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PubMed1998

Identification of suprachiasmatic melanotrope-inhibiting neurons in Xenopus laevis: a confocal laser-scanning microscopy study.

The amphibian Xenopus laevis is able to adjust its skin color to the light intensity of the environment. Paling of the skin is achieved by inhibiting the release of alpha-melanophore-stimulating hormone (alpha-MSH) from the melanotrope cells in the pars intermedia of the pituitary gland. The release of alpha-MSH is inhibited by gamma-aminobutyric acid (GABA), neuropeptide Y (NPY), and dopamine (DA). To locate and identify neurons that might be responsible for the inhibitory input, double and triple immunocytochemistry, retrograde tracing from the pars intermedia with the carbocyanine membrane probe 1,1'dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzene-sulfonate (Fast DiI), and confocal laser-scanning microscopy were combined. Glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), and NPY were found to coexist in an axonal network innervating the pars intermedia. The suprachiasmatic nucleus (SC) contained different populations of neurons that were single, double, or triple labelled for GAD, NPY, and TH. In the lateral SC, NPY+ neurons were observed. TH-immunoreactive (TH-IR) neurons occurred in the medial, dorsolateral, lateral, and ventrolateral SC. Neurons that were double labelled for NPY and TH and triple labelled for Fast DiI, NPY, and TH were present in the ventrolateral SC. This same area contained neurons that were triple labelled for GAD, NPY, and TH. It is concluded that the triple-labelled and probably the double-labelled ventrolateral SC neurons (suprachiasmatic melanotrope-inhibiting neurons) innervate the pituitary pars intermedia and are responsible for the NPY-, DA-, and GABA-mediated inhibition of melanotrope cell activity in Xenopus laevis.

Animals↗

PubMed2000

Ketoacidosis in young adults is not related to the islet antibodies at the diagnosis of Type 1 diabetes mellitus--a nationwide study.

AIMS: To test the hypothesis that there is lower prevalence of islet antibodies in subjects with newly diagnosed Type 1 diabetes mellitus in young adulthood than in children is associated with less severe diabetes at time of diagnosis. METHODS: This investigation was based on a nationwide study (Diabetes Incidence Study in Sweden) of 15-34-year-old newly diagnosed diabetic subjects. During 1992-1993, all diabetic subjects (excluding secondary and gestational diabetes) were reported on standardized forms, with information about clinical characteristics at diagnosis. The study examined islet cell antibodies (ICA) by indirect immunofluorescence, and autoantibodies to glutamic acid decarboxylase (GADA), tyrosine phosphatase-like antigen (IA-2A) and insulin (IAA) as well as C-peptide by radioimmunoassay. RESULTS: Blood samples were available from 78 patients with diabetic ketoacidosis (DKA) and 517 non-acidotic patients. The prevalence of ICA (63% vs. 57%), GADA (63% vs. 66%), IA-2A (35% vs. 44%) and IAA (20% vs. 15%) were very similar in patients with or without DKA. The median levels of the four autoantibodies did not differ between the two groups. High blood glucose (P < 0.001) and low C-peptide levels (P < 0.001) were the only parameters found to be related to DKA. CONCLUSIONS: The similarities in findings of newly diagnosed diabetic patients with or without DKA regarding ICA, GADA, IA-2A and IAA suggest that there is no relationship between the expression of antigenicity and the severity of beta-cell dysfunction. The lower prevalence of the four autoantibodies in 15-34-year-old diabetic subjects compared with previous findings in children is not explained by misclassification of diabetes type.

Adolescent↗

PubMed1994

Cardiac noradrenergic mechanisms mediate GABA-enhanced ouabain cardiotoxicity.

Peripherally administered gamma-aminobutyric acid (GABA) alters cardiovascular function and has been reported to enhance ouabain-induced cardiotoxicity in vivo. Control and reserpinized rat hearts were perfused in vitro to determine if GABA directly evokes bradycardia by GABAA receptors, interacts with ouabain, and if noradrenergic mechanisms are required. Also, double-staining immunohistochemistry was employed to determine whether GABA-ergic and noradrenergic synthetic enzymes were juxtaposed within atrial tissue. The main results were as follows. GABA produced a dose-dependent bradycardia (p < 0.05) by stimulating GABAA receptors in Langendorff-perfused hearts. Reserpinized hearts were unresponsive (p < 0.05) to GABA, except at the highest dose (20 mg/ml). Ouabain-induced cardiotoxicity was enhanced (p < 0.05) by GABA in isolated control, but not reserpinized hearts. Lastly, glutamic acid decarboxylase and tyrosine hydroxylase immunoreactivities were in close proximity in atrial slices. Collectively, the results document that GABA causes bradycardia and enhances ouabain cardiotoxicity by modulating noradrenergic mechanisms in the isolated rat heart. Since the synthetic enzymes for GABA and norepinephrine were in close proximity in atrial tissue, these transmitters may interact under physiological conditions.

Analysis of Variance↗

PubMed1998

Expression patterns conferred by tyrosine/dihydroxyphenylalanine decarboxylase promoters from opium poppy are conserved in transgenic tobacco.

Opium poppy (Papaver somniferum) contains a large family of tyrosine/dihydroxyphenylalanine decarboxylase (tydc) genes involved in the biosynthesis of benzylisoquinoline alkaloids and cell wall-bound hydroxycinnamic acid amides. Eight members from two distinct gene subfamilies have been isolated, tydc1, tydc4, tydc6, tydc8, and tydc9 in one group and tydc2, tydc3, and tydc7 in the other. The tydc8 and tydc9 genes were located 3.2 kb apart on one genomic clone, suggesting that the family is clustered. Transcripts for most tydc genes were detected only in roots. Only tydc2 and tydc7 revealed expression in both roots and shoots, and TYDC3 mRNAs were the only specific transcripts detected in seedlings. TYDC1, TYDC8, and TYDC9 mRNAs, which occurred in roots, were not detected in elicitor-treated opium poppy cultures. Expression of tydc4, which contains a premature termination codon, was not detected under any conditions. Five tydc promoters were fused to the beta-glucuronidase (GUS) reporter gene in a binary vector. All constructs produced transient GUS activity in microprojectile-bombarded opium poppy and tobacco (Nicotiana tabacum) cell cultures. The organ- and tissue-specific expression pattern of tydc promoter-GUS fusions in transgenic tobacco was generally parallel to that of corresponding tydc genes in opium poppy. GUS expression was most abundant in the internal phloem of shoot organs and in the stele of roots. Select tydc promoter-GUS fusions were also wound induced in transgenic tobacco, suggesting that the basic mechanisms of developmental and inducible tydc regulation are conserved across plant species.

Amino Acid Sequence↗

PubMed2000

Genetic and immunological characteristics of Type I diabetes mellitus in an Indo-Aryan population.

AIMS/HYPOTHESIS: Our aim was to characterise the genetic and immunological features associated with Type I (insulin-dependent) diabetes mellitus in a cohort of Indo-Aryan children resident in the United Kingdom. METHODS: Children with Type I diabetes (n = 53), unaffected first-degree relatives (n = 146) and unrelated healthy control children (n = 54) were typed for alleles of the HLA-DRB1, HLA-DQA1 and HLA-DQB1 genes. Islet cell antibodies and antibodies to glutamic acid decarboxylase, protein tyrosine phosphatase-2 (IA-2ic) and insulin were measured in the diabetic and control children. RESULTS: The DRB1*03.DQA1*05.DQB1*02 haplotype was positively associated with the disease, occurring in 78% of diabetic children compared with 22.6 % of healthy children (p(c) < 2.4 x 10(-5)). In simplex families, this haplotype was transmitted more frequently to the diabetic children than to their unaffected siblings (p < 1 x 10(-4)). The DRB1*04.DQA1* 03.DQB1*0302 haplotype was also transmitted preferentially to the diabetic probands (p < 0.025) but was not associated with disease in the case control study. Islet-related autoantibodies were detected in 89.6 % of diabetic patients compared with 11.8 % of control children (p < 1 x 10(-6)). Although protein tyrosine phosphatase-2 autoantibodies were detected more frequently among DRB1*04-positive diabetic patients compared with patients lacking this allele, the overall frequency of these autoantibodies was lower than observed in Europid diabetic subjects. This could reflect the absence of a disease association with DRB1*04 in the Indo-Aryan cohort. CONCLUSION/INTERPRETATION: Type I diabetes in our Indo-Aryan cohort is similar to the disease observed in Anglo-Europeans but has important immunogenetic differences. The low frequency of protein tyrosine phosphatase-2 autoantibodies among the Indo-Aryan diabetic children could have important implications for the design of future strategies for disease prediction in this population.

Adolescent↗

PubMed1996

Relation between cellular and humoral immunity to islet cell antigens in type 1 diabetes.

The autoimmune disease insulin-dependent diabetes is thought to result from T-cell mediated destruction of pancreatic beta cells. We analysed the relation between humoral and cellular immunity to multiple islet cell antigens, including human insulin, glutamate decarboxylase GAD65, tyrosine phosphatase (ICA512/IA2), human pancreas and RIN cells in 28 patients with newly diagnosed type 1 diabetes and 9 antibody-positive (Ab+) relatives at high risk for type 1 diabetes. Of newly diagnosed patients, all showed reactivity to at least one recombinant islet cell antigen, by elevated cellular or humoral (or both) immune responses. Fifty-seven percent of patients and relatives showed T-cell reactivity to more than one islet cell antigen and 68% revealed humoral immunity to more than one islet cell antigen. Increased T-cell response to one single islet cell antigen was observed in 32% and positive antibody response in 25% of diabetic patients and relatives. Further-more, we found that T-cell reactivity to GAD was associated with T-cell reactivity to RIN cells, whereas reactivity to ICA512 and insulin was not associated with any other T-cell response. Likewise, antibody response to ICA512/IA2 correlated with antibodies to human pancreas (ICA), whereas antibody response to GAD or insulin was not related to any other antibody response. No positive or inverse correlation, however, was detected between T cell and humoral immunity, except for a positive association of antibodies and T-cell reactivity to insulin. Our data suggest that both humoral and cellular immune reactivity to multiple islet cell antigens are present in patients with newly diagnosed type 1 diabetes and in high risk relatives, but the two immune responses are individually activated.

Adolescent↗

PubMed1979

Regulatory interrelations between GABA and polyamines. II. Effect of GABA on ornithine decarboxylase and putrescine levels in cell culture.

GABA added to rat hepatoma (HTC) cells in spinner culture at the time of induction of cell proliferation increased levels of ornithine decarboxylase (ODC) up to two- to threefold above that of control cells. The increases in ODC were also reflected by concomitant increases of intracellular putrescine levels, while spermidine and spermine were unchanged. GABA seems to have a direct stabilizing effect on ODC, since the turnover of the enzyme was slowed almost twofold when measured in cells treated with 10(-2) M GABA. The stabilizing effect is most pronounced for GABA, although some amino acids such as asparagine, glutamine, and lysine as well as some GABA analogues and homologues also tend to increase ODC but to a significantly lesser extent than GABA itself. GABA metabolites had no effect on ODC. S-Adenosylmethionine decarboxylase and tyrosine aminotransferase were not affected by the presence of GABA. The GABA effect on ODC may be important in certain types of cells for the regulation of polyamine biosynthesis.

Aminocaproates↗

PubMed1984

Prenatal ontogenesis of p-, m-octopamine and phenylethanolamine in relation to catecholamines and their metabolizing enzymes in the developing rat brain and heart.

Non-catecholamines such as phenylethanolamine and p-octopamine are present in many invertebrate nervous systems, sometimes in large amounts. These amines are normally present in the rat brain at much lower levels, p- and m-octopamine are present at trace levels in the mammalian brain. The prenatal development of these amines was studied in comparison with those of noradrenaline and dopamine. The activities of tyrosine hydroxylase, dopa decarboxylase, dopamine beta-hydroxylase and monoamine oxidase were determined in parallel. Phenylethanolamine and p-octopamine are more abundant in the brain between 13 and 17 fetal days than dopamine and noradrenaline but decrease after 17 days whereas the levels of m-octopamine and the two catecholamines increase afterwards. Dopa decarboxylase, dopamine beta-hydroxylase and tyrosine hydroxylase are detected early in fetal life (13, 15 and 14.5 days respectively) but monoamine oxidase activity was not found before 18 days.

2-Hydroxyphenethylamine↗

PubMed1979

Appearance of tyrosine hydroxylase, aromatic amino-acid decarboxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase during the ontogenesis of the adrenal medulla: an immunohistochemical study in the rat.

The cellular localization of the enzymes tyrosine hydroxylase (TH), aromatic amino-acid decarboxylase (or dopa decarboxylase, DDC), dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla of adult rats and rat fetuses (14th, 17th, 18th, 19th and 21st day) was examined. In the prenatal stages the medullary blastema and an adjacent part of the primitive sympathetic trunk were also investigated. Tissues were fixed in ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). Cryostat sections (10 micron in thickness) were stained by the indirect immunofluorescence technique. Rabbit antibodies to TH (isolated from human pheochromocytoma), DDC, DBH and PNMT (the latter three isolated from bovine adrenal medulla) were used. Sections incubated with serum of non-immunized rabbits were used as controls. In the adult adrenal medulla, two cell types can be distinguished. One cell type contains only TH, DDC and DBH. The other cell type contains PNMT in addition. It is concluded that these cells correspond to the noradrenaline-(NA-) and adrenaline- (A-)storing cells respectively. In all prenatal stages TH, DDC and DBH are found in the primitive sympathetic trunk, in the medullary blastema, and in the medullary cells which have migrated into the cortical "anlage". PNMT is observed for the first time on the 18th day. Moreover, PNMT could only be demonstrated inside the adrenal gland. From these observations it is concluded that the capacity to synthesize NA is developed even before the "medullary" cells have reached the cortical "anlage". On the contrary, the capacity to synthesize A seems to be acquired only after this contact is established. The hypothesis is put forward that this phenomenon might indicate the induction of PNMT by glucocorticoids secreted by the fetal cortex.

Adrenal Medulla↗

PubMed1996

Cellular immune response to diverse islet cell antigens in IDDM.

In IDDM, T-cells are postulated to mediate the destruction of pancreatic beta-cells. We analyzed peripheral blood mononuclear cell (PBMC) responses to human insulin, glutamate decarboxylase GAD65, tyrosine phosphatase ICA512, glucagon, membrane preparations of RIN cells and human pancreas, and three control antigens (La = nuclear cell antigen, tetanus toxoid, and phytohemagglutinin). A total of 28 patients with newly diagnosed IDDM, 9 antibody-positive (Ab+) first-degree relatives, and 16 healthy control subjects were included. Increased proliferative responses to pancreatic islet cell antigens were observed in diabetic patients and in Ab+ relatives compared with control subjects, whereas T-cell reactivity to nonpancreatic control antigens was similar between the study groups. The highest differences in the magnitude of proliferative responses were seen for ICA512, followed by membrane preparations of RIN cells, GAD65, and human pancreas. Few subjects reacted with insulin or glucagon. Interestingly, Ab+ relatives showed higher T-cell reactivity with respect to stimulation indexes and prevalences than newly diagnosed diabetic patients, and as many as 89% of Ab+ relatives showed proliferation to more than one islet cell antigen preparation in comparison to 43% of newly diagnosed diabetic patients and none of the control subjects. Statistical analysis revealed significant positive correlation of insulin autoantibody levels with the levels of insulin-specific T-cells in Ab+ relatives, but no relation of PBMC responses to age, sex, or HLA-DR haplotypes. Our results demonstrate the simultaneous existence of various autoreactive T-cells specific for islet cell antigens in the prediabetic period. These T-cells may play a significant role in the pathogenesis of the disease.

Adolescent↗

PubMed1998

Immunohistochemical and enzyme-histochemical study on the accessory olfactory bulb of the dog.

The accessory olfactory bulb (AOB) is a primary center of the vomeronasal system. In the dog, the position and morphology of the AOB remained vague for a long time. Recently, the morphological characteristics of the dog AOB were demonstrated by means of lectin-histochemical, histological, and immunohistochemical staining, although the distribution of each kind of neuron, especially granule cells, remains controversial in the dog AOB. In the present study, we examined the distribution of neuronal elements in the dog AOB by means of immunohistochemical and enzyme-histochemical staining. Horizontal paraffin or frozen sections of the dog AOB were immunostained with antisera against protein gene product 9.5 (PGP 9.5), brain nitric oxide synthase (NOS), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH), substance P (SP), and vasoactive intestinal polypeptide (VIP) by avidin-biotin peroxidase complex method. In addition, frozen sections were stained enzyme-histochemically for NADPH-diaphorase. In the dog AOB, vomeronasal nerve fibers, glomeruli, and mitral/tufted cells were PGP 9.5-immunopositive. Mitral/tufted cells were observed in the glomerular layer (GL) and the neuronal cell layer (NCL). In the NCL, a small number of NOS-, GAD-, and SP-immunopositive and NADPH-diaphorase positive granule cells were observed. In the GL, GAD-, TH-, and VIP-immunopositive periglomerular cells were observed. In the GL and the NCL, TH-, and VIP-immunopositive short axon cells were also observed. In addition to these neurons, TH- and SP-immunopositive afferent fibers were observed in the GL and the NCL. We could distinctly demonstrate the distribution of neuronal elements in the dog AOB. Since only a small number of granule cells were present in the dog AOB, the dog AOB did not display such a well-developed GCL as observed in the other mammals.

Animals↗

PubMed1990

Role of tyrosine kinases in gastrin induction of ornithine decarboxylase in colonic mucosa.

An organ culture system was utilized to evaluate the role of tyrosine kinases (Tyr-k) and tyrosine-specific phosphorylation of proteins in gastrin regulation of ornithine decarboxylase (ODC) activity in colonic mucosa. Exposure of colonic mucosal explants to gastrin (50-100 ng G-17 I/ml) resulted in a profound stimulation of both Tyr-k and ODC activities compared with the corresponding basal levels. Whereas the maximal stimulation (ranging between 70 and 150%) of Tyr-k occurred within 10-15 min of exposure to gastrin, ODC activity was significantly stimulated (180%) 2 h after exposure to the hormone, and at 4 h it was found to be 750% above the corresponding basal level. Difluoromethylornithine (DFMO; 2 mM), an irreversible inhibitor of ODC, completely abolished the gastrin-mediated stimulation of ODC but not Tyr-k activity. On the other hand, genistein (100 micrograms/ml), a specific inhibitor of Tyr-k, caused a total suppression of the gastrin-induced stimulation of both Tyr-k and ODC. Gastrin also stimulated tyrosyl phosphorylation of a colonic mucosal membrane protein with molecular mass of 57 kDa, and genistein greatly attenuated this effect. We conclude that gastrin stimulates colonic mucosal ODC in vitro, and Tyr-k may be required for the regulation of this process.

Animals↗

PubMed1983

Activity of several enzymes of amino acid catabolism in the liver of rats fed protein as a meal.

Rats having a protein-free diet available ad libitum were fed a daily casein meal at the beginning of either the light- or the dark-phase of the day. A control group received a mixed-diet ad libitum. In all three groups, daily food ingestion was the same and casein corresponded to 12% of total intake. Liver activities of alanine, aspartate, ornithine and tyrosine aminotransferase, ornithine decarboxylase and serine dehydratase were assessed. In mixed-fed controls, all activities were low. Tyrosine aminotransferase and ornithine decarboxylase exhibited clear circadian rhythms of low amplitude. Feeding casein as a concentrated meal had no effect on aspartate aminotransferase. It depressed alanine aminotransferase and serine dehydratase activities. Tyrosine aminotransferase and ornithine decarboxylase exhibited rapid and strong stimulatory responses but, within 12 hours, returned to levels similar to those observed in mixed-fed controls. Ornithine aminotransferase was increased in the group receiving the casein meal during the light phase. It is concluded that the capacity for amino acid catabolism remains low in separately-fed animals, and that only tyrosine and especially ornithine, which may become limiting for urea synthesis, are actively metabolized. Thus, when high fluxes of amino acids reach the liver following the absorption of the casein meal, more amino acids are available for incorporation into newly synthesized proteins.

Amino Acids↗

PubMed2004

Prediction of autoantibody positivity and progression to type 1 diabetes: Diabetes Autoimmunity Study in the Young (DAISY).

Diabetes Autoimmunity Study in the Young (DAISY) has followed 1972 children for islet autoimmunity and diabetes: 837 first-degree relatives of persons with type 1 diabetes and 1135 general population newborns identified through human leukocyte antigen (HLA) screening. During follow-up of 4.06 yr (range, 0.17-9 yr), serial determination of autoantibodies to glutamic acid decarboxylase, protein tyrosine phosphatase IA2, and insulin has generated approximately 20,000 results. Among 162 children with at least one positive autoantibody, in 31% the test was false positive (autoantibodies were negative twice on blinded duplicate aliquots), in 31% it was transiently positive (confirmed on blinded duplicate aliquots but negative on follow-up), and in 36% it was persistently positive. Using proportional hazards modeling, the HLA-DR3/4 DQ8 genotype, another positive autoantibody at the first positive visit, and level of autoantibody were predictive of persistent positivity. Only HLA-DR3/4 DQ8 genotype was predictive of progression to diabetes in proportional hazards modeling. This prospective study reveals that cross-sectional determination of islet autoantibodies in a population with relatively low previous probability of autoimmunity identifies as "positive" a large number of individuals who are either false or transiently positive. Predictive value of autoantibodies increases with blinded duplicate and independent sample retesting and incorporation of the level of autoantibody in the predictive algorithm.

Autoantibodies↗

PubMed1998

Delay of type I diabetes in high risk, first degree relatives by parenteral antigen administration: the Schwabing Insulin Prophylaxis Pilot Trial.

The Schwabing Insulin Prophylaxis Trial is a randomised, controlled pilot study designed to examine whether insulin therapy can delay or prevent the clinical onset of Type I diabetes in high risk first degree relatives of people with the disease. First degree relatives of patients with Type I diabetes, who were aged 4 years or more, had an islet cell antibody (ICA) value more than 20 Juvenile Diabetes Foundation Units (JDF-U), a reduced first phase insulin response (FPI) to an i.v. glucose tolerance test less than the 5th centile, and a normal oral glucose tolerance test were eligible for the trial. Between January 1989 and October 1995, 1736 relatives of patients with Type I diabetes were screened for ICA. We identified 64 cases (3.7%) with ICA values more than 20 JDF-U. Of ICA positive relatives, 17 (27%) had a low FPI and were eligible for enrolment. Of these 14 agreed to participate, of whom 7 were randomised to the treatment group and 7 to the control group. In the treatment group, human insulin was administered i.v. by continuous infusion for 7 days, followed by daily s. c. injections for 6 months. Intravenous insulin infusions were repeated every 12 months. In the treatment group 3 of the 7 individuals (follow-up from time of eligibility: 2.3 to 7.1 years) and in the control group 6 of the 7 untreated individuals (1.7 to 7.1 years) developed clinical diabetes. Life table analysis showed that clinical onset of Type I diabetes was delayed in insulin-treated subjects compared with control subjects (means+/-SEM diabetes-free survival: 5.0+/-0.9 years vs 2.3+/-0.7 years, p < 0.03). Insulin levels after i.v. glucose increased in the first year of intervention therapy. Titres of ICA, and antibodies to glutamic acid decarboxylase, and tyrosine phosphatase-like protein IA2 remained unchanged. These data suggest that insulin prophylaxis can delay the onset of overt diabetes in high risk relatives. This is encouraging in view of 1) the continuing American Diabetes Prevention Trial, which is currently testing the effect of parenteral insulin in a large nation-wide study and 2) the initiation of pilot trials to determine whether new antigen-specific intervention is more effective in delaying the clinical onset of Type I diabetes.

Adolescent↗

PubMed2000

Essential role of tyrosine 229 of the oxaloacetate decarboxylase beta-subunit in the energy coupling mechanism of the Na(+) pump.

The membrane-bound beta-subunit of oxaloacetate decarboxylase from Klebsiella pneumoniae catalyzes the decarboxylation of carboxybiotin, which is coupled to Na(+) translocation and consumes a periplasmically derived proton. Upon site-directed mutagenesis of 20 polar and/or conserved residues within putative membrane-integral regions, the specific oxaloacetate decarboxylase activities were reduced to various extents, but only the enzyme with a Y229F mutation was completely inactive. We propose that Y229 is part of the network by which the proton of S382 is delivered to carboxybiotin, where it is consumed upon catalyzing the immediate decarboxylation of this acid-labile compound. Unlike S382 or D203, Y229 appears to be not involved in Na(+) binding, because in the Y229F orY229A mutants, the beta-subunit was protected from tryptic digestion by 50 mM NaCl like in the wild-type enzyme. Oxaloacetate decarboxylase with a betaC291E mutation was unstable in the absence of Na(+) and dissociated into an alpha-gamma subcomplex and the beta-subunit. The enzyme could only be isolated in the presence of 0. 5 M NaCl. These results are consistent with the notion that the beta-subunit changes its conformation upon Na(+) binding.

Amino Acid Substitution↗

PubMed1995

Immunocytochemical localization of GTP cyclohydrolase I in the brain, adrenal gland, and liver of mice.

GTP cyclohydrolase I (GCH) is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), the cofactor of phenylalanine, tyrosine, and tryptophan hydroxylases, the enzymes that synthesize tyrosine, catecholamines (dopamine, noradrenaline, and adrenaline), and serotonin, respectively. We produced for the first time polyclonal antibody with highly sensitive immunoreactivity against an oligopeptide of rat enzyme, GEPERELPRPGA, by immunization of rabbits with the peptide conjugated to hemocyanin by glutaraldehyde. The specificity of the antibody was confirmed by Western blot analysis. Using this antibody specific for GCH, we observed strong GCH immunostaining in the liver cells, in the dopamine-, noradrenaline-, adrenaline-, or serotonin-containing cells of the brain, and in the adrenal gland of mice. Immunocytochemical studies revealed GCH to be localized in monoamine-containing perikarya in the periglomerular cells of the olfactory bulb, zona incerta, arcuate nucleus, ventral tegmental area, substantia nigra pars compacta, locus ceruleus, nucleus tractus solitarius, area postrema, and ventrolateral area of the medulla oblongata. GCH immunostaining was particularly strong in serotoninergic nuclei, such as dorsal and median raphe nuclei, nucleus raphe pallidus, and nucleus raphe magnus. By immunoelectron microscopy, GCH-labeled cytoplasm and microtubules in the processes were observed ultrastructurally, but no staining was found in the mitochondria, and Golgi apparatus. Immunostaining was observed neither in the group D neurons that contain only aromatic amino acid decarboxylase without tyrosine hydroxylase, nor in glial cells and endothelial cells. These results indicate the abundant presence of GCH in catecholaminergic and serotoninergic neurons as well as in the adrenal medulla and liver, where BH4 is synthesized as the cofactor of tyrosine, tryptophan, and phenylalanine hydroxylases.

Adrenal Glands↗

PubMed1983

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on [3H]thymidine incorporation into rat liver deoxyribonucleic acid.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on [3H]thymidine incorporation into hepatic DNA was studied in rats. In non-hepatectomized male and female animals, incorporation measured at the peak of the first round of liver DNA synthesis after TCDD treatment (10 micrograms/kg) was similar to that of control animals. In contrast, the first round of [3H]thymidine incorporation after a 1/3 hepatectomy was enhanced 3-fold in TCDD-treated rats. The enhanced response to 1/3 hepatectomy was produced by doses of TCDD ranging from 1 to 30 micrograms/kg with an apparent ED50 of 5 micrograms/kg. Enhanced incorporation was observed when the 1/3 hepatectomy was performed 5-10 days after an ED50 dose and it returned to the control level after 20 days. This enhanced response was not preceded by changes in food consumption or hepatic activities of ornithine decarboxylase (ODC), tyrosine aminotransferase (TAT) or gamma glutamyl transpeptidase (GGT) when compared to respective control values. Also, the enhanced incorporation was not necessarily due to removal of 1/3 of the liver because it was also seen in TCDD-treated rats that were laparotomized. The mechanism of enhancement in laparotomized animals does not appear to involve a diminished response of the liver to the inhibitory effects of adrenal hormones on liver DNA synthesis. This was suggested by the finding that an adrenalectomy prior to the laparotomy did not block the enhanced incorporation of [3H]thymidine into hepatic DNA. The mechanism by which TCDD enhances the first round of liver DNA synthesis after a 1/3 hepatectomy or laparotomy remains to be determined.

Animals↗