Cloning, nucleotide sequence and expression of a hemolysin gene of Clostridium septicum

Abstract A genomic library of Clostridium septicum NCTC547 strain was made in Escherichia coli by means of λgt10. The DNA insert of a hemolysin-positive (Hly⁺) λ-clone was transferred into pUC19. The resulting plasmid, pCS21, confers a Hly⁺ phenotype on E. coli . Crude lysates of E. coli (pCS21) possessed a strong lytic activity on human erythrocytes and also a lethal effect on mice, characteristic of an α toxin. Nucleotide sequence analysis revealed that the insert DNA (5.2 kb) in pCS21 included at least one open reading frame of 1380 bp. The coding frame for hemolysin was predicted to be 1329 bp in size and to encode a protein of 49.8 kDa. It coincided with the molecular mass (48 kDa) of the α toxin secreted by C. septicum . Taken together, the data indicated that plasmid pCS21 indeed encoded an α toxin gene of C. septicum .     

Production and reproductive function of intercastes inMyrmecina gvaminicola nipponica colonies (Hymenoptera: Formicidae)

Summary Many females morphologically intermediate between queens and workers were found in a northernmost population ofMyrmecina graminicola nipponica Wheeler. Dissection and morphological observation revealed that there were three categories of intercastes. Major intercastes were as large as queens in body size, with seven or more ovarioles, but had only one ocellus, unlike queens, which had three ocelli. Medium intercasts had an enlarged mesonotum, one or no ocellus and 2 to 12 ovarioles. Minor intercaste was very simlar to workers in external morphology, but had a spermatheca, unlike workers. Inseminated females constituted 75%, 40% and 28.6% in the major, medium and minor intercastes respectively. Many of the virgin medium and minor intercastes had a small disfunctional spermatheca.In queenright colonies, a single queen was inseminated and had an active ovary. In queenless colonies where the intercastes reproduced, however, some colonies were functionally monogynous, but the others polygynous. The ratio of polygynous colonies to monogynous colonies was lowest in July and highest in September, suggesting that polygyny results from newly inseminated intercastes remaining in their natal nests, although they leave those nests in the season of colonial budding. Queenless colonies containing inseminated intercastes exclusively produced intercastes, while queenright colonies almost exclusively produced queens.     

Analysis of feeding mechanism in a pitcher ofNepenthes hybrida

The process of digestion of captured feeds in a pitcher, an insect-trapping organ, ofNepenthes was studied. Changes in bacterial population, pH and NH₄ ⁺ concentrations in pitcher juice were examined. Strong activities of both acid- and alkaline phosphatase, phosphoamidase, esterase C4 and esterase C8 were found in the pitcher juice. Optimum pH of proteases in the juice and those secreted from bacteria showed pH 3.0 and pH 8.0–9.0, respectively. Twenty six strains of bacteria were isolated from 4 pitchers: 10 strains were gram positive, 16 strains were gram negative (10 strains had casein hydrolase activity). A proton excretion was induced by NH₄ ⁺ released from the added solutions, and accordingly, the pH of the solutions fell. As a simulation model of the digestion process of feeds in pitcher juice and polypeptone solution was added into the washed pitcher. A good correlation was found among the NH₄ ⁺ concentration, pH and bacterial cell titer.     

Competitive habitat utilization in the damselfly,Mnais nawai (Zygoptera: Calopterygidae) coexisting with a related species,Mnais pruinosa

Reproductive behaviors related to habitat utilization were studied in males of the damsefly,Mnais nawai, which has two male forms, territorial orange-winged males (nawai) and non-territorial pale-orange-winged males (sahoi), at the upper part of a mountain stream where they partiallycoexist with a related species,Mnais pruinosa, which also has two male forms, territorial orange-winged males (esakii) and non-territorial hyaline-winged males (strigata). These two species showed parapatric distribution; the lower part of the stream was occupied byM. nawai, and the upper part byM. pruinosa. In the present study, cross-matings occurred between bothMnais species, although normal intraspecific matings occurred more frequently than cross-matings. Territorial males of both species copulated with conspecific females that entered their territory and guarded the ovipositing females, probably to avoid sperm displacement resulting from subsequent copulations. Severe competition for oviposition sites by territorial males even occurred between the two species. On the other hand, non-territorial males of both species have alternative mating strategies (including several tactics such as sneaking, takeover and interception). The possible benefits from conflict among territorial males of both species is discussed.     

Inhibition of ATPase activity of the recA protein by ATP ribose-modified analogs

The single-stranded, DNA-dependent ATPase activity of purified recA protein was found to be inhibited competitively by ribose-modified analogs of ATP, 3'-O-anthraniloyl-ATP (Ant-ATP), and 3'-O-(N-methylanthraniloyl)-ATP (Mant-ATP). The Ki values for Ant-ATP and Mant-ATP were around 7 and 3 microM at pH 7.5, respectively. The inhibitions by these analogs were much stronger than that by ADP, which is also a competitive inhibitor for the ATPase activity of the recA protein. The Ki value for ADP is 76 microM. Ant-ATP and Mant-ATP reduced the Hill coefficient for ATP hydrolysis and thus contributed to the cooperative effect of ATP.     

A new factor from enteric bacteria of rats amplifying induction of liver enzyme by glucocorticoid. 2. Mechanism of action.

1. An amplifier of the action of glucocorticoid was purified from Proteus mirabilis as described previously. It was found that it amplified the induction of liver tyrosine aminotransferase by dexamethasone markedly with doses of dexamethasone that caused minimal enzyme induction, but had little effect with doses that caused maximal induction. Thus the amplification may represent a saving of glucocorticoid. The amplification of enzyme activity was brought about by increase in amount of enzyme. 2. The amplification was observed when the amplifier was administered before or with dexamethasone, but not when it was given 2 h after dexamethasone. These results and the finding that actinomycin D inhibited the amplification indicate that the amplifier does not act on the translational level of enzyme induction. 3. It was found that the amplifier increased both incorporation of [3H]dexamethasone into the cytosol and binding of [3H]dexamethasone of cytosol protein and that it decreased decay of the [3H]dexamethasone-protein complex.     

Antigen of "serum sickness" type of heterophile antibodies in human sera: indentification as gangliosides with N-glycolylneuraminic acid.

Antigen of “serum-sickness” type of heterophile antibodies in pathologic human sera was purified from equine and bovine erythrocyte stroma. The chemical nature of this antigen was glycosphingolipids with N-glycolylneuraminic acid. The antigen of equine erythrocytes was identified as hematoside with N-glycolylneuraminic acid, GlNeu(α, 2–3)Gal(β, 1–4)Glc(β,1-1) ceramide and the antigen of bovine erythrocytes was N-glycolylneuraminyl-paragloboside, GlNeu (α,2–3)Gal(β,1–4)GlcNAc(β,1–3)Gal(β,1–4)Glc(β,1-1) ceramide. The results indicate that “serum-sickness” antibodies react with a common disaccharide moiety of non-reducing end of the both glycosphingolipids.     

Arrest of 3T3 cells in G1 phase by low density lipoprotein

Low density lipoprotein (LDL) and high density lipoprotein (HDL) were purified from normal human serum by KBr density gradient centrifugation and gel filtration through Sepharose 4B. LDL reversibly inhibited proliferation of Swiss/3T3 cells, whereas HDL had no inhibitory effect on cell growth. The LDL-induced inhibition was LDL-dose dependent and was reversed by the addition of mevalonate, a product of the reaction of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34). These data suggest that a specific reduction in the activity of HMG-CoA reductase produced by the addition of LDL is the main cause of the inhibition of cell proliferation. Studies of the effect of LDL on the cell cycle showed that it inhibited the entry of cells arrested in G0/G1 into the S phase but that it did not affect the transition of cells at the G1/S boundary into the M phase. The cell cycle of 3T3 is arrested solely in G1 by LDL.     

Demonstration of a high affinity Ca2+ ATPase in rat liver plasma membranes

Rat liver plasma membranes contained a high affinity Ca²⁺-ATPase which had an apparent half saturation constant of 0.2 μM for calcium. The Ca²⁺-ATPase was not stimulated by adding magnesium and/or calmodulin. Conversely, the addition of these substances diminished the calcium-stimulation of the ATPase. Orthovanadate (7 nM-2 mM), mitochondrial ATPase blockers (NaN₃, KCN, dicyclohexylcarbodiimide), Na⁺, K⁺ and ouabain had no effect on the ATPase activity. The ATPase was separated from nonspecific divalent cation stimulatable ATPase (Mg²⁺-ATPase) by solubilization with Triton X-100 followed by a Sephadex G-200 column chromatography and showed an apparent molecular weight of 200,000.     

Down modulation of N-myc, heat-shock protein 70, and nucleolin during the differentiation of human neuroblastoma cells.

Cultured human neuroblastoma (GOTO) cells were induced to differentiate by dibutyryl cyclic AMP (Bt2cAMP) and/or retinoic acid (RA). A combination of Bt2cAMP (1 mM) and RA (1 microM) yielded the most significant networks of neurites after 3 to 4 days, this being associated with the reduction of N-myc mRNA levels. Next, we examined several cellular genes that were possibly linked with changes in N-myc gene expression under these conditions. Among the genes examined, both nucleolin and a major heat-shock protein (hsp70) mRNAs showed changes concomitant with those in N-myc mRNA levels when induced by Bt2cAMP and RA. Dibutyryl cAMP alone induced several short cellular processes and caused a marked decrease in N-myc mRNA within 2 days. RA alone induced a few long and straight neurites along the longitudinal axis of individual cells and a significant decrease in growth rate but showed neither network formation nor a decrease in N-myc gene expression. These results indicate differential effects of Bt2cAMP and RA on the regulatory mechanisms of both cell proliferation and differentiation and also indicate a possible association of expression of N-myc gene with those of hsp70 and nucleolin genes.