Gamma radiation induced mutagenesis in <Emphasis Type="Italic">Aspergillus niger</Emphasis> to enhance its microbial fermentation activity for industrial enzyme production

α- and β-Galactosidases find application in food processing, health and nutrition. Aspergillus niger is one of the potent producer of these enzymes and was genotypically improved using gamma-ray induced mutagenesis. The mutant-derivative produced two-fold higher α- and β-galactosidases. For testing genetic variability and its relationship with phenotypic properties of the two organisms, DNA samples of the mutant and parental strains of A. niger were amplified with 28 deca-nucleotide synthetic primers. RAPD analysis showed significantly different pattern between parental and mutant cultures. The mutant derivative yielded homogeneous while parental strain formed heterogeneous amplification patterns. Seven primers identified 42.9% polymorphism in the amplification products, indicating that these primers determined some genetic variability between the two strains. Thus RAPD was found to be an efficient technique to determine genetic variability in the mutant and wild organisms. Both wild and mutant strains were analyzed for their potential to produce galactosidases. Comparison of different carbon sources on enzyme yield revealed that wheat bran is significant (P < 0.01) effective producer and economical source followed by rice bran, rice polishing and lactose. The mutant was significantly better enzyme producer and could be considered for its prospective application in food, nutrition and health and that RAPD can be effectively used to differentiate mutant strain from the parental strain based on the RAPD patterns.     

Estimation of genetic distance based on RAPDs between 11 cotton accessions varying in heat tolerance

The genetic distance of 11 cotton genotypes varying in heat tolerance was studied using RAPD markers. Fifty-three random decamer primers were used for the estimation of genetic distance. Among the 53 RAPD primers, which were custom synthesized by GeneLink Inc., UK, 32 were polymorphic and 21 were monomorphic. The 32 polymorphic primers produced 273 fragments, with a mean of 8.3 fragments per primer. The number of polymorphic bands produced in the 11 cotton accessions ranged from 1 to 31. Primer GLC-20 produced 31 polymorphic bands, while two primers, GLB-5 and GLC-12, produced one polymorphic band each. A range of 88.89 to 42.48% genetic similarity was observed among the 11 cotton accessions. The highest genetic similarity was observed between FH-945 and BH-160 (88.89%), whereas the lowest value was found between NIAB-801/2 and FH-945 (42.48%). Unique amplification profiles were produced by most of the cultivars; the differences were sufficient to distinguish them from other genotypes. This confirms the efficacy of RAPD markers for the identification of plant genotypes. An accumulative analysis of amplified products generated by RAPDs was sufficient to assess the genetic diversity among the genotypes. This information should be helpful for formulating breeding and genome mapping programs.     

Imbalance between Neutrophil Elastase and its Inhibitor α1-Antitrypsin in Obesity Alters Insulin Sensitivity,Inflammation, and Energy Expenditure

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Highlights? Obese men and mice have decreased serum A1AT levels and increased NE activity ? Neutrophil infiltration and NE deposition occur in adipose tissue from HFD-fed mice ? NE^?/? and A1AT Tg mice have reduced HFD-induced insulin resistance and inflammation ? NE deletion increases AMPK signaling and fatty acid oxidation in liver and BAT     

Missense mutations (p.H371Y, p.D438Y) in gene CHEK2 are associated with breast cancer risk in women of Balochistan origin

CHEK2 encodes a serine/threonine-protein kinase which plays a critical role in DNA damage signaling pathways. CHEK2 directly phosphorylates and regulates the functions of p53 and BRCA1. Most women with breast and/or ovarian cancer are not carriers of mutant BRCA1 or BRCA2. Multiple studies have shown that a CHEK2*1100delC confers about a two-fold increased risk of breast cancer in unselected females and a tenfold increase in males. Moreover, studies have shown that first-degree relatives of bilateral breast cancer cases who carried the CHEK2*1100delC allele had an eight-fold increased risk of breast cancer. It has been suggested that CHEK2 functions as a low-penetrance susceptibility gene for cancers and multiplies the risks associated with other gene(s) to increase cancer risk. The main goal of this study was to evaluate and to compare the role of truncating mutations, splice junction mutations and rare missense substitutions in breast cancer susceptibility gene CHEK2. Present study was performed on 140 individuals including 70 breast cancer patients both with and without family history and 70 normal individuals. Written consent was obtained and 3 ml intravenous blood was drawn from all the subjects. DNA was extracted from all the samples through inorganic method published already. Primers were synthesized for all the 14 exons of CHEK2 gene. Coding and adjacent intronic sequences of CHEK2 gene were amplified and sequenced. Two genetic variants (p.H371Y, p.D438Y) were found in exon 10 and exon 11 of gene CHEK2 which were not found in any of the 70 control individuals from same geographical area and ethnic group. The genetic variant c.1312G>T (p.D438Y) identified in a patient with a family history of breast cancer. To our knowledge, this is first mutation scanning study of gene CHEK2 from Balochistan population.     

Molecular classification of Pakistani collared dove through DNA barcoding

Pakistan is bestowed by a diversified array of wild bird species including collared doves of which the taxonomy has been least studied and reported. DNA barcoding is a geno-taxonomic tool that has been used for characterization of bird species using mitochondrial cytochrome c oxidase I gene (COI). This study aimed to identify taxonomic order of Pakistani collared dove using DNA barcoding. Purposely herein, we present a phylogenetic analysis of Pakistani collared dove based on 650 base pairs of COI gene sequences. Analysis of phylogenetic tree revealed that Pakistani collared dove shared a common clade with Eurasian collared dove (Streptopelia decaocto) and African collared dove (Streptopelia roseogrisea) which indicated a super-species group in Streptopelia genus. This is the first report of molecular classification of Pakistani collared dove using DNA barcoding.     

Kinetics of batch single cell protein production from rice polishings with Candida utilis in continuously aerated tank reactors

Single cell protein was produced from the defatted rice polishings by fermentation with Candida utilis in an aerated 14-L fermentor to optimize bioprocess variables. Maximum values of specific growth rate coefficient (mu, h(-1)), cell mass yield (Y(X/S), g/g) and cell mass productivity (g/Lh) were 0.31, 0.65, and 1.24, respectively under optimized conditions of aeration rate (1 v.v(-1) m(-1)), dissolved oxygen (50%), corn steep liquor (5%), temperature (35 degrees C), and substrate concentration (90 g rice polishings/L) in yeast salt medium (pH 6.0). The kinetic parameters for 50-L fermentor under same conditions were 0.33 h(-1), 0.66 g/g, 1.33 g/Lh, 2.25 g/Lh, 1.23 g/Lh, 0.45 g/g substrate and 0.20 g/g cell h for mu, Y(X/S), Q(X), Q(S), Q(CP), Y(TP/S), and q(CP), respectively and were significantly higher than their respective values reported on C. utilis in batch culture studies. This biomass protein contained 23.6%, 32.75%, 11.50%, 12.95%, 10.5%, and 0.275% true protein, crude protein, crude fiber, ash, cellulose and RNA content respectively. This implied that the fermentation process could be up scaled to manufacture animal feed. Gross metabolizable energy content of dried SCP was 29,711 kcal/kg and indicated that the SCP could serve both as energy as well as a protein source. Yeast can replace expensive feed ingredients currently being incorporated in poultry feed and can reduce cost of poultry ration by 0.33 US dollars-0.51 US dollars/100 kg bag and improve the economics of feed production in our country.     

DNA fingerprinting of Pakistani buffalo breeds (Nili-Ravi, Kundi) using microsatellite and cytochrome b gene markers

Cytochrome b gene markers have been proved as an efficient and powerful tool for breed characterization and species identification of buffaloes. This study represents the substantial analysis of mitochondrial DNA variation in Pakistani buffalo breeds and provides information about their genetic diversity. In this study partial amplification of cytochrome b gene of 1,061 bp was done and sequencing results showed ten haplotypes. Comparing all fifty samples from two buffalo breeds of Pakistan, fifteen polymorphic sites were observed out of which, twelve codons 42, 71, 118, 120, 199, 235, 269, 297, 318, 327, 350, 355 of mitochondrial cytochrome b gene are monomorphic which translate same amino acids as in the reference protein sequence due to silent mutation while different in DNA sequence. Similarly three codons 163, 246, 337 of mitochondrial cytochrome b are polymorphic and different from the reference sequence with respect to DNA as well as protein sequence. For the further confirmation a panel of nine microsatellite markers was used with high polymorphism information content (PIC). The frequency distribution of these alleles varies from three to eight allele at locus CSSM66 and ILST029 respectively. The results obtained from this study may contribute to the establishment of routine genotyping service of buffalo breeds for buffalo farmers for animal forensic application in case of any dispute. Additionally this study may help for breed characterization and phylogeny of aforementioned breeds of buffalo.     

Primate-specific Long Non-coding RNAs and MicroRNAs

Non-coding RNAs (ncRNAs) are critical regulators of gene expression in essentially all life forms. Long ncRNAs (lncRNAs) and microRNAs (miRNAs) are two important RNA classes possessing regulatory functions. Up to date, many primate-specific ncRNAs have been identified and investigated. Their expression specificity to primate lineage suggests primate-specific roles. It is thus critical to elucidate the biological significance of primate or even human-specific ncRNAs, and to develop potential ncRNA-based therapeutics. Here, we have summarized the studies regarding regulatory roles of some key primate-specific lncRNAs and miRNAs.     

Mechanisms whereby glucose deprivation triggers metabolic preconditioning in the isolated rat heart

Transient glucose deprivation of the heart [GLU (-)] confers a preconditioning-like protection against subsequent ischemic/reperfusion (I/R). The mechanisms involved remain unclear. We hypothesized that GLU (-) would induce the classic ischemic preconditioning activated signaling cascade. Potential metabolic consequences and putative cell signaling events induced by transient glucose deprivation were evaluated as candidate mediators of this cardioprotection.Isolated glucose-perfused rat hearts were subjected to 30 min global ischemia followed by 30 min reperfusion (index I/R). Cardiac contractile recovery following I/R was used as the functional end-point in these studies. Metabolic preconditioning was stimulated by 15 min GLU (-) followed by 10 min glucose repletion prior to the index I/R. The potential metabolic consequences of GLU (-) were evaluated by using excess octanoate (11 mM OCT Hi) or 11 mM 2-deoxy-D-glucose (2-DG) in place of GLU (-) and by combining GLU (-) with fuels known to inhibit glycolysis supply (20 mM pyruvate or 1 mM octanoate, OCT Lo). The roles of -adrenoceptors, -adrenoceptors, adenosine receptors, protein kinase C (PKC) and mitochondrial K_ATP channels were investigated using inhibitors prazosin (10 M), propranolol (10 M), 8-(p-sulfophenyl) theophylline, (SPT 100 M), chelerythrine (CHEL 10 M) and 5-hydroxydecanoate (5 HD 100 M) respectively.GLU (-) increased mechanical recovery (59.8 ± 4.0 vs. 32.3 ± 4.7%; p < 0.01). Protection was abolished by pyruvate 26.6 ± 3.1; SPT 36.6 ± 3.0; CHEL 35 ± 4.8 or 5 HD 23.8 ± 3.3%. In a separate set of experiments, the specificity of SPT in this model was tested by preconditioning with adenosine (100 M) (34.7 ± 4 vs. control 16.8 ± 1.3%, p = 0.01) and blocking this protection with the same dose of SPT (16.3 ± 1.5%) used in the GLU (-) studies. Protection was unaltered by prazosin (50.2 ± 3.3%), propranolol (55.5 ± 4.0%), or OCT Lo (50.2 ± 2.5%). Protection was not mimicked by OCT Hi (35.6 ± 3.8%) or 2-DG (34 ± 4.3%).Transient glucose deprivation does not seem to achieve preconditioning-like cardioprotection by decreased glycolysis. Rather, the signal system may involve enhanced adenosine release, PKC, and activation of the mitochondrial K_ATP channel.