Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

Reduction of hexavalent chromium in a reconstituted system of cytochrome P-450 and cytochrome b5

The reduction of hexavalent chromium (Cr(VI] by the monooxygenase components was studied. Both a reconstituted system of cytochrome P-450 (P-450) and cytochrome b5 (b5) with NADPH was capable of reducing Na2CrO4 (30 microM) provided anaerobic atmosphere. The rates were 1.29 nmol Cr.min-1 nmol P-450(-1) and 0.73 nmol Cr.min-1 nmol b5(-1). Using NADH instead of NADPH gave very low reducing activities, confirming the enzymic nature of the P-450 dependent Cr(VI) reductase reaction. Oxygen, 22% (air) and 0.1% gave 89% and 69% inhibition of Cr(VI) reducing activity, respectively. Carbon monoxide (100%) caused an inhibition of about 37% and 44% for P-450 and b5, respectively. Externally added flavin mononucleotide (FMN) (3 microM) or Fe-ADP (10 microM) to the complete system stimulated the enzymatic reaction about 2-fold and 3-fold, respectively.     

Continuous-Culture Responses of Candida shehatae to Shifts in Temperature and Aeration: Implications for Ethanol Inhibition

Temperature and aeration shifts were used to perturb steady-state continuous cultures to determine the effects of ethanol on xylose metabolism by Candida shehatae. The accumulation of ethanol exerted a delayed inhibitory effect on the specific rate of substrate utilization. A second effect was also observed in which the specific rate of xylitol production increased at the expense of the specific rate of ethanol production. Both effects were enhanced at higher temperature. Inhibitory effects also occurred in glucose metabolism.     

Comparison of glycoconjugates at the surface of developing type II pneumocytes and Clara cells

Summary The apical surface coat of type II pneumocytes and Clara cells in pre- and post-natal rat lung was examined with lectin histochemical methods. Lectins fromHelix pomatia (HPA), peanut (PNA) andMaclura pomifera (MPA) were conjugated with horseradish peroxidase and used to stain paraffin sections of fixed lung with or without certain pre-treatments. HPA and MPA were observed to react with almost all type II pneumocytes at postnatal day 1. Type II pneumocytes that stained with a sialidase—PNA sequence increased from a few positive cells at postnatal day 5 to many in the adult. It has been reported that the surface coat of type II pneumocytes closely resembles that of Clara cells in its staining with histochemical methods employing cationic dyes or lectins including MPA and PNA. However, staining with HPA, especially after periodic acid oxidation, revealed many type II pneumocytes with strong reactivity but showed only a few Clara cells that were faintly positive. HPA also stained alveolar macrophages. The HPA affinity of macrophages, however, was labile to oxidation with periodic acid or galactose oxidase unlike that of type II pneumocytes. This difference suggests that HPA recognizes more than one type of sugar structure.To whom all correspondence and reprint requests should be addressed.     

Can we determine the biological availability of sediment-bound trace elements?

It is clear from available data that the susceptibility of biological communities to trace element contamination differs among aquatic environments. One important reason is that the bioavailability of metals in sediments appears to be altered by variations in sediment geochemistry. However, methods for explaining or predicting the effect of sediment geochemistry upon metal bioavailability are poorly developed. Experimental studies demonstrate that ingestion of sediments and uptake from solution may both be important pathways of metal bioaccumulation in deposit/detritus feeding species. Relative importance between the two is geochemistry dependent. Geochemical characteristics of sediments also affect metal concentrations in the tissues of organisms collected from nature, but the specific mechanisms by which these characteristics influence metal bioavailability have not been rigorously demonstrated. Several prerequisites are necessary to better understand the processes that control metal bioavailability from sediments. 1) improved computational or analytical methods for analyzing distribution of metals among components of the sediments; 2) improved computational methods for assessing the influences of metal form in sediments on sediment-water metal exchange; and 3) a better understanding of the processes controlling bioaccumulation of metals from solution and food by metazoan species directly exposed to the sediments. Such capabilities would allow mechanistic explanations essential to the development of practical tools sought for determining sediment quality criteria for metals.     

New antifungal agents for the systemic mycoses

The azoles are the prominent broad spectrum oral antifungal agents in use or under clinical investigation for the systemic mycoses. This class of antifungal agents is represented by the marketed drug ketoconazole (Nizoral) and the experimental triazoles furthest along in clinical trials in the United States, itraconazole and fluconazole. Ketoconazole use is limited by its side effect profile and activity spectrum. Itraconazole appears to be better tolerated and less toxic to liver function, does not cause adrenal suppression and is more active against Aspergillus and Sporothrix schenckii. Fluconazole appears to be a highly promising agent due its highly favorable pharmacokinetic profile; it is water soluble, is well tolerated, is not metabolized to inactive constituents, it has a long half-life and, unlike the other azoles, high cerebrospinal fluid levels are readily attained for consideration in meningeal mycoses. It remains to be determined what place these new triazoles have in managing immunosuppressed patients including those with acquired immune deficiency syndrome known as AIDS. Other experimental antifungal agents, including ambruticin, amphotericin B methyl ester and saramycetin are also described. Sales figures are presented of drugs marketed in the United States for the systemic mycoses and reflect the growing problem of fungal diseases in the population.Presented as part of the Everett S. Beneke Symposium in Mycology, May 27, 1988.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Enzymatic evidence for involvement of the methylmalonyl-CoA pathway in propionate oxidation by Syntrophobacter wolinii

Enzyme measurements were carried out with crude cell-free extracts of the propionate oxidizing coculture of Syntrophobacter wolinii and Desulfovibrio G11. Using cell-free extracts of a pure culture of Desulfovibrio G11 as a blank, most of the enzymes involved in the methylmalonyl-CoA pathway for propionate oxidation, including a propionyl-CoA: oxaloacetate transcarboxylase, were demonstrated in S. wolinii.