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.     

Unique regulation of anion/HCO3- exchangers by constitutive nitric oxide in rabbit small intestine

In the rabbit small intestine, there are three functionally different brush-border membrane (BBM) anion/HCO3- exchangers: 1) Cl/HCO3- exchange on the BBM of villus cells responsible for coupled NaCl absorption; 2) Cl/HCO3- exchange on the BBM of crypt cells possibly involved in HCO3- secretion; and 3) short-chain fatty acid (SCFA)/HCO3- exchange on the BBM of villus cells, which facilitates SCFA absorption. Although constitutive nitric oxide (cNO) has been postulated to alter many gastrointestinal tract functions, how cNO may specifically alter these three transporters is unknown. Inhibition of cNO synthase with NG-nitro-L-arginine methyl ester (L-NAME) 1) did not affect villus cell BBM Cl/HCO3 change, 2) stimulated crypt cell BBM Cl/HCO3- exchange, and 3) inhibited villus cell BBM SCFA/HCO3- exchange. D-NAME, an inactive analog of L-NAME, and L-N6-(1-iminoethyl)lysine, a more selective inhibitor of inducible NO, did not affect these transport processes. Kinetic studies demonstrated that 1) the mechanism of inhibition of crypt cell BBM Cl/HCO3- exchange is secondary to a decrease in the maximal rate of uptake of Cl, without an alteration in the affinity of the transporter for Cl, and 2) the mechanism of stimulation of villus cell BBM SCFA/HCO3- exchange is secondary to an increase in the affinity of the transporter for SCFA without an alteration in the maximal rate of uptake of SCFA. These results indicate that cNO uniquely regulates the three BBM anion/HCO3- transporters in the rabbit small intestine.     

Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.     

Chromosomal Location of a Gene Defining Nicotinamide Deamidase in Escherichia coli

A gene specifying the enzyme nicotinamide deamidase has been mapped, by the interrupted mating technique, at about 33 min on the chromosome of Escherichia coli K-12.     

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.     

Simple efficient methods for the isolation of malate dehydrogenase from thermophilic and mesophilic bacteria.

Malate dehydrogenase from a number of bacteria drawn from several genera and representing the mesophilic, moderately thermophilic and extremely thermophilic classes was isolated by procedures which involve only a small number of steps (in most cases only two), of which the key one is affinity chromatography on 5'-AMP--Sepharose and/or on NAD+--hexane--agarose. Electrophoretic analysis of the native enzymes in polyacrylamide gel and of the denaturated enzymes in sodium dodecyl sulphate/polyacrylamide gel revealed no significant protein impurity in the purified preparations. The yields ranged from about 40% to over 80%. The malate dehydrogenases from the extreme thermophiles and from some of the moderate thermophiles are appreciably less efficient catalytically than their mesophilic homologues.     

Morphological and molecular screening of rice germplasm lines for low soil P tolerance

Phosphorus (P) is an essential macronutrient to all crops including rice and it plays a key role in various plant activities and development. Low availability of P in the soils negatively, influences rice crop growth and causes significant yield loss. In the present study, we characterized a set of 56 germplasm lines for their tolerance to low soil P by screening them at low soil P and optimum soil P levels along with low soil P tolerant and sensitive check varieties. These lines were genotyped for the presence/absence of tolerant allele with respect to the major low soil P tolerance QTL, Pup1, using a set of locus specific PCR-based markers, viz., K46-1, K46-2, K52 and K46CG-1. High genetic variability was observed for various traits associated with low soil P tolerance. The yield parameters from normal and low soil P conditions were used to calculate stress tolerance indices and classify the genotypes according to their tolerance level. Out of the total germplasm lines screened, 15 lines were found to be tolerant to low soil P condition based on the yield reduction in comparison to the tolerant check, but most of them harbored the complete or partial Pup1 locus. Interestingly, two tolerant germplasm lines, IC216831 and IC216903 were observed to be completely devoid of Pup1 and hence they can be explored for new loci underlying low soil P tolerance.

     

Ecology and Impacts of the Invasive Species, Lantana camara, in a Social-Ecological System in South India: Perspectives from Local Knowledge

We explored how the forest-dwelling Soliga community of South India views and explains biological invasions, and how local knowledge can inform scientific knowledge on biological invasions. We used an interview schedule with open-ended questions to solicit Soliga opinion on Lantana camara (lantana) invasion. The Soliga cited three reasons for lantana spread: its prolific fruit output and wide seed dispersal, change in fire management, and historical extraction of grass and bamboo. The Soliga believe that lantana invasion has had negative effects on the ecosystem and their livelihoods. Tabling scientific knowledge with local knowledge has improved our understanding of lantana invasion. The role of existing lantana in colonizing neighboring areas, and the response of native tree communities to lantana were common to both local and scientific sources. However, the Soliga view provides a more nuanced perspective of the lantana-fire relationship (contextually based on lantana density) with fires suppressing lantana when lantana density was low. This is contrary to views held by foresters and biologists, that fires are uniformly detrimental and promote lantana. Our study shows that examining Soliga observations has improved understanding of the invasion process and presents avenues for future lantana management.     

RNAi‐mediated Ultra‐low gossypol cottonseed trait: performance of transgenic lines under field conditions

Cottonseed remains a low‐value by‐product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra‐low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ‐cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild‐type levels of gossypol and related terpenoids. Although it was a relatively small‐scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild‐type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%–8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi‐based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever‐increasing needs of humanity.