Phosphate repression of cephamycin and clavulanic acid production by Streptomyces clavuligerus

Summary Production of cephamycin and clavulanic acid by Streptomyces clavuligerus is controlled by the phosphate concentration. Phosphate represses the biosynthesis of cephamycin synthetase, expandase and clavulanic acid synthetase. In the presence of 2 mM phosphate, the specific activities of expandase, cephamycin synthetase and clavulanic acid synthetase were higher than in the presence of 75 mM phosphate. The specific activity of cephamycin synthetase is maximal with an initial phosphate concentration of 10 mM, whereas the specific activity of expandase is maximal with 1 mM phosphate. A correlation between cephamycin synthetase specific activity and expandase specific activity was established at phosphate concentrations higher than 10 mM. This shows that the expandase is an important enzyme in the mechanism by which the phosphate concentration affects the biosynthesis of cephamycin.     

Behavioural expression of D-1 receptor supersensitivity depends on previous stimulation of D-2 receptors

SKF 38393 (2 mg/kg s.c.), a reportedly selective D-1 agonist, failed to induce contralateral turning behaviour in naive rats bearing 12 days old unilateral 6-hydroxydopamine lesions. On the other hand strong contraversive turning in response to SKF 38393 was obtained if rats had been tested 2 or 7 days before with apomorphine (0.1 mg/kg s.c.) or with LY 171555 (0.2 mg/kg s.c.), a selective D-2 receptor agonist. Contraversive turning in response to SKF 38393 was blocked by a low dose (0.05 mg/kg s.c.) of the specific D-1 antagonist SCH 23390. The results indicate that the behavioural expression of D-1 receptor supersensitivity following lesion of dopaminergic neurons depends on previous exposure to a stimulation of D-2 receptors.     

The accessibility of DNA to dimethylsulfate in complexes with recA protein.

recA protein coats DNA co-operatively to form filaments approximately 100 A thick, which in the presence of ATP, and more stably so in the presence of the non-hydrolyzable analog ATP gamma S, have a helical appearance with a deep cleft in the protein coat. This protein helix follows the DNA helix, to which it imparts a new helicity of 18.5 bp per turn of 97 A pitch. Here we test the accessibility of the DNA in the complex to modification by dimethylsulfate, and find that the complexed DNA is approximately 2-fold more reactive on the major groove side than it was in B-DNA (methylation of guanine N7), while it is protected approximately 2-fold on the minor groove side (methylation of adenine N3), suggesting that the protein coats the DNA along the minor groove. Furthermore, N3 of cytosine, a residue involved in base pairing, is found exposed in complexes with single strands as it is in naked single-stranded DNA, while it remains inaccessible in complexes with double strands, suggesting that the latter is not melted at this stage of the strand exchange reaction.     

Purification and characterization of glutathione transferase of human thyroid

An anionic (pI 4.6) isoenzyme of glutathione transferase was purified to homogeneity from human thyroid by affinity chromatography followed by isoelectric focusing. The content of enzyme was calculated to constitute about 0.2% of soluble proteins. The enzyme is formed by two identical subunits of 23,000 daltons approximately. The thyroid transferase did not catalyze the reduction of peroxides. Physical, catalytic and immunological analyses demonstrated extensive similarities between the thyroid transferase and the transferase from placenta, erythrocytes and breast. On the other hand, the thyroid transferase appears catalytically different from transferase 7-7, even if both cross-react with the antibodies raised against human placenta transferase.     

Kinetics of binding of macrolides, lincosamides, and synergimycins to ribosomes

The synergistic effect of type A (virginiamycin M (VM)) and type B (virginiamycin S (VS)) synergimycins and their antagonistic effect against erythromycin (a 14-membered macrolide) for binding to the large ribosomal subunit (50 S) have been related. This investigation has now been extended to 16-membered macrolides (leucomycin A3 and spiramycin) and to lincosamides (lincomycin). A dissociation of VS-ribosome complexes was induced as well by 16-membered macrolides as by lincosamides. The observed dissociation rate constant of VS-ribosome complexes was identified with the kappa-vs in the case of 16-membered macrolides, but linearly related to lincomycin concentration, suggesting a direct binding of the latter antibiotic to VS-ribosome complexes and the triggering of a conformational change of particles entailing VS release. Two different mechanisms were also involved in the VM-promoted reassociation to ribosomes of VS previously displaced by either macrolides or lincosamides. By binding to lincosamide-ribosome complexes, VM induced a conformational change of ribosomes resulting in higher affinity for VS and lower affinity for lincosamides. On the contrary, an incompatibility for a simultaneous binding of VM and 16-membered macrolides to ribosomes was observed. These results have been interpreted by postulating specific (nonoverlapping) and aspecific (overlapping) antibiotic binding sites at the peptidyltransferase domain. All the kinetic constants of five antibiotic families (type A and B synergimycins, 14- and 16-membered macrolides, and lincosamides) and a topological model of peptidyltransferase are presently available.     

Voltage-dependent activation and inactivation of calcium channels in PC12 cells. Correlation with neurotransmitter release

The existence and mechanisms of inactivation of voltage-gated Ca2+ channels are important, but still debatable, physiological problems. By using the Ca2+ indicators quin2 and fura-2, we demonstrate that in PC12 cells voltage-gated Ca2+ channels undergo inactivation dependent on both voltage and [Ca2+]i. Inactivation, however, is never complete and a small number of channels remains open during prolonged depolarization, explaining the steady state elevation of [Ca2+]i observed in cells depolarized with high KCl. A close parallel exists between Ca2+ channel inactivation and the transient nature of neurotransmitter release: secretion is rapidly stimulated during the first 30 s of depolarization, when a transient overshoot in [Ca2+]i can be demonstrated, while it is negligible during the following period, despite the persistence of an elevated [Ca2+]i; predepolarization in Ca2+-free medium and subsequent addition of Ca2+ (a condition which allows the development of the voltage inactivation) abolishes the fast phase of secretion, while not modifying the steady state [Ca2+]i eventually attained; and increases in the intracellular Ca2+ buffering decreases the amplitude of the fast secretion phase induced by KCl without altering the steady state [Ca2+]i. We suggest that localized [Ca2+]i gradients form close to the plasma membrane shortly after depolarization and that the [Ca2+]i reached in these regions is the relevant parameter in the regulation of secretion.     

Axonal Transport Characteristics of Gangliosides in Sensory Axons of Rat Sciatic Nerve

The distribution of axonally transported gangliosides and glycoproteins along the sciatic nerve was examined from 3 h to 4 weeks following injection of[3H]glucosamine into the fifth lumbar dorsal root ganglion of adult rats. Incorporation of labeled precursor into these glycoconjugates reached a maximal level in the ganglion within 6 h. Outflow patterns of radioactivity for glycoproteins showed a well-defined crest with a transport rate of approximately 330 mm/day. In contrast, the crest of transported gangliosides was continuously attenuated, implying a significant deposition along the axon, and an alternative method of calculating velocity was required. Analysis of accumulation of labeled material at double ligatures demonstrated both anterograde and retrograde transport of glycoproteins and gangliosides and allowed for the calculation of an anterograde transport rate of about 270 mm/day for each. Additional evidence of ganglioside transport is provided in that the TLC pattern of transported radioactive gangliosides accumulating at a ligature is significantly different from the pattern seen in the dorsal root ganglion or following intraneural administration of the labeled precursor. These data indicate that gangliosides are transported at the same rapid rate as glycoproteins but are subject to a more extensive exchange with stationary material than are glycoproteins.     

Serum beta 2-microglobulin in patients with monoclonal gammopathies

Beta-2-microglobulin concentrations were determined in serum samples from 45 patients with benign and malignant monoclonal gammopathies. In the group of patients suffering from multiple myeloma or Waldenstr?m macroglobulinemia the mean beta 2-microglobulin level was significantly higher than in the group with monoclonal gammopathy of undetermined significance. Values above 3 mg/L were highly indicative of a neoplastic process and were observed in all the Waldenstr?m patients and in greater than 90% of myeloma patients. No significant correlation was noticed between beta 2-microglobulin and monoclonal protein levels in any of the groups examined.