Biomedical subjects

X Jiang

Publications and source records attributed to X Jiang.

At least 199 records · Page 11Linked to original sources

PubMed1999

Simultaneous assay of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human plasma using normal-phase liquid chromatography-tandem mass spectrometry with a silica column and an aqueous organic mobile phase.

Morphine (MOR) is an opioid analgesic used for the treatment of moderate to severe pain. MOR is extensively metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). A rapid and sensitive method that was able to reliably detect at least 0.5 ng/ml of MOR and 1.0 ng/ml of M6G was required to define their pharmacokinetic profiles. An LC-MS-MS method was developed in our laboratory to quantify all three analytes with the required sensitivity and a rapid turnaround time. A solid-phase extraction (SPE) was used to isolate MOR, M3G, M6G, and their corresponding deuterated internal standards from heparinized plasma. The extract was injected on a LC tandem mass spectrometer with a turbo ion-spray interface. Baseline chromatographic separation among MOR, M3G, and M6G peaks was achieved on a silica column with an aqueous organic mobile phase consisting of formic acid, water, and acetonitrile. The total chromatographic run time was 3 min per injection, with retention times of 1.5, 1.9 and 2.4 min for MOR, M6G, and M3G, respectively. Chromatographic separation of M3G and M6G from MOR was paramount in establishing the LC-MS-MS method selectivity because of fragmentation of M3G and M6G to MOR at the LC-MS interface. The standard curve range in plasma was 0.5-50 ng/ml for MOR, 1.0-100 ng/ml for M6G, and 10-1000 ng/ml for M3G. The inter-day precision and accuracy of the quality control (QC) samples were <7% relative standard deviation (RSD) and <6% relative error (R.E.) for MOR, <9% RSD and <5% R.E. for M6G, and <3% RSD and <6% R.E. for M3G. Analyte stability during sample processing and storage were established. Method ruggedness was demonstrated by the reproducible performance from multiple analysts using several LC-MS-MS systems to analyze over one thousand samples from clinical trials.

Chromatography, Liquid↗

PubMed1999

Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia.

Primitive subsets of leukemic cells isolated by using fluorescence-activated cell sorting from patients with newly diagnosed Ph(+)/BCR-ABL(+) chronic myeloid leukemia display an abnormal ability to proliferate in vitro in the absence of added growth factors. We now show from analyses of growth-factor gene expression, protein production, and antibody inhibition studies that this deregulated growth can be explained, at least in part, by a novel differentiation-controlled autocrine mechanism. This mechanism involves the consistent and selective activation of IL-3 and granulocyte colony-stimulating factor (G-CSF) production and a stimulation of STAT5 phosphorylation in CD34(+) leukemic cells. When these cells differentiate into CD34(-) cells in vivo, IL-3 and G-CSF production declines, and the cells concomitantly lose their capacity for autonomous growth in vitro despite their continued expression of BCR-ABL. Based on previous studies of normal cells, excessive exposure of the most primitive chronic myeloid leukemia cells to IL-3 and G-CSF through an autocrine mechanism could explain their paradoxically decreased self-renewal in vitro and slow accumulation in vivo, in spite of an increased cycling activity and selective expansion of later compartments.

Base Sequence↗

PubMed1999

Vasoactive intestinal peptide inhibits cytokine production in T lymphocytes through cAMP-dependent and cAMP-independent mechanisms.

Previous reports indicate that VIP and the structurally related peptide PACAP, inhibit IL-2 and IL-10 production in antigen-stimulated T lymphocytes. Intracellular cAMP elevation appears to be the primary transduction pathway involved. However, in the lower concentration range, an additional, cAMP-independent transduction pathway appears to mediate the VIP inhibition of cytokine production. Here, we address this question by using VIP agonists and antagonists which act through cAMP-dependent and -independent pathways. The antagonists based on the neurotensin-VIP hybrid molecule did not affect the inhibitory effect of VIP/PACAP on IL-2 and IL-10 production, confirming that astrocytes and T lymphocytes express different receptors. A lipophilic antagonist with increased membrane permeability, partially reversed the inhibitory effect of VIP/PACAP, forskolin, prostaglandin E2, and 8-bromo-cAMP without significantly affecting cAMP levels, suggesting that it acts downstream of cAMP. Two VIP agonists inhibit IL-2 and IL-10 production. One of the agonists increases cAMP, whereas the second one does not induce cAMP/cGMP. Our results indicate that VIP inhibits cytokine production in stimulated CD4+ T cells through two separate mechanisms, which involve both cAMP-dependent and cAMP-independent transduction pathways.

Animals↗

PubMed1999

Ultra-short columns for low-pressure ion chromatography.

Since the development of low-pressure ion chromatography (LPIC), many inorganic cations and anions as well as organic acids could be analyzed at a low-pressure of 1.96 x 10(5)-2.94 x 10(5) Pa. And the ultra-short columns took the place of the common long chromatographic columns. Furthermore, the ultra-short columns of LPIC not only reduced the system pressure appreciably, but also achieved high sensitivity and precision. In order to assess the characteristics of the super-short columns, much analysis, i.e., the analysis of alkali metals, alkaline earth metals, transition-metals, rare-earth elements, inorganic anions and organic acids as well as the common amino acids, were conducted. Moreover, excellent results were obtained from the LPIC columns.

Chromatography, Liquid↗

PubMed1999

Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by an increased proliferative activity of the leukemic progenitors that produce an elevated number of mature granulocytes. Nevertheless, cell cycle-active agents, even in very high doses, are alone unable to eradicate the leukemic clone, suggesting the presence of a rare subset of quiescent leukemic stem cells. To isolate such cells, we first used Hoechst 33342 and Pyronin Y staining to obtain viable G(0) and G(1)/S/G(2)/M fractions of CD34(+) cells by fluorescence-activated cell sorting (FACS) from 6 chronic-phase CML patients' samples and confirmed the quiescent and cycling status of the 2 fractions by demonstration of expected patterns of Ki-67 and D cyclin expression. Leukemic (Ph(+)/BCR-ABL(+)) cells with in vitro progenitor activity and capable of engrafting immunodeficient mice were identified in the directly isolated G(0) cells. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that many leukemic CD34(+) G(0) cells also expressed BCR-ABL mRNA. CD34(+) from 8 CML patients were also labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) before being cultured (with and without added growth factors) to allow viable cells that had remained quiescent (ie, CFSE(+)) after 4 days to be retrieved by FACS. Leukemic progenitors were again detected in all quiescent populations isolated by this second strategy, including those exposed to a combination of flt3-ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These findings provide the first direct and definitive evidence of a deeply but reversibly quiescent subpopulation of leukemic cells in patients with CML with both in vitro and in vivo stem cell properties.

Animals↗

PubMed1999

Sympathectomy decreases and adrenergic stimulation increases the release of tissue plasminogen activator (t-PA) from blood vessels: functional evidence for a neurologic regulation of plasmin production within vessel walls and other tissue matrices.

Our recent morphologic studies indicated that peripheral nervous system (PNS) adrenergic neurons synthesize, transport, and store the serene protease, tissue plasminogen activator (t-PA) in axon terminals, many of which innervate vessel walls. Sympathoadrenal stimulation induces a surge of t-PA from vessel walls into the blood. The vascular endothelium, which constitutively secretes t-PA into blood also has long been widely assumed to be the principal source of this stress-induced release, but has not been verified as such. A neurologically regulated release from adrenergic stores could thus augment the known constitutive endothelial release. To functionally test this possibility, we quantitated the effects of guanethidine-induced systemic sympathectomy on the basal and stimulated release of t-PA from isolated vessel explants in superfused organ cultures. Moment-to-moment changes in the release rate were plotted from serial assays of the t-PA free activity. The effects of endothelial and adventitial nerve plexus ablations were also tested. Sympathectomy induced 30-50% reductions in t-PA release from both arterial and microvascular explants. An acute release induced by alpha-1 adrenergic receptor stimulations was also strongly suppressed, as were basal levels of the circulating enzyme in vivo. Adventitial and endothelial ablations from normal large vessel explants produced greater reductions than small vessel endothelial ablations. Ganglion electrical stimulation also induced an acute microvascular release in vivo. These and past morphologic findings indicate a physiological infusion of t-PA into the vessel walls, blood, and other innervated matrices by sympathetic neurons.

Animals↗

PubMed1999

Characterization of NFkappaB activation by detection of green fluorescent protein-tagged IkappaB degradation in living cells.

Activation of the transcription factor NFkappaB requires rapid degradation of its inhibitor, IkappaBalpha. To facilitate the study of IkappaBalpha degradation, we fused IkappaBalpha protein to enhanced green fluorescent protein to construct IkappaBalpha-enhanced green fluorescent protein (IG). We demonstrated by both flow cytometry and Western blot analysis that the half-life of IG in the presence of human tumor necrosis factor (TNF) alpha is approximately 5 min, which is similar to the half-life of native IkappaBalpha. The degradation coincided with NFkappaB translocation from the cytoplasm to the nucleus and NFkappaB-mediated induction of transcription. Phorbol 12-myristate 13-acetate (PMA), but not forskolin, also induces degradation of IG fusion protein. The half-life of IG in the presence of PMA is approximately 15 min, longer than when induced with TNFalpha. Co-treatment with TNFalpha and PMA did not result in a synergistic effect on IG degradation, although they stimulate different kinases in two different signaling pathways. Degradation of IG was inhibited by mutations at serine residues 32 and 36, which are the target sites of the phosphorylation modification that initiates degradation of IkappaBalpha. We also demonstrated that basal degradation of IG in the presence of cycloheximide is inhibited by such mutations, suggesting that basal degradation of IkappaBalpha also requires phosphorylation as the signal for degradation. Finally, we showed that the rate of TNFalpha-induced degradation of IG remains almost constant throughout the cell cycle, except at the mitotic phase, in which IG degrades more slowly.

DNA-Binding Proteins↗

PubMed1999

3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT): a new coupling reagent with remarkable resistance to racemization.

[formula: see text] The new crystalline phosphate reagent 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) mediates amide bond formation with a remarkable resistance to racemization. Comparative racemization studies were carried out and DEPBT proved to be superior to typical phosphonium and uronium coupling reagents. DEPBT is easily prepared and is exceedingly stable with a shelf life of months at room temperature. The advantageous properties of DEPBT render it a useful and unique addition to the arsenal of coupling reagents.

Amides↗

PubMed1999

Structural and compositional determinants of cortistatin activity.

Cortistatin-14 (CST-14) is a putative novel neuropeptide that shares 11 of its 14 residues with somatostatin-14 (SRIF-14), yet its effects on sleep physiology, locomotor behavior and hippocampal function are different from those of somatostatin. We studied the structural basis for cortistatin's distinct biological activities. As with SRIF-14, CST-14 does not show any preferred conformation in solution, as determined by circular dichroism and nuclear magnetic resonance. Synthetic cortistatin analogs were designed and synthesized based on the cyclic structure of octreotide. Biological assays were carried out to determine their binding affinities to five somatostatin receptors (sstl-5) and their ability to produce changes in locomotor activity and to modulate hippocampal physiology and sleep. The results show that the compound with N-terminal proline and C-terminal lysine amide exhibits cortistatin-like biological activities, including reduction of population spike amplitudes in the hippocampal CA1 region, decrease in locomotor activity and enhancement of slow-wave sleep 2. These findings suggest that both proline and lysine are necessary for cortistatin binding to its specific receptor.

Amino Acid Sequence↗

PubMed1999

Copper and zinc ions differentially block asialoglycoprotein receptor-mediated endocytosis in isolated rat hepatocytes.

Asialoglycoprotein receptors on hepatocytes lose endocytic and ligand binding activity when hepatocytes are exposed to iron ions. Here, we report the effects of zinc and copper ions on the endocytic and ligand binding activity of asialoglycoprotein receptors on isolated rat hepatocytes. Treatment of cells at 37 degrees C for 2 h with ZnCl2 (0-220 microM) or CuCl2 (0-225 microM) reversibly blocked sustained endocytosis of 125I-asialoorosomucoid by up to 93% (t1/2 = 62 min) and 99% (t1/2 = 54 min), respectively. Cells remained viable during such treatments. Zinc- and copper-treated cells lost approximately 50% of their surface asialoglycoprotein receptor ligand binding activity; zinc-treated cells accumulated inactive asialoglycoprotein receptors intracellularly, whereas copper-treated cells accumulated inactive receptors on their surfaces. Cells treated at 4 degrees C with metal did not lose surface asialoglycoprotein receptor activity. Exposure of cells to copper ions, but not to zinc ions, blocked internalization of prebound 125I-asialoorosomucoid, but degradation of internalized ligand and pinocytosis of the fluid-phase marker Lucifer Yellow were not blocked by metal treatment. Zinc ions reduced diferric transferrin binding and endocytosis on hepatocytes by approximately 33%; copper ions had no inhibitory effects. These findings are the first demonstration of a specific inhibition of receptor-mediated endocytosis by non-iron transition metals.

Animals↗

PubMed1999

Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase.

We examined the role of A328(F330) in the binding of various inhibitors to cholinesterases (ChEs) using human butyrylcholinesterase (BChE) mutants to determine if the conclusions drawn from studies with acetylcholinesterase (AChE) mutants could be extended to BChE. For huperzine A and edrophonium, the results obtained with AChE mutants could be directly correlated with those obtained with native ChEs and site-specific mutants of human BChE. Inhibition studies of ethopropazine with BChE mutants, where A328 was modified to either F or Y, suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active-site gorge showed that the poor inhibitory activity of ethopropazine towards AChE was due to the smaller dimension of the active-site gorge. The volume of the BChE active-site gorge is approximately 200 A3 larger than that of the AChE gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations. These results suggest that, although the overall scaffolding of the two enzymes may be highly similar, the dimensions and the micro-environment of the gorge play a significant role in determining the selectivity of substrate and inhibitors for ChEs.

Acetylcholinesterase↗

PubMed1999

Altered levels of primary antioxidant enzymes in progeria skin fibroblasts.

Free radicals are involved in the aging process. In this study, the profile of primary antioxidant enzymes that scavenge reactive oxygen species (ROS) was examined for the first time in human skin fibroblasts from progeria, a premature aging disease. Altered levels of antioxidant enzymes were found in progeria cells. Basal levels of MnSOD were decreased in progeria cells as well as a blunted induction in response to chronic stress. This change may contribute to the accelerated aging process in progeria cells. In contrast, the levels of CuZnSOD showed no progeria-related change. Two H2O2 removing enzymes demonstrated a significant reduction in progeria cells: only 50% of normal CAT activity and 30% of normal GPX activity can be detected in progeria cells. This diminished H2O2 removing capacity in progeria cells may lead to an imbalance of intracellular ROS and therefore may play an important role in the development of progeria.

Adolescent↗

PubMed1999

Seroepidemiological study of genogroup I and II calicivirus infections in South and southern Africa.

Diarrhoea is associated with the daily death of between 180 and 200 children under the age of 5 years in South Africa. Until recently, many cases and outbreaks of diarrhoea were not associated with a known aetiologic agent. Previous studies using baculovirus-expressed Norwalk virus (NV) and Mexico virus (MxV) capsid antigens have shown that human calicivirus infection is common in South Africa. In this study, our surveillance was extended to different populations, as well as to four other southern African countries: Namibia, Angola, Zimbabwe, and Mozambique. More than 1,700 specimens, some involved in previous cohort studies of infectious diseases, were enrolled in the surveillance. The overall seroprevalence of antibody against NV was >90% for all cohorts except for Mozambican refugees that had 83. 8% sero-positivity. The MxV antibody prevalence was higher than NV, with >95% positivity for all cohorts, except for one in Namibia that had 81% exposure. This study is one of only a few reporting on the concurrent incidence of NV and MxV infections in a cohort study, and has determined that small round structured viruses are prevalent in the local populations of South and Southern Africa. These agents may account for a number of previously unknown or unidentified causes of diarrhoeal illness, in both adults and children, in southern Africa.

Adolescent↗

PubMed1999

Direct injection versus liquid-liquid extraction for plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry.

Direct injection versus liquid-liquid extraction for post-dose human plasma sample analysis by high performance liquid chromatography with tandem mass spectrometry (LC/MS/MS) have been studied using a drug candidate compound. For the direct-injection method, an Oasis(R) HLB column (1 x 50 mm, 30 micrometer) was used as the on-line extraction column and a conventional Waters symmetry C18 column (3.9 x 50 mm, 5 micrometer) was used as the analytical column. Each plasma sample (100 microL) was mixed with 100 microL of a working solution of the internal standard in aqueous 0.05 M ammonium acetate (pH 6.9), and portions (10 microL) of these samples were then injected into the LC/MS/MS system. For the liquid-liquid extraction method, a YMC Basic C18 column (2.0 x 50 mm, 5 micrometer) was used as the analytical column. Each sample (0.5 mL) was extracted with methyl tert-butyl ether and the extract was reconstituted and injected into the LC/MS/MS system. The total analysis time for both methods was 2.0 min per sample. The accuracy, inter-day precision and intra-day precision obtained from the quality control samples were within 8% for both methods. The analysis results of post-dose human plasma samples showed that the deviations of 91% of the concentrations obtained using the direct-injection method were within +/-20% from the concentrations obtained using the liquid-liquid extraction method, and the overall average percentage deviation was -1.5%. The results showed that the two methods were equivalent in terms of total chromatographic run time, accuracy and precision. However, for a batch of 100 samples, the sample preparation time for the direct-injection method was only about 25% of the time required for liquid-liquid extraction. This decrease in sample preparation time resulted in the doubling of the overall sample analysis throughput.

Chromatography, High Pressure Liquid↗

PubMed1999

Occupational health survey on workers exposed to 2-bromopropane at low concentrations.

BACKGROUND: Recent case studies in Korea and animal studies revealed the reproductive and hematopoietic toxicity of 2-bromopropane introduced into workplaces as an alternative to ozone-layer depleting chlorofluorocarbons. We aimed to clarify the dose-effect relationship of 2-bromopropane in workers. METHOD: The exposure concentration of 2-bromopropane and hematological indices, hormonal levels, menstruation status, and sperm indices were examined in 25 workers (11 males, 14 females) at a 2-bromopropane factory. Regression analyses of the examined indices against time-weighted average (TWA) of exposure concentration were conducted. RESULTS: Amenorrhea or polymenorrhea was observed only in older females. Hematological indices had a significant relation with TWA of exposure concentration in females with normal menstruation. However, no other indices showed any significant relation with TWA of 2-bromopropane. CONCLUSIONS: No severe cases of reproductive or hematopoietic disorders were found at less than 10 ppm (TWA), but a possible adverse effect of 2-bromopropane on hematopoiesis could not be disproved.

Adult↗

PubMed1999

TPA enhances growth hormone (GH) secretion effect of GH-releasing hormone (GHRH) by human gsp-positive pituitary somatotrophinomas.

In recent years, one of the most exciting advances in the researches of pituitary adenomas is the discovery that 30%-40% of human pituitary somatotrophinomas carry somatic mutations of the gene for the alpha-subunit of the stimulatory GTP-binding protein, Gs (Gs alpha). These mutations, termed gsp oncogenes, may play an important role in the tumorigenesis of pituitary adenomas. Of 10 somatotrophinomas examined, 3 (30%) were proved to be gsp positive, as determined by sequence analysis of DNA generated by the polymerase chain reaction (PCR). GHRH exerted a significant stimulatory effect on GH secretion in 2 of 3 gsp-positive and 4 of 7 gsp-negative tumors. Moreover, phorbol ester, 1, 2-tetradecanoylphorbol-13-acetate (TPA), enhanced stimulation of lated the GH secretion effect exerted by GHRH in gsp-positive somatotrophinomas, whereas this effect was not observed in gsp-negative tumors. This result suggests that the protein kinase C signal system as well as adenylyl cyclase-cAMP-protein kinase A intracellular signal transduction system plays a pivotal role in GH secretory control of GHRH, which may work together via a cross-talk mechanism.

Adenoma↗

PubMed1999

The clinical study and HLA genotyping of 112 familial myasthenia gravis patients.

One hundred and twelve cases of familial myasthenia gravis (MG) from 44 families selected from 2100 patients with MG diagnosed since 1983 in the Department of Neurology were studied. The clinical pictures and immunological features of the patients showed a great resemblance to those of sporadic cases. The pedigree analysis disclosed that the hereditary patterns of familial patients were basically Mendellian autosomal inheritance. Many predisposing factors such as fever, infection, use of aminoglycoside or vaccines, played an important role in presenting the phenotype of subclinical cases. The HLA genotyping suggested that the complement polymorphism C4A * 4, the complotype S42, and the genes 0901 and 1301 of DRB1 allele, were related to the pathogenesis of MG. It was concluded that the phenotype of MG may be the result of interaction between hereditary defects and environmental factors.

Alleles↗

PubMed1999

Complete nucleotide sequence and genomic organization of a primate calicivirus, Pan-1.

The primate calicivirus, Pan-1, was originally isolated from several primate species. It displayed typical calicivirus morphology by electron microscopy. We determined the genomic sequence of Pan-1 by cDNA cloning and direct RNA sequencing. Pan-1 shares a similar genomic organization and a high degree of sequence identity with feline caliciviruses. The Pan-1 genome contains 8,304 nucleotides, plus a poly-A tail, and is longer than any other calicivirus strains with a completely known sequence. The extra sequences of Pan-1 include a unique 424-nucleotide sequence at the 5' end of ORF1, additional amino acids at the N-terminus of the capsid, and a longer 3' UTR.

Animals↗