Biomedical subjects
Publications and source records attributed to Q Yang.
Raman micro-spectroanalysis was used to test and analysis micron dimension crystal grain of surface and cross-section of New stone age in two ancient stone axe from Yunnan Chuxiong. According to experimental results, the composition of ancient areas is inferred and the impact of environmental contaminants was noted.
OBJECTIVE: Clarify parkin gene deletion mutations at exons 3 to 7 in Chinese patients with Parkinson's disease (PD) and analyze them together with the clinical features of PD. METHODS: DNA was extracted from peripheral blood of 33 sporadic PD patients and 6 familial PD patients. Deletion mutations of parkin gene were identified by PCR amplification and agarose gel electrophoresis. The clinical data were analyzed together with the above information. RESULTS: Of the 33 sporadic PD patients, 1 had exons 5 and 7 deletion, 2 had exon 7 deletion. The age of onset was 46, 48, 50 respectively. In the 6 familial PD patients, 1 had exon 5 deletion. The inheritance pattern in this patient was autosomal recessive and onset of the patient was 60. All of the patients who had parkin gene deletion mutations had tremor, rigidity and bradykinesia, but athetosis was not found. In addition, deletion mutations of parkin gene at exons 3, 4 and 6, were not found. CONCLUSION: There are deletion mutations of parkin gene at exons 5 and 7 in Chinese PD patients.
CAAT/enhancer-binding proteins (C/EBPs) play an important role in the regulation of gene expression in insulin-responsive tissues. We have found that a complex containing C/EBPbeta interacts with an insulin response sequence in the insulin-like growth factor-binding protein-1 (IGFBP-1) gene and that a C/EBP-binding site can mediate effects of insulin on promoter activity. Here, we examined mechanisms mediating this effect of insulin. The ability of insulin to suppress promoter activity via a C/EBP-binding site is blocked by LY294002, a phosphatidylinositol 3-kinase inhibitor, but not by rapamycin, which blocks activation of p70(S6 kinase). Dominant negative phosphatidylinositol 3-kinase and protein kinase B (PKB) block the effect of insulin, while activated PKB suppresses promoter function via a C/EBP-binding site, mimicking the effect of insulin. Coexpression studies indicate that insulin and PKB suppress transactivation by C/EBPbeta, but not C/EBPalpha, and that N-terminal transactivation domains in C/EBPbeta are required. Studies with Gal4 fusion proteins reveal that insulin and PKB suppress transactivation by the major activation domain in C/EBPbeta (AD II), located between amino acids 31 and 83. Studies with E1A protein indicate that interaction with p300/CBP is required for transactivation by AD II and the effect of insulin and PKB. Based on a consensus sequence, we identified a PKB phosphorylation site (Ser(1834)) within the region of p300/CBP known to bind C/EBPbeta. Mammalian two-hybrid studies indicate that insulin and PKB disrupt interactions between this region of p300 and AD II and that Ser(1834) is critical for this effect. Signaling by PKB and phosphorylation of Ser(1834) may play an important role in modulating interactions between p300/CBP and transcription factors and mediate effects of insulin and related growth factors on gene expression.
Mixed halocarborane anions, 1-H-CB11Y5X6- (X, Y = Cl, Br, I), have been prepared by treatment of [Me3NH][1-H-CB11H5X6] (X = Cl, Br, I) with proper halogenating reagents at 180-220 degrees C in a sealed tube in high yield. These new anions are fully characterized by 1H, 13C, and 11B NMR, IR, and negative-ion MALDI MS spectroscopy. Some are further confirmed by single-crystal X-ray analyses. The weakly coordinating nature of these anions is probed by 29Si chemical shifts of the resulting Pri3Si(1-H-CB11Y5X6) compounds. The results suggest that the coordinating ability of these anions is mainly dependent on the substituents at 7-12 positions (namely, X atoms), and the contribution from the upper belt substituents Y is relatively small. These suggestions are consistent with the results obtained from the structural study of silver salts of mixed halo- and perhalocarborane anions.
The purpose of this report is to present a method which can be used to parameterize patterns of immunofluorescent staining in cultured neural cells. The algorithm is based on the observation that the variance in pixel intensity of the image is a power function of the magnitude of the area in immunofluorescently stained PC12 cells. This property is used to derive the fractal dimension (D) of the region of interest (ROI), and corresponds to the complexity of the pixel intensity associated with the ROI, which is analogous to a fractal surface. We show that the measure is useful in characterizing immunofluorescent staining patterns, and apply this measure to study the effects of ethanol exposure on mu-calpain and calpastatin-associated immunoreactivity. Exposure of PC12 cells to ethanol (80 mM)x48 h resulted in alterations in immunofluorescent signal (Control vs ethanol) associated with actin, calpastatin and mu-calpain: 2289+/-166 vs 1709+/-69, P<0.01; 1681+/-38 vs 2224+/-95, P<0.001; 1823+/-39 vs 2841+/-68, P<0.0001 respectively, magnitudes being pixel intensity units on a scale of 0-4095. D-values for the three proteins in the same order were: 2.32+/-0.01 vs 2.31+/-0.03, NS; 2.31+/-0.01 vs 2.32+/-0.01, NS; 2.16+/-0.03 vs 2. 24+/-0.02, P<0.01, with a possible D-value range of 2-3.
Cell cycle checkpoints are essential for the maintenance of genomic stability in response to DNA damage. We demonstrated recently that GADD45, a DNA damage-inducible protein, activates a G(2)/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivo with the G(2) cell cycle-specific kinase, Cdc2, proliferating cell nuclear antigen (PCNA), and the cell cycle kinase inhibitor p21(waf1). The ability of GADD45 to induce a G(2)/M arrest may be caused in part by the inhibition of Cdc2 kinase activity. Here, we report the identification of a region of GADD45 that is involved in this G(2)/M checkpoint. Mutants of GADD45 that lacked either the first 35 or the last 80 residues still retained an ability to induce G(2)/M arrest. A mutant with a deletion of the central region (residues 50-76), which is conserved in the family members GADD45beta and GADD45gamma, lacked such activity. This mutant also lacked an ability to bind to Cdc2, PCNA, and p21(waf1) in vivo. Consistently, either GADD45beta or GADD45gamma bind to Cdc2 in vivo. However, unlike GADD45, neither GADD45beta nor GADD45gamma inhibited the Cdc2 kinase or induced G(2)/M arrest. The unique effect of GADD45 may be caused by the presence of a region containing DEDDDR residues. Alanine substitutions in the region abolished GADD45 induction of a G(2)/M arrest and its inactivation of the Cdc2 kinase but not its binding to Cdc2, PCNA, or p21(waf1). Therefore, the binding of GADD45 to Cdc2 was insufficient to induce a G(2)/M arrest, and additional activity contributed by the DEDDDR residues may be necessary to regulate the G(2)/M checkpoint.
We have developed potent and selective thrombin inhibitors with a novel non-peptidic structure. A bicyclic lactam was used as the scaffold on which various P1 and P3 motifs were substituted. Herein, we report the in vitro and in vivo properties of four representatives of this novel class of inhibitors. Their Ki values were less than 10 nM, they inhibited equally both free and clot-bound thrombin, and they displayed high level of specificity for thrombin over other serine proteases (trypsin, factor Xa, activated Protein C, and plasmin). They prolonged the clotting time of human plasma to twice the control value in coagulation assays (TT, APTT, and PT) at a concentration below 3 microM. Their anticoagulant activities using rat plasma were similar to, although slightly weaker, than with human plasma. Furthermore, they inhibited thrombin-induced platelet aggregation (human and rat) at concentrations close to their Ki values for thrombin. These molecules demonstrated similar dose response antithrombotic efficacy in rat arterial and venous thrombosis models when given as i.v. bolus followed by infusion. Antithrombotic efficacy of 85% and greater was observed at a dose of 5-7 microM/kg/hour in each model. Bicyclic lactam inhibitor 3, at a dose which caused a complete inhibition of visible thrombus formation in the venous and arterial models of thrombosis, showed a 1.9-2.1 and a 4.0-4.8-fold shift in APTT and TT, respectively. Unfortunately, the bicyclic lactam inhibitors exhibited low oral bioavailability in rats. Therefore, this novel class of bicyclic lactam thrombin inhibitor has the potential to be promising intravenous antithrombotic agents for the treatment of arterial as well as venous thrombosis and warrants further investigation.
To target tumor cells for immunotherapy, we evaluated the feasibility of altering the epitopes on the surface polysialic acid of tumor cells. A precursor (N-propionylmannosamine), when incubated with leukemic cells, RBL-2H3 and RMA, resulted in substitution of the N-acetyl groups of surface alpha2-8 polysialic acid with N-propionyl groups. Expression of the altered alpha2-8 N-propionylpolysialic acid on the surface of tumor cells induced their susceptibility to cell death mediated by monoclonal antibody 13D9 (mAb 13D9), which specifically recognizes alpha2-8 N-propionylated polysialic acid. The expression of alpha2-8 N-propionylated polysialic acid and the lysis of tumor cells by antibody-dependent cytotoxicity depended on the time and dose of incorporation of N-propionylated mannosamine. In vivo, mAb 13D9 effectively controlled metastasis of leukemic cells RMA when mice were administered the precursor N-propionylated mannosamine.
Signaling by D(2)-dopamine receptors in neurons likely proceeds in the presence of Ca(2+) oscillations. We describe here the biochemical basis for a cross-talk between intracellular Ca(2+) and the D(2) receptor. By activation of calmodulin (CaM), Ca(2+) directly inhibits the D(2) receptor; this conclusion is based on the following observations: (i) The receptor contains a CaM-binding motif in the NH(2)-terminal end of the third loop, a domain involved in activating G(i/o). A peptide fragment encompassing this domain (D2N) bound dansylated CaM in a Ca(2+)-dependent manner (K(D) approximately 0.1 micrometer). (ii) Activation of purified Galpha(i1) by D2N, and D(2) receptor-promoted GTPgammaS (guanosine 5'-(3-O-thio)triphosphate) binding in membranes was suppressed by Ca(2+)/CaM (IC(50) approximately 0.1 micrometer). (iii) If Ca(2+) influx was elicited in D(2) receptor-expressing HEK293 cells, agonist-dependent inhibition of cAMP formation decreased. This effect was not seen with other G(i)-coupled receptors (A(1)-adenosine and Mel(1A)-melatonin receptor). (iv) The D(2) receptor was retained by immobilized CaM and radiolabeled CaM was co-immunoprecipitated with the receptor. Specifically, inhibition by CaM does not result from uncoupling the D(2) receptor from its cognate G protein(s); rather, CaM directly targets the D(2) receptor to block the receptor-operated G protein activation switch.
Aldose reductase (ALR2), a NADPH-dependent aldo-keto reductase (AKR), is widely distributed in mammalian tissues and has been implicated in complications of diabetes, including diabetic nephropathy. To identify a renal-specific reductase belonging to the AKR family, representational difference analyses of cDNA from diabetic mouse kidney were performed. A full-length cDNA with an ORF of 855 nt and yielding a approximately 1.5-kb mRNA transcript was isolated from a mouse kidney library. Human and rat homologues also were isolated, and they had approximately 91% and approximately 97% amino acid identity with mouse protein. In vitro translation of the cDNA yielded a protein product of approximately 33 kDa. Northern and Western blot analyses, using the cDNA and antirecombinant protein antibody, revealed its expression exclusively confined to the kidney. Like ALR2, the expression was up-regulated in diabetic kidneys. Its mRNA and protein expression was restricted to renal proximal tubules. The gene neither codistributed with Tamm-Horsfall protein nor aquaporin-2. The deduced protein sequence revealed an AKR-3 motif located near the N terminus, unlike the other AKR family members where it is confined to the C terminus. Fluorescence quenching and reactive blue agarose chromatography studies revealed that it binds to NADPH with high affinity (K(dNADPH) = 66.9 +/- 2.3 nM). This binding domain is a tetrapeptide (Met-Ala-Lys-Ser) located within the AKR-3 motif that is similar to the other AKR members. The identified protein is designated as RSOR because it is renal-specific with properties of an oxido-reductase, and like ALR2 it may be relevant in the renal complications of diabetes mellitus.
Given the importance of androgen and androgen receptor (AR) in the control of female breast growth and the potential association with female breast cancer, we evaluated the AR expression in 114 female breast cancers and further analyzed why AR expression was lost. Immunohistochemical analysis revealed that 14.1% (17/114) of the tumors lost the AR expression completely. To unravel the molecular mechanism for AR expression loss, we first analyzed the somatic mutations in exons 2 through 8 of the AR gene by PCR-SSCP, but no mutation was detected. As the CAG repeats within exon 1 are also microsatellite markers, we then analyzed whether allelic loss was present. Interestingly, 11 of the 17 AR-negative tumors were heterozygous and 9 of them showed allelic loss. The lost allele was further demonstrated to be the active one by X-chromosome inactivation analysis. To confirm the immunohistochemical results, Northern and Western blot hybridization was performed and neither AR mRNA nor protein was detected in these AR-negative tumors. Loss of the active AR allele was strongly correlated with the AR expression loss (P = 0.0005). We conclude that AR expression loss is attributed to the active allele loss of AR gene in female breast cancers. Our finding may be also crucial in predicting and influencing the response of breast cancer to endocrine therapy.
BACKGROUND: The prognostic value of the biochemical changes seen with proton MR spectroscopy (1H MRS) in ischemic stroke was examined. Acute diffusion-weighted imaging (DWI) was used to identify regions of ischemia for 1H MRS voxel localization. METHODS: Nineteen patients had 36 1H MRS studies, 13 patients acutely (mean, 11.1 hours), 10 subacutely (mean, 3.9 days), and 13 at outcome (mean, 82 days). Single-voxel, long-echo, timepoint-resolved spectroscopy was used to obtain lactate, n-acetylaspartate (NAA), choline, and creatine levels from the infarct core. Outcome measures were final infarct volume and clinical assessment scales (Canadian Neurological Scale, Barthel Index, and Rankin Scale). RESULTS: Acute lactate/choline ratio correlated more strongly with clinical outcome scores (r = 0.76 to 0.83; p < 0.01) and final infarct size (r = 0. 96; p < 0.01) than acute DWI lesion volume or acute NAA/choline ratio. Combination of acute lactate/choline ratio with acute DWI lesion volume improved prediction of all outcome scores (R2 = 0.80 to 0.90). The predictive effect of acute lactate/choline ratio was independent of acute DWI lesion volume (p < 0.001). In subacute and chronic infarction, both lactate/choline and NAA/choline ratios continued to correlate with outcome (p < 0.05). At the chronic stage, persistent lactate/choline ratio elevation strongly correlated with outcome measures (r = 0.71 to 0.87). CONCLUSION: Lactate/choline ratio measured in the acute infarct core by 1H MRS improves the prediction of stroke outcome and provides prognostic information complementary to DWI. Lactate/choline ratio could be used as an additional marker to select patients for acute and chronic therapies.
In cells of the mould Trichoderma viride, the existence of an antifungal protein (AFP)-like gene consisting of 285 bp was confirmed by Southern analysis that genomic DNA of T. viride could hybridize with the cDNA of mature AFP of Aspergillus giganteus MDH 18894. Except for the absence of two introns, the nucleotide sequence of the AFP-like gene was identical to that of the AFP gene of A. giganteus in positions 336-479, 568-649, and 706-765. The AFP-like gene could not be transcribed into its mRNA in T. viride cells as examined by RT-PCR using total RNAs of T. viride as template. Furthermore, AFP could not be detected either directly from the culture medium of T. viride or by Western analysis. However, the AFP-like gene could be actively expressed like the cDNA of AFP in Escherichia coli cell. Recombinant AFP exhibited similar antifungal activity as native AFP.
Stearic acid nanoparticles were prepared in this study by melt-homogenization to investigate the possibility of them as a new kind of drug carrier system. Some physicochemical properties of stearic acid nanoparticles were studied and morphology examined by transmission electron microscope. Cyclosporin A as a model drug was then encapsulated into stearic acid nanoparticles. Following the establishment of high performance liquid chromatography assay for cyclosporin A analysis in stearic acid nanoparticles or blood samples, the encapsulation ratio of cyclosporin A to stearic acid nanoparticles was estimated and pharmacokinetics as well as bioavailability of cyclosporin A stearic acid nanoparticles after oral administration to Wistar rats were studied, using the Sandimmun Neoral(R) (an available microemulsion system of cyclosporin A) as a reference. The mean diameter of cyclosporin A stearic acid nanoparticles was 316.1 nm, while the encapsulation ratio of cyclosporin A to stearic acid nanoparticles reached to 88.36%. It was demonstrated by IR spectra and differential scanning calorimetry that there was no chemical reaction occurred between the cyclosporin A and stearic acid. The relative bioavailability of cyclosporin A stearic acid nanoparticles over reference was nearly 80%, and the time to reach maximum concentration (T(max)) of cyclosporin A after oral administration of cyclosporin A stearic acid nanoparticles was delayed significantly than the reference, suggesting an obvious sustained release effect. The stearic acid nanoparticles might be a very potential drug carrier.
Sickle cell disease is caused by a variant of the beta-globin gene called sickle hemoglobin (Hb S). Inherited autosomal recessively, either two copies of Hb S or one copy of Hb S plus another beta-globin variant (such as Hb C) are required for disease expression. Hb S carriers are protected from malaria infection, and this protection probably led to the high frequency of Hb S in individuals of African and Mediterranean ancestry. Despite this advantage, individuals with sickle cell disease exhibit significant morbidity and mortality. Symptoms include chronic anemia, acute chest syndrome, stroke, splenic and renal dysfunction, pain crises, and susceptibility to bacterial infections. Pediatric mortality is primarily due to bacterial infection and stroke. In adults, specific causes of mortality are more varied, but individuals with more symptomatic disease may exhibit early mortality. Disease expression is variable and is modified by several factors, the most influential being genotype. Other factors include beta-globin cluster haplotypes, alpha-globin gene number, and fetal hemoglobin expression. In recent years, newborn screening, better medical care, parent education, and penicillin prophylaxis have successfully reduced morbidity and mortality due to Hb S.
The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) is involved in folate metabolism. The MTHFR gene is located on chromosome 1 (1p36.3), and two common alleles, the C677T (thermolabile) allele and the A1298C allele, have been described. The population frequency of C677T homozygosity ranges from 1% or less among Blacks from Africa and the United States to 20% or more among Italians and US Hispanics. C677T homozygosity in infants is associated with a moderately increased risk for spina bifida (pooled odds ratio = 1.8; 95% confidence interval: 1.4, 2.2). Maternal C677T homozygosity also appears to be a moderate risk factor (pooled odds ratio = 2.0; 95% confidence interval: 1.5, 2.8). The A 1298C allele combined with the C677T allele also could be associated with an increased risk for spina bifida. Some data suggest that the risk for spina bifida associated with C677T homozygosity may depend on nutritional status (e.g., blood folate levels, intake of vitamins) or on the genotype of other folate-related genes (e.g., cystathionine-beta-synthase and methionine synthase reductase). Studies of the C677T allele in relation to oral clefts, Down syndrome, and fetal anticonvulsant syndrome either have yielded conflicting results or have not been yet replicated.
In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled gel beads by rotary detergent dialysis. The immobilized amount was up to 80 nmol of RC and 33 micromol of lipid/g of moist gel in streptavidin-coupled Sephacryl S-1000 gel. By QAC frontal runs, retardation of mitochondrial cyt c on immobilized RC liposome columns was demonstrated. The dissociation constant for the RC-cyt c interaction was determined to be 0.20-0.57 microM. QAC studies also allowed evaluation of the orientation of reconstituted RC in immobilized liposomes by comparison of the total amount of cyt c binding sites with the amount of available binding sites obtained by QAC. It seems that the RC proteoliposomes immobilized in Sephacryl S-1000 gel exposed the cyt c binding sites on the outer surface of the liposomes due to effects of the gel network pore size and the resulting liposomal size.
The effects of zinc on c-fos gene expression and transcription in the epiphyses of fetal long bone were studied in mice. The long bones of 16-day fetal mouse were cultured for 48 hours(in medium GBJb) and then used for measuring c-fos gene expression and transcription by immunohistochemistry and in situ hybridization. The result was analyzed by an imaged-analyses system. The experiment was divided into zinc control group(ZC), zinc deficiency group(ZD), zinc deficiency replenish group(ZDR) and zinc stimulatory group(ZS) respectively. The result showed that 1) zinc deficiency caused c-fos protein and mRNA expression and the number of reactive cell decreased. 2) when zinc concentration of medium was 100 mumol/L, the expression and transcription of c-fos gene were increased in hypertrophic, proliferative and resting zone of epiphyses. The research suggested that zinc could affect the expression of c-fos gene in mouse fetal long bone.