Prevalence of vitamin B12 deficiency in healthy Indian school-going adolescents from rural and urban localities and its relationship with various anthropometric indices: a cross-sectional study.

BACKGROUND: Micronutrient deficiency is a global health burden, especially among developing countries. The present cross-sectional study aimed to determine the prevalence of vitamin B12 deficiency in healthy Indian school-going adolescents, based on area of residence, sex and body mass index (BMI). Furthermore, the relationship of serum B12 concentration with dietary vitamin B12 intake and anthropometric indices was assessed among adolescents from rural and urban India. METHODS: A total of 2403 school-going adolescents (11-17 years) from National Capital Region and rural areas of Haryana, India were selected. Serum B12 concentrations were estimated using an electrochemiluminescence immunoassay. Dietary assessments were conducted on 65% of total participants (n = 1556) by two 24-h diet recalls. RESULTS: The prevalence of vitamin B12 deficiency in the total study population was 32.4% (rural: 43.9% versus urban: 30.1%, P < 0.001; male: 34.4% versus female: 31.0%, P < 0.05; normal weight: 28.1%, versus overweight: 39.8%, versus obese: 51.2%, P < 0.001). More than half (51.2%) of obese adolescents were vitamin B12 deficient. On multiple linear regression analysis, serum B12 in rural adolescents was associated with age (ß = -0.12, P < 0.05). Among urban adolescents, serum B12 was associated with BMI (ß = -0.08, P < 0.05) and adjusted dietary vitamin B12 intake (ß = 0.14, P < 0.001). Serum vitamin B12 levels were found to be lower in rural females (ß = -0.12, P = 0.030) and urban males (ß: 0.11, P < 0.001) compared to their respective contemporaries. CONCLUSIONS: Vitamin B12 deficiency was higher among rural school-going adolescents. Boys had a higher B12 deficiency than girls. Inverse associations of serum B12 with adiposity indices were observed. Serum B12 levels were positively associated with dietary vitamin B12 intake.

De Novo Assembly of Bitter Gourd Transcriptomes: Gene Expression and Sequence Variations in Gynoecious and Monoecious Lines.

Bitter gourd (Momordica charantia L.) is a nutritious vegetable crop of Asian origin, used as a medicinal herb in Indian and Chinese traditional medicine. Molecular breeding in bitter gourd is in its infancy, due to limited molecular resources, particularly on functional markers for traits such as gynoecy. We performed de novo transcriptome sequencing of bitter gourd using Illumina next-generation sequencer, from root, flower buds, stem and leaf samples of gynoecious line (Gy323) and a monoecious line (DRAR1). A total of 65,540 transcripts for Gy323 and 61,490 for DRAR1 were obtained. Comparisons revealed SNP and SSR variations between these lines and, identification of gene classes. Based on available transcripts we identified 80 WRKY transcription factors, several reported in responses to biotic and abiotic stresses; 56 ARF genes which play a pivotal role in auxin-regulated gene expression and development. The data presented will be useful in both functions studies and breeding programs in bitter gourd.

Prospective Study of Saline Infusion Sonography and Office Hysteroscopy.

BACKGROUND: To evaluate the diagnostic potential of saline infusion sonography to pick up uterine cavity lesions and compare it with the gold standard office hysteroscopy. METHODS: Study population consisted of women scheduled for office hysteroscopy for various indications. Uterine cavity of 208 women of the study group were evaluated first by saline infusion sonography and then by office hysteroscopy by two separate examiners. Findings were recorded separately for both procedures and compared. Pain rating was also noted after each procedure. RESULT: In eight patients either or both the procedures could not be performed for various reasons, hence were excluded from the analysis. With saline infusion sonography, pathological findings were identified in 93 (46.5%) patients and hysteroscopy detected lesions in 88 (44%) patients. For all findings combined sensitivity of saline infusion sonography was 90.9%, specificity 88.3%, positive predictive value 86.0% and negative predictive value 92.5% as compared to hysteroscopy. Former was less painful and easier to perform than the latter. CONCLUSION: The findings of saline infusion sonography and office hysteroscopy did not differ significantly. Thus saline infusion sonography is an excellent option for uterine cavity evaluation.

Novel missense mutation in the coagulation factor IX catalytic domain associated with severe haemophilia B--Factor IXDelhi.

Factor IX is a vitamin K-dependent serine protease, which exists as a zymogen in the blood. On activation to factor IXa, by factor XIa or tissue factor-factor VIIa complex, it forms tenase complex with factor VIIIa, in the presence of Ca2+. This tenase complex enzymatically converts factor X to factor Xa, thereby bringing about the coagulation cascade. Mutations in factor IX gene have been shown to cause haemophilia B, which is inherited as an X-linked recessive disorder. Herein we report a novel missense mutation at the nucleotide position 30829-T > A in the exon 8 of factor IX gene. This transversion leads to the substitution of histidine 236 to glutamine. This resulting abnormal protein has been named factor IXDelhi. Molecular modelling was performed to predict the molecular pathology of this mutation. We predict that this change in the catalytic domain may affect the surface loop that accommodates Ca2+, thereby leading to severe bleeding disorder.

Serum amyloid P component binds to Fc gamma receptors and opsonizes particles for phagocytosis.

Serum amyloid P component (SAP) is a member of the pentraxin family of proteins. These proteins are characterized by cyclic pentameric structure, calcium-dependent ligand binding, and frequent regulation as acute-phase serum proteins. SAP is the serum precursor of the P component of amyloid. It binds to a broad group of molecules, including autoantigens, through a pattern recognition binding site. The related pentraxin, C-reactive protein (CRP), is a strong acute-phase reactant in man and an opsonin. We previously determined that the binding of CRP to leukocytes occurs through Fc receptors for IgG (FcgammaR). We now report that SAP also binds to FcgammaR and opsonizes particles for phagocytosis by human polymorphonuclear leukocytes (PMN). Specific, saturable binding of SAP to FcgammaRI, FcgammaRIIa, and FcgammaRIIIb expressed on transfected COS cells was detected using SAP-biotin and PE-streptavidin. Zymosan was used to test the functional consequences of SAP and CRP binding to FcgammaR. Both SAP and CRP bound to zymosan and enhanced its uptake by PMN. This enhanced phagocytosis was abrogated by treatment of PMN with wortmannin, a phosphatidylinositol-3 kinase inhibitor, or with piceatannol, a Syk inhibitor, consistent with uptake through FcgammaR. Treatment of PMN with phosphatidylinositol-specific phospholipase C to remove FcgammaRIIIb also decreased phagocytosis of SAP-opsonized zymosan, but not CRP-opsonized zymosan. These results suggest that SAP may function in host defense. In addition, as SAP binds to chromatin, a major immunogen in systemic lupus erythematosus, it may provide a clearance mechanism for this Ag through FcgammaR bearing cells.

C-reactive protein binding to FcgammaRIIa on human monocytes and neutrophils is allele-specific.

C-reactive protein (CRP) is involved in host defense, regulation of inflammation, and modulation of autoimmune disease. Although the presence of receptors for CRP on phagocytes has been inferred for years, their identity was determined only recently. FcgammaRIa, the high-affinity IgG receptor, binds CRP with low affinity, whereas FcgammaRIIa, the low-affinity IgG receptor, binds CRP with high affinity. Because the single nucleotide polymorphism in FcgammaRIIA - which encodes histidine or arginine at position 131 - strongly influences IgG2 binding, we determined this polymorphism's effect on CRP binding. CRP bound with high avidity to monocytes and neutrophils from FcgammaRIIA R-131 homozygotes, and binding was inhibited by the R-specific mAb 41H16. CRP showed decreased binding to cells from FcgammaRIIA H-131 homozygotes (which bind IgG2 with high affinity). However, IFN-gamma enhanced FcgammaRI expression by H-131 monocytes and increased CRP binding. FcgammaRIIa heterozygotes showed intermediate binding. CRP initiated increases in [Ca(2+)](i) in PMN from R-131, but not from H-131 homozygotes. These data provide direct genetic evidence for FcgammaRIIa as the functional, high-affinity CRP receptor on leukocytes while emphasizing the reciprocal relationship between IgG and CRP binding avidities. This counterbalance may affect the contribution of FcgammaRIIA alleles to host defense and autoimmunity.

The major receptor for C-reactive protein on leukocytes is fcgamma receptor II.

C-reactive protein (CRP) is an acute phase serum protein that shares several functions with immunoglobulin (Ig)G including complement activation and binding to receptors on monocytes and neutrophils. The identity of the receptor for CRP has been the target of extensive research. We previously determined that CRP binds to the high affinity receptor for IgG, FcgammaRI (CD64). However, this interaction could not account for the majority of binding of CRP to neutrophils or monocytic cells. We now determine that CRP also interacts with FcgammaRIIa (CD32), the low affinity receptor for IgG on monocytes and neutrophils. COS-7 cells were transfected with a construct containing the human FcgammaRIIA cDNA. CRP binding and the presence of CD32 were detected by mAb and analyzed by two-color flow cytometry. Cells expressing CD32 bound CRP in a dose-dependent and saturable manner consistent with receptor binding. CRP bound to transfectants and K-562 cells with similar kinetics, and in both cases binding was completely inhibited by aggregated IgG. On monocytic cell lines, treatment with Bt(2)cAMP increased FcgammaRII expression and enhanced CRP binding. CRP also specifically precipitated FcgammaRI and FcgammaRII from the monocytic cell line, THP-1. It is suggested that the major receptor for CRP on phagocytic cells is FcgammaRII.

Factor VII central. A novel mutation in the catalytic domain that reduces tissue factor binding, impairs activation by factor Xa, and abolishes amidolytic and coagulant activity.

Factor VII is a vitamin K-dependent zymogen of a serine protease that participates in the initial phase of blood coagulation. A factor VII molecular variant (factor VII Central) was identified in a 24-year-old male with severe factor VII deficiency and whose plasma factor VII antigen was 38% of normal, but expressed <1% factor VII procoagulant activity. DNA sequence analysis of the patient's factor VII gene revealed a thymidine to cytidine transition at nucleotide 10907 in exon VIII that results in a novel amino acid substitution of Phe328 to Ser. The patient was homozygous for this mutation, whereas each parent of the patient was heterozygous for this mutation. To investigate the molecular properties of this variant, a recombinant F328S factor VII mutant was prepared and analyzed in relation to wild-type factor VII. F328S factor VII exhibited <1% factor VII procoagulant activity and a 2-fold decreased affinity for tissue factor and failed to activate factor X or IX in the presence of tissue factor following activation by factor Xa. In addition, F328S factor VIIa exhibited no detectable amidolytic activity in the presence of tissue factor. The rate of F328S factor VII activation by factor Xa was markedly decreased relative to the rate of wild-type factor VII activation as revealed by densitometry scanning of SDS gels. Temporal analysis of this reaction by SDS-polyacrylamide gel electrophoresis also revealed the formation of two novel F328S factor VII degradation products (40 and 9 kDa) resulting from factor Xa proteolysis of the Arg315-Lys316 peptide bond in intact F328S factor VII. Computer modeling and molecular dynamics simulations of the serine protease domain of factor VIIa suggested that the inability of F328S factor VIIa to cleave substrates may result from the apparent formation of a hydrogen bond between Tyr377 and Asp338, a residue at the bottom of the substrate-binding pocket important for the interaction of substrate arginine side chains with the enzyme. These findings suggest that Phe328, which is conserved in prothrombin, factor IX, factor X, factor VII, and trypsin, is important for factor VIIa catalysis.

Characterization of a membrane protease from rat submaxillary-gland mitochondria that possess thrombin-like activity.

A membrane protease possessing thrombin-like activity was purified to homogeneity from mitochondria of rat submaxillary gland. The molecular mass of the enzyme was determined to be 45 kDa by SDS/PAGE under reducing conditions and by gel filtration on a Sephadex G-100 column. The enzyme is a glycoprotein and has an isoelectric point of 3.25. Maximum activity was observed at pH 10.5. Inhibition by di-isopropyl fluorophosphate, benzamidine, aprotinin and antipain suggested the enzyme to be a serine protease. Other inhibitors such as EDTA, soya-bean trypsin inhibitor, lima-bean trypsin inhibitor, TosLysCH2Cl and chymostatin did not alter the activity. The enzyme showed affinity towards different synthetic substrates (p-nitroanilide derivatives) containing arginine at the P1 position. Kinetic studies revealed that Kcat./Km was highest with the substrate N-Bz-Phe-Val-Arg-p-nitroanilide. The enzyme exhibits significant plasma-coagulating activity. The coagulation initiated by the enzyme was not altered by concanavalin A, indicating that the carbohydrate moiety of the enzyme is not essential for this reaction. Further, this enzyme can catalyse the formation of fibrin clots from purified fibrinogen, which describes its thrombin-like activity. However, an antibody raised against the purified enzyme inhibited the plasma-clotting as well as fibrinogen-clotting activity of the enzyme. Fibrinogen coagulation by the enzyme was blocked in the presence of aprotinin, a protease inhibitor. Release of fibrinopeptides A and B from bovine fibrinogen by the enzyme has been shown by HPLC analysis. Our studies reveal that the enzyme reported here differs from trypsin, chymotrypsin and other mitochondrial proteases reported so far.

Enzymatic removal of sialic acid from human factor IX and factor X has no effect on their coagulant activity.

Factor IX and factor X have sialic acid in O-linked and N-linked oligosaccharides on their activation peptides, and a terminal sialic acid is found on a recently described O-linked tetrasaccharide at Ser-61 in the light chain of human factor IXa. In studies presented here, the potential role of sialic acid residues in mediating activity of human coagulation factors IX and X was tested after enzymatic removal of sialic acid residues. In contrast to previous reports, treatment of factor IX or factor IXa with recombinant sialidase did not decrease the rate of factor IX activation or proteolytic properties of human factor IXa. The activation rates of factor IX and desialated factor IX were indistinguishable when treated with factor XIa, with factor VIIa/tissue factor complex, and with the factor X activating enzyme from Russell's viper venom. Desialated human factor IXa showed full activity in the non-activated partial thromboplastin time assay and retained full "tenase" activity in a coupled amidolytic assay. Similar experiments with human factor X showed no detectable loss of clotting activity in the prothrombin time assay after desialation. Additionally, desialated human factor X was cleaved by the factor X activating enzyme from Russell's viper venom and intrinsic tenase at the same rate as untreated factor X when analyzed by SDS-polyacrylamide gel electrophoresis. These studies have shown that factor IX and factor X clotting activity are not dependent on sialic acid content. Further studies are needed to determine whether desialated factor IX binds to endothelial cells, and whether factors IX and X are more rapidly cleared from circulation or have altered susceptibility to proteolysis after enzymatic removal of sialic acid.

A new blood-coagulating protease in mitochondrial membranes of rat submaxillary glands. Purification and characterization of protease and its blood-coagulating activity.

An integral membrane protease was solubilized and purified to homogeneity from rat submaxillary mitochondria. The purified enzyme could coagulate rabbit plasma. The molecular mass of the enzyme is 22 kDa on SDS-polyacrylamide gel electrophoresis under reducing conditions and 24 kDa on gel filtration on a Sephadex G-100 column. Its isoelectric point is 4.2-4.25. Enzyme activity is strongly inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, benzamidine, aprotinin, and antipain, suggesting the enzyme as a serine protease. Its pH optimum for activity is 8.5. Zn2+ is strongly inhibitory; at 1 mM concentration it produced 72% inhibition. The enzyme is active toward different synthetic substrates (p-nitroanilide derivatives) containing Arg at the P1 position with blocked NH2 terminus. Kcat/Km was highest with the substrate N-Bz-Pro-Arg-pNa (where Bz is benzoyl and pNA is paranitroanilide). The purified enzyme coagulates rabbit plasma in a dose-dependent manner. Plasma coagulation by the enzyme is completely blocked in the presence of aprotinin or soybean trypsin inhibitor, suggesting that protease activity is required for this coagulation reaction. Antibody raised against the purified enzyme inhibits the plasma coagulation initiated by the enzyme. The enzyme can correct the prolonged clotting time of factor X-deficient human plasma but is unable to convert purified fibrinogen to fibrin clots, indicating factor Xa-like activity of the enzyme. The enzyme has the ability to activate prothrombin. Several properties of the enzyme distinguish it from other reported submaxillary proteases.

Impairment of natural killer cell activity in Indian kala-azar: restoration of activity by interleukin 2 but not by alpha or gamma interferon.

Indian kala-azar patients have normal numbers of peripheral blood NK cells but impaired functional activity due to decreased binding and lysis of target cells. This impairment of NK activity could not be corrected by exogenous recombinant human alpha or gamma interferon. However, recombinant human interleukin 2 was able to restore this activity by augmenting conjugate formation and lysis of target cells.

Pyroglutamate aminopeptidase in rat submaxillary gland.

A significant amount of pyroglutamate aminopeptidase (PGAP) activity was found to be present in 27,000 x g supernatant of rat submaxillary gland, maximum activity being at pH 6.5. EDTA stimulated the enzyme activity by 95% at pH 8.0 while at pH 6.5 it did not have any significant effect. On comparison of its properties submaxillary PGAP appears to be different from brain, pituitary and other reported PGAPs. Submaxillary PGAP could also catalyze efficiently the formation of cyclo (His-Pro) from TRH. Cyclo (His-Pro) formation by submaxillary enzyme was more pronounced than that by liver PGAP.

Silanized silica bound trypsin as analytical probe.

Trypsin immobilized by covalent coupling to silanized silica shows significant activity (30-38%) and greater thermostability as compared to soluble trypsin. Proteolytic processing of albumin at varying periods suggest that the enzyme matrix can be used efficiently for limited proteolysis. Repeated use of the immobilized enzyme in protein digestion produces similar products as seen by electrophoretic analysis. Also, digestion of albumin by the immobilized enzyme follows similar pattern as that by soluble enzyme. The enzyme matrix can be easily removed from the incubation mixture. The results indicate the possibility of the immobilized enzyme for its effective application as analytical tool in peptide mapping and limited proteolytic processing.

Chymotrypsinogen: an activating enzyme in the mitochondrial membrane of rat submaxillary gland.

A peripheral membrane protease was purified from mitochondria of rat submaxillary gland. On non-denaturing PAGE the purified enzyme showed a single protein band with the enzyme activity. It yielded two protein bands with molecular weights of 39 KDa and 20 KDa on SDS-PAGE, indicating that the enzyme is composed of two protein components. The enzyme activity was strongly inhibited by SBTI, aprotinin and benzamidine. PMSF, TLCK and EDTA did not produce inhibition. The enzyme could hydrolyze different synthetic substrates having arginine at the P1 position with highest affinity for the substrate Bz-Phe-Val-Arg-p-nitroanilide was noted. The enzyme showed significant activation of chymotrypsinogen A.