Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.     

Cell-membrane phospholipase C is involved in inducing the antiviral effect of interferon

A monospecific inhibitory antibody directed to phospholipase C (phosphoinositidase C) blocked the antiviral effect of human interferons alpha and beta when tested on human quiescent fibroblasts challenged with the vesicular stomatitis virus. This action was due to specific inhibition of polyphosphoinositide hydrolysis because (a) the F(ab)₂ fragment of the antibody molecule was also inhibitory; (b) excess antibodies directed to phospholipase A₂ and to a phosphatidylcholine-preferring phospholipase C did not have any inhibitory effect, and (c) the combination of 12-O-tetradecanoylphorbol-acetate and calcium ionophore A23187 had an interferon-like antiviral effect which was not influenced by the inhibitory anti-phospholipase C antibodies. To avoid an interferon-like effect due to induction of interferon by second messengers, Vero cells, which lack interferon biosynthesis, were also used. Liposomes containing inositol 1,4,5-triphosphate and 1-oleoyl-2-acetyl-rac-glycerol protected Vero cells against the infection with the vesicular stomatitis virus. These results taken together show that phosphoinositide-derived second messengers are involved in triggering the antiviral effect of interferons alpha and beta.     

Prenatal diagnosis of familial amyloidotic polyneuropathy: evidence for an early expression of the associated transthyretin methionine 30

Summary Transthyretin methionine 30 (TTR Met 30), which is associated with familial amyloidotic polyneuropathy, originates in a single base substitution (A for G) in the second exon of the TTR gene. This autosomal dominant disease can be diagnosed by RFLP analysis of NsiI-digested DNA. The amplification of DNA by PCR improves the diagnosis method, making it suitable for prenatal diagnosis. Using PCR-amplified DNA, prenatal diagnosis of two at-risk fetuses was performed. Control Met 30 and normal DNA (either genomic or produced by site directed mutagenesis) were processed in parallel. The diagnosis was made by hybridization with allele-specific oligonucleotide probes, and later confirmed by screening of the mutant protein in the amniotic fluid and, when possible, in the sera from the newborns. TTR Met 30 was detected in the amniotic fluid of a positive fetus whose father was the carrier of the mutation. This indicates that the mutant protein is expressed very early in development.     

Chromium in plants

Chromium is an essential trace element and is associated with some biological pathways, especially with glucose tolerance. For these reasons, we decided to determine the concentration of chromium in two sets of Brazilian medicinal plants. The first group consisted of plants that are considered as antidiabetic, whereas the second included plants that do not have this therapeutic property. The concentration of chromium was determined by flameless atomic absorption. All the plants analyzed contain chromium in the normal range for this element, but the hypoglycemic plants contain more chromium than the others (1–4 μg/g compared to 0.5–1.5 μg/g).     

Homologous and heterologous β-adrenergic desensitization in hepatocytes. Additivity and effect of pertussis toxin

In hepatocytes obtained from hypothyroid rats, phorbol myristate acetate (PMA) and vasopressin diminished the accumulation of cyclic AMP and the stimulation of ureagenesis induced by isoprenaline or glucagon without altering significantly the accumulation of cyclic AMP induced by forskolin. Pretreatment with PMA markedly reduced the stimulation of ureagenesis and the accumulation of cyclic AMP induced by isoprenaline or glucagon. In membranes from cells pretreated with PMA, the stimulation of adenylate cyclase induced by isoprenaline + GTP, glucagon + GTP or by Gpp[NH]p were clearly diminished as compared to the control, whereas forskolin-stimulated activity was not affected. The data indicate heterologous desensitization of adenylate cyclase. It was also observed that the homologous (García-Sáinz J.A. and Michel, B. (1987) Biochem. J. 246, 331–336) and this heterologous β-adrenergic desensitizations were additive. Pertussis toxin treatment markedly reduced the heterologous desensitization of adenylate cyclase but not the homologous β-adrenergic desensitization. It is concluded that the homologous and heterologous desensitizations involve different mechanisms. The homologous desensitization seems to occur at the receptor level, whereas the heterologous probably involves the guanine nucleotide-binding regulatory protein, Ns.     

Xenopus spermatozoon: Is there any correlation between motility and oxygen consumption?

The motility status of Xenopus laevis spermatozoa does not affect their respiration rate. Oxygen consumption for 10⁹ spermatozoa is approximately 0.4 μmol/minute. Oxygen consumption is not increased by gramicidin D, an uncoupler, and it is not blocked by KCN or NaN₃. The adenosine triphosphate (ATP) content of spermatozoa that have been activated is definitely less than that in the spermatozoa that remained immotile. Incubation in KCN, NaN₃, and gramicidin decreases the ATP content and impairs motility. The conclusions of the present study are that in Xenopus spermatozoa motility and oxygen consumption are not correlated, and the composition of the respiratory chain of these spermatozoa presents noteworthy peculiarities.     

Antibacterial activity of rufloxacin in theStaphylococcus aureus rat granuloma pouch model

The protective effects of rufloxacin againstStaphylococcus aureus-induced infections were compared with those of ciprofloxacin in the granuloma pouch model in the rat. Two strains with different in vitro sensitivity to the drugs were studied. Rufloxacin concentrations persisted longer than ciprofloxacin in the exudate in the pouch cavity and were about eight times higher. Equal doses of rufloxacin and ciprofloxacin had similar antibacterial activities. However, rufloxacin inhibitedStaphylococcus aureus bacterial growth significantly longer than did ciprofloxacin.     

Morphological changes and induced sporulation in HmBR transformants ofCochliobolus lunatus

The phenomena following the transformation of the fungusCochliobolus lunatus by plasmid-encoded HmB resistance were investigated. All of the 16 tested transformants had markedly altered morphology. Unlike the untransformed fungus, the transformants produced both conidia and arthrospores, did not excrete slime, lost their purple color, and had an altered progesterone-bioconverting pathway.