Control of circadian rhythm in nitrate reductase activity in Wolffia microscopica Griff

In Wolffia microscopica, the activity of nitrate reductase showedstrong diurnal rhythmicity with a peak in the forenoon. Therhythmicity persists under extended dark conditions as wellas under continuous light. The phase of the rhythm shifts underinverted light-dark cycles. Studies with various inhibitorsof macromolecular synthesis suggest that the increase in enzymeactivity depends on the new synthesis of both RNA and protein. (Received February 14, 1979; )     

Molecular Cloning and Nucleotide Sequence of the Gene for an Alkaline Protease from Bacillus circulans MTCC 7906

Bacillus circulans MTCC 7906, an extracellular alkaline protease producer was genetically characterized. B. circulans genomic DNA was isolated, oligonucleotide primers specific to alkaline protease gene of B. circulans were designed and its PCR amplification was done. The purified PCR product and pTrcHisA vector were subjected to restriction digestion with NcoI and HindIII and transformed into Escherichia coli DH5-α competent cells. The recombinant expression of alkaline protease gene studied by inducible expression and analysis by SDS-PAGE, established that the alkaline protease protein had an estimated molecular size of 46 kDa. Gene sequencing of the insert from selected recombinant clone showed it to be a 1329 bp gene encoding a protein of 442 amino acids. The sequence was blasted and aligned with known alkaline protease genes for comparison with their nucleotide and amino acid sequences. This identified major matches with three closely related subsp. of B. subtilis (B. subtilis subsp. subtilis strain 168, B. subtilis BSn5 and B. subtilis subsp. spizizenii strain W23). The insert also showed a number of substitutions (mutations) with other sp. of Bacillus which established that alkaline protease of B. circulans MTCC 7906 is a novel gene. The phylogenetic analysis of alkaline protease gene and its predicted amino acid sequences also validated that alkaline protease gene is a novel gene and the same has been accessioned in GenBank with accession number {"type":"entrez-nucleotide","attrs":{"text":"JN645176.1","term_id":"353260575","term_text":"JN645176.1"}}JN645176.1.     

Protein name tagging guidelines: lessons learned

Interest in information extraction from the biomedical literature is motivated by the need to speed up the creation of structured databases representing the latest scientific knowledge about specific objects, such as proteins and genes. This paper addresses the issue of a lack of standard definition of the problem of protein name tagging. We describe the lessons learned in developing a set of guidelines and present the first set of inter-coder results, viewed as an upper bound on system performance. Problems coders face include: (a) the ambiguity of names that can refer to either genes or proteins; (b) the difficulty of getting the exact extents of long protein names; and (c) the complexity of the guidelines. These problems have been addressed in two ways: (a) defining the tagging targets as protein named entities used in the literature to describe proteins or protein-associated or -related objects, such as domains, pathways, expression or genes, and (b) using two types of tags, protein tags and long-form tags, with the latter being used to optionally extend the boundaries of the protein tag when the name boundary is difficult to determine. Inter-coder consistency across three annotators on protein tags on 300 MEDLINE abstracts is 0.868 F-measure. The guidelines and annotated datasets, along with automatic tools, are available for research use.     

Ataxia and pancytopenia caused by a mutation in TINF2

Disseminated superficial actinic porokeratosis (DSAP) is a chronic autosomal dominant cutaneous disorder with high genetic heterogeneity. Two genetic loci for DSAP were identified, but no specific genes were reported to date. The pathogenic mechanism of this disorder remains to be elucidated. In this study, a large, five-generation Chinese family with DSAP was genetically characterized. Two known DSAP loci, DSAP1 and DSAP2, two DSAP candidate genes (SART3 and SSH1), one DSP-linked locus and one PPPD-linked locus were first excluded in the family. The family was then characterized by genome-wide linkage analysis and a new DSAP locus was identified on chromosome 1p31.3–p31.1 with a maximum two-point LOD score of 5.09 with marker D1S2897 (θ = 0). Fine mapping showed that the disease gene was located within an 8.2 cM or 11.9 Mb region between markers D1S438 and D1S464. This is the third locus identified for DSAP (DSAP3). Eight candidate genes including GNG12, IL12RB2, ITGB3BP, DNAJ6, PIN1L, GADD45A, RPE65 and NEGR1 were sequenced, but found to be negative for functional sequence variants. Further mutational analysis of the candidate genes in the region will identify the specific gene for DSAP, which will provide insights into the pathogenesis of DSAP.     

LncRNA VEAL2 regulates PRKCB2 to modulate endothelial permeability in diabetic retinopathy

Long non‐coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell function. Here, we investigated the role of a novel vascular endothelial‐associated lncRNA (VEAL2) in regulating endothelial permeability. Precise editing of veal2 loci in zebrafish (veal2 ^gib005Δ8/+) induced cranial hemorrhage. In vitro and in vivo studies revealed that veal2 competes with diacylglycerol for interaction with protein kinase C beta‐b (Prkcbb) and regulates its kinase activity. Using PRKCB2 as bait, we identified functional ortholog of veal2 in humans from HUVECs and named it as VEAL2. Overexpression and knockdown of VEAL2 affected tubulogenesis and permeability in HUVECs. VEAL2 was differentially expressed in choroid tissue in eye and blood from patients with diabetic retinopathy, a disease where PRKCB2 is known to be hyperactivated. Further, VEAL2 could rescue the effects of PRKCB2‐mediated turnover of endothelial junctional proteins thus reducing hyperpermeability in hyperglycemic HUVEC model of diabetic retinopathy. Based on evidence from zebrafish and hyperglycemic HUVEC models and diabetic retinopathy patients, we report a hitherto unknown VEAL2 lncRNA‐mediated regulation of PRKCB2, for modulating junctional dynamics and maintenance of endothelial permeability.     

The molecular genetic basis of age-related macular degeneration: an overview

Age-related macular degeneration (AMD) is a complex disorder of the eye and the third leading cause of blindness worldwide. With a multifactorial etiology, AMD results in progressive loss of central vision affecting the macular region of the eye in elderly. While the prevalence is relatively higher in the Caucasian populations, it has gradually become a major public health issue among the non-Caucasian populations (including Indians) as well due to senescence, rapidly changing demographics and life-style factors. Recent genome-wide association studies (GWAS) on large case-control cohorts have helped in mapping genes in the complement cascade that are involved in the regulation of innate immunity with AMD susceptibility. Genes involved with mitochondrial oxidative stress and extracellular matrix regulation also play a role in AMD pathogenesis. Majority of the associations observed in complement (CFH, CFB, C2 and C3) and other (ARMS2 and HTRA1) genes have been replicated in diverse populations worldwide. Gene-gene (CFH with ARMS2 and HTRA1) interactions and correlations with environmental traits (smoking and body mass index) have been established as significant covariates in AMD pathology. In this review, we have provided an overview on the underlying molecular genetic mechanisms in AMD worldwide and highlight the AMD-associated-candidate genes and their potential role in disease pathogenesis.     

Activation of cPLA2, PKC, and ERKs in the Rat Cerebral Cortex During Ischemia/Reperfusion

Release of the excitotoxic amino acid, glutamate, into the extracellular space during ischemia/reperfusion contributes to neuronal injury and death. To gain insights into the signal transduction pathways involved in glutamate release we examined the time course of changes in enzyme levels and activities of cPLA₂, PKC and ERKs in the rat cerebral cortex after four vessel (4VO) ischemia followed by reperfusion. Measurement both by enzymatic assay and Western blot analysis showed significant increases in the activity and protein levels of cPLA₂ during 10–20 min of ischemia. Activity remained elevated at 10 min and 20 min of reperfusion, whereas cPLA levels had returned to base line levels after 20 min of reperfusion. PKC activity increased significantly in the particulate, but not in the cytosolic, fractions both during ischemia and reperfusion. Increases in PKC levels were recorded in the particulate fraction during ischemia and reperfusion, and in the cytosolic fraction during ischemia. Western blot analysis with a phosphospecific antibody for characterization of MAPK (ERKs) activation revealed significantly increased phosphorylation of ERK₁, and ERK₂ in the particulate fraction, of ERK₂ in the cytosolic fraction, during ischemia and of both enzymes in the particulate and cytosolic fractions after 10 min of reperfusion. The relevance of the results to glutamate release is discussed.     

A comparative study on the graft copolymerization of acrylic acid onto rayon fibre by a ceric ion redox system and a γ-radiation method

Functionalization of rayon fibre has been carried out by grafting acrylic acid (AAC) both by a chemical method using a Ce⁴⁺-HNO₃ redox initiator and by a mutual irradiation (γ-rays) method. The reaction conditions affecting the grafting percentage have been optimized for both methods, and the results are compared. The maximum percentage of grafting (50%) by the chemical method was obtained utilizing 18.24 × 10⁻³ moles/L of ceric ammonium nitrate (CAN), 39.68 × 10⁻² moles/L of HNO₃, and 104.08 × 10⁻² moles/L of AAc in 20 mL of water at 45 °C for120 min. For the radiation method, the maximum grafting percentage (60%) was higher, and the product was obtained under milder reaction conditions using a lower concentration of AAc (69.38 × 10⁻² moles/L) in 10 mL of water at an optimum total dose of 0.932 kGy. Swelling studies showed higher swelling for the grafted rayon fibre in water (854.54%) as compared to the pristine fibre (407%), while dye uptake studies revealed poor uptake of the dye (crystal violet) by the grafted fibre in comparison with the pristine fibre. The graft copolymers were characterized by IR, TGA, and scanning electron micrographic methods. Grafted fibre, prepared by the radiation-induced method, showed better thermal behaviour. Comparison of the two methods revealed that the radiation method of grafting of acrylic acid onto rayon fibre is a better method of grafting in comparison with the chemical method.     

Detection of hereditary haemochromatosis in an HLA-identical pedigree showing discordance between HLA class I genes and the disease locus

Summary Hereditary haemochromatosis is a recessive disease in which primary hepatocellular carcinoma, complicating cirrhosis, is responsible for about one-third of deaths in affected homozygotes. We describe a unique HLA haplo-identical pedigree showing parent-to-off-spring transmission of hereditary haemochromatosis in whom HLA typing studies, including class I and class II allogenotype analysis, were of no benefit in identifying affected homozygotes. However, affected siblings in the pre-cirrhotic stage of haemochromatosis, with apparent discordance between the haemochromatosis allele and class I loci on chromosome 6, were detected by undertaking a family study, using analysis of serum parameters of iron status in combination with magnetic resonance imaging (MRI). This pedigree emphasises the critical importance of genetic and non-invasive methods for the identification of asymptomatic homozygotes before cirrhosis develops.