results give on the secondary-structural differences between different classes of 5S rRNA are discussed. Supporting evidence is found for tertiary interactions between hairpin loop c and internal loop d of eubacterial 5S rRNA.     

Changes in the cell wall of Clostridium species following passage in animals

Morphological changes in clostridial isolates after animal passage with other flora in mixed infections were studied by utilizing a subcutaneous abscess model in mice. We used 26 isolates of 7 clostridial species, and one isolate each of Bacteroides fragilis and Klebsiella pneumoniae. Abscesses were induced by all 7 Clostridium perfringens and 3 Clostridium butyricum isolates and by some of the other isolates. A thick granular wall prior to animal inoculation was shown only in C. perfringens, C. butyricum, and C. difficile. This structure was observed in other clostridia only following their animal passage alone or when co-inoculated with K. pneumoniae or B. fragilis.     

Activity of maize leaf phosphoenolpyruvate carboxylase in relation to tautomerization and nonenzymatic decarboxylation of oxaloacetate

The keto form of oxaloacetate (OAA), a product of phosphoenolpyruvate carboxylase (PEPC) activity, can undergo various nonenzymatic conversions which make conventional methods of assaying the enzyme difficult, because the products may either act as inhibitors or go undetected. In studies with PEPC isolated from leaves of maize, an assay coupled with reduction of OAA to malate was compared with product analysis using high-performance liquid chromatography and an assay based on Pi release. The results show that activity of the enzyme in the assay coupled to malate dehydrogenase is underestimated, to varying extents, depending on magnesium concentration, buffer, and pH. In the assay coupled to malate dehydrogenase, inaccuracies occur due to conversion of the keto form of OAA to the enol form, which is not utilized as a substrate, and due to loss of OAA by decarboxylation to pyruvate. The assay based on Pi formation is considered to give the true rate of catalysis. With this assay the pH optimum is 7.8, compared to 8.3-8.5 for the assay coupled to malate dehydrogenase. The metal enol complex of oxaloacetate (M-OAAenol) is an inhibitor of PEPC and conditions which are favorable for forming this tautomer, high pH with divalent metal ions or high concentrations of Tris buffer at a pH below its pKa value, limit catalysis. Glycine stimulates enzyme activity, and it may have its effect by preventing the formation of the hydrated M-OAAenol complex and maintaining more of the OAA in the keto form. This interpretation is consistent with glycine stimulation of malate synthesis in the assay of PEPC coupled to malate dehydrogenase, with glycine stimulation of the decarboxylation of OAA, and with a reduction in the level of the M-OAAenol complex in the presence of glycine.     

Rotavirus-associated diarrhea in a commercial rabbitry

An epizootic of diarrheal disease occurred in a commercial specific-pathogen-free rabbitry, and was characterized by sudden onset, rapid spread, and high morbidity and mortality among sucklings. Affected rabbits rapidly became dehydrated and most died within two days of the onset of diarrhea. Eight of these rabbits were necropsied. Five had blunted and fused small intestinal villi with attenuated villous enterocytes. A rotavirus was isolated from four rabbits, and five survivors of affected litters had strong antibody responses to rotavirus.     

Defect in expression of heat-shock proteins at high temperature in xthA mutants.

Escherichia coli mutants lacking exonuclease III (xthA) are defective in the induction of heat-shock proteins upon severe heat-shock treatment (upshift from 30 to 50 degrees C) but not mild heat-shock treatment (upshift from 30 to 42 degrees C). We show that this defect is due to the xthA mutation by complementation. Furthermore, increasing the gene dosage of xthA+ prolongs the synthesis of heat shock proteins seen after a shift to 42 degrees C. Increasing the gene dosage of htpR+ partially suppresses the defect of xthA mutants in the synthesis of heat-shock proteins at 50 degrees C. When an xthA strain was incubated at 42 degrees C before a shift to 50 degrees C, it was then able to carry out the synthesis of heat-shock proteins at 50 degrees C.     

Thermoregulation, metabolism, and stages of sleep in cold-exposed men

Four naked men, selected for their ability to sleep in the cold, were exposed to an ambient temperature (Ta) of 21 degrees C for five consecutive nights. Electrophysiological stages of sleep, O2 consumption (VO2), and skin (Tsk), rectal (Tre), and tympanic (Tty) temperatures were recorded. Compared with five nights at a thermoneutral Ta of 29 degrees C, cold induced increased wakefulness and decreased stage 2 sleep, without significantly affecting other stages. Tre and Tty declined during each condition. The decrease in Tre was greater at 21 degrees C than at 29 degrees C, whereas Tty did not differ significantly between conditions. Increases in Tty following REM sleep onset at 21 degrees C were negatively correlated with absolute Tty. VO2 and forehead Tsk also increased during REM sleep at both TaS, whereas Tsk of the limb extremities declined at 21 degrees C. Unsuppressed REM sleep in association with peripheral vasoconstriction and increased Tty and VO2 in cold-exposed humans, do not signify an inhibition of thermoregulation during this sleep stage as has been observed in other mammals.     

The role of encapsulation in the pathogenesis of anaerobic gram-positive cocci

The pathogenicity of 22 anaerobic and facultative Gram-positive cocci (AFGPC) was investigated by inoculating them into mice and determining their ability to cause subcutaneous abscesses. Only 11 heavily encapsulated isolates (greater than 50% of the cells were encapsulated) induced abscesses. However, when the other 11 isolates were injected with Bacteroides sp. or facultative and aerobic bacteria, abscesses were formed in 8 of the 11 combinations. The AFGPC recovered from the mixed infections contained many encapsulated cells. Encapsulation also occurred in cocci injected with capsular material or with Formalin-killed cells of Klebsiella pneumoniae or capsule-positive Bacteroides sp. After acquisition of capsules, these strains could induce abscesses on reinoculation in mice.     

Yolk polypeptide gene expression in Minute Drosophila females

Minutes have been considered for some time to be mutant at the sites of synthesis of some components of the protein synthetic apparatus. To study the hypothetical relationship between Minutes and suboptimal translation, a group of abundant proteins, the yolk polypeptides, was assayed in outcrossed females bearing M(3)w, M(3)h ^y , or M(1)n mutations. Recently emerged Minute females contained a lower amount of yolk polypeptides, in both ovarian and nonovarian tissues, than their non-Minute sisters. This low level correlated with the lower abundance of cytoplasmic RNA in Minutes compared to control females. By 1 week of age, both M(3)w and their non-Minute sibs contained the same amount of yolk polypeptides and the corresponding mRNA. The double heterozygote, ap ⁴/+;M(3)w/+, did not differ in yolk polypeptide content from control flies. M(3)w females demonstrated reduced fecundity during the period of low yolk polypeptide content but gradually increased egg deposition as yolk polypeptide levels rose. These results suggest that the low protein levels are due to the slower maturation of M(3)w, and not to less efficient translation machinery.This work was supported by the NSERC (Canada) and a Queen's University ARC grant.     

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gk^a and Gk^b, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent K _m values for MgATP^2– and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.This work has been supported in part by a grant from the British Diabetic Association and a Training Studentship to PAJ from the Medical Research Council (U.K.).     

The skeleton of early Eocene Cantius, oldest lemuriform primate

A recently discovered partial skeleton of the adapid Cantius trigonodus from the early Eocene Willwood Formation of the Bighorn Basin, Wyoming, documents substantial new information about the anatomy of the oldest lemuriform primates. It is very similar in all features to its descendant, middle Eocene Notharctus, and both exhibit numerous resemblances to certain extant Malagasy lemurs, particularly Lepilemur, Propithecus, Lemur, and Hapalemur griseus. Like these forms, Cantius had relatively long hind limbs and short forelimbs. Forelimb traits (prominent brachialis flange of the humerus, well-developed olecranon process of the ulna, and strong shafts of the ulna and radius) suggest active use of the forelimbs in progression. Specializations in the hind limb (e.g., expanded articular surface of the femoral head, narrow and elevated patellar trochlea and prominent lateral trochlear ridge, posteriorly oriented femoral and tibial condyles, narrow and elongate talus, and hallucal metatarsal with prominent peroneal tubercle) indicate capabilities for leaping and for powerful grasping with an opposable hallux. Cantius was presumably primitive in having a relatively long ischium and much more distal inferior tibial tuberosity than most extant lemurs--traits suggesting that powerful extension of the thigh and flexion at the knee were important in its locomotion and posture. We interpret Cantius as an active arboreal quadruped with a propensity for leaping. The existence of this skeletal structure in one of the oldest primates of modern aspect suggests that it represents the primitive lemuriform morphology.