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Biomedical subjects

E Mezey

Publications and source records attributed to E Mezey.

At least 73 records · Page 4Linked to original sources
PubMed1998

Plasma metanephrines are markers of pheochromocytoma produced by catechol-O-methyltransferase within tumors.

This study examined whether the high sensitivity of plasma free metanephrines for diagnosis of pheochromocytoma may result from production of free metanephrines within tumors. Presence in pheochromocytomas of catechol-O-methyltransferase (COMT), the enzyme responsible for conversion of catecholamines to metanephrines, was confirmed by Western blot analysis, enzyme assay, and immunohistochemistry. Western blot analysis and enzyme assay indicated that membrane-bound and not soluble COMT was the predominant form of the enzyme in pheochromocytoma. Immunohistochemistry revealed colocalization of COMT in the same chromaffin cells where catecholamines are translocated into storage vesicles by the vesicular monoamine transporter. Levels of free metanephrines in pheochromocytoma over 10,000 times higher than plasma concentrations in the same patients before removal of tumors indicated production of metanephrines within tumors. Comparisons of the production of metanephrines in patients with pheochromocytoma with production from catecholamines released or infused into the circulation indicated that more than 93% of the consistently elevated levels of circulating free metanephrines in patients with pheochromocytoma are derived from metabolism before and not after release of catecholamines into the circulation. The data indicate that the elevated plasma levels of free metanephrines in patients with pheochromocytoma are derived from catecholamines produced and metabolized within tumors. Some tumors do not secrete catecholamines, but all appear to metabolize catecholamines to free metanephrines, thus explaining the better sensitivity of plasma free metanephrines over other tests for diagnosis of pheochromocytoma.

Adult↗
PubMed1998

Identification of elf1, a beta-spectrin, in early mouse liver development.

Beta-spectrins play essential roles in cell-cell interactions and in the maintenance of cell polarity. Our aim was to identify beta-spectrin genes important for the establishment of hepatocyte polarity and differentiation. Using subtractive screening of cDNA libraries from early embryonic mouse livers (post-coital days 10, 11, and 12), we have isolated elf1 (embryonic liver fodrin 1), a differentially expressed beta-spectrin or fodrin (betaSpIIsigmaI). Elf1 encodes a 220-amino acid protein with an NH2 terminal actin-binding domain. In situ hybridization studies demonstrate elf1 expression initially in day 10 embryonic heart tissue, then in day 11-11.5 hepatic tissue. These studies suggest that elf1 may play a role in the emergence of hepatocyte polarity during liver development.

Amino Acid Sequence↗
PubMed1997

Praja1, a novel gene encoding a RING-H2 motif in mouse development.

As part of a cloning strategy to identify genes involved in early mouse liver development we have isolated Praja1, a gene with similar sequences to the Drosophila melanogaster gene goliath (gl) which is involved in the fate of mesodermal cells ultimately forming gut musculatures, fat body, and the heart. Praja1 is a 2.1 kb gene encoding a putative 396 amino acid ORF and includes a COOH-terminal RING-H2 domain. Using the Jackson Laboratory BSS panel, we have localized Praja1 on chromosome X at 36 cM, which may be a candidate gene for mouse sla (sex linked sideroblastic anemia), near the X inactivation center gene, Xist. Northern blot analysis demonstrated three transcripts (3.1, 2.6 and 2.1 kb) in mRNA from adult mouse tissues brain, liver, and kidney as well as in mRNA from developing mouse embryos (days 7, 11, 15 and 17 post coitus, p.c.). In vitro transcription/translation yielded a product with an Mr of 59 kD. Immunohistochemical staining of in vitro liver explant cultures using a heterologous antibody against praja1 demonstrated cytoplasmic staining of cuboidal cells that have hepatocyte morphology and organization. The presence of the RING-H2 domain, a proline-rich region at the COOH-end, and regions rich in acidic amino acids, leads to the hypothesis that the Praja1 product is possibly involved in mediating protein-protein interactions, possibly as part of a protein sorting or transport pathway. This is strengthened by the similarity of Praja1 to rat Neurodap1, whose product has been shown to localize to the endoplasmic reticulum and golgi in brain.

Amino Acid Sequence↗
PubMed1997

Tyrosine hydroxylase assay for detection of low levels of enzyme activity in peripheral tissues.

A nonisotopic assay for tyrosine hydroxylase, with optimized signal-to-noise ratios, enables determination of low levels of enzyme activity in peripheral tissues. DOPA produced by the enzyme is measured using HPLC with electrochemical detection. Increased signal-to-noise ratios are obtained by including in the reaction mixture glycerol for reduction of blank values and dihydropteridine reductase and NADPH for regeneration of the tetrahydropteridine cofactor. With this method, tyrosine hydroxylase activity can be detected in as few as 200 PC12 cells and in peripheral tissues at levels as low as 4.5 fmol/min/mg wet weight. The assay permits activity to be assessed in a variety of peripheral tissues.

Animals↗
PubMed1997

Effect of acetaldehyde on Sp1 binding and activation of the mouse alpha 2(I) collagen promoter.

Acetaldehyde activates the mouse alpha 2(I) collagen promoter and this effect is mediated in part by increased binding of nuclear factor I (NF-I). Additional mechanisms may exist since deletions in the promoter upstream to the NF-I binding site prevented enhancement by acetaldehyde. Three adjacent areas of binding by nuclear proteins from activated hepatic stellate cells were identified at -568 to -554 (region 1), -542 to -518 (region 2), and -473 to -453 (region 3) of the promoter using DNase I protection analyses. Multiple DNA-protein complexes were formed in electrophoretic mobility shift assays with oligonucleotide probes specifying the three regions. Sp1 and NF-1 bound to all three regions, while Sp3 bound to region 2. Acetaldehyde decreased nuclear protein binding to all three regions. Mutations of regions 1, 2, and 3 reduced basal activity of the promoter and inhibited acetaldehyde stimulation in transfected stellate cells. Acetaldehyde inhibited the stimulatory effect of the Sp1 vector pPacSp1 on the promoter in transfected Drosophila cells. In conclusion, three regions of Sp1 binding were identified and are required for optimal activity of the alpha 2(I) collagen promoter. Sp1 is required for basal activity of the alpha 2(I) collagen promoter; however, the enhancing effect of acetaldehyde on the promoter is not mediated by Sp1.

Acetaldehyde↗
PubMed1997

Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice.

Glial cells are thought to derive embryologically from either myeloid cells of the hematopoietic system (microglia) or neuroepithelial progenitor cells (astroglia and oligodendrocytes). However, it is unclear whether the glia in adult brains free of disease or injury originate solely from cells present in the brain since the fetal stage of development, or if there is further input into such adult brains from cells originating outside the central nervous system. To test the ability of hematopoietic cells to contribute to the central nervous system, we have transplanted adult female mice with donor bone marrow cells genetically marked either with a retroviral tag or by using male donor cells. Using in situ hybridization histochemistry, a continuing influx of hematopoietic cells into the brain was detected. Marrow-derived cells were already detected in the brains of mice 3 days after transplant, and their numbers increased over the next several weeks, exceeding 14,000 cells per brain in several animals. Marrow-derived cells were widely distributed throughout the brain, including the cortex, hippocampus, thalamus, brain stem, and cerebellum. When in situ hybridization histochemistry was combined with immunohistochemical staining using lineage-specific markers, some bone marrow-derived cells were positive for the microglial antigenic marker F4/80. Other marrow-derived cells surprisingly expressed the astroglial marker glial fibrillary acidic protein. These results indicate that some microglia and astroglia arise from a precursor that is a normal constituent of adult bone marrow.

Animals↗
PubMed1997

Mutations in SOD1 associated with amyotrophic lateral sclerosis cause novel protein interactions.

A subset of familial and sporadic amyotrophic lateral sclerosis (ALS-a fatal disorder characterised by progressive motor neuron degeneration) cases are due to mutations in the gene encoding Cu,Zn superoxide dismutase (SOD1). Two mutations which have been successfully used to generate transgenic mice that develop an ALS-like syndrome are glycine 85 to arginine (G85R) and glycine 93 to alanine (G93A) with the mutant SOD1 allele overexpressed in a normal mouse genetic background. No ALS-like phenotype is observed in mice overexpressing wild-type SOD1 or mice without any SOD1 activity. These dominant mutations, which do not necessarily decrease SOD1 activity, may confer a gain of function that is selectively lethal to motor neurons. The yeast interaction trap system allowed us to determine whether these mutations in SOD1 caused novel protein interactions not observed with wild-type SOD1 and which might participate in the generation of the ALS phenotype. Two proteins, lysyl-tRNA synthetase and translocon-associated protein delta, interact with mutant forms of SOD1 but not with wild-type SOD1. The specificity of the interactions was confirmed by the coimmunoprecipitation of mutant SOD1 and the expressed proteins. These proteins are expressed in ventral cord, lending support to the relevance of this interaction to motor neuron disease.

12E7 Antigen↗
PubMed1997

Distribution of somatostatin receptor messenger RNAs in the rat gastrointestinal tract.

BACKGROUND & AIMS: The gastrointestinal (GI) tract is a major source and target of somatostatin (SRIF). Recently, five pharmacologically different SRIF receptors (sst1-5) were cloned. The cellular and tissue distribution of the sst1-5 messenger RNAs (mRNAs) were studied in the rat GI tract using in situ hybridization histochemistry (ISHH). METHODS: Two sets of (35)S-uridine triphosphate (UTP)-labeled antisense and sense riboprobes were prepared for each sst. ISHH was conducted on frozen tissue samples from rat stomach, duodenum, jejunum, ileum, colon, and pancreas. RESULTS: mRNAs of all five sst-s are widely expressed in the rat GI tract. The distribution pattern for each sst mRNA was identical with both antisense probes. No specific signal was found with any of the sense probes. Each layer of the different parts of the gut expressed mRNAs of multiple sst subtypes. All organs expressed sst3 mRNA very intensely. The lowest levels of mRNA expression for all five subtypes within the GI tract were found in the pancreas. CONCLUSIONS: The widespread expression of sst mRNAs suggests a significant role for SRIF in the regulation of GI function.

Animals↗
PubMed1997

Substantial production of dopamine in the human gastrointestinal tract.

Considerable urinary excretion of dopamine metabolites indicates that large amounts of dopamine are produced in unknown locations of the body. This study assessed the contribution of mesenteric organs (gastrointestinal tract, spleen, and pancreas) to the total body production of dopamine in humans and examined the presence of the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase, in gastrointestinal tissues. Blood sampled from an artery and portal and hepatic veins in eight subjects and from arterial and renal venous sites in other subjects was analyzed for plasma concentrations of dopamine and its metabolites. The activity and distribution of tyrosine hydroxylase was also examined in tissue samples from the stomach and duodenum. Higher concentrations of dopamine and its metabolites in portal venous than arterial plasma indicated substantial production of dopamine by mesenteric organs (12.0 nmol/min) amounting to 42-46% of the renal removal of circulating dopamine metabolites. Tissue samples showed immunoreactive tyrosine hydroxylase in nonneuronal cell bodies and detectable levels of tyrosine hydroxylase in nonneuronal cell bodies and detectable levels of tyrosine hydroxylase enzyme activity. The results show that mesenteric organs produce close to half of the dopamine formed in the body, most of which is unlikely to be derived from sympathetic nerves but may reflect production in a novel nonneuronal dopaminergic system.

Aged↗
PubMed1997

Immunohistochemical localization of somatostatin receptor SST2A in the rat pancreas.

Somatostatin (SRIF) acts on specific membrane receptors to inhibit exocrine and endocrine pancreatic functions. Five SRIF receptor genes have been cloned, producing six receptor proteins (sst-s). We used a recently developed antibody to localize the sst2A splice variant in the rat pancreas. Western blots identified the sst2A receptor as an 90 kDa glycosylated protein in pancreatic tissue. In tyramide-amplified immunostainings all acinar cells, and the glucagon and pancreatic polypeptide immunoreactive cells (A and PP, respectively) were intensely labeled for sst2A, while no signal was detected in SRIF producing (D) cells. A very few insulin immunoreactive (B) cells were also labeled for sst2A, but the signal in these cells was lower than in exocrine, A or PP cells. Absorption of the sst2A antibody with the receptor peptide abolished specific staining in both immunoblots and tissue sections (negative control). These studies are the first to localize any SRIF receptor subtype in the rat pancreas. The specific localization of sst2A receptor in acinar, A and PP cells if confirmed in humans, would suggest that subtype specific analogs will be useful for the therapeutic regulation of exocrine and/or endocrine pancreatic secretion.

Animals↗
PubMed1997

Immunohistochemical localization of somatostatin receptor SST2A in the rat pancreas.

Somatostatin (SRIF) acts on specific membrane receptors to inhibit exocrine and endocrine pancreatic functions. Five SRIF receptor genes have been cloned, producing six receptor proteins (sst-s). We used a recently developed antibody to localize the sst2A splice variant in the rat pancreas. Western blots identified the sst2A receptor as an 90 kDa glycosylated protein in pancreatic tissue. In tyramide-amplified immunostainings all acinar cells, and the glucagon and pancreatic polypeptide immunoreactive cells (A and PP, respectively) were intensely labeled for sst2A, while no signal was detected in SRIF producing (D) cells. A very few insulin immunoreactive (B) cells were also labeled for sst2A, but the signal in these cells was lower than in exocrine, A or PP cells. Absorption of the sst2A antibody with the receptor peptide abolished specific staining in both immunoblots and tissue sections (negative control). These studies are the first to localize any SRIF receptor subtype in the rat pancreas. The specific localization of sst2A receptor in acinar, A and PP cells if confirmed in humans, would suggest that subtype specific analogs will be useful for the therapeutic regulation of exocrine and/or endocrine pancreatic secretion.

Alternative Splicing↗
PubMed1996

A novel nonneuronal catecholaminergic system: exocrine pancreas synthesizes and releases dopamine.

Cells of the exocrine pancreas produce digestive enzymes potentially harmful to the intestinal mucosa. Dopamine has been reported to protect against mucosal injury. In looking for the source of dopamine in the small intestine, we found that the duodenal juice contains high levels of dopamine and that the pancreas itself has a high dopamine [and dihydroxyphenylalanine (dopa)] content that does not change significantly after chemical sympathectomy. Furthermore, we were able to demonstrate tyrosine hydroxylase (TH) activity in control pancreas as well as in pancreas from rats after chemical sympathectomy. Immunostaining and in situ hybridization histochemistry confirmed both the presence of TH, dopamine, and the dopamine transporter, and the mRNAs encoding TH and dopamine transporter, and the presence of both types of vesicular monoamine transporters in the exocrine cells of the pancreas. Since there are no catecholaminergic enteric ganglia in the pancreas, the above results indicate that pancreatic cells have all the characteristics of dopamine-producing cells. We suggest that the pancreas is an important source of nonneuronal dopamine in the body, and that this dopamine has a role in protecting the intestinal mucosa and suggests that dopamine D1b receptor agonists might be used to help mucosal healing in the gastrointestinal tract.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1996

Activation by acetaldehyde of the promoter of the mouse alpha2(I) collagen gene when transfected into rat activated stellate cells.

The effect of acetaldehyde in activating the mouse alpha2(I) collagen promoter in transiently transfected rat activated stellate cells and the possible mediating effect of transforming growth factor beta1 (TGFbeta1) on type I collagen gene expression were determined. Acetaldehyde and TGFbeta1, each had a similar effect in activating the wild-type promoter, but failed to activate the promoter with a -352 to -104 deletion, or the promoter containing a 3-bp substitution between -305 and -303 in the putative nuclear factor I (NF-I) binding site. The effects of acetaldehyde and TGFbeta1 are therefore mediated by a similar factor or factors that bind to the NF-I consensus sequence within the region -352 to -104. Additional factors may also play a role in the effects of acetaldehyde and TGFbeta1, which have similar effects on the wild-type promoter, but become additive in activating the promoter with a more distal deletion containing a cis-repressor element. Pretreatment of activated stellate cells with antibodies to TGFbeta1 suppressed the effect of acetaldehyde in increasing the alpha1(I) collagen message, indicating that TGFbeta1 mediates the effect of the acetaldehyde-induced increase in the expression of the alpha1(I) collagen gene which also contains NF-I binding sites.

Acetaldehyde↗
PubMed1996

Identification of endogenous peroxidase-containing cells as eosinophils in the gastrointestinal system.

Endogenous peroxidase (EPX) activity in certain cells in the gastrointestinal system interferes with immunohistochemical methods based on the horseradish peroxidase-catalyzed substrate deposition. We studied the distribution and characteristics of these cells. We also report an effective and antigen-preserving EPX blocking method, to make possible the evaluation of immunoperoxidase stainings in cryostat sections. The EPX-containing cells (EPX cells) are present in every part of the gastrointestinal tract, predominantly in the tunica propria. We identified them as eosinophil cells in May-Grünwald-Giemsa stained sections. The complete match was confirmed by different fluorescence techniques. Firstly, the EPX cells were labeled by a red fluorochrome-conjugated substrate of peroxidase enzymes, rhodamine-tyramide, whereas the eosinophil cells were labeled by the green fluorochrome, l-hydroxy-3,6,8-pyrenetrisulfonic acid, which is known to label exclusively eosinophilic granules at pH 10. Secondly, all the EPX cells reacted with a monoclonal antibody against the eosinophil peroxidase enzyme. Finally, a set of commercially available leukocyte markers was used to characterize the EPX cells colabeled by fluorochrome-tyramides. Neither macrophages nor mast cells showed EPX activity. Increased numbers and altered distribution were seen in stressed rats and in ulcerated human stomach.

Animals↗
PubMed1996

Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostaining.

The biotinyl-tyramide substrate of the horseradish peroxidase enzyme has been recently introduced to amplify immunohistochemical signals. We applied either fluorochromeor biotin-conjugated tyramine to improve the detection of different antigens in sections of rat stomach, pancreas, and hypothalamus. A ten- to 100-fold increase in staining efficiency was achieved, depending on the antibody, with either fluorescent or peroxidase detection systems. The amplification method was particularly useful for increasing a weak signal of conventional immunostaining caused by suboptimal tissue fixation. At a very low concentration of the primary antibody, the antigen can no longer be detected by a conventional fluorescent secondary antibody but is still detectable after amplification. When an antibody is used at this very low concentration and is detected by a fluorescent amplification method, another primary antibody, raised in the same host species, can be used and demonstrated with a different fluorochrome in subsequent conventional immunostaining of the same section. In this way it becomes possible to immunostain the same section with two different primary antibodies raised in the same host species. Samples for such double immunostaining are demonstrated here using pairs of monoclonal antibodies (to tyrosine hydroxylase and oxytocin) in the hypothalamus and polyclonal antibodies (to glucagon and neurofilament M) in sections of rat pancreas. Because in many cases the availability of antibodies is limited, the amplification method can be a quick and efficient tool for double immunostaining with antibodies from the same host species.

Animals↗
PubMed1996

Distribution of parathyroid hormone-2 receptor messenger ribonucleic acid in rat.

The PTH2 receptor is a recently identified G protein-coupled receptor activated by PTH. Its amino acid sequence is most similar to the PTH/PTHrP receptor, but unlike the PTH/PTHrP receptor, it is activated by PTH and not by PTH-related peptide. We previously demonstrated using Northern blots that expression of PTH2 receptor messenger RNA was greatest within the brain and occurred at lower levels in pancreas, testis, and placenta. We have now obtained a complementary DNA encoding the rat PTH2 receptor and used it to study the distribution of the PTH2 receptor using in situ hybridization histochemistry. PTH2 receptor messenger RNA is abundantly expressed in arterial and cardiac endothelium and at lower levels in vascular smooth muscle. It is also abundant in the lung, both within bronchi and in the parenchyma, and is present within the exocrine pancreas. It is expressed by sperm in the head of the epididymis. A small number of cells associated with the vascular pole of renal glomeruli express the receptor. These data suggest that the PTH2 receptor may be responsible for PTH effects in a number of physiological systems.

Animals↗
PubMed1996

Food-dependent Cushing's syndrome resulting from abundant expression of gastric inhibitory polypeptide receptors in adrenal adenoma cells.

We studied a 45-yr-old woman with food-dependent Cushing's syndrome. Plasma cortisol levels were subnormal (4-47 nmol/L) after an overnight fast and increased after a mixed meal to values between 500-1000 nmol/L. There was a close correlation between circulating gastric inhibitory polypeptide (GIP) and cortisol levels during normal food intake (r = 0.92; P < 0.0002). Plasma corticotropin (ACTH) levels were undetectable. Nonfasting plasma cortisol levels were not suppressed by low or high doses of dexamethasone. Plasma ACTH and cortisol levels did not increase after human CRH administration, but fasting plasma cortisol levels increased after ACTH treatment. The infusion of GIP increased plasma cortisol levels to 7.8 times above baseline. Radiological and cholesterol uptake studies pointed to a unilateral adrenal adenoma. Treatment with octreotide initially prevented the meal-induced increases in cortisol and GIP levels and decreased urinary cortisol excretion. Unilateral adrenalectomy was performed. Cortisol production by cultured adrenal adenoma cells from the patient was stimulated by GIP and ACTH. In situ hybridization studies using a GIP receptor probe showed an abundant expression of GIP receptor messenger ribonucleic acid in the adrenocortical adenoma. We conclude that food-dependent Cushing's syndrome results from the expression of GIP receptors on adrenocortical adenoma cells.

Adenoma↗