Biomedical subjects

R Sakthivel

Publications and source records attributed to R Sakthivel.

13 recordsLinked to original sources

PubMed2005

Uric acid: an abettor or protector in calcium oxalate urolithiasis? Biochemical study in stone formers.

BACKGROUND: Free radical induced renal damage leads to crystal retention and formation of large stones. However, the scenario behind uric acid (UA) stone formation is still a mystery, as uric acid, a risk factor of stone formation, seems to be a potent antioxidant that can protect cells from damage by reactive oxygen species. This study was intended to evaluate the role of uric acid in stone formers by assessing the oxidative stress status of the stone patients. METHODS: Determination of urinary stone forming risk factors and oxidative stress factors like plasma lipid peroxidation, protein carbonyls of stone formers and histopathological changes and uric acid deposition in stone patients kidney biopsy were studied. RESULTS: Increased concentrations of urinary uric acid and oxalate in both uric acid as well as calcium oxalate stone formers were observed, whereas calcium is increased in calcium stone formers and not in the uric acid stone patients. Inhibitors such as citrate and glycosaminoglycans (GAGs) were found to be significantly decreased in all the stone patients. Histopathological studies confirmed the deposition of crystals in the damaged tubules and De Galantha staining authenticates that the damage is caused due to uric acid crystals. Increased oxidative stress is dictated by the concentrations of lipid peroxidation and protein carbonyls in stone formers. Moreover, increased activities of urinary marker enzymes substantiate the tubular damage. CONCLUSION: We speculated that uric acid acts as a calculi forming salt rather than an antioxidant and it has no role in preventing oxidative stress pertaining to urolithiasis.

Adult↗

PubMed2004

Nuclear pore complex oxalate binding protein p62: expression in different kidney disorders.

BACKGROUND: Urolithiasis is a multifactorial process that starts with the formation of microcrystals in the urine and terminates as mature renal calculi. The oxalate binding protein plays a vital role in the transport of oxalate. The physiological significance of the presence of oxalate binding protein in the nuclear pore complex is not well understood. METHODS: The nuclear envelope was extracted from human cadaver kidneys. 14C oxalate was labeled, nuclear pore complex proteins were extracted and loaded onto Sephadex G-200, and further purified in DEAE-Sephadex A-50 column. The radioactive protein peak was pooled, concentrated and checked for purity in SDS-PAGE. The purified protein showed cross-reactivity with the monoclonal antibody (MAb 414) and was homogeneous. Urine samples of healthy individuals with no history of kidney disease served as control. Blood and urine samples were collected from kidney and autoimmune disorder patients and checked for the expression of p62 protein by ELISA. RESULTS: Extracted and purified nuclear pore complex oxalate binding protein had a molecular weight of 62 kDa. A threefold increase in oxalate excretion was observed in hyperoxaluric patients compared to control subjects. The protein expression was found to be higher in hyperoxaluric patients vs. controls, chronic renal failure (CRF) and acute renal failure (ARF), whereas decreased expression was observed in nephrotic syndrome (NS) patients. p62 autoantibodies was observed in hyperoxaluria (HO), systemic lupus erythematosus (SLE) and primary biliary cirrhosis (PBC), whereas it was absent in controls. CONCLUSION: Increased expression of p62 may be due to membrane damage induced by oxalate stress, and may be used as a diagnostic marker. This study also confirms the presence of p62 autoantibodies in HO patients.

Autoantibodies↗

PubMed2004

Expression of nuclear pore complex oxalate binding protein p62 in experimental hyperoxaluria.

Proteins are thought to play a major role in stone formation. Oxalate binding protein plays a vital role in the transport of oxalate. This study was aimed at determining whether hyperoxaluria induces the expression of nuclear pore complex oxalate binding protein p62 which has the transport function. Hyperoxaluria was induced in male Wistar rats by feeding 0.75% ethylene glycol in water. The oxalate binding activity of the nuclear pore complex protein increased markedly during experimental hyperoxaluria, whereas nuclear lamina had no binding at all. There was an alteration in the elution profile of the nuclear pore complex oxalate binding protein during the hyperoxaluric condition. The protein was purified and had a molecular weight of 62 kDa (data not shown). The purified protein showed cross-reactivity with the monoclonal antibody (MAb 414) and it showed homogeneity. The expression of this protein (p62) during the hyperoxaluric condition was determined by ELISA and a 3-fold increase was observed when compared to control rats. The increased expression is further confirmed by Western blotting and immunohistochemistry. The increase in p62 protein expression may be either due to increased expression of certain genes or degradation of the cell membrane by oxalate-induced cell injury. Thus, the present study suggests that the increased expression of this protein (p62) may be due to the oxalate induction.

Animals↗

PubMed2001

Regulation of the ligand binding activity of the human very low density lipoprotein receptor by protein kinase C-dependent phosphorylation.

The very low density lipoprotein receptor (VLDL-R) binds and internalizes several ligands, including very low density lipoprotein (VLDL), urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 complexes, lipoprotein lipase, and the 39-kDa receptor-associated protein that copurifies with the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor. Although several agonists regulate VLDL-R mRNA and/or protein expression, post-transcriptional regulation of receptor activity has not been described. Here, we report that the ligand binding activity of the VLDL-R in THP-1 monocytic cells, endothelial cells, smooth muscle cells, and VLDL-R-transfected HEK 293 cells is diminished after treatment with phorbol 12-myristate 13-acetate. This response was blocked by inhibitors of protein kinase C (PK-C), including a specific inhibitor of the PK-C beta II isoform, and was associated with phosphorylation of serine residues in the cytoplasmic domain of the receptor. Culture of endothelial cells in the presence of high glucose concentrations, which stimulate diacylglycerol synthesis and PK-C beta II activation, also induced a PK-C-dependent loss of VLDL-R ligand binding activity. Taken together, these studies demonstrate that the ligand binding activity of the VLDL-R is regulated by PK-C-dependent phosphorylation and that hyperglycemia may diminish VLDL-R activity.

Cell Line↗

PubMed2000

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.

We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by approximately 10 nM HKa. This activity was Zn(2+) dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.

Amino Acid Sequence↗

PubMed1998

The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor regulates cell surface plasminogen activator activity on human trophoblast cells.

The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP/alpha2MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/alpha2MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/alpha2MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/alpha2MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous 125I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA.PAI complexes) in an LRP/alpha2MR-dependent manner, which was inhibited by the LRP/alpha2MR receptor-associated protein. Receptor-associated protein also caused a approximately 50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/alpha2MR antibodies. These results demonstrate that LRP/alpha2MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA.PAI complexes and regeneration of unoccupied cell surface uPAR.

Biological Transport↗

PubMed1994

Human breast cancers respond to growth factors in vivo but not in vitro.

Growth response of human breast cancer cells to epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) was tested both in culture and in vivo in nude mice. Human breast cancer cells were obtained from palpable tumors resulting from xenografted primary breast cancers in nude mice. In collagen gel culture, the breast cancer cells grew autonomously as expanding spherical masses of loosely adherent cells in the basal medium and the supplementation of growth factors had no additional stimulatory effect. To determine whether this in vitro response is reflected in vivo, the collagen gel embedded human breast cancer cells were transplanted into athymic nude mice and the growth response to EGF was studied in vivo. In contrast to the situation in vitro, exogenous EGF was growth promoting in vivo. Our results demonstrate the importance of the combined in vitro-in vivo approach in studying physiologically relevant growth regulation. In addition, the use of collagen gel embedded human breast cancer cells for transplantation studies may more closely model the clinical situation in view of the close histopathological resemblance of the recovered gels to the surgical breast specimens.

Animals↗

PubMed1994

Human SP-A: genotype and single-strand conformation polymorphism analysis.

We have previously characterized two surfactant protein A (SP-A) cDNAs termed 1A and 6A, as well as a 6A allelic variant termed 6A1. These sequences are quite heterogeneous at the 3' untranslated region (3'UT). Differences between 6A and 6A1 alleles include an 11-bp insertion/deletion 407 bases downstream from the start of the translation termination codon and a base pair polymorphism (C or G) in exon 1 (position 1,193; White, Damm, Miller, Spratt, Schilling, Hawgood, Benson, and Cordell. Nature Lond. 317: 361-363, 1985). The 11-bp (GCCCACTGCCT) segment is present in 6A1 and absent in 6A. The 6A/6A genotype, in a small number of specimens, showed a trend toward a higher frequency in the black Nigerian population compared with Caucasians. In this report, we examine the frequency of the 6A genotype in a larger number of samples from Caucasians and black Nigerians as well as the meiotic stability of the 3'UT heterogeneity. Slot-blot analysis and allele-specific oligonucleotide probes have confirmed that the 6A/6A genotype is more frequent in the Nigerian population. Single-strand conformation polymorphisms in the 3'UT appear to be stably inherited by members of a three-generation family, suggesting that these nucleotide variants represent natural polymorphisms in the population.

Base Sequence↗

PubMed1993

Effect of TGF-alpha on growth of normal human breast epithelial cells in serum-free primary culture using 3-dimensional collagen gels.

The mitogenic effect of TGF-alpha, acidic-FGF, basic-FGF and lithium on normal human breast epithelial cells was studied in a collagen gel culture system using a serum-free 1:1 mixture of Ham's F12 and DME medium containing insulin, cholera toxin and bovine serum albumin. TGF-alpha elicited a strong mitogenic response in a dose dependent manner. Addition of cortisol to TGF-alpha stimulated growth over and above that achieved with TGF-alpha alone. A consistent observation has been the effect of a combination of TGF-alpha and cortisol on growth stimulation of normal human breast epithelial cells resulting in 3-12 fold growth after 11-13 days in culture. Acidic-FGF, basic-FGF and lithium were not growth promoting.

Breast↗

PubMed1992

Incidence of c-Ki-ras activation in N-methyl-N-nitrosourea-induced mammary carcinomas in pituitary-isografted mice.

We found previously that mouse mammary epithelial cells cultured in the presence of the mammogenic hormones progesterone and prolactin and treated with the carcinogen N-methyl-N-nitrosourea produced a high frequency of hyperplastic alveolar nodules and carcinomas with squamous metaplasia upon transplantation to syngeneic mice. The majority of these mammary transformants had an activated c-Ki-ras proto-oncogene with a specific point mutation in codon 12 (G35 to A35). To determine whether these in vitro findings parallel mammary carcinogenesis in vivo, virgin female mice were pituitary isografted to increase their circulating levels of progesterone and prolactin. The pituitary isograft results in an increase in proliferation, leading to lobulo-alveolar development and differentiation of the mammary epithelial cells. Five weeks after pituitary isografting, the mice were treated with a single injection of N-methyl-N-nitrosourea (50 micrograms/g body weight). Greater than 90% of the N-methyl-N-nitrosourea-treated mice developed mammary carcinomas between 3 and 7 months after treatment. The majority (75%) of the carcinomas had histopathology identical to that of tumors induced in vitro in the presence of progesterone and prolactin. A number of the mammary cancers (17%) induced in pituitary-isografted mice also had the identical point mutation in the c-Ki-ras proto-oncogene found in the in vitro studies. These results suggest that the hormonal milieu around the time of carcinogen exposure affects not only the incidence and phenotype of the mammary transformants but also the molecular events associated with mammary carcinogenesis.

Animals↗

PubMed1990

Expression of Epstein-Barr virus-encoded proteins and B-cell markers in fatal infectious mononucleosis.

We assessed 33 lymphoid tissues from 15 patients, including 7 with X-linked lymphoproliferative disease (XLP) and 8 patients with sporadic fatal infectious mononucleosis (IM), to determine whether the cellular infiltrate had the immunophenotype and expressed Epstein-Barr virus (EBV)-encoded proteins characteristic of either EBV-immortalized lymphoblastoid cell lines (LCL) or EBV-carrying Burkitt lymphoma (BL) cells. The results of these studies revealed that in 13 cases the proliferating B cells were polyclonal, LCL-like, and in 2 cases they were monoclonal, malignant lymphoma-like.

Adult↗

PubMed1989

Immunophenotypic characterization of follicle-center-cell-derived non-Hodgkin's lymphomas.

The distribution of the BLA, CALLA (CD 10), AC-2 (CD 39), MHM-6 (CD 23), LB-I, and 351C5 (CD 45R) antigens in 40 non-Hodgkin's lymphomas was demonstrated by immunohistochemical staining of frozen tissue sections. Nine out of 10 centroblastic and centrocytic follicular and diffuse type of lymphomas (CB/CC F/D) and all 10 cases of CB/CC follicular lymphomas were BLA+ and CALLA+. A few cases also showed weak expression of activation antigens (AC-2, MHM-6 and LB-I) and 351C5. In contrast, 3 CC and 3 lymphoblastic (non-Burkitt) lymphomas showed a heterogeneous pattern of distribution with dominating activation antigen expression. A single case of lymphoblastic lymphoma of Burkitt-like type expressed BLA and CALLA but not activation antigens. In reactive follicular center and FCC lymphomas different cell populations appeared to express BLA and activation antigens, respectively. Assessment of staining intensity and proportion of the stained cells indicated that almost all BLA+ cells are CALLA+. CALLA+ BLA- cells were regularly present, in addition. The co-expression of BLA and CALLA in the same cell was confirmed by double immuno-enzymatic staining. By the same technique, BLA+ and CALLA+ cells were shown to be activation-antigen negative.

Antigens, Differentiation, B-Lymphocyte↗

PubMedDate not supplied

Lymphoma associated antigen (LAA)--a unique biomarker for lymphomas.

A lymphoma associated antigen (LAA) isolated from pooled lymph nodes of confirmed Hodgkin's and non-Hodgkin's lymphomas has been purified and characterized. Using a xenogenic rabbit anti-serum, enzyme-linked immunosorbent assay (ELISA) and RIA were developed for LAA. LAA was detected in the sera of all confirmed lymphomas, the test being negative for normals, for patients with benign lymphadenitis and various other types of cancers. Except for a very few false positive results, no false negative was observed. LAA was identified in urine, CSF, saliva and gastric juice of a few lymphoma patients, and the test proved to be of diagnostic potential, as for a few patients it had a lead time of a few months over the histological diagnosis. In order to render the LAA test more precise and specific, monoclonal antibodies were generated by both in vitro and in vivo immunization procedures. Seven monoclonals were generated, viz. 7D6, 7D2, 7G2, 7C5, 6G2, 23B7 and 23G11, which exhibited cytoplasmic staining of frozen sections of malignant lymphoid tissues of B cell derived non-Hodgkin's lymphomas. Two of these monoclonal antibodies, 7D6 and 23B7, revealed strong cytoplasmic staining of frozen sections, impression smears and cytospin specimens of B cell non-Hodgkin's lymphomas. The reactivity was very weak or negative for T cell lymphomas. The test was negative for Hodgkin's disease and controls. These results were confirmed by dot blotting, immunoprecipitation and immunofluorescence study. By ELISA with a sensitivity of 15 ng/ml, serum LAA levels for lymphomas were in the range 72-1250 ng/ml. LAA could not be detected in the sera of normals and controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗