The effect of substrates on the kinetics and the in vivo threshold activity of mutant HIV-1 proteases.
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Binding Sites↗
Biomedical subjects
X Lin
Publications and source records attributed to X Lin.
Expression of functional domain of chicken gizzard calponin.
A full-length cDNA of the function domain of wild-type chicken gizzard calponin was cloned into expression vector pAED4 and the recombinant function domain of wild-type calponin was expressed in an Escherichia coli expression system. The actin domain of calponin (CaP-B) can bind with actin and it is a requisite for its inhibition of ATPase and vasoconstriction of smooth muscle. In this study, the cDNA of CaP-B was inserted into vector pAED4 by direction-cloning method. The cDNA of CaP-B was obtained with PCR cloning technique. The recombinant DNA pAED4-Cap-B was transformed into E. coli BL21 (DE3) and identified with the restriction analysis. The bacterial clones containing transformants were induced to be highly expressed in E. coli BL21 (DE3). The target protein was detected and identified by Western Blot analysis. The content of target protein was as high as 10% of the whole protein after overnight (16 h) culture. The results confirmed that Cap-B was relatively highly expressed in E. coli.
Animals↗
Effects of temperature and deltaGo on electron transfer from cytochrome c2 to the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides.
The kinetics of electron transfer from cytochrome c2 to the primary donor (P) of the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides have been investigated by time-resolved absorption spectroscopy. Rereduction of P+ induced by a laser pulse has been measured at temperatures from 300 K to 220 K in a series of specifically mutated reaction centers characterized by altered midpoint redox potentials of P+/P varying from 410 mV to 765 mV (as compared to 505 mV for wild type). Rate constants for first-order electron donation within preformed reaction center-cytochrome c2 complexes and for the bimolecular oxidation of free cytochrome c2 have been obtained by multiexponential deconvolution of the kinetics. At all temperatures the rate of the fastest intracomplex electron transfer increases by more than two orders of magnitude as the driving force -deltaGo is varied over a range of 350 meV. The temperature and deltaGo dependences of the rate constant fit the Marcus equation well. Global analysis yields a reorganization energy lambda = 0.96 +/- 0.07 eV and a set of electronic matrix elements, specific for each mutant, ranging from 1.2 10(-4) eV to 2.5 10(-4) eV. Analysis in terms of the Jortner equation indicates that the best fit is obtained in the classical limit and restricts the range of coupled vibrational modes to frequencies lower than approximately 200 cm(-1). An additional slower kinetic component of P+ reduction, attributed to electron transfer from cyt c2 docked in a nonoptimal configuration of the complex, displays a Marcus type dependence of the rate constant upon deltaGo, characterized by a similar value of lambda (0.8 +/- 0.1 eV) and by an average electronic matrix element smaller by more than one order of magnitude. In all of the mutants, as the temperature is decreased below 260 K, both intracomplex reactions are abruptly inhibited, their rate being negligible at 220 K. The free energy dependence of the second-order rate constant for oxidation of cyt c2 in solution suggests that the collisional reaction is partially diffusion controlled, reaching the diffusion limit at exothermicities between 150 and 250 meV over the temperature range investigated.
Cytochrome c Group↗
Differential expression of insulin receptor tyrosine kinase inhibitor (fetuin) gene in a model of diet-induced obesity.
The Differential Display technique has been used to identify differences in mRNA expression in adipose tissue after the introduction of a high fat diet to two strains of rat (OM and S5B/PI) that differ in their susceptibility to develop obesity on this diet. The insulin receptor tyrosine kinase inhibitor protein (fetuin) was shown to be differentially expressed in OM but not S5B/PI rats. This circulating protein may play a role in the development of peripheral insulin resistance associated with high fat diets.
Animals↗
CD95L confers immune priviledge to liver grafts which are spontaneously accepted.
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Animals↗
Spontaneous liver graft acceptance is mediated by intragraft FAS-ligand expression and viable passenger leukocytes.
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Animals↗
GvHD after small bowel transplantation: the role of caspases, FAS-L, and galectin-1.
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Animals↗
Effect of berbamine on T-cell mediated immunity and the prevention of rejection on skin transplants in mice.
Berbamine, an ingredient of Berberis, which itself is widely utilized in Chinese folk-medicine has been used as a source of leukogenics, anti-arrhythmics and anti-hypertensives. In recent years, the immunosuppressive effects of berbamine has been demonstrated. In order to further investigate the value of berbamine as an immunosuppressive agent, the delayed type hypersensitivity reaction (DTH) response with sheep red blood cells (SRBC), the mixed lymphocyte reaction (MLR) and a skin model of allograft rejection on mice were studied. Berbamine showed suppressive effects on DTH and MLR and significantly prolonged allograft survival compared with untreated transplanted mice. The results indicate that berbamine may be a potential agent in clinical transplantation.
Alkaloids↗
WOFIE augments the immunosuppressive potency of FK-506. Window of opportunity for immunological engagement.
UNLABELLED: Bidirectional recognition of donor- and recipient-derived immunocompetent cells has been proven to play a pivotal role for the induction of long-term unresponsiveness to allogeneic grafts. This study investigated the fate of heterotopic heart grafts with respect to the timing of subtherapeutic doses of FK-506 and with respect to the time point and type of donor antigen application, leaving space for mutual adaptation of alloreactive lymphocytes, designated as the 'WOFIE-concept' (window of opportunity for immunological engagement), originally described by R Calne. METHODS: Heterotropic heart transplantation was performed using male DA (RT1.a) donor and LEW (RT1.1) recipient rats in the following groups (n = 6). FK-506 was applied intramuscularly (i.m.) using doses of 2 mg/kg x body weight per day. Donor antigen application was performed either by DA blood transfusion, 2 ml intravenously (i.v.), or by i.v. transfusion of 5 x 10(7) DA splenocytes. (i) LEW --> LEW, untreated; (ii) DA --> LEW, untreated; (iii) DA --> LEW, FK-506 days 0, 4-7; (iv) DA --> LEW, FK-506 as group (iii) plus 2 ml of DA blood 6 h post-Tx; (v) same as group (iv) but DA blood transfusion 24 h post-Tx; (vi) DA --> LEW, FK-506 as group (iii) plus DA splenocytes 6 h post-Tx; (vii) same as group (vi) but DA splenocyte transfusion 24 h post-Tx; (viii) DA --> LEW, FK-506 days 0-4 and (ix) DA --> LEW, FK-506 as group (viii) plus DA blood 6 h post-Tx. Immunohistochemical stainings (APAAP-method) of the allografts and flow cytometric analysis of recipient spleens were performed electively 3, 7 and 14 days after organ reperfusion. RESULTS: The mean graft survival differed significantly between groups and comprised (mean +/- SD days): (i) >100, (ii) 6.5 +/- 1.0, (iii) 31.6 +/- 12.1, (iv) 44.8 +/- 10.1, (v) 29.8 +/- 14.2, (vi) 27.2 +/- 4.7, (vii) 14.6 +/- 4.2, 17.5 +/- 4.2, (viii) 17.5 +/- 4.2 and (ix) 18.8 +/- 2.8 days. Prolongation of graft survival and long-term unresponsiveness (group iv) revealed a substantially different pattern of graft infiltration. CONCLUSIONS: Effective treatment with unspecific immunosuppressants like FK-506 can be substantially improved if (i) mutual antigen recognition between donor and recipient immunocompetent cells is warranted, (ii) donor-derived blood-borne antigens are given immediately after graft reperfusion, and (iii) the type of inoculated donor antigen has a strong impact on graft survival as splenocytes which contain a large population of professional antigen-presenting cells failed to prolong graft survival after interrupted FK-506 treatment.
Animals↗
Large-scale sequencing of plant genomes.
The large number of ESTs generated for Arabidopsis and rice in recent years now act as an important complement to whole genome sequencing projects. The Arabidopsis Genome Initiative has begun a coordinated effort to sequence the entire genome and, as a result, increasing numbers of large sequence entries can be found in the public databases. In addition, the mitochondrial genome of Arabidopsis has been completely sequenced. Genome sequencing studies and the public sequence databases have begun to influence the direction of diverse areas of research from physiology to evolution.
Base Sequence↗
Nucleotide and predicted amino acid sequences of all genes encoded by the 3' genomic portion (9.5 kb) of respiratory bovine coronaviruses and comparisons among respiratory and enteric coronaviruses.
The 3'-ends of the genomes (9538 bp) of two wild-type respiratory bovine coronavirus (RBCV) isolates LSU and OK were obtained by cDNA sequencing. In addition, the 3'-end of the genome (9545) of the wild-type enteric bovine coronavirus (EBCV) strain LY-138 was assembled from available sequences and by cDNA sequencing of unknown genomic regions. Comparative analyses of RBCV and EBCV nucleotide and deduced amino acid sequences revealed that RBCV-specific nucleotide and amino acid differences were disproportionally concentrated within the S gene and the genomic region between the S and E genes. Comparisons among virulent and avirulent BCV strains revealed that virulence-specific nucleotide and amino acid changes were located within the S and E genes, and the 32 kDa open reading frame.
Amino Acid Sequence↗
Impaired postprandial gastric slow waves in patients with functional dyspepsia.
The aim of this study was to investigate gastric myoelectrical activity in patients with functional dyspepsia. Thirteen healthy subjects and 14 patients with functional dyspepsia participated in the study. The electrogastrogram (EGG) recording was made in each subject for 30 min in the fasting state and 120 min after a standard test meal of 475 calories. Spectral analysis methods were applied to derive quantitative EGG parameters. There was no difference in the EGG between the patients and controls in the fasting state. However, abnormalities in the postprandial EGG were found in the patients. The percentage of 2-4 cpm waves was significantly lower (74.4+/-4.0% vs 85.7+/-1.6%, P < 0.03) and the postprandial increase in EGG dominant power was significantly less (-0.52+/-0.92 dB vs 2.24+/-0.88 dB, P < 0.03) in patients than in controls. It was also found that the percentage of postprandial 2-4 cpm waves could be used to differentiate the patients with functional dyspepsia from the healthy controls with a specificity of 100% and a sensitivity of 43%. It was concluded that a subset of patients with functional dyspepsia have impaired gastric myoelectrical activity in the fed state.
Adult↗
Gastric myoelectrical activity in healthy children and children with functional dyspepsia.
The aim of this study was to investigate abnormalities in pediatric patients with functional dyspepsia. Fifteen symptomatic pediatric patients diagnosed with functional dyspepsia and 17 aged-matched healthy controls were studied. Gastric myoelectrical activity was recorded using surface electrogastrography for 1 hr in the fasting state and 1 hr after a test meal. It was found that, in comparison with the controls, the children with functional dyspepsia had a lower percentage of 2- to 4-cpm slow waves in both fasting state (66.0+/-4.7% vs 79.7+/-3.1%, P < 0.07) and fed state (72.4+/-5.4% vs 85.0+/-2.9%, P < 0.04), and a significantly higher instability of the dominant frequency in both fasting state (0.50+/-0.05 vs 0.31+/-0.04, P < 0.01) and fed state (0.39+/-0.05 vs 0.25+/-0.03, P < 0.05). It was also found the postprandial increase in EGG dominant power in the patients was inversely correlated with the total symptom score (r = 0.63, P = 0.03). It was concluded that abnormal gastric myoelectrical activity may play a role in the pathogenesis of pediatric functional dyspepsia.
Adolescent↗
Construction of new retroviral producer cells from adenoviral and retroviral vectors.
A combination of adenoviral and retroviral vectors was used to construct second generation packaging cells that deliver marker genes to target cells. A vector based upon Moloney murine leukemia virus (MoMLV) was used to deliver marker genes, and an adenovirus-based delivery system was used to deliver MoMLV structural genes (gag pol and env) to cultured cells. The procedure transformed the cells into new retroviral producer cells, which generate replication-incompetent retroviral particles in the culture supernatant for transferring marker genes to target cells. The titer of the retroviral-containing supernatant generated from the second generation producer cells reached above 10(5) c.f.u./ml, which is comparable to the MoMLV-based producer cell lines currently used in human gene therapy trials. These observations suggest that this new gene transfer scheme is technically feasible. The vector and procedures may be adapted for experimental human gene therapy in which the new producer cells are transplanted into patients for continuous gene transfer.
3T3 Cells↗
Synthesis and adenosine receptor affinity of 7-beta-D-ribofuranosylxanthine.
7-beta-D-Ribofuranosylxanthine, a previously unreported isomer of xanthosine, was prepared in four steps from 7-benzylxanthine. The procedure, which involves the use of pivaloyloxymethyl groups to protect the xanthine ring, was also applied to preparation of some 1-N-alkyl derivatives of 7-ribosylxanthine. Adenosine receptor affinity for these compounds was determined. 7-beta-D-Ribofuranosylxanthine was found to have higher affinity and greater selectivity for the A1 receptor than previously reported xanthine nucleosides, and to be a partial agonist.
Animals↗
The effects of a high fat diet on leptin mRNA, serum leptin and the response to leptin are not altered in a rat strain susceptible to high fat diet-induced obesity.
Osborne-Mendel (OM) and S5B/Pl rats differ in their sensitivity to develop obesity when fed a high fat (HF) diet; OM rats become obese, whereas S5B/Pl rats remain thin. We have investigated the possibilities that either an impaired leptin response or resistance to leptin action underlies the sensitivity to this form of obesity in OM rats. In Experiment 1, OM and S5B/Pl rats fed a nonpurified diet were killed at d 0 or were fed either a HF (56% fat energy) or a low fat (LF, 10% fat energy) diet for 2 or 7 d. The HF diet increased serum leptin significantly by d 2 to levels that were similar in both rat strains. At 7 d, leptin levels were lower than at d 2 but remained higher than levels in the d 0 control groups. The leptin mRNA:18S RNA ratio in epididymal adipose tissue increased to higher levels in HF-fed OM rats than in S5B/Pl rats fed that diet. However, although the LF diet had no effect in S5B/Pl rats, it increased leptin mRNA levels in epididymal adipose tissue of OM rats compared with the controls fed the nonpurified diet. In Experiment 2, OM and S5B/Pl rats were fed HF or LF diets for 5 wk. At that time, their feeding response to a range of leptin doses (0, 1, 5 or 10 microgram) given intracerebroventricularly was tested after overnight food deprivation. There was a similar dose-dependent reduction in energy intake in response to leptin in both OM and S5B/Pl rats. These responses were independent of the diet. The data suggest that the susceptibility of OM rats to HF diet-induced obesity is not related to either a loss of central sensitivity to leptin or a failure to enhance leptin production acutely, although the failure to maintain chronically increased levels of serum leptin could contribute to the obesity.
Adipose Tissue↗
Mutations at the consensus phosphorylation sites in the third intracellular loop of the rat gonadotropin-releasing hormone receptor: effects on receptor ligand binding and signal transduction.
In this study, site-directed mutagenesis of potential phosphorylation sites (Thr238, Ser253, and Thr264) for protein kinase C and C-terminal portion (Ala260-Leu265) of the third intracellular loop of the rat GnRH receptor (rGnRHR) was performed to assess the significance of these regions in the function of the GnRHR. Mutation at one or all of the three potential phosphorylation sites had differential effects on receptor ligand binding. Mutation of Ser253 or Thr264 to Ala did not significantly affect the receptor-binding affinity but decreased the number of measurable binding sites. Mutation of Thr238 to Ala or triple mutation of Thr238, Ser253, and Thr264 impaired or abolished receptor-binding affinity. Mutations of the potential phosphorylation sites affected receptor-mediated inositol phospholipid (IP) production and correlated with alterations in receptor binding after mutation, but they did not significantly affect receptor-mediated cAMP production or cAMP-mediated prolactin release. In addition, mutation of Ser253 or Thr264 to Ala did not affect the GnRH-provoked desensitization in terms of GnRH agonist-stimulated IP production. Deletion of the C-terminal portion (Ala260-Leu265) of the third intracellular loop of the rGnRHR, including a potential phosphorylation site (Thr264), abolished the receptor-binding affinity and receptor-mediated signal transduction. Replacement of the deleted C-terminal portion with a C-terminal portion (Ala-Ala-Arg-Thr-Leu-Ser) of the third intracellular loop of the Gq/11-coupled rat M1 muscarinic acetylcholine receptor did not restore receptor function. These results suggest that the potential phosphorylation sites or the region around the phosphorylation site of the third intracellular loop of the GnRHR is important for the structural integrity and expression of the receptor but that phosphorylation at these sites is not required for desensitization.
Animals↗
Charged beads enhance cutaneous wound healing in rhesus non-human primates.
Enhanced cutaneous wound healing by positively charged cross-linked diethylaminoethyl dextran beads (CLDD) was studied in a standardized incisional wound model in 20 adult and 20 geriatric Macaca mulatta (rhesus) partitioned equally over five time periods. Physiologic saline served as a control. Soft-tissue linear incisions were prepared between and 1 cm inferior to the scapulae. There were four incisions per rhesus; each incision was 1.5 cm long with 1 cm of undisturbed tissue between incisions, and both the experimental CLDD and physiologic saline treatments were administered to each rhesus. The incision treatments were either CLDD and soft-tissue closure with 4-0 BioSyn sutures or sterile physiologic saline and closure with 4-0 BioSyn sutures. The hypothesis was CLDD would enhance cutaneous wound repair. Verification of the hypothesis consisted of clinical examinations and histologic and tensiometric evaluations on biopsy specimens at 10 and 15 days, whereas 5-day and 2- and 4-month groups were assessed clinically and biopsy specimens were assessed histologically. The clinical course of healing for all groups was unremarkable. At 10 days, incisions in adult rhesus treated with CLDD had a 30-percent greater tensile strength compared with the physiologic saline-treated incisions (p = 0.01), whereas for geriatric rhesus, the CLDD treatment proved to be 15 percent greater in tensile strength compared with the physiologic saline cohort (p = 0.11). By day 15, incisions in adult rhesus were 26 percent stronger than the saline treatment group (p = 0.07), and the difference was 36 percent (p = 0.02) for the geriatric rhesus. From 5 through 15 days, histologic observations revealed a gradual decrease in quantity and integrity of CLDD, with no remnants of CLDD at either 2 or 4 months. Macrophages and multinucleated giant cells were localized in the dermis and were associated with the CLDD. These cells decreased commensurately with the decrease of CLDD beads. The data suggest that CLDD can enhance significantly the tensile properties of healing cutaneous wounds in both adult and geriatric rhesus. Moreover, if the wound healing is enhanced in geriatric patients, this finding may be clinically germane to conditions where wound healing is compromised, such as in diabetics and patients on steroids.
Animals↗