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Biomedical subjects

X Peng

Publications and source records attributed to X Peng.

At least 19 recordsLinked to original sources

Quantitative description of analyte migration behavior based on dynamic complexation in capillary electrophoresis with one or more additives.

A comprehensive theory is proposed to describe the migration behavior of analytes in capillary electrophoresis (CE) when one or more additives are present in the buffer solution. This theory amalgamates and extends the previous work done by others. The capacity factor (k') in this theory is defined as the product of the equilibrium constant and the additive concentration, thus, k' changes linearly with additive concentration. The net electrophoretic mobility of an analyte is a function of k', therefore, it can be changed by varying the additive concentration. Three parameters are needed to predict the mobility of an analyte in a one-additive CE system: the mobility of the free analyte, the mobility of the complex, and the equilibrium constant for the analyte-additive interaction (which determines the fraction of the free analyte at different additive concentrations). When additives are used, the change in viscosity obscures this relationship, therefore, a viscosity correction factor is required to convert all mobilities to an ideal state where the viscosity remains constant. The migration behavior of an analyte in a solution with multiple additives can be predicted and controlled, once the equilibrium constants of the interactions between the analyte and each of the additives are obtained separately. beta-Cyclodextrin and hydroxypropyl-beta-cyclodextrin are used as additives and the migration behavior of phenol, p-nitrophenol, and benzoic acid are studied as a model system to verify this theory. When the necessary viscosity correction factor is included, the net electrophoretic mobilities of the analytes obtained from experimental results agree with the values predicted by the theory based on dynamic complexation. Although only experiments with one and two additives were carried out to verify the theory, the equations apply to situations when more than two additives are used. The relationship between the theories of electrophoresis and chromatography is clarified.

2-Hydroxypropyl-beta-cyclodextrin

Interleukin 12 and B7/CD28 interaction synergistically upregulate interleukin 10 production by human T cells.

Interleukin 12 (IL-12) is a heterodimeric cytokine which promotes the development of Th1 cells and their interferon gamma (IFN-gamma) production after TCR/CD3 triggering. Previous reports indicate that IL-12 synergizes with accessory signalling through B7/CD28 interaction in inducing proliferation and IFN-gamma production by human T cells. In this study, we investigated the capacity of IL-12 to modify cytokine synthesis by freshly purified human peripheral blood T cells stimulated with anti-CD3 as the primary signal and with CD80 on transfected mouse cells as an accessory signal. Our data demonstrate that IL-12 indeed synergizes with B7/CD28 interaction, not only in inducing IFN-gamma production, but also in enhancing IL-10 synthesis in a dose-dependent manner. In contrast, IL-4 and IL-5 production were slightly inhibited by IL-12. The effect of IL-12 on the secretion of IL-10 was confirmed by stimulating T cells in the absence of accessory cells with immobilized anti-CD3 mAb and soluble anti-CD28 mAb. CD80 and IL-12 mainly costimulated CD4+CD45RO+ T cells but not CD8+ or CD45RA+ T cells to produce IL-10. Cyclosporin A (CsA) partially inhibited, and a neutralizing anti-IL-2 mAb in combination with anti-IL-2R mAbs (anti-Tac and Mik beta1) strongly reduced IL-10 production. On the other hand, IL-12 did not affect IL-2 production. The data thus suggest a model in which optimal IL-10 production by stimulated peripheral blood T cells results from the co-operation of IL-12, B7/CD28 interaction and the ensuing IL-2 activity.

Adult

Life events and anxiety in Chinese medical students.

The authors studied a sample of 537 Chinese medical students aged 15-21 years using Zung's Self-Rating Anxiety Scale (SAS) and a life events checklist. The results showed that test pressure, less free time, peer competition, failure in a test, and financial problems were the most common stressful experiences for medical students during the previous 12 months. Social and personal problems were rated the highest on scores of perceived stress. Of all students, 12.5% scored over the cut-off point on the SAS previously established to indicate risk of psychiatric disorder. Using a stepwise regression analysis, it was shown that poor health status, little physical exercise, financial problems in the family, test pressure, conflict with classmates, the personality trait of introversion, getting up late in the morning, and freshman status were independently associated with the presence of anxiety.

Adolescent

Chronic nicotine treatment up-regulates alpha3 and alpha7 acetylcholine receptor subtypes expressed by the human neuroblastoma cell line SH-SY5Y.

Chronic exposure to nicotine has been reported to increase the number of nicotinic acetylcholine receptors (AChRs) in brain. The mechanism of up-regulation for the alpha4beta2 AChR subtype, which accounts for the majority of high affinity nicotine binding in mammalian brain, has previously been shown to involve a decrease in the rate of alpha4beta2 AChR turnover. Here, we report an investigation of the extent and mechanism of nicotine-induced up-regulation of alpha3 AChRs and alpha7 AChR subtypes expressed in the human neuroblastoma cell line SH-SY5Y. Up-regulation of human alpha3 AChRs and alpha7 AChRs, unlike alpha4beta2 AChRs, requires much higher nicotine concentrations than are encountered in smokers; the extent of increase of surface AChRs is much less; and the mechanisms of up-regulation are different than with alpha4beta2 AChRs. The mechanisms of up-regulation may be different for alpha3 AChRs or alpha7 AChRs. Chronic treatment with nicotine or carbamylcholine, but not d-tubocurarine, mecamylamine, or dihydro-beta-erythroidine, induced a 500-600% increase in the number of alpha3 AChRs but only a 30% increase in alpha7 AChRs. Chronic nicotine treatment did not increase affinity for nicotine or increase the amount of RNA for alpha3 or alpha7 subunits. The effect of nicotine on up-regulation of alpha7 AChRs was partially blocked by either d-tubocurarine or mecamylamine. The effect of nicotine treatment on the number of alpha3 AChRs was only slightly blocked by the antagonists d-tubocurarine, mecamylamine, or dihydro-beta-erythroidine at concentrations that efficiently block alpha3 AChR function. Most of the nicotine-induced increase in alpha3 AChRs was found to be intracellular. The alpha3 AChRs, which accumulate intracellularly, were shown to have been previously exposed on the cell surface by their susceptibility to antigenic modulation. The data suggest that chronic exposure to nicotine may induce a conformation of cell surface alpha3 AChRs that at least in this cell line are consequently internalized but not immediately destroyed.

Endocytosis

Analysis of west African hunters for foamy virus infections.

Foamy viruses are a genus of complex retroviruses that infect a wide variety of mammals. However, a clear association with any disease process has yet to be proven for these viruses. A higher human seroprevalence was reported in African populations, perhaps due to exposure to simian foamy viruses (SFV) endemic in primates. However, the earlier serologic surveys were not confirmed by studies employing nucleic acid amplification. Foamy virus infections of humans clearly do occur as rare zoonoses among primate center or laboratory workers exposed to captive primates or their blood. We sought to detect foamy virus infections in a cohort of humans also presumed to be exposed to SFV, i.e., West African hunters. We constructed recombinant vaccinia viruses that expressed human foamy virus (HFV) Gag or Env polyproteins in mammalian cells. The sera from 17 monkey hunters or several controls were tested in radioimmunoprecipitation assays (RIPAs) against the recombinant HFV proteins. Chimpanzee sera or HFV-positive human sera immunoprecipitated gp130, the HFV Env precursor, as well as p74, the HFV Gag polyprotein. None of the hunters' sera recognized both of these recombinant proteins. We then employed a nested polymerase chain reaction (PCR) analysis of the hunters' DNA but also failed to detect foamy virus infections. Therefore, by utilizing a recombinant RIPA and a nested PCR assay, we have not identified foamy virus infections occurring naturally in hunters exposed to wild monkeys in West Africa.

Africa, Western

Immortalization of inbred rabbit keratinocytes from a Shope papilloma and tumorigenic transformation of the cells by EJ-ras.

An immortalized cell line of keratinocytes, named SPG1-3, was established from a papilloma induced from cottontail rabbit papillomavirus (CRPV)-infected inbred rabbit skin. The cells have reached 60 passages in culture and are still growing well, but they are not tumorigenic in athymic mice. Although CRPV DNA was present as extrachromosomal episomes in the papilloma from which the cell line was derived from a single colony of keratinocytes, there was no CRPV DNA detectable in the cells. Three sub-cell lines of SPG1-3EJ, SPG1-3EJ1 and SPG1-3EJ2 were then established from the EJ-ras transfected SPG1-3 cells. All of the three sub-lines contained both EJ-ras DNA and a 1.2 kb transcript of EJ-ras, and they are malignantly tumorigenic in athymic mice. These data indicate that CRPV genome and its expression might be essential for the initiation and maintenance of neoplasia, but not for the maintenance of immortalization of the tumor-derived cells. In addition, some oncogenes such as EJ-ras may play an essential role in tumorigenic and malignant conversion of the immortalized cells. These cell lines derived from inbred rabbit skin may provide a useful in vitro system for better understanding of the oncogenic processes of papillomavirus-involved neoplastic progression by transfecting the cells with CRPV genes and serial transplantation to the inbred rabbits for studying host immune responses to the viral oncogenic potential.

Animals

Organization of 'nanocrystal molecules' using DNA.

Patterning matter on the nanometre scale is an important objective of current materials chemistry and physics. It is driven by both the need to further miniaturize electronic components and the fact that at the nanometre scale, materials properties are strongly size-dependent and thus can be tuned sensitively. In nanoscale crystals, quantum size effects and the large number of surface atoms influence the, chemical, electronic, magnetic and optical behaviour. 'Top-down' (for example, lithographic) methods for nanoscale manipulation reach only to the upper end of the nanometre regime; but whereas 'bottom-up' wet chemical techniques allow for the preparation of mono-disperse, defect-free crystallites just 1-10 nm in size, ways to control the structure of nanocrystal assemblies are scarce. Here we describe a strategy for the synthesis of 'nanocrystal molecules', in which discrete numbers of gold nanocrystals are organized into spatially defined structures based on Watson-Crick base-pairing interactions. We attach single-stranded DNA oligonucleotides of defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addition of a complementary single-stranded DNA template. We anticipate that this approach should allow the construction of more complex two- and three-dimensional assemblies.

Base Sequence

Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2, and beta4 subunits.

Nicotinic acetylcholine receptors formed from combinations of alpha3, beta2, beta4, and alpha5 subunits are found in chicken ciliary ganglion neurons and some human neuroblastoma cell lines. We studied the co-expression of various combinations of cloned human alpha3, beta2, beta4, and alpha5 subunits in Xenopus oocytes. Expression on the surface membrane was found only for combinations of alpha3beta2, alpha3beta4, alpha3beta2alpha5, and alpha3beta4alpha5 subunits but not for other combinations of one, two, or three of these subunits. alpha5 subunits assembled inside the oocyte with beta2 but not with alpha3 subunits or other alpha5 subunits. alpha5 subunits coassembled very efficiently with alpha3beta2 or alpha3beta4 combinations. The presence of alpha5 subunits had very little effect on the binding affinities for epibatidine of receptors containing also alpha3 and beta2 or alpha3 and beta4 subunits. The presence of alpha5 subunits increased the rate of desensitization of both receptors containing also alpha3 and beta2 or alpha3 and beta4 subunits. In the case of receptors containing alpha3 and beta4 subunits, the addition of alpha5 subunits had little effect on the responses to acetylcholine or nicotine. However, in the case of receptors containing alpha3 and beta2 subunits, the addition of alpha5 subunits reduced the EC50 for acetylcholine from 28 to 0.5 microM and the EC50 for nicotine from 6.8 to 1.9 microM, while increasing the efficacy of nicotine from 50% on alpha3beta2 receptors to 100% on alpha3beta2alpha5 receptors. Both alpha3beta2 and alpha3beta2alpha5 receptors expressed in oocytes sedimented at the same 11 S value as native alpha3-containing receptors from the human neuroblastoma cell line SH-SY5Y. In the receptors from the neuroblastoma alpha3, beta2, and alpha5 subunits were co-assembled, and 56% of the receptor subtypes containing alpha3 subunits also contained beta2 subunits. The beta2 subunit-containing receptors from SH-SY5Y cells exhibited the high affinity for epibatidine characteristic of receptors formed from alpha3 and beta2 or alpha3, beta2, and alpha5 subunits rather than the low affinity exhibited by receptors formed from alpha3 and beta4 or alpha3, beta4, and alpha5 subunits. Nicotine, like the structurally similar toxin epibatidine, also distinguishes by binding affinity two subtypes of receptors containing alpha3 subunits in SH-SY5Y cells. The affinities of alpha3beta2 receptors expressed in oocytes were similar to the affinities of native alpha3 containing receptors from SH-SY5Y cells for acetylcholine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium.

Acetylcholine

Accessory signaling by CD40 for T cell activation: induction of Th1 and Th2 cytokines and synergy with interleukin-12 for interferon-gamma production.

The interaction of CD40 ligand (CD40L) on activated T cells with CD40 on B cells, monocytes and dendritic cells is essential for humoral immunity and for up-regulation of antigen-presenting cell (APC) functions, as a result of signaling through CD40. There are also some indications that after interaction with CD40, CD40L can directly signal T cells. In this study we demonstrate that upon stimulation of human peripheral blood T cells through the T cell receptor (TCR)/CD3 complex, CD40/CD40L interaction strongly enhances the production of Th1 cytokines such as interleukin (IL)-2 and interferon (IFN)-gamma and Th2 cytokines such as IL-4, IL-5 and IL-10 by a direct effect on T cells. Furthermore, CD40/CD40L interaction synergizes with IL-12 in selectively enhancing IFN-gamma production by purified anti-CD3-stimulated T cells. These effects were observed at both the protein and the mRNA level. Both CD4+ and CD8+ T cells were able to produce IFN-gamma in the presence of helper signals from IL-12 and CD40, although CD8+ T cells were less active. Since CD40/CD40L interaction also up-regulates IL-12 production and B7 expression by APC, our results suggest that CD40/CD40L interaction is bidirectional, and promotes activation of both APC and T cells.

Adult

Tyrosine phosphorylation of extracellular signal-regulated protein kinase 4 in response to growth factors.

Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this re-agent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.

Animals

Immunological evidence for the expression of the Fas antigen in the infant and adult human ovary during follicular regression and atresia.

Immunohistochemical localization of the Fas antigen in the infant and adult human ovary during follicular growth, regression, and atresia was examined by the avidin/biotin immunoperoxidase method with a monoclonal antibody to the Fas antigen. Western blotting was used to confirm the presence of the Fas antigen protein. In primordial and primary follicles within the normal adult ovary, only the oocyte showed moderate immunostaining for the Fas antigen. In secondary and antral follicles, only the oocyte showed weak staining for the Fas antigen, while in preovulatory follicles, neither the oocyte nor the granulosa and theca cells were immunostained for the Fas antigen. In corpora lutea, the Fas antigen staining became apparent in the granulosa lutein cells during the early luteal phase and intensified during the mid luteal phase, while the theca lutein cells became positive for the Fas antigen staining during the mid luteal phase. During the late luteal phase, the staining intensity of the Fas antigen in the regressing corpora lutea further increased. As the regressing corpora lutea were converted into corpora albicans, the staining intensity decreased, and the corpora albicans and stromal cells were negative for the Fas antigen. In atretic primordial and primary follicles, only the degenerating oocyte showed the Fas antigen staining. By contrast, in atretic antral follicles, the Fas antigen staining was profound in the degenerating granulosa cells at the early stage of atresia, and at the mid stage of atresia it was intensified in the cell surface of the scattered granulosa cells and became apparent in the theca cells. At the late stage of atresia the Fas antigen remained only in the hypertrophied theca cells. In the infant ovary, only the oocyte in primordial and primary follicles exhibited intense staining for the Fas antigen. In the postmenopausal ovary, the Fas antigen staining was entirely negative. Western blot analysis revealed the presence of the Fas antigen protein with a molecular mass of 45 kDa in luteal tissues. On the basis of the recent evidence, that the Fas antigen mediates an apoptotic signal in a variety of cells, the abundant expression of the Fas antigen in the regressing corpora lutea and atretic follicles suggests that the Fas antigen participates in luteal regression and follicular atresia through the apoptotic process. Furthermore, notable expression of the Fas antigen in the oocyte of primordial and primary follicles within the infant and adult human ovary followed by the decrease in the Fas antigen expression in the oocyte with the advance of follicular maturation suggests that the Fas antigen expression in the oocyte may play a role in follicular selection.

Adult

Comparison of immunocytologic localization of insulin-like growth factor binding protein-4 in normal and polycystic ovary syndrome human ovaries.

The cytologic localization and cellular levels of insulin-like growth factor binding protein-4 (IGFBP-4) in follicular and stromal compartments of normal and polycystic ovary syndrome (PCOS) ovaries during follicular growth and regression were investigated by the avidin/biotin immunoperoxidase method with a polyclonal antibody to human IGFBP-4, and a comparative assessment of IGFBP-4 expression in normal and PCOS ovaries was provided. In normal human ovaries, IGFBP-4 was immunolocalized to the oocyte throughout follicular growth, while the surrounding granulosa and theca cells were negligible for IGFBP-4 immunostaining in primordial, preantral and antral follicles. IGFBP-4 immunostaining became apparent, however, in the lutein cells of corpora lutea and the granulosa and theca cells of atretic follicles. In PCOS ovaries, prominent immunostaining for IGFBP-4 was apparent not only in the oocyte, but also in the surrounding granulosa cells in preantral follicles. In antral follicles from PCOS women without hyperinsulinemia, IGFBP-4 immunostaining was more prominent in the granulosa cells than the theca cells, whereas in antral follicles from PCOS women with hyperinsulinemia IGFBP-4 immunostaining was more prominent in the theca cells than the granulosa cells. Furthermore, in atretic follicles within PCOS ovaries IGFBP-4 immunostaining was prominent in the theca cells, regardless of the association of hyperinsulinemia. These results demonstrate for the first time that there is a great difference in cellular expression of IGFBP-4 between normal and PCOS human ovaries. In light of the high affinity of IGFBP-4 for IGF-1, the abundant expression of IGFBP-4 in granulosa and theca cells of preantral and antral follicles of PCOS ovaries may lead to decreases in the bioavailability of IGF-I in those follicles. The decrease in IGF-I-mediated stimulation of gonadotropin actions on granulosa and theca cells in preantral and antral follicles may impair the induction of aromatase activity, causing an androgenic microenvironment which is characteristic of atretic follicles and PCOS follicles.

Adult

Elimination of glycosylation heterogeneity affecting heparin affinity of recombinant human antithrombin III by expression of a beta-like variant in baculovirus-infected insect cells.

In order to promote homogeneity of recombinant antithrombin III interactions with heparin, an asparagine-135 to alanine substitution mutant was expressed in baculovirus-infected insect cells. The N135A variant does not bear an N-linked oligosaccharide on residue 135 and is therefore similar to the beta isoform of plasma antithrombin. Purified bv.hat3.N135A is homogeneous with respect to molecular mass, charge and elution from immobilized heparin. Second-order rate constants for thrombin and factor Xa inhibition determined in the absence and presence of heparin are in good agreement with values established for plasma antithrombin and these enzymes. Based on far- and near-UV CD, bv.hat3.N135A has a high degree of conformational similarity to plasma antithrombin. Near-UV CD, absorption difference and fluorescence spectroscopy studies indicate that it also undergoes an identical or very similar conformational change upon heparin binding. The Kds of bv.hat3.N135A for high-affinity heparin and pentasaccharide were determined and are in good agreement with those of the plasma beta-antithrombin isoform. The demonstrated similarity of bv.hat3.N135A and plasma antithrombin interactions with target proteinases and heparins suggest that it will be a useful base molecule for investigating the structural basis of antithrombin III heparin cofactor activity.

Animals

NMR study of the cold, heat, and pressure unfolding of ribonuclease A.

The reversible cold, heat, and pressure unfolding of RNase A and RNase A--inhibitor complex were studied by 1D and 2D 1H NMR spectroscopy. The reversible pressure denaturation experiments in the pressure range from 1 bar to 5 kbar were carried out at pH 2.0 and 10 degrees C. The cold denaturation was carried out at 3 kbar, where the protein solution can be cooled down to -25 degrees C without freezing. Including heat denaturation experiments, the experimental data obtained allowed us to construct the pressure--temperature phase diagram of RNase A. The experimental results suggest the possibility that all three denaturation processes (cold, heat, and pressure) lead to non-cooperative unfolding. The appearance of a new histidine resonance in the cold-denatured and pressure-denatured RNase A spectra, compared to the absence of this resonance in the heat-denatured state, indicates that the pressure-denatured and cold-denatured states may contain partially folded structures that are similar to that of the early folding intermediate found in the temperature-jump experiment reported by Blum et al. [Blum, A. D., et al. (1978) J. Mol. Biol. 118, 305]. A hydrogen-exchange experiment was performed to confirm the presence of partially folded structures in the pressure-denatured state. Stable hydrogen-bonded structures protecting the backbone amide hydrogens from solvent exchange were observed in the pressure-denatured state. These experimental results suggest that the pressure-denatured RNase A displays the characteristics of a the inhibitor 3'-UMP show that the RNase A-inhibitor complex is more stable than RNase without the inhibitor.

Animals

One and two dimensional 1H-NMR studies of pressure and tetracaine effects on sonicated phospholipid vesicles.

One and two dimensional 1H-NMR experiments have been performed to study the molecular order and dynamics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles under the influence of high pressure and the local anesthetic, tetracaine (TTC), which can also be considered as a model-charged amphiphile. The TTC molecules have an ordering effect on the headgroups but a disordering effect on the acyl chains, whereas, pressure has an ordering effect on the acyl chains. The results from 2D NOESY experiments on pure DPPC vesicles show that the intensities of NMe3/CH3, NMe3/(CH2)n, and CH3/(CH2)n cross-peaks increase with increasing pressure in the liquid-crystalline phase but decrease with pressure in the gel phase, further suggesting that the appearance of the cross-peaks between the two extremes of the DPPC molecules is due to spin-diffusion. For the DPPC/TTC vesicles, the 2D NOESY experiments confirm that charged TTC molecules are located in the headgroup region of the DPPC bilayers and, in the gel phase, also suggest the formation of an interdigitated gel phase.

1,2-Dipalmitoylphosphatidylcholine

Methylation of cottontail rabbit papillomavirus DNA and tissue-specific expression in transgenic rabbits.

Transgenic rabbits carrying multiple copies of CRPV genomic DNA were described previously (Peng et al. (1993) J. Virol. 67, 1698-1701). CRPV DNA was detectable in all tissues of the transgenic rabbits, however, the transcripts of CRPV genes only were found in skin and skin tumors. Tumor development was also restricted to skin. To study the mechanism involving tissue-specific expression of CRPV genes in rabbits, cellular DNAs, isolated from different normal tissues and skin tumors, were digested with the two isoschizomeric restriction endonucleases MspI (methylation resistant) and HpaII (methylation sensitive), respectively, and analyzed by Southern blot. CRPV DNA, especially its upstream regulatory region (URR), was extensively methylated in all normal tissues, but methylation was remarkably reduced in skin tumors. These data suggest that extensive methylation of CRPV genome, especially in the URR, might be a factor in controlling its tissue-specific expression.

5-Methylcytosine