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Biomedical subjects

S H Wong

Publications and source records attributed to S H Wong.

At least 19 recordsLinked to original sources
PubMed1992

Biochemical fractionation and characterization of proteins from Golgi-enriched membranes.

Fractions enriched in Golgi membranes were prepared from rat liver by sucrose gradient ultracentrifugation. These enriched membranes were further subfractionated on the basis of their solubilities in EGTA, 150 mM sodium carbonate, pH 11.5, sodium deoxycholate, Triton X-100, or sodium dodecyl sulfate. This led to isolation of peripheral, luminal, and integral membrane proteins of the Golgi-enriched membranes. Luminal and membrane proteins were further purified by wheat germ agglutinin and concanavalin A lectin affinity chromatographies. Some proteins from these lectin columns were resolved by preparative gel electrophoresis and microsequenced. Subsequently, antibodies were produced for two proteins by immunization of either mice or rabbits. Immunofluorescence microscopy suggests that these proteins are confined to Golgi apparatus-like structures. The protocol described is well suited for the study of organelle structure and function.

Amino Acid Sequence↗
PubMed1992

The transmembrane domain of N-glucosaminyltransferase I contains a Golgi retention signal.

The enzyme N-acetylglucosaminyltransferase I (NT, EC 2.4.1.101) is a resident type II transmembrane protein of the Golgi apparatus. To delineate the portion of its primary sequence that is responsible for the Golgi retention of this protein, we constructed chimeras containing different N-terminal portions of NT joined to a reporter sequence, the ectodomain of a type II surface membrane protein. These chimeric proteins were found to be retained in the Golgi apparatus as assessed by cell surface biotinylation and immunofluorescence. We found that the transmembrane domain of NT is sufficient to confer Golgi retention of the fusion proteins and propose that it contains the Golgi retention signal of the parent molecule.

Animals↗
PubMed1992

Retention of a type II surface membrane protein in the endoplasmic reticulum by the Lys-Asp-Glu-Leu sequence.

Soluble luminal proteins of the endoplasmic reticulum (ER) are known to be retained by a tetrapeptide retention signal, KDEL. We report in this communication that the KDEL sequence when appended to the carboxy terminus of a cell surface membrane protein, dipeptidyl peptidase IV (DPPIV), resulted in its retention in the endoplasmic reticulum of transfected Madin-Darby canine kidney cells as assessed by indirect immunofluorescence. Selective surface biotinylation revealed that about 90-95% of the expressed DPPIV was retained in the ER. Appendance of the sequence KDEV did not, however, result in ER retention, illustrating the functional specificity of the retention signal. The ER retention was not due to misfolding of the mutant protein, as the mutant proteins remained enzymatically active. Our data suggest that the KDEL receptor is able to recognize and recycle type II membrane proteins containing a carboxyl-terminal KDEL sequence and postulates the existence of such yet to be identified endogenous proteins.

Amino Acid Sequence↗
PubMed1992

Disturbances in the composition of plasma lipoproteins during gram-negative sepsis in the rat.

Hyperlipidemia is associated with gram-negative sepsis. In this study we characterized the plasma lipoproteins of fasted and fed septic and control rats with respect to their lipid and apolipoprotein composition. Sepsis was induced by i.v. injection of 8 x 10(7) live Escherichia coli colonies/100 g body wt. Food was removed from fasted control and fasted E. coli-treated rats after injection. Fed rats were infused intragastrically with a nutritionally complete diet for 5 days prior to E. coli treatment. 24 h after treatment with E. coli, lipid and protein concentrations of very-low-density lipoprotein (VLDL) were over 2-fold higher in the fasted E. coli-treated rats than those of the fasted control rats. This appears to be due to a decrease in the clearance of VLDL. The relative composition of apolipoprotein B-48 and apolipoprotein E were lower while that of apolipoprotein B-100 was higher in fasted E. coli-treated rats than in fasted controls. Low-density lipoprotein (LDL) and high-density lipoprotein lipids were also significantly elevated, indicating greater synthesis of these particles during sepsis and food deprivation. By contrast, VLDL-triacyglycerol from fed, E. coli-treated did not differ from that of their respective controls although the total cholesterol remained elevated. Percentages of apolipoprotein B-48 and apolipoprotein B-100 increased while apolipoprotein E contributed significantly less to the total protein of VLDL from the E. coli-treated rats compared with controls. LDL lipids were also increased. In conclusion, gram-negative sepsis leads to marked changes in the plasma lipoprotein composition which may be attributed to altered hepatic synthesis, peripheral metabolism or hepatic uptake of lipoproteins and their remnants. These in turn may be a function of the nutritional status.

Animals↗
PubMed1992

The 17-residue transmembrane domain of beta-galactoside alpha 2,6-sialyltransferase is sufficient for Golgi retention.

beta-Galactoside alpha 2,6-sialyltransferase (ST) is a type II integral membrane protein of the Golgi apparatus involved in the sialylation of N-linked glycans. A series of experiments has shown that the 17-residue transmembrane domain of ST is sufficient to confer localization to the Golgi apparatus when transferred to the corresponding region of a cell surface type II integral membrane protein. Lectin affinity chromatography of chimeric proteins bearing this 17-residue sequence suggests that these chimeric proteins are localized in the trans-Golgi cisternae and/or trans-Golgi network. Further experiments suggest that this 17-residue sequence functions as a retention signal for the Golgi apparatus.

Amino Acid Sequence↗
PubMed1992

Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A.

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.

Animals↗
PubMed1992

An evaluation of the staphylococcal co-agglutination test for the detection of group A rotavirus in human faeces.

The group A rotavirus staphylococcal co-agglutination test was evaluated and its sensitivity and specificity compared with an in-house enzyme-linked immunosorbent assay (ELISA) and a commercial latex agglutination test (Rotalex). In addition, the storage stability of the staphylococcal reagents was ascertained. Examination of 136 clarified suspensions of diarrhoeal faeces by the staphylococcal co-agglutination test revealed a high proportion of false positives (26%) and uninterpretable results (34%) due to non-specific agglutination. Non-specific agglutination could be removed effectively by prior absorption of the clarified faecal specimens with unsensitized staphylococci. The staphylococcal co-agglutination test was less sensitive and specific than the in-house enzyme-linked immunosorbent assay but was comparable to the Rotalex slide latex agglutination test. The staphylococcal reagents have a shelf life of at least 29 weeks.

Agglutination Tests↗
PubMed1991

Involvement of both vectorial and transcytotic pathways in the preferential apical cell surface localization of rat dipeptidyl peptidase IV in transfected LLC-PK1 cells.

Dipeptidyl peptidase IV (DPPIV) is a membrane glycoprotein with type II orientation. It is predominantly localized to the apical surface in epithelial cells. Previous studies (Bantles, J. P., Feracci, H. M., Shinger, B., and Hubbard, A. L. (1987) J. Cell Biol. 105, 1241-1251) using cellular fractionation and immunoprecipitation in rat liver suggest that DPPIV is targeted to the apical surface by an indirect pathway through transient appearance in the basolateral surface followed by specific transcytosis to the apical domain. In transfected Madin-Darby canine kidney (MDCK) cells using domain-selective biotinylation and streptavidin absorption, it was, however, shown that DPPIV is directly sorted to the apical surface (Low, S. H., Wong, S. H., Tang, B. L. Subramaniam, V. N., and Hong, W. (1991) J. Biol. Chem, 266, 13391-13396). These studies suggest that the sorting pathway for DPPIV may be cell type-specific, but it cannot be ruled out that the observed difference in the DPPIV sorting pathway may be due to different methods employed for dissecting the sorting pathway. In this study, we have expressed rat DPPIV, using an expression system driven by the Rous sarcoma virus enhancer and the SV40 early promoter region, in another epithelial cell line, LLC-PK1. As in MDCK cells, DPPIV is preferentially (about 90%) localized to the apical surface. Employing identical methods used previously in MDCK cells, it was found that both direct and transcytotic pathways are involved in the apical surface localization of DPPIV in this epithelial cell type. These observations clearly illustrate that the sorting pathway of rat DPPIV is cell type-specific.

Animals↗
PubMed1991

Inhibition by brefeldin A of protein secretion from the apical cell surface of Madin-Darby canine kidney cells.

The effect of brefeldin A (BFA) on total and polarized protein secretion was examined in MDCK cells. Increasing concentrations of BFA have increasingly inhibitory effects on total protein secretion. The total protein secretion was essentially unaffected by BFA at 0.5 microgram/ml. When the BFA concentration was increased to 10 and 30 micrograms/ml, the total protein secretion was reduced to about 70 and 25%, respectively, of the control level. Consistent with this effect on total protein secretion, the Golgi structure as revealed by C6-NBD-ceramide (a fluorescent ceramide analog) staining was essentially unaltered by 0.5 microgram/ml BFA, while 10 and 30 micrograms/ml BFA significantly dispersed the Golgi apparatus. When the polarity of protein secretion was examined, it was found that the ratio of proteins secreted from the apical to those from the basolateral surface was reduced from 1.5-2.0 to 0.4-0.7 by all three BFA concentrations. Furthermore, several proteins which are preferentially released from the apical surface were found to be released without apparent surface polarity, while several other proteins which were preferentially released from the basolateral surface were unaffected. This study suggests that BFA, at 0.5 microgram/ml, can selectively inhibit protein secretion from the apical surface without affecting total protein secretion. The inhibition of apical secretion results in enhanced protein secretion from the basolateral surface.

4-Chloro-7-nitrobenzofurazan↗
PubMed1991

Apical cell surface expression of rat dipeptidyl peptidase IV in transfected Madin-Darby canine kidney cells.

Dipeptidyl peptidase IV (DPPIV) is a type II membrane glycoprotein that is predominantly localized to the apical plasma membrane in various epithelial cells. In order to understand in more detail the biogenesis and sorting of DPPIV, the cDNA for rat DPPIV was inserted into a mammalian plasmid expression vector so that DPPIV expression was driven by a control region composed of the SV40 early promoter region fused to the enhancer of the Rous sarcoma virus. Madin-Darby canine kidney cells transfected with this construct were found to express the DPPIV protein. In these transfected cells, the majority of DPPIV was present on the apial cell surface. This observation suggests that the information for apical surface localization is inherent in the DPPIV molecule itself and that this sorting information is decipherable in the epithelial cells of a different species. DPPIV is transported efficiently from the endoplasmic reticulum to the Golgi apparatus as assessed by pulse-chase experiments. Furthermore, evidence is presented which suggests that the majority of DPPIV is sorted intracellularly to the apical cell surface. The same protein has, however, been reported to be sorted by an indirect pathway through transcytosis from the basolateral to the apical cell surface in hepatocytes (Bartles, J.R., Feracci, H., M., Stinger, B., and Hubbard, A.L. (1987) J. Cell Biol. 105, 1241-1251). This study suggests that the same protein can take two different pathways in different cell types for its correct apical cell surface localization.

Animals↗
PubMed1991

Calcium and ethanol-induced gastric mucosal damage in rats.

The effects of graded doses of ethanol on stomach mucosal damage and calcium levels were studied in rats. The influence of verapamil and/or calcium chloride on these changes was also investigated. Orally administered ethanol (20, 50 or 80% v/v) markedly decreased gastric glandular tissue calcium and it concentration dependently produced mucosal lesions. Pretreatment with verapamil (2.5 or 5 mg/kg, i.p.) dose dependently lessened glandular wall calcium levels and worsened ethanol-induced mucosal damage. Calcium chloride (50 mg/kg, i.p.) significantly prevented ethanol-induced gastric calcium depletion; it also dose dependently antagonized the damaging effect of ethanol as well as the lesion-intensifying action of verapamil. The findings that verapamil potentiated, whereas calcium chloride prevented, ethanol-induced glandular mucosal damage and tissue calcium changes indeed suggest that altered gastric cell calcium levels could be closely related to the mucosal lesions produced by ethanol in rats.

Animals↗
PubMed1991

Nitrous oxide and internal tamponade during vitrectomy.

We analysed the nitrous oxide composition of the intraocular gas bubble following vitrectomy and fluid-air exchange in 12 patients. Samples were taken under standardised conditions at 20 minutes after completion of the fluid-air exchange. Analysis was conducted by a Medishield MS2 mass spectrometer. The percentage composition of nitrous oxide in the samples varied between 4 and 21% (mean 9%). This influx of nitrous oxide was associated with an increase of intraocular pressure. Pressure rises of greater than 20 mm Hg were not seen owing to venting of gas through the sclerostomies. An inverse relationship was noted between the extent of retinal detachment preoperatively and the amount of nitrous oxide entering the eye. A possible explanation for this relationship is proposed. The importance of nitrous oxide movement is stressed.

Adolescent↗
PubMed1990

Determination of fluoxetine and norfluoxetine by high-performance liquid chromatography.

A high-performance liquid chromatographic assay was developed for a recently introduced atypical antidepressant, fluoxetine and its demethylated metabolite, norfluoxetine. Prior to analysis, aliquots of alkalinized plasma were extracted with n-hexane and isoamyl alcohol, followed by back-extraction with diluted phosphoric acid. These extracts were injected into a 10 microns, reversed-phase C18 column with phosphate and acetonitrile as the mobile phase and detection at 214 nm. Peak height ratios were linearly correlated up to 800 micrograms/l. Acceptable coefficients of variation were demonstrated for both within-run and day-to-day studies. Selected drugs were checked for interference. The assay was used to monitor nine patients receiving 20 to 80 mg of fluoxetine per day. Plasma concentrations of fluoxetine and norfluoxetine ranged from 37 to 301 micrograms/l and 29 to 326 micrograms/l respectively.

Chromatography, High Pressure Liquid↗
PubMed1990

Novel strategies for clinical drug analysis with new column technology in liquid chromatography.

Clinical drug analysis has been the focus of much attention and controversy because of the increase in substance abuse testing of addicts, patients, employees and others, in addition to the rapidly growing fields of therapeutic drug monitoring and clinical toxicology. This review focuses on the latest methodological and technological advances in liquid chromatography (LC) as it is applied to clinical drug analysis. Based on the author's experience, key chromatographic parameters such as carbon load, functionalities and temperature are proposed for the separation of structurally similar metabolites and for resolving chromatographic interferences by other drugs and metabolites. Novel sample preparation for cyclosporine and gradient elution of its metabolites are reviewed, followed by an update on monitoring of 3'-azido-3'-deoxythymidine with emphasis on automated sample preparation. Various approaches of direct sample analysis are advocated for increased efficiency as a result of minimal sample preparation and potential advantages such as decreased exposure of personnel to infectious samples. An update of microbore LC indicates that 2-mm columns may be readily used for clinical paediatric and neonatal analysis without dedicated chromatographs. Potential applications of multidimensional-multimodal chromatography include analyses of a cocaine impurity, anticonvulsants, antidepressants, and five ingredients of a common cold medication.

Body Fluids↗
PubMed1990

Detection of lipid peroxidation in lung and in bronchoalveolar lavage cells and fluid.

Inhalation of toxic materials such as asbestos, silica, 100% oxygen, ozone, or nitrogen dioxide may lead to an increased production of reactive oxygen metabolites which may initiate lipid peroxidation. Measurement of lipid peroxidation in cells and fluid obtained by bronchoalveolar lavage (BAL), as well as in lung tissue, may aid in monitoring the development and extent of pulmonary damage after inhalation of a toxic substance. In this study, we employed a sensitive assay for detection of malondialdehyde (MDA), a breakdown product of lipid peroxidation. By separation of the adduct with thiobarbituric acid, using a reverse phase high pressure liquid chromatographic technique, we accurately and sensitively measured the content of MDA in BAL cells, lavage fluid, and lavaged lung tissue homogenates of rats. The amounts of sample required for detection of MDA were small enough possibly to be applied to use with human specimens; in addition, recovery of added MDA was acceptable with all types of samples. Inclusion of a metal chelator in the preparation of samples appeared necessary to prevent metal-catalyzed propagation of lipid peroxidation during the assay. Overall, the method described here using samples from rats may be applicable to detecting lipid peroxidation in BAL samples from humans.

Animals↗
PubMed1990

The role of serotonin in ethanol-induced gastric glandular damage in rats.

The effects of serotonin (5-HT) or methysergide (a 5-HT antagonist), given intraperitoneally 30 min beforehand, on ethanol-induced mucosal injury and mucosal blood flow were studied in rats. 5-HT itself dose dependently decreased the gastric mucosal mucus content and induced gastric damage in conscious animals. It also worsened ethanol-induced lesion formation but not mucus depletion. Methysergide pretreatment only prevented the former action. In the ex vivo chamber preparation, 5-HT lowered the gastric mucosal blood flow and produced mucosal damage in unconscious animals. It also potentiated ethanol-induced gastric injury and 5-HT release. Methysergide significantly prevented lesion formation and 5-HT release in ethanol-treated rats. Ethanol decreased the gastric mucosal blood flow in the mucosa which had been preincubated with HCl. This depression of gastric mucosal blood flow was further reduced by 5-HT, but was reversed by methysergide. The lesion-potentiating or -protecting actions of 5-HT or methysergide, respectively, suggest that the amine is involved in gastric mucosal damage by ethanol in rats.

Animals↗
PubMed1989

Toxicity to alveolar macrophages in rats following parenteral injection of nickel chloride.

Alveolar macrophages collected by pulmonary lavage from male Fischer-344 rats at intervals (1-72 hr) after NiCl2 injection (62-500 mumol/kg, sc) were tested by several techniques. Within 1 to 4 hr, the macrophages showed morphological and biochemical signs of activation (hypertrophy, ruffled plasma membrane, increased cyclic AMP concentration, and markedly diminished 5'-nucleotidase activity, assayed by concanavalin A inhibition). Functional impairment (reduced phagocytic activity) was first seen at 24 hr; lipid peroxidation (increased malondialdehyde concentration) was not detected until 48 hr. Dose- and time-related effects of NiCl2 on 5'-nucleotidase activity, phagocytic activity, malondialdehyde concentration, and nickel content of alveolar macrophages were observed 24 to 72 hr postinjection. Diminished cell viability occurred only at 72 hr after the highest dosage of NiCl2. In alveolar macrophages from 63NiCl2-treated rats, 63Ni was located primarily in the cytoplasm, based upon liquid scintillation counting and autoradiography; fractionations of macrophage cytosol by gel filtration chromatography showed that 63Ni was bound to several high- and low-molecular-weight constituents. This study demonstrates that sc administration of NiCl2 to rats caused nickel uptake into and activation of alveolar macrophages, followed by reduced phagocytic capacity. The alveolar macrophage was a cellular target for nickel toxicity following parenteral exposure to NiCl2.

Animals↗