Biomedical subjects

H Pan

Publications and source records attributed to H Pan.

At least 73 records · Page 4Linked to original sources

PubMed1999

Cloning, expression, and purification of Bacillus stearothermophilus DNA primase and crystallization of the zinc-binding domain.

The dnaG gene encoding DNA primase has been isolated from chromosomal DNA of Bacillus stearothermophilus and its entire nucleotide sequence determined. The deduced amino acid sequence comprised 597 amino acid residues and the molecular mass was calculated to be 67068 Da. B. stearothermophilus primase was overexpressed in Escherichia coli and purified to homogeneity. The N-terminal 12 kDa zinc-binding domain has been crystallized. The crystals are of the monoclinic space group P21 with cell dimensions a=36 A, b=59 A, c=46 A, beta=91.8 degrees and diffract to 1.7 A resolution.

Amino Acid Sequence↗

PubMed1999

cDNA cloning and expression of acutin.

Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies. Its first 15 N-terminal amino acid residues sequence was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR. Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for the first time. The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms. Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic residues, His41, Asp84 and Ser179; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys231, Cys118-Cys185, Cys150-Cys164, Cys175-Cys200. The recombinant acutin has been expressed in E. coli and purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase.

Agkistrodon↗

PubMed1999

Application of FTA sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based Southern blotting.

Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7deaza-dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM.

Blood Specimen Collection↗

PubMed1999

[Expression of chimeric single-chain antibody with specificity for HBsAg in E. coli].

The Vk gene of MAb with specificity for HBsAg and human Ck gene have been combined to form chimeric light chain (Vk-Ck) by recombinant PCR, which has combined with VH to construct chimeric single-chain antibody(ScFv-Ck) by a linker encoding a flexible peptide[(Gly3 Ser)3]. The ScFv-Ck has been expressed in the heat-induced expression system and secreted expression system of E. coli respectively. Analysis by Western-blot and indirect ELISA shows that the ScFv-Ck product in two systems both have HBsAg-binding ability, and ScFv-Ck has been secreted from E. coli in the direction of secretion peptide.

Blotting, Western↗

PubMed1999

[The genotype-based haplotype relative risk and transmission disequilibrium test analyses of familial febrile convulsions].

OBJECTIVE: To confirm the linkage of familial febrile convulsions to the short arm of chromosome 6(6p) or the long arm of chromosome 8(8q). METHODS: The authors finished genotyping of Pst I locus on the coding region of heat shock protein (HSP) 70, 5'untranslated region of HSP70-1, 3' untranslated region of HSP70-2, D8S84 and D8S85. The data were processed by the genotype-based haplotype relative risk(GHRR) and transmission disequilibrium test(TDT) methods in PPAP. RESULTS: Some signs of association and disequilibrium between D8S85 and FC were shown by GHRR and TDT. CONCLUSION: A suspect linkage of familial febrile convulsions to the long arm of chromosome 8 has been proposed.

Chromosomes, Human, Pair 6↗

PubMed1999

[Demonstration for periodicity of leptospirosis in Yichang City of Hubei Province during 1960-1997].

OBJECTIVE: To study the periodicity of leptospirosis incidence. METHODS: Data of leptospirosis epidemics in Yichang City collected during the past 38 years from 1960 to 1997 were analyzed with periodic graphics method. RESULTS: Cause-specific incidence rate of leptospirosis was 15.43 per 100,000 in average, with an epidemic interval of 10 years, with statistical significance in periodic vibration (J = 4.179, P < 0.05). CONCLUSION: The results mentioned above provided scientific basis for the effective control and prevention of the disease.

China↗

PubMed1999

[Screening of beta zero-thalassemia in cord blood from 2,423 newborns in Xilin county of Guangxi].

OBJECTIVE: To investigate the incidence of beta zero-thalassemia in newborns in Xilin county of Guangxi and identify the common type of the gene mutation. METHODS: Cord blood samples from successive 2,423 newborns were screened for beta zero-thalassemia by PAGE, and the gene mutation was assayed by PCR and dot blot hybridization. RESULTS: Seven cases (0.29%) of beta zero-thalassemia were revealed in the 2,423 newborns. The gene mutation types were as follows: three cases of CD17/CD17, two of CD17/CD41-42, one of CD41-42/CD41-42 and one of CD17/IVS-I-1. CONCLUSION: These data might be of help to prenatal diagnosis of beta zero-thalassemia in Guixi district.

China↗

PubMed1999

[Protection of PNH red blood cells from lysis by transferring GPI-anchored protein vesicles of normal human RBCs in vitro].

OBJECTIVE: To observe the transfer of GPI-anchored protein CD59 from vesicles of normal RBCs to paroxysmal nocturnal hemoglobinuria(PNH) RBCs can correct the susceptibility to complement attack in vitro. METHODS: Vesicles released from normal RBCs under ATP depletion or storage, are rich in CD59. PNH CD59- RBCs were separated by elution from immunoaffinity column bound with monoclonal antibody CD59, and then incubated with normal RBCs vesicles. The content of CD59 in vesicles and RBCs was detected by Western blot and flowcytometric analysis respectively and hemolysis of PNH CD59- RBCs was detected by cobra venom factor hemolysis test. RESULTS: The fluorescence intensity of PNH CD59- RBCs were increased from (1.68 +/- 0.57)% to (58.42 +/- 8.25)% and the hemolysis was significantly decreased from (27.13 +/- 9.69)% to (19.49 +/- 7.61)% after incubation with vesicles prepared from normal donors (P < 0.05), while normal RBCs had no obvious change before and after the same treatment. (P > 0.05). CONCLUSIONS: It seems that CD59 protein molecules could be transfered from normal RBCs vesicles to PNH CD59- RBCs and retaining the complement regulatory function. CD59- RBCs could be rendered less liable to hemolyze during complement attack.

CD59 Antigens↗

PubMed1999

[Monitoring glucoamylase fermentation with infrared spectroscopy].

The glucoamylase fermentation was investigated with Fourier transform infrared spectroscopy. During the cultivation, the variation of carbon and nitrogen source can be reflected from the infrared spectra. In most cases, the complex media are used to cultivate cells or bacteria. It is difficult to construct calibration samples with synthetic mixtures which can represent the composition of real broth. The infrared spectral data of the real broth and off-line chemical analysis results were used to establish the calibration model. The model is successfully used to predict the concentrations of total sugar, reduced sugar, and nitrogen in real broth. The correlation coefficients between the infrared analysis results and chemical analysis are 0.999, 0.990 and 0.974 for total sugar, reduced sugar and nitrogen, respectively. The bacteria, culture and operating condition are relatively stable during the practical productive processes. The method can be used to monitor the industrial processes.

Aspergillus niger↗

PubMed1998

Multi-level repeated measures growth modelling using extended spline functions.

This paper explores the fitting of multi-level models to growth data over a wide age range using a new class of extended spline models. These extend conventional splines based on a '+' function representation by allowing variable order functions and by including fractional polynomial terms. The work focuses on modelling human growth in height and head circumference with example data sets. The procedures can be used with covariates and for comparing population parameters.

Adolescent↗

PubMed1998

Expression of membrane-bound transferrin-like protein p97 on the cell surface of chondrocytes.

A concanavalin-A-binding protein of 76 kDa was purified from the plasma membrane fraction of rabbit chondrocyte cultures. Amino acid sequencing of the N-terminal region and of tryptic peptides of the protein, in addition to sequencing of its cDNA revealed that this protein is highly similar to the tumour-associated antigen p97. Hence, it was concluded that this protein is the rabbit form of p97. Western blotting, Northern blotting and reverse-transcription PCR analyses indicated that rabbit p97 is expressed at high levels in cartilage and chondrocytes, but is barely detectable in the bone, liver, kidney, small intestine, eye, pancreas, heart, testis, skeletal muscle, spleen and fibroblasts. Immunocytochemical and immunohistochemical analyses demonstrated that p97 is expressed in the plasma membrane of chondrocytes. p97 transcript was detected in all zones of the cartilage but the level was relatively low in the hypertrophic zone. These findings suggest that p97 is involved in maintaining the cell surface characteristics of chondrocytes.

Amino Acid Sequence↗

PubMed1998

Diversity of cDNAs encoding phospholipase A2 from Agkistrodon halys pallas venom, and its expression in E. coli.

As a step toward understanding the structure and function of phospholipase A2(PLA2), we isolated several novel cDNAs encoding Agkistrodon halys Pallas PLA2 isoenzymes including B-PLA2, Asn49-PLA2, A-PLA2, A'-PLA2 and BA1-PLA2 by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminus of these enzymes. The amino acid sequences of A-PLA2 deduced from cDNA are consistent with that isolated from venom except for four residues. Asn49-PLA2 and B-PLA2 are highly similar (> 95%), but the critical residue Asp49 in the active centre of B-PLA2 is replaced by Asn49 in Asn49-PLA2. The N-terminal residues (1-24) of BA1-PLA2 shows high similarity to that of B-PLA2 which has strong ability to hemolyze erythrocytes, while its C-terminal residues (72-125) are the same as that of A-PLA2 which can inhibit platelet aggregation. The successful cloning of these isoenzymes not only provide excellent native material to study the structure-function relationship of PLA2s, but also to disclose the genesis of structural diversity of PLA2s, namely DNA modification and gene rearrangement. The cloned cDNA for A-PLA2 has been expressed in E. coli. By Q-Sepharose column chromatography, denaturation-renaturation and FPLC, we obtained the active recombinant protein with the initiator Met. This is the first report of the production of an active recombinant PLA2 with the initiator Met.

Agkistrodon↗

PubMed1998

Key roles for E2F1 in signaling p53-dependent apoptosis and in cell division within developing tumors.

Apoptosis induced by the p53 tumor suppressor can attenuate cancer growth in preclinical animal models. Inactivation of the pRb proteins in mouse brain epithelium by the T121 oncogene induces aberrant proliferation and p53-dependent apoptosis. p53 inactivation causes aggressive tumor growth due to an 85% reduction in apoptosis. Here, we show that E2F1 signals p53-dependent apoptosis since E2F1 deficiency causes an 80% apoptosis reduction. E2F1 acts upstream of p53 since transcriptional activation of p53 target genes is also impaired. Yet, E2F1 deficiency does not accelerate tumor growth. Unlike normal cells, tumor cell proliferation is impaired without E2F1, counterbalancing the effect of apoptosis reduction. These studies may explain the apparent paradox that E2F1 can act as both an oncogene and a tumor suppressor in experimental systems.

Animals↗

PubMed1998

Rapid, transient changes at the env locus of plasma human immunodeficiency virus type 1 populations during the emergence of protease inhibitor resistance.

Plasma human immunodeficiency virus type 1 (HIV-1) populations were genetically analyzed at their most variable locus, the envelope gene, during the rapid emergence of resistance to protease inhibitor monotherapy. Plasma virus populations remained genetically constant prior to drug treatment and during the 1 to 2 weeks following initiation of therapy, while viremia fell 10- to 100-fold. Concomitant with rapid plasma viremia rebounds associated with the emergence of drug-resistant virus, marked alterations were then detected at the env locus. Plasma population changes lasted only a few weeks before the reappearance of the pretreatment envelope variants. The emergence of resistance to single protease inhibitors was therefore associated with major but transient changes at a nonselected locus. Selection for resistance to single protease inhibitors thus appears to be more complex than the continued replication of a large, random, and therefore genetically representative sampling of the pretreatment plasma population. The possibility that drug-privileged anatomical sites containing distinct envelope variants and/or a small effective HIV-1 population size account for these results is discussed.

Drug Resistance, Microbial↗

PubMed1998

Guinea pig 5-HT transporter: cloning, expression, distribution, and function in intestinal sensory reception.

Studies of the guinea pig small intestine have suggested that serotonin (5-HT) may be a mucosal transmitter that stimulates sensory nerves and initiates peristaltic and secretory reflexes. We tested the hypothesis that guinea pig villus epithelial cells are able to inactivate 5-HT because they express the same 5-HT transporter as serotonergic neurons. A full-length cDNA, encoding a 630-amino acid protein (89.2% and 90% identical, respectively, to the rat and human 5-HT transporters) was cloned from the guinea pig intestinal mucosa. Evidence demonstrating that this cDNA encodes the guinea pig 5-HT transporter included 1) hybridization with a single species of mRNA ( approximately 3.7 kb) in Northern blots of the guinea pig brain stem and mucosa and 2) uptake of [3H]5-HT by transfected HeLa cells via a saturable, high-affinity (Michaelis constant 618 nM, maximum velocity 2.4 x 10(-17) mol . cell-1 . min-1), Na+-dependent mechanism that was inhibited by chlorimipramine > imipramine > fluoxetine > desipramine > zimelidine. Expression of the 5-HT transporter in guinea pig raphe and enteric neurons and the epithelium of the entire crypt-villus axis was demonstrated by in situ hybridization and immunocytochemistry. Inhibition of mucosal 5-HT uptake potentiates responses of submucosal neurons to mucosal stimulation. The epithelial reuptake of 5-HT thus appears to be responsible for terminating mucosal actions of 5-HT.

Amino Acid Sequence↗

PubMed1998

Downstream technique study of human tumor necrosis factor alpha and its mutant.

The study of downstream techniques for recombinant human necrosis factor alpha (rhTNF alpha) and its mutant (Lys2)-rhTNF alpha led to the results of approximately 50 g wet recombinant E. coli per liter with high expression level (> 50%) harvested from autocontrolled fed-batch culture in 15 L fermentor (B. Braun). The rhTNF alpha and (Lys2)-rhTNF alpha expressed are totally soluble. Followed by the process of ultrasonication, ammonium sulfate precipitation, ion-exchange chromatography (DEAE-sepharose FF, CM-sepharose FF), and molecular sieve (Sephacryl S-200), a yield of approximately 1 g pure recombinant protein from 1 L broth is obtained. The purity is up to 98%. The specific activity of rhTNF alpha and (Lys2)-rhTNF alpha are approximately 1.5 x 10(8) IU/mg and approximately 6 x 10(8) IU/mg, respectively.

Escherichia coli↗

PubMed1998

[The effect of antioxidant sodium ferulate on human lymphocytes apoptosis induced by H2O2].

OBJECTIVE: Sodium ferulate is a potent antioxidant purified from ferula assafoetida L. The effect of sodium ferulate on apoptosis of human lymphocytes induced by H2O2 in vitro was studied. METHODS: Apoptosis was detected by light and electron microscopic, DNA electrophoresis and Flow cytometric cell cycle analysis. mitochondrial function was detected by MTT method. BCL-2 and Bax expression was detected by confocal laser scanning microscope. RESULTS: The morphological and biochemical changes characteristic of apoptosis were partially restored after treatment with sodium ferulate, Immunofluorescence assay showed a decrease in BCL-2 and an increase in Bax oncogene proteins in lymphocytes treated with H2O2 and an improvement of cell apoptosis was found after sodium ferulate treatment. CONCLUSION: Oxidative stress can induce apoptosis in lymphocytes, and this induction can be partly prevented by sodium ferulate.

Apoptosis↗

PubMed1998

Aging of human mature erythrocytes is like a process of apoptosis in enucleated cell.

Apoptosis of nucleated cells is well known, but how about the unnucleated cells is still not elucidated. In the present paper, the morphological and biochemical features of the aged erythrocytes were observed and compared with the characteristic events of apoptosis. Membrane of aged erythrocytes tends to shrink, protrude, from vesicle and lose lipid asymmetry. Aged erythrocytes were removed by phagocytosis. Both of the events are very similar to the apoptotic nucleated cells. The authors suggested that aging of erythrocytes is also a process of apoptosis.

Apoptosis↗