Biomedical subjects

Y Wei

Publications and source records attributed to Y Wei.

At least 19 recordsLinked to original sources

PubMed2006

The LIM-only protein FHL2 is a negative regulator of E4F1.

The E1A-targeted transcription factor E4F1 is a key player in the control of mammalian embryonic and somatic cell proliferation and survival. Mouse embryos lacking E4F die at an early developmental stage, whereas enforced expression of E4F1 in various cell lines inhibits cell cycle progression. E4F1-antiproliferative effects have been shown to depend on its capacity to repress transcription and to interact with pRb and p53. Here we show that full-length E4F1 protein (p120(E4F1)) but not its E1A-activated and truncated form (p50(E4F1)), interacts directly in vitro and in vivo with the LIM-only protein FHL2, the product of the p53-responsive gene FHL2/DRAL (downregulated in rhabdomyosarcoma Lim protein). This E4F1-FHL2 association occurs in the nuclear compartment and inhibits the capacity of E4F1 to block cell proliferation. Consistent with this effect, ectopic expression of FHL2 inhibits E4F1 repressive effects on transcription and correlates with a reduction of nuclear E4F1-p53 complexes. Overall, these results suggest that FHL2/DRAL is an inhibitor of E4F1 activity. Finally, we show that endogenous E4F1-FHL2 complexes form in U2OS cells upon UV-light-induced nuclear accumulation of FHL2.

Active Transport, Cell Nucleus↗

PubMed2006

Dichloroacetate causes toxic neuropathy in MELAS: a randomized, controlled clinical trial.

OBJECTIVE: To evaluate the efficacy of dichloroacetate (DCA) in the treatment of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). BACKGROUND: High levels of ventricular lactate, the brain spectroscopic signature of MELAS, correlate with more severe neurologic impairment. The authors hypothesized that chronic cerebral lactic acidosis exacerbates neuronal injury in MELAS and therefore, investigated DCA, a potent lactate-lowering agent, as potential treatment for MELAS. METHODS: The authors conducted a double-blind, placebo-controlled, randomized, 3-year cross-over trial of DCA (25 mg/kg/day) in 30 patients (aged 10 to 60 years) with MELAS and the A3243G mutation. Primary outcome measure was a Global Assessment of Treatment Efficacy (GATE) score based on a health-related event inventory, and on neurologic, neuropsychological, and daily living functioning. Biologic outcome measures included venous, CSF, and 1H MRSI-estimated brain lactate. Blood tests and nerve conduction studies were performed to monitor safety. RESULTS: During the initial 24-month treatment period, 15 of 15 patients randomized to DCA were taken off study medication, compared to 4 of 15 patients randomized to placebo. Study medication was discontinued in 17 of 19 patients because of onset or worsening of peripheral neuropathy. The clinical trial was terminated early because of peripheral nerve toxicity. The mean GATE score was not significantly different between treatment arms. CONCLUSION: DCA at 25 mg/kg/day is associated with peripheral nerve toxicity resulting in a high rate of medication discontinuation and early study termination. Under these experimental conditions, the authors were unable to detect any beneficial effect. The findings show that DCA-associated neuropathy overshadows the assessment of any potential benefit in MELAS.

Action Potentials↗

PubMed2006

Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1alpha eradicates gliomas.

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible-factors responsible for stimulating tumor angiogenesis and glycolysis, and targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to tumorigenesis and development of sporadic renal cell carcinomas and central nervous system hemangioblastomas. In the present study, we investigated whether engineered overexpression of pVHL in C6 glioma cells, which already express endogenous pVHL, would suppress the tumorigenicity of this particular tumor cell type. C6 cells overexpressing VHL displayed a reduced growth rate (70% inhibition) compared to the parental cell line when subcutaneously implanted in athymic (nu/nu) mice. Growth inhibition was associated with a 50% reduction in the number of tumor vessels and a 60% increase in tumor cell apoptosis, due in part to downregulation of HIF-1, VEGF, and the antiapoptotic factor Bcl-2, respectively. Gene transfer of VHL suppressed the growth of established C6 gliomas, and synergized with antisense HIF-1 to completely eradicate tumors. The data suggest that VHL gene therapy and/or agents that increase VHL expression could have utility in the treatment of gliomas, particularly when combined with agents that inhibit the expression or function of HIF-1.

Animals↗

PubMed2006

Controlled synthesis and field emission properties of ZnO nanostructures with different morphologies.

By simply controlling atmosphere, rods, tetraleg-rods, and arrays of ZnO nanostructures have been fabricated respectively through pure zinc powder evaporation without catalyst at temperature of 650 - 700 degrees C. Investigations through HRTEM and XRD showed that the growth of the synthesized ZnO nanostructures was controlled by vapor-solid mechanism. Field emission measurements revealed that all of the structures, owing to their very low turn-on voltage, sufficient emission current and proper linearity of 1/V - Ln(l/V2), are likely to be potential candidates as a field emitter. The results also indicated that field emission properties are relative to morphology and size of the tips of ZnO nanostructures, and the nanorods with sharp tips possess the first-class FE property.

Crystallization↗

PubMed2005

Polychlorobiphenyls and polycyclic aromatic hydrocarbons in the sea-surface micro-layer and the water column at Gerlache Inlet, Antarctica.

The enrichment of PCBs (polychlorobiphenyls) and PAHs (polycyclic aromatic hydrocarbons) in the sea-surface micro-layer and depth profile of these pollutants in the water column were investigated at Gerlache Inlet, Terra Nova Bay, Antarctica. Depth profile samplings were repeated three times during the Antarctic summer (from November to February). PCBs and PAHs showed a concentration range in the water column of 30-120 pg l(-1) and 150-400 pg l(-1), respectively, and these values were very much dependent on the suspended matter content. A nearly two-fold decrease in the pollutant concentration was also observed in the depth profile obtained in February, i.e. late summer, which might be correlated both with the high content of suspended matter and the reduction of the pollutant input. Moreover, isomer ratios of PAHs, such as LMW/HMW and PHE/ANT, highlight that the main PAH source might be petrogenic in nature, whereas the pyrolytic source seems to be less important. Sea surface micro-layer (SML) and sub-surface sea water (SSW) samples were simultaneously collected in the same site by a remote controlled rotating drum-based sampling system, a prototype named MUMS (Multi-User Micro-layer Sampler). Sea surface micro-layer samples showed a total content of PCBs and PAHs in the range 400-450 pg l(-1) and 2000-3000 pg l(-1), respectively, whereas the mean content of the sub-surface sea water samples was 48 pg l(-1) and 325 pg l(-1), respectively. The mean enrichment factors of PCBs and PAHs in sea-surface micro-layer were about 10 and 7, respectively. The surface excess concentrations of PCBs and PAHs were about 35 000 and 200 000, respectively. A fairly good correlation was observed between the concentration of pollutants and water solubility. Based on the assumption that POPs are confined in a very thin top layer of the SML about 0.01-0.001 microm thick, namely the sea-surface nano-layer, and also on an estimated thickness of the sampled sea-surface layer of about 100 microm, an enrichment factor of 10(5)-10(6) for the sea-surface nano-layer was calculated. Such a very high concentration increase was related to the two-fold increase of PAH concentration observed in the underlying 20 cm of the water column in late summer.

Antarctic Regions↗

PubMed2005

Akt is essential for insulin modulation of amphetamine-induced human dopamine transporter cell-surface redistribution.

Uptake by the dopamine transporter (DAT) is the primary pathway for the clearance of extracellular dopamine (DA) and consequently for regulating the magnitude and duration of dopaminergic signaling. Amphetamine (AMPH) has been shown to decrease simultaneously DAT cell-surface expression and [(3)H]DA uptake. We have shown that insulin and its subsequent signaling through the phosphatidylinositol 3-kinase (PI3K)-dependent pathway oppose this effect of AMPH by promoting increased cell-surface expression. Here, we used human embryonic kidney 293 cells stably expressing the human DAT (hDAT cells) to investigate the downstream cellular components important for this effect of insulin. Akt is a protein kinase effector immediately downstream of PI3K. Both overexpression of a dominant-negative mutant of Akt (K179R) and the addition of 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML9), a pharmacological inhibitor of Akt, decreased cell-surface expression of DAT, suggesting a role of basal Akt signaling in the homoeostasis of DAT. Moreover, expression of a constitutively active Akt mutant reduced the ability of AMPH to decrease hDAT cell-surface expression as well as [(3)H]DA uptake. In contrast, overexpression of K179R blocked the ability of insulin to oppose AMPH-induced reduction of hDAT cell-surface expression and [(3)H]DA uptake, as did ML9. Our data demonstrate that hDAT cell-surface expression is regulated by the insulin signaling pathway and that Akt plays a key role in the hormonal modulation of AMPH-induced hDAT trafficking and in the regulation of basal hDAT cell-surface expression.

Amphetamine↗

PubMed2005

Selecting peptide ligands of microcystin-LR from phage displayed random libraries.

In the present study, we investigated to find novel ligands for low molecular weight environmental toxin, microcystin-LR (MC-LR) by using phage display technology. Two random libraries, displaying linear 12-mer peptides and cyclic 7-mer peptides, were screened against the immobilized target respectively. After three rounds of panning, phage clones that recognized microcystin-LR specifically were obtained from both the linear and the constrained libraries, proved by enzyme-linked immumosorbent assays and immunoprecipitation assays. DNA sequencing indicated that peptides displayed on some of the selected clones shared consensus sequences. Compared with traditional methods, this approach provided a cheaper and more rapid alternative to screen specific ligands for microcystin-LR. Moreover, since it is rather difficult to take small molecules as targets of phage display libraries, the success of this experiment expanded the applications of phage display technology, and provided a new avenue to study environmental small molecular toxins.

Bacterial Toxins↗

PubMed2005

Non-specific tolerance induced by staphylococcal enterotoxin B in treating high risk corneal transplantation in rats.

AIMS: To explore the role of staphylococcal enterotoxin B (SEB) in treating high risk corneal keratoplasty in rats. METHODS: Rat corneal high risk transplantation rejection models were set up using Fisher 344 and Lewis rats. The experimental rats were injected intraperitoneally with 0.2 ml SEB at different concentrations before keratoplasty. The rejection indexes of the allograft were recorded and the lymphocyte infiltration in the allograft and the percentage of the lymphocyte subpopulation in the lymphatic organs were also examined. Lymphocyte proliferation ability and the concentration of IL-2 and IL-10 in the serum were also evaluated. RESULTS: Compared with the control group, SEB prolonged the survival time of the allograft significantly from 7 to 12 days. It could also reduce CD4(+) and CD8(+) lymphocyte infiltration in the allograft and minimise the percentage of CD4(+) and CD8(+) lymphocytes in the lymphatic organs. The lymphocyte proliferation ability was also weakened. However, the percentage of CD4(+) NK T lymphocytes in the lymphatic organs was raised. The serum concentration of IL-10 was higher but IL-2 was lower in the SEB treated groups. CONCLUSIONS: SEB prolonged the survival time of the allograft in high risk rat corneal allo-transplantation, which may be caused by T cell deletion and acquisition of non-specific tolerance.

Animals↗

PubMed2005

The discharged excess sludge treated by Oligochaeta.

To overcome unstable worm growth, a new worm-reactor was developed for oligochaete growth. The bench scale of this worm-reactor was used to treat the discharged excess sludge from a pilot activated sludge system, and experiments were carried out to investigate the sludge reduction induced by Oligochaeta. Due to difficult getting free-swimming worms such as Aeolosoma hemprichicii and Nais elinguis, Tubifex tubifex was thus selected and inoculated in Reactor 1 at the start-up phase except the control reactor (Reactor 2). Tubifex occurred in Reactor 1 throughout the operation period after its inoculation, and mainly attached on the carriers and aggregated on the bottom of the worm-reactor. Free-swimming worms such as Aeolosoma hemprichicii, Nais elinguis, and Aulophorus furcatus were found in both reactors since the 35th day. Microscopic investigation showed that these free-swimming worms naturally produced in the pilot activated sludge system, and entered into both reactors along the discharged sludge. Results clearly showed that the average sludge reduction in Reactor 1 was 59%, much higher than that in the control. The characteristics of sludge settling was improved by worm growth, but was not too much. High ammonia nitrogen concentration in influent sludge was toxic to worms, and then inhibited worm growth. Both the total inorganic nitrogen and phosphorus releases into effluent sludge were observed in Reactor 1, but such increases were not heavy.

Animals↗

PubMed2004

Protein structure to function: insights from computation.

Computation plays an important role in functional genomics. THEMATICS is a computational method that predicts chemical and electrostatic properties of residues in enzymes and utilizes information contained in those predictions to identify active sites. The only input required is the three-dimensional structure of the query protein. The identification of residues involved in catalysis and in recognition is discussed. The two serine proteases Kex2 from Saccharomyces cerevisiae and subtilisin from Bacillus subtilis are used as examples to illustrate how the method finds the catalytic residues for both enzymes. In addition, Kex2 is specific for dibasic sites and THEMATICS finds the recognition residues for both the S1 and S2 sites of Kex2. In contrast, no such recognition sites are found for the non-specific enzyme subtilisin. The ability to identify sites that govern recognition opens the door to better understanding of specificity and to the design of highly specific inhibitors.

Binding Sites↗

PubMed2004

SEB combined with IL-1ra could prolong the survival of the rat allografts in high-risk corneal transplantation.

PURPOSE: To determine whether the superantigen Staphylococcal enterotoxin B (SEB) combined with interleukin-1 receptor antagonist (IL-1ra) prolong allograft survival better than individual agents in high-risk corneal transplantation in a rat model. METHODS: Fisher 344 donor corneas were transplanted into Lewis recipients. High-risk transplantation meant that the transplants were sutured into the recipient beds with corneal neovascularization induced by placing three interrupted sutures in the host cornea. All of the recipients were divided in blinded fashion into four groups. Group I was injected with saline buffer. Group II was injected intraperitoneally with 0.2 mL SEB (75 microg/kg) at 4-day intervals on three occasions before transplantation. Group III was injected with 0.1 mL IL-1ra (1 mg/mL) subconjunctivally from the first day after transplantation for 2 weeks. Group IV received both SEB and IL-1ra. All transplants were evaluated for signs of rejection for 4 weeks after surgery. Ten days after transplantation, two recipients in each group were sacrificed for histopathological and immunological evaluation. RESULTS: The mean survival time of the allografts in the control group was 5.89 +/- 0.79 days; in SEB group, 10.70 +/- 2.52 days; in IL-1ra group, 8.25 +/- 0.71 days; in the SEB and IL-1ra group, 17.36 +/- 2.39 days. CD4+ and CD8+ lymphocyte infiltration into the allografts and the percentage of the lymphocytes in the spleen and mandibular lymphatic nodes was significantly decreased among the treated groups with dampened lymphocyte reactivity. The SEB plus IL-1ra combination group showed the strongest inhibition. CONCLUSION: SEB and IL-1ra are most effective in combination to treat high-risk corneal transplants.

Animals↗

PubMed2004

Mifepristone-induced abortion and birth weight in the first subsequent pregnancy.

OBJECTIVES: To assess the effect of a medically induced abortion (MA) on birth weight in the first subsequent pregnancy. METHODS: Pregnant women who had had a MA, a surgical abortion (SA), or primigravidas with no history of abortion (NA), were recruited for a prospective cohort study between July 1998 and February 2001. The sample for the present analysis included 12995 singleton live births at term. RESULTS: The overall incidence rate of low birth weight (LBW) was 1.0%. The rates of LBW in the MA, SA, and NA groups were 1.0%, 0.9%, and 1.2, respectively. There were no significant differences in LBW rates between the MA and SA groups, neither between the MA and NA. Logistic regression analysis was used to estimate the effects of MA on birth weight, and we found no relationship between risk of LBW and MA. CONCLUSIONS: We did not find a statistically significant relationship between a history of one medically induced abortion and LBW for the first subsequent term pregnancy.

Abortifacient Agents, Steroidal↗

PubMed2002

A gene therapy for cancer based on the angiogenesis inhibitor, vasostatin.

The growth and persistence of solid tumors and their metastasis are angiogenesis-dependent. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is a potent angiogenesis inhibitor. To investigate whether intramuscular administration of vasostatin gene has the antitumor activity in mouse tumor models, we constructed a plasmid DNA encoding vasostatin and a control vector. Production and secretion of vasostatin protein by COS cells transfected with the plasmid DNA encoding vasostatin (pSecTag2B-vaso) were confirmed by Western blot analysis and ELISA. Conditioned medium from vasostatin-transfected COS cells apparently inhibited human umbilical vein endothelial cell (HUVEC) and mouse endothelial cell (SVEC4-10) proliferation, compared with conditioned medium from the COS cells transfected with control vector or non-transfected cells. Treatment with pSecTag2B-vaso twice weekly for 4 weeks resulted in the inhibition of tumor growth and the prolongation of the survival of tumor-bearing mice. The sustained high level of vasostatin protein in serum could be identified in ELISA. Angiogenesis was apparently inhibited in tumor by immunohistochemical analysis. Angiogenesis was also inhibited in the chicken embryo CAM assay and mouse corneal micropocket assay. The increased apoptotic cells were found within the tumor tissues from the mice treated with plasmid DNA encoding vasostatin. Taken together, the data in the present study indicate that the cancer gene therapy by the intramuscular delivery of plasmid DNA encoding vasostatin, is effective in the inhibition of the systemic angiogenesis and tumor growth in murine models. The present findings also provide further evidence of the anti-tumor effects of the vasostatin, and may be of importance for the further exploration of the application of this molecule in the treatment of cancer.

Angiogenesis Inhibitors↗

PubMed2001

Role of active site binding interactions in 4-chlorobenzoyl-coenzyme A dehalogenase catalysis.

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA (4-HBA-CoA) via a multistep mechanism involving initial attack of Asp145 on C(4) of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of the chloride ion to form an arylated enzyme intermediate (EAr) and then ester hydrolysis in the EAr to form product. This study examines the role of binding interactions in dehalogenase catalysis. The enzyme and substrate groups positioned for favorable binding interaction were identified from the X-ray crystal structure of the enzyme-4-HBA-3'-dephospho-CoA complex. These groups were individually modified (via site-directed mutagenesis or chemical synthesis) for the purpose of disrupting the binding interaction. The changes in the Gibbs free energy of the enzyme-substrate complex (DeltaDeltaG(ES)) and enzyme-transition state complex (DeltaDeltaG) brought about by the modification were measured. Cases where DeltaDeltaG exceeds DeltaDeltaG(ES) are indicative of binding interactions used for catalysis. On the basis of this analysis, we show that the H-bond interactions between the Gly114 and Phe64 backbone amide NHs and the substrate benzoyl C=O group contribute an additional 3.1 kcal/mol of stabilization at the rate-limiting transition state. The binding interactions between the enzyme and the substrate CoA nucleotide moiety also intensify in the rate-limiting transition state, reducing the energy barrier to catalysis by an additional 3.3 kcal/mol. Together, these binding interactions contribute approximately 10(6) to the k(cat)/K(m).

Acyl Coenzyme A↗

PubMed2001

The structure of anti-Gal immunoglobulin genes in naïve and stimulated Gal knockout mice.

BACKGROUND: Naturally occurring antibodies (Nabs) that bind to terminal galactose alpha1,3-galactose carbohydrate structures (Gal) are present in humans and Old World monkeys but are negatively regulated in other mammalian species because they express Gal epitopes on their cell surfaces. A Gal knockout mouse (Gal-/-) model, generated by homologous disruption of alpha1,3-galactosyltransferase gene, is capable of producing natural anti-Gal Abs. METHODS: To study the genetic control of the anti-Gal response, we have generated anti-Gal hybridomas from Gal-/- mice and analyzed VH genes of anti-Gal Abs from naïve animals and from mice stimulated by rat heterotopic heart transplantation. RESULTS: Six immunoglobulin (Ig)M anti-Gal hybridomas derived from naïve Gal-/- mice exhibited anti-Gal binding activity with some cross-reactivity to related carbohydrate structures. These naïve anti-Gal Abs used five different VH genes in a germline configuration. Anti-Gal IgM hybridomas isolated after a rat heterotopic heart xenograft (4 days) utilized germline VH gene segments from the VH7183 family and exhibited less cross-reactivity. In contrast to mice 4 days after xenograft, we have predominantly isolated IgG anti-Gal hybridomas from mice 21 days after rat heterotopic heart xenografts, indicating an isotype switch. Nine of the IgG anti-Gal hybridomas secreted IgG3 subclass and one produced IgG1. Sequence analysis of the VH gene usage from the induced anti-Gal IgG antibodies demonstrated a restricted gene utilization (VHJ606-V14A). CONCLUSION: Our results demonstrate that the anti-Gal response in naïve Gal-/- mice is encoded by multiple germline progenitors. In response to a xenograft, the induced anti-Gal Abs exhibited a restricted gene usage and somatic mutations, indicating a positive selection.

Animals↗

PubMed2001

Role of TGFbeta in development of spontaneous autoimmune thyroiditis in NOD.H-2h4 mice.

Nearly 100% of NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) and produce anti-mouse thyroglobulin autoantibodies when they receive 0.05% NaI in their drinking water beginning at 8 wk of age. Our previous studies showed that TGFbeta1 mRNA was constitutively expressed in thyroids and spleens of normal NOD.H-2h4 mice but not other strains of mice. To determine whether TGFbeta might have a role in SAT, mice were given anti-TGFbeta mAb at various times during development of SAT. Anti-TGFbeta markedly inhibited development of SAT and production of anti-mouse thyroglobulin IgG1 autoantibodies. Anti-TGFbeta was most effective in inhibiting SAT when given during the time thyroid lesions were developing, i.e., starting 4 wk after administration of NaI water. The active form of the TGFbeta1 protein was present in thyroids of mice with SAT but not in normal NOD.H-2h4 thyroids. However, thyrocytes of normal NOD.H-2h4 thyroids did express latent TGFbeta1. TGFbeta1 protein expression in the thyroid correlated with SAT severity scores, and administration of anti-TGFbeta inhibited TGFbeta1 protein expression in both the thyroid and spleen. TGFbeta1 was produced primarily by inflammatory cells and was primarily localized in areas of the thyroid containing clusters of CD4(+) T and B cells. Depletion of CD8(+) T cells had no effect on TGFbeta1 protein expression. Activation of splenic T cells was apparently not inhibited by anti-TGFbeta, because up-regulation of mRNA for cytokines and other T cell activation markers was similar for control and anti-TGFbeta-treated mice. TGFbeta1 may function by promoting migration to, or retention of, inflammatory cells in the thyroid.

Animals↗

PubMed2001

Expression and regulation of Fas and Fas ligand on thyrocytes and infiltrating cells during induction and resolution of granulomatous experimental autoimmune thyroiditis.

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin-sensitized spleen cells activated in vitro with mouse thyroglobulin, anti-IL-2R, and IL-12. G-EAT lesions reach maximal severity 19-21 days after cell transfer, and lesions almost completely resolve by day 35. Depletion of CD8+ cells delays resolution and reduces Fas ligand (FasL) mRNA expression in thyroids. This study was undertaken to analyze Fas and FasL protein expression in the thyroid during induction and resolution of G-EAT and to determine whether CD8+ cells might regulate Fas or FasL expression in the thyroid. Fas and FasL expression was analyzed by immunohistochemical staining or in situ hybridization in thyroids of mice with or without depletion of CD8+ cells. Fas and FasL proteins were not detectable in normal thyroids, but expression of both proteins increased during development of G-EAT. Fas was expressed primarily by inflammatory cells; some enlarged thyrocytes were also Fas+. Thyrocytes had intense FasL immunoreactvity, and many CD8+ cells were also FasL positive. Depletion of CD8+ cells resulted in decreased FasL expression by thyrocytes and inflammatory cells, but had no effect on Fas expression. TUNEL assay detected many apoptotic inflammatory cells in proximity to thyrocytes. CD8-depleted thyroids had ongoing inflammation with fewer apoptotic infiltrating cells at day 35. Administration of a neutralizing anti-FasL mAb had no apparent effects on development of G-EAT, but anti-FasL was as effective as anti-CD8 in preventing G-EAT resolution. These results suggested that CD8+ T cells and thyrocytes may kill inflammatory cells through the Fas pathway, contributing to G-EAT resolution.

Animals↗

PubMed2001

Histidine 90 function in 4-chlorobenzoyl-coenzyme a dehalogenase catalysis.

4-chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA by attack of Asp145 on the C4 of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of chloride ion to form an arylated enzyme intermediate (EAr) and, finally, ester hydrolysis in EAr to form 4-hydroxybenzoyl-CoA (4-HBA-CoA). This study examines the contribution of the active site His90 to catalysis of this reaction pathway. The His90 residue was replaced with glutamine by site-directed mutagenesis. X-ray crystallographic analysis of H90Q dehalogenase complexed with 4-HBA-CoA revealed that the positions of the catalytic groups are unchanged from those observed in the structure of the 4-HBA-CoA-wild-type dehalogenase complex. The one exception is the Gln90 side chain, which is rotated away from the position of the His90 side chain. The vacated His90 site is occupied by two water molecules. Kinetic techniques were used to evaluate ligand binding and catalytic turnover rates in the wild-type and H90Q mutant dehalogenases. The rate constants for 4-CBA-CoA (both 7 microM(-1) x s(-1)) and 4-HBA-CoA (33 and 11 microM(-1) x s(-1)) binding to the two dehalogenases are similar in value. For wild-type dehalogenase, the rate constant for a single turnover is 2.3 s(-1) while that for multiple turnovers is 0.7 s(-1). For H90Q dehalogenase, these rate constants are 1.6 x 10(-2) and 2 x 10(-4) s(-1). The rate constants for EMc formation in wild-type and mutant dehalogenase are approximately 200 s(-1) while the rate constants for EAr formation are 40 and 0.3 s(-1), respectively. The rate constant for hydrolysis of EAr in wild-type dehalogenase is 20 s(-1) and in the H90Q mutant, 0.13 s(-1). The 133-fold reduction in the rate of EAr formation in the mutant may be the result of active site hydration, while the 154-fold reduction in the rate EAr hydrolysis may be the result of lost general base catalysis. Substitution of the His90 with Gln also introduces a rate-limiting step which follows catalysis, and may involve renewing the catalytic site through a slow conformational change.

Binding Sites↗