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PubMed2000

RT-PCR ELISA method for the analysis of neurotrophin mRNA expression in brain and peripheral tissues.

The levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) in brain and periphery are susceptible to changes during development and as result of different physiopathological conditions, such as stress and aging and during the onset and progression of neurological and autoimmune diseases. Despite the sensitive methods for measurement of neurotrophin protein levels in different tissues, no easily applicable methods to evaluate changes in the level of NGF and BDNF mRNA expression within physiological range have been described. This study reports the development of a reproducible and simple procedure for measurement of neurotrophin mRNA expression in brain and peripheral tissues based upon an enzyme linked immunosorbent assay (ELISA) detection system of reverse transcriptase-polymerase chain reaction (RT-PCR) products. The major advantages of this RT-PCR ELISA procedure is to allow the co-amplification of diverse mRNAs starting from small amounts of tissues; to contemporaneously test a large number of samples; to be rapid and to use only commercial reagents and widely available equipment. The procedure could also be useful in studies addressed to measure the pattern of expression of molecules involved in the pathogenesis of neurodegenerative and inflammatory diseases, such as neuropeptides and cytokines.

Animals↗
PubMed1981

Glutamate as a precursor of GABA in rat brain and peripheral tissues.

The formation of GABA from L-glutamate was investigated in homogenates of rat brain, liver, and kidney, using highly purified [14C]-L-glutamic acid as substrate and a thin-layer chromatographic separation of products. In agreement with other workers, liberation of [14C]-CO2 was found to be stoichiometric with GABA formation in brain homogenates, but not in liver or kidney extracts. Subcellular fractionation and dialysis experiments suggested that most of the GABA synthesis in these peripheral tissues, unlike brain, does not occur via a direct decarboxylation of glutamate and requires one or more cofactors other than pyridoxal phosphate. NAD stimulated GABA formation in dialyzed extracts, and inhibition of GABA-transaminase, both in vitro and in vivo, caused marked inhibition of GABA formation from glutamate in peripheral extracts. Although a very low GAD activity in liver and kidney cannot be excluded, these experiments suggest a major pathway from glutamate to GABA in these homogenates which includes (1) conversion of glutamate to alpha-ketoglutarate by glutamate dehydrogenase or transaminases, (2) conversion of alpha-ketoglutarate to succinic semialdehyde, and (3) formation of GABA from succinic semialdehyde and glutamate by GABA-transaminase.

4-Aminobutyrate Transaminase↗
PubMedDate not supplied

Effect of a single lethal dose of TCDD on the levels of monoamines, their metabolites and tryptophan in discrete brain nuclei and peripheral tissues of Long-Evans rats.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent anorexigens in rats with a yet unidentified mechanism of action. Since biogenic amines are known to essentially participate in the control of body weight and food intake, their levels were determined in various hypothalamic and other brain sites together with selected peripheral tissues after TCDD administration to adult male Long-Evans rats. Rats were given a single lethal dose of TCDD (1000 micrograms/kg intraperitoneally, in dimethylsulphoxide) or vehicle alone and they were decapitated at 1, 5, 25 hr or 8 days after TCDD administration. The samples were analyzed for concentrations of biogenic amines and their metabolites by HPLC-EC. Administration of TCDD increased the concentration of tryptophan at 8 days after exposure by about 20% in almost all nuclei examined, with the change reaching statistical significance in the lateral hypothalamic area and in lateral and medial accumbens nuclei. Importantly, this elevation was not seen in pair-fed control animals. Although not statistically significant, there was a tendency to 5-10% diminished dopamine, serotonin and/or 5-hydroxyindoleacetic acid levels in most brain sites during the first day postexposure. The present results argue against a crucial role for catecholamines as mediators of TCDD toxicity. However, the delayed changes in brain tryptophan do not appear to be secondary to TCDD hypophagia.

Adrenal Glands↗
PubMed1978

Intraventricular administration of nerve growth factor induces ornithine decarboxylase in peripheral tissues of the rat.

Intraventricular administration of nerve growth factor causes an increase in the activity of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) in liver, kidney, and adrenal as well as in brain itself. An increase in the concentration of corticosterone in the blood was also observed. Adrenalectomy, hypophysectomy, or pituitary stalk section inhibits the increase of ornithine decarboxylase activity in the peripheral tissues. Ornithine decarboxylase activity in the brain, however, responds to nerve growth factor in these animals. The data indicate that nerve growth factor causes an acitivation of the hypothalamo-hypophyseal endocrine system.

Adrenal Glands↗
PubMed1996

Cloning and pharmacological characterization of a fourth P2X receptor subtype widely expressed in brain and peripheral tissues including various endocrine tissues.

We have isolated cDNA encoding a fourth member (P2X-4) of the ATP receptor P2X receptor family from a rat pancreatic islet cDNA library. Rat P2X-4 is a protein of 388 amino acids which shares 50%, 49%, and 47% identity with P2X-1, P2X-2, and P2X-3, respectively, and has two putative transmembrane segments. Rat P2X-4 mRNA is widely expressed in brain and peripheral tissues, including various endocrine tissues, and it is also expressed in various hormone-secreting cell lines. We have heterologously expressed the cloned P2X-4 in Xenopus laevis oocytes and have characterized its pharmacological properties. ATP, its analogs and ADP activate cation-selective ion channels. The order of agonist potency is ATP ADP 2-methyl- thioATP(2MeSATP) >> alpha beta-methelene-ATP (alpha betameATP). ATP-evoked currents are only partially blocked by suramin, reactive blue-2, or H2DIDS. The present study suggests that P2X-4, with pharmacological properties distinct from those of P2X-1+, P2X-2, and P2X-3, mediates extracellular ATP-induced biological effects in non-neuronal cells, including endocrine cells, as well as in neuronal cells.

Amino Acid Sequence↗
PubMed2003

Distribution of the VPAC2 receptor in peripheral tissues of the mouse.

The neuropeptide vasoactive intestinal peptide (VIP) exerts its actions through two structurally related G protein-coupled receptors (VPAC(1) and VPAC(2)). Pituitary adenylate cyclase-activating polypeptide (PACAP) is also a potent agonist of VPAC(1) and VPAC(2) receptors as well as of a third, PACAP-specific receptor (PAC(1)). We report here the distribution of the VPAC(2) receptor in peripheral tissues of the mouse, determined by receptor autoradiography using [(125)I]VIP and the selective VPAC(2) receptor agonist [(125)I]Ro25-1553 in wild-type and VPAC(2) receptor-null mice. In addition, displacement experiments with the VPAC(2)-selective agonist Ro25-1553 and the VPAC(1)-selective agonist [K(15),R(16),L(27)]VIP(1-7)/GRF(8-27) were performed using the universal radioligand [(125)I]VIP. The VPAC(2) receptor is found predominantly in smooth muscle (in blood vessels and in the smooth muscle layers of the gastrointestinal and reproductive systems), the basal part of the mucosal epithelium in the colon, lung, the vasculature of the kidney, adrenal medulla, and retina. Unexpectedly, the receptor was also present in thyroid follicular cells and acinar cells of the pancreas, tissues that have not been found to express the receptor in other species, and in very large amounts in the lung. Our data suggest novel functions of the VPAC(2) receptor and additional potential therapeutic uses of drugs acting at the receptor (including the treatment of erectile dysfunction), but our results also indicate that caution should be exercised in using the mouse as an animal model for the evaluation of VIP analogs intended for diagnostic or therapeutic use in man.

Animals↗
PubMed2001

Induction of immunity in peripheral tissues combined with intracerebral transplantation of interleukin-2-producing cells eliminates established brain tumors.

Cytokine gene therapy for the induction of potent immune responses against central nervous system tumors has proven to have significant potential. However, this strategy needs improvement in the process of antigen presentation and/or insufficient recruitment of immunocompetent cells to achieve successful eradication of established brain tumors. We investigated the therapeutic potential of induced systemic immunity in peripheral tissues combined with interleukin-2 (IL-2) production in the vicinity of brain tumors to treat established brain tumors. Sequential magnetic resonance image monitoring showed that the combinatory therapy consisting of intracerebral (i.c.) transplantation of IL-2-producing rat gliosarcoma 9L (9L/IL-2) cells and s.c. vaccination using irradiated 9L or 9L/IL-2 cells could cure 9L-bearing rats, whereas either the i.c. injection of 9L/IL-2 cells or the s.c. vaccination produced little or marginal antitumor effects, respectively. Xenogeneic murine neuroblastoma cells secreting IL-2 could substitute for 9L/IL-2 cells, producing significant antitumor effects in the vaccinated rats. Tumor-specific cytotoxic activity was induced in the vaccinated rats but not fully in the rats treated only with i.c. injection of 9L/IL-2 cells. Immunohistochemical analysis revealed that a number of CD4(+) and CD8(+) T cells infiltrated into the brain tumors which were treated with the combinatory therapy. The level of cell infiltration was similar to that found in s.c. 9L/IL-2 tumors which were subsequently rejected. In contrast, the brain tumors treated with either i.c. transplantation of 9L/IL-2 cells or the s.c. vaccination showed only moderate infiltration of T cells. The combinatory strategy, i.c. grafting of IL-2-producing cells, and s.c. immunization of irradiated whole tumor cell vaccine, is, thus, effective for recruiting activated T cells into the brain tumor site and could be a potential therapy for brain tumors.

Animals↗
PubMed1992

Effects of selenium and iodine deficiency on iodothyronine deiodinases in brain, thyroid and peripheral tissue.

Long term nutritional selenium (Se) deficiency had only marginal effects on the thyroid T4 and T3 content and on the activity of the selenoenzyme type I deiodinase (5'D-I) in the thyroid gland. These findings reveal a remarkable resistance of the thyroid to Se-deficiency which may substantially contribute to the observed maintenance of T4 and T3 levels in circulating blood. In contrast to its maintained thyroidal activity, 5'D-I in peripheral tissues like liver and kidney was strongly decreased by Se-deficiency. The observed decrease of type II deiodinase (5'D-II) in the cerebral cortex of Se-deficient rats was obviously caused by the suppressing regulatory effect of elevated cortex T4 concentrations. The severalfold 5'D-II enhancement in iodine depleted animals was not abolished by additional Se-deficiency, suggesting that brain type II deiodinase is not a selenoenzyme. The role of selenium for cortex type III 5-deiodinase, which was moderately decreased in selenium as well as iodine-deficient rats, awaits definite evaluation by further studies. The different responsiveness to thyroidal and hepatic 5'D-I to Se restriction is further evidence for priorities in the selenium supply to different tissues.

Animals↗
PubMed1996

Comparison of hypervariable regions (HVR1 and HVR2) in positive- and negative-stranded hepatitis C virus RNA in cancerous and non-cancerous liver tissue, peripheral blood mononuclear cells and serum from a patient with hepatocellular carcinoma.

Hepatitis C virus (HCV) infection is associated with a wide spectrum of liver diseases including cirrhosis and hepatocellular carcinoma (HCC). Although the biological relation between the virus and cirrhosis or HCC is unclear, such variable pathogenicity may be related to the genetic heterogeneity of HCV. Genetic variability of HCV was assessed by determining the nucleotide sequence corresponding to the hypervariable regions (HVR1 and HVR2) of the putative envelope protein (E2/NS1) in positive- and negative-stranded HCV RNA from the cancerous and surrounding non-cancerous liver tissue, peripheral blood mononuclear cells and serum of a patient with HCC. Nineteen distinct HVR1 amino acid sequences (deduced from the nucleotide sequences) were obtained from the patient and could be classified into 5 groups on the basis of the site and time of detection. Some viral isolates with the same HVR1 sequence were shown to replicate in both cancerous and non-cancerous liver tissue, whereas others replicated in HCC tissue only.

Amino Acid Sequence↗
PubMed1998

Changes of peripheral tissue thyroid hormone metabolism in rats fed with selenium- and vitamin E-deficient artificial semisynthetic diet.

OBJECTIVE: To examine the relationship between selenium (Se)- and vitamin E(VE)-deficiency and thyroid hormone (TH) metabolic disturbance, especially type I iodothyronine 5'-deiodinase (ID-I) activity. METHODS: We observed the metabolic changes of TH-3, 3',5-triidothyronine (T3) and thyroxin (T4) and their free radicals-glutathione peroxidase (GSH-Px) and lipid peroxides (LPO) in the peripheral tissues (liver, kidney and blood) of Wistar rats maintained on Se- and VE-deficient artificial semi-synthetic diet for 8 weeks. RESULTS: In the Se- and VE-deficient rats (compared with Se- and VE-supplement rats): 1). Hepatic and renal ID-I activities decreased by 60% and 50% respectively, serum levels of T3 reduced 36%, and T4 increased by 32%. Hepatic ID-I activity and serum T3 concentration in Se- and VE-supplement rats were significantly higher than those in the Se-supplement and VE-deficient rats. 2). GSH-Px activities in the whole blood and liver decreased by 61% and 82% respectively. LPO concentrations in serum and liver increased by 53% and 40% respectively. When Se content remained the same, changes of VE content did not significantly affect GSH-Px activity, but the combined supplement of Se and VE decreased significantly the LPO concentration. CONCLUSIONS: The data suggests that GSH-Px activity is influenced mainly by Se level, but ID-I activity is influenced by VE concentration in addition to Se level. VE seemed to play a protective role on ID-I and its mechanism might be related to the fact that VE protects the stability of microsomal membrane in which ID-I exists, avoiding from free radical damage as well as the coordinated action and mutual sparing effect of Se and VE.

Animals↗
PubMed1990

Measurement of human nerve growth factor binding sites in brain and in peripheral tissues by a specific immunoprecipitation assay.

Using a monoclonal antibody against the human nerve growth factor (NGF) receptor (20.4 IgG), a specific immunoprecipitation assay for the quantitation of human NGF binding sites has been established. The procedure involves specific labeling of NGF receptors by covalent crosslinking to 125I-NGF with a carbodiimide reagent, and subsequent immunoprecipitation of the detergent-extracted 125I-NGF-receptor complexes with the 20.4 IgG. This two-site assay provides a specific method to measure NGF binding sites in peripheral tissues and central nervous system. Moreover, it allows analysis of the immunoprecipitated receptor species by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The finding that specific NGF binding sites are expressed on human neuronal and nonneuronal tissues, including lymphoid tissues, indicates that NGF exerts a broader physiological function than originally ascribed.

Aged↗
PubMed1993

Two glycine transporter variants with distinct localization in the CNS and peripheral tissues are encoded by a common gene.

We have isolated a cDNA encoding a high affinity, Na+/Cl(-)-dependent glycine transporter, GLYT-2, which is distinct from another glycine transporter, GLYT-1. While the 3' sequences of these two cDNAs are identical, the 5' noncoding regions and the N-termini are completely different. GLYT-1 is found only in the white matter of the CNS, while GLYT-2 is found in the gray matter of the CNS as well as in macrophages and mast cells in peripheral tissues. Our findings suggest that tissue-specific alternative splicing or alternative promoter usage from a single gene results in two mRNA products encoding similar but distinct glycine transporters. The anatomic distribution of GLYT-2 mRNA supports the emerging status of glycine as a supraspinal neurotransmitter and suggests that glycine may function as a chemical messenger outside the CNS.

Amino Acid Sequence↗
PubMed2000

Extra-pituitary growth hormone in peripheral tissues of early chick embryos.

Early embryonic growth is independent of pituitary growth hormone (GH), since it occurs prior to the differentiation of pituitary somatotrophs. Embryogenesis is therefore thought to be regulated by local growth factors. As GH is now known to be produced in many extrapituitary sites, in which it acts in an autocrine or paracrine manner, the possibility that extra-pituitary GH may participate in embryogenesis and organogenesis was assessed by determining the immunocytochemical presence and location of GH- and GH-receptor (GHR)-like proteins in the peripheral tissues of chick embryos during their 21-day incubation period. Immunoreactive (IR)-GH, detectable by a monoclonal and two polyclonal antibodies for chicken GH, was specifically and ubiquitously present in tissues of 3-day-old embryos. At embryonic day (ED) 5, IR-GH was widespread in ectodermal, mesodermal and endodermal tissues, but it was not present in every cell of each tissue. IR-GH was particularly abundant i! n the neural tube, notochord, limb bud, somites, heart, stomach, liver, kidney, Wolffian duct and the amnion. By ED8, IR-GH was still widespread and was now present in limb bud cartilage, although the heart and liver were no longer GH immunoreactive. GH receptor immunoreactivity was also present in most tissues and cells of ED3-ED8 embryos. These results demonstrate that extrapituitary GH is abundantly present during early embryogenesis, prior to the differentiation of pituitary somatotrophs (at ED12). Since GH- and GHR-like proteins are present in most tissues of the chick embryo, it is proposed that extrapituitary GH may act as a local growth factor during embryonic development.

Amnion↗
PubMed2003

Anatomical location of macrophage migration inhibitory factor in urogenital tissues, peripheral ganglia and lumbosacral spinal cord of the rat.

BACKGROUND: Previous work suggested that macrophage migration inhibitory factor (MIF) may be involved in bladder inflammation. Therefore, the location of MIF was determined immunohistochemically in the bladder, prostate, major pelvic ganglia, sympathetic chain, the L6-S1 dorsal root ganglia (DRG) and the lumbosacral spinal cord of the rat. RESULTS: In the pelvic organs, MIF immunostaining was prominent in the epithelia. MIF was widely present in neurons in the MPG and the sympathetic chain. Some of those neurons also co-localized tyrosine hydroxylase (TH). In the DRGs, some of the neurons that stained for MIF also stained for Substance P. In the lumbosacral spinal cord, MIF immunostaining was observed in the white mater, the dorsal horn, the intermediolateral region and in the area around the central canal. Many cells were intensely stained for MIF and glial fibrillary acidic protein (GFAP) suggesting they were glial cells. However, some cells in the lumbosacral dorsal horn were MIF positive, GFAP negative cells suggestive of neurons. CONCLUSIONS: Therefore, MIF, a pro-inflammatory cytokine, is localized to pelvic organs and also in neurons of the peripheral and central nervous tissues that innervate those organs. Changes in MIF's expression at the end organ and at peripheral and central nervous system sites suggest that MIF is involved in pelvic viscera inflammation and may act at several levels to promote inflammatory changes.

Animals↗
PubMed1992

Fungemia observations of peripheral tissue clearance in humans.

Patients with fungemia, mainly due to Candida albicans, had cultures repeated from arterial and venous sites to determine yeast cell clearance during fungemia. Of the 48 patients, 37 had repeat positive cultures (36 arterial and venous samples; one venous culture). Additionally, 24 patients had arterial and venous samples cultured quantitatively. An average of 9.1 colony forming units (CFU) ml-1 was isolated from arterial samples and 5.5 CFU ml-1 from venous samples (60% of arterial, P < 0.01). This suggest that arterial fungal densities exceed venous densities and that peripheral tissues clear 40% of yeasts.

Arteries↗
PubMed1998

Intracerebroventricular passive immunization. II. Intracerebroventricular infusion of neuropeptide antisera can inhibit neuropeptide signaling in peripheral tissues.

The findings of the preceding article suggest that intracerebroventricular (i.c.v.) administration of small amounts (5 microl) of antisera to rats may produce effectual immunoneutralization of peptides in blood/tissues outside of the central nervous system (CNS). In the present work we sought to test this hypothesis by determining the titers of corresponding antibodies in jugular venous plasma after i.c.v. infusion of three different antisera: a sheep anti-CRF, a rabbit anti-CRF, and a rabbit anti-GnRH. For all antisera tested, corresponding antibodies were detected in systemic plasma within 30 min of i.c.v. infusion of 5 microl antiserum. By 8 h, blood levels of the corresponding antibodies were similar whether the antisera had been infused i.c.v. or i.v. When the dilutions of antibodies equivalent to those in systemic blood 1-24 h after i.c.v. infusion of 5 microl antiserum were employed in rat anterior pituitary cell culture assays, they proved effective at inhibiting CRF- or GnRH-induced hormone secretion. Furthermore, in rats pretreated i.c.v. with 5 microl anti-CRF (at -4 h), pituitary ACTH secretion induced by i.v. CRF (0.3 nmol/kg) was reduced by 88%. Collectively, these data demonstrate that shortly after i.c.v. infusion of neuropeptide antisera, the levels of corresponding antibodies found in systemic blood are sufficient to inhibit neuropeptide signaling within peripheral tissues. As i.c.v. passive immunization procedures have been used extensively in the investigation of the biological roles of neuropeptides within the CNS, these findings indicate a critical reevaluation of the peripheral vs. CNS functions of neuropeptides.

Adrenocorticotropic Hormone↗
PubMed1991

Sympathetic reflex control of blood flow in human peripheral tissues.

Sympathetic vasoconstrictor reflexes are essential for the maintenance of arterial blood pressure in upright position. It has been generally believed that supraspinal sympathetic vasoconstrictor reflexes elicited by changes in baroreceptor activity play an important role. Recent studies on human skeletal muscle, cutaneous and subcutaneous tissues of the limbs indicate that the situation is more complex. Measurements have been carried out during acute as well as chronic sympathetic denervation. Spinal sympathetic reflex mechanisms have been evaluated in tetraplegic patients, where supraspinal sympathetic vasoconstrictor reflexes are blocked. Blood flow has been measure by the local 133Xe-technique. The results indicate the presence of spinal as well as supraspinal sympathetic vasoconstrictor reflexes to human peripheral tissues. Especially is emphasized the presence of a local sympathetic veno-arteriolar axon reflex which is elicited by increase in venous transmural pressure of more than about 25 mm Hg. Finally, centrally elicited vasoconstrictor activity is modified by simultaneously activating the local veno-arteriolar axon reflex or vice versa by means of an intraneuronal impulse inhibition due to collision of normodromically and antidromically conducted impulses in efferent sympathetic vasoconstrictor fibers. The evidence obtained suggests that sympathetic vasoconstrictor reflexes to postural changes are complex and highly differentiated.

Blood Pressure↗
PubMed1985

Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets.

The relative amount of apolipoprotein (apo-) E mRNA in 12 different tissues of the rat and marmoset was examined by dot blot hybridization using cloned cDNA probes. As expected, it was found to be most abundant in the liver. However, substantial amounts of apo-E mRNA were found in the brain and adrenals at relative levels about one-third of that found in the liver. Significant quantities of apo-E mRNA were detected in all of the other peripheral tissues as well. The apo-E mRNA levels in these tissues were 2-10% of that found in the liver of the rat and 10-30% of that found in the liver of the marmoset. Apo-E mRNA was also abundant in human brain and in each species examined; it was distributed throughout all major areas of this organ. In contrast, apo-A-I mRNA was detected in abundant amounts only in the small intestine and in the liver. Extrahepatic apo-E mRNA appears to be functional, generating a translation product similar or identical to that generated by the liver. During fetal and neonatal development, apo-E mRNA is rapidly induced from low levels to approximately equal to 60% of adult levels in liver at parturition. The fetal yolk sac contains more apo-E mRNA than the fetal liver, suggesting a significant role for the yolk sac as a source of apo-E during gestation.

Adrenal Glands↗