Metronidazole and the isolation of temperature-sensitive photosynthetic mutants in cyanobacteria

A procedure has been developed for use of metronidazole (2-methyl-5-nitroimidazole-1-ethanol) as an enrichment agent during the isolation of temperature-sensitive, photosynthetic mutants in the cyanobacteriumSynechococcus cedrorum. The protocol includes incubation with this drug following mutagenesis withN-methyl-N-nitro-N-nitrosoguanidine. Incubation of photosynthetically activeS. cedrorum cells with 1 mM metronidazole causes a light-dependent reduction of cell viability. Maximum reduction in cell viability occurred following 6 h of incubation. Cessation of electron transport reduced the impact of the drug by five orders of magnitude. Yet during the time of incubation, metronidazole did not influence the electron transport capacities of theS. cedrorum cells, suggesting that the thylakoid membrane was not the target of the toxic effects of this drug. In addition, this drug was found to be an effective electron acceptor to photosystem I although high concentrations were required to observe maximum rates of electron transfer. Metronidazole interacted in a noncompetitive manner with methyl viologen, which suggested that those two acceptors to photosystem I have unique reduction sites on theS. cedrorum thylakoid membrane. The temperature-sensitive strains that were isolated using the procedure presented here were assessed for photosynthetic electron transport and chlorophyll fluorescence (induction kinetics and low-temperature emission spectra) characteristics. Approximately one-half of the temperature-sensitive mutants isolated possessed abnormal photosynthetic properties when shifted to the restrictive temperature (40°C). A total of 31 strains have been characterized and initially classified, showing abnormalities throughout the photosynthetic electron-transport chain.     

Chromosomal variation and zoogeography inAteles

Karyotypes are reported from 21 spider monkeys distributed among five taxa of the genus Ateles.G- and C- banding techniques revealed variations between taxa. Two variants were discovered for chromosome 5, for chromosome 7, and for the Y chromosome. Four forms of chromosome 6 were seen. The variations are all probably pericentric inversions. One individual was heteromorphic at position 6. The data are compared with prebanding reports of Ateleschromosomes and reports of five animals studied with banding techniques. The variation in Ateleschromosomes is similar in degree to that found in other ceboid genera. The variants may be related to forest refugia formed during the Plio-Pleistocene. Karyotyping of many New World primates is required to ensure a homogeneous experimental group.     

Time-dependent changes of calcium influx and efflux rates in rabbit skeletal muscle sarcoplasmic reticulum

Unfractionated and low buoyant density sarcoplasmic reticulum vesicles released calcium spontaneously after ATP- or acetyl phosphate-supported calcium uptake when internal Ca²⁺ was stabilized by the use of 50 mM phosphate as calcium-precipitating anion. This spontaneous calcium release could not be attributed to falling Ca²⁺ concentration outside the vesicles (Ca₀²⁺), substrate depletion, ADP accumulation, nonspecific membrane deterioration or the attainment of a high vesicular calcium content. Instead, spontaneous calcium release was directly proportional to Ca₀²⁺ at the time that calcium content was maximal. A causal relationship between high Ca₀²⁺ and spontaneous calcium release was suggested by the finding that elevation of Ca₀²⁺ from less than 1 μM to 3–5 μM increased the rate and extent of calcium release.The spontaneous calcium release was due both to acceleration of calcium efflux and slowing of calcium influx that was not accompanied by a significant change in the rate of ATP hydrolysis. Neither reversal of the transmembrane KCl gradient nor incubation with cation and proton ionophores abolished the spontaneous calcium release. The persistence of calcium release under conditions where the membrane was permeable to both anions and cations makes it unlikely that this phenomenon is due to a changing transmembrane potential.     

THE LACK OF SPECIFICITY TOWARDS SALTS IN THE ACTIVATION OF CHOLINE ACETYLTRANSFERASE FROM HUMAN PLACENTA

Abstract— The effects of monovalent and divalent anions on the choline acetyltransferase reaction have been determined at high (5.0 mM) and low (0.58 mM) choline. At 0.58 mM-choline, both monovalent and divalent anions activate the enzyme ±9 fold; however, at 5.0mM-choline, monovalent anions activate the enzyme ±25 fold, while divalent anions activate ±9 fold. Both monovalent and divalent anions show uncompetitive activation with respect to choline. When either dimethylaminoethanol, N -(2-hydroxyethyl)- N -methyl piperidinium iodide, or N -(2-hydroxyethyl)- N -propyl pyrrolidinium iodide was substituted for choline, activation by monovalent or divalent anions was only 2.5-4 fold. With AcCoA as substrate the ChA reaction can be increased ±20 fold by increased salts; however, with acetyl dephosphoCoA as substrate, the reaction is insensitive to the salt concentration. Similar salt effects on the ChA reaction, as measured in the direction of acetylcholine synthesis, have been demonstrated in the reverse reaction. In addition, inhibition of the forward reaction by acetylcholine has been measured as a function of sodium chloride concentration. Although the K₁ for acetylcholine increases with increasing salt, this change in K ₁, parallels the increase in the K _m for choline. These results support the hypothesis that both monovalent and divalent anions activate choline acetyltransferase by the same singular mechanism; which is to increase the rate of dissociation of coenzyme A from the enzyme.     

meoA is the structural gene for outer membrane protein c of Escherichia coli K12

Summary The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Mel resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptorcomplex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c^*. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.     

Metabolisme lipidique chez l'Aspergillus versicolor (Vuill.) tiraboschi. Relations avec la biogenese de la sterig matocystine

Résumé L' Aspergillus versicolor est cultivé sur un milieu synthétique pendant 22 jours. Les productions de lipides et de stérigmatocystine sont comparées. Les acides gras des fractions neutres et polaires sont essentiellement: C 160, C 180, C 181 C 182, et C 183. Les quantités maximales de mycélium sec, de lipides neutres et de stérigmatocystine apparaissent respectivement au 4e, 7e et 20e jours. Une diminution de la teneur en lipides précède la phase de concentration maximale en métabolites secondaires du type polycéto-acide. Il semble que les lipides, et tout particulièrement l'acide palmitique, participent à la biogenèse de ces dérivés.

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Summary Aspergillus versicolor is cultivated in a synthetic medium for 22 days. Bioproduction of lipids and sterigmatocystin are compared. The fatty acids of the neutral lipid and polar lipids fractions are mainly: C 160, C 180, C 181, C 182, C 183. Maximal yields of dry weight, neutral lipids and sterigmatocystin occur, respectively, on the 4th, the 7th and the 20th days. These results and their comparison with other works emphasize that a fall of concentration in lipids precedes the phase of highest concentration in secondary metabolites of polyketide type; it appears that fats and particularly palmitic acid are present in biogenesis of these derivatives.

     

Cardiac allografts in mice congenic at non-H-2 histocompatibility loci

Neonatal mouse heart fragments were grafted under the ear skin of adult recipients. Cardiac allograft survival was evaluated by visual observation of pulsation, electrocardiography, and histology. Employing a series of congenic resistant strains differing from C57BL/10Sn at theH-1, H-3,H-4, H-7, H-8, H-9, H-10, H-11, andH-12 loci, the median survival times of the heart grafts to and from C57BL/10Sn were obtained. The various interallelic combinations resulted in a wide variation of graft survival. Reciprocal transplants frequently showed different survival times.H-1 ^c grafts were rejected by B10.129(5M)/nSn female mice with a median survival time of 90 days.H-1 ^b grafts were not rejected by C57BL/10Sn mice for the experiment's duration of 200 days. The weaker the histocompatibility barrier, the more variable the survival times and the smaller the ratio of rejected to total grafted heart fragments. Female recipients were observed to reject their grafts more rapidly and to reject a higher proportion than males of the same strain. Although the strength of the different non-H-2 barriers generally paralleled that determined by skin transplants, the rankings of the strongest minor barriers were not the same for both tissues.     

Evidence for variation in the number of functional gene copies at the AmaR locus in chinese hamster cell lines

The hypothesis of functional hemizygosity has been examined for the α-amanitin resistant (Ama^R, a codominant marker) locus in a series of Chinese hamster cell lines. Ama^R mutants were obtained from different cell lines, e.g., CHO, CHW, M3-1 and CHO-Kl, at similar frequencies. After fractionation of different RNA polymerase activities in the extracts by chromatographic procedures, the sensitivity of the mutant RNA polymerase II towards α-amanitin was determined. While all of the RNA polymerase II activity in mutant CHO and CHO-Kl lines became resistant to α-amanitin inhibition, only about 50% of the activity is highly resistant in Ama^R mutants of CHW and M3-1 cell lines. The remaining activity in the latter cell lines shows α-amanitin sensitivity similar to that seen with the wild-type enzyme. This behaviour is similar to that observed with a 1:1 mixture of resistant and sensitive enzymes from CHO cells. These results, therefore, strongly indicate that while only one functional copy of the gene affected by α-amanitin is present in CHO and CHO-Kl cells, two copies of this gene are functional in the CHW and M3-1 cell lines.