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Biomedical subjects

S Kumar

Publications and source records attributed to S Kumar.

At least 865 records · Page 48Linked to original sources
PubMed1998

Regulation of DNA-dependent protein kinase by the Lyn tyrosine kinase.

The Src-like protein-tyrosine kinase Lyn is activated by ionizing radiation and certain other DNA-damaging agents, whereas the DNA-dependent protein kinase (DNA-PK), consisting of the catalytic subunits (DNA-PKcs) and Ku DNA-binding components, requires DNA double-stranded breaks for activation. Here we demonstrate that Lyn associates constitutively with DNA-PKcs. The SH3 domain of Lyn interacts directly with DNA-PKcs near a leucine zipper homology domain. We also show that Lyn phosphorylates DNA-PKcs but not Ku in vitro. The interaction between Lyn and DNA-PKcs inhibits DNA-PKcs activity and the ability of DNA-PKcs to form a complex with Ku/DNA. These results support the hypothesis that there are functional interactions between Lyn and DNA-PKcs in the response to DNA damage.

DNA-Activated Protein Kinase↗
PubMed1998

Real-time magnetic resonance image-guided interstitial brachytherapy in the treatment of select patients with clinically localized prostate cancer.

PURPOSE: This study was performed to establish the dose-localization capability and acute toxicity of a real-time intraoperative magnetic resonance (MR) image-guided approach to prostate brachytherapy in select patients with clinically localized prostate cancer. METHODS AND MATERIALS: Nine patients with 1997 American Joint Commission on Cancer (AJCC) clinical stage T1cNxM0 prostate cancer, prostate-specific antigen (PSA) < 10 ng/ml, biopsy Gleason score not exceeding 3 + 4, and endorectal coil MR stage T2 disease were enrolled into this study. The prescribed minimum peripheral dose was 160 Gy to the clinical target volume (CTV), which was the MR-defined peripheral zone (PZ) of the prostate gland. Using a real-time 0.5 Tesla intraoperative MR imaging unit, 5-mm image planes were obtained throughout the prostate gland. The PZ of the prostate gland, anterior rectal wall, and prostatic urethra were identified on the T2 weighted axial images by an MR radiologist. An optimized treatment plan for catheter insertion was generated intraoperatively. Each catheter containing the 125Iodine sources was placed under real-time MR guidance to ensure that its position in the coronal, sagittal, and axial planes was in agreement with the planned trajectory. Real-time dose- volume histogram analyses were used intraoperatively to optimize the dosimetry. RESULTS: For the 9 study patients, 89-99% (median 94%) of the CTV received a minimum peripheral dose of 160 Gy and > or = 95% of the volume of the prostatic urethra and 42-89% (median 70%) of the volume of the anterior rectal wall received doses that were below the reported tolerance. All patients voided spontaneously within 3 h after discontinuation of the Foley catheter and no patient required more than a limited course (< or = 3 weeks) of oral alpha-1 blockers for postimplant urethritis. CONCLUSIONS: Real-time MR-guided interstitial radiation therapy provided the ability to achieve the planned optimized dose-volume histogram profiles to the CTV and healthy juxtaposed structures intraoperatively, with minimal acute morbidity.

Aged↗
PubMed1998

Insulin-like growth factor II and PAX3-FKHR cooperate in the oncogenesis of rhabdomyosarcoma.

The mouse myoblast C2C12 cell line transfected singly with cDNA for Pax-3, PAX3-FKHR, or insulin-like growth factor (IGF) II or cotransfected with IGF-II plus Pax-3 or with IGF-II plus PAX3-FKHR genes showed an altered morphology, a lack of differentiation, and higher proliferation rates in vitro. On s.c. injection into nude mice, tumors grew from transfected cell lines but not from cells transfected with the empty vector. Tumors derived from IGF-II/PAX3-FKHR- and IGF-II-transfected cells grew most rapidly. Cotransfection of IGF-II plus Pax-3 induced tumors comprised highly differentiated striated muscle cells; Pax-3, PAX3-FKHR, or IGF-II transfection produced tumors at varying stages of differentiation. Tumors derived from IGF-II plus PAX3-FKHR-cotransfected cells were composed of undifferentiated cells. This was the only tumor type to infiltrate the underlying muscle. The most angiogenesis and the least apoptosis were observed in the latter tumors. These results support the hypothesis that PAX3-FKHR interacts with IGF-II to play a critical role in the oncogenesis of rhabdomyosarcoma.

Animals↗
PubMed1998

Prodomain-dependent nuclear localization of the caspase-2 (Nedd2) precursor. A novel function for a caspase prodomain.

Caspases are cysteine proteases that play an essential role in apoptosis by cleaving several key cellular proteins. Despite their function in apoptosis, little is known about where in the cell they are localized and whether they are translocated to specific cellular compartments upon activation. In the present paper, using Aequorea victoria green fluorescent protein fusion constructs, we have determined the localization of Nedd2 (mouse caspase-2) and show that both precursor and processed caspase-2 localize to the cytoplasmic and the nuclear compartments. We demonstrate that the nuclear localization of caspase-2 is strictly dependent on the presence of the prodomain. A caspase-2 prodomain-green fluorescent protein localized to dot- and fiber-like structures mostly in the nucleus, whereas a protein lacking the prodomain was largely concentrated in the cytoplasm. We also show that an amino-terminal fusion of the prodomain of caspase-2 to caspase-3 mediates nuclear transport of caspase-3, which is normally localized in the cytoplasm. These results suggest that, in addition to roles in dimerization and recruitment through adaptors, the caspase-2 prodomain has a novel function in nuclear transport.

3T3 Cells↗
PubMed1998

Immunodetection and characterisation of soluble CD105-TGFbeta complexes.

CD105 (endoglin) is a receptor for transforming growth factor beta (TGFbeta). Although methods to measure soluble forms of TGFbeta and CD105 have been published, no assay is available to quantify the receptor-ligand complexes. We describe both an indirect enzyme-linked immunosorbent assay for the quantitation of soluble CD105-TGFbeta1 and the characterization of the complexes by immunoprecipitation and immunoblotting. Mab E9, specifically reactive with CD105, was utilised as the capture reagent in the ELISA system. Detection of complexes was achieved using chicken antibody against TGFbeta1 and the subsequent detection of bound antibody demonstrated by the addition of anti-species antiserum conjugated to horseradish peroxidase (HRP). By using enhanced chemiluminescence and optimised antibodies, the assay was made sufficiently sensitive and reproducible to detect low levels of circulating complexes. Whether the assay had any practical applications was evaluated in breast cancer patients. Plasma levels of CD105-TGFbeta1 were significantly elevated in 59 patients with breast cancer compared to 52 age matched normal women (p < 0.001). Immunoprecipitation using a rabbit anti-CD105 antibody, which reacts with both dimeric and monomeric CD105, and immunoblotting showed that three molecular forms of CD105-TGFbeta1 complexes > 200, 195, and 125 kDa existed in the plasma. We believe these represent the oligomer, dimer and probably the protease degraded form of CD105 complexed to TGFbeta1. The resistance to hypertonic solution, SDS and heat treatment suggested that the soluble CD105-TGFbeta1 complex may be linked by covalent bonds. The measurement of CD105-TGFbeta complexes in the circulation may have important clinical applications not only in cancer but also in patients with other angiogenic diseases such as rheumatoid arthritis, myocardial infarction and stroke.

Antigens, CD↗
PubMed1998

Endothelial and fibroblast cell-derived heparan sulphate bind with differing affinity to basic fibroblast growth factor.

Heparan sulphate from endothelial cells (ECHS) has been shown to bind to bFGF with a lower affinity than that seen for 3T3 fibroblast HS (FHS). To investigate the structural reasons for the low affinity binding of ECHS to bFGF, enzymatic degradation of intact ECHS and FHS chains was undertaken. Filter binding assays showed ECHS heparinase III-resistant fragments 6-7 disaccharides in length and had affinity for bFGF equivalent to that of the intact ECHS chains. The largest resistant fragments from FHS, again 6-7 disaccharides in length, bound to bFGF with a similar affinity to the largest ECHS oligosaccharides, and they therefore have considerably lower affinity than seen for the intact FHS chains. Disaccharide compositional analysis of both ECHS and FHS oligosaccharides showed them to contain similar amounts of 2-O-, 6-O-, and N-sulphated disaccharides. These results suggest that the sulphation pattern within sulphated HS domains and their overall length are not the sole contributors to the binding of intact HS chains to bFGF. It is suggested that domain organisation and frequency of occurrence of large heparinase III-resistant oligosaccharides within intact chains play an important role not only in governing the maximum observed binding affinity of intact chains in the assay system used, but also in the regulation of other biological properties of HS.

3T3 Cells↗
PubMed1998

Sodium.

Disorders of serum sodium are both the most common and probably most the poorly understood electrolyte disorders in clinical medicine. In the past few years increased knowledge about the non-osmotic release of vasopressin and the cloning of vasopressin receptors and of vasopressin-regulated water channels (AQP2) has enhanced our understanding of these disorders. Also controversies surrounding the treatment of hyponatraemic patients have led to well-accepted therapeutic guidelines.

Diabetes Insipidus↗
PubMed1998

Deletion of a recombined Ig heavy chain transgene in B-lineage cells of transgenic mice.

Fully recombined transgenes are stable in their transmission in the germline of transgenic mice, in common with the endogenous genetic complement of most mammalian somatic tissues, including the genes for lymphoid Ag receptors somatically generated from germline minigenes. There have, however, been isolated reports of unusual low frequency transgene losses in various transgenic mice. Here we show, using Southern blots and PCR-based assays, that plasmablast hybridomas and B cells from three independently derived founder lines of transgenic mice bearing a recombined heavy chain Ig transgene we have been studying show a significant net loss of transgene copies. This loss is more marked in the B cells expressing endogenous heavy chains than in those expressing transgenic heavy chains. We have also examined cells of the B lineage in the bone marrow, and a small degree of deletion is also evident in CD19+ CD23- IgM- immature B-lineage cells. As greater deletion is observed in mature B cells, it is possible that the deletion process either continues into B cell maturity and/or provides a selective advantage. We have investigated the relationship between transgene expression and deletion, and we find that while thymocytes in these mice express the transgene well, T cell hybridomas derived from transgenic thymus do not show any loss of the transgene. Thus, a recombined Ig heavy chain transgene prominently undergoes somatic deletion in B-lineage cells independent of its insertion site or expression. This transgenic instability is significant to the analysis of genomic stability as well as to the design of gene therapy strategies.

Animals↗
PubMed1998

SB 203580 inhibits p38 mitogen-activated protein kinase, nitric oxide production, and inducible nitric oxide synthase in bovine cartilage-derived chondrocytes.

Nitric oxide (NO) is implicated in a number of inflammatory processes and is an important mediator in animal models of rheumatoid arthritis and in in vitro models of cartilage degradation. The pyridinyl imidazole SB 203580 inhibits p38 mitogen-activated protein (MAP) kinase in vitro, blocks proinflammatory cytokine production in vitro and in vivo, and is effective in animal models of arthritis. The purpose of this study was to determine whether SB 203580 could inhibit p38 MAP kinase activity, NO production, and inducible NO synthase (iNOS) in IL-1 stimulated bovine articular cartilage/chondrocyte cultures. The results indicated that SB 203580 inhibited both IL-1 stimulated p38 MAP kinase activity in isolated chondrocytes and NO production in bovine chondrocytes and cartilage explants with an IC50 value of approximately 1 microM. To inhibit NO production, SB 203580 had to be present in cartilage explant cultures during the first 8 h of IL-1 stimulation, and activity was lost when it was added 24 h following IL-1. SB 203580 did not inhibit iNOS activity, as measured by the conversion of arginine to citrulline, when added directly to cultures where the enzyme had already been induced, but had to be present during the induction period. Using a 372-bp probe for bovine iNOS we demonstrated inhibition of IL-1-induced mRNA by SB 203580 at both 4 and 24 h following IL-1 treatment. The iNOS mRNA levels were consistent with NO levels in 24-h cell culture supernatants of the IL-1-stimulated bovine chondrocytes used to obtain the RNA.

Animals↗
PubMed1998

Acquisition of sensitivity of stress-activated protein kinases to the p38 inhibitor, SB 203580, by alteration of one or more amino acids within the ATP binding pocket.

Pyridinyl imidazole inhibitors of p38 mitogen-activated protein kinase compete with ATP for binding. Mutation of 23 residues in the ATP pocket indicated that several residues which affected binding of pyridinyl imidazole photoaffinity cross-linker 125I-SB 206718 did not affect kinase activity, and vice versa, suggesting that pyridinyl imidazoles bind p38 differently than ATP. Two close homologues of p38, SAPK3 and SAPK4, are not inhibited by SB 203580 and differ from p38 by three amino acids near the hinge of the ATP pocket. Substitution of the three amino acids in p38 by those in SAPK3/4 (Thr-106, His-107, and Leu-108 to Met, Pro, and Phe) resulted in decreased 125I-SB 206718 cross-linking and loss of inhibition by SB 203580. Substitution of just Thr-106 by Met resulted in incomplete loss of inhibition. Conversely, substitution of the three amino acids of p38 into SAPK3, SAPK4, or the more distantly related JNK1 resulted in inhibition by SB 203580, whereas mutation of just Met-106 to Thr resulted in weaker inhibition. These results indicate that these three amino acids can confer specificity and sensitivity to SB 203580 for at least two different classes of MAPKs.

Adenosine Triphosphate↗
PubMed1998

Nedd4 mediates control of an epithelial Na+ channel in salivary duct cells by cytosolic Na+.

Epithelial Na+ channels are expressed widely in absorptive epithelia such as the renal collecting duct and the colon and play a critical role in fluid and electrolyte homeostasis. Recent studies have shown that these channels interact via PY motifs in the C terminals of their alpha, beta, and gamma subunits with the WW domains of the ubiquitin-protein ligase Nedd4. Mutation or deletion of these PY motifs (as occurs, for example, in the heritable form of hypertension known as Liddle's syndrome) leads to increased Na+ channel activity. Thus, binding of Nedd4 by the PY motifs would appear to be part of a physiological control system for down-regulation of Na+ channel activity. The nature of this control system is, however, unknown. In the present paper, we show that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. We further show that Nedd4 operates downstream of Go in this feedback pathway. We find, however, that Nedd4 is not involved in the feedback control of Na+ channels by intracellular anions. Finally, we show that Nedd4 has no influence on Na+ channel activity when the Na+ and anion feedback systems are inactive. We conclude that Nedd4 normally mediates feedback control of epithelial Na+ channels by intracellular Na+, and we suggest that the increased Na+ channel activity observed in Liddle's syndrome is attributable to the loss of this regulatory feedback system.

Animals↗
PubMed1998

Dissecting alpha-helices: position-specific analysis of alpha-helices in globular proteins.

An analysis of the amino acid distributions at 15 positions, viz., N", N', Ncap, N1, N2, N3, N4, Mid, C4, C3, C2, C1, Ccap, C', and C" in 1,131 alpha-helices reveals that each position has its own unique characteristics. In general, natural helix sequences optimize by identifying the residues to be avoided at a given position and minimizing the occurrence of these avoided residues rather than by maximizing the preferred residues at various positions. Ncap is most selective in its choice of residues, with six amino acids (S, D, T, N, G, and P) being preferred at this position and another 11 (V, I, F, A, K, L, Y, R, E, M, and Q) being strongly avoided. Ser, Asp, and Thr are all more preferred at Ncap position than Asn, whose role at helix N-terminus has been highlighted by earlier analyses. Furthermore, Asn is also found to be almost equally preferred at helix C-terminus and a novel structural motif is identified, involving a hydrogen bond formed by N delta 2 of Asn at Ccap or C1 position, with the backbone carbonyl oxygen four residues inside the helix. His also forms a similar motif at the C-terminus. Pro is the most avoided residue in the main body (N4 to C4 positions) and at C-terminus, including Ccap of an alpha-helix. In 1,131 alpha-helices, no helix contains Pro at C3 or C2 positions. However, Pro is highly favoured at N1 and C'. The doublet X-Pro, with Pro at C' position and extended backbone conformation for the X residue at Ccap, appears to be a common structural motif for termination of alpha-helices, in addition to the Schellman motif. Main body of the helix shows a high preference for aliphatic residues Ala, Leu, Val, and Ile, while these are avoided at helix termini. A propensity scale for amino acids to occur in the middle of helices has been obtained. Comparison of this scale with several previously reported scales shows that this scale correlates best with the experimentally determined values.

Amino Acid Sequence↗
PubMed1998

Biphasic effect of aluminium on cholinergic enzyme of rat brain.

The cholinotoxic effect of aluminium has been widely reported. In vitro aluminium has a biphasic effect on acetylcholinesterase activity. The present study analyses its in vivo effect in brain regions. Rats were exposed to aluminium chloride by the oral route at a dose of 320 mg/kg body weight for shorter (4 and 14 days) and longer (60 days) duration. Acetylcholinesterase activity in olfactory bulb, striatum and hypothalamus brain regions increased after 4 and 14 days and decreased after 60 days of aluminium exposure. Aluminium level in the brain regions studied increased significantly. No significant change in body weight of rats exposed to aluminium was found. The biphasic change in acetylcholinesterase activity may be due to slow accumulation of aluminium in the brain regions and its effect on the enzyme.

Acetylcholinesterase↗
PubMed1998

Caspase-mediated cleavage of the ubiquitin-protein ligase Nedd4 during apoptosis.

The onset of apoptosis is coupled to the proteolytic activation of a family of cysteine proteases, termed caspases. These proteases cleave their target proteins after an aspartate residue. Following caspase activation during apoptosis, a number of specific proteins have been shown to be cleaved. Here we show that Nedd4, a ubiquitin-protein ligase containing multiple WW domains and a calcium/lipid-binding domain, is also cleaved during apoptosis induced by a variety of stimuli including Fas-ligation, gamma-radiation, tumor necrosis factor-alpha, C-8 ceramide, and etoposide treatment. Extracts from apoptotic cells also generated cleavage patterns similar to that seen in vivo, and this cleavage was inhibited by an inhibitor of caspase-3-like proteases. In vitro, Nedd4 was cleaved by a number of caspases, including caspase-1, -3, -6, and -7. By site-directed mutagenesis, one of the in vitro caspase cleavage sites in mouse Nedd4 was mapped to a DQPD237 downward arrow sequence, which is conserved between mouse, rat, and human proteins. This is the first report demonstrating that an enzyme of the ubiquitin pathway is cleaved by caspases during apoptosis.

Animals↗
PubMed1998

Endoglin, a component of the TGF-beta receptor system, is a differentiation marker of human choriocarcinoma cells.

Endoglin is an integral membrane glycoprotein that binds transforming growth factor-beta1 (TGF-beta1) with high affinity and it is strongly expressed on syncytiotrophoblasts throughout pregnancy. Here, we describe the expression of endoglin by the choriocarcinoma cell line JAR as evidenced by flow cytometry, immunoprecipitation, Western blot and reverse transcriptase polymerase chain reaction analyses. Cross-linking experiments of [125I]-labeled TGF-beta1 to JAR cells indicated that endoglin expressed at the surface of these cells binds TGF-beta. Furthermore, staining of human choriocarcinoma tissue sections with a polyclonal antibody to endoglin demonstrated a high expression of endoglin in syncytiotrophoblast-like areas, as opposed to a negative staining of cytotrophoblast-like cells. This pattern of endoglin expression was confirmed by experiments with methotrexate, an inducer of giant, multinucleated, non-proliferative cells, morphologically indistinguishable from the naturally occurring syncytiotrophoblasts. Thus, treatment of the JAR and JEG-3 choriocarcinoma cell lines with methotrexate led to an increase in endoglin expression, as demonstrated by Western and Northern blot analyses. Taken together, our results suggest that endoglin, in addition to being involved in placental development, may also be a cellular differentiation marker.

Antigens, CD↗
PubMed1998

A molecular timescale for vertebrate evolution.

A timescale is necessary for estimating rates of molecular and morphological change in organisms and for interpreting patterns of macroevolution and biogeography. Traditionally, these times have been obtained from the fossil record, where the earliest representatives of two lineages establish a minimum time of divergence of these lineages. The clock-like accumulation of sequence differences in some genes provides an alternative method by which the mean divergence time can be estimated. Estimates from single genes may have large statistical errors, but multiple genes can be studied to obtain a more reliable estimate of divergence time. However, until recently, the number of genes available for estimation of divergence time has been limited. Here we present divergence-time estimates for mammalian orders and major lineages of vertebrates, from an analysis of 658 nuclear genes. The molecular times agree with most early (Palaeozoic) and late (Cenozoic) fossil-based times, but indicate major gaps in the Mesozoic fossil record. At least five lineages of placental mammals arose more than 100 million years ago, and most of the modern orders seem to have diversified before the Cretaceous/Tertiary extinction of the dinosaurs.

Animals↗
PubMed1998

Autosomal-dominant branchio-otic (BO) syndrome is not allelic to the branchio-oto-renal (BOR) gene at 8q13.

The manifestations of branchio-oto-renal syndrome (BOR), Treacher Collins syndrome, tricho-rhino-phalangeal syndrome, van der Woude syndrome, and Langer-Giedion syndrome are well-defined; these conditions represent clinically and genetically separate syndromes. Autosomal-dominant branchio-oto-renal syndrome comprises preauricular pits, branchial fistulas, hearing loss, and renal anomalies. However, several families have been described without one or more of these clinical findings. In some families, the phenotypic expression is limited to branchial anomalies, preauricular pits, and hearing loss, with no renal dysplasia (branchio-otic or BO syndrome). In other families, branchial and renal anomalies occur without hearing impairment. It is not known whether the variable clinical manifestations are due to the effect of a single gene or whether these represent different syndromes. We investigated BO syndrome in a large family to determine whether BOR and BO syndromes are allelic to each other. The genetic linkage analysis provides evidence that BO syndrome is not allelic to the BOR gene at 8q13.

Alleles↗