Estimation of the effect of the acidosis and alkalosis on the anesthetic potency of local anesthetics by biopartitioning micellar chromatography and micellar electrokinetic chromatography.

Local anesthetics are hydrophobic compounds and weak bases with protonation constants ranged between 7.5 and 8.8. These drugs block reversibly nerve conduction near their site of application or injection and thus produce temporary loss of feeling or sensation in a limited area of the body. The efficacy of anesthetic blockade of local anesthetics depends on the charged/uncharged form ratio and the hydrophobicity of the compounds. In addition their toxicological effects have been reported to be highly dependent on the physiological pH. Biopartitioning micellar chromatography (BMC) and micellar electrokinetic chromatography (MEKC), that use micellar solutions as mobile phases, have proven to be useful for describing the biological behavior of different kind of compounds. In this paper, relationships between the retention data in BMC and MEKC using Brij35 as surfactant (at pH 7.4) and some pharmacodynamic parameters of local anesthetics are obtained. These models are compared with those obtained using an immobilized artificial column (IAM). Finally, the effect of the corporal pH in situations of acidosis and alkalosis on the pharmacological and toxicological properties of local anesthetics is studied using the retention of compounds in BMC at different mobile phase pH values.

Induction of L-lysine epsilon-aminotransferase by L-lysine in Streptomyces clavuligerus, producer of cephalosporins.

L-Lysine epsilon-aminotransferase (LAT) catalyzes the first reaction in the two-step conversion of L-lysine (Lys) to 1-alpha-aminoadipic acid (Aaa), a direct precursor of cephalosporins (including cephamycin C) in Streptomyces clavuligerus. Previous work showed that addition of Lys to chemically defined medium improved antibiotic production. We show that in S. clavuligerus cultures supplemented with high concentrations of Lys, Lys enhances antibiotic production by a dual effect, i.e. as a substrate of LAT thus providing Aaa and also as an inducer of LAT yielding even more Aaa. On the other hand, LAT is not induced by Aaa.

Buffering capacity and H+ membrane conductance of gram-negative bacteria.

Buffering capacity and membrane H+ conductance were examined in seven Gram-negative species: Aquaspirillum serpens, Pseudomonas aeruginosa, Alcaligenes faecalis, Escherichia coli, Salmonella typhimurium, Proteus mirabilis and Aeromonas hydrophila. All strains of Enterobacteriaceae studied here showed a decrease in both parameters as the external pH increased, over the pH range studied. The other four species presented an increase in buffering capacity and membrane conductance to protons as the external pH increased from 5.5 to 7.0.

Lysine epsilon-aminotransferase, the initial enzyme of cephalosporin biosynthesis in actinomycetes.

Streptomyces clavuligerus, Streptomyces lipmanii and Nocardia (formerly Streptomyces) lactamdurans are Gram-positive mycelial bacteria that produce medically important beta-lactam antibiotics (penicillins and cephalosporins including cephamycins) that are synthesized through a series of reactions starting from lysine, cysteine and valine. L-lysine epsilon-aminotransferase (LAT) is the initial enzyme in the two-step conversion of L-lysine to L-alpha-aminoadipic acid, a specific precursor of all penicillins and cephalosporins. Whereas S. clavuligerus uses LAT for cephalosporin production, it uses the cadaverine pathway for catabolism when lysine is the nitrogen source for growth. Although the cadaverine path is present in all examined streptomycetes, the LAT pathway appears to exist only in beta-lactam-producing strains. Genetically increasing the level of LAT enhances the production of cephamycin. LAT is the key rate-limiting enzyme in cephalosporin biosynthesis in S. clavuligerus strain NRRL 3585. This review will summarize information on this important enzyme.

Buffering capacity and membrane H+ conductance of Halobacterium halobium.

Buffering capacity and membrane H+ conductance were measured in Halobacterium halobium suspensions in the light and in the dark over a wide range of external pH. The values of both variables for this archaeobacterium were significantly higher than those found for eubacteria in other reports. It appears from our results that the special chemical composition of the cell envelope and the movement of ions, mainly protons, may influence the magnitude of the buffering power and the H+ membrane conductance of these cells.

Buffering capacity and membrane H+ conductance of neutrophilic and alkalophilic gram-positive bacteria.

Buffering capacity and membrane H+ conductance were examined in three gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis, and Bacillus alcalophilus. An acid pulse technique was used to measure both parameters. The buffering capacity and membrane H+ conductance of B. alcalophilus are influenced by the pH of the medium and the culture conditions. Suspensions of B. alcalophilus cells from both H. A. medium and L-malate medium cultures grown at pH 10.5 exhibited higher values for these parameters than cells grown at pH 8.5. B. alcalophilus grown aerobically had a lower buffering capacity and a lower membrane conductance for protons than the neutrophilic bacteria S. aureus and B. subtilis. Fermenting cells exhibited significantly higher values for both variables than respiring cells.

Buffering Capacity of Pigmented and Nonpigmented Strains of Serratia marcescens.

The pigmented strain Serratia marcescens ATCC 274 had a higher buffering capacity and a higher membrane H conductance than S. marcescens GP, a spontaneous nonpigmented mutant of ATCC 274. The data suggest that mutations which apparently affect only the synthesis of a secondary metabolite can modify buffering capacity and passive H conductance.

Rapid extracellular acidification induced by glucose metabolism in non-proliferating cells of Serratia marcescens.

The addition of glucose or other sugars to resting cells of Serratia maurcescens induced rapid acidification of the extracellular medium. This acidification was due to the catabolism of sugars. The rate of acidification depended on the carbon source and its concentration. HPLC analysis of the supernatants demonstrated that the progressive fall in pH resulted from the rapid production of lactic, acetic, pyruvic and citric acids. Other microorganisms were tested for their ability to produce this rapid acidification of the medium. This study may provide a rapid and simple method for metabolism studies.

The effect of pH on prodigiosin production by non-proliferating cells of Serratia marcescens.

The synthesis of prodigiosin by non-proliferating cells of Serratia marcescens was examined at various pH values between 5.5 and 9.5. During incubation in unbuffered medium, pH changed and prodigiosin production was similar regardless of the initial pH. Variations in pigment production were noted when buffers were employed in cultures of non-proliferating cells. The optimum pH for prodigiosin production was 8.0-8.5. Proline oxidase was also measured. The results suggest that the effect of pH may be related to the amount of proline which can be incorporated into prodigiosin.

Clonal population structure of Pseudomonas stutzeri, a species with exceptional genetic diversity.

Genetic diversity and genetic relationships among 42 Pseudomonas stutzeri strains belonging to several genomovars and isolated from different sources were investigated in an examination of 20 metabolic enzymes by multilocus enzyme electrophoresis analysis. Forty-two distinct allele profiles were identified, indicating that all multilocus genotypes were represented by a single strain. All 20 loci were exceptionally polymorphic, with an average of 15.9 alleles per locus. To the best of our knowledge, this P. stutzeri sample exhibited the highest mean genetic diversity (H = 0.876) found to date in all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles was identified. The index of association (I(A)) for the P. stutzeri strains analyzed was 1.10. The I(A) values were always significantly different from zero for all subgroups studied, including clinical and environmental isolates and strains classified as genomovar 1. These results suggest that the population structure of P. stutzeri is strongly clonal, indicating that there is no significant level of assortative recombination that might destroy linkage disequilibrium.

Partial purification, characterization and nitrogen regulation of the lysine epsilon-aminotransferase of Streptomyces clavuligerus.

The L-lysine epsilon-aminotransferase (LAT) of Streptomyces clavuligerus was partially purified and characterized. The 51.3-kDa enzyme exhibited optimal activity at pH 7.0-7.5 and 30 degrees C. It catalyzed transfer of the terminal amino group of L-lysine or L-ornithine to alpha-ketoglutarate. Oxalacetate and pyruvate were also used as acceptors of the amino group but with very low efficiency. Increasing ammonium concentrations added to chemically-defined medium MM enhanced the formation of LAT and decreased production of cephalosporins by S. clavuligerus. In cultures grown in the absence of lysine, greater enhancement of LAT formation by ammonium and less repression of cephalosporin biosynthesis were observed. In the chemically-defined GSPG medium, ammonium ions decreased cephalosporin production without showing an effect on LAT formation.