Carbon source transport, nucleotide levels, and stable RNA synthesis in a mutant strain of Escherichia coli

We have previously described a temperature-sensitive mutant of Escherichia coli, 2S142 (rel-, met-, rns-, ilv-, ts-) which shows specific inhibition of stable RNA synthesis at 42 degrees C. This mutation mimics a carbon source downshift in that the decay of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) is inhibited at the restrictive temperature. In this paper we show that the temperature-sensitive lesion in 2S142 does affect the uptake of glucose or alpha-D-methylglucopyranoside (alpha DMG) at 42 degrees C. However, restoration of glucose or alpha DMG uptake by the insertion of a constitutive galactose permease gene or further restriction of glucose uptake by insertion of a ptsG mutation into 2S142 have no effect on rRNA synthesis at 42 degrees C (although ppGpp levels are lowered in both cases). Furthermore, while restriction of uptake at 42 degrees C varies widely from carbon source to carbon source, severe restriction of rRNA synthesis is observed on all carbon sources tested at 42 degrees C. Levels of glycolytic intermediates, adenylate energy charge, ATP levels, and cAMP levels are all unaffected at the restrictive temperature. GTP levels decrease at 42 degrees C in glucose grown cells but that also does not appear to be related to the decrease in rRNA synthesis. These data were interpreted to suggest that the restriction of stable RNA synthesis in 2S142 at 42 degrees C can not be explained on the basis of decreased uptake and/or metabolism of carbon source. "Phantom spot" levels do decrease in 2S142 at 42 degrees C. In fact, "phantom spot" is the only putative regulatory molecule which correlates with restriction of rRNA synthesis on all carbon sources tested.     

Estimation of the coancestry coefficient: basis for a short-term genetic distance

A distance measure for populations diverging by drift only is based on the coancestry coefficient θ, and three estimators of the distance D = -ln(1 - θ) are constructed for multiallelic, multilocus data. Simulations of a monoecious population mating at random showed that a weighted ratio of single-locus estimators performed better than an unweighted average or a least squares estimator. Jackknifing over loci provided satisfactory variance estimates of distance values. In the drift situation, in which mutation is excluded, the weighted estimator of D appears to be a better measure of distance than others that have appeared in the literature.     

Autoradiographic and cytochemical studies of phagocytic cells in selected fibre tracts of the mouse periodontium

Proliferative and protein synthetic activities of phagocytic cells of specific fibre tracts of the periodontium of C57Bl mice were employing autoradiographic techniques; these were combined with a histochemical technique for horseradish peroxidase (HRP) as a marker for phagocytic activity. Animals were injected either with [3H]thymidine as a marker for proliferative activity, or with [3H]proline as a marker for protein synthetic activity prior to HRP injection. Blocks from the maxillae of experimental and control animals were fixed, decalcified, and sectioned at 50 micrometers. These were incubated with HRP localization media, dehydrated and flat embedded in Epon 812 wafers. The entire length of the periodontium, including adjacent tooth and bone, were selectively cut from the wafers, mounted on epoxy blocks and serially sectioned at 2 micrometers. Slides containing these sections were then dipped in NTB-3 nuclear track emulsion, and after appropriate exposure times, were developed and post-stained. Sections were examined microscopically, employing an ocular grid, and phagocytic cells within each area examined were delineated as either 'fibroblast-like' (FL cells) or 'endothelial/macrophage-like' (EML cells) according to criteria such as morphology, location, orientation and proximity to a vascular channel. They were then subclassified as labelled or unlabelled with respect to the autoradiographic markers. The thymidine labelling index obtained for non-phagocytic FL cells was 3.09%; this was more than twice that for phagocytic FL cells (1.35%). Similarly phagocytic FL cells in all regions studied incorporated less than half as much [3H]proline as did their non-phagocytic counterparts. This was determined by silver grain counts over HRP-stained and unstained cells using a matched pair system. In addition, the variation of the relative number of phagocytic FL cells in specific fibre tracts suggested a relationship to functional demand. The distribution of these cells was closely related to experimentally determined rates of protein turnover. Phagocytic FL cells have a markedly reduced proliferative rate and synthesize proline-containing proteins at a reduced rate. This may reflect protein production primarily for the purpose of cell maintenance. These findings are consistent with the presence of subpopulations of fibroblasts (or fibrocytes) developmentally or functionally modified for phagocytosis; alternatively, this could signify modulation of fibroblasts from primarily biosynthetic activities to degradative functions in response to varying microenvironmental conditions.     

Insulin's structural behavior and its relation to activity

This paper discusses the hypothesis that insulin undergoes a conformational change either before or during its binding to the receptor. The evidence for this is not conclusive but allows us to reconcile the following observations: (1) no chemical modification or deletion of invariant surface residues has abolished the hormone's activity—only reduced its potency. (2) Reduction in potency follows many modifications to different side chains, both variant and invariant. (3) There are insulins with perfectly preserved structure (by the criteria of aggregation, spectroscopy, and x-ray analysis) that have markedly reduced potency. (4) Insulins with disturbed structure still exhibit real, sometimes substantial activity.     

Bursicon in the mealworm, Tenebrio molitor L. and its role in the control of postecdysial tanning

Using the adult Calliphora bioassay, we found that the tanning hormone, bursicon, is present in the blood of pupal and adult Tenebrio only at the time of ecdysis, when it is released massively from the thoracic and abdominal central nervous system. The hormone's half life in the blood is short (about 1–2 h). Contrary to the findings of other workers, we could find no evidence for the presence of the hormone in the haemolymph during pharate adult development, before ecdysis begins. When newly ecdysed pupae were ligated about the neck, adult development of the thorax and abdomen proceeded normally, but postecdysial tanning of the adult cuticle was almost completely prevented. This failure to tan was not due to lack of bursicon as the hormone was released normally in the ligated animals at the time of ecdysis. This suggests that a pre-ecdysial signal may be required for the development of epidermal competence to respond to bursicon.     

Effect of acidosis on glutamine transport by isolated rat renal brush-border and basolateral-membrane vesicles.

Glutamine uptake was examined in isolated renal brush-border and basolateral-membrane vesicles from control and acidotic rats. In brush-border vesicles from acidotic animals, there was a significant increase in the initial rate of glutamine uptake compared with that in controls. Lowering the pH of the medium increased the initial rate of glutamine uptake in brush-border vesicles from acidotic, but not from control, rats. In brush-border vesicles from both groups of animals, two saturable transport systems mediated glutamine uptake. There was a 2-fold increase in the Vmax. of the low-affinity high-capacity system in the brush-border vesicles from the acidotic animals compared with that from control animals, with no alteration in the other kinetic parameters. There was no difference in glutamine uptake by the two saturable transport systems in basolateral vesicles from control and acidotic animals. Lowering the incubation-medium pH increased the uptake of glutamine by basolateral vesicles from both control and acidotic rats to a similar extent. The data indicate that during acidosis there are alterations in glutamine transport by both the basolateral and brush-border membrane which could enhance its uptake by the renal-tubule cell for use in ammoniagenesis.     

ERYTHROID CELL DIFFERENTIATION AND THE INHIBITION OF CYTOKINESIS BY CYTOCHALASIN

Cytochalasin B produces multinucleated erythroid cells in tissue cultures of very young chick blastoderms. There is no apparent qualitative interference with differentiation and maturation of erythroid cells, but the amounts produced are reduced 4- and 10-fold. These effects of cytochalasin are readily reversible.     

Biology of the small pelagic fishes in the New Volta Lake in Ghana. Part III: Sex and reproduction

Reproductive patterns of five species, from three families, of small pelagic fishes in the new Volta Lake in Ghana showed several similar features. These included sex size defferences, males maturing and reaching final sizes smaller than females; heterogenous sex ratios in four species, being most markedly unequal usually at peak breeding times; demersal eggs; and extended fractional spawning patterns. The clupeids Pellonula and Cynothrissa and the cyprinid Barilius all breed in the dry season, whereas the schilbeids Physailia and Siluranodon are wet-season spawners. Only Pellonula and Physailia have extended their breeding season in the new lake to cover most of the year; the other species have not, although Siluranodon spawns into the early dry season. In the lake environment Pellonula's pelagic larva is no longer endangered by seasonal floods, whereas Physailia can use the lake's continuous “high waters” for extended breeding. Relaxation of breeding patterns and protracted fractional spawning has allowed best exploitation of the new regime.     

Binding of β-lactam antibiotics to the exocellular dd-carboxypeptidase–transpeptidase of Streptomyces R39

Benzylpenicillin and cephaloridine reacted with the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R39 to form equimolar and inactive antibiotic–enzyme complexes. At saturation, the molar ratio of chromogenic cephalosporin 87-312 to enzyme was 1.3:1, but this discrepancy might be due to a lack of accuracy in the measurement of the antibiotic. Spectrophotometric studies showed that binding of cephaloridine and cephalosporin 87-312 to the enzyme caused opening of their β-lactam rings. Benzylpenicillin and cephalosporin 87-312 competed for the same site on the free enzyme, suggesting that binding of benzylpenicillin also resulted in the opening of its β-lactam ring. In Tris–NaCl–MgCl₂ buffer at pH7.7 and 37°C, the rate constants for the dissociation of the antibiotic–enzyme complexes were 2.8×10⁻⁶, 1.5×10⁻⁶ and 0.63×10⁻⁶s⁻¹ (half-lives 70, 130 and 300h) for benzylpenicillin, cephalosporin 87-312 and cephaloridine respectively. During the process, the protein underwent reactivation. The enzyme that was regenerated from its complex with benzylpenicillin was as sensitive to fresh benzylpenicillin as the native enzyme. With [¹⁴C]benzylpenicillin, the released radioactive compound was neither benzylpenicillin nor benzylpenicilloic acid. The Streptomyces R39 enzyme thus behaved as a β-lactam-antibiotic-destroying enzyme but did not function as a β-lactamase. Incubation at 37°C in 0.01m-phosphate buffer, pH7.0, and in the same buffer supplemented with sodium dodecyl sulphate caused a more rapid reversion of the [¹⁴C]benzylpenicillin–enzyme complex. The rate constants were 1.6×10⁻⁵s⁻¹ and 0.8×10⁻⁴s⁻¹ respectively. Under these conditions, however, there was no concomitant reactivation of the enzyme and the released radioactive compound(s) appeared not to be the same as before. The Streptomyces R39 enzyme and the exocellular dd-carboxypeptidase–transpeptidase from Streptomyces R61 appeared to differ from each other with regard to the topography of their penicillin-binding site.