Analysis of the sequence of a new cryptic plasmid, pRJF2, from a rumen bacterium of the genus Butyrivibrio: Comparison with other Butyrivibrio plasmids and application in the development of a cloning vector

Abstract A small cryptic plasmid, pRJF2, from Butyrivibrio fibrisolvens strain OB157 was isolated and sequenced. The plasmid is similar in organisation to the previously sequenced Butyrivibrio plasmid, pRJF1, with two open reading frames, ORF1 and ORF2, flanking a region tentatively identified as the replication origin, and a region of unknown function defined by terminal 79 bp invert repeats. The sequences of ORF1, ORF2, and the presumptive replication origin are highly conserved. The sequence between the 79 bp invert repeats is not, and is therefore presumed to be of lesser functional significance, although the 5' and 3' termini are still highly conserved. The functional importance for plasmid replication of these regions was tested by constructing potential shuttle vectors, each lacking one or more of the regions of interest. When the region between the invert repeats was deleted and replaced by the erythromycin resistance gene from pAM β1 together with pUC18, to produce the 7.9 kb chimaeric plasmid pYK4, the construct was successfully transformed into E. coli and B. fibrisolvens by electroporation, and was stably maintained in both hosts. Both ORF1 and ORF2 were required for successful transformation of B. fibrisolvens .     

Additive stimulation of hepatic putrescine production by glucagon and insulin after partial hepatectomy in rats

When rats received glucagon or insulin every 2 h after partial hepatectomy (Hx), hepatic putrescine content was increased above control levels at 6 and 12 h, respectively. When the two hormones were combined, the increased levels were additive. Hepatic ornithine decarboxylase activity was above control levels at 12 h after insulin treatment. Hepatic spermidine N1-acetyltransferase activity was enhanced at 6 h only when glucagon was dosed. Putrescine administration from 0 to 4 h or from 6 to 10 h increased hepatic DNA synthesis to similar levels 22 h after Hx. These results suggest that glucagon and insulin additively stimulate hepatic putrescine production after Hx. This may explain the cooperative stimulation of liver regeneration by both hormones.     

Low-pressure microwave-induced helium plasma emission spectrometry: Vaporization characteristics of metal chlorides,nitrates, and sulfates

A low-pressure microwave-induced helium plasma serves as an excitation source for metal chlorides, nitrates, and sulfates vaporized from a filament, resulting in fractional vaporization and differential sensitivities of detection of the elements depending on the vapor pressures of their salts. The shapes of the single emission peaks, which are simple in the presence of potassium chloride, become complex and may double in number.     

Effects of synthetic and naturally occurring flavonoids on Na+,K+-ATPase: aspects of the structure-activity relationship and action mechanism

A comparative study was made of the effects of 15 synthetic and naturally occurring flavonoids on the hydrolytic activity of Na+, K+-adenosine triphosphatase (ATPase). Twelve of the flavonoids examined were mono-hydroxy or mono-methoxy derivatives. All inhibited Na+, K+-ATPase from dog kidney cortex when present at concentrations from 40-1000 microM. Flavones possessing cyclohexyl instead of the phenyl group (i.e., 2-cyclohexyl-benzopyran-4-one derivatives), were the most potent with IC50 at 257-320 microM. Structure-activity relationships were observed among the following mono-substituted flavones as: i) 2-cyclohexyl-benzopyran-4-one much greater than 2-phenyl-benzopyran-4-one; ii) 2-cyclohexyl-7-hydroxybenzopyran-4-one greater than 2-cyclohexyl-6-hydroxybenzopyran-4-one greater than 2-cyclohexyl-5-hydroxybenzopyran-4-one. Some flavonoids showing potent inhibitory activity were also examined for ouabain-displacement activity on human erythrocytes. Hardly any of the flavonoids were able to block [3H]ouabain binding to erythrocytes. These results suggest that the mechanism by which flavonoid block Na+, K+-ATPase is not related to the cardiac glycoside-specific binding site(s) of this enzyme.     

Quantitative analysis of development of mitochondrial ultrastructure in differentiating mouse hepatocytes during postnatal period

Between birth and 10 days of age, the volume density (volume/unit cytoplasmic volume) of the matrix, and the surface density (area/unit cytoplasmic volume) of the inner membrane and cristae increased in both periportal and perihepatic hepatocytes, and did not differ significantly between the cells of the two zones. After 10 days of age, however, the volume density of the matrix decreased in perihepatic cells and remained unchanged in periportal cells, and, therefore, it became greater in periportal cells than in perihepatic cells in 20-day-old and adult animals. The surface density of the inner membrane and cristae decreased in the cells of both zones. Further, the hepatocyte volume increased markedly, especially in perihepatic zones between 20 days of age and the adult. The results show that, in postnatally differentiating hepatocytes, mitochondria are likely to develop during early postnatal period, then the structural heterogeneity of mitochondria arises, and hepatocyte volume increases markedly during late postnatal period after weaning. Thus, the process of postnatal hepatocyte differentiation includes such several phases of development.     

Impairment and restoration of response to TSH in dog thyroid slices after treatment with phospholipase A and lubrol PX.

Incubation of dog thyroid slices with phospholipase A (10-40 U/Ml) or Lubrol PX (0.08-0.4%) caused a diminution in the subsequent TSH effect on the tissue cyclic AMP level and glucose oxidation. The same treatment had no effect on the basal level of these parameters. When the phospholipase A or Lubrol PX-treated slices were rinsed intensively with a Krebs-Ringer bicarbonate buffer and then incubated at 37degreesC in the same buffer for a further 1 to 3 hours, responsiveness to TSH recovered progressively reaching almost completely that of the control slices. Again, these procedures were without any significant effect on the responsiveness of the control slices. The above results together with those reported previously suggest strongly that phospholipids are an essential component of the plasma membrane system by which TSH stimulates adenylate cyclase activity. In addition, these essential lipids in the membrane appear to be renewed rather efficiently in this tissue, thus securing the functional integrity of the thyroid in the face of various deleterious situations.     

Effects of calcium ionophore (A-23187) on glucose oxidation and iodide transport in dog thyroid slices.

A calcium ionophore (A-23187, 20 mug/ml) stimulted 14C-1-glucose oxidation in dog thyroid slices to an extent equivalent to that obtained by the optimal concentration of dibutyryl cyclic AMP (1mM). Furthermore, the ionophore augmented the stimulation by dibutyryl cyclic AMP much more than the simple additive effect. The ionophore also enhanced the effect of TSH, but to a lesser extent. Under conditions where organic binding was blocked, T/M ratio of radioiodine concentration was lowered in slices by the ionophore; the findings similar to those obtained with TSH and dibutyryl cyclic AMP. The ionophore exhibited a slightly depressive effect on the basal cyclic AMP level. The elevation by TSH of cyclic AMP levels was also slightly depressed by the ionophore, but statistically insignificant in most cases. These results indicate that calcium ion may play an important role in the TSH regulation of iodide transport and glucose metabolism in the thyroid, in some cases by augmenting the effects of cyclic AMP.     

GPR35 is a novel lysophosphatidic acid receptor

GPR35 is a rhodopsin-like G protein-coupled receptor identified in 1998. It has been reported that kynurenic acid, a tryptophan metabolite, may act as an endogenous ligand for GPR35. However, the concentrations of kynurenic acid required to elicit the cellular responses are usually high, raising the possibility that another endogenous ligand may exist. In this study, we searched for another endogenous ligand for GPR35. Finally, we found that the magnitude of the Ca²⁺ response induced by 2-acyl lysophosphatidic acid in the GPR35-expressing HEK293 cells was markedly greater than that in the vector-transfected control cells. Such a difference was not apparent in the case of 1-acyl lysophosphatidic acid. 2-Acyl lysophosphatidic acid also caused the sustained activation of RhoA and the phosphorylation of extracellular signal-regulated kinase, and triggered the internalization of the GPR35 molecule. These results strongly suggest that 2-acyl lysophosphatidic acid is an endogenous ligand for GPR35.