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Biomedical subjects

M Saji

Publications and source records attributed to M Saji.

At least 19 recordsLinked to original sources
PubMed1992

Hormonal regulation of major histocompatibility complex class I genes in rat thyroid FRTL-5 cells: thyroid-stimulating hormone induces a cAMP-mediated decrease in class I expression.

Thyrocytes normally express major histocompatibility complex (MHC) class I, but not class II, cell surface antigens. A rat thyrocyte cell line, FRTL-5, also expresses MHC class I antigens, in addition to a variety of thyroid-specific genes. Treatment of FRTL-5 thyrocytes with physiological concentrations of thyroid-stimulating hormone (TSH) has been shown to induce increased expressed of thyroglobulin and thyroid peroxidase but to simultaneously decrease expression of the TSH receptor. The reduction in TSH receptor expression by TSH is cAMP mediated. In the present study, it is demonstrated that, in thyrocytes treated with TSH, MHC class I expression decreases concomitant with the decrease in TSH receptor expression. This decreased expression is evidenced by reduced cell surface levels of MHC class I antigens, by reduced steady-state RNA levels, and by reduced transcription of the class I genes. TSH-mediated reduction of MHC class I gene transcription in FRTL-5 cells was mapped to a region within 135 base pairs of the promoter.

Animals↗
PubMed1992

Muscimol suppresses rCGU increase in the substantia nigra after destruction of the caudate nucleus.

Injection of ibotenic acid into the caudate nucleus caused an increase in the uptake of 2-deoxyglucose (2DG) in the ipsilateral substantia nigra pars reticulata (SNr). Increased 2DG uptake was completely suppressed by chronic infusion of muscimol, the gamma-aminobutyric acid (GABA) agonist. While delayed shorter infusion of muscimol from the 3rd to the 7th day, when 2DG accumulation was the most prominent, partially prevented this increase. These data suggest that GABA-mediated transneuronal processes play an important role in the delayed elevation in 2DG uptake in SNR but hyperexcitation due to disinhibition by the loss of GABAergic inputs may play only a partial role in this increase.

Animals↗
PubMed1992

[Effect of gentiana violet against methicillin-resistant Staphylococcus aureus (MRSA)].

The bactericidal effect of gentiana violet against MRSA isolated from clinical specimens was studied both in vitro and in vivo. The results obtained are as follows: 1) Minimum bactericidal concentration (MBC) of gentiana violet to MRSA was between 0.0025% and 0.08% and the MBC was not influenced even if 25% human whole serum exists in the medium. 2) The number of cells were 2.1 x 10(7) CFU/ml in medium which decreased to 5.4 x 10(4) CFU/ml within 5 min by existing 0.1% gentiana violet in the medium as the final concentration, and also decreased under 10(3) CFU/ml 15 min later. 3) Minimum inhibitory concentration (MIC) of gentiana violet to MRSA was between 0.00015% and 0.00063%, and the inhibitory activity was not influenced even if gentiana violet was incubated with the bacteria in the medium for 72 hr at 37 degrees C. 4) By using an ointment containing 0.1% gentiana violet to 12 cases of patients with the MRSA infected skin lesions, MRSA was eliminated completely from the infected areas of the skin within 4 weeks. 5) The side effects of gentiana violet were not observed in all cases during the use of the ointment containing gentiana violet. It is suggested that gentiana violet may be one of the useful drugs for the treatment of the skin lesions infected with MRSA.

Adult↗
PubMed1992

Regulation of thyrotropin receptor gene expression in rat FRTL-5 thyroid cells.

TSH receptor mRNA levels in FRTL-5 thyroid cells are autoregulated at a transcriptional level by the same hormones required for the growth and function of the cells: TSH, insulin, and insulin-like growth factor-I (IGF-I). Thus, the ability of TSH, via its cAMP signal, to down-regulate steady state receptor mRNA levels is preceded by the action of TSH to decrease pre-mRNA levels in nuclear run-on assays to the same quantitative level as evident in Northern analyses. In contrast, the receptor mRNA half-life is shown not to change when down-regulation is reversed by withdrawing TSH in the presence or absence of actinomycin-D. Evidence is additionally provided that TSH receptor mRNA levels are increased by insulin, IGF-I, or calf serum in both Northern and run-on assays. This action cannot be duplicated by hydrocortisone and is evident at more than 20-fold lower concentrations of IGF-I than insulin. Moreover, insulin, IGF-I, and/or calf serum are required for the autoregulatory negative transcriptional regulation of the TSH receptor by TSH/cAMP, as is the case for thyroglobulin. This occurs despite the opposite actions of TSH/cAMP on the two genes, positive in the case of thyroglobulin and negative with TSH receptor. The positive and negative regulatory actions, respectively, of insulin/IGF-I and TSH on receptor gene expression are associated with coincident increases or decreases in cell surface receptors measured by [125I]TSH binding. The autoregulation additionally involves the interplay of a second cAMP-modulated regulatory factor, one which up-regulates TSH receptor mRNA levels rather than causing down-regulation. Thus, cycloheximide inhibits the transcriptional action of both TSH/cAMP and insulin/IGF-I/serum within 4 h, i.e. a rapidly synthesized protein is an intermediate in both cases. The presence of cycloheximide for as little as 1 h, however, uncovers the ability of TSH/cAMP to increase TSH receptor mRNA levels. This activity is the result of the action of a stable cAMP-induced activator which can be detected physiologically, i.e. in the absence of cycloheximide. For example, low levels of a cAMP analog (0.2 mM), as opposed to high levels (greater than 1 mM), can increase TSH receptor RNA levels. Low levels also accelerate the insulin/IGF-I-dependent return of receptor mRNA to normal levels after TSH withdrawal.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Major histocompatibility complex class I gene expression in rat thyroid cells is regulated by hormones, methimazole, and iodide as well as interferon.

Autoimmune thyroid disease is associated with enhanced expression of major histocompatibility complex class I antigens on thyrocytes. To better understand this phenomenon, we have studied the normal expression of class I genes in FRTL-5 rat thyroid cells. A variety of hormones and growth factors that regulate the growth and function of these thyroid cells were found to decrease class I RNA levels: serum, insulin or insulin-like growth factor-I (IGF-I), and hydrocortisone. Antibody preparations from Graves' patients (thyroid-stimulating antibodies), which increase cAMP levels and stimulate the thyroid, also decrease class I RNA levels. This is consistent with the fact that TSH, via its cAMP signal, reduces class I transcripts. The class I response to TSH, serum, insulin, IGF-I, or hydrocortisone is specific, in that the same agents do not similarly affect TSH receptor, thyroglobulin, thyroid peroxidase, malic enzyme, or beta-actin RNA levels. Both gamma- and alpha-interferon increase class I RNA levels in FRTL-5 cells, even in the presence of the serum, IGF-I, or hormones noted above, i.e. they overcome hormonal negative regulation in normal thyrocytes. In contrast, methimazole treatment of rat FRTL-5 thyroid cells, but not rat fibroblasts or rat FRT thyroid cells, which have no TSH receptor and no TSH-regulated function, results in reduced class I RNA levels. The action of methimazole can inhibit interferon action, is transcriptional, is duplicated by iodide, and is additive with the negative regulatory action of hormones and serum factors, including TSH.

Animals↗
PubMed1992

Molecular basis for the autoreactivity against thyroid stimulating hormone receptor.

The present report identifies an important immunogenic region of the TSH receptor and determinants on the TSH receptor for the two types of autoantibodies seen in hyperthyroid Graves' disease and hypothyroid idiopathic myxedema, TSAbs and TSBAbs, respectively. The immunogenic domain with no important functional determinants, is contained within residues 303-382 and involves residues 352-366 in particular. There are determinants flanking the immunogenic domain on the C-terminal portion of the receptor which are the TSBAb and high affinity TSH binding sites: residues 295-306, 387-395, and tyrosine 385. Determinants on the N-terminal portion of the external domain, centered on residues 38-45, are TSAb interactions linked to low affinity TSH binding important for signal generation: threonine 40 and residues 30-33, 34-37, 42-45, 52-56, and 58-61. These determinants are conserved in human and rat receptors, are not present in gonadotropin receptors, and are each related to separate actions of TSH: binding vs. signal generation. They can, therefore, account for organ specific autoimmunity and the different disease expression effected by TSBAbs vs TSAbs, i.e. hypo- vs. hyperthyroidism, respectively. It is proposed that, in the thyroid, hormonal (TSH, insulin, hydrocortisone, IGF-I) suppression of class I genes might be one means of preserving self-tolerance in the face of the hormone action to increase the expression of tissue specific genes such as thyroglobulin and thyroid peroxidase. Inappropriately high class I expression in the thyroid, i.e. if induced by interferon, viruses, or some as yet unknown agent, would contribute to the generation of autoimmune disease. Thus, it would result in increased antigen presentation to the immune system, particularly those autoantigens increased by TSH and its cAMP signal such as thyroglobulin or thyroid peroxidase, or whose turnover is increased by TSH and its cAMP signal, such as the TSH receptor. In the case of the latter, peptide 352-366, known to be near a protease sensitive site on the receptor [41,49], would now act as a potent self-antigen and induce the formation of receptor autoantibodies. It is further proposed that methimazole and high doses of iodide are therapeutically effective agents in thyroid autoimmune disease because they, in part, decrease MHC class I gene expression. Speculation is presented which suggests that elimination of negative regulation of MHC class I and the TSH receptor is an important factor in the development of autoimmune thyroid disease.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Increases in cytosolic Ca++ down regulate thyrotropin receptor gene expression by a mechanism different from the cAMP signal.

Thyrotropin (TSH) receptor mRNA levels in rat FRTL-5 thyroid cells are decreased by treatment with the calcium ionophores, A23187 or ionomycin, as well as with TSH, cholera toxin, forskolin, and 8-bromo-cAMP. Down regulation is, in each case, associated with a decrease in [125I]TSH binding and a decreased ability of TSH to increase cAMP levels. The ionophore does not alter cAMP levels and ethylene glycol-bis-(beta-aminoethyl ether) N, N'-tetraacetic acid (EGTA) in the medium prevents down regulation of TSH receptor mRNA levels by the ionophore, but not by TSH; the EGTA action is reversed by the simultaneous addition of Ca++. Whereas down regulation by TSH and its cAMP signal requires the presence of insulin and/or serum in the medium; down regulation by a calcium ionophore is still evident in their absence. Down regulation of TSH receptor mRNA levels and receptor desensitization by TSH/cAMP or an ionophore is lost in cells transfected with a full length TSH receptor cDNA devoid of regulatory elements, but able to reconstitute TSH receptor signal generation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Coexistence of glutamate and choline acetyltransferase in a major subpopulation of laryngeal motoneurons of the rat.

Glutamate immunoreactivity was found in 88% of the laryngeal motoneurons which were located in the ambiguus nucleus and the retrofacial nucleus. The glutamate-containing laryngeal motoneurons were identified by double labeling using the retrograde tracing of True blue from the cut end of the recurrent laryngeal nerve and immunocytochemistry of glutamate. On the other hand, choline acetyltransferase (ChAT) immunoreactivity was found in all the laryngeal motoneurons which were similarly identified as the origin of the recurrent laryngeal nerve using retrograde tracing of True blue. Since glutamate coexists richly with ChAT in a major subpopulation of laryngeal motoneurons, it is probable that glutamate may play a role as a co-transmitter in the cholinergic motoneurons.

Animals↗
PubMed1991

Regulation of prostaglandin synthesis by thyrotropin, insulin or insulin-like growth factor-I, and serum in FRTL-5 rat thyroid cells.

The present report shows that thyrotropin (TSH) regulates all three steps involved in prostaglandin synthesis in FRTL-5 rat thyroid cells, i.e. arachidonic acid release from membrane phospholipids, cyclooxygenase (prostaglandin H synthase) action, and individual prostaglandin formation; however, its action at specific steps may require the presence of, or can be duplicated by, insulin, insulin-like growth factor-I (IGF-I), and/or a serum factor. Thus, TSH releases free arachidonic acid from rat FRTL-5 thyroid cells whose phospholipid fraction is radiolabeled with [3H]arachidonic acid; this action involves a pertussis toxin-sensitive G protein, is not cAMP mediated, and does not require insulin or 5% serum. To quantitate TSH effects on cyclooxygenase activity and on individual prostaglandin formation, a homogenate system and a rapid reversed-phase high pressure liquid chromatography procedure have been developed to measure cyclooxygenase metabolites. TSH increased cyclooxygenase activity in homogenates only if the cells were also exposed to insulin, IGF-I, and/or 5% calf serum; TSH alone had no apparent effect on the activity. Maximal activation, 4-fold over basal/micrograms of DNA, took 36 h to achieve and reflected, at least in part, an increase in cyclooxygenase gene expression. Like cyclooxygenase activity, induction of prostaglandin E2 production required 2 or more factors, i.e. TSH plus insulin/IGF-I or TSH plus insulin/IGF-I plus serum. Increased production of prostaglandin D2, could, however, be detected if cells were treated with TSH alone and the TSH activity could be duplicated by insulin, IGF-I, or calf serum alone.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1991

The TSH receptor in autoimmune Basedow's disease.

The cloning approaches of the past two years have opened new doors to the pursuit of our understanding Basedow's disease. The cloning of the TSH receptor is the most dramatic step; nevertheless, all the proteins mentioned in the following appear to be important molecules in the bioactivity of the thyroid cell and are implicated as autoantigens.

Amino Acid Sequence↗
PubMed1991

Insulin and insulin-like growth factor-I inhibit thyrotropin-increased iodide transport in serum-depleted FRTL-5 rat thyroid cells: modulation of adenosine 3',5'-monophosphate signal action.

Insulin enhances the ability of TSH to induce iodide uptake in FRTL-5 rat thyroid cells maintained in 5% serum; however, in cells maintained in 0.2% serum, insulin inhibits the ability of TSH to induce iodide uptake. Since the inhibitory action of insulin is duplicated by 100-fold lower concentrations of insulin-like growth factor-I (IGF-I), inhibition appears to be mediated by the IGF-I receptor. Insulin and IGF-I inhibit the action of a cAMP analog to induce iodide uptake in a manner identical to TSH, but do not inhibit basal or TSH-increased cAMP levels; inhibition, thus, results from regulation of cAMP signal action rather than cAMP signal generation. Inhibition is associated with a more than 2-fold decrease in the maximum velocity of iodide influx, a less than 15% change in the rate of iodide efflux, and no change in the Km for iodide influx, i.e. inhibition effectively results from a decrease in the number of iodide porters. The inhibitory action of insulin/IGF-I is not additive with hydrocortisone, which, under the same conditions, also inhibits TSH- or cAMP-induced iodide porter activity. Actinomycin-D, given 24 h after TSH, superinduces TSH-induced iodide porter activity and abolishes the inhibition by insulin, IGF-I, and/or hydrocortisone; a similar paradoxical effect of actinomycin-D under these conditions has been explained by its ability to inhibit the action of a cAMP-induced factor that increases mRNA degradation. The inhibitory actions of insulin, IGF-I, and hydrocortisone on cAMP-induced iodide porter activity contrast with their simultaneous and synergistic stimulation of the transcriptional action of cAMP on DNA and thyroglobulin synthesis under these conditions.

Animals↗
PubMed1991

Methimazole regulation of thyroglobulin biosynthesis and gene transcription in rat FRTL-5 thyroid cells.

Methimazole (MMI) increases thyroglobulin (Tg) mRNA levels in FRTL-5 rat thyroid cells. The increase reflects a transcriptional action of the antithyroid agent and is inhibited by cycloheximide, as is the transcriptional action of TSH. It takes several hours to be apparent, is maximal between 24-48 h, and is specific, in that thyroid peroxidase and beta-actin mRNA levels are not increased simultaneously. The increased mRNA levels are associated with increased recovery of immunoprecipitable Tg in the medium of cells exposed to [35S]methionine. The MMI effect appears to be independent of the action of TSH or its cAMP signal, since the MMI-induced increase in Tg mRNA levels is evident in cells treated with TSH or maintained in its absence and is associated not with increases in cAMP levels but, rather, under some circumstances with a decrease. The effect is evident under conditions in which the ability of insulin or insulin-like growth factor-I to increase Tg mRNA levels is already maximal. The MMI-induced increase is inhibited by concentrations of iodide associated with autoregulation of FRTL-5 rat thyroid cells, is inhibited but not mimicked by propylthiouracil, and is not altered by T3. The increase in Tg mRNA levels does not correlate with increased DNA synthesis as a function of MMI concentration either in cells treated with TSH or in those maintained in its absence. A concentration of MMI (5 mM) that increases Tg mRNA levels can also inhibit 8-bromo-cAMP- or phorbol ester-induced increases in [3H]thymidine incorporation into DNA.

Animals↗
PubMed1990

Evidence that glutamate is the transmitter mediating respiratory drive from medullary premotor neurons to phrenic motoneurons: a double labeling study in the rat.

Glutamate immunoreactivity was found in 81-84% of the bulbospinal neurons projecting to the phrenic motoneuron pool, which were located in the para-ambiguous nucleus and the ventral vicinity of the retrofacial nucleus. Their glutamate contents were identified by a double labeling technique in combination with the retrograde transport of horseradish peroxidase and immunocytochemistry of the glutamate. The remaining 16-19% of the bulbospinal neurons projecting to the phrenic motoneuron pool did not contain glutamate immunoreactivity. Thus it appears that glutamate may be a major transmitter of the inspiratory drive from the medullary respiratory neurons to the phrenic motoneurons.

Animals↗
PubMed1990

A microsequencing approach to identify proteins which appear to interact with thyrotropin in rat FRTL-5 thyroid cells.

In order to resolve questions concerning the in situ structure of the thyrotropin (TSH) receptor, [35S]methionine-labeled thyroid cell preparations were detergent solubilized and proteins exhibiting TSH-dependent binding to TSH-Sepharose were identified. Two such proteins, 43 and 70 kd, are identified in this report as gamma-actin and a member of the heat shock 70 protein family, respectively, based on the microsequence of two peptides from each. Identification of the former was confirmed by Western blotting and immunostaining using anti-actin, the latter by its ability to bind [32P]ATP, a characteristic feature of this family of proteins. The results suggest that TSH-cross linking reports defining TSH receptor subunits should be viewed with caution in the absence of comparative sequence data; consideration must, however, be given to the existence of receptor associated proteins.

Actins↗
PubMed1990

Thoracic expiratory motor neurons of the rat: localization and sites of origin of their premotor neurons.

The expiratory motor neurons of the representative thoracic segment of the rat were examined as to their localization and sites of origin of their premotor neurons in the lower brainstem. Either T6 or T7 was selected as a representative target segment for horseradish peroxidase (HRP) injection. The intercostal motor neurons in the T6 or T7 were doubly labeled by HRP placed in the cut end of the internal intercostal nerve and True blue placed in the cut end of the external intercostal nerve. The thoracic expiratory motor neurons labeled by HRP were concentrated in the oblique zone running along the dorsal to ventrolateral direction in both the T6 and T7 segments. By contrast, the thoracic inspiratory motor neurons labeled by True blue were concentrated in the horizontal zone running along the bottom of the ventral horn in both the T6 and T7 segments. A small amount of HRP was iontophoretically injected through a double-barrel coaxial electrode to sites of the expiratory motor neurons which had been identified electrophysiologically. In 5 successful experiments, the HRP-labeled cells were bilaterally distributed in the para-ambiguus nucleus (59.0%), ventral subnucleus of the paramedian reticular nucleus (13.6%), and raphe nuclei (10.8%). In another experiment, it was found that the expiratory premotor neurons in the para-ambiguus nucleus were present in a narrow column over the entire length of the nucleus at a level between 1.0 mm rostral and 1.4 mm caudal to the obex.

Animals↗
PubMed1990

Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies.

A rat thyrotropin (thyroid-stimulating hormone, TSH) receptor cDNA was isolated that encoded a protein of 764 amino acids, Mr 86,528. Transfection of the cDNA caused COS-7 cells to develop a TSH-sensitive adenylate cyclase response and the ability to bind 125I-labeled TSH; both activities were similar to those of rat FRTL-5 thyroid cells and not duplicated by lutropin. The gene represented by the cDNA was assigned to mouse chromosome 12 and human chromosome 14. Northern analyses identified two species of mRNA, 5.6 and 3.3 kilobases, in FRTL-5 thyroid cells; the transcripts appeared to differ only in the extent of their 3' noncoding sequences. There were minimal amounts of the two mRNAs in rat ovary, and neither was detected in RNA preparations from rat testis, liver, lung, brain, spleen, and FRT thyroid cells, which do not have a functional TSH receptor. TSH decreased both mRNA species 3- to 4-fold within 8 hr in FRTL-5 thyroid cells; down-regulation was dependent on TSH concentration and duplicated by forskolin, cholera toxin, or 8-bromo-cAMP but not by a phorbol ester. Down-regulation was also duplicated by thyroid-stimulating autoantibodies, which increased cAMP levels, but not by thyrotropin binding-inhibiting auto-antibodies, which actually increased TSH receptor mRNA levels.

Amino Acid Sequence↗
PubMed1990

Effect of lithium on deoxyribonucleic acid synthesis and iodide uptake in porcine thyroid cells in culture.

We investigated the direct effect of lithium on porcine thyroid cells in culture to exclude the secondary regulatory factors. First we have studied the effect of lithium on TSH-induced iodide uptake. Significant suppression was seen at 0.1 mmol/liter, and half-maximal suppression was obtained at the pharmacological concentration reported in patient serum. The suppression was dose dependent and reversible. Besides the suppression of cAMP production stimulated by TSH, lithium also inhibited iodide uptake stimulated by forskolin or 8-bromo-cAMP. These results demonstrated that lithium inhibits TSH-induced iodide uptake not only by reducing cAMP production, but also by acting on the steps of post-cAMP production. Next, we studied the effect of lithium on DNA synthesis of the cultured porcine thyroid cells. Lithium stimulated [3H]thymidine incorporation of the thyroid cells in the basal condition (0.5% fetal calf serum) as well as those stimulated by insulin-like growth factor-I (100 micrograms/liter). The minimal concentrations for the significant increase were 0.5 and 0.1 mmol/liter, respectively. These results suggest that lithium might contribute to the formation of the goiter directly at the cellular levels in patients treated with the agent.

Animals↗