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Biomedical subjects

S Zhou

Publications and source records attributed to S Zhou.

At least 451 records · Page 25Linked to original sources

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02.

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.

Cell Differentiation↗

Explant culture of rabbit tracheobronchial epithelium: structure and prostaglandin metabolism.

This study examines the potential usefulness of explant culture of rabbit tracheal epithelium as a model for the study of epithelial function under normal and potentially pathologic conditions. Accordingly, we assessed the structure and prostaglandin E2 (PGE2) release of tracheal epithelial explants obtained from adult pathogen-free rabbits. Epithelial cells attached to their native connective tissue substratum were maintained in culture for 5 days in serum-free medium, under bipolar conditions (air-liquid interface) on a permeable membrane (pore size, 0.2 mm), and nourished from the basolateral surface. At 5 days in culture, scanning and transmission electron microscopy and light microscopy demonstrated a pseudostratified, ciliated columnar epithelium with prominent folds and mucus secretion identical in appearance to the mucosa before culture. On the day of dissection (day 0) and after 4 days in culture (day 4), explants released PGE2 into the medium spontaneously. However, day 4 explants produced 3- to 4-fold greater amounts of PGE2 than day 0 explants. Moreover, day 4 explants demonstrated increased PGE2 release in response to bradykinin, a receptor-dependent agonist, and ionomycin, a calcium ionophore, while day 0 explants did not. Primary tracheal epithelial cell cultures grown to confluence (day 9) on a collagen substrate demonstrated PGE2 responses to bradykinin and ionomycin that qualitatively resembled those of day 4 explants. We conclude that rabbit tracheal explants cultured in vitro under the above conditions maintain cellular differentiation, in situ three-dimensional organization, and PGE2 synthetic pathways over several days in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Ozonolysis products of membrane fatty acids activate eicosanoid metabolism in human airway epithelial cells.

When inhaled, ozone reacts at the airway luminal surface with unsaturated fatty acids contained in the extracellular fluid and plasma membrane to form an aldehyde and hydroxyhydroperoxide. The resulting hydroxyhydroperoxide degrades in aqueous systems to yield a second aldehyde and hydrogen peroxide (H2O2). Previously, we demonstrated that ozone can augment eicosanoid metabolism in bovine airway epithelial cells. To examine structure-activity relationships of ozone-fatty acid degradation products on eicosanoid metabolism in human airway epithelial cells, 3-, 6-, and 9-carbon saturated aldehydes and hydroxyhydroperoxides were synthesized and purified. Eicosanoid metabolism was evaluated by determination of total 3H-activity release from confluent cells previously incubated with [3H]arachidonic acid and by identification of specific metabolites with high performance liquid chromatography and radioimmunoassay. The major metabolites detected were prostaglandin E2, prostaglandin F2 alpha, and 15-hydroxyeicosatetraenoic acid. The 9-carbon aldehyde, nonanal, in contrast to 3- or 6-carbon aldehydes, stimulated release at concentrations > or = 100 microM, suggesting that the stimulatory effect increases with increasing chain length. When tested under identical conditions, the 3-, 6-, and 9-carbon hydroxyhydroperoxides were more potent than the corresponding aldehydes. Again, a greater effect was noted when the chain length was increased. One possible explanation for the increased potency of the hydroxyhydroperoxides over the aldehydes could be due to degradation of the hydroxyhydroperoxide into H2O2 and aldehyde. We consider this an unlikely explanation because responses varied with chain length (although each hydroxyhydroperoxide would produce an equivalent amount of H2O2) and because exposure to H2O2 alone or H2O2 plus hexanal produced a response dissimilar to 1-hydroxy-1-hexanehydroperoxide.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehydes↗

[Dot immunobinding assay for detection of three types of rheumatoid factor and its clinical significance].

We set up a dot immunobinding assay method for determination of IgM-, IgG- and IgA-rheumatoid factor (RF) and report here the results in 86 patients with rheumatoid arthritis (RA). It was found that the positive rates of IgM-, IgG- and IgA-RF were 59.7%, 54.7% and 17.4% respectively. The total positive rate was 72.8%. The positive rate using the Latex method was only 43.0%. Our results reveal that dot immunobinding assay for RF is more sensitive than the Latex method. Furthermore, there was a profound correlation of IgG-RF with active stage and the frequency of extra-artivular manifestations in RA patients.

Adolescent↗

A 9-year follow-up study of the immunogenicity and long-term efficacy of plasma-derived hepatitis B vaccine in high-risk Chinese neonates.

The long-term immunogenicity of and protection provided by a plasma-derived hepatitis B vaccine was determined in a cohort of 98 susceptible Chinese neonates immunized in 1982-1983. Within 24 hours of birth, 89 infants received the first of three 30-micrograms doses of vaccine (at 0, 1, and 6 months) and were subsequently followed up by testing for serological markers of hepatitis B virus and by determining levels of alanine aminotransferase annually for 9 years. After the primary series of vaccine doses, 74 (83%) of 89 subjects developed antibody to hepatitis B surface antigen (anti-HBs). At the 9-year follow-up, 51% of vaccinees still had levels of antibody of > or = 10 mIU/mL, considered the protective level. Seven responders to vaccine (9.4% of 74 evaluable patients) developed antibody to hepatitis B core antigen, and in six of these responders, levels of anti-HBs increased transiently. None of the vaccinees developed chronic carriage of hepatitis B surface antigen or clinical hepatitis. Immunization of high-risk neonates with a plasma-derived hepatitis B vaccine can induce long-lasting protective immunity that can prevent or modify primary infection for at least 9 years. Booster doses are not necessary during this period.

Child↗

[The changes in free radical and prostaglandin metabolism in newborn infants with asphyxia].

Blood plasma thromboxane-B2 (TXB2) and 6-keto-PGF1 alpha levels were determined by radioimmunoassay technique during the first six to sixteen hours of life in 16 newborn infants with severe asphyxia, 18 newborn infants with mild asphyxia and 27 normal term neonates. Plasma lipid peroxidation (LPO) content was measured by TBA-colour-contrast method in 15 infants with severe asphyxia, 17 infants with mild asphyxia and 24 healthy term newborn infants. The results showed that blood plasma LPO, TXB2 and 6-keto-PGF1 alpha levels in infants suffering from severe asphyxia were higher than those in infants with mild asphyxia and normal infants (P < 0.01), but no significant difference was noted between the mild asphyxia group and normal control group (P > 0.01). These suggest that production of free radicals is increased and prostaglandin metabolism is triggered in the infants with severe asphyxia, that cerebral ischemia and hypoxia caused by asphyxia contributes to the augmented production of prostaglandins and free radicals, and that accumulation of free radicals and prostaglandin enhances brain damage and the metabolism of arachidonic acid appears to be an important source of the free radicals in infants with intrauterine asphyxia.

6-Ketoprostaglandin F1 alpha↗

[The effects of phenytoin and carbamazepine on the cognitive function of epileptic patients].

We made a comparative study of the cognitive function of 60 epileptic patients before and after the use of phenytoin (DPH) and carbamazepine (CBZ). Five types of neuropsychological tests were adopted. Although both DPH and CBZ affected cognitive function, the degrees of their effects were not the same. DPH affected cognitive function in a broad field, including concentration, remembrance, psychological motor speed and mental processing, whereas CBZ had only slight effect on perception and motor speed. It is stressed that cognitive function disorder caused by antiepileptic drugs should be early detected by synchronous examinations of the level of antiepileptic drugs and the neuropsychological functions.

Attention↗

Hepatitis B virus capsid particles are assembled from core-protein dimer precursors.

Our studies on the assembly of hepatitis B virus capsids or core particles in Xenopus oocytes have demonstrated that unassembled p21.5 core proteins ("free p21.5") provide a pool of low-molecular-mass precursors for core-particle assembly. Here we have characterized this material. Free p21.5 sedimented through gradients of 3-25% sucrose (wt/vol) as a single protein species of approximately 40 kDa, corresponding to a p21.5 dimer. On nonreducing SDS/polyacrylamide gels, free p21.5 migrated as disulfide-linked p21.5 dimeric species of 35 and 37 kDa. Truncated core proteins lacking most or all of the 36-amino acid protamine region at the p21.5 carboxyl terminus were also found to behave as disulfide-linked dimers with appropriately reduced molecular masses. Our experiments failed to reveal monomeric core proteins or stable intermediates between dimers and capsids along the assembly pathway. We conclude that hepatitis B virus core particles are most likely assembled by aggregating 90 (or possibly 180) disulfide-linked p21.5 dimers. We discuss similarities between the assembly of hepatitis B virus capsids and simple T = 3 plant virus and bacteriophage structures.

Animals↗

Parvovirus B19 replication in human umbilical cord blood cells.

The human parvovirus B19 is now known to be one of the causative agents of nonimmune hydrops fetalis and spontaneous abortions in pregnant women. The presence of the viral proteins and antibodies in fetuses of B19-infected women suggests that the virus can cross the placental barrier. In order to gain an insight into the mechanism of intrauterine fetal infection and the virus-induced hydrops fetalis, we examined whether human umbilical cord blood cells were permissive for B19 replication. Cord blood cells were infected with B19 in vitro, and Southern blot analyses of low M(r) DNA isolated from these cells revealed the presence of the characteristic replicative intermediates of B19 DNA. In addition, B19 genome expression in cord blood cells was detected by Northern blot analysis. Quantitative DNA dot blot analysis of culture supernatants documented complete assembly and release of B19 progeny virions in these cells. The progeny virions were biologically active in secondary infections of normal human bone marrow cells. The human umbilical cord blood cells may be a useful alternative to bone marrow and fetal liver culture systems for further studies on B19 since the need for bone marrow donors is obviated and, unlike fetal tissues, there are no ethical questions associated with the experimental use of cord blood because it is normally discarded. These studies also suggest that the umbilical cord blood may be a site for active replication of parvovirus B19 in vivo and may thus provide a means for transmission of the virus during intrauterine fetal infections.

Cells, Cultured↗

Inhibitory effects of diallyl sulfide on the metabolism and tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mouse lung.

Diallyl sulfide (DAS), a component of garlic oil, has been shown to inhibit tumorigenesis by several chemical carcinogens. Our previous work demonstrated that DAS inhibited the metabolic activation of carcinogenic nitrosamines, including the tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in rat lung and nasal mucosa microsomes. In the present study, the effects of DAS on the tumorigenicity and the metabolism of NNK in A/J mouse lung were examined. Female A/J mice at 7 weeks of age were pretreated with DAS (200 mg/kg body wt in corn oil, p.o) daily for 3 days. Two hours after the final DAS treatment, the mice were either given a single dose of NNK (2 mg/mouse, i.p.) and kept for an additional 16 weeks for determining the production of pulmonary tumors, or were killed immediately so as to measure the microsomal activity in metabolizing NNK. In comparison to the vehicle control group, DAS pretreatment significantly decreased the incidence of NNK-induced lung tumors (37.9 versus 100%) and the tumor multiplicity (0.6 versus 7.2 tumors/mouse). In pulmonary metabolism of NNK, DAS pretreatment reduced the rates of formation of keto aldehyde, keto alcohol, NNAL-N-oxide, and NNK-N-oxide by 70-90%. In addition, the formation of NNK oxidative metabolites from NNK in the liver microsomes from DAS-pretreated mice was remarkably reduced. DAS also inhibited the metabolism of NNK in mouse lung microsomes in vitro. These results demonstrate that DAS is an effective chemopreventive agent against NNK-induced lung tumorigenesis, probably by inhibiting the metabolic activation of NNK.

Allyl Compounds↗

Characterization of hepatitis B virus capsid particle assembly in Xenopus oocytes.

Little is known about the assembly of the 28-nm nucleocapsid or core particle of hepatitis B virus. Here we show that this assembly process can be reconstituted in Xenopus oocytes injected with a synthetic mRNA encoding the hepatitis B virus capsid protein (p21.5). Injected oocytes produce both a nonparticulate p21.5 species (free p21.5) and capsid particles. We describe rapid and simple methods for fractionating these species on a small scale either with step gradients of 10 to 60% (wt/vol) sucrose or by centrifugation to pellet the particles, and we characterize the oocyte core particles. Free p21.5 exhibits chemical and physical properties distinctly different from those of particles. Free p21.5 is partially cleaved by proteinase K, whereas core particles are almost completely resistant to cleavage. This suggests that the carboxyl-terminal protamine region, the main target for proteases within p21.5, is exposed in free p21.5 but faces the interior of the p21.5 core particle. Finally, pulse-chase experiments demonstrated that free p21.5 can be chased almost quantitatively into core particles, establishing that free p21.5 is fully competent to form particles and represents an assembly intermediate on the pathway for core particle formation. However, core particle assembly appears very dependent on p21.5 concentration and is rapidly compromised if the p21.5 concentration is lowered. The advantages of oocytes for studying assembly are discussed.

Animals↗

RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication.

The hepatitis B virus capsid or core protein (p21.5) binds nucleic acid through a carboxy-terminal protamine region that contains nucleic acid-binding motifs organized into four repeats (I to IV). Using carboxy-terminally truncated proteins expressed in Escherichia coli, we detected both RNA- and DNA-binding activities within the repeats. RNA-binding and packaging activity, assessed by resolving purified E. coli capsids on agarose gels and disclosing their RNA content with ethidium bromide, required only the proximal repeat I (RRRDRGRS). Strikingly, a mutant in which four Arg residues replaced repeat I was competent to package RNA, demonstrating that Arg residues drive RNA binding. In contrast, probing immobilized core proteins with 32P-nucleic acid revealed an activity which (i) required more of the protamine region (repeats I and II), (ii) appeared to bind DNA better than RNA, and (iii) was apparently modulated by phosphorylation in p21.5 derived from Xenopus oocytes. Deletion analysis suggested that this activity may depend on an SPXX-type DNA-binding motif in repeat II. Similar motifs found in repeats III and IV may also function to bind DNA. On the basis of these observations, together with a reinterpretation of recent studies showing that capsid protein mutants cause defects in viral genome replication, we propose a model suggesting that hepadnavirus capsid proteins participate directly in the intracapsid reverse transcription of RNA into DNA. In this model, repeat I binds RNA whereas the distal repeats are progressively recruited to bind elongating DNA strands. The latter motifs may be required for replication to be energetically feasible.

Amino Acid Sequence↗

Cys residues of the hepatitis B virus capsid protein are not essential for the assembly of viral core particles but can influence their stability.

In the spherical capsid of hepatitis B virus (HBV), intermolecular disulfide bonds cross-link the approximately 180 p21.5 capsid protein subunits into a stable lattice. In this study, we used mutant capsid proteins to investigate the role that disulfide bonds and the four p21.5 Cys residues (positions 48, 61, 107, and 185) play in capsid assembly and/or stabilization. p21.5 Cys residues were either replaced by Ala or removed (Cys-185) by carboxyl-terminal truncation, creating Cys-minus mutants which were expressed in Xenopus oocytes via microinjected synthetic mRNAs. Fractionation of radiolabeled oocyte extracts on 10 to 60% sucrose gradients revealed that Cys-minus core proteins resolved into the nonparticulate and capsid forms seen for wild-type p21.5. On 5 to 30% sucrose gradients, nonparticulate Cys-minus core proteins sedimented as dimers of approximately 40 kDa. We conclude that Cys residues and disulfides are not required for the assembly of either HBV capsids or the dimers that provide the precursors for capsid assembly. Since assembly presumably demands an appropriate p21.5 tertiary structure, it is unlikely that Cys residues are required for proper p21.5 folding. However, Cys residues stabilize isolated p21.5 structures, as evidenced by the marked reduction in stability of Cys-minus dimers and capsids (i) in nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and (ii) upon protease digestion. We discuss these results in the context of the HBV life cycle and the role of Cys residues in other proteins.

Alanine↗

A technique to harvest viable tracheobronchial epithelial cells from living human donors.

The ability to obtain airway epithelial cells from the lower respiratory tract in living human donors will facilitate study of the biologic properties of these cells. We report our experience harvesting tracheobronchial epithelial cells from living human donors by brushing the mucosal surface of the trachea and mainstem bronchi. Cells were obtained on 21 occasions from 18 healthy adult subjects under direct vision with a brush-tipped catheter during fiberoptic bronchoscopy. The average number of cells harvested per subject was 14 +/- 2 x 10(6), and cell viability determined by trypan blue exclusion averaged 36 +/- 4%. Of note, cell viability was significantly enhanced when lidocaine was confined to the nares. Lidocaine was also observed to diminish cell viability in vitro in a dose-dependent fashion. Morphologic and staining properties were used to classify harvested cells into the three major cell types present in the mucosa (i.e., ciliated, secretory, and basal cells). All three subtypes were obtained. The percentage of ciliated, secretory, and basal-like cells was 24 +/- 2%, 11 +/- 1%, 29 +/- 1%, respectively, while the remaining 36% were difficult to type. In one subject in whom brushing was performed on three occasions over a 7-wk period, the percentage of each of the three subtypes was similar across procedures. Harvested cells could be successfully placed in primary culture with a plating efficiency of 50 to 60% and could be subcultured for up to seven passages. Acutely dissociated cells could be used to study the beta-adrenergic receptor adenylyl cyclase system since they produced cAMP in response to isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗