Decreased total carbonic anhydrase esterase activity and decreased levels of carbonic anhydrase 1 isozyme in erythrocytes of type II diabetic patients

In this exploratory study, we investigated total erythrocyte carbonic anhydrase (CA) estrase activity as well as CA I isozyme concentration in patients with diabetes mellitus type II (DM) and healthy individuals of Howard University Hospital community. Total estrase activity of CA was measured spectrophotometrically using p-nitrophenol acetate before and after inhibition with acetazolamide. CA I isozyme was measured by radial immunodiffusion using monoclonal antibody (CA I) in agarose plates. The study involved 20 consented participants; 10 normal (N) and 10 (DM), 21 to 84 years of age. The study was approved by the Howard University Institution Review Board. The CA activity was measured following lysis of cells as U/min/mL and CA I concentration as mg/l. We observed CA activity as 46.3±4(N) and 25±2.1 (DM) whereas CA I concentration as 1896±125 (N) and 1104 ±63 (DM). We speculate that the change in the CA activity may of fundamental importance in the regulation of intracellular; pH_i for the basic control of metabolism in diabetes mellitus. Further, we propose that CA activity is a good candidate for a biomarker of diabetes mellitus for the early detection of insulin resistance because the CA activity variation was proportional to the severity of the diabetes. Jehan Ornasir—these studies were undertaken as a partial requirement of her M.S. Degree, Graduate School, Howard University, Washington, DC, USA     

Immunohistochemistry of carbonic anhydrase isozymes VI and II during development of the rat salivary glands

 Secreted carbonic anhydrase (isozyme VI; CA VI) was localized by immunohistochemistry in the developing postnatal rat submandibular and parotid glands using a specific monoclonal antibody to the rat enzyme. CA VI immunostaining was not detectable in the glands before birth. In the submandibular gland, granular immunostaining for CA VI was detectable in several terminal tubule cells of 1-day-old rats. At 1 week, the CA VI-positive cells were located at the periphery of the terminal tubules and appeared to be budding off the tubules. These cellular buds gradually increased, and, by 4 weeks, formed acini. CA VI was also detected in the duct lumen from day 1. The immunostaining in the parotid gland was detected sporadically in the acinar cells at 2 or 3 weeks. By 4 weeks, when the gland was almost indistinguishable from the adult one, the number of positive acinar cells had increased. Their number, however, was far smaller than in the adult gland, and the enzyme could not be detected in the duct lumen. CA II was also localized using specific antibodies to the rat isozyme. CA II was detectable in the inter- and intralobular striated ducts at 2 weeks after birth in the submandibular gland and at 3 weeks in the parotid gland. These results suggset that CA VI is secreted into saliva from soon after birth and that CA II appears in parallel with the functional maturation of the ducts. In addition, CA II was transiently expressed by the cellular buds of the submandibular gland at 2 and 3 weeks. Accepted: 7 January 1998     

Structure and exon to protein domain relationships of the mouse carbonic anhydrase II gene

We have isolated a cosmid clone containing the entire mouse (YBR strain) carbonic anhydrase (CA) II gene in 38 kilobase pairs of genomic DNA. The gene was found to be composed of seven exons and six introns. A TATA box (TATAAAA) and a possible CCAAT box (CCACT) have been located beginning 92 and 142 base pairs, respectively, upstream from the initiation codon ATG. When the regions encoded by exons and protein domains are examined, all but 1 of the 30 putative active site residues are encoded by four exons: exons 2 and 3 mainly code for hydrophilic residues and exons 4 and 6 mostly hydrophobic residues. Two intron splice positions, one between the codons for Glu-116 and Leu-117 and the other interrupting the codon for Gly-143, are located at the bottom of the active site cavity, and the former separates two of the three histidine residues forming ligands to the active site zinc ion. The other four splice sites map to the exterior of the molecule. Thus, except for the possible association of the 29 active site residues encoded by four exons, no obvious correspondence is seen between the regions coded by exons and the functional or secondary structural domains of the mouse CA II molecule. During this study, the possible basis for the two electrophoretic types, CA IIa and CA IIb, of inbred mouse strains was detected as a Gln/His interchange at position 38.     

Association between osteoprotegerin gene polymorphisms and cardiovascular disease in type 2 diabetic patients

Osteoprotegerin (OPG) gene polymorphisms (T245G, T950C and G1181C) have been associated with osteoporosis and early predictors of cardiovascular disease. The aim of this study was to evaluate whether these polymorphisms contribute to cardiovascular disease (CVD) in type 2 diabetic patients. We performed a case-control study with 178 CVD subjects with diabetes and 312 diabetic patients without CVD to assess the impact of variants of the OPG gene on the risk of CVD. The OPG gene polymorphisms were analyzed by using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). There was no significant association between the T245G and G1181C polymorphisms and CVD in the additive genetic model (OR = 0.96, 95% CI 0.64–1.45, p = 0.79; OR = 1.06, 95% CI 0.81–1.39, p = 0.65, respectively). However, the C allele of the T950C polymorphism was independently associated with a risk of CVD in type 2 diabetic patients in this genetic model (OR = 1.38, 95% CI 1.07–1.80, p = 0.01). This study provides evidence that the C allele of the T950C polymorphism is associated with increased risk of CVD in diabetic patients. However, well-designed prospective studies with a larger sample size are needed to validate these results.     

Activation parameters for the carbonic anhydrase II-catalyzed hydration of CO2

We have determined the activation parameters of kcat and kcat/Km for the carbonic anhydrase II-catalyzed hydration of CO2. The enthalpy and entropy of activation for kcat is 7860 +/- 120 cal mol-1 and -3.99 +/- 0.42 cal mol-1 K-1, respectively, for the human enzyme. Results for the bovine enzyme were statistically indistinguishable from those of the human enzyme. The entropy of activation of kcat for the human enzyme was further decomposed into partially compensating electrostatic(es) (delta S*es = +15.1 cal mol-1 K-1) and nonelectrostatic(nes) (delta S*nes = -19.1 cal mol-1 K-1) terms. Computer simulations of a formal kinetic mechanism for carbonic anhydrase II-catalyzed CO2 hydration show that 82% of the temperature effect on kcat can be attributed to the temperature effect on the intramolecular proton transfer step. The reported activation parameters are consistent with a substantial enzyme or active site solvent conformational change in the transition state of the intramolecular proton transfer step, and is consistent with the mechanism of proton transfer proposed by Venkatasubban and Silverman (Venkatasubban, K. S., and Silverman, D. N. (1980) Biochemistry 19, 4984-4989).     

Adiponectin gene polymorphisms in Egyptian type 2 diabetes mellitus patients with and without diabetic nephropathy

Recently, several reports addressed the associations of adiponectin (ADIPOQ) gene polymorphisms with abnormal adiponectin serum levels, type 2 diabetes mellitus (T2DM), and diabetic nephropathy (DN); however, results are inconsistent. This study aimed to investigate the possible association of ADIPOQ gene polymorphisms with T2DM and/or DN and whether they affect serum adiponectin levels in Egyptian population. Two hundred and ninety-six T2DM patients (100 normoalbuminuric patients, 103 microalbuminuric patients, and 93 macroalbuminuric patients) and 209 controls were enrolled in the present study. Polymorphisms of +45, ?11391, and +276 of the ADIPOQ gene were detected using polymerase chain reaction restriction fragment length polymorphism. Serum adiponectin was measured using ELISA. Our results revealed that ADIPOQ +45 TG and GG genotypes and G allele were significantly associated with T2DM, micro/macroalbuminuria, and decreased serum adiponectin level. ADIPOQ ?11391 AA genotype frequency was significantly increased in T2DM group. Moreover, GA and AA genotypes and A allele of ADIPOQ ?11391 were significantly associated with susceptibility to macroalbuminuria despite increased serum adiponectin concentrations. While, ADIPOQ +276 TT genotype and T allele were protective factors regarding the susceptibility to T2DM and micro/macroalbuminuria, and they were significantly associated with increased adiponectin levels. We observed also that the decrease of the serum Adiponectin level was accompanied by an insulin resistance, albuminuria, as well as an increase of serum creatinine. We concluded that ADIPOQ +45; ADIPOQ ?11391 gene polymorphisms are associated with T2DM and/or DN in Egyptian population. While, ADIPOQ +276 gene polymorphism is a protective factor regarding T2DM and/or DN susceptibility.     

Gene polymorphisms in patients with type 2 diabetes and diabetic nephropathy

A considerable variability in the incidence and prevalence of diabetic nephropathy (DN) coheres with an important contribution of multigenetic predisposition in the development of DN. Some genes, which probably participate in the pathogenesis of diabetic nephropathy, also play a role in the regulation of blood pressure, familial hyperlipidemia, familial hypertension and other diseases of the cardiovascular system. We have examined the association of diabetic nephropathy, nephropathy of non-diabetic origin, hypertension and of type 2 diabetes itself with several genetic polymorphisms (the insertion/deletion polymorphism in the gene for angiotensin-converting enzyme, the G/T polymorphism in the glucose transporter 1 gene, the G/T (894) polymorphism and the T/C (−786) polymorphism in the eNOS gene in three groups of patients with diabetes mellitus: 1) patients without diabetic nephropathy (DM); 2) patients with DN; 3) patients with nephropathy of non-diabetic origin (NDRD). Angiotensin-converting enzyme is an important factor in a development of arterial hypertension, but in our groups of Central European diabetic patients the I/D polymorphism was not associated with diabetic nephropathy. Furthermore, we have confirmed that the T/C (T786C) polymorphism in the eNOS gene is associated with metabolic syndrome including type 2 diabetes.     

Localization of carbonic anhydrase in the salivary glands of the cockroach,Periplaneta americana

Carbonic anhydrase (CA) activity was localized in the salivery glands of the cockroach, Periplaneta americana, by (1) Hansson's histochemical technique, and (2) the use of the fluorescent sulphonamide, 5-dimethyl-amino-naphthalene-1-sulphonamide (DNSA). Both techniques reveal the same distribution pattern of CA in the four morphologically different cell types of the glands: peripheral cells, central cells, inner acinar duct cells, and distal duct cells. Positive reactions with Hansson's cobalt/phosphate technique were found in the apical regions of the peripheral cells and the distal duct cells, and were inhibited by 10^–5 M acetazolamide in control experiments. No staining could be detected in the central cells and the inner acinar duct cells. The fluorescent CA inhibitor DNSA (10^–4 M) specifically stained the peripheral cells and the distal duct cells in methanolfixed cryostat sections, whereas the central cells and the inner acinar duct cells remained unstained. The role of CA in the peripheral cells is not clear. CA activity in the distal duct cells may provide the protons needed to run the vacuolar-type H⁺-ATPase on the apical infoldings of the cells. This ATPase may be involved in modification of the primary saliva.     

The gene for human carbonic anhydrase VI(CA6) is on the tip of the short arm of chromosome 1

The gene encoding the human secreted carbonic anhydrase isozyme CAVI(CA6) maps to chromosome 1 by Southern analysis of a somatic cell hybrid panel and to 1p36.22----p36.33 by in situ hybridization. CA6 is therefore not linked to the cytoplasmic carbonic anhydrase genes on chromosome 8 or to CA7 on chromosome 16.     

线粒体基因组D-Loop区基因多态性与2型糖尿病的相关性

随机选取199例浙江地区2型糖尿病患者与102例正常对照, 采用聚合酶链反应(Polymerase chain re-action, PCR)、基因片段直接测序来检测线粒体基因组D-Loop区域基因变异情况, 同时分析其与主要临床指标的关系。结果显示: 线粒体基因组D-Loop区域为一高变异区, np73A-G、np263A-G、np16223C-T、np16519T-C为4个高变异位点; 发现29个未见报道的新变异位点; np193A-G、np234A-G、np16108C-T等变异与糖尿病家族史有关。这表明浙江籍汉族人线粒体基因组D-Loop区存在大量基因多态性现象, 此区域的某些变异可能与糖尿病的发生发展等具有一定相关性。     

Nucleotide sequence and structure of the mouse carbonic anhydrase III gene

At least 14 distinct isozymes of carbonic anhydrase have been identified in mammals. These enzymes catalyze the hydration of carbon dioxide and are essential for regulation of cellular pH and carbon dioxide transport. Carbonic anhydrase III is highly expressed in certain tissues, including muscle and fat where it constitutes up to 25% of the soluble protein. We cloned a cDNA encoding mouse carbonic anhydrase III. This cDNA contains 1653 bp, consisting of 79 bp in the 5' UTR, a 780 bp open reading frame, and 794 bp of the 3' UTR, including two potential polyadenylation signals. Fluorescent in situ hybridization confirmed the existence of a single copy of the gene on chromosome 3. We then isolated the genomic DNA for mouse carbonic anhydrase III and analyzed its structure. The gene consists of seven exons and six introns which span 10.5 kb. The 5' flanking region of the genomic DNA is notable for a pyrimidine rich region consisting of two dinucleotide repeats containing 23 and 20 TC pairs separated by the same 15 bp spacer.     

A pilot study on genetic variation in purine-rich elements in the nephrin gene promoter in type 2 diabetic patients

Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls.     

Screening of mutations and polymorphisms in the glucokinase gene in Czech diabetic and healthy control populations

Glucokinase (GCK) plays a key role in glucose metabolism. GCK mutations are known as a pathogenic cause of maturity-onset diabetes of the young type 2 (MODY2). These mutations are also found in gestational diabetics. The aim of our study was to assess the variability of the GCK gene in the Czech diabetic and control populations. We screened all 10 exons specific for the pancreatic isoform of glucokinase (1a and 2-10) including the intron flanking regions in 722 subjects (in 12 patients with an unrecognised type of MODY and their 10 family members, 313 patients with diabetes mellitus type 2 (DM2), 141 gestational diabetics (GDM), 130 healthy offspring of diabetic parents, and 116 healthy controls without family history of DM2). In two MODY families we identified two mutations in exon 2 of the GCK gene: a novel mutation Val33Ala and the previously described mutation Glu40Lys. In other subgroups (excluding MODY families) we detected only intronic variants and previously described polymorphisms in exons 6 (Tyr215Tyr) and 7 (Ser263Ser), we did not find any known GCK pathogenic mutation. We observed no difference in the frequencies of GCK polymorphisms between Czech diabetic (DM2, GDM) and non-diabetic populations.     

Association of RAGE gene polymorphisms with type 2 diabetes mellitus,diabetic retinopathy and diabetic nephropathy

A meta-analysis was performed to assess the associations of the receptor for advanced glycation end products (RAGE) gene polymorphisms [Gly82Ser (rs2070600), 1704 G/T (rs184003), 429 T/C (rs1800625)] with type 2 diabetes mellitus (T2DM), diabetic retinopathy (DR) and diabetic nephropathy (DN). A comprehensive research was conducted to identify all case-control or cohort studies. The fixed or random effect pooled measure was selected based on the homogeneity test among studies that was evaluated with I². Meta-regression was used to explore the potential sources of between-study heterogeneity. Publication bias was estimated using Peters test. Twenty-nine articles were included. Overall, after excluding articles deviating from Hardy–Weinberg equilibrium in controls and sensitive analysis, no significant association was found between RAGE gene polymorphisms (Gly82Ser, 1704 G/T, 429 T/C) and any of T2DM, DR and DN risk, respectively. Subgroup analysis stratified by ethnicity (Asian and Caucasian) also found no significant association between the above-mentioned three polymorphisms and T2DM risk, respectively. This meta-analysis suggested that there might be no association of RAGE gene polymorphisms (Gly82Ser, 1704 G/T, 429 T/C) with T2DM, DR and DN risk.     

Immunohistochemical demonstration of carbonic anhydrase isoenzyme VI (CA VI) expression in rat lower airways and lung.

Carbonic anhydrase isoenzyme VI (CA VI), which is transported in high concentrations in saliva and milk into the alimentary tract, is an important element of mucosal protection in the upper alimentary tract. Like alimentary tract mucosa, the respiratory tract mucosa is also exposed to heavy microbial, physical, and chemical stress. The protective and renewal-promoting factors present in the surface mucus of the respiratory tract are mainly produced by the seromucous tracheobronchial glands. Here we studied the secretion of CA VI by these glands in adult and developing rats using immunohistochemical techniques. The serous acinar and duct cells of the tracheobronchial glands stained for CA VI. The presence of the enzyme also in the duct content indicates its active secretion into the surface mucus. CA VI was also visible in the secretory cells and at the base of the ciliated cells of the tracheobronchial surface epithelium. Moreover, the Clara cells of the bronchiolar surface epithelium stained for CA VI. These findings are consistent with the hypothesis that CA VI has a mucosa-protective role not only in the gastrointestinal tract but also in the respiratory tract, where CA VI may act as a pivotal pH neutralizer and growth factor.     

Detection of exon polymorphisms in the human lactoferrin gene.

We previously demonstrated that lactoferrin gene polymorphisms occur in cancer cells of patients with leukemia and breast cancer. In this study, we established a non-radioactive polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, one of the most sensitive and simplest methods to detect polymorphisms and mutations of the human lactoferrin gene. We optimized the PCR conditions for nine different DNA templates and 16 pairs of exon primers for SSCP analysis. The DNA templates used in the analyses were prepared from a cosmid clone (CT6-1) that contains the human lactoferrin gene, human placental tissue, leukocytes from 10 normal volunteers, leukemic cells of two patients, and previously established three breast and two leukemic cell lines. With the appropriate exon-primer sets, PCR products from exon I to exon 16 of the lactoferrin gene were generated from the DNA templates and analyzed by SSCP. Compared with the homogenous cloned DNA, lactoferrin gene polymorphisms were detected within exons 2, 5, 7, 9, 13, 14, and 15 of the normal placental and leukocyte DNA. In addition, abnormal migration patterns of the lactoferrin gene in cancer cells were detected in exons 4, 5, 13, 14, and 15. The PCR-SSCP band migration patterns can be attributed either to gene polymorphism in normal cells or to DNA mutations in cancer cells and the employed method cannot distinguish between them. Nonetheless, the present analysis suggests that genetic polymorphisms of the lactoferrin gene exist in selected exons and additional mutations of the lactoferrin gene do occur in the cancer cells.