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Biomedical subjects

M Srinivasan

Publications and source records attributed to M Srinivasan.

At least 109 records · Page 6Linked to original sources
PubMed1995

Autoimmunity to a cornea-associated stromal antigen in patients with Mooren's ulcer.

PURPOSE: To purify and characterize a cornea-associated antigen (CO-Ag) and to determine antibody levels to CO-Ag in patients with Mooren's ulcer. METHOD: Standard ion exchange and gel filtration chromatographies were used to isolate and purify CO-Ag from crude bovine stromal extracts. The serum of a patient with Mooren's ulcer, containing a high level of antibodies directed against CO-Ag, was used to monitor isolation procedures. Using this newly purified CO-Ag, an enzyme-linked immunoabsorbent assay was used to detect the presence of antibodies to CO-Ag in the sera of other patients with Mooren's ulcer. RESULTS: CO-Ag was purified to apparent homogeneity from bovine corneal stromal extracts by a series of ion exchange chromatographies and gel filtration. Polyacrylamide gel electrophoresis showed that CO-Ag was a tetramer with a molecular weight of 30,000 d that may dissociate under denaturing conditions into a monomer of 7000 d. Strong indirect immunofluorescent staining was demonstrated of the stroma by guinea pig anti-CO-Ag antibody. A statistically significant difference in the level of specific antibodies to CO-Ag between patients with Mooren's ulcer and controls was found (P < 0.001). The antibody level was elevated in patients with Mooren's ulcer (mean antibody level, 0.58 +/- 0.13) compared with the controls (mean antibody level, 0.22 +/- 0.04). CONCLUSION: These results suggest that an autoantigen exists in the corneal stroma that reacts with serum antibodies from patients with Mooren's ulcer. The availability of a purified corneal antigen could facilitate the diagnosis and define the pathogenetic mechanisms in Mooren's ulcer.

Animals↗
PubMed1994

Mitochondrial carbonic anhydrase (isozyme V) in mouse and rat: cDNA cloning, expression, subcellular localization, processing, and tissue distribution.

When the human cDNA, isolated on the basis of homology to the murine carbonic anhydrase (CA) "Y" was expressed in COS cells, the human CA was targeted to and processed in mitochondria, as expected for CA-V. However, tissue distribution reported for the corresponding mouse CA Y mRNA was much more limited than that reported for the distribution of CA-V immunostaining in rat tissues. To determine whether the murine cDNA actually encodes a mitochondrial CA activity and to compare the tissue distribution of the homologous murine and rat gene products, we used reverse transcription-PCR to reisolate the murine CA-V candidate cDNA and used the murine cDNA probe to isolate the homologous rat cDNA. We compared the two cDNA sequences, the activities they expressed after transfection of COS cells, and the sites of N-terminal processing of expressed products. In addition, we used antibodies to the C-terminal peptides predicted from each cDNA to compare distribution of CA-V in mouse and rat tissues and to identify CA-Vs in mitochondria isolated from mouse and rat liver. From these studies, we conclude that both mouse and rat CA-V candidate cDNAs encode active CAs that are targeted to and processed in mitochondria and that there are real differences in tissue distribution of CA-V between mouse and rat. However, the findings that are M(r) of CA-V in rat tissues is smaller than that previously reported and that the tissue distribution also differs lead us to conclude that the antibody used in prior reports most likely misidentified another antigen in rat tissues as CA-V.

Amino Acid Sequence↗
PubMed1994

Extrasynaptic localization of taurine-like immunoreactivity in the lamprey spinal cord.

Taurine is an endogenous amino acid that can occur in nerve terminals in the central nervous system and that can produce inhibitory neuronal responses. It is unclear, however, whether this amino acid can function as a synaptic transmitter. To examine the distribution of taurine at high anatomical resolution in a vertebrate, light and electron microscopic immunocytochemical postembedding techniques were applied to the lamprey spinal cord (Ichtyomyzon unicuspis and Lampetra fluviatilis), which contains many large, unmyelinated axons. The most intense immunolabeling occurred in a population of liquor-contacting cells (tanycytes), located around the central canal, which extended processes to the dorsal, lateral, and ventral margins of the spinal cord. In addition, a proportion of the taurine-immunoreactive cells contained gamma-aminobutyric acid (GABA)-like immunoreactivity. A moderate level of taurine immunoreactivity was also present in ependymal cells, located around the central canal, as well as in astrocytes throughout all regions of the spinal cord. At the ultrastructural level, the taurine immunoreactivity showed an even distribution in the cytoplasm of the labeled cells. In contrast to the glial labeling, neuronal cell bodies and axons exhibited very low levels of taurine labeling, which were similar to the level of background labeling. The synaptic vesicle clusters within the axons did not show any clear accumulation of taurine immunoreactivity. These results suggest that taurine may have metabolic roles in the lamprey spinal cord, and, as in other systems, it may take part in osmoregulation. However, the lack of immunolabeling in presynaptic elements is not consistent with a role of taurine as a synaptic transmitter.

Animals↗
PubMed1994

Stimulation of protein phosphatase-1 activity by phorbol esters. Evaluation of the regulatory role of protein kinase C in insulin action.

In this study, we examined the role of insulin, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) cascade in activation of protein phosphatase-1 (PP-1) by using three complementary approaches. First, differentiated L6 cells were acutely exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA, 400 nM) to activate PKC. In these cells, TPA caused 32% stimulation of PP-1 activity. The PP-1 stimulation by TPA was comparable to stimulation by insulin (t1/2 = 1 min and EC50 = 5 nM) with a maximum effect in 5 min. The effects of insulin and TPA were not additive. Insulin and TPA also stimulated MAPK (> 2-fold increase over basal, with myelin basic protein as a substrate). ML-9, a myosin light chain kinase inhibitor, blocked the effects of insulin and TPA on both MAPK and PP-1 activation. In the second approach, PKC was down-regulated by chronic treatment with TPA. In these cells subsequent effects of insulin on MAPK and PP-1 activation were blocked, without an effect on basal enzyme levels. In the third approach, two selective inhibitors of PKC, calphostin and chelerythrine chloride, were used to inhibit PKC. These inhibitors completely prevented insulin and TPA stimulation of MAPK and PP-1 and blocked insulin-induced translocation of PKC to the plasma membranes. We conclude that PKC plays an important role in insulin stimulation of PP-1 via the activation of MAPK cascade.

Alkaloids↗
PubMed1994

Gastric relaxation in response to chemical stimulation of the area postrema in the rabbit.

Microinjections of DL-homocysteic acid into the area postrema (AP) of anesthetized rabbits provoked gastric relaxations associated with small changes in blood pressure and marked excitatory effects on respiration. Both gastric and cardiovascular effects failed to occur after bilateral vagotomy. Comparable gastric relaxations were induced before and after treatment with atropine or atropine and guanethidine. The AP appears to play a role in gastric motility via vagus nerves and nonadrenergic noncholinergic intramural inhibitory neurons.

Animals↗
PubMed1994

Regulation of protein phosphatase 1 and 2A activities by insulin during myogenesis in rat skeletal muscle cells in culture.

In this study, we examined protein phosphatase 1 (PP-1) and protein phosphatase 2A (PP-2A) activities during various stages of myogenesis and their regulation by insulin in rat skeletal muscle cells. Protein phosphatase activities were measured using 32P-labeled phosphorylase a, glycogen synthase, and phosphorylase kinase as substrates. Spontaneous PP-1 activity increased progressively in cultures from 2 to 5 days, PP-2A activities remained constant in days 2-4 cultures and increased sharply on day 5. Most of the times in culture, a significant proportion (approximately 65%) of PP-1 was in a form that could be activated by trypsin. Insulin stimulated PP-1 activity (40-80% increase over basal) in a time (t1/2 approximately 5 min)- and dose (EC50 approximately 0.1 nM)-dependent manner. Insulin activation of PP-1 was accompanied by a corresponding inhibition in PP-2A activity. The effects of insulin on PP-1 and PP-2A were differentiation dependent and were observed only in cells at fusion (day 5) and post-fusion. The insulin's effect on PP-1 correlated with the gradual appearance of PP-1 G subunit in cells at fusion. Immunoprecipitation of PP-1 from 32P-labeled cells with an antibody directed against the site 1 sequence of rabbit skeletal muscle PP-1G detected a 160-kDa protein, phosphorylation of which was significantly increased by insulin. This correlated well with the increase observed in immunoprecipitated PP-1G activity. Treatment of cells with a cAMP agonist (SpcAMP) completely blocked activation of PP-1 by insulin and diminished insulin-stimulated phosphorylation of the 160-kDa protein. The likely identity of the 160-kDa band as the regulatory subunit of PP-1 was confirmed by assay of PP-1 activity in the immunoprecipitates and by competition studies with the site 1 peptide against which the antibody was made. From these studies, we conclude that insulin activates PP-1 in L6 cells by increasing the phosphorylation of its regulatory subunit.

Animals↗
PubMed1994

Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus.

Intracellular targeting may enable protein kinases with broad substrate-specificities, such as multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) to achieve a selectivity of action in vivo. We have examined the intracellular targeting of three delta-CaM kinase isoforms. The delta B-CaM kinase isoform is targeted to the nucleus in transfected cells while the delta A- and delta C-CaM kinase isoforms are cytosolic/cytoskeletal. A chimeric construct of alpha-CaM kinase containing the delta B-CaM kinase variable domain is rerouted to the nucleus while the native alpha-CaM kinase and chimeras of alpha-CaM kinase which contain the delta A- or delta C-CaM kinase variable domains are retained in the cytoplasm. Using site-directed mutagenesis, we have defined a nuclear localization signal (NLS) within an 11-amino acid sequence, likely inserted by alternative splicing, in the variable domain of delta B-CaM kinase. Isoform-specific nuclear targeting of CaM kinase is probably a key mechanism in the selective regulation of nuclear functions by CaM kinase. CaM kinase is a multimer that can be composed of several isoforms. We find that when cells express two different isoforms of CaM kinase, cellular targeting is determined by the ratio of the isoforms. When an excess of the cytoplasmic isoform of CaM kinase is coexpressed along with the nuclear isoform, both isoforms are localized in the cytoplasm. Conversely an excess of the nuclear isoform can reroute the cytoplasmic isoform to the nucleus. The nuclear isoform likely coassembles with the cytosolic isoform, to form a heteromultimeric holoenzyme which is transported into the nucleus. These experiments demonstrate isoform-specific targeting of CaM kinase and indicate that such targeting can be modified by the expression of multiple isoforms of the enzyme.

Alternative Splicing↗
PubMed1994

Psychological sequelae following the Gulf War. Factors associated with subsequent morbidity and the effectiveness of psychological debriefing.

BACKGROUND: The aim was to study the effect of brief counselling and psychological debriefing following a trauma on subsequent morbidity. METHOD: We investigated psychological morbidity in 62 British soldiers whose duties included the handling and identification of dead bodies of allied and enemy soldiers during the Gulf War. Of these soldiers, 69% received a psychological debriefing on completion of their duties. The subjects completed by post a demographic questionnaire, the General Health Questionnaire (GHQ-28) and the Impact of Events Scale. RESULTS: After nine months 50% had evidence of some psychological disturbance suggestive of posttraumatic stress disorder (PTSD); 18% had sought professional help; 26% reported relationship difficulties. Neither prior training nor the psychological intervention appeared to make any difference to subsequent psychiatric morbidity. Morbidity at nine months was more likely in those with a history of psychological problems and those who believed their lives had been in danger in the Gulf. CONCLUSIONS: These findings show that a psychological debriefing following a series of traumatic events or experiences does not appear to reduce subsequent psychiatric morbidity and highlights the need for further research in military and civilian settings.

Adult↗
PubMed1993

Endothelin concentrations in respiration-related structures of the medulla during the perinatal period of the rat.

Endothelin-like immunoreactivity (ET-LI) was quantified in the developing (foetal and postnatal) rat brain stem and cerebellum using radioimmunoassay. The brain stem structures chosen for this study were (a) dorsal medullary region (DMR) including the region of nucleus tractus solitarius where the peripheral chemoreceptor afferents are known to terminate, (b) ventral medullary region (VMR) where the central chemoreceptors are thought to be located and (c) cerebellum (CER), as a control area. Compared to the prenatal period, significantly elevated concentrations of ET-LI were detected in the early postnatal period and thereafter the concentrations decreased: DMR and VMR: in comparison to the prenatal concentrations, a two-fold increase was found on the day of birth which further increased significantly (P < 0.001) on postnatal day 1 only in the region of DMR; CER: low concentrations of ET-LI were found in the early postnatal period which were not significantly different from the prenatal values. No ET-LI could be detected in any of the three regions in the adult rats. The results are discussed in view of the hypothesis that (1) endothelin appears to play an important role in the perinatal period and (2) it is involved in the chemoreceptor pathway.

Animals↗
PubMed1993

Functional replacement of the Saccharomyces cerevisiae Trg1/Pdi1 protein by members of the mammalian protein disulfide isomerase family.

The TRG1/PDI1 gene of Saccharomyces cerevisiae is essential for growth and encodes a lumenal endoplasmic reticulum (ER) glycoprotein that is structurally related to thioredoxin and is involved in the secretory pathway. We have tested whether the yeast Trg1/Pdi1 protein can be replaced in vivo by three members of the mammalian thioredoxin-related protein family, protein disulfide isomerase (PDI), ERp72, and ERp61. Multicopy plasmids containing galactose-inducible rodent PDI and ERp72 genes support germination and growth of haploid trg1 null mutants in galactose-containing media, whereas the ERp61 gene is inactive. Strains expressing PDI or ERp72 instead of Trg1 are thermosensitive. An overproduced mutant Trg1 protein lacking the HDEL retention signal supports growth, whereas a truncated version of the protein containing only one thioredoxin-like domain is inactive. The mammalian proteins were localized to both the soluble and microsomal membrane fraction of yeast cells. Our observations indicate that the two unglycosylated mammalian proteins PDI and ERp72 are capable of replacing at least some of the critical functions of Trg1, in spite of the fact that the three proteins diverge considerably in sequences surrounding the thioredoxin-related domains.

Animals↗
PubMed1993

Peptides of 23 residues or greater are required to stimulate a high affinity class II-restricted T cell response.

Helper T cells recognize fragments of antigen bound to the class II molecules on the surface of antigen-presenting cells. Naturally processed antigenic fragments have been isolated from the class II molecules and shown to be heterogeneous in length, ranging from 13 to 25 residues, and to vary at both the N and C termini. A 15-residue peptide in an extended conformation is predicted to fit in an open peptide-binding cleft of the class II molecules. Thus, the longer peptides observed bound to class II presumably have regions which reside outside the cleft. It is not known if the additional length contributes significantly to T cell activation. We have carried out a systematic analysis of the antigenicity of peptides of increasing length beyond the minimally defined T cell antigenic peptide. Here we show that the full functional activities of peptides representing the major antigenic determinant of the protein antigen, cytochrome c, minimally require that the peptides be 23 amino acids long. The long peptides do not require processing and are presented by purified class II molecules incorporated into synthetic membranes, indicating that such peptides associate directly with class II and require no additional cellular machinery for presentation. We also show that a hybrid peptide, 51 residues in length, containing a 29-residue cytochrome c peptide and a "promiscuous" peptide of tetanus toxoid, is more antigenic than the 23-residue peptide alone and significantly, does not require processing. Thus, the additional peptide length, although not predicted to bind in the peptide-binding groove of the MHC class II molecule, has a significant impact on the ability of the peptides to stimulate T cell responses maximally.

Amino Acid Sequence↗
PubMed1993

Identification of genomic sequences that mediate the induction of the endoplasmic reticulum stress protein, ERp72, by protein traffic.

ERp72, a resident protein of the endoplasmic reticulum (ER) is both a stress protein and a member of the protein disulfide isomerase family of proteins. Analysis of the murine ERp72 promoter region revealed the presence of potential transcriptional control elements characteristic of the promoters of mammalian ER proteins. These include multiple CCAAT elements and Sp1 and AP-2 consensus sequences. Functional analysis of mutations in the ERp72 promoter and 5'-flanking region revealed an 82-bp fragment that is sufficient to mediate the stimulation observed for ERp72 either by stress or by the expression of incompletely assembled immunoglobulin mu heavy chain in the ER. This 82-bp fragment contains two CCAAT elements but little additional homology to protein traffic-responsive sequences of other members of the ER stress family. This suggests that the ERp72 gene contains a novel element that is the target of an intracellular signaling pathway initiated by protein traffic in the ER.

3T3 Cells↗
PubMed1993

Respiratory responses to electrical and chemical stimulation of the area postrema in the rabbit.

1. The respiratory role of the area postrema (AP) has been investigated in pentobarbitone- or alpha-chloralose-anaesthetized, vagotomized, paralysed and artificially ventilated rabbits, by means of electrical stimulation and microinjections of DL-homocysteic acid (DLH). Phrenic nerve activity was used as an index of central respiratory drive. 2. Bipolar electrical or chemical stimulation (microinjections of DLH, 5-30 nl; 160 mM) of the caudal compact portion of the AP provoked excitatory effects on the inspiratory motor output, without apparent changes in the arterial blood pressure. 3. Depressant effects on inspiratory activity, accompanied on some occasions by changes in arterial blood pressure (as a rule, increases > or = 30 mmHg) were induced by DLH microinjections in close neighbouring areas (including the medial part of the nucleus tractus solitarii) or in the IV ventricle. 4. These results support a role for the AP in the neural control of respiration. The findings are discussed in connection with other autonomic functions to which the AP has been reported to contribute, in different animal species.

Animals↗
PubMed1992

The expression of murine protein disulfide isomerase in Escherichia coli.

Protein disulfide isomerase (PDI), a luminal enzyme of the endoplasmic reticulum (ER), is thought to be involved in the process that assures that the correct disulfide bonds form as a newly synthesized protein folds into its appropriate three-dimensional structure (Freeman, 1984). In recent years, the ER has been shown to have at least two additional, distinct PDI-related luminal proteins (Bennett et al., 1988; Mazzarella et al., 1990). As a potential first step toward an investigation of the structure and function of PDI and of the PDI-related proteins as well, we have developed a bacterial expression system in Escherichia coli capable of synthesizing significant levels of enzymatically active PDI under the control of the inducible tac promoter. We have observed that the use of this bacterial expression system is complicated by the fact that there is a significant amount of internal initiation of protein synthesis within the PDI coding sequence and the fact that all of the PDI-related expression products are found equally distributed between the cytoplasmic and periplasmic fractions due to a single peptide-independent mechanism. Our studies with this system have demonstrated that at least some truncated PDI molecules containing the carboxy-terminal most active site have significant PDI activity.

Amino Acid Sequence↗