Biomedical subjects
Publications and source records attributed to L Zhang.
Numerous 1,2-diaryl(heteroaryl)pyrroles and -3-methylpyrroles were prepared in a two-step procedure from N-allylbenzotriazoles via intramolecular oxidative cyclization in the presence of Pd(II) catalyst.
With the coexistence of the mobile phase, the stationary phase, and the electric field in capillary electrochromatography, the chromatographic zone-sharpening effect and field-enhanced sample-stacking technique were utilized to improve detection sensitivity. By the former means, with less organic modifier in the sample solution compared to that in the mobile phase, the concentration factors of neutral solutes benzoin and mephenytion were 134 and 219, respectively. Through the latter one, without electrolyte in the sample solution, the detection sensitivity of propatenene with positive charge was improved by 1600 times. While with the combination of these two methods, improvement of over 17,000 times for the sensitivity of propatenene was obtained. By the combined means, the analysis of basic pharmaceutical compounds at concentrations of nanograms per milliliter by UV detection was realized. In addition, parameters that might affect the efficiency of on-line concentration were studied and equations that described the on-line concentration procedure were deduced.
To assess the role of BAX in drug-induced apoptosis in human colorectal cancer cells, we generated cells that lack functional BAX genes. Such cells were partially resistant to the apoptotic effects of the chemotherapeutic agent 5-fluorouracil, but apoptosis was not abolished. In contrast, the absence of BAX completely abolished the apoptotic response to the chemopreventive agent sulindac and other nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs inhibited the expression of the antiapoptotic protein Bcl-XL, resulting in an altered ratio of BAX to Bcl-XL and subsequent mitochondria-mediated cell death. These results establish an unambiguous role for BAX in apoptotic processes in human epithelial cancers and may have implications for cancer chemoprevention strategies.
Hepatocyte growth factor (HGF) is a multifunctional protein that exerts trophic effects on neural cells. HGF is expressed in normal brains and increased after brain injury. Recent studies suggest that neurons and astrocytes are the main producers of HGF in the brain. Here we report that microglia also produce HGF both in vitro and in vivo. Treatment of cultured microglia with prostaglandin E(2) (PGE(2)), one of the major inflammatory mediators in the brain, induced significant production of HGF, and this induction was suppressed by pretreatment with the adenylate cyclase inhibitor SQ22536, suggesting that the induction of HGF by PGE(2) in microglia proceeds via a cAMP-mediated pathway. We further investigated whether microglia also produce HGF in vivo under the pathological condition of cerebral ischemia. We found that HGF expression was increased after permanent occlusion of the middle cerebral artery (MCA), and double immunohistochemical staining revealed that the most of HGF-positive cells were microglia. PGE(2) level was increased 8 hr after start of MCA occlusion, and this enhancement is in parallel with the increase in HGF expression, suggesting that PGE(2) not only may induce HGF production in microglia in vitro but may also be an inducer in vivo.
In the vascular endothelium of human beings, telomere length is negatively related while the frequency of aneuploidy is positively related to donor age. Both in culture and in vivo the frequency of aneuploidy increases as telomere length is shortened. In this study we explored the relation between telomere length and aneuploidy in cultured human umbilical vein endothelial cells (HUVEC) by: (a) karyotype analysis and fluorescent in situ hybridization (FISH), (b) measurement of the terminal restriction fragments (TRF), and (c) assessment of replicative senescence by the expression of beta-galactosidase. Of 8 HUVEC strains, 7 cell strains lost chromosome 13, as shown by metaphase analysis and FISH of interphase cells. Five strains gained chromosome 11. In addition, five HUVEC strains became hypotetraploid shortly after the loss of chromosome 13. The loss of chromosome 13 was observed as early as PD 20, when mean TRF length was greater than 9 kb and the percentage of cells positive for beta-galactosidase was relatively low. The almost uniform loss of chromosome 13 suggests that this unique type of aneuploidy of HUVEC is the result of a progressive expression of clones with survival advantage.
The fluorescence property of zwitterionic surfactant tetradecylbetaine (C14BE) solutions in the presence of polyvinylpyrrolidone (PVPk30 and PVPk90), or Xanthan (Xc) have been investigated by steady-state fluorescence probe. The intensity ratio of the first and third peaks (I1/I3) of the monomer and that of the excimer to monomer (Ie/Im), and the aggregation number of the aggregates have been monitored. The results showed that C14BE/PVP and C14BE/Xc interactions mostly occur at the micelle surface. The C14BE clusters or micelles are bound to macromolecule chains by electrostatic force for PVP and by hydrogen-bonding for Xc, respectively.
We have targeted the activation of an endogenous chromosomal locus including the human erythropoietin gene using synthetic transcription factors. These transcription factors are targeted to particular DNA sequences in the 5'-flanking region of the erythropoietin gene through engineering of a zinc finger DNA binding domain. The DNA binding domain is linked to a VP16 transcriptional activation domain. We find that these synthetic transcription factors invariably activate transiently transfected templates in which sequences within the 5' flank of the erythropoietin gene are fused to a luciferase reporter. The efficiency of activation under these circumstances at a defined site is dependent on DNA binding affinity. In contrast, only a subset of these same zinc finger proteins is able to activate the endogenous chromosomal locus. The activity of these proteins is influenced by their capacity to gain access to their recognition elements within the chromatin infrastructure. Zinc finger transcription factors will provide a powerful tool to probe the determinants of chromatin accessibility and remodeling within endogenous chromosomal loci.
The c-myc gene is translocated to one of the immunoglobulin genes in Burkitt's lymphoma resulting in deregulated expression of c-myc. Several enhancers have been shown to be important for expression of the immunoglobulin heavy chain gene. Four enhancer regions (murine-hypersensitive sites (MHS) 1, 2, 3, and 4) located 3' of the murine immunoglobulin heavy chain gene play a role in activating expression of the translocated c-myc gene. The enhancer regions also result in a shift in transcriptional initiation from the P2 promoter to P1 that is characteristic of the translocated c-myc allele. We found that the most 3' enhancer region (MHS4) activated the c-myc promoter by 46-fold in the Raji Burkitt's lymphoma cell line, and it was the most active enhancer in these cells. The addition of enhancer regions MHS1,2 and 3 to MHS4 increased c-myc transcription by an additional 3-fold and resulted in the full promoter shift from P2 to P1. By deletion analysis of enhancer region MHS4, we located a region that was critical for the transcriptional activity of MHS4. Electrophoretic mobility shift assay analysis revealed that NF-kappaB/Rel family members bound to this region. Mutation of the NF-kappaB binding site abolished both the enhancer activity and the promoter shift activity of MHS4. An active NF-kappaB site was also identified in the human HS4 enhancer. Inhibition of c-myc promoter activity driven by the immunoglobulin enhancers was observed with expression of a super-repressor IkappaBalpha construct. These results indicate that the NF-kappaB/Rel transcription factors play an important role in the deregulation of the translocated c-myc gene in Burkitt's lymphoma and suggest that interference with NF-kappaB function may represent a new approach to the treatment of Burkitt's lymphoma.
Vacuolar H(+)-ATPase (V-ATPase) binds actin filaments with high affinity (K(d) = 55 nm; Lee, B. S., Gluck, S. L., and Holliday, L. S. (1999) J. Biol. Chem. 274, 29164-29171). We have proposed that this interaction is an important mechanism controlling transport of V-ATPase from the cytoplasm to the plasma membrane of osteoclasts. Here we show that both the B1 (kidney) and B2 (brain) isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain. In pelleting assays containing actin filaments and partially disrupted V-ATPase, B subunits were found in greater abundance in actin pellets than were other V-ATPase subunits, suggesting that the B subunit contained an F-actin binding site. In overlay assays, biotinylated actin filaments also bound to the B subunit. A fusion protein containing the amino-terminal half of B1 subunit bound actin filaments tightly, but fusion proteins containing the carboxyl-terminal half of B1 subunit, or the full-length E subunit, did not bind F-actin. Fusion proteins containing the amino-terminal 106 amino acids of the B1 isoform or the amino-terminal 112 amino acids of the B2 isoform bound filamentous actin with K(d) values of 130 and 190 nm, respectively, and approached saturation at 1 mol of fusion protein/mol of filamentous actin. The B1 and B2 amino-terminal fusion proteins competed with V-ATPase for binding to filamentous actin. In summary, binding sites for F-actin are present in the amino-terminal domains of both isoforms of the B subunit, and likely are responsible for the interaction between V-ATPase and actin filaments in vivo.
3-Alkoxylmethyl-5-alkylpyrazoles undergo regioselective N-arylation with 4-fluoronitrobenzene in the presence of base to yield the corresponding 1-(4-nitro-phenyl)pyrazoles. Further elaboration of these intermediates furnishes a practical synthesis of unsymmetrical 3,5-dialkyl-1-arypyrazoles. A tentative explanation of the observed regioselectivities is provided.
A solid-phase method for the synthesis of 1,2,3, 4-tetrahydro-beta-carboline-containing peptidomimetics has been developed. The key step in the strategy is the Pictet-Spengler condensation of a resin-bound tryptophan-containing fragment with an Fmoc-amino aldehyde.
BACKGROUND: Gastric cancer is generally thought to arise through a series of gastric mucosal changes, but the determinants of the precancerous lesions are not well understood. To identify such determinants, we launched a follow-up study in 1989-1990 among 3433 adults in Linqu County, China, a region with very high rates of gastric cancer. METHODS: Data on cigarette smoking, alcohol consumption, and other characteristics of the participants were obtained by interview in 1989-1990, when an initial endoscopy was taken. At study entry, antibodies to Helicobacter pylori were assayed in 2646 adults (77% of people screened), and levels of serum micronutrients were measured in approximately 450 adults. Follow-up endoscopic and histopathologic examinations were conducted in 1994. Antibodies to H. pylori, levels of serum micronutrients, and other baseline characteristics were compared between subjects whose condition showed progression to dysplasia or gastric cancer from study entry to 1994 and subjects with no change or with regression of their lesions over the same time frame. All P: values are two-sided. RESULTS: The presence of H. pylori at baseline was associated with an increased risk of progression to dysplasia or gastric cancer (odds ratio [OR] = 1.8; 95% confidence interval [CI] = 1.2-2.6). The risk of progression to dysplasia or gastric cancer also was moderately increased with the number of years of cigarette smoking. In contrast, the risk of progression was decreased by 80% (OR = 0.2; 95% CI = 0.1-0.7) among subjects with baseline ascorbic acid levels in the highest tertile compared with those in the lowest tertile, and there was a slightly elevated risk in those individuals with higher levels of alpha-tocopherol. CONCLUSIONS: H. pylori infection, cigarette smoking, and low levels of dietary vitamin C may contribute to the progression of precancerous lesions to gastric cancer in this high-risk population.
BACKGROUND: The renin-angiotensin system is thought to be critical for the development of cardiac hypertrophy, whereas the role of the angiotensin II type 2 (AT(2)) receptor in the process is not defined. Using the AT(2) receptor-null (Agtr2-) mouse, we tested the hypothesis that the AT(2) receptor could exert an antigrowth effect in cardiac hypertrophy. METHODS AND RESULTS: Cardiac hypertrophy was induced by suprarenal abdominal aortic banding in 10- to 12-week-old Agtr2- and wild-type (Agtr2+) mice for 6 or 12 weeks. Carotid arterial pressure was not different between the strains, although aortic banding increased arterial pressure by approximately 40 mm Hg. Aortic banding increased the heart-weight/body-weight ratio and the cross-sectional area of cardiomyocytes by 15%, resulting in comparable cardiomyocyte hypertrophy in the 2 strains. In contrast, coronary arterial thickening and perivascular fibrosis, determined by the media/lumen-area ratio and the collagen/vessel-area ratio, respectively, were 50% greater in Agtr2- than in Agtr2+ mice after banding, whereas these parameters were similar in sham-operated mice. Radioligand binding studies using the whole heart and immunohistochemistry showed that AT(2) receptor expression was limited and localized in the coronary artery and perivascular region. CONCLUSIONS: These results suggest that the AT(2) receptor mediates an inhibitory effect on coronary arterial remodeling, such as medial hypertrophy and perivascular fibrosis in response to pressure overload, and an activation of the renin-angiotensin system.
To determine the feasibility of cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) studies using cryopreserved cells, we compared lymphocyte proliferation assays (LPA), responder cell frequency (RCF), interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) production using fresh and cryopreserved peripheral blood mononuclear cells (PBMCs) from 53 HIV-infected patients and 15 uninfected controls. Qualitative CMV LPA results were concordant in >/=84% of the specimens from either HIV-infected patients or controls. Proliferation-based RCF, IL-2, and IFN-gamma comparisons showed that cryopreservation reduces the number of CMV-specific responders and decreases cytokine secretion, without changing the rank order of the results (p <.01). In contrast, the number of flow cytometry-enumerated IFN-gamma-producing CD4+ cells was not significantly changed by cryopreservation. In HIV-infected patients, the differences between fresh and frozen cell assays were not influenced by CD4 cell numbers or HIV viral load. These data indicate that cryopreserved cells are suitable for longitudinal studies of the CMV-specific immune response in HIV-infected patients and uninfected controls.
BACKGROUND AND PURPOSE: To simulate human stroke, we developed a model of focal cerebral embolic ischemia in the unanesthetized rat. Using this model, we tested the hypothesis that intra-arterial administration of TNK-tPA, a fibrin specific second generation thrombolytic agent, is effective in reducing ischemic volume without increasing intra-cerebral hemorrhage. METHODS: Under anesthesia, a catheter was inserted to the origin of the MCA of male Wistar rats. Forty-five minutes after recovery from anesthesia, the MCA was occluded in the awake rat by a single fibrin rich clot placed via the catheter. TNK-tPA (1.5 mg/kg) was administered intraarterially via the catheter at either 2 h or 4 h after stroke. All rats were sacrificed at 48 h after ischemia. Neurological deficits, gross hemorrhage and ischemic lesion volume were measured. RESULTS: A clot was detected at the origin of the MCA 4 h after MCA occlusion in the awake rats (n=4). Rats (n=12) subjected to MCA occlusion showed immediate neurological deficits which persisted for 48 h of ischemia. Ischemic rats had a lesion volume of 38.2+/-3.8% and 25% of rats exhibited gross hemorrhage. Ischemic rats (n=10) treated with TNK-tPA at 2 h showed a significant (P<0.05) reduction of neurological deficits, body weight loss and infarct volume (22.8+/-2.1%) without an increase in gross hemorrhage (10%) compared with the non treated ischemic rats (25%). Although treatment with TNK-tPA of ischemic rats (n=12) at 4 h did not significantly (P=0.06) reduce infarct volume (28.6+/-3.0%), it also did not increase gross hemorrhage (25%) compared with the control group (25%). CONCLUSIONS: This study demonstrates that intraarterial administration of TNK-tPA at 2 h of ischemia in the unanesthesthetized rat is effective in reducing neurological deficits and ischemic lesion volume without increasing hemorrhagic transformation and that administration of TNK-tPA at 4 h of ischemia does not increase the incidence of hemorrhagic transformation.
The presenilin proteins are involved in the proteolytic processing of transmembrane proteins such as Notch/lin-12 and the beta-amyloid precursor protein (betaAPP). Mutation of a conserved cysteine (Cys60Ser) in the C. elegans presenilin sel-12 has a loss-of-function effect on Notch/lin-12 processing similar to that of null mutations in sel-12. In contrast, in mammalian cells, most missense mutations increase gamma-secretase cleavage of betaAPP. We report here that mutation of this conserved cysteine (Cys92Ser) in human presenilin 1 confers a loss-of-function effect in C. elegans, but causes increased A beta42 secretion in mammalian cells. These data suggest that the role of presenilins in Notch/lin-12 signalling and betaAPP processing are either separately regulated activities or independent activities of the presenilins.
Transposon mutagenesis and complementation studies previously identified a gene (xabB) for a large (526kDa) polyketide-peptide synthase required for biosynthesis of albicidin antibiotics and phytotoxins in the sugarcane leaf scald pathogen Xanthomonas albilineans. A cistron immediately downstream from xabB encodes a polypeptide of 343aa containing three conserved motifs characteristic of a family of S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases. Insertional mutagenesis and complementation indicate that the product of this cistron (designated xabC) is essential for albicidin production, and that there is no other required downstream cistron. The xabB promoter region is bidirectional, and insertional mutagenesis of the first open reading frame (ORF) in the divergent gene also blocks albicidin biosynthesis. This divergent ORF (designated thp) encodes a protein of 239aa displaying high similarity to several IS21-like transposition helper proteins. The thp cistron is not located in a recognizable transposon, and is probably a remnant from a past transposition event that may have contributed to the development of the albicidin biosynthetic gene cluster. Failure of 'in trans' complementation of thp indicates that a downstream cistron transcribed with thp is required for albicidin biosynthesis.
Selectively regulating gene expression in bacteria has provided an important tool for studying gene function. However, well-regulated gene control systems have been restricted primarily for use in laboratory non-pathogenic strains of bacteria (e.g. Escherichia coli, Bacillus subtilis). The development of analogous systems for use in bacterial pathogens such as Staphylococcus aureus would significantly enhance our ability to examine the contribution of any given gene product to pathogen growth and viability. In this report, we adapt, examine and compare three regulated gene expression systems in S. aureus, which had previously been used in B. subtilis. We demonstrate that all three systems function and exhibit titratable induction, together covering a dynamic range of gene expression of approximately 3000-fold. This dynamic range correlates well with the physiological expression levels of cellular proteins. Importantly, we show that one of these systems, the Spac system, is particularly useful for examining gene essentiality and creating specific conditional lethal phenotypes. Moreover, we find that titration of selective target gene products using this system allows direct demonstration of antibiotic mode of action.