Carbonic anhydrase II deficiency: diagnosis and carrier detection using differential enzyme inhibition and inactivation.

Carbonic anhydrase (CA) I and II are soluble isozymes that represent the major nonhemoglobin proteins in the erythrocyte. We recently identified a deficiency of CA II as the enzymatic basis for the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Virtual absence of the CA II peak on high-performance liquid chromatography, of CA II esterase activity, and of immunoprecipitable CA II were demonstrated on extracts of red cell lysates from all patients studied. Reduced levels of CA II were found in obligate heterozygotes. Here, we present evidence that CA II in red cell lysates can be quantitated by measuring CO2 hydratase activity in the presence of inhibitors that selectively inhibit the activity of CA I to a much greater extent than that of CA II. This was done with iodide (anion binding) and bromopyruvic acid (alkylation), and the respective assays evaluated as diagnostic tools for CA II deficiency in human red cells. These techniques greatly simplify the quantitation of CA II in hemolysates and should make genetic diagnosis and counseling for the newly described inborn error of metabolism due to CA II deficiency generally available. They also allow quantitation of CA I in red cell lysates.     

Nature of the Complementation Products Formed by a Complementing Mutant of Neurospora crassa

The mutant am-14 produces no active nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase (GDH) and no protein showing immunological cross-reaction with the enzyme. Nevertheless, it shows complementation with several other am mutants in heterokaryons. Active GDH can be extracted from heterokaryons formed from am-14 and other mutants which, by themselves, produce more or less inactive varieties of the enzyme. The enzyme from am-14 + am-3 heterokaryons can be partially separated from am-3 mutant GDH on a diethylaminoethyl cellulose column. It is characterized by abnormally high thermolability and by a capacity for activation by glutamate. By the same procedure as brings about hybridization between mutant GDH proteins, it has been possible to recover enzyme with the properties of pure am-3 GDH from a partially purified am-14 + am-3 GDH preparation which was initially substantially free of unhybridized am-3 enzyme. This is interpreted as evidence that the active complementation product is a hybrid oligomer containing am-3 monomers and also am-14 monomers, the latter being unable to aggregate by themselves. Heterokaryons formed from am-14 and wild type produce GDH of abnormally high thermolability, presumably due to the formation of am-14 + am(+) hybrids.     

Nonenzymatic Glycosylation of Lepidopteran-Active Bacillus thuringiensis Protein Crystals

We used high-pH anion-exchange chromatography with pulsed amperometric detection to quantify the monosaccharides covalently attached to Bacillus thuringiensis HD-1 (Dipel) crystals. The crystals contained 0.54% sugars, including, in decreasing order of prevalence, glucose, fucose, arabinose/rhamnose, galactose, galactosamine, glucosamine, xylose, and mannose. Three lines of evidence indicated that these sugars arose from nonenzymatic glycosylation: (i) the sugars could not be removed by N- or O-glycanases; (ii) the sugars attached were influenced both by the medium in which the bacteria had been grown and by the time at which the crystals were harvested; and (iii) the chemical identity and stoichiometry of the sugars detected did not fit any known glycoprotein models. Thus, the sugars detected were the product of fermentation conditions rather than bacterial genetics. The implications of these findings are discussed in terms of crystal chemistry, fermentation technology, and the efficacy of B. thuringiensis as a microbial insecticide.     

Suppressors of a Lin-12 Hypomorph Define Genes That Interact with Both Lin-12 and Glp-1 in Caenorhabditis Elegans

The lin-12 gene of Caenorhabditis elegans is thought to encode a receptor which mediates cell-cell interactions required to specify certain cell fates. Reversion of the egg-laying defective phenotype caused by a hypomorphic lin-12 allele identified rare extragenic suppressor mutations in five genes, sel-1, sel-9, sel-10, sel-11 and sel(ar40) (sel = suppressor and/or enhancer of lin-12). Mutations in each of these sel genes suppress defects associated with reduced lin-12 activity, and enhance at least one defect associated with elevated lin-12 activity. None of the sel mutations cause any obvious phenotype in a wild-type background. Gene dosage experiments suggest that sel-1 and sel(ar40) mutations are reduction-of-function mutations, while sel-9 and sel-11 mutations are gain-of-function mutations. sel-1, sel-9, sel-11 and sel(ar40) mutations do not suppress amorphic lin-12 alleles, while sel-10 mutations are able to bypass partially the requirement for lin-12 activity in at least one cell fate decision. sel-1, sel-9, sel-10, sel-11 and sel(ar40) mutations are also able to suppress the maternal-effect lethality caused by a partial loss-of-function allele of glp-1, a gene that is both structurally and functionally related to lin-12. These sel genes may therefore function in both lin-12 and glp-1 mediated cell fate decisions.     

1,25-dihydroxyvitamin D3 and macrophage colony-stimulating factor-1 synergistically phosphorylate talin

Macrophage colony stimulating factor (CSF-1) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are potent inducers of macrophage differentiation. Both appear to modulate protein phosphorylation, at least in part, through protein kinase C (PKC) raising the question as to whether they concurrently impact on macrophage-like cells. In this regard, we utilized the CSF-1 dependent murine macrophage-like line BAC 1.25F5. CSF-1 treatment of these cells for 30 min leads to particular phosphorylation of a 165 kDa protein, the putative CSF-1 receptor, and a 210 kDa moiety. 1,25(OH)₂D₃ exposure for 24 h prior to addition of CSF-1 enhances phosphorylation of the 165 kDa species and, especially, the 210 kDa protein. Phosphorylation of the latter protein is 1,25(OH)₂D₃ dose- and time-dependent and the molecule is specifically immunoprecipitated with a rabbit polyclonal anti-talin antibody. Experiments with okadaic acid show that the enhanced phosphorylation of talin does not result from serine phosphatase inhibition. CSF-1 and 1,25(OH)₂D₃, alone or in combination, do not increase talin protein expression. The tyrosine kinase inhibitor, genestein, blocks 1,25(OH)₂D₃/CSF-1 induced phosphorylation of the putative CSF-1 receptor but has no effect on talin phosphorylation which occurs exclusively on serine. In contrast to genestein, staurosporin, an inhibitor of PKC, inhibits phosphorylation of talin. Moreover, exposure of 1,25(OH)₂D₃ pretreated cells to phorbol 12-myristate 13-acetate (PMA) in place of CSF-1 also prompts talin phosphorylation. Finally, 1,25(OH)₂D₃ enhances ³[H]PDBu binding, indicating that the steroid increases PMA receptor capacity. Thus, CSF-1 and 1,25(OH)₂D₃ act synergistically via PKC to phosphorylate talin, a cytoskeletal-associated protein.     

Fungal spores are an important component of library air

The airborne fungal spore types were studied in different libraries in Delhi, using an Andersen sampler and a Burkard personal sampler, for culturable and non-culturable fungi respectively. The concentration inside the libraries, before and after the agitation of books, were compared with outside air. The major contributors to the library air areCladosporium, aspergilli/penicillia, smuts andAlternaria, varying from 50 to 14%. Some fungi (Cladosporium, Alternaria, smut,Penicillium chrysogenum andnigricans) showed seasonal occurrence, corresponding to their occurrence in the extramural environment. Aspergilli/penicillia,Drechslera, Curvularia andAspergillus flavus had a significantly higher concentration (P<0.01) inside the library, and recorded a significant increase in concentration after agitation of books. Air-conditioned libraries have low fungal spore concentrations, as compared to naturally ventilated libraries.     

Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp.

Isocitrate lyase was isolated in homogeneous state from a thermophilic Bacillus. The enzyme has a mol.wt. of 180000 and a pI of 4.5 and contains threonine as the N-terminal residue. It resembles in size the cognate enzyme from the mesophilic bacterium Pseudomonas indigofera, but is smaller than the enzyme from the eukaryotic fungus Neurospora crassa. All three lyases are tetramers and similar in amino acid composition, but the thermophile enzyme is distinctive from its mesophilic coutnerparts in possessing a lower catalytic-centre activity, greater resistance to chemical and thermal denaturation and fewer thiol groups and in being strongly activated by salts. Salt activation, by 0.4M-KCl, is about 3-fold at 30 degrees C and pH 6.8 and weakens progressively as the temperature or pH is raised. The activation is probably due to a change in the enzyme conformation caused by the electrolyte modifying the interaction between charged groups or between hydrophobic groups in protein. The possible significance of the salt activation, of the relative paucity of thiol groups and of the greater resistance to chemical denaturants is discussed. Besides its effect on the Vmax., KCl produces large increases in the magnitude of several kinetic parameters. A rise in reaction temperature from 30 to 55 degrees C produces a somewhat similar result. In view of these peculiar features, the patterns of inhibition of enzyme activity by compounds such as succinate and phosphoenolpyruvate were examined at 30 and 55 degrees C in the presence and absence of KCl.     

Tryptophan uptake by Mycobacterium tuberculosis H37Rv--inhibition by isoniazid.

The effect of various sub-inhibitory concentrations of isoniazid on tryptophan uptake by $\text{Mycobacterium tuberculosis}$ H₃₇Rv grown $\text{in vitro}$ and $\text{in vivo}$ was studied. Uptake, measured after 3 minutes of drug exposure was inhibited mildly by 0.1 μg/ml and 0.2 μg/ml concentration and completely by 0.3 μg/ml. However, with the minimal inhibitory concentration (MIC)⁷ of 0.5 μg/ml, not only inhibition but also a strong efflux of the preformed tryptophan pool were observed. The results are discussed in the light of the theory that isoniazid interferes with the cell wall mycolate synthesis.