Jasim M. Radhi

ANTIOXIDANT DEFENSE SYSTEM IN DIABETIC KIDNEY : A TIME COURSE STUDY

Oxygen free radicals (OFRs) have been suggested to be a contributory factor in complications of diabetes mellitus. In the present study, we investigated the lipid peroxide level measured as thiobarbituric acid reactive substances (TBARS) and activities of antioxidant enzymes viz., [superoxide dismutase (SOD), catalase (CAT) and glutathione-peroxidase (GSH-Px)] in the kidney of streptozotocin induced diabetic rats at various stages of development of diabetes. Sprague Dawley rats were divided into two groups: group I, control (n = 42) and group II, diabetic (n = 42). Each group was further subdivided into seven groups each consisting of six rats. Rats in subgroups were studied at weekly intervals (0 to 6 weeks). Blood glucose levels were estimated at the time of sacrifice. TBARS levels and activity of antioxidant enzymes were measured in kidney. The levels of TBARS in the diabetic group increased initially, dropped to baseline level after 2 weeks and then progressively increased at 5th and 6th week (p < 0.05). There was an increase in catalase activity at first week after that it decreased as compared to control group. However, GSH-Px activity in the diabetic group increased after 1 week and then remained at the same level except a small drop in the 2nd week. Total SOD and CuZn-SOD activity increased significantly in diabetic kidney as compared to controls at all time intervals, while Mn-SOD activity showed no change. The present findings suggest that oxidative stress accompanies at early onset of diabetes mellitus and the susceptibility of the kidney to oxidative stress during the early stages may be an important factor in the development of diabetic nephropathy.

Increased Expression of N-Myristoyltransferase in Gallbladder Carcinomas

Activated Src, which has intrinsic protein tyrosine kinase activity, has been found in human solid tumors such as colorectal and breast carcinomas. The Src gene encodes a cytoplasmic tyrosine kinase p60src, which attaches to the inner surface of the membrane after N‐terminal myristoylation and is implicated in transduction of signals to the nucleus. N‐myristoyltransferase (NMT) catalyzes the biochemical modification process called N‐myristoylation. To investigate whether, through Src, NMT contributes to the pathogenesis of gallbladder carcinoma, the authors investigated expression of NMT and p53 in in situ and invasive carcinomas.

Malignant melanoma arising from nevi, p53, p16, and Bcl-2: expression in benign versus malignant components.

Background: The etiology of malignant melanoma has been the subject of much study. Tumour suppressor genes p53 and pl6 and the antiapoptotic, Bcl-2, are implicated in the development and progression of malignant melanoma. Objective: To compare the expression of p53, pl6, and the Bcl-2 genes in both benign and malignant components of malignant melanoma arising from pre-existing nevocellular nevi. Methods: Twenty cases of malignant melanoma arising from pre-existing nevi were selected and studied by immuno-histochemistry for the expression of p53 (D07) CDK4I/MTS-1/INK ←4, which detects both wild and mutant type, pl6 CDK4I/MTS-1/INK ←4, and Bcl-2 using an avidin-biotin technique on formalin-fixed, paraffin-embedded sections. Results: Fifteen cases demonstrated p53 immunoreactivity in the malignant components ranging from 5 to 20% with no expression being seen in the benign components. The pl6 gene showed strong nuclear reactivity in the benign components of 14 cases and weak reaction in 6 cases; the malignant components expressed weak nuclear staining in 18 cases and cytoplasmic staining in all cases. The Bcl-2 gene was expressed strongly to moderately in benign components and weakly in malignant components of nine cases, and was negative in 11 cases. Conclusion: Immunostaining for p53 is expressed only in the malignant component, whereas p16 and Bcl-2 showed decreased staining and a different pattern in malignant compared with benign components. These findings suggest that expression and alterations in the subcellular localization of the cell cycle regulators may contribute to the mechanism of tumourigenesis.

Altered expression of high-molecular-weight calmodulin-binding protein in human ischaemic myocardium.

A high‐molecular‐weight calmodulin‐binding protein (HMWCaMBP) was previously identified and purified from the cytosolic fraction of bovine heart. Based on the sequence homology, amino acid analysis, antibody reactivity, and calpain inhibition, HMWCaMBP has been identified as a homologue of the calpain inhibitor calpastatin. In the present study the expression of HMWCaMBP was investigated in normal and ischaemic human myocardium. Western blot analysis of normal human cardiac muscle extract with the polyclonal antibody raised against bovine HMWCaMBP indicated a prominent immunoreactive band with a molecular mass of 140 kD. HMWCaMBP was localized in the cytoplasm and myofilaments of cardiac myocytes. Furthermore, Western blot analysis of normal and ischaemic cardiac tissues indicated a decrease in the expression of HMWCaMBP in ischaemic tissues. These studies were further substantiated by immunohistochemical studies, indicating strong to moderate HMWCaMBP immunoreactivity in normal cardiac muscle and poor to negative immunoreactivity in ischaemic muscle. The results obtained from the rat ischaemic model suggested that the expression of cardiac HMWCaMBP was significantly decreased during ischaemia/reperfusion. In addition, μ‐calpain and m‐calpain expression was higher in ischaemic cardiac tissue samples than in normal controls. The calpain inhibitory activity of ischaemic cardiac tissues was significantly lower than normal cardiac tissue samples. In some cases of cardiac ischaemia, HMWCaMBP highlighted the contraction band necrosis seen at the margins of a myocardial infarct. In vitro, HMWCaMBP was proteolysed by μ‐calpain and m‐calpain. These results indicate that HMWCaMBP could be susceptible to proteolysis by calpains during ischaemia or reperfusion and may play a contributory role in myocardial injury. Copyright

Fibromuscular dysplasia of the aorta presenting as multiple recurrent thoracic aneurysms

A 17-year-old boy, who had undergone resection of aortic coarctation with a large saccular aneurysm 10 years previously, developed recurrent aneurysms above and below the site of a Gore-Tex interposition graft. These were resected using femoral-femoral bypass, and the upper thoracic aorta was replaced with a Hemashield Dacron tube. Histology of the aorta showed fibromuscular dysplasia. In addition to aortic dissection and aortic rupture, such patients may be at risk of forming further aneurysms.

Biological significance of phosphorylation and myristoylation in the regulation of cardiac muscle proteins

Post-translational modification has long been recognized as a way in which the properties of proteins may be subtly altered after synthesis of the polypeptide chain is complete. Amongst the moieties most commonly encountered covalently attached to proteins are oligosaccharides, phosphate, acetyl, formyl and nucleosides. Protein phosphorylation and dephosphorylation is one of the most prevalent and best understood modifications employed in cellular regulation. The bovine heart calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPEDE) can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzymes affinity for Ca2+ and calmodulin (CaM). The phosphorylation of CaMPDE is blocked by Ca2+ and CaM and reversed by the CaM-dependent phosphatase (calcineurin). The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for CaM. Analysis of the complex regulatory properties of CaMPDE has led to the suggestion that fluxes of cAMP and Ca2+ during cell activations are closely coupled and that the CaMPDE play a key role in the signal coupling phenomenon. The high molecular weight calmodulin binding protein (HMWCaMBP) was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of HMWCBP was higher in the absence of Ca2+/CaM then in the presence of Ca2+/CaM and reversed by the CaM-dependent phosphatase. Recently, it has become apparent that the binding of myristate to proteins is also widespread in eukaryotic cells and viruses and certainly is of great importance to the correct functioning of an organism. Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyses the attachment of myristate to the amino-terminal glycine residue of various signal transduction proteins. Cardiac tissue express high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated. The subcellular localization of bovine cardiac muscle NMT indicated a majority of the activity was localized in cytoplasm. Under native conditions the enzyme exhibited an apparent molecular mass of 50 kDa. Recovery of NMT activity, from both cytosol and particulate fractions, was found to be higher than the total activity in crude homogenates, suggesting that particulate fraction may contain an inhibitory activity towards NMT. Research in our laboratory has been focusing on the covalent modification of proteins and regulation of various signal transduction proteins. This special review is designed to summarize some aspects of the current work on co- and post-translational modification of proteins in cardiac muscle.

Immunohistochemical analysis of thyroid adenomas with Hurthle cells.

Summary Twenty-five cases of thyroid adenomas with Hurthle cell changes, both pure and focal, were studied histologically and immunohistochemically with two objectives: first to elucidate the relationship between the normal uninvolved thyroid and the adenoma; and second, to evaluate the role of immunohistochemical studies in adenomas with Hurthle cell changes. Representative sections were stained with a panel of nine antibodies directed against thyroglobulin (TG), high molecular weight keratin (HMK), low molecular weight keratin (LMK), p53, bcl-2, epithelial membrane antigen (EMA), S100, carcinoembryonic antigen (CEA) and HMB45. In all cases, uniform strong positive staining (+ + +) with TG and bcl-2 was seen in the normal thyroid tissue while the adenoma stained moderately positive (+ +). The reverse pattern was observed with LMK staining. Non-adenomatous thyroid cells were p53-negative, the majority of the Hurthle cells, however, were p53-positive and adenomas with an increased number of Hurthle cells had an increased percentage of p53 staining. The expression of EMA was variable. All thyroid cells both outside and within the adenoma were S100-, CEA-4 and HMB45-negative in all cases. The exact significance of p53 overexpression in the Hurthle cells needs further evaluation.Abbreviations: CEA, carcinoembryonic antigen; EMA, epithelial membrane antigen; HMK, high molecular weight keratin, LMK, low molecular weight keratin; TG, thyroglobulin.

Dedifferentiated chondrosarcoma with features of telangiectatic osteosarcoma

We describe a 44-year-old female with a known history of a solitary osteochondroma of the scapula followed on X-ray for five years. She then presented with a rapidly growing lump. Imaging studies confirmed the presence of an aggressive looking lesion. Excision was performed and pathology showed a dedifferentiated chondrosarcoma with features of a telangiectatic osteosarcoma.

Altered expression and localization of N-myristoyltransferase in experimentally induced rat model of ischemia-reperfusion.

N‐myristoyltransferase (NMT) catalyzes the attachment of myristate onto the amino‐terminal glycine residue of select polypeptides. In the present study, we investigated the expression and activity of NMT in rat heart after ischemia and reperfusion. Western blot analysis of rat heart samples indicated a prominent immunoreactive band of 66 kDa probed with human NMT antibody. Both the expression and activity of NMT were increased by ischemia‐reperfusion. Immunohistochemical studies showed cytosolic localization of NMT in normal rat heart and predominant nuclear localization after ischemia followed by reperfusion. The pre‐ischemic perfusion and post‐ischemic reperfusion of hearts with a cell‐permeable calpain inhibitor (N‐Ac‐Leu‐Leu‐methioninal) suppressed the increase in calpain expression and reversed the localization of NMT from nucleus to cytoplasm. This is the first study demonstrating the expression and alteration of NMT localization in cardiac ischemia and pertaining to a possible role of co‐translational modification of proteins in cardiac functions and injury. J. Cell. Biochem. 86: 509–519, 2002.

Lobular carcinoma in situ and phyllodes tumor arising within a small fibroadenoma.

Abstract: A small fibroadenoma with florid lobular carcinoma in situ and foci of phyllodes tumor is presented.