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Biomedical subjects

A Waheed

Publications and source records attributed to A Waheed.

At least 19 recordsLinked to original sources
PubMed2006

Expression of transmembrane carbonic anhydrases IX and XII in ovarian tumours.

AIMS: Carbonic anhydrase (CA) isozymes IX and XII have been suggested to play a role in oncogenic processes. The aim of the present study was to investigate CA IX and XII expression in patients with ovarian tumours. METHODS AND RESULTS: A series of ovarian tumours was immunostained for CA IX and XII and the results were correlated with histopathological and clinical parameters. Most cases of borderline mucinous cystadenomas, mucinous cystadenocarcinomas and serous cystadenocarcinomas were moderately or strongly positive for CA IX. In malignant tumours, the staining was most prominent in hypoxic regions. Expression of CA XII was detected in all tumour categories, although the mean staining intensity was weaker than for CA IX in all groups except for clear cell carcinomas. CONCLUSIONS: The wide expression of CA IX and XII in ovarian tumours suggests that these isozymes could represent potential targets in ovarian cancer therapy. The expression pattern of CA IX suggests that it could also serve as a useful histopathological marker protein for hypoxia in malignant ovarian tumours.

Antigens, Neoplasm↗
PubMed2006

Sodium and potassium excretion in normotensive and hypertensive population in Kashmir.

One hundred thirty five hypertensive patients and equal number of age and sex matched healthy controls were taken up for studying the relationship of 24 hour urinary sodium and potassium excretion, sodium-potassium molar ratio and body mass index (BMI) with blood pressure in normotensive and hypertensive population in Kashmir. There was statistically significant elevated 24 hour urinary sodium excretion (p < .001), increased Na+-K+ molar ratio, significantly higher BMI in hypertensive population as compared to controls whereas there was a lower 24 hour urinary excretion of potassium (p > .20) in patients with hypertension. Thus sodium and potassium excretion, Na+-K+ molar ratio and body mass index has direct bearing in perpetuation or causation of hypertension in Kashmir which may be related to intake of salt tea.

Adult↗
PubMed2004

Cardiovascular health--behaviour of medical students in Karachi.

OBJECTIVE: To assess the health behaviour and perceptions of medical students towards cardiovascular disease. METHODS: This descriptive cross-sectional survey was conducted on 203 Pakistani medical students enrolled in a private medical college in Karachi, Pakistan using systematic random sampling. It was based on a self-administered questionnaire. RESULTS: Eight percent smoked, 9% were overweight, 33% had a family history of coronary artery disease, 32% regulated dietary fat intake, 28% exercised regularly, 62.1% knew personal blood pressure and 5.4% personal cholesterol levels. Regarding developing cardiovascular disease in the future, 62% showed concern but only 54% of these adopted preventive practices. About 46% believed medical college life had a harmful effect on their health. Gender, family history and personal health status perception were behavior modifying influences (p<0.05). Reasons reported for their behavior were: no need of prevention at their age (38.3%) and never thinking about these issues (37.0%). CONCLUSIONS: The study shows a high prevalence of coronary artery disease family history, inappropriate dietary intake, physical inactivity; poor screening practices and lack of awareness. The results underscore the urgent need to promote preventive knowledge and practices among medical students, if they are to become prevention oriented physicians and counsel patients on preventive strategies to counter the rapidly increasing burden of cardiovascular diseases effectively.

Adult↗
PubMed2002

Effect of purinergic agonists and antagonists on insulin secretion from INS-1 cells (insulinoma cell line) and rat pancreatic islets.

The effects of purinergic agonists on insulin release are controversial in the literature. In our studies (mainly using INS-1 cells, but also using rat pancreatic islets), ATP had a dual effect on insulin release depending on the ATP concentration: increasing insulin release (EC50 approximately/= 0.0032 microM) and inhibiting insulin release (EC50 approximately/= 0.32 microM) at both 5.6 and 8.3 mM glucose. This is compatible with the view that either two different receptors are involved, or the cells desensitize and (or) the effect of an inhibitory degradation product such as adenosine (ectonucleotidase effect) emerges. The same dual effects of ATP on insulin release were obtained using rat pancreatic islets instead of INS-1 cells. ADPbetaS, which is less degradable than ATP and rather specific for P2Y1 receptors, had a dual effect on insulin release at 8.3 mM glucose: stimulatory (EC50 approximately/= 0.02 microM) and inhibitory (EC50 approximately/= 0.32 microM). The effectiveness of this compound indicates the possible involvement of a P2Y1 receptor. 2-Methylthio-ATP exhibited an insulinotropic effect at very high concentrations (EC50 approximately/= 15 microM at 8.3 mM glucose). This indicated that distinct P2X or the P2Y1 receptor may be involved in these insulin-secreting cells. UTP increased insulin release (EC50 approximately/= 2 microM) very weakly, indicating that a P2U receptor (P2X3 or possibly a P2Y2 or P2Y4) are not likely to be involved. Suramin (50 microM) antagonized the insulinotropic effect of ATP (0.01 microM) and UTP (0.32 microM). Since suramin is not selective, the data indicated that various P2X and P2Y receptors may be involved. PPADS (100 microM), a P2X and P2Y1,4,6 receptor antagonist, was ineffective using either low or high concentrations of ATP and ADPbetaS, which combined with the suramin data hints at a P2Y receptor effect of the compounds. Adenosine inhibited insulin release in a concentration-dependent manner. DPCPX (100 microM), an adenosine (A1) receptor antagonist, inhibited the inhibitory effects of both adenosine and of high concentrations of ATP. Adenosine deaminase (1 U/mL) abolished the inhibitory effect of high ATP concentrations, indicating the involvement of the degradation product adenosine. Repetitive addition of ATP did not desensitize the stimulatory effect of ATP. U-73122 (2 microM), a PLC inhibitor, abolished the ATP effect at low concentrations. The data indicate that ATP at low concentrations is effective via P2Y receptors and the PLC-system and not via P2X receptors; it inhibits insulin release at high concentrations by being metabolized to adenosine.

Adenosine Deaminase↗
PubMed2001

Crystal structure of the dimeric extracellular domain of human carbonic anhydrase XII, a bitopic membrane protein overexpressed in certain cancer tumor cells.

Overexpression of the zinc enzyme carbonic anhydrase (CA; EC ) XII is observed in certain human cancers. This bitopic membrane protein contains an N-terminal extracellular catalytic domain, a membrane-spanning alpha-helix, and a small intracellular C-terminal domain. We have determined the three-dimensional structure of the extracellular catalytic domain of human CA XII by x-ray crystallographic methods at 1.55-A resolution. The structure reveals a prototypical CA fold; however, two CA XII domains associate to form an isologous dimer, an observation that is confirmed by studies of the enzyme in solution. The identification of signature GXXXG and GXXXS motifs in the transmembrane sequence that facilitate helix-helix association is additionally consistent with dimeric architecture. The dimer interface is situated so that the active site clefts of each monomer are clearly exposed on one face of the dimer, and the C termini are located together on the opposite face of the dimer to facilitate membrane interaction. The amino acid composition of the active-site cleft closely resembles that of the other CA isozymes in the immediate vicinity of the catalytic zinc ion, but differs in the region of the nearby alpha-helical "130's segment." The structure of the CA XII-acetazolamide complex is also reported at 1.50-A resolution, and prospects for the design of CA XII-specific inhibitors of possible chemotherapeutic value are discussed.

Acetazolamide↗
PubMed2001

Mouse strain differences determine severity of iron accumulation in Hfe knockout model of hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is a common disorder of iron metabolism caused by mutation in HFE, a gene encoding an MHC class I-like protein. Clinical studies demonstrate that the severity of iron loading is highly variable among individuals with identical HFE genotypes. To determine whether genetic factors other than Hfe genotype influence the severity of iron loading in the murine model of HH, we bred the disrupted murine Hfe allele onto three different genetically defined mouse strains (AKR, C57BL/6, and C3H), which differ in basal iron status and sensitivity to dietary iron loading. Serum transferrin saturations (percent saturation of serum transferrin with iron), hepatic and splenic iron concentrations, and hepatocellular iron distribution patterns were compared for wild-type (Hfe +/+), heterozygote (Hfe +/-), and knockout (Hfe -/-) mice from each strain. Although the Hfe -/- mice from all three strains demonstrated increased transferrin saturations and liver iron concentrations compared with Hfe +/+ mice, strain differences in severity of iron accumulation were striking. Targeted disruption of the Hfe gene led to hepatic iron levels in Hfe -/- AKR mice that were 2.5 or 3.6 times higher than those of Hfe -/- C3H or Hfe -/- C57BL/6 mice, respectively. The Hfe -/- mice also demonstrated strain-dependent differences in transferrin saturation, with the highest values in AKR mice and the lowest values in C3H mice. These observations demonstrate that heritable factors markedly influence iron homeostasis in response to Hfe disruption. Analysis of mice from crosses between C57BL/6 and AKR mice should allow the mapping and subsequent identification of genes modifying the severity of iron loading in this murine model of HH.

Animals↗
PubMed2001

Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain.

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Amino Acid Sequence↗
PubMed2001

Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer.

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

Biomarkers, Tumor↗
PubMed2001

The biosynthesis of prostate-specific antigen in non prostatic cell lines.

The biosynthesis of prostate specific antigen (PSA) was studied in human epidermoid carcinoma, (KB) cells and in normal human embryonic lung (WI-38)cells. The prostate carcinoma cell line, PC-35F12 was used as a control. PSA specific antibodies were used to precipitate the immunologically reactive peptides from cell extracts and conditioned media. The immunoprecipitates were analyzed by electrophoresis followed by fluorgraphy. Human PSA is initially synthesized as 32-kDa secretory glycopeptide containing one N-linked oligosaccharide and then processed to a 34-kDa secretory glycoprotein in KB cells. PSA is also expressed in normal human embryonic lung cells, WI-38. These results confirm that PSA expression is not prostate specific, but is also found in some nonprostatic cells. From these results, we conclude that PSA may play an important physiologic role in several tissues.

Carcinoma, Squamous Cell↗
PubMed2001

Differential expression of cytoplasmic carbonic anhydrases, CA I and II, and membrane-associated isozymes, CA IX and XII, in normal mucosa of large intestine and in colorectal tumors.

This study compares the localization of carbonic anhydrase isozymes (CA) I and II and that of IX and XII in normal large intestine and in colorectal tumors. Immunohistochemical studies were performed on 69 colorectal lesions. While the normal mucosa of the large intestine showed high expression for CA I and II, the intensity of the immunostaining for both isozymes decreased in benign lesions and was very weak in malignant tumors. The reciprocal pattern of expression observed for these cytoplasmic isozymes and transmembrane CA IX and XII in intestinal tissue specimens supports the suggestion that CA IX and XII may be functionally involved in tumor progression to malignancy and/or in invasion. By contrast, while CA I and II are prominent in normal colorectal mucosa, where they play a role in regulation of pH homeostasis and water and ion transport, loss of expression of these cytoplasmic isozymes consistently accompanies progression to malignant transformation.

Adenocarcinoma↗
PubMed2001

Molecular aspects of iron absorption and HFE expression.

Hereditary hemochromatosis, a disease of iron overload, occurs in about 1 in 200-400 Caucasians. The gene mutated in this disorder is termed HFE. The product of this gene, HFE protein, is homologous to major histocompatibility complex class I proteins, but HFE does not present peptides to T cells. Based on recent structural, biochemical, and cell biological studies, transferrin receptor (TfR) is a ligand for HFE. This association directly links HFE protein to the TfR-mediated regulation of iron homeostasis. Although evidence is accumulating that binding of HFE to TfR is critical for the effects of HFE, the final pieces in the HFE puzzle have not been established. This review focuses on recent advances in HFE research and presents a hypothetical model of HFE function.

Absorption↗
PubMed2001

Expression of the transmembrane carbonic anhydrases, CA IX and CA XII, in the human male excurrent ducts.

Testicular fluid is concentrated and acidified during its passage through the excurrent ducts. These processes involve bicarbonate absorption, in which carbonic anhydrases are implicated. In this study, the distribution of two transmembrane carbonic anhydrase isozymes (CA IX and CA XII) in the human excurrent ducts was investigated using isozyme-specific antibodies in conjunction with immunohistochemical and immunoblotting techniques. Specific staining for CA XII was present in the basolateral plasma membrane of the epithelial cells in the efferent ducts, predominantly in the non-ciliated cells. In the epididymal duct, CA XII was detected only in sporadic cells, which also contained CA II, thus suggesting that they are apical mitochondria-rich cells. CA IX was also localized to the basolateral plasma membrane of the epithelium in the efferent ducts, but its staining was weaker and less uniform compared to CA XII. No signal for CA IX was detected in the epididymal duct. Western blot analysis from efferent duct samples revealed specific bands for CA IX and CA XII, confirming that the immunohistochemical stainings represent these isozymes. The expression of CA XII and CA IX in the excurrent duct system and co-expression of CA XII with Aquaporin-1 in the same efferent duct epithelial cells suggest their functional involvement in ion transport and concentration processes of testicular fluid.

Blotting, Western↗
PubMed2000

Purification and kinetic analysis of recombinant CA XII, a membrane carbonic anhydrase overexpressed in certain cancers.

Carbonic anhydrase XII (CA XII) is a transmembrane glycoprotein with an active extracellular CA domain that is overexpressed on cell surfaces of certain cancers. Its expression has been linked to tumor invasiveness. To characterize its catalytic properties, we purified recombinant secretory forms of wild-type and mutant CA XIIs. The catalytic properties of these enzymes in the hydration of CO(2) were measured at steady state by stopped-flow spectrophotometry and at chemical equilibrium by the exchange of (18)O between CO(2) and water determined by mass spectrometry. The catalysis of CO(2) hydration by soluble CA XII has a maximal value of k(cat)/K(m) at 34 microM(-1) small middle dots(-1), which is similar to those of the membrane-associated CA IV and to soluble CA I. The pH profiles of this catalysis and the catalyzed hydrolysis of 4-nitrophenylacetate indicate that the pK(a) of the zinc-bound water in CA XII is 7.1. His64 in CA XII acts as a proton shuttle residue, as evidenced by the reduced rate constant for proton transfer in the mutants containing the replacements His64 --> Ala and His64 --> Arg, as well as by the selective inhibition of the proton transfer step by cupric ions in wild-type CA XII. The catalytic rate of CO(2) hydration by the soluble form of CA XII is identical with that of the membrane-bound enzyme. These observations suggest a role for CA XII in CO(2)/HCO(3)(-) homeostasis in cells in which it is normally expressed. They are also compatible with a role for CA XII in acidifying the microenvironment of cancer cells in which CA XII is overexpressed, providing a mechanism for CA XII to augment tumor invasiveness and suggesting CA XII as a potential target for chemotherapeutic agents.

Animals↗
PubMed2000

Nuclear NonO/p54(nrb) protein is a nonclassical carbonic anhydrase.

The growing carbonic anhydrase (CA) gene family includes 11 enzymatically active isozymes in mammals. Each of them has a characteristic cellular and subcellular distribution pattern. In this report, we demonstrate for the first time a nuclear protein with CA activity. A polypeptide recognized by CA II antibodies was purified from several rat tissues using CA inhibitor affinity chromatography. This polypeptide of apparent 66 kDa mass was characterized using amino acid sequencing and CA activity measurements. It appeared to be identical to nonO/p54(nrb), a previously cloned and characterized RNA and DNA binding nuclear factor. Recombinant nonO generated in baculovirus bound to the CA inhibitor affinity chromatography matrix and revealed detectable CA activity (25 units/mg). Hansson's histochemical staining of rat lymph nodes followed by light and electron microscopy showed nuclear CA activity in lymphocytes, suggesting that the nuclear nonO protein is catalytically active in vivo. These results demonstrate that a previously known transcription factor is a novel, nonclassical CA. Through its CA activity, the nonO may function in the maintenance of pH homeostasis in the nucleus.

Animals↗
PubMed2000

Transferrin receptor 2: continued expression in mouse liver in the face of iron overload and in hereditary hemochromatosis.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by excess absorption of dietary iron and progressive iron deposition in several tissues, particularly liver. Liver disease resulting from iron toxicity is the major cause of death in HH. Hepatic iron loading in HH is progressive despite down-regulation of the classical transferrin receptor (TfR). Recently a human cDNA highly homologous to TfR was identified and reported to encode a protein (TfR2) that binds holotransferrin and mediates uptake of transferrin-bound iron. We independently identified a full-length murine EST encoding the mouse orthologue of the human TfR2. Although homologous to murine TfR in the coding region, the TfR2 transcript does not contain the iron-responsive elements found in the 3' untranslated sequence of TfR mRNA. To determine the potential role for TfR2 in iron uptake by liver, we investigated TfR and TfR2 expression in normal mice and murine models of dietary iron overload (2% carbonyl iron), dietary iron deficiency (gastric parietal cell ablation), and HH (HFE -/-). Northern blot analyses demonstrated distinct tissue-specific patterns of expression for TfR and TfR2, with TfR2 expressed highly only in liver where TfR expression is low. In situ hybridization demonstrated abundant TfR2 expression in hepatocytes. In contrast to TfR, TfR2 expression in liver was not increased in iron deficiency. Furthermore, hepatic expression of TfR2 was not down-regulated with dietary iron loading or in the HFE -/- model of HH. From these observations, we propose that TfR2 allows continued uptake of Tf-bound iron by hepatocytes even after TfR has been down-regulated by iron overload, and this uptake contributes to the susceptibility of liver to iron loading in HH.

Amino Acid Sequence↗
PubMed2000

Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells in vitro.

Acidification of the extracellular milieu of malignant tumors is reported to increase the invasive behavior of cancer cells. In normal tissues, production of acid is catalyzed by carbonic anhydrases (CAs), some of which are known to be overexpressed in certain cancers. To investigate the functional role of CA activity in such cancer cells, we analyzed the effect of acetazolamide, a potent CA inhibitor, on the invasive capacity of four renal carcinoma cell lines (Caki-1, Caki-2, ACHN, and A-498). We found that 10 microM acetazolamide inhibited the relative invasion rate of these cell lines between 18-74%. The Caki-2 and ACHN cell lines displayed the highest responsiveness, and their responses clearly depended on the acetazolamide concentration in the culture medium. Immunocytochemical and Western blotting results identified the presence of CA isoenzyme II in the cytoplasm of all four cell lines and CA XII on the plasma membrane in three of four cell lines. Because acetazolamide alone reduced invasiveness of these cancer cells in vitro, we conclude that the CAs overexpressed in these renal cancer cells contribute to invasiveness, at least in vitro, and suggest that CA inhibitors may also reduce invasiveness in other tumors that overexpress one or more CAs.

Acetazolamide↗
PubMed2000

Mitochondrial carbonic anhydrase CA VB: differences in tissue distribution and pattern of evolution from those of CA VA suggest distinct physiological roles.

A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitochondria isolated from transfected COS cells. Northern blot analyses of total RNAs from mouse tissues and Western blot analyses of mouse tissue homogenates showed differences in tissue-specific expression between CA VB and CA VA. CA VB was readily detected in most tissues, while CA VA expression was limited to liver, skeletal muscle, and kidney. The human orthologue of murine CA VB was recently reported also. Comparison of the CA domain sequence of human CA VB with that reported here shows that the CA domains of CA VB are much more highly conserved between mouse and human (95% identity) than the CA domains of mouse and human CA VAs (78% identity). Analysis of phylogenetic relationships between these and other available human and mouse CA isozyme sequences revealed that mammalian CA VB evolved much more slowly than CA VA, accepting amino acid substitutions at least 4.5 times more slowly since each evolved from its respective human-mouse ancestral gene around 90 million years ago. Both the differences in tissue distribution and the much greater evolutionary constraints on CA VB sequences suggest that CA VB and CA VA have evolved to assume different physiological roles.

Amino Acid Sequence↗