Characterization of an antibody-binding epitope from the 18-kDa protein on Mycobacterium leprae

A murine mAb, designated L5, appears to be specific for an epitope on a protein from Mycobacterium leprae of restricted distribution within the mycobacteria. This protein, of Mr 18,000 (18 kDa) is of interest because monoclonal antibodies raised against it do not appear to cross-react with other mycobacterial pathogens. The L5 antibody-binding epitope has been mapped by two complementary methods; expression of gene fragments and synthesis of short peptides. This L5-binding region of the 18-kDa protein (amino acids 109 to 115) shows some homology to a region of the GroEL heat shock family of proteins. Characterization of this antibody-binding epitope may lead to a reagent of use in early diagnosis of infection.     

Regionally selective alterations in enzymatic activities and metabolic fluxes during thiamin deficiency

To further elucidate the molecular basis of the selective damage to various brain regions by thiamin deficiency, changes in enzymatic activities were compared to carbohydrate flux through various pathways from vulnerable (mammillary bodies and inferior colliculi) and nonvulnerable (cochlear nuclei) regions after 11 or 14 days of pyrithiamin-induced thiamin deficiency. After 11 days,large decreases (–43 to –59%) in transketolase (TK) occurred in all 3 regions; 2-ketoglutarate dehydrogenase (KGDHC) declined (–45%), but only in mammillary bodies; pyruvate dehydrogenase (PDHC) was unaffected. By day 14, TK remained reduced by 58%–66%; KGDHC was now reduced in all regions (–48 to –55%); PDHC was also reduced (–32%), but only in the mammillary bodies. Thus, the enzyme changes did not parallel the pathological vulnerability of these regions to thiamin deficiency.¹⁴CO₂ production from¹⁴C-glucose labeled in various positions was utilized to assess metabolic flux. After 14 days, CO₂ production in the vulnerable regions declined severely (–46 to 70%) and approximately twice as much as those in the cochlear nucleus. Also by day 14, the ratio of enzymatic activity to metabolic flux increased as much as 56% in the vulnerable regions, but decreased 18 to 30% in the cochlear nuclei. These differences reflect a greater decrease in flux than enzyme activities in the two vulnerable regions. Thus, selective cellular responses to thiamin deficiency can be demonstrated ex vivo, and these changes can be directly related to alterations in metabolic flux. Since they cannot be related to enzymatic alterations in the three regions, factors other than decreases in the activity of these TPP-dependent enzymes must underlie selective vulnerability in this model of thiamin deficiency.Abbreviations KGDHC 2-ketoglutarate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.61, EC 1.6.4.3. - PDHC pyruvate dehydrogenase complex EC 1.2.4.2., EC 2.3.1.12, EC 1.6.4.3 - TK transketolase (EC 2.2.1.1) - TPP thiamin pyrophosphate     

Acute and Chronic Effects of Ethanol on Transbilayer Membrane Domains

Alcohols, including ethanol, have a specific effect on transbilayer and lateral membrane domains. Recent evidence has shown that alcohols in vitro have a greater effect on fluidity of one leaflet as compared to the other. The present study examined effects of chronic ethanol consumption on fluidity of synaptic plasma membrane (SPM) exofacial and cytofacial leaflets using trinitrobenzenesulfonic acid (TNBS) labeling and differential polarized fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). Mice were administered ethanol or a control liquid diet for 3 weeks. Animals were killed and SPM prepared. The exofacial leaflet of SPM was significantly more fluid than the cytofacial leaflet in both groups, as indicated by limiting anisotropy of DPH. However, differences between the two leaflets were much smaller in the ethanol-treated group. Ethanol at concentrations seen clinically had a greater effect in vitro on the more fluid exofacial leaflet. This asymmetric effect of ethanol was significantly diminished in the exofacial leaflet of the ethanol-treated mice. Chronic ethanol consumption has a specific effect on membranes. Membrane functions that may be regulated by asymmetry of fluidity and lipid distribution may be altered by chronic ethanol consumption.     

Metabolism of phenanthrene by the marine cyanobacterium Agmenellum quadruplicatum PR-6.

Under photoautotrophic growth conditions, the marine cyanobacterium Agmenellum quadruplicatum PR-6 metabolized phenanthrene to form trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) and 1-methoxyphenanthrene as the major ethyl acetate-extractable metabolites. Small amounts of phenanthrols were also formed. The metabolites were purified by high-pressure liquid chromatography and identified from their UV, infrared, mass, and proton magnetic resonance spectral properties. A. quadruplicatum PR-6 formed phenanthrene trans-9,10-dihydrodiol with a 22% enantiomeric excess of the (-)-9S,10S-enantiomer. Incorporation experiments with 18O2 showed that one atom of oxygen from O2 was incorporated into the dihydrodiol. Toxicity studies, using an algal lawn bioassay, indicated that 9-phenanthrol and 9,10-phenanthrenequinone inhibit the growth of A. quadruplicatum PR-6.     

Site-directed mutagenesis of beta-lactamase I. Single and double mutants of Glu-166 and Lys-73.

Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave 'burst' kinetics.     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.     

Molecular structure of rare but geographically widespread sn-glycerol-3-phosphate dehydrogenase ‘ultra-fast’ electrophoretic alleles in Drosophila melanogaster

Six naturally occurring but rare alleles of sn-glycerol-3-phosphate dehydrogenase (Gpdh) in Drosophila melanogaster have been investigated in this study. They all belong to a class of Gpdh ^UF (ultra-fast) alleles, because their electrophoretic mobilities are faster than that of the Gpdh ^F (fast) allele. The Gpdh ^UF variants are widespread, and have been reported from five continents. DNA sequence analysis has shown that the change in electrophoretic mobility was in each allele caused by a single amino acid residue substitution in the encoded protein. In the Xiamen ^UF allele it is a substitution of lysine (AAA) to asparagine (AAT) in exon 1 (residue 3). An asparagine (AAT) to aspartate (GAT) change was found in exon 6 (residue 336) in the Iowa ^UF and Netherlands ^UF alleles. The mobility of the Raleigh ^UF allele was altered by a valine (GTG) to glutamate (GAG) substitution in exon 3 (residue 76). Two mutations were detected in the Brazzaville ^UF allele: a lysine (AAG) to methionine (ATG) substitution in exon 2 (residue 68) is responsible for the ultra-fast phenotype of this variant, while a tyrosine (TAT) to phenylalanine (TTT) substitution in exon 4 (residue 244) is not expected to alter the electrophoretic mobility of the encoded protein. These results indicate that the Gpdh ^UF alleles originate from different mutational events, and only two of them — Iowa ^UF and Netherlands ^UF — might share a common ancestry. The GPDH activity of the Iowa ^UF allele is intermediate between those of the Gpdh ^S and Gpdh ^F control stocks. The other Gpdh ^UF variants have lower activities than the controls: Xiamen ^UF -83%, Raleigh ^UF -80% and Brazzaville ^UF -73% of the Gpdh ^F control.     

An experimental demonstration of trophic interactions affecting water quality of Rice Lake, Ontario (Canada)

Eight cylindrical enclosures (3 m diameter, 2.7 m long, V = 20m³) were installed in eutrophic Rice Lake (Ontario, Canada) in late spring of 1987. Fish (yearling yellow perch (Perca flavescens) and macrophytes (Potamogeton crispus) presence and absence were set at the beginning of the experiment to yield four combinations of duplicate treatments. The purpose of the experiment was to determine if the phytoplankton, zooplankton, macrophytes and fish species resident in the lake interact to influence water quality (major ions, phosphorus, algal densities and water clarity).The presence of fish was associated with: (1) decreased biomass of total zooplankton, (2) decreased number of species in the zooplankton, (3) decreased average size of several zooplankton taxa, (4) higher total phosphorus concentrations, (5) higher phytoplankton and chlorophyll a concentrations, (6) lower water clarity, (7) lower potassium levels during macrophyte die-back, (8) lower pH and higher conductivity in the presence of macrophytes. Biomass of large Daphnia species (but not total zooplankton) was highly correlated with the algal response (r ² = 0.995) and was associated with reduced biomass of several algal taxa including some large forms (Mougeotia, Oedogonium) and several colonial blue-green algae. However, no significant control of late summer growth of the bloom-forming blue-green alga Anabaena planctonica Brun. was achieved by the Daphnia presence-fish absence treatment. Release of phosphorus to the water column during the die-back of P. crispus was not an important phenomenon.