Tyrosine Hydroxylase Regulation: Apparent Kinetic Alterations Following Incubation of Brain Slices in a Sodium-free Medium

In an attempt to determine if alterations in intraneuronal Ca2+ may regulate tyrosine hydroxylase activity, brain slices were subjected to experimental manipulations known to increase the intraneuronal concentration of free Ca2+ ions. Incubation of either striatal or olfactory tubercle slices in a Na+-free medium for 15 min at 37 degrees resulted in a marked increase in the activity of tyrosine hydroxylase present in the 20,000 g supernatant fraction of homogenates prepared from the slices. Tyrosine hydroxylase isolated from slices previously incubated in a Na+-free, choline-enriched medium or in a Na+-free, sucrose-enriched medium exhibited maximal activities when assayed at pH 6.0 and 7.0, respectively. However, the percentage stimulation of enzyme activity induced by incubation of the slices in a Na+-free medium was maximal when the enzyme assays were performed at pH 7.0. The observed increase in enzyme activity seems to be mediated by a decrease in the apparent Km of the enzyme for pteridine cofactor, regardless of whether the kinetic enzyme analyses were conducted at pH 6.0 or 7.0, and by an increase in the Ki of the enzyme for end-product inhibitor dopamine. The apparent kinetic changes in the enzyme do not seem to result from alterations in the endogenous dopamine content of the slices, and they are independent of any increase in dopamine release that might have occurred as a response to the augmented intraneuronal Ca2+ concentration. Furthermore, the activation of tyrosine hydroxylase produced by incubating slices in a Na+-free medium is observed even in slices depleted of dopamine by pretreatment of rats with reserpine 90 min before preparation of brain slices. The activation of tyrosine hydroxylase observed under these experimental conditions does not seem to be mediated by cAMP or by a cAMP-dependent phosphorylation process. It is suggested that the changes in tyrosine hydroxylase reported are mediated primarily by a rise in the free Ca2+ concentration within the nerve tissue. These observations are consistent with the hypothesis that the kinetic activation of tyrosine hydroxylase produced after depolarization of central dopaminergic neurons may occur through a Ca2+-dependent even other than transmitter release.     

Site-specific integration of an F'' lac pro factor in the region of the replication origin (oriC) of E. coli

Summary An episome, F 128, which carries approximately 8x10⁴ base pairs of chromosomal DNA homologous to the lac pro region of the E. coli chromosome, has been found to integrate into the oriC region of the chromosome in a site specific reaction. While the event appears to be recA-dependent, no homology between the episome and this region of the chromosome was detected. The Hfr strains formed result from the integration of intact F 128 molecules. The structure of the Hfr strains generated has been determined and their transfer properties analyzed.     

Terpenes from pittosporaceae

The monoterpenes of the fruits of Pittosporum resiniferum and P. undulatum have been identified. The essential oil of P. resiniferum contai     

Gliding motility in Aphanothece halophytica: analysis of wall proteins in mot mutants.

The unicellular cyanobacterium Aphanothece halophytica (PCC 7418) is motile, and spontaneous nonmotile (mot) mutants accumulate when the organism is subcultured. Analysis of mot mutants suggests that a glycoprotein in the cell wall is involved in the motility mechanism. Proteins from the wall fraction of the wild type and five mot clones were analyzed by gradient sodium dodecyl sulfate-acrylamide gel electrophoresis. Four clones were similar to the wild type, and one clone, mot-3, was missing a high-molecular-weight protein (approximately 200,000) and had at least one new polypeptide (160,000). The high-molecular-weight protein stained with periodic acid-Schiff reagent, suggesting that it was a glycoprotein. The absence of the protein in mot-3 did not affect the mechanical strength of the wall, since both mot-3 and wild-type cells were broken at the same rate by controlled cavitation. Several other cyanobacteria were also screened for the presence of glycoproteins. All motile strains have such proteins, although none had an apparent molecular weight as high as that in Aphanothece sp. Some motile strains, such as Oscillatoria limnetica and Phormidium sp., showed very large amounts of glycoproteins; whereas some nonmotile strains, such as Synechococcus sp. (UTEX 625) and Microcystis sp. (PCC 7820), showed no high-molecular-weight glycoproteins.     

Homology between the invertible deoxyribonucleic acid sequence that controls flagellar-phase variation in Salmonella sp. and deoxyribonucleic acid sequences in other organisms.

The invertible deoxyribonucleic acid (DNA) segment cloned from Salmonella sp. was radioactively labeled and used as a probe to search for homologous sequences by Southern hybridization. Only one copy of the invertible segment could be found on the Salmonella sp. genome. Partial sequence homology with the invertible region was detected in bacteriophage Mu and P1 DNA by low-stringency hybridization. Under these conditions, no homology was detected with Escherichia coli DNA. A strain of Salmonella sp. defective in phase variation carrying the vH2- allele was also analyzed by DNA-DNA hybridization. The results show that there is sequence divergence between diphasic and vH2- strains within the invertible sequence.     

Hydrogen peroxide causes the fatal injury to human fibroblasts exposed to oxygen radicals

Oxygen radicals are suspected as being a cause of the cellular damage that occurs at sites of inflammation. The phagocytic cells that accumulate in areas of inflammation produce superoxide, hydrogen peroxide, hydroxyl radical, and probably singlet oxygen in the extracellular fluid. The mechanism by which these oxygen molecules kill cells is unknown. To determine which of the oxygen species is responsible for the cellular killing, we exposed human fibroblasts in culture to oxygen radicals generated by the enzymatic action of xanthine oxidase upon acetaldehyde. Using the amount of chromium-51 released from labeled fibroblasts as an index of cellular death, we found that cells were protected only by interventions that reduce hydrogen peroxide concentration. Agents that inactivate superoxide, hydroxyl radical, and singlet oxygen were ineffective in limiting oxygen radical-induced cellular death.     

Multiple electrophoretic forms of methyl-accepting chemotaxis proteins generated by stimulus-elicited methylation in Escherichia coli.

The tsr and tar genetic loci of Escherichia coli determine the presence in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of methyl-accepting chemotaxis proteins (MCPs) I and II, respectively, each of which consists of a distinct group of multiple bands. Synthesis of the tsr and tar products was directed in ultraviolet-irradiated bacteria by lambda transducing phages. The addition of appropriate chemotactic stimuli to these cells resulted in the appearance of additional, faster migrating electrophoretic forms of the Tsr and Tar polypeptides which disappeared upon removal of the stimulus. The stimulus-elicited forms comigrated with component bands of the corresponding MCPs. These results indicate that methylation itself caused shifts in electrophoretic mobility and hence led to the observed MCP band patterns. The number of Tsr species suggested that there were at least three methylated sites on the Tsr polypeptide. The conclusion that methylation generates multiplicity was supported by the results of experiments in which the tsr product was synthesized in mutant bacteria defective in specific chemotaxis functions concerned with methylation or demethylation of MCPs. Thus, the presence of a cheX defect blocked the stimulus-elicited appearance of faster migrating forms of the tsr product; conversely, the presence of a cheB defect resulted in a pronounced shift toward these forms in the absence of a chemotactic stimulus.     

Effects of chemical group specific reagents on sodium entry and the amiloride binding site in frog skin: Evidence for separate sites

Summary Previously we have shown that the inhibition of active transport by amiloride is noncompetitive with sodium inRana catesbeiana skin, suggesting that amiloride acts at a site separate from the sodium entry site (Benos, D.J., Mandel, L.J., Balaban, R.S. 1979,J. Gen Physiol. 73: 307). In the present study, the effects of a number of sulfhydryl, amino, and carboxyl group selective reagents were studied on short-circuit current (I _sc) as well as the efficacy of amiloride in bullfrog skin, to determine those functional ligands which may be involved with either of these processes.Addition of the sulfhydryl reagent PCMBS (1mm) to the outside bathing medium produced biphasic effects, initially reversibly increasingI _sc by an average 56% followed by a slower, irreversible decay to levels below baseline. In contrast, the addition of 0.1mm PCMB always resulted in a rapid, irreversible decrease inI _sc. When a 40,000 mol wt dextran molecule was attached to PCMB, a stable, reversible increase inI _sc was observed. These observations suggest that at least two populations of-SH groups are involved in Na translocation through the entry step. Amiloride was equally effective in inhibitingI _sc before and after treatment with PCMBS both during the stimulatory as well as the inhibitory phase. The sulfhydryl reducing agent DTT, and oxidizing agent DTNB had only minor influence onI _sc and did not alter the effectiveness of amiloride.Similarly, the amino reagents, SITS and TNBS did not affectI _sc. However, TNBS decreased the ability of amiloride to inhibit Na entry. These results suggest that an amino group may be involved in the interaction of amiloride and its site, without affecting Na entry.The carboxyl reagents EEDQ, TMO, and three separate carbodiimides were without effect onI _sc or amiloride inhibition. Methylene blue (MB), a molecule that interacts with both carboxyl and hydroxylspecific groups, inhibitedI _sc by 20% and decreased amiloride's ability to inhibitI _sc. These effects, however, are likely to occur from the cytoplasmic side as MB appears to enter into the cells.These results support the notion that amiloride and Na interact with the entry protein at different regions on the membrane.     

Effect of suramin (Bayer 205) on renal ornithine decarboxylase activity and polyamine concentrations in rats.

After a single high dose (20 mg/100 g i.v.) of suramin ornithine decarboxylase activity was increased rapidly in the rat kidney. Enzyme kinetic measurements indicate that on the basis of K_m values renal ornithine decarboxylases from control or suramin treated rats are indistinguishable. Renal nucleic acid and polyamine levels were also enhanced in response to suramin. Changes observed in this study are considered as biochemical signs of induced renal growth.     

Restricted randomization designs in clinical trials.

Though therapeutic clinical trials are often categorized as using either "randomization" or "historical controls" as a basis for treatment evaluation, pure random assignment of treatments is rarely employed. Instead various restricted randomization designs are used. The restrictions include the balancing of treatment assignments over time and the stratification of the assignment with regard to covariates that may affect response. Restricted randomization designs for clinical trials differ from those of other experimental areas because patients arrive sequentially and a balanced design cannot be ensured. The major restricted randomization designs and arguments concerning the proper role of stratification are reviewed here. The effect of randomization restrictions on the validity of significance tests is discussed.