Determination of the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium.

Pulsed-field gel electrophoresis was used to determine the chromosomal size of three different strains of Enterococcus faecalis and one strain of Enterococcus faecium. The size determinations of OG1X, a strain of E. faecalis widely used in many laboratories for genetic studies, using Sma I, Not I, and Sfi I alone or in combination, ranged from 2,750 to 2,761 kb. Using the same enzymes as with OG1X, the size of HH-67, a plasmid-free clinical isolate of E. faecalis, was determined to be 2,170-2,288 kb and the size of JH2-2, an E. faecalis recipient strain, ranged from 2,008 to 2,135 kb. The size range generated for GE-1, a plasmid-free E. faecium strain, with the use of Sma I, Not I, and Apa I was 2,045-2,155 kb. Although OG1X differed in size from the other three enterococci, each individual enterococcal strain generated reproducible results in different experiments. However, for both E. faecalis OG1X and E. faecium GE-1, one of the enzymes used generated a considerably smaller molecular size than that generated by the other two enzymes. The discrepancy was due to visually undiscernible comigrating fragments, and serves to point out a potential source of error if fewer than two enzymes are used to size a genome. The size discrepancies were resolved by digesting individual fragments with a second enzyme. The molecular sizes of these enterococcal strains are larger than that recently reported for Campylobacter, smaller than that of Escherichia coli and Pseudomonas aeruginosa, and similar (OG1X) or smaller (JH2-2, HH67, and GE-1) than the 2,819-kb reported for Streptococcus mutans.     

Regulation of gene expression in preimplantation mouse embryos: effects of the zygotic clock and the first mitosis on promoter and enhancer activities

Previous studies have reported that promoters requiring enhancers for full activity in mammalian somatic cells also require enhancers when injected into mouse two-cell embryos, whereas the same promoters can be expressed just as efficiently in the absence of an enhancer when injected into arrested one-cell embryos. Experiments were designed to determine whether this phenomenon reflected normal developmental changes at the beginning of mammalian development, or simply differences in the physiological states of these cells under the experimental conditions employed. The activity of three different promoters that function in a wide variety of mammalian cells was measured both in embryos whose morphological development was arrested and in embryos that continued development in vitro. Expression of the injected gene was related to the onset of zygotic gene expression ("zygotic clock"), the phase of the cell proliferation cycle, the use of aphidicolin to arrest cell proliferation, and formation of two-cell embryos in vitro and in vivo. The results demonstrated that promoter activity was tightly linked to zygotic gene expression, while the need for enhancers to stimulate promoter activity depended only on formation of a two-cell embryo. These results further support the hypothesis that the first mitosis induces a general repression of promoters prior to initiation of zygotic gene expression that is relieved specifically by enhancers.     

IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.     

Effect of methabenzthiazuron on growth and nitrogenase activity in Vicia faba

The effects of the herbicide methabenzthiazuron (175 and 220 g ha^-1) on vegetative and reproductive growth, nodulation and nitrogenase activity of Vicia faba were studied in the field under Mediterranean conditions. Nitrogenase activity of excised nodules was estimated using the acetylene reduction assay four times during the developmental period. Leaf area index, dry weight and nitrogen content of the different parts of the plants were measured. Methabenzthiazuron-treated plants showed an increase in nodulation, nitrogenase activity and vegetative growth at early pod fill. Methabenzthiazuron also caused an increase in leaf N content and fruits. These were transient effects found during early and mid pot fill. Nevertheless, plants treated with these sublethal doses of herbicide improved seed production and nitrogen content of seeds at harvest time. The stimulatory effect of methabenzthiazuron on N₂ fixation and vegetative growth seems not be related with the transient stimulatory effect on photosynthetic capacity, also caused by the herbicide, since the stimulatory effect on N₂ fixation was apparent during pod fill, when photosynthetic capacity declined and was not modified by methabenzthiazuron.     

Flavin-containing monooxygenase: a major detoxifying enzyme for the pyrrolizidine alkaloid senecionine in guinea pig tissues

Evidence based on optimal pH, thermal stability, and enzyme inhibition data suggests that the NADPH-dependent microsomal N-oxidation of the pyrrolizidine alkaloid senecionine is carried out largely by flavin-containing monooxygenase in guinea pig liver, lung, and kidney. In contrast, the hepatic microsomal conversion of senecionine to the pyrrole metabolite (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) is catalyzed largely by cytochrome P450. However, the rate of senecionine N-oxide formation (detoxication) far exceeded the rate of DHP formation (activation) in guinea pig liver microsomes over a range of pHs (pH 6.8 to 9.8). In guinea pig lung and kidney microsomes, N-oxide was the major metabolite formed from senecionine with little or no production of DHP. The high rate of detoxication coupled with the low level of activation of senecionine in liver, lung, and kidney may help explain the apparent resistance of the guinea pig to intoxication by senecionine and other pyrrolizidine alkaloids.     

Comparison of rainbow trout and mammalian cytochrome P450 enzymes: evidence for structural similarity between trout P450 LMC5 and human P450IIIA4

Studies were undertaken to determine the immunochemical relationship between constitutive trout cytochrome P450s and mammalian cytochrome P450IIIA enzymes. Polyclonal antibodies (IgG) generated against trout P450 LMC5 reacted strongly with P450IIIA1 in dexamethasone-induced rat liver microsomes and with P450IIIA4 in human liver microsomes in immunoblots. In contrast, rabbit anti-P450 LMC1 IgG did not recognize these proteins in rat and human liver microsomes. Reciprocal immunoblots using anti-rat P450IIIA1 showed that this antibody does not recognize trout P450 LMC1 or LMC5. However, anti-human P450IIIA4 IgG was found to cross react strongly with P450 LMC1 and LMC5. Progesterone 6 beta-hydroxylase activity of trout liver microsomes, a reaction catalyzed by P450 LMC5, was markedly inhibited by anti-P450IIIA4 and by gestodene, a mechanism-based inactivator of P450IIIA4. These results provide evidence for a close structural similarity between trout P450 LMC5 and human P450IIIA4.     

Genetic variation in isozymes of wild boar (Sus scrofa ferus Linné)

Eleven isozyme systems were investigated in wild boar (Sus scrofa ferus L.) by means of horizontal starch gel-electrophoresis using liver and kidney extracts. Samples of 103 specimen that originated from three adjunct localities from Rhineland-Palatinate (Fed. Rep. Germany) were analysed. AAT, ACO, GDH, GPDH, LDH, ME, MPI, MR, PGDH, and PGI were invariant. Genetic polymorphism is described for PGM. The genetic polymorphism at Pgm2 is explained by a biallelic model. Observed genotypic structure did not differ significantly from Hardy-Wein-berg-proportions at this gene locus. The degree of heterozygosity is 58,3 % and Wright's Fixation Index F =–0.166 at Pgm2. The allelic structure at this gene locus differed to a great extent from that found in domestic swine, i. e. in domestic swine the faster migrating Pgm2 allozyme was only found in relative low frequencies compared to wild boar. The genetic polymorphisms were moderate in the wild boar population compared to electrophoretic data of other animal species.     

A study on melanogenesis and catecholamine biosynthesis during Bufo bufo development

Tyrosinase and L-DOPA decarboxylase activities have been investigated during Bufo bufo development since catecholamines and melanin are formed from common substrates in homologous cells. Catecholamines first appear at stage 13 (neural plate), but tyrosinase, at a very low level, and L-DOPA decarboxylase are present throughout all of prior development. Hence, L-DOPA decarboxylase activity is not likely to be correlated with the control of catecholamine synthesis, although at stage 17 it is mainly localized in the nonneural part of the embryo. The distribution of young melanosomes and L-DOPA decarboxylase suggest a separation between melanogenesis and catecholamine synthesis.     

Use of activated clotting time for monitoring anticoagulation during cardiopulmonary bypass in infants and children with congenital heart disease

The use of a fixed dosage schedule was compared with the use of activated clotting time (ACT) for determining heparin and protamine dosages during and after cardiopulmonary bypass disease. Use of the ACT resulted in a statistically significant increase in heparin dosage and a statistically significant reduction of postoperative blood loss. With ACT use, chest tubes were retained for a shorter period of time, and the incidence of serious postoperative hemorrhage was reduced from 44% to 18%. These results confirm the superiority of the ACT method for monitoring intraoperative anticoagulation in pediatric patients with congenital heart disease.