Immunohistochemical localization of arginase II and other enzymes of arginine metabolism in rat kidney and liver

Arginine is a precursor for the synthesis of urea, polyamines, creatine phosphate, nitric oxide and proteins. It is synthesized from ornithine by argininosuccinate synthetase and argininosuccinate lyase and is degraded by arginase, which consists of a liver-type (arginase I) and a non-hepatic type (arginase II). Recently, cDNAs for human and rat arginase II have been isolated. In this study, immunocytochemical analysis showed that human arginase II expressed in COS-7 cells was localized in the mitochondria. Arginase II mRNA was abundant in the rat small intestine and kidney. In the kidney, argininosuccinate synthetase and lyase were immunostained in the cortex, intensely in proximal tubules and much less intensely in distal tubules. In contrast, arginase II was stained intensely in the outer stripes of the outer medulla, presumably in the proximal straight tubules, and in a subpopulation of the proximal tubules in the cortex. Immunostaining of serial sections of the kidney showed that argininosuccinate synthetase and arginase II were colocalized in a subpopulation of proximal tubules in the cortex, whereas only the synthetase, but not arginase II, was present in another subpopulation of proximal tubules. In the liver, all the enzymes of the urea cycle, i.e. carbamylphosphate synthetase I, ornithine transcarbamylase, argininosuccinate synthetase and lyase and arginase I, showed similar zonation patterns with staining more intense in periportal hepatocytes than in pericentral hepatocytes, although zonation of ornithine transcarbamylase was much less prominent. The implications of these results are discussed.     

Immunohistochemical localization of the neural cannabinoid receptor in rat brain

A sample of UK consumers (N = 311) was interviewed in order to identify the attitudinal, cognitive and involvement characteristics of probable early adopters of polyunsaturated fatty acid (PUFA) fed fish. Attitude to fish significantly influenced PUFA fish, premium price PUFA fish, PUFA salmon, PUFA eel and PUFA sturgeon purchase. Involvement in healthy eating influenced PUFA fish, premium price PUFA fish and PUFA salmon purchase. Cognitive style did not influence PUFA fish and premium price PUFA fish purchase; nor, contrary to earlier research, did cognitive style and involvement interact to influence intended PUFA fish purchases.     

Immunohistochemical localization of UDP-glucuronosyltransferases in rat brain during early development

OBJECTIVE: Benign tumors account for less than 1% of testicular tumors and the incidence is even lower in children. A rare case of epidermoid cyst of the testis in a child is described. The differential diagnosis and treatment options are discussed. METHODS/RESULTS: A case of unilateral epidermoid cyst of the testis in an 11-year-old boy is presented. The clinical and diagnostic aspects are discussed. Definitive diagnosis could be made only after surgical excision. CONCLUSIONS: Pathological analysis of the entire testis is warranted to make the definitive diagnosis of epidermoid cyst. However, preservation of the testis can be considered, particularly in those cases with bilateral involvement, if supported by solid, consistent diagnostic evidence, including intraoperative biopsy.     

Immunohistochemical localization of chromogranin A in endocrine tissues and endocrine tumors of dogs

BACKGROUND: Research on health care quality and effectiveness often relies on global health status measures, such as functional status, but little is known about the functional status of patients in the primary care setting (without limitation to specific diseases) and even less about the function of the poor or ethnic minorities. In preparation for a planned practice-based research network, we administered a functional-status survey to patients visiting an inner-city family practice center. METHODS: Over 9 weeks, 555 established patients older than 18 years, as well as adolescents accompanied by a parent or guardian, completed a survey that included the SF-36 Health Survey and questions about demographic variables and cigarette use. The survey was self-administered in the waiting area and examination room, and patients received no assistance from staff. RESULTS: Functional-status scores reported by this primary care cohort were significantly lower than those of the general population (P < .001) and comparable with those reported nationally for patients with chronic diseases (e.g., congestive heart failure, diabetes). Functional-status scores were associated with age, sex, and, most strikingly, socioeconomic status. For example, patients with a yearly income of less than $15,000 had lower mean physical function scores than those reported nationally for patients with hypertension, diabetes, depression, recent myocardial infarction, or hypertension (P < .05). Patients who currently smoked reported lower physical function (P = .004) and strikingly lower mental function (P < .001) than nonsmokers. CONCLUSIONS: Although patients completing the survey included healthy persons seeking preventive care and sick patients with acute and chronic illnesses, their overall functional status resembled that reported nationally for patients with chronic disease, perhaps reflecting the influence of poverty. Few studies have reported the association we observed between smoking and lower functional status. Further longitudinal studies in the primary care setting are necessary to fully interpret these associations and to evaluate the true impact of interventions on outcomes.     

Immunohistochemical localization of parathyroid hormone-related protein in developing mouse Meckel''s cartilage and mandible

In order to clarify the role of parathyroid hormone-related protein (PTHrP) during Meckel's cartilage and mandibular development, an immunohistochemical study of PTHrP and its receptor, PTH/PTHrP receptor, was designed to examine their localization in the anterior region of Meckel's cartilage including the rostrum, which is known to contribute to the development of the mandible. Meckel's cartilage was first observed on day 13 of gestation and PTHrP was faintly localized in the chondrocytes. On day 16 of gestation, at the stage of elongation and initiation of endochondral ossification in Meckel's cartilage, PTHrP was localized in the chondrocytes located in the area showing interstitial growth and in and around the nuclei of hypertrophic chondrocytes undergoing endochondral ossification. At day 18 of gestation, endochondral ossification was spread over the entire area proximal to the molar region in Meckel's cartilage, except in the mesial fusion site formed by immature chondrocytes. PTHrP was localized in the osteoblasts adjacent to the calcified matrix, but had disappeared from the chondrocytes forming Meckel's cartilage. The localization of PTH/PTHrP receptor was similar to that of PTHrP. These results show that localization of PTHrP is spatially and temporally related to the growth of Meckel's cartilage.     

Immunohistochemical localization of enkephalin in peripheral sensory axons in the rat

Impaired energy metabolism plays an important role in neuronal cell death after brain ischemia, and apoptosis has been implicated in cell death induced by metabolic impairment. In the present study, metabolic impairment was induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. In order to clarify the involvement of poly(ADP-ribosyl)ation and apoptotic pathway in 3-NP induced cell death, we examined poly(ADP-ribosyl)ation and the apoptosis related gene protein expression after systemic administration of 3-NP by immunohistochemistry. Poly(ADP-ribosyl)ation was evidently detected in the striatal lesion but not in any other region. Immunoreactive ratio of Bcl-2 to Bax significantly increased both in the striatum and cortex. The data suggest that striatal cell death involves poly(ADP-ribosyl)ation and also apoptotic pathway in part following administration of 3-NP.     

Immunohistochemical localization of caffeine-induced c-Fos protein expression in the rat brain

Although caffeine is the most widely used central nervous system stimulant, the neuronal populations and pathways mediating its stimulant effects are not well understood. Using c-Fos protein as a marker for neuronal activation, the present study investigated the pattern of c-Fos induction at 2 hours after low locomotor-stimulant doses (1, 5, 10, and 30 mg/kg, i.p.) of caffeine and compared them with those after a higher dose (75 mg/kg, i.p.) or saline injection in adult male rats. Fos-immunoreactive neurons were counted in selected nuclei across the entire brain. Caffeine induced an increase in locomotor activity in a dose-dependent manner up to doses of 30 mg/kg and a decline at 75 mg/kg. Quantitative analysis of Fos-immunoreactive neurons indicated that no structures showed significant Fos expression at doses below 75 mg/kg or a biphasic pattern of Fos expression, as in locomotion. In contrast, caffeine at 75 mg/kg induced a significant increase compared with the saline condition in the number of Fos-immunoreactive neurons in the majority of structures examined. The structures included the striatum, nucleus accumbens, globus pallidus, and substantia nigra pars reticulata and autonomic and limbic structures including the basolateral and central nuclei of the amygdala, paraventricular and supraoptic hypothalamic nuclei, periventricular hypothalamus, paraventricular thalamic nuclei, parabrachial nuclei, locus coeruleus, and nucleus of the solitary tract. The locomotor-enhancing effects of low doses of caffeine did not appear to be associated with significant Fos expression in the rat brain.     

Stored renin isoforms in the developing rat kidney

Each of 5 drugs, i.e., 4 different vasodilator drugs (captopril, enalapril, hydralazine and prazosin) and a cardiotonic drug (digoxin), was administered to dogs with mitral regurgitation (MR) for 1-72 days in order to quantitatively evaluate the influence of therapeutic agents on blood flow in heart disease. Hemodynamic changes were assessed before and after administration of each drug by determining mitral regurgitant jet mapping area (MRMA) and aortic forward flow mapping area (AFMA), which were displayed by the color Doppler method, and the ratio of MRMA to AFMA (MRMA/AFMA) as parameters. When the four vasodilator drugs were used appropriately, MRMA and MRMA/AFMA decreased in all cases, compared with the values before the administration. These two parameters showed dose-dependent changes after administration of captopril, enalapril and hydralazine. When the cardiotonic drug was used. MRMA and MRMA/AFMA increased in 4 of 5 cases. The MRMA/AFMA values were slightly more reproducible than the MRMA values, whereas the AFMA values showed no constant tendency when any vasodilator drug or the cardiotonic drug was used. These results suggest that the efficacy of cardiotonic and vasodilator drugs in MR can be quantitatively evaluated by determining MRMA/AFMA in particular, and MRMA.     

Activation of angiotensin-generating systems in the developing rat kidney

The present study was designed to determine the developmental changes in intrarenal angiotensin (Ang) peptides in the rat. Kidney Ang I and II levels were threefold and sixfold higher in newborn than adult kidneys, respectively (Ang I, 678 +/- 180 versus 243 +/- 38 fmol/g, P < .01; Ang II, 667 +/- 75 versus 103 +/- 6 fmol/g, P < .001). Intrarenal Ang II levels correlated positively with the temporal changes in renin gene expression (r = .93, P < .001). However, no correlation was found between renal Ang II content and angiotensin-converting enzyme (ACE) expression during development, which prompted us to evaluate whether renal enzymes, other than renin and ACE, contribute to Ang II formation in the developing kidney. Angiotensin peptide levels were measured in newborn and adult kidney homogenates incubated with human angiotensinogen (a poor rat renin substrate) for 30 minutes at 37 degrees C. Inhibitors of aspartyl proteases and metalloproteases were ineffective in preventing the formation of Ang II in either newborn or adult kidneys. However, addition of the serine protease inhibitors soybean trypsin inhibitor and phenylmethylsulfonyl fluoride inhibited Ang II generation in the newborn kidneys only. In contrast, Ang I generation was not affected by inhibition of serine proteases in either newborn or adult kidneys. We conclude that Ang I and II synthesis is activated in the developing rat kidney. In addition to renin and ACE, the newborn rat kidney expresses serine protease activity that is capable of generating Ang II directly from angiotensinogen. This putative enzyme is induced in the newborn kidney and may cooperate with renin in the activation of Ang II synthesis during early development.     

Cinnamyl anthranilate causes coinduction of hepatic microsomal and peroxisomal enzymes in mouse but not rat

Cinnamyl anthranilate is a synthetic food flavoring and fragrance agent, formerly used at low levels. Although it is not genotoxic, very high doses have been shown to cause liver tumors in mice but not rats. In this report we characterize hepatic changes brought about by cinnamyl anthranilate in rats and mice. Groups of male CD1 mice and Fischer 344 rats received 0, 100, or 1000 mg/kg cinnamyl anthranilate by intraperitoneal injection daily for 3 days. After euthanization on the 4th day, plasma lipids and relative liver weight, tissue DNA, the peroxisome marker CN(-)-insensitive palmitoyl-CoA oxidation, cytochrome P450, microsomal lauric acid hydroxylation, aminopyrine N-demethylase and ethoxyresorufin O-deethylase, bilirubin UDP-glucuronosyltransferase, microsomal and cytosolic epoxide hydrolase, and the peroxisome/mitochondria ratio in liver sections were monitored. In mice a pattern of change pointing to peroxisomal proliferation was seen at both doses of cinnamyl anthranilate, but in rats fewer and smaller changes were seen with little or no evidence of peroxisomal proliferation at the doses used. Groups of male CD1 mice were given 0-200 mg/kg cinnamyl anthranilate daily for 3 days. At doses of 20 mg/kg and above, there were dose-dependent increases in relative liver weight, total cytochrome P450, and CN(-)-insensitive palmitoyl-CoA oxidation. The hepatic effects of cinnamyl anthranilate are apparently due to the intact ester, since neither its expected metabolites alone nor an equimolar mixture of the hydrolysis products, cinnamyl alcohol and anthranilic acid, had any significant effect on the weight or marker enzyme content of mouse liver. The data are discussed in relation to the species specificity of the hepatocarcinogenicity of cinnamyl anthranilate and to metabolic studies in rats and mice. It is suggested that in mice, peroxisomal proliferation occurs only at doses high enough to prevent complete metabolic hydrolysis.     

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.     

Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.     

Immunohistochemical localization of annexin VI in the endocytic compartment of rat liver hepatocytes

Annexin VI has been isolated from rat liver endosomes and affinity purified antibodies have been produced. By Western blotting, in rat liver subcellular fractions, anti-annexin VI was demonstrated to recognise a 68 kDa band in the three endosomal fractions. In the present study, immunogold labeling of ultrathin Lowicryl sections of rat liver has been used to get insights into the ultrastructural hepatocyte localization. Although at the immunofluorescence level the staining seemed located at the apical, canalicular plasma membrane, domain of the hepatocytes, the electron microscopy revealed that 80% of the labeling, with the anti-annexin VI antibody was specifically localized not at the plasma membrane but in the close subapical endocytic compartment surrounding the bile canalicular plasma membrane of the hepatocyte. Double immunogold labeling with an anti peptide antibody to Rab5 and anti-annexin VI showed that 80% of the Rab5 positive apical endosomes were also labeled with anti-annexin VI antibodies. However, there was no significant colocalization of annexin VI and structures labeled with antibodies to the polymeric immunoglobulin receptor. The results suggest that annexin VI could be involved in regulating the functioning of this apical compartment in the hepatocyte.     

Immunohistochemical localization of cytosolic sialidase in the epithelium of rat cornea and conjunctiva

OBJECTIVE: To compare the hemodynamic change, course of recovery and adverse reaction in desflurane, sevoflurane and enflurane inhalation under low flow for patients undergoing selective abdominal surgery. METHODS: Following thiopental induction, 42 patients were divided into three groups: the first group received desflurane, the second sevoflurane and the third enflurane. During surgery, one of the agents around 1 minimum alveolar concentration (MAC) was used for maintenance, with fresh gas flow of 0.3-0.5 L/min for either desflurane or enflurane, and (0.8-1.0) L/min for sevoflurane. Heart rate (HR), blood pressure and end-tidal anesthetic concentration were monitored continuously. Time intervals from cutting off anesthetic to patient opening eyes, following commands, stating the time and location and recalling date of birth were all recorded. In addition, postoperative nausea or vomiting was traced. RESULTS: Desflurane caused the least cardiovascular depression. with mean arterial pressure (MAP) maintained significantly better at 10, 30 and 60 minutes of surgery and with HR stabilized right after incision as well. Its emergence was 2 times faster than sevoflurane, and 5-6 times quicker than enflurane. However, nausea or vomiting was found the lowest in patients receiving sevoflurane, though no distinct difference was shown between desflurane and enflurane. Nevertheless, patients under desflurane suffered less. CONCLUSIONS: Desflurane offers significant advantages for clinical anesthesia maintenance over sevoflurane and enflurane. It provides minimal cardiovascular depression, much quicker recovery, yet still causes some nausea during emergence.     

Immunohistochemical localization of ORL-1 in the central nervous system of the rat

A novel member of the opioid receptor family (ORL-1) has been cloned from a variety of vertebrates. ORL-1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N-terminus of ORL-1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II-V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1-CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL-1 was observed in telencephalic areas such as caudate-putamen. In the cerebellum, ORL-1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL-1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL-1 receptor in the rat CNS.     

Immunohistochemical demonstration of poly(adenosine diphosphate-ribose) in nuclei of various rat tissues

Natural distribution of poly(adenosine diphosphate-ribose), a novel macromolecule in eukaryotes, was investigated using an indirect immunofluorescence technique. The antibody, produced in a rabbit toward poly(ADP-ribose), was most reactive with polymers having the chain length of about 25 ADP-ribose units and weakly reactive with short oligomers; it was totally inert with monomers. Immunostaining with this antibody revealed the existence of the polymer in various rat tissues. The immunostaining seems to be specific for poly(ADP-ribose), as judged by its disappearance by preabsorption of the antiserum with purified poly(ADP-ribose) or pretreatment of tissue sections with poly(ADP-ribose)-degrading enzymes. Intensification of the fluorescence by preincubation with nicotinamide adenine dinucleotide (NAD), a substrate for poly(ADP-ribose) synthesis, also supported this view. The immunofluorescence of poly(ADP-ribose) was found exclusively in the nucleus of almost all tissues tested, including liver (adult, newborn, regenerating, and hepatoma), brain, heart, intestine, pancreas, kidney, spleen, testis, thyroid gland, and skeletal muscle. Exceptions were blood cells; little fluorescence was detectable in nuclei of peripheral leukocytes. Only after preincubation with NAD, did lymphocytes and monocytes exhibit fluorescence, however, granulocytes never did exhibit fluorescence. The cells appeared to represent the first instance where poly(ADP-ribose) synthesis activity among eukaryotic cells was missing.     

Immunohistochemical localization of novel CART peptides in rat hypothalamus, pituitary and adrenal gland

CART peptide specific polyclonal antisera were raised in rabbits. The antisera were raised to CART peptide fragments that span most of the predicted CART protein. The specificity of each antisera was demonstrated by blockade of immunostaining by the immunizing peptide but not by the other CART peptide fragments. In the hypothalamus and pituitary of colchicine and noncolchicine treated rats, immunostaining was observed in cell bodies, fibers and varicosities. Clusters of cells were also stained in the adrenal medulla. It is noteworthy that cellular immunostaining was only found in areas previously shown to express CART mRNA. These findings indicate the presence of CART peptide(s) in the hypothalamus, pituitary, and adrenal gland. Furthermore, we also present evidence for the possible processing of the CART pro-peptide into smaller peptide fragments. These neuroanatomical findings suggest a role of CART peptides in hypothalamic, pituitary and adrenal function.