Biomedical subjects

N Swaminathan

Publications and source records attributed to N Swaminathan.

At least 19 recordsLinked to original sources

PubMed2001

Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development.

Mammalian cells harbor three highly homologous and widely expressed members of the ras family (H-ras, N-ras, and K-ras), but it remains unclear whether they play specific or overlapping cellular roles. To gain insight into such functional roles, here we generated and analyzed H-ras null mutant mice, which were then also bred with N-ras knockout animals to ascertain the viability and properties of potential double null mutations in both loci. Mating among heterozygous H-ras(+/-) mice produced H-ras(-/-) offspring with a normal Mendelian pattern of inheritance, indicating that the loss of H-ras did not interfere with embryonic and fetal viability in the uterus. Homozygous mutant H-ras(-/-) mice reached sexual maturity at the same age as their littermates, and both males and females were fertile. Characterization of lymphocyte subsets in the spleen and thymus showed no significant differences between wild-type and H-ras(-/-) mice. Analysis of neuronal markers in the brains of knockout and wild-type H-ras mice showed that disruption of this locus did not impair or alter neuronal development. Breeding between our H-ras mutant animals and previously available N-ras null mutants gave rise to viable double knockout (H-ras(-/-)/N-ras(-/-)) offspring expressing only K-ras genes which grew normally, were fertile, and did not show any obvious phenotype. Interestingly, however, lower-than-expected numbers of adult, double knockout animals were consistently obtained in Mendelian crosses between heterozygous N-ras/H-ras mice. Our results indicate that, as for N-ras, H-ras gene function is dispensable for normal mouse development, growth, fertility, and neuronal development. Additionally, of the three ras genes, K-ras appears to be not only essential but also sufficient for normal mouse development.

Animals↗

PubMed1999

Assessment of all-trans retinoic acid (ATRA) efficacy as a single agent in primary lymphoid neoplasia.

All-trans retinoic acid (ATRA) is currently widely used in the therapy of acute promyelocytic leukemia and is being tested in vitro and in vivo on several other malignancies. Previously ATRA has been shown to inhibit the growth in vitro, of established human myeloma cell lines as well as cultured primary myeloma cells from patients. ATRA acts by down-regulating IL-6-receptor-alpha or gp130 on the surface of the myeloma cells. However, despite its in vitro effects on myeloma cells, ATRA therapy on advanced stage multiple myeloma (MM) patients has so far largely been ineffective. In current studies, we have assessed the efficacy of ATRA therapy against primary murine plasma cell tumors, which are an animal model for human MM. These tumors are induced at about 50% incidence in pristane-primed BALB/c mice by injection of v-raf/v-myc- containing retroviruses and are IL-6 dependent. Using this animal model, we assessed the effect of ATRA as a therapeutic agent against primary tumors at two early time points in disease development. ATRA was administered in liposomal vesicles (ATRAGEN), since liposomal-ATRA has been shown to circumvent clearance mechanisms by hepatic microsomes, which normally occur with free ATRA. In addition, ATRAGEN was previously shown to be less toxic in mice than free ATRA. ATRAGEN was administered beginning on day 25 or day 45 after virus injection and continued twice weekly for 8-11 weeks. ATRAGEN administration begun at either time point did not alter the incidence or the latency of plasma cell tumors compared with control animals. These results suggest that ATRA may not be an effective sole therapy against early MM.

Animals↗

PubMed1998

Thermal cycle labeling: zeptomole detection sensitivity and microgram probe amplification using CviJl* restriction-generated oligonucleotides.

A new method for efficiently labeling and amplifying DNA probes from anonymous samples has been developed. The two/three base recognition endonuclease CviJI* restricts DNA to numerous small fragments primarily 20-60 bp in size. Thermal denaturation of these fragments results in sequence-specific oligonucleotides complementary to their cognate template. Repeated cycles of denaturation, annealing, and extension of such a multiprimed template by a thermostable DNA polymerase results in a significant amplification of the starting material. This method of amplification, referred to as thermal cycle labeling (TCL), appears to generate a large fraction of rearranged and presumably branched products. The inclusion of nucleotide analogs in the TCL reaction generates microgram amounts of haptentagged probe with a detection limit of 25 zmol (2.5 x 10(-20) mol). Reactions containing [alpha-33P]dCTP yield high-specific-activity probes (2.6 x 10(9) cpm/microgram) with reduced radiolytic decay and a useful shelf life of 1 month. CviJI* -generated primers circumvent the need for synthetic oligos while providing microgram amounts of amplified and labeled probes using the described TCL protocol.

Deoxyribonucleases, Type II Site-Specific↗

PubMed1996

Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A.

R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence. Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends. In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli. The primary E.coli cviJIR gene product is a 278 amino acid protein initiated from a GTG codon, rather than the expected 358 amino acid protein initiated from an in-frame upstream ATG codon. Interestingly, the 278 amino acid protein displays the normal restriction activity but not the R.CviJI* activity of the native enzyme. Nine restriction and modification proteins which recognize a central GC or CG sequence share short regions of identity with R.CviJI amino acids 144-235, suggesting that this region is the recognition and/or catalytic domain.

Amino Acid Sequence↗

PubMed1996

Elevated epidermal growth factor receptor levels in hypertensive Lyon rat kidney and aorta.

1. The adult spontaneously hypertensive Lyon rats (LH strain) exhibited increased maximal epidermal growth factor (EGF) binding in freshly prepared kidney and aortic tissue membranes compared with age-matched normotensive (LN) or hypotensive (LL) strains. However, the binding affinity of the receptors to EGF was the same in all the three strains studied. These findings indicate an increased number of EGF receptors (EGFR) in the hypertensive LH strain. 2. Protein tyrosine kinase activity associated with the EGFR was also elevated in the LH strain compared with LN or LL strains, indicating that these receptors are functionally active. 3. There was a correlation between maximal EGF binding by aortic membranes and blood pressure in individual animals (r = 0.55; P < 0.001). 4. Taken together with previously reported similar findings in other models of genetic hypertension, the present results suggest a possible role for increased levels of EGFR in the development and maintenance of genetic hypertension.

Analysis of Variance↗

PubMed1996

Induction of high affinity epidermal growth factor binding in the aorta of Dahl hypertensive rats fed with high salt diet.

Dahl salt sensitive rats (DS) developed severe hypertension on four weeks of high salt feeding while the Dahl salt resistant rats (DR) remained normotensive under the same conditions. The specific maximal binding of epidermal growth factor (EGF) in the freshly prepared kidney membranes of high salt fed DS rats was higher than those from DR rats (5.3 +/- 1.9 vs. 1.6 +/- 0.62 fmoles/mg protein, p<0.001). Scatchard analysis of EGF binding in the kidney showed one class of receptors in the DR (K(d) = 0.75 +/- 0.05 nM) as well as in the DS rats (K(d)=0.69 +/- 0.06 nM). The EGF binding in the aortic membranes of DS rats was also high compared to DR rats (24.98 +/- 5.52 vs. 13.20 +/- 4.10 fmoles/mg protein, p < 0.001). Scatchard analysis of EGF binding in the aorta showed one class of receptors in the DR aorta with a K(d) of 0.70 +/- 0.06 nM. On the other hand, in the DS rat aorta two classes of receptors, a high affinity form (K(d)=0.05 +/- 0.01 nM) and a low affinity form (K(d)=3.5 +/- 0.3 nM) were noted. The induction of a high affinity species of EGF receptors in the aorta, appears to be a mechanism unique to the salt fed DS rats.

Animals↗

PubMed1995

Cloning and applications of the two/three-base restriction endonuclease R.CviJI from IL-3A virus-infected Chlorella.

The gene (cviJIR) encoding the two/three-base R.CviJI eukaryotic restriction endonuclease (ENase) from IL-3A virus-infected Chlorella was cloned into Escherichia coli. A high frequency of DNA cleavage by R.CviJI required overexpression of the gene encoding the M.CviJI methyltransferase prior to cloning the gene for the ENase. Both genes were sequenced and their organization was determined to be in head-to-tail order. The open reading frame coding for R.CviJI can potentially translate a 41.4-kDa protein; however, in the E. coli host, a truncated version of the enzyme is produced (32.5 kDa). The recombinant ENase does not exhibit ATP-induced 'star' activity (R.CviJI cleaves at RGCY, while R.CviJI* also cleaves at RGCR and YGCY, but not at YGCR), as is characteristic for native R.CviJI. The very high frequency of DNA cleavage by R.CviJI* was exploited in the development of a quasi-random shotgun library method. R.CviJI*-generated oligodeoxyribonucleotides were applied to improve certain molecular biology applications, i.e., DNA labeling, detection, high-resolution restriction mapping, amplification and epitope mapping.

Amino Acid Sequence↗

PubMed1995

Interferon-alpha inhibits erythropoietin-induced proliferation, but not differentiation, and restricts erythroleukemia development.

The immature erythroid cell line J2E responds to erythropoietin (Epo) by proliferating and terminally differentiating into hemoglobin-synthesizing red blood cells. These cells produce a rapid, fatal erythroleukemia in mice characterized by hepatosplenomegaly and severe anemia. The aim of this study was to investigate the effects of murine interferons-alpha (MuIFN-alpha) on J2E cells in vitro and in vivo. Here we show that in culture MuIFN-alpha inhibited the Epo-induced proliferation of J2E cells but did not interfere with differentiation. When mice with J2E erythroleukemias were treated with MuIFNs in vivo, an extension of their life span was observed. Moreover, numerous necrotic lesions of infiltrating leukemic cells were detected in the spleens of these mice. Finally, ex vivo treatment of leukemic bone marrow cells with Epo and MuIFNs delayed mortality even further. It was concluded that MuIFNs (1) suppressed the proliferation of J2E cells in vitro but did not affect Epo-induced differentiation, and (2) inhibited the progress of erythroleukemias, especially in combination with Epo.

Animals↗

PubMed1994

Restriction generated oligonucleotides utilizing the two base recognition endonuclease CviJI*.

The conversion of an anonymous DNA sample into numerous oligonucleotides is enzymatically feasible using an unusual restriction endonuclease, CviJI. Depending on reaction conditions, CviJI is capable of digesting DNA at a two or three base recognition sequence. CviJI normally cleaves RGCY sites between the G and C to leave blunt ends. Under 'relaxed' conditions CviJI* cleaves RGCY, and RGCR/YGCY, but not YGCR sites. In theory, CviJI* restriction of pUC19 (2686 bp) should produce 157 fragments, 75% of which are smaller than 20 bp. Instead, 96% of the CviJI* fragments were 18-56 bp long and none of the fragments were smaller than 18 bp. Thermal denaturation of these fragments generates sequence specific oligonucleotides homologous for the cognate template. The enzymatic conversion of anonymous DNA into sequence specific oligomers has implications for several conventional and novel molecular biology procedures.

Base Sequence↗

PubMed1993

Possible mechanism of interleukin-2-induced decline of serum cholesterol during adoptive cellular immunotherapy in cancer patients.

The serum levels of total, LDL, and HDL cholesterol of patients receiving intravenous infusion of interleukin-2 as part of adoptive cellular immunotherapy were analyzed. The total, LDL, and HDL cholesterol significantly decreased to about one-half of the pretreatment levels after 5 days of infusion (183 +/- 34 to 110 +/- 19, 112 +/- 40 to 48 +/- 24, and 41 +/- 10 to 16 +/- 7 mg/dl, respectively). The decrease was gradual during each day of the treatment. Lymphocyte concentration increased markedly during treatment (4.0 +/- 0.52 to 12.3 +/- 2.95 million cells/ml) and the low-density lipoprotein receptor levels in the lymphocytes also increased significantly (1188 +/- 240 to 1442 +/- 276 ng of bound LDL/million cells). The decrease in cholesterol levels may be related to the cholesterol needed for membrane synthesis during lymphocyte proliferation.

Cholesterol↗

PubMed1992

Structure/function studies of murine interferon-alpha 1 using site-directed mutagenesis followed by in vitro synthesis.

Site-directed in vitro mutagenesis followed by in vitro transcription and translation has been used to study structure/function relationships for murine interferon-alpha 1 (MuIFN-alpha 1). The mature form of the MuIFN-alpha 1 protein was expressed as well as analogue forms with amino acid substitutions at positions 33, 71, 72, 123 and 133. These positions were chosen on the basis of known human interferon-alpha structure/function relationships. Biological assays for antiviral activity on murine cells and natural killer cell activation have been performed for each of the proteins produced. The data obtained have been interpreted in the light of previous human and murine interferon-alpha structure/function work and the recently published three-dimensional structure of murine type I interferon.

Animals↗

PubMed1992

Biological activities of recombinant murine interferons alpha 1 and alpha 4: large difference in antiproliferative effect.

The mature forms of two recombinant murine interferons alpha, alpha 1 and alpha 4, have been expressed in vitro using an established transcription and translation system. The relative specific antiviral activity, antiproliferative activity and the natural killer cell stimulating activity of both subtypes were compared in vitro. While the antiviral and natural killer cell stimulating activities of the 2 subtypes were similar, the relative antiproliferative activities varied markedly. On the basis of equal molar inputs, MuIFN-alpha 1 had less than 8% of the antiproliferative activity of MuIFN-alpha 4. This data shows that a large functional difference exists between these two subtypes which are known to be expressed at different levels in mouse L-cells in vitro.

Animals↗

PubMed1992

Increased EGF binding and EGFR mRNA expression in rat aorta with chronic administration of pressor angiotensin II.

This study examines the changes in the mRNA expression of epidermal growth factor (EGF), EGF receptor (EGFR), platelet derived growth factor (PDGF-B), and transforming growth factor beta (TGF-beta 1) before and after sustained pressor infusion of angiotensin II (Ang II) for 4 weeks. A threefold increase occurred in the levels of EGFR mRNA (17,240 +/- 827 vs 6403 +/- 1372 units, P less than 0.01) and TGF-beta 1 mRNA (1644 +/- 584 vs 475 +/- 30 units, P less than 0.01) only in the aorta and not in the heart and kidney tissues. This increase in both of the above mRNA transcripts highly correlated (r = 0.96 and 0.92, P less than 0.01) with the elevation of blood pressure. The specific binding of 125I-labeled EGF to aortic membranes also increased (11,429 +/- 728 vs 8630 +/- 420 cpm/mg protein, P less than 0.05) with a parallel increase in the protein tyrosine kinase activity of the membranes indicating that the enhanced EGFR mRNA expression resulted in increased activity of a functional receptor. No significant changes were observed in either EGF mRNA or PDGF-B mRNA levels. These findings suggest that EGFR and TGF-beta 1 participate in the long-term progressive pressor response to Ang II and thus potentially in the progression and the maintenance of chronic hypertension.

Angiotensin II↗

PubMed1991

Antibodies to the retina N-acetylgalactosaminylphosphotransferase inhibit neurite outgrowth.

The neural retina N-acetylgalactosaminylphosphotransferase (GalNAcPTase) is a cell surface molecule (Balsamo and Lilien, 1980, 1983; Balsamo et al., 1986a) that is tightly associated with, and glycosylates, the calcium-dependent, cell-cell adhesion molecule, N-cadherin (Balsamo and Lilien, 1990). N-cadherin has been implicated in neuronal attachment and neurite outgrowth when at the surface of cells (Bixby et al., 1987, 1988; Matsunaga et al., 1988; Neugebauer et al., 1988; Tomaselli et al., 1988). The intimate association of the GalNAcPTase and N-cadherin prompted us to test the possibility that the GalNAcPTase is also involved in the process of neurite outgrowth. We tested the effect of one polyclonal and two monoclonal anti-GalNAcPTase antibodies in cultures of chick neural retina cells extending neurites on substrates requiring N-cadherin, beta integrin receptors, or the chicken homologue of L1, G4. The length and number of neurites produced were dramatically reduced on all of these substrates by the polyclonal and one of the monoclonal anti-GalNAcPTase antibodies. The second monoclonal antibody bound to the cell surface but was not inhibitory, indicating that it reacts with a different epitope. The mechanism through which the retina cell surface GalNAcPTase may modulate neurite outgrowth on many substrates is discussed.

Animals↗

PubMed1991

Antibodies to the retina N-acetylgalactosaminylphosphotransferase modulate N-cadherin-mediated adhesion and uncouple the N-cadherin transferase complex from the actin-containing cytoskeleton.

Embryonic chick neural retina cells have at their surface an N-Acetylgalactosaminylphosphotransferase (GalNAcPTase) which is associated with, and glycosylates, the calcium-dependent cell-cell adhesion molecule, N-cadherin (Balsamo, J., and J. Lilien. 1990. J. Biol. Chem. 265:2923-2928). In this manuscript, we demonstrate that antibodies directed against the GalNAcPTase, as well as anti-N-cadherin antibodies, are able to inhibit adhesion of chick neural retina cells to a cell monolayer, to immobilized N-cadherin, or to immobilized anti-N-cadherin antibody. These results indicate that anti-GalNAcPTase antibodies modulate the function of N-cadherin, interfering with the formation of N-cadherin-mediated adhesions. We also demonstrate that actin is associated with the N-cadherin/GalNAcPTase complex and that binding of anti-GalNAcPTase antibodies to intact cells results in dissociation of actin from the complex. We suggest that the GalNAcPTase modulates N-cadherin function by altering its interaction with the cytoskeleton.

Actins↗

PubMed1991

Relative antiviral activity of in vitro-synthesized murine interferon-alpha 4 and -alpha 1.

Murine interferon-alpha 4 (MuIFN-alpha 4) is notable among the MuIFN-alpha subtypes because it lacks 5 amino acids corresponding to positions 103-107 of the other subtypes, yet is the most highly expressed subtype. Site-directed in vitro mutagenesis has been used to modify the genes coding for MuIFN-alpha 4 and MuIFN-alpha 1. The modifications have allowed (i) the in vitro expression of the mature form of each MuIFN-alpha subtype and (ii) the insertion of five amino acids, corresponding to amino acid positions 103-107 of MuIFN-alpha 1, into the MuIFN-alpha 4 sequence. In contrast to previously published data MuIFN-alpha 4 and MuIFN-alpha 1 show only a twofold difference in antiviral activity, with MuIFN-alpha 4 being the more active subtype. In keeping with this observation, it was also found that insertion of the five "missing" amino acids into MuIFN-alpha 4 resulted in an analogue MuIFN-alpha with antiviral activity equivalent to MuIFN-alpha 1. It may be inferred from this work that the deletion of amino acids 103-107 (QVGVQ) is solely responsible for the difference in antiviral activity between MuIFN-alpha 4 and MuIFN-alpha 1.

Animals↗

PubMed1988

Murine cytomegalovirus binds reversibly to mouse embryo fibroblasts: implications for quantitation and explanation of centrifugal enhancement.

In a study of the infection of mouse embryo fibroblasts with murine cytomegalovirus (MCMV), we found that plaque number is directly related to virus concentration and not to the total amount of virus contained in the inoculum. These results suggested that virus binding was reversible and that during infection a binding equilibrium is established which limits the amount of bound virus. Further analysis revealed three categories of plaque based on reversibility after virus adsorption. One group was removed simply by washing cell monolayers after virus removal. A second group of plaques was lost gradually with time, giving complete reversal after 5 min at 37 degrees C. The rate of reversal was temperature dependent, and probably represented true virus dissociation. The final group was irreversible plaques, the number of which increased with increasing infection time. The number of reversible plaques remained constant with time of infection, and represented about 70% of the total plaques after 1 h of virus adsorption. Centrifugation of the virus inoculum onto the fibroblast monolayer at 1000 X g increased plaque numbers up to 100-fold, but had little effect on plaque number when carried out after the virus inoculum was removed. In contrast centrifugation increased the number of reversible plaques, suggesting an increase in the number of virus particles attached to the cell monolayers. We suggest that centrifugation enhances MCMV infection by three mechanisms related to reversibility of binding: (1), it increases the rate of virus association; (2), it decreases the rate of dissociation; (3), by increasing the length of time each virus particle is bound it increases the probability of virus being taken into the cell.

Adsorption↗

PubMed1984

Isolation of guinea pig basophils using anti-leukocyte antibody and density gradient centrifugation on Percoll.

Peripheral blood basophils were isolated from guinea pigs using a 2-step procedure. Initial enrichment of basophils was achieved by treating leukocyte preparations obtained from whole blood with antibody to contaminating granulocytes and mononuclear cells. Basophil populations of 82% purity (mean; range 65-99%) were obtained by subsequent application of the antibody treated preparation to discontinuous Percoll gradients. The isolated basophils were viable and shown to be functional by histamine release upon stimulation with various secretogogues.

Animals↗