Biomedical subjects

A Chen

Publications and source records attributed to A Chen.

At least 73 records · Page 4Linked to original sources

PubMed2004

Search for extraterrestrial point sources of neutrinos with AMANDA-II.

We present the results of a search for point sources of high-energy neutrinos in the northern hemisphere using AMANDA-II data collected in the year 2000. Included are flux limits on several active-galactic-nuclei blazars, microquasars, magnetars, and other candidate neutrino sources. A search for excesses above a random background of cosmic-ray-induced atmospheric neutrinos and misreconstructed downgoing cosmic-ray muons reveals no statistically significant neutrino point sources. We show that AMANDA-II has achieved the sensitivity required to probe known TeV gamma-ray sources such as the blazar Markarian 501 in its 1997 flaring state at a level where neutrino and gamma-ray fluxes are equal.

Journal Article↗

PubMed2004

Molecular cloning and functional expression of a serotonin receptor from the Southern cattle tick, Boophilus microplus (Acari: Ixodidae).

A full-length cDNA encoding a 5-hydroxytryptamine (5-HT) receptor from the Southern cattle tick, Boophilus microplus, was isolated using a strategy based on sequence homology among G protein-coupled receptors. The deduced amino acid sequence revealed highest identity with Drosophila melanogaster 5HT-dro2A (Z11489, 50.8%) and 5HT-dro2B (Z11490, 49.5%) receptors. The receptor was transiently expressed in mammalian HEK293 cells, and Western blot analysis showed the expected 43.3 kDa band. In these cells, application of 5-HT (10 microm) inhibited forskolin-induced cAMP synthesis by 26%. The results indicate that the tick receptor is an invertebrate 5-HT1-like receptor that couples to Galphai protein.

Amino Acid Sequence↗

PubMed2004

Ciguatera fish poisoning in industrial ship crewmembers: a retrospective study in a seaport general practice in Trinidad and Tobago.

The objective of this study was to outline the epidemiology of Ciguatera fish poisoning as seen in a general practice serving two industrial seaports in Trinidad and Tobago, in order to highlight the potential public health implications. A retrospective study was undertaken of all the cases of Ciguatera fish poisoning identified between November 1, 1992 and October 31, 1998 in a seaport general practice to identify signs, symptoms and treatment. An investigation of one outbreak was undertaken. Four outbreaks affecting 42 male ship crewmembers were identified. The suspect fish were caught in northern Caribbean waters en route to Trinidad and Tobago. The most common early symptoms were diarrhoea, vomiting, abdominal cramps, pruritus and tiredness. In the third outbreak, dysaesthesia was common. Progression to muscular weakness, ataxic gait, unsteadiness and other neurotoxic signs were seen in moderate to severe disease. Hypotension was an important prognostic sign in the initial case. Treatment was symptomatic and supportive and included vitamins B12 and BCO, folic acid, prostigmine, steroids and antihistamines as indicated. In the investigation of the second outbreak, the relative risk of 'eating fish meat' was 5 (95% CI 1.45, 17.27, p < 0.0001). Abdominal symptoms, pruritus, and muscle weakness with a history of consuming a fish-meal were diagnostic indicators of 'ciguatera fish poisoning.' All cases were industrial ship crewmembers. It is suggested that increased clinician awareness with early and appropriate treatment, and focussed public health intervention may help limit the potential public health impact of ciguatera poisoning in industrial ship crewmembers and other fish-consuming communities in the future.

Adult↗

PubMed2003

The cytopathic 18f strain of Hepatitis A virus induces RNA degradation in FrhK4 cells.

The mechanism responsible for the induction of apoptosis by the rapidly replicating HM175/18f strain of Hepatitis A virus (HAV) was investigated. Full length HAV RNA and viral capsid protein VP1 were detected in 18f infected cells at earlier times post-infection than in HM175/clone 1 infected cells. Analysis of total cellular RNA from HM175/18f infected FrhK4 cells by denaturing agarose gel electrophoresis and Northern blot hybridization revealed extensive degradation of both the 28S and 18S ribosomal RNA (rRNA) molecules. Similar degradation was observed when these cells were infected with Human coxsackievirus B1, a fast replicating enterovirus. In contrast, the parental strain of 18f, HM175/clone 1 did not induce RNA degradation. Inhibition of RNA degradation correlated with inhibition of virus replication. The pattern of rRNA degradation resembled degradation of rRNAs by RNase L, an enzyme activated in interferon-treated cells following infection with certain viruses. Ribosomal RNA degradation was accompanied by the reduction in the levels of several cellular RNAs including those for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. Interferon mRNAs could not be detected in either infected or mock-infected control cells, and STAT1, a key regulator of interferon action was not phosphorylated following virus infection. These results reveal a heretofore-undescribed pathway that involves the regulation of RNA degradation and apoptosis following HAV/18f replication in FrhK4 cells.

Animals↗

PubMed2003

Recurrent minimal change disease post-allograft renal transplant.

A young adult was diagnosed with steroid-resistant minimal change disease (MCD) without evidence of focal segmental glomerulosclerosis (FSGS) despite serial and detailed sectioning and screening of the renal biopsy. He received initial treatment with steroids and then cyclosporine plus low-dose steroids for 2 years. Renal function progressively deteriorated due to resistance to steroid and cyclosporine. Two months after initiation of hemodialysis, the patient received a living-related allogenic kidney transplant. However, recurrent nephrotic syndrome and renal insufficiency occurred after transplantation. In spite of aggressive treatment, renal function showed no significant improvement. The kidney graft was removed 2 weeks after transplantation. Serial sectioning and thorough sampling and screening revealed no evidence of FSGS, but light microscopy and electron microscopy showed the typical morphologic pattern of MCD, corresponding to the pretransplant diagnosis. We believe that this is the first reported case of recurrent MCD after renal transplant.

Adult↗

PubMed2003

Serotonin type II receptor activation facilitates synaptic plasticity via N-methyl-D-aspartate-mediated mechanism in the rat basolateral amygdala.

The modulation of synaptic plasticity by serotonin type II (5-hydroxytryptamine type II (5-HT(2)))-receptor stimulation was explored using intracellular, field potential and Fura-2 fluorescence image recordings in a rat amygdala slice preparation. Bath application of 5HT(2) receptor agonist 1-(2,5)-dimethoxy-4-iodophen-2-aminopropane (DOI) transformed theta-burst-stimulated (TBS) synaptic plasticity from short-term potentiation to long-term potentiation. DOI enhanced N-methyl-D-aspartate (NMDA) receptor-mediated potentials and calcium influx without affecting the resting membrane potential or input resistance of the neurons. In contrast, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor-mediated excitatory synaptic responses were unaffected by DOI. The facilitating effects of DOI were blocked by the 5-HT(2) receptor antagonist, ketanserin, and by the 5-HT(2C)-receptor selective antagonist, RS102221. These results indicate that 5-HT(2)-receptor activation enhances NMDA receptor-mediated synaptic function in the basolateral amygdala (BLA).

Amygdala↗

PubMed2003

Low dose and gene gun immunization with a hepatitis C virus nonstructural (NS) 3 DNA-based vaccine containing NS4A inhibit NS3/4A-expressing tumors in vivo.

The hepatitis C virus (HCV) protease and helicase encompasses the nonstructural (NS) 3 protein and the cofactor NS4A, which targets the NS3/4A-complex to intracellular membranes. We here evaluate the importance of NS4A in NS3-based genetic immunogens. A full-length genotype 1 NS3/4A gene was cloned into a eucaryotic expression vector in the form of NS3/4A and NS3 alone. Transient transfections revealed that the inclusion of NS4A increased the expression levels of NS3. Subsequently, immunization with the NS3/4A gene primed 10- to 100-fold higher levels of NS3-specific antibodies as compared to immunization with the NS3 gene. Humoral responses primed by the NS3/4A gene had a higher IgG2a/IgG1 ratio (>20) as compared to the NS3 gene (3.0), suggesting a T helper 1-skewed response. Low dose i.m. (10 microg) immunization with the NS3/4A gene inhibited the growth of NS3/4A-expressing tumor cells in vivo, whereas the NS3 gene alone or NS3 protein did not. We then evaluated the efficiency of the NS3/4A gene administered by the gene gun, at the same doses used for humans, in priming cytotoxic T lymphocyte (CTL) responses. Three to four 4 microg doses of the NS3/4A gene primed CTL at a precursor frequency of 2-4%, which inhibited the growth of NS3/4A-expressing tumor cells in vivo. Thus, NS4A enhances the expression levels and immunogenicity of NS3, and an NS3/4A gene delivered transdermally could be a therapeutic vaccine candidate.

Animals↗

PubMed2002

Pulmonary function changes and immunomodulation of cytokine expression by zafirlukast after sensitization and allergen challenge in brown Norway rats.

BACKGROUND: The cysteinyl leukotrienes are known important mediators in bronchial asthma. OBJECTIVE: Our purpose was to evaluate the effect of zafirlukast on the late-phase reaction, bronchial hyper-responsiveness (BHR) and T cell-related cytokine mRNA expression in ovalbumin (OA)-sensitized brown Norway rats (BNRs). METHODS: Thirty BNRs were equally divided into three groups. Group I and II animals were sensitized and then provoked with OA. Zafirlukast was given intraperitoneally (i.p.) to group I animals prior to provocation. Group II animals received i.p. normal saline. Group III animals (controls) were not sensitized and breathed aerosolized saline. After OA provocation, the animals were anaesthetized. Pulmonary function tests (PFT) were performed at baseline and after varying doses of acetylcholine. Thereafter, bronchoalveolar lavage (BAL) was performed and the lungs were examined histologically. Total RNA was extracted from lung tissue and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using primers for IL-2, IL-4, IL-5, IL-6, IL-10, TNF-alpha, IFN-gamma, iNOS and beta-actin. RESULTS: Group II OA-treated BNRs had worse PFT results, more severe bronchoconstriction in response to acetylcholine, and more severe inflammation in lung tissue than the other two groups. Group II had higher IL-2, IL-4, IL-10 and IFN-gamma cytokine levels in BAL fluid and higher IL-2, IL-4, IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha and iNOS mRNA levels when compared with group I. CONCLUSION: Zafirlukast is effective in preventing late-phase bronchoconstriction and BHR, reducing inflammatory response, and decreasing IL-2, IL-4, IL-5, IL-6, IL-10 and IFN-gamma and iNOS mRNA expression.

Adjuvants, Immunologic↗

PubMed2002

Penetration of levofloxacin into skin tissue after oral administration of multiple 750 mg once-daily doses.

OBJECTIVE: To probe the pharmacokinetic basis for the use of levofloxacin for complicated skin and skin-structure infections (SSSIs) at a once-daily dosage of 750 mg by investigating its penetration into skin tissue. METHOD: Ten healthy volunteers were administered three oral, once-daily 750 mg doses of levofloxacin, and levofloxacin concentrations were subsequently measured over time (0.5-24 h) in skin-punch biopsy tissue and plasma. RESULTS: Skin tissue concentrations consistently exceeded those in plasma at every time point, with tissue/plasma ratios of 1.37 +/- 0.81 for peak concentration and 1.97 +/- 0.35 for area under the concentration versus time curve. Three of the ten subjects reported treatment-emergent adverse events (AEs) that were considered unrelated to treatment. An 11th subject who had enrolled in the study withdrew after AEs of mild severity that were possibly related to the study drug. CONCLUSION: The results support the clinical usage of levofloxacin 750 mg once-daily for complicated SSSIs.

Administration, Oral↗

PubMed2002

Powder compaction properties of sodium starch glycolate disintegrants.

The compaction behavior of three "as supplied" commercially available grades of sodium starch glycolate (SSG), Explotab, Primojel, and Vivastar P, was investigated at compression speeds of 0.17 and 30 mm/sec. The results suggested that the three "as supplied" materials exhibit different compression and compaction behavior. Primojel and Explotab exhibited similar compactibility, whereas Vivastar P produced compacts of poor integrity. This behavior was not mirrored in the compressibility of the powders, where Vivastar P and Explotab exhibited similar performance. The materials were studied using x-ray diffraction, scanning electron microscopy, Carr's compressibility index, and swelling volume. In terms of material characteristics, all the products exhibited similar swelling in water. Primojel and Explotab retained most of the crystallographic order from the parent potato starch and exhibited comparable particle surface topographies. Vivastar P contained the lowest moisture level. However, it is not clear if the poor compactibility of Vivastar P is due to differences in moisture content, the reduced surface topography, or subtle differences in the SSG polymer structures (substitution, cross-linking, and crystallinity). Overall, even though the three commercial grades of sodium starch glycolate are successfully used as disintegrants, they do exhibit differences in their "as supplied" powder mechanical properties: Primojel and Explotab exhibit similar compactibility, whereas Vivastar P is poorly compactable but exhibits similar compressibility to Explotab. These observations may have implications when formulating poorly compactable or moisture-sensitive drugs.

Chemistry, Pharmaceutical↗

PubMed2001

A rheostatic mechanism for T-cell inhibition based on elevation of activation thresholds.

The activation of discrete T-cell responses depends on the triggering of individualized threshold numbers of T-cell receptors (TCRs). The results of this study indicate that the lipocalin placental protein 14 (PP14), a T-cell inhibitor produced by cells of the reproductive and hematopoietic systems, mediates its anti-inflammatory activity by elevating the T-cell activation threshold, thereby rendering T cells less sensitive to stimulation. Significantly, the data demonstrate hierarchical sensitivity of selected cytokine responses to PP14-mediated inhibition, with the hierarchy reflecting their respective activation thresholds. These findings suggest a novel paradigm for immunoinhibition wherein negative regulators can finely tune, rather than inactivate, T-cell responses, and thereby skew the cytokine output of immunologic responses.

Amniotic Fluid↗

PubMed2001

Geranylgeranylglyceryl phosphate synthase. Characterization of the recombinant enzyme from Methanobacterium thermoautotrophicum.

Geranylgeranylglyceryl diphosphate synthase (GGGP synthase) catalyzes alkylation of (S)-glyceryl phosphate [(S)-GP] by geranylgeranyl diphosphate (GGPP) to produce (S)-geranylgeranylglyceryl phosphate [(S)-GGGP]. This reaction is the first committed step in the biosynthesis of ether-linked membrane lipids in Archaea. The gene encoding GGGP synthase from Methanobacterium thermoautotrophicum was cloned using probes designed from the N-terminal sequence determined from the purified enzyme. The open reading frame, which encoded a protein of 245 amino acids, was inserted into a pET expression vector and expressed in Escherichia coli. The recombinant GGGP synthase was purified to homogeneity. The enzyme is active as a homopentamer, as determined by size exclusion chromatography and equilibrium sedimentation experiments. GGGP synthase has optimal activity at 55 degrees C in pH 8.0 buffer containing 1 mM MgCl(2). V(max) = 4.0 +/- 0.1 micromol min(-1) mg(-1) (k(cat) = 0.34 +/- 0.03 s(-1) for pentameric GGGP synthase assuming all subunits are fully active), K(m)((S)-GP) = 13.5 +/- 1.0 microM, and K(m)(GGPP) = 506 +/- 47 nM. These steady-state catalytic constants were identical to those for enzyme isolated from cell extracts of M. thermoautotrophicum [Chen, A., Zhang, D., and Poulter, C. D. (1993) J. Biol. Chem. 268, 21701-21705]. Alignment of seven putative archaeal GGGP synthase sequences revealed a number of highly conserved residues consisting of five aspartate/glutamates, three serine/threonines, two prolines, and five glycines, including a conserved GGG motif.

Alkyl and Aryl Transferases↗

PubMed2001

Neoceptor concept based on molecular complementarity in GPCRs: a mutant adenosine A(3) receptor with selectively enhanced affinity for amine-modified nucleosides.

Adenosine A(3) receptors are of interest in the treatment of cardiac ischemia, inflammation, and neurodegenerative diseases. In an effort to create a unique receptor mutant that would be activated by tailor-made synthetic ligands, we mutated the human A(3) receptor at the site of a critical His residue in TM7, previously proposed to be involved in ligand recognition through interaction with the ribose moiety. The H272E mutant receptor displayed reduced affinity for most of the uncharged A(3) receptor agonists and antagonists examined. For example, the nonselective agonist 1a was 19-fold less potent at the mutant receptor than at the wild-type receptor. The introduction of an amino group on the ribose moiety of adenosine resulted in either equipotency or enhanced binding affinity at the H272E mutant relative to wild-type A(3) receptors, depending on the position of the amino group. 3'-Amino-3'-deoxyadenosine proved to be 7-fold more potent at the H272E mutant receptor than at the wild-type receptor, while the corresponding 2'- and 5'-amino analogues did not display significantly enhanced affinities. An 3'-amino-N(6)-iodobenzyl analogue showed only a small enhancement at the mutant (K(i) = 320 nM) vs wild-type receptors. The 3'-amino group was intended for a direct electrostatic interaction with the negatively charged ribose-binding region of the mutant receptor, yet molecular modeling did not support this notion. This design approach is an example of engineering the structure of mutant receptors to recognize synthetic ligands for which they are selectively matched on the basis of molecular complementarity between the mutant receptor and the ligand. We have termed such engineered receptors "neoceptors", since the ligand recognition profile of such mutant receptors need not correspond to the profile of the parent, native receptor.

Adenosine↗

PubMed2001

Induction of antitumor immunity via intratumoral tetra-costimulator protein transfer.

Our group recently described a novel two-step Fc(gamma1) fusion protein transfer method, which entails the docking of Fc(gamma1) fusion proteins onto cells precoated with chemically palmitated protein A (pal-prot A). In the present study, we have adapted this protein transfer method, originally used in an ex vivo context, for in situ tumor cell engineering, and in so doing, we have evaluated its utility for the induction of antitumor immunity via combinatorial costimulator protein transfer on to tumor cell surfaces. The feasibility of "painting" cells with preformed conjugates of a murine B7-1 costimulator derivative, B7-1.Fc(gamma1), and pal-prot A in a single step was first established ex vivo. Next, B7-1.Fc(gamma1):pal-prot A transfer was accomplished in vivo by directly injecting the preformed conjugates into highly aggressive L5178Y-R lymphomas grown intradermally in syngeneic mice. The presence of cell surface-associated B7-1 epitopes on cells of the injected tumors was documented by flow cytometric analysis of cells recovered subsequently from the injected tumors. B7-1.Fc(gamma1), along with Fc(gamma1) fusion protein derivatives of three additional costimulators (Fc(gamma1).4-1BBL, CD48.Fc(gamma1), and Fc(gamma1).CD40L) geared toward a variety of immune effectors, were together preconjugated with pal-prot A and injected directly into tumor beds. Significantly, this "tetra-costimulator" combination, delivered intratumorally, induced complete tumor regression in approximately 45% of treated mice, whereas control injections of pal-prot A alone had no therapeutic effect. Furthermore, there was evidence for systemic antitumor immunity in that tumor-specific CTLs were detected in spleens recovered from cured mice, and these mice were uniformly protected against tumor rechallenge at distant tumor sites. Hence, combinatorial costimulator transfer, coupled to intratumoral delivery, may have special advantages for the induction of antitumor immunity.

4-1BB Ligand↗

PubMed2001

Methylation in hMLH1 promoter interferes with its binding to transcription factor CBF and inhibits gene expression.

Microsatellite instability (MSI) is caused by the dysfunction of mismatch repair genes, such as hMLH1, hMSH2. Loss of hMLH1 expression and methylation of CpG sites in hMLH1 promoter are frequently present in sporadic colorectal cancer with MSI. In this study, by transient transfection assay with constructs containing different lengths of hMLH1 promoter and a luciferase reporter gene, we located a proximal region of hMLH1 promoter, which plays a main role in regulating the gene. The fact that luciferase activities were high in all host cell lines regardless of their hMLH1 expression levels indicates that the transcription machinery is intact even in non-expressing cells. When hMLH1 promoter was in vitro methylated before transfection, the luciferase activities in the transfectants were significantly reduced. This observation indicates that methylation causes the inhibition of hMLH1 promoter activity. By electrophoretic mobility shift assay (EMSA), we identified a CCAAT box in this region, which specifically bound transcription factor CBF. Mutations in CCAAT box not only inhibited its binding to CBF factor, but also reduced its ability to drive the expression of luciferase gene. The role of CBF in activating transcription was further substantiated by inhibition of promoter activity with a plasmid expressing a dominant negative CBF-B mutant. Methylation at a CpG site two base pairs upstream of the CCAAT box inhibited the binding of CBF to CCAAT box. We conclude that methylation of an adjacent CpG site inhibits binding of the CBF transcription to the corresponding CCAAT box, and is one of the causes of hMLH1 gene silencing in colon cancer cells.

Adaptor Proteins, Signal Transducing↗

PubMed2001

The RING domains of the promyelocytic leukemia protein PML and the arenaviral protein Z repress translation by directly inhibiting translation initiation factor eIF4E.

The promyelocytic leukemia protein (PML) is a mammalian regulator of cell growth which is characteristically disrupted in acute promyelocytic leukemia and by a variety of viruses. PML contains a RING domain which is required for its growth-suppressive and antiviral properties. Although normally nuclear, in certain pathogenic conditions, including arenaviral infection, PML is relocated to the cytoplasm, where its functions are poorly understood. Here, we observe that PML and arenavirus protein Z use regions around the first zinc-binding site of their respective RING domains to directly interact, with sub-micromolar affinity, with the dorsal surface of translation initiation factor eIF4E, representing a novel mode of eIF4E recognition. PML and Z profoundly reduce the affinity of eIF4E for its substrate, the 5' 7-methyl guanosine cap of mRNA, by over 100-fold. Association with the dorsal surface of eIF4E and direct antagonism of mRNA cap binding by PML and Z lead to direct inhibition of translation. These activities of the RING domains of PML and Z do not involve ubiquitin-mediated protein degradation, in contrast to many RINGs which have been observed to do so. Although PML and Z have well characterized physiological functions in regulation of growth and apoptosis, this work establishes the first discrete biochemical mechanism which underlies the biological activities of their RING domains. Thus, we establish PML and Z as translational repressors, with potential contributions to the pathogenesis of acute promyelocytic leukemia and variety of viral infections.

Arenavirus↗

PubMed2001

Changes in forest biomass carbon storage in China between 1949 and 1998.

The location and mechanisms responsible for the carbon sink in northern mid-latitude lands are uncertain. Here, we used an improved estimation method of forest biomass and a 50-year national forest resource inventory in China to estimate changes in the storage of living biomass between 1949 and 1998. Our results suggest that Chinese forests released about 0.68 petagram of carbon between 1949 and 1980, for an annual emission rate of 0.022 petagram of carbon. Carbon storage increased significantly after the late 1970s from 4.38 to 4.75 petagram of carbon by 1998, for a mean accumulation rate of 0.021 petagram of carbon per year, mainly due to forest expansion and regrowth. Since the mid-1970s, planted forests (afforestation and reforestation) have sequestered 0.45 petagram of carbon, and their average carbon density increased from 15.3 to 31.1 megagrams per hectare, while natural forests have lost an additional 0.14 petagram of carbon, suggesting that carbon sequestration through forest management practices addressed in the Kyoto Protocol could help offset industrial carbon dioxide emissions.

Biomass↗

PubMed2001

Constitutive activation of A(3) adenosine receptors by site-directed mutagenesis.

The objective of this study was to create constitutively active mutant human A(3) adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors. A(3) ARs mutated in transmembrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in both functional assays. The potency of the A(3) agonist Cl-IB-MECA (1-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide) in PLC activation was enhanced by at least an order of magnitude over wild type (EC(50) 951 nM) in R108A and A229E mutant receptors. Cl-IB-MECA was much less potent (>10-fold) in C88F, Y109F, and Y282F and mutants or inactive following double mutation of the DRY motif. The degree of constitutive activation was more pronounced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cytosolic region were responsible for constraining the receptor in a G protein-uncoupled conformation.

Adenosine↗