Biomedical subjects

K Natarajan

Publications and source records attributed to K Natarajan.

At least 19 recordsLinked to original sources

PubMed1996

Mutations in the Dictyostelium heterotrimeric G protein alpha subunit G alpha5 alter the kinetics of tip morphogenesis.

Tip morphogenesis during the Dictyostelium developmental life cycle is a process by which prestalk cells sort to form the anterior region of the multicellular organism. We show that the temporal regulation of this morphological process is dependent on the copy number of the Dictyostelium G alpha5 gene. Tip formation is delayed in aggregates of g alpha5 null mutant cells and accelerated in aggregates overexpressing the G alpha5 gene compared to tip formation in wild-type cells. The onset of cell-type-specific gene expression associated with mound formation and tip morphogenesis is also temporally altered in G alpha5 mutants. Tip morphogenesis in chimeric organisms of G alpha5 mutants and wild-type cells is dependent on the copy number of the G alpha5 gene, indicating that G alpha5 function plays an integral role in the intercellular signaling of this stage of development. The G alpha5 gene encodes a G alpha subunit that has 51% identity to the Dictyostelium G alpha4 subunit. Like the G alpha4 gene, the G alpha5 gene is expressed in a subset of cells distributed throughout the multicellular organism, with a distribution that is similar to the anterior-like cell population. Amino acid substitutions in the G alpha5 subunit analogous to substitutions altering guanine nucleotide binding and hydrolysis in other G alpha subunits had no apparent effect on the rate of tip formation when a single copy of the mutant gene was used to replace the wild-type gene. Overexpression of these mutant G alpha5 genes by increased gene dosage resulted in cell death, suggesting that high levels of the altered subunits have detrimental effects during vegetative growth.

Amino Acid Sequence↗

PubMed1995

Correlation of peptide specificity and IgG subclass with pathogenic and nonpathogenic autoantibodies in pemphigus vulgaris: a model for autoimmunity.

Pemphigus vulgaris (PV) is a rare, potentially fatal, autoimmune disease that affects the skin and mucous membranes. The PV antigen (PVA) has been characterized as desmoglein 3. PV patients carry HLA-DR4- or HLA-DR6-bearing extended haplotypes. We recently demonstrated that patients with active disease have high titers of PV autoantibodies of the IgG1 and IgG4 subclasses. Patients in remission, healthy unaffected relatives, and some MHC-matched normal individuals have low levels of PV autoantibodies, which are IgG1 only. Furthermore, intraperitoneal injection of IgG from patients with active disease caused clinical disease in mice, but IgG from patients in remission, healthy relatives, or MHC-matched normal individuals did not. We prepared 12 peptides of 30 amino acids each (peptides Bos 1-12) spanning the extracellular domain of PVA. Patients with active disease recognize peptides Bos 1 and Bos 6 with high titers of IgG1 and IgG4 autoantibodies. Patients in remission have IgG1 autoantibodies to peptide Bos 1 only, in statistically significantly lower titers (P < 0.01). They no longer have IgG4 subclass autoantibodies to peptide Bos 6. Healthy relatives and normal unrelated individuals have low levels of only IgG1 autoantibodies that recognize only Bos 1. In vitro studies indicate that Bos 6-specific IgG and, to a lesser extent, Bos 1-specific IgG can cause acantholysis. Our data suggest that Bos 6-specific IgG4 is probably the main acantholytic autoantibody, while Bos 1-specific IgG4 may act as a facilitator or enhancer of the process. In this study we illustrate some of the paradigms that demonstrate the interactions between the MHC, subclass of autoantibodies, and peptide specificities of the autoantibodies in the autoimmune process. Thus, PV provides an important model to study the pathogenesis of autoimmunity.

Amino Acid Sequence↗

PubMed1994

Evaluation of laboratory tests for diagnosis of chlamydial infections in conjunctival specimens.

We evaluated the chlamydial direct diagnostic tests and McCoy cell culture methods for detection of Chlamydia trachomatis in 234 conjunctival scrapings from 127 patients. Direct (rapid) diagnostic methods of Giemsa stain cytology, fluorescent antibody test (FAT) and immunoperoxidase (IPO) stain and McCoy cell culture isolation method using IPO staining and FAT for detection of C. trachomatis antigen were carried out. McCoy cell culture isolation using FAT antigen detection method was considered as the gold standard. The specificity of all the direct diagnostic tests were above 90 per cent but sensitivity of Giemsa cytology, FAT and IPO stain were 36, 54 and 53 per cent respectively showing that as direct diagnostic tests Giemsa stain cytology was inefficient and FAT and IPO were useful. Specificity and sensitivity of McCoy cell culture using IPO stain for detection C. trachomatis antigen were above 90 per cent and it is suggested that this test can be used as a routine diagnostic method.

Antigens, Bacterial↗

PubMed1993

Molecular cloning and analysis of the NAG1 cDNA coding for glucosamine-6-phosphate deaminase from Candida albicans.

Candida albicans and other pathogenic Candida species can use N-acetylglucosamine as a sole carbon source for growth. GlcNAc induces the enzymes of GlcNAc catabolic pathway; besides, under certain conditions, GlcNAc also induces a change from the yeast to germ tube morphology. Glucosamine-6-phosphate deaminase (EC 5.3.1.10) is the terminal enzyme of the GlcNAc catabolic pathway. We have purified the deaminase from C. albicans and studied its characteristics. The size of the deaminase estimated from SDS-polyacrylamide gel electrophoresis is 28 kDa. N-Acetylglucosamine 6-phosphate, an allosteric activator of the Escherichia coli deaminase, has no effect on the activity of the C. albicans enzyme. The deaminase is induced over 100-fold by GlcNAc and its level is about 0.3-0.5% of the proteins in crude extract. Three cDNA clones were obtained from a lambda gt11 expression library by immunoscreening with deaminase antiserum. C. albicans genomic DNA blot hybridization revealed that the NAG1 gene, encoding the glucosamine-6-phosphate deaminase, is present in a single copy. Hybrid-selected translation and immunoprecipitation experiments revealed that the purified deaminase and the protein encoded by the clones were similar in size and in their antigenicity. DNA sequencing revealed that the largest cDNA clone contained the complete open reading frame, which can code for a 27.5-kDa protein. The NH2-terminal sequence (35 residues) determined from the purified deaminase was identical to the sequence of the deduced protein. The Nag1 protein has about 47% identity with the sequence of the E. coli glucosamine-6-phosphate deaminase. Furthermore, RNA blot hybridization showed that GlcNAc induces the expression of NAG1 gene.

Aldose-Ketose Isomerases↗

PubMed1993

Discrete subvalvular aortic stenosis in the Beckwith-Wiedemann syndrome.

Various congenital cardiac malformations have been described in patients with Beckwith-Wiedemann (BW) syndrome, including reversible obstructive subaortic stenosis in one patient. We herein present a case of a 2.5-year-old black boy with BW syndrome and discrete subvalvular aortic stenosis of the membraneous type. Such association of these two entities has previously not been documented.

Aortic Stenosis, Subvalvular↗

PubMed1992

Major histocompatibility complex determinants select T-cell receptor alpha chain variable region dominance in a peptide-specific response.

Dominant expression of T-cell receptor (TCR) alpha or beta chain variable region (V alpha or V beta) gene families has been observed in the T-cell response to some conventional peptide antigens. Current models for the interaction of TCR V region elements with different determinants of a major histocompatibility complex (MHC)-peptide complex, the normal TCR ligand, suggest that the TCR V-J junctional region (CDR3, where J is joining) is the primary contact with a peptide epitope and that other TCR V region segments may interact directly with neighboring MHC determinants. This suggests that V alpha or V beta dominance in a specific response can be MHC-selected. In this case, if related peptides bind to an MHC molecule in a similar orientation, they could select for identical V alpha or V beta dominance even if they are noncrossreactive at the level of T-cell activation. We have screened for this possibility by introducing minimal conservative substitutions in a synthetic peptide, YYEELLKYYEELLK, that is presented to T cells in association with an uncommon A beta E alpha d mixed Ia isotype. We report here that the peptide variant FFEELLKFFEELLK is noncrossreactive with YYEELLKYYEELLK but appears to preserve the same MHC binding motif since T-cell responses are restricted to the same mixed A beta E alpha isotype. Although the two peptides are noncrossreactive in either direction, the same members of the V alpha 4 gene family are dominantly expressed in T cells specific for either peptide. We conclude that the similar topography of the two MHC-peptide complexes gives functional significance to a unique A beta E alpha determinant that selects for V alpha 4 dominance.

Amino Acid Sequence↗

PubMed1992

A free-radical hypothesis for the instability and evolution of genotype and phenotype in vitro.

It has been known for several decades that cultured murine cells undergo a defined series of changes, i.e., an in vitro evolution, which includes crisis, spontaneous transformation ('immortalization'), aneuploidy, and spontaneous neoplastic transformation. These changes have been shown to be caused by the in vitro environment rather than an inherent instability of the murine phenotype or genotype. Serum amine oxidases were recently identified as a predominant cause of crisis. These enzymes generate hydrogen peroxide from polyamine substrates that enter the extracellular milieu. This finding implicates free-radical toxicity as the underlying cause of in vitro evolution. We propose an oxyradical hypothesis to explain each of the stages of in vitro evolution and discuss its significance for cytotechnology and long-term cultivation of mammalian cell types.

Aneuploidy↗

PubMed1991

Specific identification and subcellular localization of three calmodulin-binding proteins in the rat gonadotrope: spectrin, caldesmon, and calcineurin.

In an effort to characterize the second messenger system for LH release, we have previously identified five calmodulin-binding proteins in rat gonadotropes of Mr greater than 205,000, 200,000, 135,000, 60,000, and 52,000. In the present study, we have used a calmodulin overlayer assay combined with Western blotting to determine the molecular identity of three calmodulin-binding proteins in rat gonadotropes: the alpha subunit of spectrin (Mr greater than 205,000), caldesmon (Mr 84,000), and the alpha subunit of calcineurin (Mr 60,000). The Mr greater than 205,000 and Mr 60,000 components or rat pituitary which bind calmodulin are immunoreactive with spectrin and calcineurin antisera, respectively. Rat pituitary also contains an Mr 84,000 component, which is immunoreactive with polyclonal sera and monoclonal antibody raised to chicken gizzard caldesmon (Mr 150,000). Like caldesmon from other sources, the Mr 84,000 component remains soluble after heat treatment and preferentially binds either filamentous actin or calmodulin, depending on the Ca2+ concentration. The three calmodulin-binding proteins were localized specifically in gonadotropes using indirect immunofluorescence microscopy or by Western-blotting cell fractions enriched for gonadotropes. After differential centrifugation of pituitary homogenate, spectrin immunoreactivity was found associated with the nuclear and secretory granule fractions, whereas caldesmon immunoreactivity was seen in the cytosolic fraction and calcineurin in the cytosolic and nuclear fractions. Although the precise role for these proteins remains unknown, the apparent requirement for calmodulin and the small number of calmodulin-binding proteins in the gonadotrope suggest their involvement in mediating GnRH actions.

Animals↗

PubMed1991

Caldesmon: a bifunctional (calmodulin and actin) binding protein which regulates stimulated gonadotropin release.

Calmodulin (CaM) serves as an intracellular Ca2+ receptor in the gonadotrope and appears to mediate GnRH-stimulated gonadotropin release. Recently we have specifically identified three CaM binding proteins of the gonadotrope as calcineurin, caldesmon, and spectrin. Caldesmon (identified by seven polyclonal and a monoclonal antibody, as well as by functional characteristics) appears to be a CaM-regulated, F-actin binding, protein. This 84,000 mol wt component (CaD84) is heat stable and cosediments with F-actin in the absence of Ca2+. In the presence of Ca2+ (greater than 1 microM) this protein disassociates from F-actin and reassociates with calmodulin. We have prepared an antibody which blocks the caldesmon-actin interaction. In the present study, we have loaded this antibody into cells to prevent the (re-)association of caldesmon with F-actin. This treatment synergistically augments the ability of GnRH and other secretogogues (maitotoxin, phorbol myristyl acetate) to stimulate gonadotropin release from the pituitary. This finding, along with the previous observations that GnRH provokes a sufficient rise in intracellular Ca2+ to allow CaM to redistribute and bind proteins which it regulates, suggests a role for caldesmon in GnRH-stimulated gonadotropin release from the pituitary.

Actins↗

PubMed1990

Imprint of thymic selection on autoreactive repertoires.

We have focussed on the differences in origin and physiological properties of two classes of self-reactive T cells. Autoreactive T cells described in many laboratories are activated in the course of normal immune responses to foreign antigen. These T cells can be shown under well-defined conditions to be the direct progeny of antigen-stimulated precursors. This, together with evidence that their activation requirements can be distinguished from those of antigen-specific, MHC-restricted T cells, leads us to suggest that they represent a particular physiological state that recapitulates the conditions of thymic selection and is induced in many antigen-specific, MHC-restricted peripheral T cells as a result of normal antigen-dependent activation. Although it appears that the associated physiological properties can be stable in some in vitro maintained lines, it is possible that this is normally a transient state in vivo. Available evidence concerning the specificity of these T cells indicates only that they can be activated in the absence of any identifiable foreign antigen by class II MHC-syngeneic but not MHC-allogeneic stimulators. We have suggested that such T cells are specific for the same elements, possibly an association of MHC and other self-peptides (Singer et al. 1987), that are the basis for positive selection in the thymus. The properties of these autoreactive T cells need to be distinguished from those of T cells associated with autoimmune pathology. It is presumed that autoimmune T cells are directly activated in a resting state by specific self-peptides. Our interest in distinguishing these self-reactive T-cell populations has focussed on different predictions concerning the diversity of their associated self-reactive repertoires. The relative complexity of the immune repertoire expressed in autoreactive T cells expanded by positive selection and restimulated in the course of normal antigen-specific immune responses should be considerably greater than that of autoimmune T cells constrained by negative selection and a narrow window of escape from self-tolerance. We were greatly hindered in our initial efforts in this analysis by the considerable effort required to characterize any specific immune repertoire. A published technique employing poly(A) tailing (Frohman et al. 1988) did not work efficiently in our hands, although others (Loh et al. 1989) have apparently had some success. We describe above an alternative approach, linker-facilitated PCR, which we have employed for efficient repertoire analysis. Using this method we have been able to identify dominant utilization of the Va4 family in T cells specific for the synthetic peptide YYEELLKYYEELLK.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1989

Current trends in Candida albicans research.

Candida albicans is an opportunistic pathogen of human beings and other mammals. Two other features, besides its pathogenicity, have made it a popular organism of study. It exists in different cellular forms and can change from one form to another, depending on growth conditions. Thus, it is being used as a model system to study cellular differentiation. It can also heritably and reversibly switch its cellular and colony morphologies. The yeast is diploid and lacks a sexual cycle. Thus, it has not been possible to apply the powerful methods of genetic analysis to understand morphogenesis or pathogenesis. Few clinical isolates are haploid, but they do not form hyphae and are not yet well characterized. Recombinant DNA techniques are increasingly being applied to C. albicans to solve many of the unanswered questions of morphogenesis and pathogenesis. Genetic transformation and gene-disruption techniques were recently developed for the yeast. Thus it is possible to study the role of any cloned gene through directed mutagenesis. However, the difficulty is to clone the putative genes involved in morphogenesis or pathogenesis. Candida albicans exists in four different cellular forms, namely blastospores, pseudohyphae, hyphae and chlamydospores. Blastospore-to-hypha conversion is well studied. A variety of conditions can induce this transition. It is not clear how cells sense such varied conditions and respond appropriately. In other systems where differentiation is well understood, regulatory genes which control differentiation have been uncovered. These genes cause differential expression of other genes, and ultimately differentiated phenotypes. Thus, it is likely that differential gene expression is involved in the bud-to-hypha transition in C. albicans. Certain proteins are expressed exclusively on the cell surface of hyphae. It should be possible to clone genes coding for these proteins. A study of the expression of these genes might allow us to identify the regulatory gene which determines differentiation. Another approach to understanding morphogenesis is to study how the difference in the shape of buds and hyphae is generated. This difference appears to be due to the differential activity of apical and general growth zones, which determine growth of the cell wall. Activity of these growth zones is apparently determined by actin localization. It remains a possibility that conditions which induce hyphae formation may directly affect actin localization or cell-wall growth zones and cause differences in cell shape. Candida albicans can also heritably switch its cellular phenotype. This has come to light from a study of colony-morphology switching. Some strains can switch their colony morphology, both heritably and reversibly.(ABSTRACT TRUNCATED AT 400 WORDS)

Candida albicans↗

PubMed1988

Characterization of a specific inhibitor of IL2-mediated proliferation from serum of Plasmodium berghei infected mice.

Serum pools from mice undergoing lethal infection with Plasmodium berghei inhibit the growth of an IL2-dependent mouse cytotoxic T cell line (CTLL). A partially purified preparation of the inhibitory factor specifically inhibited IL2-mediated events such as IL2-dependent CTLL growth and the Con A mitogenic response of normal mouse spleen cells. Production of and response to IL1, as well as growth of myeloma lines, was not affected. Administration of the partially purified preparation to normal mice resulted in a significant depression in IL2 production, thereby indicating a role for the inhibitory factor in maintaining immune depression in malaria-infected mice.

Animals↗

PubMed1987

Polyclonal B-cell activation and autoantibody formation during the course of mosquito-transmitted Plasmodium berghei infection in mice.

The time course of polyclonal B cell activation, as measured by titers of antibodies to DNP, FITC, and haemocyanin, as well as the time course of autoantibody formation, was followed in mice infected with Plasmodium berghei via Anopheles stephensi. IgM class antibodies to DNP, FITC, and haemocyanin appeared earlier than IgG class antibodies and persisted until death. Only IgM class anti-DNP peaked, the others remained high until death. Although IgM class autoantibodies also appeared earlier than IgG class, they peaked sharply at days 14 to 15, as did antibodies to mouse RBC. Polyclonal B cell activation, as measured by spleen plaque-forming cells (PFC) using SRBC and TNP-SRBC in the direct test, peaked at day 13. This latter event could be correlated with the time course of hypergammaglobulinemia.

Animals↗

PubMed1986

Intestinal calcium transport in the spontaneously hypertensive rat: response to calcium depletion.

We previously found intestinal Ca2+ transport to be lower in the spontaneously hypertensive (SH) as compared with the Wistar-Kyoto control (WKY) rat. These animals were fed a relatively high (1%) Ca2+ diet, and the concentration of 1 alpha,25-dihydroxycholecalciferol [1 alpha,25(OH)2D3] in serum was the same in both groups. In the present experiment we tested the possibility that the lower Ca2+ transport in the SH rat was the result of unresponsiveness to 1 alpha,25(OH)2D3. We fed diets high and low in Ca2+ and measured serum 1 alpha,25(OH)2D3 and Ca2+ transport. Serum 1 alpha,25(OH)2D3 increased in response to Ca2+ depletion at both 5 and 12 wk in both the WKY and SH rat. With high-Ca2+ diet, Ca2+ transport was lower in SH than in WKY when studied 1) in vitro in duodenum at 5 wk of age, and 2) in vivo in proximal and distal small intestine at 12 wk of age. Ca2+ transport increased in SH in response to Ca2+ depletion, but not in WKY, except in distal small intestine in vivo at 12 wk. In summary, although Ca2+ transport is lower in the SH as compared with the WKY rat when vitamin D activity is basal through feeding a high-Ca2+ diet, Ca2+ transport increases in the SH rat in response to the increase in 1 alpha,25(OH)2D3 produced by feeding a low-Ca2+ diet. We conclude that 1) the vitamin D-regulated component of mediated Ca2+ transport is intact in the SH rat and is unrelated to hypertension, and 2) mediated Ca2+ transport under basal conditions, i.e., nonvitamin D-regulated, differs in the SH and WKY rats and may be related to hypertension.

Animals↗

PubMed1985

Distribution and ontogeny of B cells in the garden lizard, Calotes versicolor.

Surface immunoglobulin bearing (sIg+) cells were identified in the lizard Calotes versicolor by immunofluorescence using a polyvalent antiserum to lizard immunoglobulins and class-specific antibodies to lizard IgM and IgY. 53.3 +/- 1.6% of spleen cells, 23.6 +/- 0.8% of peripheral blood mononuclear cells, 21.5 +/- 1.8% of bone marrow mononuclear cells and less than 1% of thymus cells were found to bear immunoglobulin (Ig) on their surface. A similar proportion of cells in each tissue was stained with rabbit anti-lizard mu (specific for IgM) whereas only a small proportion of cells were stained with rabbit anti-lizard (specific for IgY). Adult thymectomy significantly increased the proportion of sIgM+ cells in spleen and blood whereas high dose cyclophosphamide (300mg/kg body weight) decreased the proportion of sIgM+ cells thus suggesting that sIg+ cells in the lizard are of B cell lineage as in higher vertebrates. Ontogenetic studies indicate that embryonic liver is an organ enriched for sIgM+ cells at certain stages of development and thus suggest liver to be a site for differentiation of sIg+ cells in lizard embryos.

Animals↗

PubMed1984

Immunoglobulin classes in the garden lizard, Calotes versicolor.

Two classes of immunoglobulins have been purified from lizard serum using a combination of ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 or on Sepharose 6B. Lizard IgM is 2-ME sensitive and has an intact molecular weight similar to human IgM. On SDS-PAGE, reduced IgM dissociates into heavy and light chains of molecular weight 70,000 and 23,500 daltons respectively. Lizards also possess a 2-ME resistant, low molecular weight immunoglobulin designated as IgY similar to avian and amphibian IgY. IgY dissociates on SDS-PAGE to yield 59,500 dalton heavy and 26,000 dalton light chains. Antisera raised in rabbits to each of the two Ig classes could be made class-specific by cross-absorption, thus indicating that IgM and IgY represent distinct isotypes.

Animals↗

PubMed1984

Induction of N-acetyl-D-glucosamine catabolic enzymes and germinative response in Candida albicans.

The regulation of N-acetylglucosamine catabolic enzymes was studied in both yeast and germ tube forms of the dimorphic fungus Candida albicans. The induction pattern of these enzymes was the same for yeast cells incubated at 28 degrees C and in cells incubated at 37 degrees C which formed germ tubes. However, the level of activity of these enzymes in germ tube stage is lower as compared to yeast phase cells. A strain of C. albicans that did not form germ tubes was endowed with a pronounced ability for induction of N-acetylglucosamine catabolic enzymes. This result suggests that germ tube formation and N-acetylglucosamine metabolism are mutually exclusive events.

Acetylglucosamine↗

PubMed1983

Comparison of techniques for the estimation of the prevalence of poliomyelitis in developing countries.

Although reliable data on the prevalence and trends of paralytic poliomyelitis are necessary for health planning, they are often difficult to obtain in developing countries. Comparisons were made of the cost and the time taken to obtain prevalence rates of residual poliomyelitis paralysis in a single community by (1) a surveillance and reporting system using physicians and other practitioners, (2) a school survey covering grades 1 to 12, (3) annual surveys of grade 1 schoolchildren, and (4) a house-to-house questionnaire survey. These techniques gave poliomyelitis prevalence rates of 3-5 per 1000 school-age children, and poliomyelitis incidence rates of 12-29 per 100 000 population per year. The annual grade 1 school survey was the cheapest and easiest to carry out and appears reliable in areas with a high rate of school enrolment.

Adolescent↗