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Biomedical subjects

Xiang Xu

Publications and source records attributed to Xiang Xu.

At least 19 recordsLinked to original sources

Neutrophil histamine contributes to inflammation in mycoplasma pneumonia.

Mycoplasmas cause chronic inflammation and are implicated in asthma. Mast cells defend against mycoplasma infection and worsen allergic inflammation, which is mediated partly by histamine. To address the hypothesis that mycoplasma provokes histamine release, we exposed mice to Mycoplasma pulmonis, comparing responses in wild-type and mast cell-deficient KitW-sh/KitW-sh (W-sh) mice. Low histamine levels in uninfected W-sh mice confirmed the conventional wisdom that mast cells are principal sources of airway and serum histamine. Although mycoplasma did not release histamine acutely in wild-type airways, levels rose up to 50-fold above baseline 1 week after infection in mice heavily burdened with neutrophils. Surprisingly, histamine levels also rose profoundly in infected W-sh lungs, increasing in parallel with neutrophils and declining with neutrophil depletion. Furthermore, neutrophils from infected airway were highly enriched in histamine compared with naive neutrophils. In vitro, mycoplasma directly stimulated histamine production by naive neutrophils and strongly upregulated mRNA encoding histidine decarboxylase, the rate-limiting enzyme in histamine synthesis. In vivo, treatment with antihistamines pyrilamine or cimetidine decreased lung weight and severity of pneumonia and tracheobronchitis in infected W-sh mice. These findings suggest that neutrophils, provoked by mycoplasma, greatly expand their capacity to synthesize histamine, thereby contributing to lung and airway inflammation.

Animals↗

Anti-inflammatory effects of a novel peptide designed to bind with NF-kappaB p50 subunit.

AIM: To explore the anti-inflammatory effects of a novel peptide designed to bind with the NF-kappaB p50 subunit. METHODS: The affinity of the peptide binding with p50 was measured with a biosensor. Levels of tumor necrosis factor-alpha(TNF-alpha) and interleukin 6 (IL-6) from a human acute monocytic leukemia cell line (THP-1) treated with lipopolysaccharide (LPS) were measured using the ELISA method. In vivo anti-inflammatory effects of the peptide were tested with phorbol myristate acetate (PMA)-induced ear edema and zymosan A-induced peritonitis in mice. RESULTS: The peptide has the ability to interact with the NF-kappaB p50 subunit and can effectively inhibit TNF-alpha and IL-6 production in the THP-1 cell line, PMA-induced ear edema and zymosan A-induced peritonitis in mice. CONCLUSION: The peptide may have therapeutic potential for the treatment of local acute inflammation.

Animals↗

[Determination of acacetin in Xiangjuganmao Keli (no sweet) by HPLC].

OBJECTIVE: To establish a method for the determination of acacetin in Xiangjuganmao Keli (no sweet). METHODS: Acacetin in powdered herb was extracted by ultrasonator with methanol and was hydrolyzed with hydrochloric acid. Separation was accomplished on an ODS reversed phase column (5 microm, 4.6 x 250 mm) with a mobile phase of methanol-water-acetic acid(350: 150: 2). The detective wavelength was at 340 nm. RESULTS: The method was accurate, the results were stable and reproducible. The linear range of calibration cure was within the concentration of 2.00 - 10.00 microg/ml (r = 0.9998). The average extraction recovery was 99.9% (n = 6), RSD = 0.41% (n = 6). CONCLUSION: The method is simple, convenient, sensitive, and reproducible for quality control of Xiangjuganmao Keli (no sweet).

Chromatography, High Pressure Liquid↗

Effects of nuclear factor-kappaB on rat hepatocyte regeneration and apoptosis after 70% portal branch ligation.

AIM: To detect the DNA binding activity of nuclear factor-kappaB (NF-kappaB) in rat hepatocyte and to investigate the effects of NF-kappaB on rat hepatocyte regeneration and apoptosis after 70% portal branch ligation. METHODS: Sixty Wistar rats were randomly divided into control group and portal branch ligation group. The animals were killed 12 h, 1, 2, 3, 7, and 14 d after surgery to determine the contents of plasma ALT. Hepatocytes were isolated and nuclear protein was extracted. DNA binding activity of NF-kappaB was measured by EMSA. Hepatocyte regeneration and apoptosis were observed under microscope by TUNEL staining. The ultrastructural changes of liver were observed under electron microscope. RESULTS: Seventy percent portal branch ligation produced atrophy of the ligated lobes and the perfused lobes underwent compensatory regeneration, the total liver weight and plasma ALT levels were maintained at the level of sham-operated animals throughout the experiment. After 2 d of portal branch ligation, DNA binding activity of NF-kappaB in hepatocyte increased and reached its peak, the number of apoptotic hepatocyte in the ligated lobes and the number of mitotic hepatocyte in the perfused lobes also reached their peak. Typical apoptotic changes and evident fibrotic changes in the ligated lobes were observed under electron microscope. CONCLUSION: After 70% portal branch ligation, DNA binding activity of NF-kappaB in hepatocyte is significantly increased and NF-kappaB plays an important role in hepatocyte regeneration and apoptosis.

Animals↗

Clinical characteristics and distribution of hepatitis B virus genotypes in Guangxi Zhuang population.

AIM: To investigate the distribution of HBV genotypes and their YMDD mutations in Guangxi Zhuang population, China, and to study the relationship between HBV genotypes and clinical types of HB, ALT, HBV DNA, HBe system as well as the curative effect of Lamivudine (LAM) on hepatitis B. METHODS: A total of 156 cases were randomly chosen as study subjects from 317 patients with chronic hepatitis B (CHB). HBV genotypes were determined by PCR-microcosmic nucleic acid cross-ELISA. YMDD mutations were detected by microcosmic nucleic acid cross-nucleic acid quantitative determination. HBV DNA was detected by fluorescence ratio PCR analysis. LAM was given to 81 cases and its curative effect was observed by measuring ALT, HBV DNA load, HBeAg, and HBeAg/HBeAb conversion rate. RESULTS: HBV genotypes B, C, D, and non-classified genotypes were found in Guangxi Zhuang population. accounting for 25.6%, 47.4%, 58.3%, and 16.0%, respectively. Seventy-four cases were CD-, CB-, BD-mixed genotypes (47.7%). Forty-six (29.5%) cases had YMDD mutations. Genotype B was mostly found in mild and moderate CHB patients. Genotypes C, D and mixed genotype mostly occurred in severe CHB cases. Genotypes D and CD HBV-infected patients had higher ALT and HBV DNA than patients with other types of HBV infection. There was no significant difference among the genotypes in YMDD mutations, clinical types, ALT and HBV DNA level. Non-classified types geno had a significantly lower positive rate of HBeAg than other genotypes (c2=12.841, P<0.05). There was no significant difference in ALT recovery rate, HBV DNA load, HBeAg, and HBeAg/HBeAb conversion rate, 48 wk after LAM treatment between groups of genotypes D, CD, and non-classified type. CONCLUSION: Genotypes B, C, and D, non-classified and mixed genotype of HBV are identified in the Guangxi Zhuang population. Variations in genotypes are associated with clinical severity and serum ALT levels, but not with YMDD mutation or HBV DNA load. Therapeutic effects of LAM on clinical parameters are not influenced by differences in genotypes. Further studies are needed to gain an in-depth understanding of the relationship between HBV genotypes and serum HBeAb and HBeAg.

Adolescent↗

Mast cells protect mice from Mycoplasma pneumonia.

RATIONALE: As the smallest free-living bacteria and a frequent cause of respiratory infections, mycoplasmas are unique pathogens. Mice infected with Mycoplasma pulmonis can develop localized, life-long airway infection accompanied by persistent inflammation and remodeling. OBJECTIVE: Because mast cells protect mice from acute septic peritonitis and gram-negative pneumonia, we hypothesized that they defend against mycoplasma infection. This study tests this hypothesis using mast cell-deficient mice. METHODS: Responses to airway infection with M. pulmonis were compared in wild-type and mast cell-deficient Kit(W-sh)/Kit(W-sh) mice and sham-infected control mice. MEASUREMENTS AND MAIN RESULTS: Endpoints include mortality, body and lymph node weight, mycoplasma antibody titer, and lung mycoplasma burden and histopathology at intervals after infection. The results reveal that infected Kit(W-sh)/Kit(W-sh) mice, compared with other groups, lose more weight and are more likely to die. Live mycoplasma burden is greater in Kit(W-sh)/Kit(W-sh) than in wild-type mice at early time points. Four days after infection, the difference is 162-fold. Titers of mycoplasma-specific IgM and IgA appear earlier and rise higher in Kit(W-sh)/Kit(W-sh) mice, but antibody responses to heat-killed mycoplasma are not different compared with wild-type mice. Infected Kit(W-sh)/Kit(W-sh) mice develop larger bronchial lymph nodes and progressive pneumonia and airway occlusion with neutrophil-rich exudates, accompanied by angiogenesis and lymphangiogenesis. In wild-type mice, pneumonia and exudates are less severe, quicker to resolve, and are not associated with increased angiogenesis. CONCLUSIONS: These findings suggest that mast cells are important for innate immune containment of and recovery from respiratory mycoplasma infection.

Animals↗

Structural basis for the unique biological function of small GTPase RHEB.

The small GTPase Rheb displays unique biological and biochemical properties different from other small GTPases and functions as an important mediator between the tumor suppressor proteins TSC1 and TSC2 and the mammalian target of rapamycin to stimulate cell growth. We report here the three-dimensional structures of human Rheb in complexes with GDP, GTP, and GppNHp (5'-(beta,gamma-imide)triphosphate), which reveal novel structural features of Rheb and provide a molecular basis for its distinct properties. During GTP/GDP cycling, switch I of Rheb undergoes conformational change while switch II maintains a stable, unusually extended conformation, which is substantially different from the alpha-helical conformation seen in other small GTPases. The unique switch II conformation results in a displacement of Gln64 (equivalent to the catalytic Gln61 of Ras), making it incapable of participating in GTP hydrolysis and thus accounting for the low intrinsic GTPase activity of Rheb. This rearrangement also creates space to accommodate the side chain of Arg15, avoiding its steric hindrance with the catalytic residue and explaining its noninvolvement in GTP hydrolysis. Unlike Ras, the phosphate moiety of GTP in Rheb is shielded by the conserved Tyr35 of switch I, leading to the closure of the GTP-binding site, which appears to prohibit the insertion of a potential arginine finger from its GTPase-activating protein. Taking the genetic, biochemical, biological, and structural data together, we propose that Rheb forms a new group of the Ras/Rap subfamily and uses a novel GTP hydrolysis mechanism that utilizes Asn1643 of the tuberous sclerosis complex 2 GTPase-activating protein domain instead of Gln64 of Rheb as the catalytic residue.

Amino Acid Sequence↗

YMDD mutations in patients with chronic hepatitis B untreated with antiviral medicines.

AIM: To polymerase P region (YMDD) mutations of hepatitis B virus gene (HBV DNA) in patients with chronic hepatitis B (CHB) untreated with antiviral medicines and to explore its correlation with pre-c-zone mutations, HBV genotypes and HBV DNA level, and to observe its curative effect. METHODS: A total of 104 cases (38 cases in group of familial aggregation and 66 cases in group of non-familial aggregation) were randomly chosen from 226 patients with CHB who did not receive the treatment of lamivudine (LAM) and any other antivirus drugs within the last one year. Their serum YMDD mutations were detected by microcosmic nucleic acid and cross-nucleic acid quantitative determination, HBV genotypes by PCR-microcosmic nucleic acid cross-ELISA, HBV DNA quantitative determination and fluorescence ration PCR analysis, hepatitis B virus markers (HBVM) by ELISA. LAM was taken by 10 patients with YMDD mutations and its curative effect was observed. RESULTS: Twenty-eight cases (26.9%) had YMDD mutations, of them 11 cases (28.9%) were in familial aggregation group (38 cases) and 17 cases (25.8%) in non-familial aggregation group (66 cases) with no significant difference between the two groups. Twenty-seven point one percent (16/59) cases were positive for HBeAg YMDD mutations, and 26.7% (12/45) cases were negative for HBeAg and positive for anti-HBe. There was also no significant difference between the two groups. Different YMDD incidence rate existed in different HBV genotypes. HBV DNA level did not have a positive correlation with the incidence of YMDD mutations. LAM was effective for all patients with mutations. CONCLUSION: Wild mutant strains in HBV and their incidence rate have no significant difference between familial aggregation and non-familial aggregation. It may have no significant relationship between YMDD mutations and pre-c-zone mutations. HBV DNA level may not have a positive correlation with YMDD mutations. LAM is clinically effective for CHB patients with YMDD mutations.

Adolescent↗

Transcript signatures of lymphocytic bronchitis in lung allograft biopsy specimens.

BACKGROUND: Rejection and obliterative bronchiolitis are barriers to sustained graft function in recipients of transplanted lungs. Early detection is hindered by inadequate tests and an incomplete understanding of the molecular events preceding or accompanying graft deterioration. METHODS: Hypothesizing that genes involved in immune responses and tissue remodeling produce biomarkers of rejection, we measured the expression of 192 selected genes in 72 sets of biopsy specimens from human lung allografts. Gene transcripts were quantified using a 2-step, multiplex, real-time polymerase chain reaction approach in endobronchial and transbronchial biopsy specimens from transplant recipients without acute infections undergoing routine surveillance bronchoscopy. RESULTS: Comparisons of histopathology in parallel biopsy specimens identified 6 genes correlating with rejection as manifested by lymphocytic bronchitis, a suspected harbinger of obliterative bronchiolitis. For example, beta2-defensin and collagenase transcripts in inflamed bronchi increased 37-fold and 163-fold, respectively. By contrast, these transcripts did not correlate with acute rejection in transbronchial specimens. Further, no correspondence was noted between histopathologic bronchitis and parenchymal rejection when endobronchial and transbronchial samples were obtained from the same patient. CONCLUSIONS: Our highly sensitive method permits quantitation of many gene transcripts simultaneously in small, bronchoscopically acquired biopsy specimens of allografts. Transcript signatures obtained by this approach suggest that airway and alveolar responses to rejection differ and that endobronchial biopsy specimens assess lymphocytic bronchitis and chronic rejection but are not proxies for transbronchial biopsy specimens. Further, they reveal changes in airway expression of the specific genes involved in host defense and remodeling and suggest that the measurement of transcripts correlating with lymphocytic bronchitis may be diagnostic adjuncts to histopathology.

Adult↗

[Changes in glucocorticoid receptor and heat shock protein 70 in liver tissue after trauma with hemorrhagic shock in rats].

OBJECTIVE: To investigate changes and functions of glucocorticoid (GR) and heat shock protein 70 (HSP70) at the level of cellular receptor in liver in hemorrhagic shock after trauma. METHODS: Adult Wistar rats were used and a rat model of shock was reproduced by hemorrhaging accompanied by bilateral femur fracture. Changes in hepatic tissue GR, HSP70, pathology of liver, hepatic function markers in serum were dynamically observed. The expression of GR and HSP70 in hepatic tissue was assayed by Western blot and then analyzed with computer imaging system. RESULTS: Protein content of GR gradually decreased in hepatic tissue after trauma, reduced to the nadir at 6 hours after trauma, and it was decreased even more when trauma was added to bleeding. Protein content of HSP70 was gradually increased in hepatic tissue after severe trauma, peaking at 6 hours after trauma, and it maintained at a rather high level at 8 hours after trauma. It was even more increased in when hemorrhagic shock was followed by trauma. Alterations in hepatic function markers and pathology were not obvious after simple trauma. But after trauma was added to hemorrhagic shock, significant increase in alanine aminotransferase (ALT) and total bilirubin (TB) in serum was noted at 4 and 2 hours after trauma respectively (all P<0.01), and albumin content was obviously decreased (P<0.01). There were more inflammatory cell infiltrations in hepatic sinusoids at 6 hours after trauma. CONCLUSION: GR may play an important role in anti-injury mechanism of hepatic tissue cell after trauma with hemorrhagic shock. HSP70 may participate in initiating anti-injury mechanism of hepatic cells.

Animals↗

Identification and functional characterization of a novel human misshapen/Nck interacting kinase-related kinase, hMINK beta.

Misshapen/NIKs-related kinase (MINK) is a member of the germinal center family of kinases that are homologous to the yeast sterile 20 (Ste20) kinases and regulate a wide variety of cellular processes, including cell morphology, cytoskeletal rearrangement, and survival. Here, we present the cloning and functional characterization of a novel human Misshapen/NIKs-related kinase beta (hMINK beta) that encodes a polypeptide of 1312 amino acids. hMINK beta is ubiquitously expressed in most tissues with at least five alternatively spliced isoforms. Similar to Nck interacting kinase (NIK) and Traf2 and Nck-interacting kinase (TNIK), hMINK beta moderately activates c-Jun N-terminal kinase (JNK) and associates with Nck via the intermediate domain in the yeast two-hybrid system and in a glutathione S-transferase (GST) pull-down assay. Interestingly, overexpression of the kinase domain deleted and kinase-inactive mutants of hMINK beta in human fibrosarcoma HT1080 cells enhanced cell spreading, actin stress fiber formation, and adhesion to extracellular matrix, as well as decreased cell motility and cell invasion. Furthermore, these mutants also promoted cell-cell adhesion in human breast carcinoma MCF7 cells, evidenced with cell growth in clusters and increased membrane localization of beta-catenin, a multifunctional protein involved in E-cadherin-mediated cell adhesion. Finally, hMINK beta protein was found to colocalize with the Golgi apparatus, implicating that hMINK beta might exert its functions, at least in part, through the modulation of intracellular protein transport. Taken together, these results suggest that hMINK beta plays an important role in cytoskeleton reorganization, cell adhesion, and cell motility.

Adaptor Proteins, Signal Transducing↗

Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity.

Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, and regulation of the enzymatic activity of IDHs is crucial for their biological functions. Bacterial IDHs are reversibly regulated by phosphorylation of a strictly conserved serine residue at the active site. Eukaryotic NADP-dependent IDHs (NADP-IDHs) have been shown to have diverse important biological functions; however, their regulatory mechanism remains unclear. Structural studies of human cytosolic NADP-IDH (HcIDH) in complex with NADP and in complex with NADP, isocitrate, and Ca2+ reveal three biologically relevant conformational states of the enzyme that differ substantially in the structure of the active site and in the overall structure. A structural segment at the active site that forms a conserved alpha-helix in all known NADP-IDH structures assumes a loop conformation in the open, inactive form of HcIDH; a partially unraveled alpha-helix in the semi-open, intermediate form; and an alpha-helix in the closed, active form. The side chain of Asp279 of this segment occupies the isocitrate-binding site and forms hydrogen bonds with Ser94 (the equivalent of the phosphorylation site in bacterial IDHs) in the inactive form and chelates the metal ion in the active form. The structural data led us to propose a novel self-regulatory mechanism for HcIDH that mimics the phosphorylation mechanism used by the bacterial homologs, consistent with biochemical and biological data. This mechanism might be applicable to other eukaryotic NADP-IDHs. The results also provide insights into the recognition and specificity of substrate and cofactor by eukaryotic NADP-IDHs.

Amino Acid Sequence↗

Computational and experimental studies on human misshapen/NIK-related kinase MINK-1.

We have studied the structure and function of Human Misshapen/NIK-related kinase (MINK-1) through a combination of computational methods and experimental approaches, including (1) fold recognition and sequence-structure alignment for each structural domain using the threading program PROSPECT, (2) gene expression and protein-protein interaction analysis of yeast homologs of human MINK-1 domains, and (3) yeast two-hybrid screening for proteins that interact with human MINK-1. Our structure prediction dissects MINK-1 into four domains: a conserved N-terminal kinase domain, followed by a coiled-coil region and a proline-rich region, and a C-terminal GCK domain. Gene expression and yeast two-hybrid analysis of yeast homologs of the MINK-1 domains suggest that MINK-1 may be involved in cell-cycle progression and cytoskeletal control. Consistent with these predicted functions, our in-house yeast two-hybrid screen for proteins that interact with human MINK-1 provides strong evidence that the coiled-coil and proline-rich domains of MINK-1 participate in the regulation of cytoskeletal organization, cell-cycle control and apoptosis. A homology model of the MINK-1 kinase domain was used to screen the NCI open compound database in DOCK, and chemical compounds with pharmaceutically acceptable properties were identified. Further medicinal chemistry compound structure optimization and kinase assays are underway.

Amino Acid Sequence↗

[Changes in human leukocyte antigen-DR expression on monocytes and its value of prediction on infection complication in trauma patients].

OBJECTIVE: To observe the changes of human leukocyte antigen DR (HLA-DR) expression in monocytes of trauma patients and its value of prediction on infection complications. METHODS: Fifty-four trauma patients were divided into three groups according to severity of injury: severe trauma group injury severity score (ISS) >or=25, moderate trauma group (16 or=16) were divided into three groups according to infection or not: no infection group, localized infection group and systemic infection group. Blood samples were collected immediately after admission and serially at 8:30 to 9:00 a.m. on days 1, 2, 4, 6, 8, 14 after admission, and monocyte HLA-DR expression was determined with monoclonal staining and flow cytometry. RESULTS: The HLA-DR expression in monocytes was reduced in the trauma patients. The lowest levels of HLA-DR were recorded on day 2 after trauma. Subsequently HLA-DR expression in monocytes increased gradually. During the whole observation, the HLA-DR expression was significantly decreased in both severe trauma group and moderate trauma group versus control group, but no significant differences were found between severe trauma group and moderate trauma group, mild trauma group and control group, Immediately after trauma, HLA-DR expression in monocytes was significantly lower in the localized infection group than that in the patients without infection, and lasted until day 4 after trauma. The mean fluorescence intensity of HLA-DR expression in monocytes on the day 2 to 14 after trauma, the percentage of HLA-DR monocytes on the day 1 to 14 after trauma were significantly lower in the systemic infection group than those in the localized infection group. The level of HLA-DR expression in monocyte in the 2 died trauma patients was lowered till died. CONCLUSION: In severe trauma patients, the HLA-DR expression in monocytes is significantly decreased, and decreased levels of HLA-DR expression in monocytes might be the early indicators of an immune deviation associated with the development of infection complications and prognosis.

Adult↗

[Cloning and identification of NF-kappaB p50 Rel homology domain and detection of its autonomous reporter gene activity].

AIM: To clone NF-kappaB p50 Rel homology domain (RHD) gene and construct the "bait" vector in yeast two-hybrid system, and detect the yeast cell toxicity and autonomous reporter gene activity of target gene. METHODS: Total RNA was extracted from human peripheral blood mononuclear cells and NF-kappaB p50 RHD gene was amplified by RT-PCR, and cloned into pGBKT7. The recombinant plasmid was transformed into yeast AH109. The growth condition of the transformants was observed in the selected medium SD/-Trp. The reporter gene activity of target gene in the yeast cells was verified by filter blotting. RESULTS: Using restriction enzyme digestion analysis and PCR, the length of inserted gene was confirmed correct. Sequencing result indicated that the sequence of the inserted gene and its open reading frame were completely correct, and then the recombinant plasmid was named pGBKT7-p50. p50 RHD gene had neither autonomous reporter gene activity nor yeast cytotoxicity. CONCLUSION: As a bait plasmid, pGBKT7-p50 could be used in yeast two-hybrid system to screen and capture the polypeptides which interact with p50 RHD.

Cloning, Molecular↗

Molecular model of SARS coronavirus polymerase: implications for biochemical functions and drug design.

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The RNA-dependent RNA polymerase (RdRp) of SARS-CoV plays a pivotal role in viral replication and is a potential target for anti-SARS therapy. There is a lack of structural or biochemical data on any coronavirus polymerase. To provide insights into the structure and function of SARS-CoV RdRp, we have located its conserved motifs that are shared by all RdRps, and built a three-dimensional model of the catalytic domain. The structural model permits us to discuss the potential functional roles of the conserved motifs and residues in replication and their potential interactions with inhibitors of related enzymes. We predict important structural attributes of potential anti-SARS-CoV RdRp nucleotide analog inhibitors: hydrogen-bonding capability for the 2' and 3' groups of the sugar ring and C3' endo sugar puckering, and the absence of a hydrophobic binding pocket for non-nucleoside analog inhibitors similar to those observed in hepatitis C virus RdRp and human immunodeficiency virus type 1 reverse transcriptase. We propose that the clinically observed resistance of SARS to ribavirin is probably due to perturbation of the conserved motif A that controls rNTP binding and fidelity of polymerization. Our results suggest that designing anti-SARS therapies can benefit from successful experiences in design of other antiviral drugs. This work should also provide guidance for future biochemical experiments.

Amino Acid Motifs↗

Crystal structure of human tryptophanyl-tRNA synthetase catalytic fragment: insights into substrate recognition, tRNA binding, and angiogenesis activity.

Human tryptophanyl-tRNA synthetase (hTrpRS) produces a full-length and three N terminus-truncated forms through alternative splicing and proteolysis. The shortest fragment that contains the aminoacylation catalytic fragment (T2-hTrpRS) exhibits the most potent angiostatic activity. We report here the crystal structure of T2-hTrpRS at 2.5 A resolution, which was solved using the multi-wavelength anomalous diffraction method. T2-hTrpRS shares a very low sequence homology of 22% with Bacillus stearothermophilus TrpRS (bTrpRS); however, their overall structures are strikingly similar. Structural comparison of T2-hTrpRS with bTrpRS reveals substantial structural differences in the substrate-binding pocket and at the entrance to the pocket that play important roles in substrate binding and tRNA binding. T2-hTrpRS has a wide opening to the active site and adopts a compact conformation similar to the closed conformation of bTrpRS. These results suggest that mammalian and bacterial TrpRSs might use different mechanisms to recognize the substrate. Modeling studies indicate that tRNA binds with the dimeric enzyme and interacts primarily with the connective polypeptide 1 of hTrpRS via its acceptor arm and the alpha-helical domain of hTrpRS via its anticodon loop. Our results also suggest that the angiostatic activity is likely located at the alpha-helical domain, which resembles the short chain cytokines.

Adenosine Triphosphate↗

Distinct requirements for the naturally occurring splice forms Stat4alpha and Stat4beta in IL-12 responses.

Signal transducer and activator of transcription (Stat)4 is a signaling molecule required for normal responses to interleukin-12 (IL-12) and is critically involved in inflammatory responses. We have isolated an alternatively spliced isoform of Stat4, termed Stat4beta, which lacks 44 amino acids at the C-terminus, encompassing the putative transcriptional activation domain. To assess the in vivo roles of these Stat4 isoforms, we generated transgenic Stat4-deficient mice expressing Stat4alpha or Stat4beta. Our results indicate that T-cell-specific expression of Stat4alpha or Stat4beta can mediate many aspects of IL-12 signaling including the differentiation of Th1 cells. However, Stat4alpha is required for normal levels of IL-12-induced interferon-gamma production from Th1 cells. Microarray analysis identified 98 genes induced by both Stat4 isoforms, 32 genes induced only by Stat4alpha and 29 genes induced only by Stat4beta. Some induced genes correlate with specific functions including the ability of Stat4beta, but not Stat4alpha, to mediate IL-12-stimulated proliferation. Thus, Stat4alpha and Stat4beta have distinct roles in mediating responses to IL-12.

Adjuvants, Immunologic↗