Biomedical subjects

X Qi

Publications and source records attributed to X Qi.

At least 19 recordsLinked to original sources

PubMed1999

New technique for vagal nerve stimulation.

INTRODUCTION: The vagus nerve travels in a neurovascular bundle with the carotid artery and internal jugular vein. The present study was designed to assess whether transvascular stimulation through the carotid artery of the dog can be used to directly stimulate the vagus nerve and increase parasympathetic tone. METHODS: In five anesthetized dogs, a steerable electrode catheter was positioned under fluoroscopic guidance in the right carotid artery in the mid neck via the femoral artery. Multipolar catheters were positioned transvenously through the femoral vein in the right atrium, across the tricuspid valve to record a His-bundle electrogram, and in the right ventricle. RESULTS: In all five animals, vagal nerve stimulation was successfully achieved with outputs ranging between 10 and 30 mA. Sinus cycle length increased from 473 +/- 113 ms at baseline to 894 +/- 315 ms (P < 0.025) during stimulation from the right carotid artery. There was an increase in the AH interval from 55 +/- 14 to 77 +/- 23 ms (P < 0.03), a shortening of the atrial effective refractory period from 136 +/- 8 to 126 +/- 6 ms (P < 0.01), and a fall in the systolic blood pressure from 135 +/- 20 to 117 +/- 20 mmHg (P < 0.005) with stimulation from the right carotid artery. A prolongation of the AV and VA block cycle lengths and the AV nodal effective refractory period was also noted with stimulation from the right carotid artery. Atrial fibrillation was not induced at baseline in any animal. During stimulation from the right carotid artery, atrial fibrillation was induced in three of five animals and persisted for the duration of stimulation from the right carotid artery. CONCLUSION: Cardiac parasympathetic stimulation can be achieved by positioning a catheter in the neurovascular bundle in the neck adjacent to the vagus nerve with resultant effects on cardiac electrophysiology.

Animals↗

PubMed1999

Conformational and amino acid residue requirements for the saposin C neuritogenic effect.

Prosaposin is the precursor of four activator proteins, termed saposins A, B, C, and D, that are required for much of glycosphingolipid hydrolysis. The intact precursor also has neurite outgrowth activity ex vivo and in vivo that is localized to amino acid residues 22-31 of saposin C. Across species, this saposin C region has a high degree of identity and similarity with amino acids in the analogous region of saposin A. Wild-type and mutant saposins C and A from human and mouse were expressed in E. coli. Pure proteins, synthetic peptide analogues, conformation-specific antibodies, and CD spectroscopy were used to evaluate the basis of the ex vivo neuritogenic effect. Wild-type saposin A had no neuritogenic activity whereas reduced and alkylated saposin A did. Introduction of the conserved saposin A Tyr 30 (Y30) into saposin C at the analogous position 31, a conserved Ala(A)/Gly(G)31, diminished neuritogenic activity by 50-60%. Nondenatured saposin A with an introduced A30 acquired substantial neuritogenic activity. Polyclonal antibodies directed against the NH2-terminus of saposin C cross-reacted well with reduced and alkylated saposins C and A, wild-type saposin C, and saposin A [Y30A], poorly with saposin C [A31Y], and not at all with wild-type saposin A. CD spectra of wild-type and mutant saposins C and A, the corresponding neuritogenic region of saposin C, and the analogous region of saposin A showed that more "saposin C-like" molecules had neuritogenic properties. Those with more "saposin A-like" spectra did not. These studies show that the neuritogenic activity of saposin C requires specific placement of amino acids, and that Y30 of saposin A significantly alters local conformation in this critical region and suppresses neuritogenic activity.

Amino Acid Sequence↗

PubMed1999

Effect of phosphorylation on activities of Rap1A to interact with Raf-1 and to suppress Ras-dependent Raf-1 activation.

Rap1A is phosphorylated by cAMP-dependent protein kinase (PKA), and this phosphorylation has been shown to modulate its interaction with other proteins. However, it is not known whether Rap1A phosphorylation is involved in regulation of its cellular functions, including suppression of Ras-dependent Raf-1 activation. We have previously shown that this suppressive activity of Rap1A is attributable to its greatly enhanced ability to bind to the cysteine-rich region (CRR, residues 152-184) of Raf-1 compared with that of Ras. Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR. Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation residue, Ser-180, was substituted by an acidic residue, Glu, to mimic its phosphorylated form, failed to suppress Ras-dependent Raf-1 activation in COS-7 cells. These results indicate that the CRR binding activity and the Ras-suppressive function of Rap1A can be modulated through phosphorylation and suggest that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor.

Amino Acid Sequence↗

PubMed1999

Adenoviral gene therapy with catalase suppresses experimental optic neuritis.

OBJECTIVE: To determine if adenoviral-mediated transfer of the gene for catalase (CAT), the reactive oxygen species scavenger, suppresses experimental optic neuritis. CLINICAL RELEVANCE: Gene therapy with CAT delivered by an adeno-associated viral vector was previously shown to suppress experimental optic neuritis. Because the transduction of protein expression with recombinant adeno-associated viral vector is relatively slow, taking weeks to reach full levels, we studied the effects of replication-deficient adenovirus containing CAT in suppressing experimental optic neuritis. Transduction with adenovirus occurs within days of inoculation, thus, it may be more applicable for the treatment of patients with acute optic neuritis. MATERIALS AND METHODS: Replication-deficient adenovirus containing CAT was injected above the right optic nerve heads of SJL/J mice that were simultaneously sensitized for experimental allergic encephalomyelitis. For controls, the left eyes were injected with the replication-deficient adenovirus without CAT or no virus. The histological effects of CAT on the lesions of experimental allergic encephalomyelitis were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), the extent of the cellular infiltrate, extravasated serum albumin labeled with immunogold (for disruption of the blood-brain barrier), and the in vivo hydrogen peroxide reaction product. RESULTS: After 1 month, cell-specific catalase activity, evaluated by the quantitation of catalase immunogold, was increased about 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the CAT-inoculated right optic nerves compared with the control left optic nerves. The increased cellular levels of catalase reduced demyelination by 30%, optic nerve head swelling by 25%, cellular infiltration by 26%, disruption of the blood-brain barrier by 61%, and in vivo levels of hydrogen peroxide by 81%. CONCLUSIONS: Adenoviral-mediated gene transfer increased catalase levels in all optic nerve cell types, and it persisted for 1 month after inoculation. The increased cellular levels of catalase suppressed demyelination and blood-brain barrier disruption at the foci in the optic nerve where prior magnetic resonance imaging and histopathologic studies have demonstrated the demyelinating inflammation of experimental and human optic neuritis. Together, they suggest that gene therapy with CAT may be helpful in the treatment of patients with optic neuritis.

Adenoviridae↗

PubMed1999

Reporter expression persists 1 year after adeno-associated virus-mediated gene transfer to the optic nerve.

OBJECTIVE: To determine the foci and duration of protein expression following virus-mediated gene transfer to the optic nerve. METHODS: A cytomegalovirus (CMV) promoter was linked to a lacZ-SV40 polyA reporter gene or a humanized green fluorescent protein (hgfp) reporter gene, then inserted into a bacterial plasmid containing adeno-associated virus (AAV) terminal repeat sequences. The CMV-lacZ or the CMV-hgfp construct were injected into the vitreous cavity of strain-13 guinea pigs. Controls consisted of eyes injected with AAV without the promoter and reporter elements or eyes that received no injections. The eyes and optic nerves were processed for beta-galactosidase immunohistochemistry and hgfp fluorescence analyses. Cellular transduction at the messenger RNA (mRNA) level was evaluated by in situ reverse transcription-polymerase chain reaction. RESULTS: Weekly fundus photography, done for 1 month, documented the absence of any ocular abnormality due to the viral injections. No in vivo hgfp fluorescence of the retina was visualized. Beta-galactosidase histochemical analysis of eye cups that received the lacZ gene construct showed blue lacZ staining of the optic nerve head at 2 weeks. Light microscopy revealed the blue beta-galactosidase reaction product in fibers, glial cells, and blood vessels of the optic nerve head and retrobulbar nerve. Histochemistry showed absence of beta-galactosidase in the optic nerve at 3 to 12 months, but immunochemistry showed the persistence of beta-galactosidase in fibers, glial cells, and blood vessels as late as 1 year after a single ocular injection. In the retina, histochemical staining showed evidence of lacZ at 3 months, but not later. In situ reverse transcription-polymerase chain reaction revealed brown lacZ mRNA reaction product in ganglion cells of the retina. Control eyes that received AAV without the promoter and reporter elements and the eyes that received no viral injections and were processed for beta-galactosidase showed no reporter gene expression in any ocular tissue or cell type. CONCLUSIONS: Viral-mediated gene transfer can be successfully accomplished in the optic nerve. Further evaluation is needed to determine whether the level of protein expression at 1 year after injection, which is clearly reduced relative to shorter postinjection time, is sufficient for therapeutic purposes. CLINICAL RELEVANCE: We have previously shown that gene therapy with catalase suppressed experimental optic neuritis at 1 month after injection. Viral-mediated gene transfer may be a powerful technique for the treatment of optic neuropathies, particularly for recurrences of optic neuritis, if long-term expression of transduced protein can be demonstrated in the optic nerve.

Animals↗

PubMed1999

[Study of transcription and cleavage in vitro of HDV with HBV-specific hammerhead ribozyme].

OBJECTIVE: To study the effect of hepatitis B virus(HBV) specific ribozyme(RZ) and recombinant hepatitis D virus(HDV) inserting hammerhead ribozyme(rHDVRZP and rHDVRZA). METHODS: 831 bp HBV C gene fragment was cloned under the control of T7 promoter, 32P-labeled HBV transcript was incubated with gel-purified RZ, rHDVRZA, rHDVRZP at different temperature and autoradiographed after denaturing gel-electrophoresis. RESULT: These results show that rRZ, rHDVRZA, rHDVRZP were active at 37 degrees C and more so at higher temperatures. CONCLUSION: Recombinant Delt virus could serve as a vector for the delivery of a ribozyme specific for hepatitis B virus cleavage. Our data demonstrate the value of recombinant ribozyme as potential therapeutic agents for treatment of HBV infection. Further study about cleavage in vitro and in vivo will continue.

Cloning, Molecular↗

PubMed1998

Adeno-associated viral-mediated catalase expression suppresses optic neuritis in experimental allergic encephalomyelitis.

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood-brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood-brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

Animals↗

PubMed1998

Acid beta-glucosidase: intrinsic fluorescence and conformational changes induced by phospholipids and saposin C.

Acid beta-glucosidase is a lysosomal membrane protein that cleaves the O-beta-D-glucosidic linkage of glucosylceramide and aryl-beta-glucosides. Full activity reconstitution of the pure enzyme requires phospholipids and saposin C, an 80 aa activator protein. The deficiency of the enzyme or activator leads to Gaucher disease. A conformational change of acid beta-glucosidase is shown to accompany activity reconstitution by selected phospholipids or, particularly, phospholipid/saposin C complexes by intrinsic fluorescence spectral shifts, fluorescence quenching, and circular dichroism (CD). Negatively charged phospholipid (NCP) interfaces with unsaturated fatty acid acyl chains (UFAC) induced concordant blue-shifts in tryptophanyl fluorescence spectra and a loss of beta-strand structure by CD. The enzyme required an unsaturated fatty acid acyl chain in proximity (10-11 A) within liposomal membranes for activation, fluorescence blue-shifts, and changes in CD spectra. Activity enhancements were greatest when UFAC and the negatively charged headgroup were present on the same phospholipid. NCPs with UFAC protected the enzyme from fluorescence quenching by aqueous agents (I-, Cs+, acrylamide, TEMPO). Phosphatidylcholine with doxyl spin-labeled fatty acid acyl chains at carbons 7, 10, or 16 quenched enzyme fluorescence only when in NCP/PC liposomes. Saposin C (Trp-free) induced additional activity and fluorescence spectral changes in the enzyme only in the presence of NCP liposomes containing UFA. CD spectral changes indicated saposin C and acid beta-glucosidase interaction only in the presence of NCPs with UFA. These studies show that acid beta-glucosidase requires interfaces composed of NCPs, containing UFAC, for penetration into the outer leaflet of membranes. Furthermore, this interaction induces essential conformational changes for saposin C binding and further enhancement of acid beta-glucosidase catalytic activity.

Animals↗

PubMed1998

Correlation between cardiac remodelling, function, and myocardial contractility in rat hearts 5 weeks after myocardial infarction.

Early after infarction, ventricular dysfunction occurs as a result of loss of myocardial tissue. Although papillary muscle studies suggest that reduced myocardial contractility contributes to this ventricular dysfunction, in vivo studies indicate that at rest, cardiac output is normal or near normal, suggesting that contractility of the remaining viable myocardium of the ventricular wall is preserved. However, this has never been verified. To explore this further, 100 rats with various-sized myocardial infarctions had ventricular function assessed by Langendorff preparation or by isolated papillary muscle studies 5 weeks after infarction. Morphologic studies were also done. Rats with large infarctions (54%) had marked ventricular dilatation (dilatation index from 0.23 to 0.75, p < 0.01) and papillary muscle dysfunction (total tension from 6.7 to 3.2 g/mm2, p < 0.01) but only moderate left ventricular dysfunction (maximum developed tension from 206 to 151 mmHg (1 mmHg = 133.3 Pa), p < 0.01), a decrease less than one would expect with an infarct size of 54%. The contractility of the remaining viable myocardium of the ventricle was also moderately depressed (peak systolic midwall stress 91 to 60 mmHg, p < 0.01). Rats with moderate infarctions (32%) had less marked but still moderate ventricular dilatation (dilatation index 0.37, p < 0.001) and moderate papillary muscle dysfunction (total tension 4.2 g/mm2, p < 0.01). However, their decrease in ventricular function was only mild (maximum developed pressure 178 mmHg, p < 0.01) and less than one would expect with an infarct size of 32%. The remaining viable myocardium of the ventricular wall appeared to have normal contractility (peak systolic midwall stress = 86 mmHg, ns). We conclude that in this postinfarction model, in large myocardial infarctions, a loss of contractility of the remaining viable myocardium of the ventricular wall occurs as early as 5 weeks after infarction and that papillary muscle studies slightly overestimate the degree of ventricular dysfunction. In moderate infarctions, the remaining viable myocardium of the ventricular wall has preserved contractility while papillary muscle function is depressed. In this relatively early postinfarction phase, ventricular remodelling appears to help maintain left ventricular function in both moderate and large infarctions.

Animals↗

PubMed1997

Development of AFLP markers in barley.

To investigate the application of amplified fragment length polymorphism (AFLP) markers in barley, 96 primer combinations were used to generate AFLP patterns with two barley lines, L94 and Vada. With seven primer combinations, only a few intense bands were obtained, probably derived from repeated sequences. With the majority of the remaining 89 primer combinations, on average about 120 amplification products were generated, and the polymorphism rate between the two lines was generally over 18%. Based on the number of amplified products and the polymorphism rate, the 48 best primer combinations were selected and tested on 16 barley lines, again including L94 and Vada. Using a subset of 24 primer combinations 2188 clearly visible bands within the range from 80 to 510 bp were generated; 55% of these showed some degree of polymorphism among the 16 lines. L94 versus Vada showed the highest polymorphism rate (29%) and Proctor versus Nudinka yielded the lowest (12%). The polymorphism rates per primer combination showed little dependence on the barley lines used. Hence the most efficient and informative primer combinations identified for a given pair of lines turned out to be highly efficient when applied to others. Generally, more than 100 common markers (possibly locus specific) among populations or crosses were easily identified by comparing 48 AFLP profiles of the parent lines. The existence of such a large number of markers common to populations will facilitate the merging of molecular marker data and other genetic data into one integrated genetic map of barley.

Chromosome Mapping↗

PubMed1997

Distribution of prohormone convertase-6 expression in the gastrointestinal tract and effects of a fasting-refeeding regimen and a high-fat diet on ileal prohormone convertase-6 expression.

Proforms of gastrointestinal peptide hormones and neuropeptides are processed, in part, by prohormone convertases (PCs) to mature, biologically active peptides. The purpose of this study was to characterize the mRNA levels of PC-6 isoforms, PC-6A and PC-6B, in the gastrointestinal tract and pancreas of the rat, and to investigate the effects of a fasting and refeeding regimen, and a high-fat diet on ileal PC-6A expression. PC-6A mRNA is expressed throughout the entire gastrointestinal tract with the highest levels in the small intestine. Multiple-sized transcripts are present. PC-6B mRNA is expressed in the antrum and fundus of the stomach, in the small intestine, and colon. Ileal PC-6A mRNA expression increases significantly with fasting and then declines with refeeding toward control levels. Increased dietary fat increases PC-6A mRNA levels in the ileum. Since PC-6 is found throughout the entire gastrointestinal tract, it is likely that PC-6 participates in the processing of proforms of gastrointestinal peptides. The two isoforms of PC-6 have different patterns of distribution in the gastrointestinal tract and pancreas, suggesting that they process proforms of different gut peptides.

Animals↗

PubMed1997

[Construction of Escherichia coli-Mycobacteria shuttle plasmid and the stable expression of human interleukin-2 in BCG and Escherichia coli].

OBJECTIVE: To construct and identify Escherchia coli (E. coli)-Mycobacteria shuttle plasmid and to detect stable expression of foreign gene in E. coli and BCG. METHOD: With a genetic engineering technique to construct the E. coli-Mycobacteria shuttle plasmid, the human interleukin-2 (IL-2) gene was electrophoreted into BCG with recombinant plasmid PZSIII-I and positive clones selected using polymerase chain reaction (PCR) technique. The expression of foreign gene of human IL-2 in BCG was identified by ELISA and SDS-PAGE. RESULT: Human IL-2 cytokine was steadily expressed in recombinant BCG and E. coli and could secrete outside the cell. CONCLUSION: M. bovis BCG recombinant constructed can produce and secrete the human IL-2. A secretion of the active cytokine was accomplished through the combined use of the BCG HSP65 promoter and a secretion signal from the BCG Ag-85B. The BCG HSP65 promoter is active in both BCG as well as E. coli which can not secrete foreign protein.

BCG Vaccine↗

PubMed1997

[The role of CDDP on cytotoxicity of TILs obtained from colorectal tumors].

The effects of intravenous injection of cisplatin (CDDP) and the incubated with CDDP in vitro on the epitopes and cytotoxicity of TIL obtained from eight colorectal tumor patients were detected respectively by flow cytometer. There was a significant increase in CD3+/CD8+ subset in TIL in patients receiving intravenous injection of CDDP. And the cytotoxicity of these TILs increased significantly. The cytotoxities were positively correlated with the amount of CD3+/CD8+ subset in TIL. Raji cells incubated with CDDP in vitro showed increased susceptibility to TIL induced lysis.

Antineoplastic Agents↗

PubMed1997

Increase of manganese superoxide dismutase, but not of Cu/Zn-SOD, in experimental optic neuritis.

PURPOSE: To evaluate the role of manganese superoxide dismutase (Mn-SOD) and copper/zinc superoxide dismutase (Cu/Zn-SOD) in cellular protection of the optic nerve against the oxidative injury that contributes to demyelination in experimental allergic encephalomyelitis (EAE). METHODS: Immunocytochemistry for Mn-SOD and Cu/Zn-SOD and ultracytochemical localization of hydrogen peroxide (H2O2) were performed on the optic nerves of guinea pigs with EAE and normal guinea pigs. Cell-specific enzyme expression of SOD was quantitated by computerized morphometric analysis. RESULTS: Light microscopy showed a perivascular distribution of Mn-SOD-positive cells in the optic nerves of animals with EAE. Electron microscopy showed that the Mn-SOD immunogold was confined exclusively to mitochondria, whereas Cu/Zn-SOD immunogold was found in the cytoplasmic matrix and nucleus of cells of the optic nerve in both animals with EAE and normal animals. Results of quantitative analysis of the optic nerves of animals with EAE showed an 8-fold increase in Mn-SOD immunogold in astroglial cells and a 13-fold increase in microglial/phagocytic cells in comparison with that of normal animals. Increases in Mn-SOD immunogold were contiguous to H2O2-derived reaction product. No increases in Cu/Zn-SOD immunogold were detected in EAE. CONCLUSIONS: Increases in Mn-SOD activity in astroglial cells and microglial/phagocytic cells may contribute to the relative sparing of these cells from injury in EAE, whereas the low level of Mn-SOD in oligodendroglial cells and axons may increase their vulnerability to the effects of superoxide-induced oxidative injury that results in demyelination.

Animals↗

PubMed1996

Influence of nicotine on gastrin and peptide YY in the rat.

The objective of this study was to examine the effects of nicotine and high-fat diets on gastrin and peptide YY (PYY) homeostasis in the rat. Antral levels of gastrin mRNA and peptide and ileal and colonic levels of PYY mRNA and peptide were examined. Serum levels of gastrin in response to food were also measured. Control rats were ad-lib fed or pair-fed according to the daily food intake of nicotine-treated rats. The results of this study indicate that nicotine treatment and fat diets can influence gastrin and PYY gene expression in the gastrointestinal tract.

Animals↗

PubMed1996

Proteolytic processing patterns of prosaposin in insect and mammalian cells.

Prosaposin is a multifunctional protein encoded at a single locus in humans and mice. The precursor contains, in tandem, four glycoprotein activators or saposins, termed A, B, C, and D, that are essential for specific glycosphingolipid hydrolase activities. Prosaposin appears to be a potent neurotrophic factor. To explore the proteolytic processing from prosaposin to mature activator proteins, metabolic labeling was done with human prosaposin expressed in insect cells, human fibroblasts, neuronal stem cells (NT2) and retinoic acid-differentiated NT2 neurons. In all cell types, the major processing pathway was through a tetrasaposin, A-B-C-D, from which saposin A was then removed. In mammalian cells monosaposins were derived from the trisaposin B-C-D by cleavage to the disaposins, B-C and C-D, that were processed to monosaposins. In insect cells the major end products were the disaposins, with A-B and C-D derived from the tetrasaposin, A-B-C-D, or with B-C and C-D derived from the trisaposin, B-C-D. In insect and mammalian cells, the nonsignal NH2-terminal peptide preceding saposin A (termed Nter) was usually removed prior to saposin A cleavage. In NT2-derived differentiated neurons, precursor tetrasaposins containing A-B-C-D were secreted with and without Nter. Immunofluorescence studies using prosaposin-specific antisera showed large steady state amounts of uncleaved prosaposin in Purkinje cells, cortical neurons, and other specific cell types in adult mice. These studies indicate that prosaposin processing is highly regulated at a proteolytic level to produce prosaposin, tetrasaposins, or mature monosaposins in specific mammalian cells.

Animals↗

PubMed1996

Localization of NADPH diaphorase/nitric oxide synthase in the optic nerve of the normal guinea pig: a light and electron microscopic study.

The aim of this study is to determine the presence and subcellular distribution of NADPH diaphorase (NADPH-d)/nitric oxide synthase (NOS) in the optic nerve of the normal guinea pig. Optic nerve specimens were stained by NADPH-d histochemistry, and double labeled by combining NADPH-d histochemistry with immunostaining for (a) anti-glial fibrillary acidic protein (GFAP) antibody for recognition of astrocytes, (b) griffonia simplicifilia B4-isolectin (GSA-IB4) horse radish peroxidase (HRP)-conjugate for identification of microglia, or (c) oligodendrocyte-associated antibodies to carbonic anhydrase isoenzyme II (CA-II) or to galactocerebroside (GalC) for visualization of oligodendrocytes. In addition, constitutive NOS (cNOS) and inducible NOS (iNOS) immunostaining were used for colocalization with NADPH-d histochemistry. Light microscopy revealed NADPH-d reaction product in the blood vessels and neuroglia of the unmyelinated optic nerve head and myelinated retrobulbar optic nerve. Double labeling with GFAP immunoperoxidase combined with NADPH-d histochemistry revealed both activities in astrocytes. Microglia were labeled with GSA-IB4 isolectin HRP-conjugate, but they did not have NADPH-d activity. Oligodendroglia were immunolabeled with anti CA-II or anti GalC antibodies, but they did not have NADPH-d activity. Both iNOS and cNOS immunoperoxidase labeled astrocytes, but not microglia or oligodendroglia. Under transmission electron microscopy, NADPH-d reaction product appeared as electron-dense particles. These particles were seen in the cytoplasm of endothelial cells, perivascular smooth muscle cells and fibrous astrocytes. Axons and myelin were devoid of NADPH-d activity. This study demonstrates the existence and cellular distribution of NADPH-d/NOS activity in endothelial cells, perivascular smooth muscle cells and fibrous astrocytes of the optic nerve of the normal guinea pig. The presence of these non-neuronal sources of NOS in the optic nerve provides the foundation for future comparative studies of the functional role of reactive oxygen induced toxicity in disorders affecting the optic nerve.

Animals↗

PubMed1996

Efficient catalysis by beta-lactamase from Staphylococcus aureus PC1 accompanied by accumulation of an acyl-enzyme.

The pH- and temperature-dependence of steady-state kinetic parameters for 6-beta-(2-furyl)-acryloylamido-penicillanic acid showed it to be a good substrate of staphylococcal PC1 beta-lactamase, and the viscosity-dependence of K(m)/k(cat) indicated that steps up to the formation of the acyl-enzyme were partially diffusion-limited. In the pH range 4-9, a pre-steady-state transient blue shift in the UV absorption spectrum of the bound furyl-acryloylamido chromophore was of constant amplitude and decayed to the spectrum of the product with a first-order rate constant equal to k(cat). The spectrum of the isolated denatured acyl-enzyme was similar to that of the methyl ester of furyl-acryloylpenicilloic acid, pointing to non-covalent interactions with the folded protein, possibly associated with the charge on Glu-166, as the source of the blue-shifted spectrum. Taken together, these results point to a rapid acylation and slower deacylation at Ser-70 and imply that ionization of groups affecting enzyme activity at alkaline pH, for which likely candidates are Lys-73 and Lys-234, affect the rate of deacylation.

Acylation↗