Prevalence and risk factors of hypocalcemia among pregnant and non-pregnant women in Maiduguri, Nigeria: A cross-sectional study.

BACKGROUND: Pregnancy is a period of heightened calcium demand necessary for the optimum growth and development of the fetus and placenta. Women with low calcium intake may manifest with hypocalcemia in pregnancy. OBJECTIVE: The aim of this study was to determine the prevalence and risk factors of hypocalcemia among pregnant and non-pregnant women in Maiduguri, Nigeria. SUBJECTS AND METHODS: A comparative cross-sectional study was conducted at the University of Maiduguri Teaching Hospital, Maiduguri from 1st January 2017 to 31st December 2018. For each patient, sociodemographic and clinical characteristics were noted. Serum calcium, phosphate, total protein, and albumin were determined using spectrophometric methods with their specific reagents. The data obtained was analyzed using the SPSS statistical software for windows version 20. Chi-square test, Student t-test and ANOVA were used as appropriate. Odd ratio with multiple logistic regression was used to determine risk factors for hypocalcemia. P value < 0.05 was considered statistically significant. RESULTS: A total of 1,500 (1,000 pregnant and 500 non-pregnant) women were analyzed. The mean age, total protein, and albumin were similar in the pregnant and non-pregnant women. However, the non-pregnant women were of higher parity than the pregnant women (3.75 ± 2.79 versus 2.45 ± 2.24, P < 0.001). The staple food of both group were mainly high carbohydrate diets. The prevalence of hypocalcemia was 29.20% among the pregnant women and 14.20% among the non-pregnant women (X2 = 64.34, P < 0.001). The mean serum calcium was lower among the pregnant compared to the non-pregnant women (2.08 ± 0.22 mmol/L versus 2.2 ± 0.19 mmol/L, F = 5.73, P = 0.02). There were no statistically significant differences in the mean serum calcium across the trimesters of pregnancy (F = 1.58, P = 0.21). CONCLUSION: Hypocalcemia in pregnancy is common in our environment and it is associated with anemia in pregnancy, anorexia, and primigravidity while attainment of at least basic education is protective. We recommend routine calcium supplementation in pregnancy and preconception period.

Turbot (Scophthalmus maximus) Nk-lysin induces protection against the pathogenic parasite Philasterides dicentrarchi via membrane disruption.

P. dicentrarchi is one of the most threatening pathogens for turbot aquaculture. This protozoan ciliate is a causative agent of scuticociliatosis, which is a disease with important economic consequences for the sector. Neither vaccines nor therapeutic treatments are commercially available to combat this infection. Numerous antimicrobial peptides (AMPs) have demonstrated broad-spectrum activity against bacteria, viruses, fungi, parasites and even tumor cells; an example is Nk-lysin (Nkl), which is an AMP belonging to the saposin-like protein (SAPLIP) family with an ability to interact with biological membranes. Following the recent characterization of turbot Nkl, an expression plasmid encoding Nkl was constructed and an anti-Nkl polyclonal antibody was successfully tested. Using these tools, we demonstrated that although infection did not clearly affect nkl mRNA expression, it induced changes at the protein level. Turbot Nkl had the ability to inhibit proliferation of the P. dicentrarchi parasite both in vivo and in vitro. Moreover, a shortened peptide containing the active core of turbot Nkl (Nkl71-100) was synthesized and showed high antiparasitic activity with a direct effect on parasite viability that probably occurred via membrane disruption. Therefore, the nkl gene may be a good candidate for genetic breeding selection of fish, and either the encoded peptide or its shortened analog is a promising antiparasitic treatment in aquaculture.

A novel rapid-onset high-penetrance plasmacytoma mouse model driven by deregulation of cMYC cooperating with KRAS12V in BALB/c mice.

Our goal is to develop a rapid and scalable system for functionally evaluating deregulated genes in multiple myeloma (MM). Here, we forcibly expressed human cMYC and KRAS12V in mouse T2 B cells (IgM(+)B220(+)CD38(+)IgD(+)) using retroviral transduction and transplanted these cells into lethally irradiated recipient mice. Recipients developed plasmacytomas with short onset (70 days) and high penetrance, whereas neither cMYC nor KRAS12V alone induced disease in recipient mice. Tumor cell morphology and cell surface biomarkers (CD138(+)B220(-)IgM(-)GFP(+)) indicate a plasma cell neoplasm. Gene set enrichment analysis further confirms that the tumor cells have a plasma cell gene expression signature. Plasmacytoma cells infiltrated multiple loci in the bone marrow, spleen and liver; secreted immunoglobulins; and caused glomerular damage. Our findings therefore demonstrate that deregulated expression of cMYC with KRAS12V in T2 B cells rapidly generates a plasma cell disease in mice, suggesting utility of this model both to elucidate molecular pathogenesis and to validate novel targeted therapies.

The use of plasma lipid and lipoprotein ratios in interpreting the hyperlipidaemia of pregnancy.

The aim of this study was to determine the lipid and lipoproteins ratio in pregnant mothers and to evaluate their role in the interpretation of hyperlipidaemias. A total of 222 pregnant women who registered for ANC and 222 non-pregnant healthy women of the sameage and parity as control were recruited for the study. A sample of venous blood after an overnight fast was collected for analysis and interpretation. The mean ± SD age (years) of pregnant women, 27.317 ± 7.283 years and that of the non-pregnant women, 26.234 ± 6.234 years are not significantly different, p = 0.429. Total cholesterol, HDL-c and TGs were significantly higher in pregnant women (5.29 ± 1.04 mmol/l, 1.64 ± 0.42 mmol/l and 1.74 ± 0.42 mmol/l) compared with that of non-pregnant women (4.64 ± 0.92 mmol/l, 1.25 ± 0.35 mmol/l and 1.37 ± 0.45 mmol/l, respectively) All showed p < 0.000. The frequencies of hypercholesterolaemia, 96(43.2%) and hypertriglyceridaemia, 82 (36.9%), are significantly higher in the pregnant women than in the non-pregnant women, 58 (26.1%) and 26 (11.7%), respectively. TC/HDL-C ratio, 3.33 ± 1.01 and LDL/HDL-C ratio, 1.91 ± 0.85 are significantly lower in pregnant women compared with non-pregnant women counterparts, 3.89 ± 0.97 and 2.35 ± 0.84, respectively. Similarly the frequencies of increased TC/HDL-C ratio, 22 (9.9%) and LDL/HDL-C ratio, 16 (7.2%) are significantly less in the pregnant compared with the non-pregnant women, 54 (24.3%) and 28 (12.6%), respectively.

Inhibition of lysine acetyltransferase KAT3B/p300 activity by a naturally occurring hydroxynaphthoquinone, plumbagin.

Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.

Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain.

Ubiquitin is ligated to L28, a component of the large ribosomal subunit, to form the most abundant ubiquitin-protein conjugate in S. cerevisiae. The human ortholog of L28 is also ubiquitinated, indicating that this modification is highly conserved in evolution. During S phase of the yeast cell cycle, L28 is strongly ubiquitinated, while reduced levels of L28 ubiquitination are observed in G1 cells. L28 ubiquitination is inhibited by a Lys63 to Arg substitution in ubiquitin, indicating that L28 is modified by a variant, Lys63-linked multiubiquitin chain. The K63R mutant of ubiquitin displays defects in ribosomal function in vivo and in vitro, including a dramatic sensitivity to translational inhibitors. L28, like other ribosomal proteins, is metabolically stable. Therefore, these data suggest a regulatory role for multiubiquitin chains that is reversible and does not function to target the acceptor protein for degradation.

Molecular basis of glutathione synthetase deficiency and a rare gene permutation event.

Glutathione synthetase (GS) catalyses the production of glutathione from gamma-glutamylcysteine and glycine in an ATP-dependent manner. Malfunctioning of GS results in disorders including metabolic acidosis, 5-oxoprolinuria, neurological dysfunction, haemolytic anaemia and in some cases is probably lethal. Here we report the crystal structure of human GS (hGS) at 2.1 A resolution in complex with ADP, two magnesium ions, a sulfate ion and glutathione. The structure indicates that hGS belongs to the recently identified ATP-grasp superfamily, although it displays no detectable sequence identity with other family members including its bacterial counterpart, Escherichia coli GS. The difficulty in identifying hGS as a member of the family is due in part to a rare gene permutation which has resulted in a circular shift of the conserved secondary structure elements in hGS with respect to the other known ATP-grasp proteins. Nevertheless, it appears likely that the enzyme shares the same general catalytic mechanism as other ligases. The possibility of cyclic permutations provides an insight into the evolution of this family and will probably lead to the identification of new members. Mutations that lead to GS deficiency have been mapped onto the structure, providing a molecular basis for understanding their effects.

The structure of the human glutathione synthetase gene.

In this study we have isolated two overlapping cosmid clones which contain the sequence of the human glutathione synthetase gene. The size of the gene is approximately 32 kb and it is composed of 13 exons with the intron sizes ranging from 102 bp to 6 kb. We have also shown an alignment of the amino acid sequences of all currently known eukaryotic glutathione synthetases. This alignment highlights conserved regions that may be of structural or functional significance.

Missense mutations in the human glutathione synthetase gene result in severe metabolic acidosis, 5-oxoprolinuria, hemolytic anemia and neurological dysfunction.

Severe glutathione synthetase (GS) deficiency is a rare genetic disorder with neonatal onset. The enzymatic block of the gamma-glutamyl cycle leads to a generalized glutathione deficiency. Clinically affected patients present with severe metabolic acidosis, 5-oxoprolinuria, increased rate of hemolysis and defective function of the central nervous system. The disorder is inherited in an autosomal recessive mode and, until recently, the molecular basis has remained unknown. We have sequenced 18 GS alleles associated with enzyme deficiency and we detected missense mutations by direct sequencing of cDNAs and genomic DNA. In total, 13 different mutations were identified. Four patients were found to be compound heterozygotes and two individuals were apparently homozygous. Reduced enzymatic activities were demonstrated in recombinant protein expressed from cDNAs in four cases with different missense mutations. The results from biochemical analysis of patient specimens, supported by the properties of the expressed mutant proteins, indicate that a residual activity is present in affected individuals. Our results suggest that complete loss of function of both GS alleles is probably lethal. It is postulated that missense mutations will account for the phenotype in the majority of patients with severe GS deficiency.

Identification of an essential cysteine residue in human glutathione synthase.

Glutathione is essential for a variety of cellular functions, and is synthesized from gamma-glutamylcysteine and glycine by the action of glutathione synthase (EC 6.3.2.3). Human glutathione synthase is a dimer of two identical subunits, each composed of 474 amino acids. Little is known about the structure-function relationships of mammalian glutathione synthases and, in order to gain a greater understanding of this critical enzyme, we have probed the role of cysteine residues by chemical modification and site-directed mutagenesis. Preincubation with thiol reagents such as p-chloromercuribenzoate, N-ethylmaleimide, iodoacetate and 5,5'-dithiobis-(2-nitrobenzoate) resulted in significant inhibition of recombinant human glutathione synthase. Each subunit contains cysteine residues at positions 294, 409 and 422, and we have prepared four different mutants by replacing individual cysteine residues, or all of the cysteine residues, with alanine. The C294A and C409A cysteine mutants retained significant residual activity, indicating that these two cysteine residues are not essential for activity. In contrast, substantial decreases in enzymic activity were detected with the C422A and cysteine-free mutants. This suggests that Cys-422 may play a significant structural or functional role in human glutathione synthase.

The gene encoding human glutathione synthetase (GSS) maps to the long arm of chromosome 20 at band 11.2.

Two forms of glutathione synthetase deficiency have been described. While one form is mild, causing hemolytic anemia, the other more severe form causes 5-oxo-prolinuria with secondary neurological involvement. Despite the existence of two deficiency phenotypes, Southern blots hybridized with a glutathione synthetase cDNA suggest that there is a single glutathione synthetase gene in the human genome. Analysis of somatic cell hybrids showed the human glutathione synthetase gene (GSS) to be located on chromosome 20, and this assignment has been refined to subband 20q11.2 using in situ hybridization.

Sequencing and expression of a cDNA for human glutathione synthetase.

A human brain cDNA clone encoding glutathione synthetase (EC 6.3.2.3) has been sequenced and expressed in Escherichia coli. The protein is 474 amino acids in length with a subunit molecular mass of 52,352 Da. The recombinant protein exhibits glutathione synthetase activity and occurs as a homodimer. The recombinant glutathione synthetase was purified to homogeneity and had a specific activity of 1.73 mumol/min per mg of protein, an isoelectric point of 5.35 and a pH optimum between 7.0 and 7.5. Southern blots of human genomic DNA hybridized with the glutathione synthetase cDNA revealed a relatively simple pattern of strongly hybridizing fragments, indicating the absence of a large gene family and suggesting that there may be only one glutathione synthetase gene in the human genome.

Reciprocal effects of the protein kinase C inhibitors staurosporine and H-7 on the regulation of glycogen synthase and phosphorylase in the primary culture of hepatocytes.

The effects of the protein kinase C inhibitors staurosporine and H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine] on glucose-induced regulation of glycogen synthase and phosphorylase activities were investigated in the primary culture of hepatocytes. Glycogen synthesis as measured by the incorporation of [14C]glucose into glycogen was enhanced up to 78% (P < .001) by 100 nmol/L staurosporine. In contrast, H-7 inhibited glycogen synthesis in a dose-dependent manner, with an IC50 value of 70 mumol/L. Activation of glycogen synthase by 30 mmol/L glucose was enhanced significantly (P < .02 and less) by staurosporine at 20 nmol/L and higher concentrations whereas the activity of this enzyme was inhibited by H-7 (IC50 = 50 mumol/L). The inactivation of phosphorylase by glucose was significantly greater when staurosporine was included in the medium. However, H-7 increased the phosphorylase activity ratio by 1.5- to 2.5-fold at concentrations of 20 to 100 mumol/L. The time course of synthase activation and phosphorylase inactivation showed that the effect of glucose was enhanced by staurosporine and inhibited by H-7. These novel reciprocal effects of protein kinase C inhibitors were also observed at different concentrations of glucose. The effects of H-8, a compound with structural resemblance to H-7 and an inhibitor of protein kinase A, were similar to those of staurosporine but not to those of H-7. Staurosporine blocked the effects of vasopressin and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), whereas H-7 in combination with these protein kinase C activators acted in the same direction. The effects of staurosporine, a relatively more specific inhibitor of protein kinase C, indicated that this enzyme plays a role in the regulation of glycogen metabolism in liver. However, H-7, which is known to have protein kinase C-independent effects in intact cells, seems to alter the activities of glycogen synthase and phosphorylase by a different mechanism.

Differential effects of calyculin A and okadaic acid on the glucose-induced regulation of glycogen synthase and phosphorylase activities in cultured hepatocytes.

The effects of the phosphatase inhibitors calyculin A and okadaic acid were investigated to determine the roles of protein phosphatases type 1 and 2A in the regulation of the activities of glycogen synthase and phosphorylase by glucose in a primary culture of hepatocytes. Glycogen synthesis, as measured by the incorporation of labelled glucose into glycogen, was inhibited in a dose-dependent manner by calyculin A (IC50 = 2.2 nM) and okadaic acid with (IC50 = 14 nM). Glucose-induced activation of glycogen synthase was inhibited by calyculin A and okadaic acid with IC50 values of 3.7 nM and 90 nM, respectively. Phosphorylase was simultaneously activated by these inhibitors with calyculin A again being more active (P < 0.001) than okadaic acid. The differing potencies (P < 0.001) of these inhibitors on the activities of glycogen synthase and phosphorylase were also observed with varying concentrations of glucose (5.6-60 mM) in the medium and at different incubation periods upto 120 min. It has been previously shown that both inhibitors inhibit protein phosphatase-2A with equal potency and calyculin A is a more potent inhibitor of protein phosphatase-1 than okadaic acid. Heat- and proteinase-treated cytosolic fractions from hepatocytes incubated with calyculin A and okadaic acid showed similar differential inhibitory activities towards purified types 1 and 2-A protein phosphatases. Hence, these data provide further evidence that protein phosphatase type-1 plays a major role in the control of glycogen synthesis by regulating the activities of glycogen synthase and phosphorylase.