Biomedical subjects

H Peng

Publications and source records attributed to H Peng.

At least 127 records · Page 7Linked to original sources

PubMed1997

Enhancement or inhibition of HIV-1 replication by intracellular expression of sense or antisense RNA targeted at different intermediates of reverse transcription.

OBJECTIVES: To construct retroviral vectors expressing sense or antisense RNA targeted at HIV reverse transcription intermediates, and to test the anti-HIV properties of these constructs in transduced T cells. DESIGN: Five double-copy retroviral vectors were constructed, in which the expression of the sense or antisense RNA corresponding to HIV minus- or plus-strand strong-stop DNA was driven by the human tRNA(met) promoter. METHOD: The templates for the sense or antisense RNA were polymerase chain reaction-cloned from HIV pNL43 into a murine leukaemia virus-based vector and corresponding defective virions were packaged in PA317 cells. Human Jurkat T cells transduced with these vectors were challenged with HIV and monitored for viral RNA, viral DNA and p24 production for 23 weeks. RESULTS: Intracellular expression of HIV sense RU5 sequences (RNA complementary to minus-strand strong-stop DNA) enhanced HIV replication in T cells. Expression of HIV sense or antisense U3RU5 sequences (identical or complementary to plus-strand strong-stop DNA) conferred long-term inhibition of HIV replication, despite continuous presence of viral challenge in the transduced cell cultures. CONCLUSION: Plus-strand strong-stop DNA as an intermediate in the early process of viral reverse transcription can be explored as an additional target for anti-HIV gene therapy.

Cell Line↗

PubMed1997

Suppression by platelet factor 4 of the myogenic activity of basic fibroblast growth factor.

The effect of platelet factor 4 (PF4) on myoblast cultures with or without basic fibroblast growth factor (bFGF) or other growth factors was investigated in the present in vitro experiments, with reference to bFGF binding to myoblast membrane fraction. When PF4 was added to the culture medium 1 day after myoblast cultivation, the nuclei of both myoblasts and myotubes were markedly reduced in number in a dose-dependent manner, whereas the inhibitory effect of PF4 on myoblast development was not observed when PF4 was added to the culture medium 3, 7, or 14 days after myoblast cultivation. In contrast, bFGF significantly increased the numbers of myoblast and myotube nuclei. When bFGF and PF4 were simultaneously added to the culture medium, PF4 abolished the facilitatory effects of bFGF on myogenesis. The real-time biospecific interaction analysis (BLA) core system showed that the myoblast membrane fraction at 1 day after cultivation contains bFGF-binding elements which are blocked by PF4 in a dose-dependent manner. Moreover, [126I]-bFGF binding experiments indicated the existence of both high and low affinity binding sites on myoblast membranes, although the high affinity binding sites decreased in number and the dissociation constant increased in value as the culture period was prolonged. Among the six other growth factors examined, acidic fibroblast growth factor and platelet-derived growth factor-BB stimulated myogenesis, and their effects were blocked by PF4 treatment. These findings suggest that: 1) PF4 inhibits myoblast proliferation and myotube formation only for a limited initial period of cultivation, possibly because of the time-dependent down-regulation of high affinity bFGF receptors: and 2) PF4 may be used as a tool to investigate the function of endogenous heparin-binding growth factors upregulated transiently at a certain developmental stage or in case of tissue damage and repair, even though it is not monospecific to bFGF.

Animals↗

PubMed1997

[An inquiry on the normal value of Chinese' ENoG and the influencing factors].

The normal value of the ENoG of 172 facial nerves from 86 Chinese has been estimated with the method of regulating the strength of stimulating current. It showed that the CAPs of the Chinese' ENoG were of normal distribution. The mean of ampitudes was 3.94 +/- 1.02 mV. The coefficient of variation was 25.8%; The mean of latencies was 3.18 +/- 0.42 ms. The coefficient of variation was 13.2%. The variance from age to age, sex to sex and side to side were not significant. It was concluded that the interpretation of ENoG should be based on the fact that the CAP obtained from one certain subject was symmetrical between the opposite sides. The effecting factors which influenced the ENoG of normal facial nerve were also discussed in this paper.

Action Potentials↗

PubMed1997

Immersion-histo polymerase chain reaction: a practical tool for visualization of single-copy genes in tissue sections.

A method, immersion-histo polymerase chain reaction (IH-PCR), was developed for visualization of single-copy DNA sequences in cells in paraffin-embedded tissue sections. Sections were mounted on coverslips, cut into small pieces, and immersed in reaction mixtures in micro-tubes for specific DNA amplification using a conventional thermal cycler. This was followed by in situ hybridization, in micro-tubes, with PCR-generated, digoxigenin-labeled probes. Epstein-Barr virus, chromosomal translocations, and rearranged immunoglobulin genes were clearly demonstrated in individual cells in human lymphomas. The use of a standard thermal cycler, the ease of probe preparation, and the high specificity and sensitivity of target localization make IH-PCR a practical and powerful tool for the study of genetic events within individual cells.

DNA Primers↗

PubMed1997

[The value of detecting CVB-IgM antibodies in diagnosing viral myocarditis of children].

By using the indirect immunofluorescence assay and the tissue cells infected with coxackie virus Group B as antigen, the authors detected the antibodies type IgM to coxsackie virus Group B (CVB-IgM) of the sera of 105 children with viral myocarditis (VMC), 59 children with other diseases (COD) and 67 healthy children (HC). The results showed that in VMC, the genometric mean titre (GMT) of CVB-IgM in female (1:16.58) was higher than that in male (1:9.28, P < 0.05); not only in female, but also in male the GMT of VMC was higher than that of HC; also the GMT in female of VMC was higher than the GMT in female of COD, but no considerable difference existed between male of VMC and COD. Without sex distinction the GMT of HC was 0.289, its standard variance was 0.9335, the upper limit titre of normal range was 1:8.6. The authors took 1:10 as the positive criterion, the sensitivity of CVB-IgM in diagnosing VMC was 79.05%, the specialities of differentiating VMC from COD and HC were 42.3% and 91.04%, the consistancy rates of diagnosing VMC from COD and HC were 65.85% and 83.72% respectively.

Antibodies, Viral↗

PubMed1996

Long-term protection against HIV-1 infection conferred by tat or rev antisense RNA was affected by the design of the retroviral vector.

We have constructed a series of retroviral vectors in which the expression of antisense RNA targeted at the full length coding sequence of HIV-1 tat or rev was driven by three different promoters and in the context of double-copy or single-copy vectors. Jurkat cells transduced by these vectors were shown to express the expected tat or rev antisense RNA without alteration in cell proliferation or surface CD4 expression. After challenge with HIV, four patterns of protection were identified, with the degree of protection being determined primarily by the design of the expression system. In those patterns showing long-term complete protection, we could detect no HIV p24 in the culture supernatants or in the cells, and no HIV RNA or HIV proviral DNA (by PCR), during a 23-week follow-up. Experiments designed to rescue any live virus still formed in the culture after 20 weeks' challenge demonstrated that, with some constructs, infectious virus could no longer be isolated, while with other constructs, only a low level of infectious virus was still being formed and providing a continuing virus challenge, although all other markers of infection remained undetectable. Our results demonstrated that antisense RNA expression driven by tRNA promoter in the context of a double-copy vector conferred better long-term protection against HIV infection compared to that driven by HIV LTR or MLV LTR promoters, and that the optimized vectors may be useful in developing a gene therapy against HIV-1 infection and AIDS.

Base Sequence↗

PubMed1996

Preliminary crystallographic analysis of the ParE subunit of Escherichia coli topoisomerase IV.

The ParE subunit of Escherichia coli topoisomerase IV has been crystallized in the presence of the non-hydrolyzable ATP analogue, 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP). The crystals are of the orthorhombic space group, P2(1)2(1)2(1), with unit-cell dimensions a = 92.6, b = 119.1, c = 135.3 A. Data have been collected to 3.5 A resolution from frozen native crystals. Self-rotation function analysis of these data indicate the position of a molecular twofold axis. Higher resolution native data are being collected and a derivative search is underway.

Journal Article↗

PubMed1996

Replication error phenotype and p53 gene mutation in lymphomas of mucosa-associated lymphoid tissue.

Low grade mucosa-associated lymphoid tissue (MALT) lymphomas commonly arise from a background of chronic inflammatory lesions and can transform into high grade tumors at a late stage. Because chronic inflammation is closely associated with genetic instability, which is one of the mechanisms leading to activation of oncogenes and inactivation of tumor suppressor genes, it is possible that genetic instability plays an important role in MALT lymphomagenesis. In this study, we have examined the frequency of replication error (RER+) phenotype, a newly defined manifestation of genetic instability, and its relationship to p53 mutations in 40 MALT lymphomas (16 high grade and 24 low grade). RER+ phenotype was detected in 21/40 (52.5%) MALT lymphomas (12/24, 50% in low grade; 9/16, 56.2% in high grade). Five of seven reactive lymphoid infiltrates adjacent to tumors also showed one microsatellite alteration, four of which were identified in the corresponding lymphoma lesions in the same patient. In five RER+ high grade lymphomas with low grade lesions, homogeneous and heterogeneous microsatellite alterations were observed between the two components. The same 40 cases were investigated for p53 gene mutations at exons 5 to 8 by PCR-SSCP and direct sequencing. p53 point mutations were found in 11 (27.5%) of the 40 cases. These mutations were statistically related to RER+ phenotype (P < 0.05). Our results demonstrate that the RER+ phenotype is a common genetic feature of MALT lymphomas. Genetic instability occurs throughout the spectrum of the lymphoma development and may be related to the accumulation of genetic aberrations such as p53 mutations. The observation of identical microsatellite alterations between the adjacent lymphoid infiltrates and their corresponding lymphomas provides genetic evidence for evolutionary link of the two lesions. The homogeneous and heterogeneous microsatellite alterations observed between low and high grade components indicate their clonal lineage and genetic diversity.

Base Sequence↗

PubMed1996

Ongoing mutation in MALT lymphoma immunoglobulin gene suggests that antigen stimulation plays a role in the clonal expansion.

Indirect antigenic stimulation by H. pylori-specific T cells is implicated in the development of low-grade gastric lymphoma of mucosa-associated lymphoid tissue (MALT), however, the role of direct antigen stimulation is unknown. To study the role of direct antigen stimulation in MALT lymphomagenesis and its relationship with the pathogenesis of distinct pathological lesions, which represent different stages of the tumour progression, we cloned and sequenced the rearranged immunoglobulin (Ig) heavy chain gene in three low-grade (two from the lung, one from the stomach) and one high-grade (from the stomach) cases. In the low-grade gastric case, we studied the Ig sequence in primary as well as its disseminated and recurrent tumours. In the high-grade gastric case, we analysed the Ig sequence in tumour cell populations microdissected from the residual diffuse low-grade lesions, diffuse high-grade areas from follicles colonized by high-grade blasts. Compared with the published germline sequences, the heavy chain variable (VH) genes of three MALT lymphomas, in which the putative germline was identified, contained frequent somatic mutations, showing a much higher ratio of replacement/silent mutations in the complementarity determining regions (CDRs) than the framework regions (FRs). Ongoing mutation as indicated by intraclonal variation of the Ig sequence clearly existed in low-grade tumour including its dissemination and recurrence, but was not evident in high-grade tumour cell populations including those microdissected from independent colonized follicles. In addition, the germlines of VH genes used by the three MALT lymphomas are frequently found in autoreactive antibodies. Our results suggest that MALT lymphoma derives from postgerminal centre memory B cells, possibly autoreactive B cell clones, and that direct antigen stimulation may play an important role in the clonal expansion of low-grade MALT lymphoma.

Animals↗

PubMed1996

Persistent c-fos expression in the brains of mice with chronic social stress.

The present study was conducted to demonstrate immunohistochemically, the sites of c-fos protein expression in the brains of mice subjected to acute and chronic social defeat stress. To induce social stress, mice were placed in situations of species-specific intermale aggression either only once or five times at 24 h intervals. Two hours after the single or fifth defeat stress, many c-fos immunoreactive neurons were observed in a variety of brain regions including the limbic system and sensory relay nuclei. The c-fos immunoreactive neurons in the brains of acute defeat mice decreased in number with time and the c-fos staining pattern of acute defeat mice became indistinguishable from that of normal control mice by 24 h after the single defeat stress. In contrast, chronic defeat stress induced persistent c-fos expression in the forebrain and brainstem even 24 h after the fifth defeat stress. In the forebrain of chronic defeat mice, the olfactory bulb, cingulate cortex, hippocampus, entire hypothalamus, septal nuclei and the amygdaloid complex, except for the central nucleus, were labeled intensely with c-fos antiserum. In the lower brainstem, nerve cells with c-fos immunoreactivity were seen mainly in ascending and descending sensory relay nuclei relevant to auditory and vestibular transmission, epicritic sensation (gracile and external cuneate nuclei), pain inhibition (central gray and raphe nuclei), and viscerosensory transmission (solitary tract nucleus). The differences in c-fos expression among the normal control, acute and chronic defeat mice were evaluated by an enumeration of the immunopositive neurons within each brain nucleus examined, and they were confirmed subsequently by statistical analysis. There was little c-fos expression in the nucleus putamen, lateral, ventral and posterior thalamic nuclei, pretectal nuclei, medial geniculate nucleus, red nucleus, substantia nigra, cerebellum, spinal cord, or cranial nerve nuclei. These findings suggest that chronic but not acute defeat stress causes persistent c-fos expression in more widespread brain regions than do any other stresses so far investigated. The present study may shed light on the central mechanisms by which behavioral abnormalities and/or chronic sociopsychological stress leads to the occurrence of abnormal behavior and/or psychosomatic disorders in experimental animals and humans.

Animals↗

PubMed1995

The accumulation of p53 abnormalities is associated with progression of mucosa-associated lymphoid tissue lymphoma.

The genetic mechanisms underlying the genesis of low-grade mucosa-associated lymphoid tissue (MALT) lymphomas and their transformation into high-grade lymphoma are poorly understood. p53 inactivation, commonly caused by mutation and allele loss, has been shown to play an important role in the early development and/or the late disease progression of many human tumors including lymphoid malignancies and, thus, may also be important in MALT lymphomagenesis. We examined 75 cases (48 low grade and 27 high grade) of MALT lymphoma for p53 allele loss and mutation as well as protein accumulation. DNA samples prepared from microdissected cell populations were used for the detection of p53 gene abnormalities. Loss of heterozygosity (LOH) of the gene was detected by polymerase chain reaction-based analysis of p53 CA repeat polymorphism, whereas p53 mutation was studied by single-strand conformation polymorphism analysis and direct sequencing. p53 expression was assessed by immunostaining with CM1 polyclonal antibody. p53 allele loss and mutation, which resulted in the alteration in the amino acid sequence, were found in both low-grade (LOH, 3 of 44 [6.8%]; mutation, 9 of 48 [18.8%]) and high-grade (LOH, 6 of 21 [28.6%]; mutation, 9 of 27 [33.3%]) MALT lymphomas, particularly in the latter group. p53 staining was not observed in any low-grade tumors but in 6 high-grade cases that harbored missense mutations. There were also differences in the extent of p53 abnormalities, between low- and high-grade tumors. Of the 11 low-grade tumors showing p53 abnormalities, only 1 tumor showed the concomitance of p53 mutation and allele loss, whereas in high-grade tumors, 6 of 9 affected cases displayed both p53 mutation and allele loss. Our results suggest that p53 partial inactivation may play an important role in the development of low-grade MALT lymphomas, whereas complete inactivation may be associated with high-grade transformation.

Alleles↗

PubMed1995

The interaction of Escherichia coli topoisomerase IV with DNA.

The two type II topoisomerases in Escherichia coli, DNA gyrase and topoisomerase (Topo) IV, share considerable amino acid sequence similarity, yet they have distinctive topoisomerization activities. Only DNA gyrase can supercoil relaxed DNA, whereas during oriC DNA replication in vitro, only Topo IV can support the final stages of replication, processing of the late intermediate and decatenation of the daughter molecules. In order to develop an understanding for the basis of the differential activities of these two enzymes, we have initiated a characterization of Topo IV binding to DNA. We find that unlike gyrase, Topo IV neither constrains DNA in a positive supercoil when it binds nor protects a 150-base pair region of DNA from digestion with micro-coccal nuclease. Consistent with this, DNase I footprinting experiments showed that Topo IV protected a 34-base pair region roughly centered about the topoisomerase-induced cleavage site. In addition, Topo IV preferentially bound supercoiled rather than relaxed DNA. Thus, the DNA binding characteristics of Topo IV are more akin to those of the type II eukaryotic enzymes rather than those of its prokaryotic partner.

Base Sequence↗

PubMed1995

Regulation of Na(+)-coupled glucose transport in LLC-PK1 cells. Message stabilization induced by cyclic AMP elevation is accompanied by binding of a M(r) = 48,000 protein to a uridine-rich domain in the 3'-untranslated region.

In an exploration of the molecular basis of cyclic AMP-induced stabilization of Na+/glucose cotransporter mRNA (SGLT1 isoform) accompanying cell differentiation in the pig kidney cell line LLC-PK1, we have identified a 48-kDa cytoplasmic protein factor, designated SG-URBP, which specifically binds a 120-nucleotide sequence within the 3'-untranslated region of the SGLT1 message. A 46-nucleotide uridine-rich element within this region appears necessary for specific binding, and the presence of the 3'-untranslated region is necessary for message stabilization by cyclic AMP. The binding activity of SG-URBP is up-regulated after cyclic AMP elevation and protein kinase A activation, whereas protein dephosphorylation either in vivo or in vitro is associated with loss of binding activity. The increase in SG-URBP binding activity correlates with an increase in the half-life of the SGLT1 message, suggesting a cause and effect relationship.

Animals↗

PubMed1995

Post-transcriptional regulation of Na+/glucose cotransporter (SGTL1) gene expression in LLC-PK1 cells. Increased message stability after cyclic AMP elevation or differentiation inducer treatment.

We have further investigated the molecular basis of increased differentiation-regulated expression of SGTL1, a Na+/glucose cotransporter, in the renal epithelial cell line LLC-PK1. Treatment of confluent monolayers either with the differentiation inducer hexamethylene bisacetamide (HMBA) or with cyclic AMP-elevating agents promoted increased levels of the SGLT1 mRNA, the immunodetectable 75-kDa cotransporter subunit, and the transport activity. Two molecular species of SGLT1 mRNA (2.2 and 3.9 kilobases (kb)) are transcribed from the same gene in LLC-PK1 cells and differ only in the length of the 3'-untranslated region. The larger transcript is less stable (t1/2 = 2 h) than the smaller one (t1/2 = 10 h) in control, confluent monolayers. The 3.9-kb species was stabilized from degradation after either cyclic AMP elevation (t1/2 = 14 h) or HMBA addition (t1/2 = 8 h), with negligible effects on the stability of the 2.2-kb species (t1/2 = 11 h). Inhibition of translation by cycloheximide resulted in a 10-fold increase in the t1/2 of the 3.9-kb transcript and a 2-fold increase in that of the 2.2-kb species in control monolayers. Our results demonstrate that post-transcriptional regulation of message stability plays a major role in differentiation-dependent SGTL1 expression promoted by either HMBA or cyclic AMP.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Single cell transcript analysis of human immunodeficiency virus gene expression in the transition from latent to productive infection.

In the lymph nodes of individuals infected with human immunodeficiency virus (HIV), there is evidence that points to three kinds of virus-cell relationships. Virions may be associated with CD4+ lymphocytes that are actively producing virus or may be bound at the surfaces of follicular dendritic cells like other antigens. HIV is also harbored in CD4+ lymphocytes and monocytes/macrophages in a latent form as transcriptionally silenced provirus. To ultimately investigate in vivo these and other HIV-cell interactions that play such critical roles in the persistence of virus, immune dysregulation, and depletion, we have developed an in situ hybridization method that discriminates multiply spliced from singly or unspliced viral transcripts. In this report we describe the method and the results obtained with it in an analysis of the switch from latent to productive infection of chronically infected T lymphocytes in culture. We found with this single-cell technique that there are two subpopulations in the culture, a minor one of productively infected cells and a major one of latently infected cells in which only low levels of viral transcripts terminated close to the 5' end of the viral genome were detected. Shortly after activation of viral gene expression with phorbol ester, transcripts encoding Tat and Rev increase in abundancy in individual latently infected cells and this is followed by increases in and cytoplasmic export of singly or unspliced mRNAs encoding structural proteins. These studies provide insights into the regulation of HIV gene expression from a single-cell perspective and, from that perspective, transcript profiles of productively infected cells as a frame of reference for defining HIV-cell relationships in individual cells in tissue sections.

Gene Expression Regulation, Viral↗

PubMed1995

High-resolution SSCP analysis using polyacrylamide agarose composite gel and a background-free silver staining method.

We describe here an improved methodology for single-strand conformation polymorphism (SSCP) analysis of PCR products. The method utilizes a polyacrylamide agarose composite gel and background-free silver staining. In comparison with conventional pure polyacrylamide gel for PCR-SSCP analysis, the composite gels have a much greater mechanical strength and improved resolution. The background staining commonly seen in many silver staining protocols has been eliminated by incorporation of thiosulfate, which can prevent nonspecific deposits of silver salts. As shown in our titration tests, the composite gel and background-free silver staining together have allowed clear identification of point mutations in samples containing as little as 5% of the target sequences, the same sensitivity achieved by radioactive labeling methods.

DNA↗

PubMed1994

Identification of a domain of Escherichia coli primase required for functional interaction with the DnaB helicase at the replication fork.

Primase plays a key role in governing the sequence of events required on the lagging strand during a cycle of Okazaki fragment synthesis. To begin to probe the protein-protein interactions necessary for primase function at the replication fork, we have used limited trypsinolysis to separate primase into two functional domains, an N-terminal domain of 49 kDa (p49) and a carboxyl-terminal domain of 16 kDa (p16). p49 retained primase activity in replication assays that utilized bacteriophage M13 DNA carrying the bacteriophage G4 origin of DNA replication as the template, but was inactive during general priming or the conversion of phi X174 single-stranded circular (ss(c))-DNA to the replicative form (RF) and could not support lagging-strand DNA synthesis at replication forks reconstituted with the phi X-type primosomal proteins and the DNA polymerase III holoenzyme. On the other hand, p16 inhibited those replication reactions that included the replication fork helicase, DnaB (general priming, phi X174 ss(c)-->RF, and at the replication fork), but had no effect on those that did not (M13Gori ss(c)-->RF). These results demonstrate that p49 defines a domain of primase required for catalytic activity, that p16 defines a domain of primase required for functional interaction with DnaB, and that it is a protein-protein interaction with DnaB that attracts primase to the replication fork.

Bacterial Proteins↗

PubMed1994

Regulation of Na+/glucose cotransporter (SGLT1) mRNA in LLC-PK1 cells.

The porcine kidney epithelial cell line LLC-PK1 expresses a sodium-coupled glucose cotransporter (SGLT1) together with other differentiation markers of renal proximal tubule such as trehalase and gamma-glutamyltranspeptidase. Expression is regulated by cell density and exogenous differentiation inducers such as hexamethylene bisacetamide (HMBA). Northern blot and PCR analysis of clonal cell populations indicated SGLT1 mRNA was not detectable in subconfluent cultures, but 2.2 and 3.9 kb SGLT1 mRNA species appeared after cell confluence, accompanying expression of the transport activity. SGLT1 mRNA levels were significantly increased after treatment of confluent cultures with HMBA, paralleling increases in the transport activity and immunodetectable 75 kD cotransporter subunit. SGLT1 mRNA was also increased after treatment of cultures with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), an inducer of Na+/glucose cotransport activity. The 3.9 kb SGLT1 transcript showed the largest increase after either HMBA or IBMX treatment. HMBA treatment also resulted in increased mRNA levels of two other differentiation markers--trehalase and gamma-glutamyltranspeptidase. By contrast, trehalase and gamma-glutamyltranspeptidase mRNA levels were not increased by IBMX. Regulation of Na+/glucose symporter expression by either cell density, cyclic AMP elevation, or differentiation inducer treatment occurs, at least in part, at the level of SGLT1 mRNA and can be dissociated from regulation of other differentiation markers.

1-Methyl-3-isobutylxanthine↗