Biomedical subjects

B Shen

Publications and source records attributed to B Shen.

At least 163 records · Page 9Linked to original sources

PubMed1995

Site-directed mutagenesis of Azotobacter vinelandii ferredoxin I: cysteine ligation of the [4Fe-4S] cluster with protein rearrangement is preferred over serine ligation.

The [4Fe-4S] cluster of Azotobacter vinelandii ferredoxin I receives three of its four ligands from a Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys sequence at positions 39-45 while the fourth ligand, Cys20, is provided by a distal portion of the sequence. Previously we reported that the site-directed mutation of Cys20 to Ala (C20A protein) resulted in the formation of a new [4Fe-4S] cluster that obtained its fourth ligand from Cys24, a free cysteine in the native structure. That ligand exchange required significant protein rearrangement. Here we report the conversion of Cys20 to Ser (C20S protein), which gives the protein the opportunity either to retain the native structure and use the Ser20 O gamma as a ligand or to rearrange and use Cys24. X-ray crystallography demonstrates that the cluster does not use the Ser20 O gamma as a ligand; rather it rearranges to use Cys24. In the C20S protein the [4Fe-4S] cluster has altered stability and redox properties relative to either C20A or the native protein.

Amino Acid Sequence↗

PubMed1995

Malonyl-coenzyme A:acyl carrier protein acyltransferase of Streptomyces glaucescens: a possible link between fatty acid and polyketide biosynthesis.

Streptomyces glaucescens, a Gram-positive soil bacterium, produces the polyketide antibiotic tetracenomycin (Tcm) C. To study possible biochemical connections between the biosynthesis of bacterial fatty acids and polyketides, the abundant acyl carrier protein (ACP) detected throughout the growth of the tetracenomycin (Tcm) C-producing S. glaucescens was purified to homogeneity and found to behave like many other ACPs from bacteria and plants (apparent M(r) of 20,000 on gel filtration chromatography, apparent M(r) of 3400-4800 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, and pI approximately 3.8). By using an oligodeoxynucleotide synthesized in accordance with the sequence of residues 25-36 of the ACP, the fabC gene encoding this protein was cloned, and expression of this gene in Escherichia coli yielded the ACP entirely as the active holoenzyme. Sequence analysis of 4.3 kilobases (kb) of DNA flanking fabC revealed the presence of three other genes oriented in the same transcriptional direction in the order fabD, fabH, fabC, and fabB. Each of the four genes is predicted to encode proteins with high sequence similarity to the following components of the E. coli fatty acid synthase (FAS): the FabD malonyl-coenzyme A:ACP acyltransferase (MAT), FabH 3-oxoacyl:ACP synthase III, AcpP ACP, and FabB 3-oxoacyl:ACP synthase I. Expression of the S. glaucescens fabD gene in E. coli produced active MAT able to catalyze in vitro the transfer of radioactive malonate from malonyl-coenzyme A to the E. coli AcpP and S. glaucescens FabC ACPs, as well as to the TcmM ACP component of the Tcm type II polyketide synthase [Shen, B., et al. (1992) J. Bacteriol 174, 3818-3821]. Expression of fabD also restored the high-temperature growth of the E. coli fabD89 mutant that bears a temperature-sensitive MAT. The latter finding and the close similarity between the organization of the S. glaucescens fabDHCB and E. coli FAS-encoding genes (fabH/fabD/fabG/acpP/fabF) suggest that the S. glaucescens genes encode FAS enzymes. Moreover, on the basis of its in vitro activity, it is possible that the S. glaucescens FabD MAT is responsible for charging the TcmM ACP with malonate in vivo, a key step in the synthesis of the deca(polyketide) precursor of Tcm C. This implies the existence of a functional connection between fatty acid and polyketide metabolism in this bacterium.

Acyl-Carrier Protein S-Malonyltransferase↗

PubMed1995

Creation of a high cytotoxic active human tumor necrosis factor having the truncated and more basic amino terminus.

In order to define the structure-functional relationship of tumor necrosis factor(TNF), a mutant TNF gene was created by site-specific mutagenesis based on the PCR technique. This gene was highly expressed in E.coli cells. The amount of the recombinant protein was up to about 80% of the total cellular proteins. Through one-step ion exchange chromatography, the mutant TNF could be purified to homogeneity. This mutein showed the molecular weight of a dimer but not a trimer. It bears the features of truncated amino terminus and increase of the basicity of amino terminal residues. Compared with the wild type TNF, the specific activity of mutant TNF was increased by fourfold.

Amino Acid Sequence↗

PubMed1995

Multiple autoantibodies in patients with silicone breast implants.

Diverse immunologic abnormalities have been described in women who received silicone breast implants. However, most studies have focused on either a limited number of patients or a small panel of autoantibodies. We report the analysis of 20 autoantibodies in 116 women with implants and 134 controls. The patients ranged from 26- to 66-years-old, with a mean of 45.7 +/- 8.3 years; breast prostheses were in place for a mean of 15 +/- 5.6 years, with a range of 4 to 30, the chief complaints of the 116 patients included polyarthralgias, fatigue, myalgias, morning stiffness, and decreased memory. All 250 sera were tested blindly using a panel of 20 autoantigens including SS-A, SS-B, RNP, cardiolipin (CL), collagen types I, II and IV, phosphatidylserine (PS), myeloperoxidase (MPO), sulfatides (sulf), thyroglobulin (TG), gangliosides (GDIa;GM2), proteinase-3 (PR3), Jo-1, Sm, HPRPP-ribosomal phosphate, histones (H2AH2B), Scl-70 and glomerular basement membrane (NC-1). Values from individual patients were considered positive only when greater than 3 SD above the control mean. There was a statistically significant greater frequency of autoantibodies in women with implants for 15 of the 20 autoantigens; these were particularly striking for anti-H2AH2B, HPRPP, SS-A, SS-B, Scl-70, CL, PS, GM2, and NC-1. Many patients harbored several autoantibodies; 20% had four autoantibodies; 8% had six autoantibodies. The association of autoantibodies and implants suggests an adjuvant action of silicon/silicone byproducts.

Adjuvants, Immunologic↗

PubMed1995

Autoantibody profile in the sera of women with hyperprolactinemia.

Prolactin (PRL) has been implicated as an important in vivo modulator of cellular and humoral immunity. In order to elucidate the impact of elevated serum PRL levels on the immune system, we measured circulating autoantibodies in the serum of 33 hyperprolactinemic (HPRL) women and in 19 healthy women with normal PRL levels. All sera were examined for the presence of autoantibodies against 15 different antigens, including: ssDNA, dsDNA, histones (H2AH2B), Sm, RNP, SS-A/Ro, SS-B/La, cardiolipin, Scl-70, Jo1, collagen, glomerular basement membrane (GBM), pyruvate dehydrogenase (PDH), proteinase-3 (PR3) and MPO. Twenty-five of 33 (75.7%) HPRL women were found to have at least one autoantibody, while none of the 19 women with normal PRL had any. Eight HPRL women had seven or more (up to nine) different autoantibodies. Some of the autoantibodies were more frequently expressed than others, namely: anti-ss-DNA, anti-dsDNA, anti-Sm, anti-PDH and anti-SS-A/Ro. Autoantibodies to the autoantigens tested are common in a variety of autoimmune and rheumatic disorders including systemic lupus erythematosus (SLE), Sjögren's syndrome, mixed connective tissue disease (MCTD), scleroderma, primary biliary cirrhosis (PBC), polymyositis/dermatomyositis and vasculitis. Yet none of the HPRL women whose serum was found to contain high titers of autoantibodies presented with symptoms related to the respective autoimmune disorders. Our results support the role of PRL in the regulation of immune responses in man.

Adult↗

PubMed1995

XPG protein has a structure-specific endonuclease activity.

Biochemically active human DNA repair protein, xeroderma pigmentosum G (XPG), was overexpressed in insect cells by a recombinant baculovirus. The recombinant baculovirus produced XPG with a mobility of approximately 185 kDa in a denaturing polyacrylamide gel. Indirect immunofluorescence studies demonstrated that the recombinant full-length XPG protein was expressed predominantly as a nuclear protein. The recombinant XPG protein was purified to apparent homogeneity using Q-sepharose, S-300 size exclusion, and Mono Q column chromatography. XPG protein showed a structure-specific DNA endonuclease activity, and a preferential affinity to single-stranded DNA and RNA compared to double-stranded DNA.

Animals↗

PubMed1995

Long-term potentiation at the apical and basal dendritic synapses of CA1 after local stimulation in behaving rats.

1. We recorded the extracellular excitatory postsynaptic potentials (EPSPs) in CA1 in the freely behaving rats after stimulation of the apical dendritic and basal dendritic afferents. The apical dendritic population EPSP was negative at the apical dendritic layers and positive at stratum oriens and alveus. The basal dendritic population EPSP was negative at basal dendritic layer (stratum oriens) and positive at the cell body and apical dendritic layers. 2. We delivered various tetanic stimulus patterns in an attempt to elicit long-term potentiation (LTP) at the apical dendritic synapse of CA1 pyramidal cells. A 200-Hz high-frequency train (HF) 1 s in duration was more successful than theta-frequency primed bursts (PBs; 1 or 4 priming pulses followed by 10 or 4 pulses at 100 Hz) in eliciting LTP of the apical dendritic population EPSP, recorded either ipsilaterally or contralaterally. 3. Tetanus applied to the apical dendritic afferents occasionally elicited long-term depression (LTD) at an intensity lower than the LTP threshold. LTD persisted to > or = 1 day after a tetanus. 4. High-intensity tetanic stimulations often elicited hippocampal afterdischarges (ADs). If the AD duration was brief (< 15 s), apical dendritic LTP could follow the AD. However, if the AD duration was long, no apical dendritic LTP was observed. Tetanus that evoked an apical dendritic LTP typically evoked a short hippocampal AD < 15 s in duration. 5. LTP of the basal dendritic population EPSP was readily elicited either after an HF or a PB tetanus of low (1.5-2 times threshold) stimulus intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗

PubMed1995

Effects of TNF alone or in combination with chemotherapeutic agents on human ovarian cancers in vitro and in nude mice.

Using the tetrazolium (MTT) assay, we examined the cytotoxicities of recombinant human tumor necrosis factor (rhTNF) and five chemotherapeutic agents, namely CTX, 5-FU, VCR, DDP and KSM, on human ovarian cancer cell lines OVCAR3 and CAOV3. The results showed that the cytotoxicities of rhTNF at concentrations of 50-50,000 U/ml on OVCAR3 cell line and CAOV3 cell line exposed to rhTNF for 24 hours were from 14.2% +/- 6.8% to 67.2% +/- 3.0% and from 8.2% +/- 4.3% to 60.9% +/- 1.3%, respectively. The cytotoxicities of all five chemotherapeutic agents tested on the two cell lines were much lower than that of rhTNF. We also studied the combined antitumor potential of rhTNF with the five chemotherapeutic agents and the results showed that there were various degrees of synergism in cytotoxicities of rhTNF in combination with DDP or KSM on the two cell lines. Based on experiments in vitro, the in vivo antitumor activities of rhTNF, both alone and in combination with KSM, were examined in OVCAR3 cancer transplanted in nude mice. The results showed a considerable antitumor effect of rhTNF when it was used alone and a marked synergistic effect when it was used in combination with KSM on the xenograft tumors.

Animals↗

PubMed1995

Metabolic approach for regulation of IL-1 beta in vivo.

IL-1 beta, the autoantibodies to IL-1 beta and IL-1 beta-containing immune complex in pleural effusion and cerebrospinal fluid were detected using anti-recombinant human IL-1 beta monoclonal antibody. The results indicate that IL-1 beta is detectable in some cases, and the amounts decrease gradually with the chemotherapy following the autoantibodies and the specific detectable immune complex. These autoantibodies may have a feedback effect, inhibiting the IL-1 beta activity, and forming the immune complex to speed the clearance of activated IL-1 beta.

Antibodies, Monoclonal↗

PubMed1995

Scanning electron microscope observation on tegumental alteration of Schistosoma japonicum induced by levo- and dextro-praziquantel.

When mice infected with Schistosoma japonicum for 35 d were treated ig with levo-praziquantel (L-Pra) 150 mg.kg-1 or dextro-praziquantel (D-Pra) 150-600 mg.kg-1, the alterations of the worm tegument were examined by scanning electron microscopy within 1-24 h after administration. The results showed that L-Pra induced apparent and extensive damage on the worm surface including severe swelling, fusion, erosion and peeling of tegument accompanied by attachment of the host leukocytes. The discoid-like sensory structures usually showed swelling, deformation and collapse. With D-Pra 150 mg.kg-1, only slight damage of tegument was seen. When the dose was increased to 600 mg.kg-1, the damage of worm tegument was similar to that induced by L-Pra 150 mg.kg-1 but the intensity of the damage was slighter as compared to that induced by L-Pra. The results suggested that the actions of the 2 optical enantiomers of praziquantel were different in quantity but not in quality.

Animals↗

PubMed1995

Expression of human stem cell factor in the baculovirus expression system.

A partial cDNA of human Stem Cell Factor (SCF) was isolated and transferred into the genome of Bombyx Mori Nuclear Polyhedrosis Virus adjacent to the polyhedrin promoter. The soluble form of the SCF protein was expressed in Bombyx Mori cells and silkworm larvae and was secreted into the medium in an active state. The recombinant SCF was similar to natural SCF with respect to its interaction with monoclonal antibody and its synergistic biological activity in conjunction with colony-stimulating factor.

Baculoviridae↗

PubMed1995

Experimental studies of the effect of Forskolin on the lowering of intraocular pressure.

PURPOSE: To determine the effect of domestic Forskolin on lowering of intraocular pressure (IOP) in rabbits. METHODS: Measure the normal IOP and ocular hypertension by water load in rabbits using Perkins tonometer. Measure the adenylate cyclase (AC) activity by Ho & Sutherland's method. RESULTS: The results show that Forskolin significantly lowered the normal IOP of rabbits and blocked the ocular hypertension induced by water load in rabbits (P < 0.01). The maximum decrease value of 2%, 1% and 0.5% of the Forskolin was 0.59, 0.36 and 0.19 kPa (1kPa = 7.5 mmHg), which showed the noticeable dose-effect relationship. Topical ocular application of Forskolin lowered IOP in half an hour, reached to a peak in 2-3 hours and remained significantly for 10 hours. The pupillary diameter did not change when IOP was reduced. The Forskolin had potent stimulative properties to AC. The greater the ability of the Forskolin to stimulate AC, the stronger the effect of IOP lowering. CONCLUSIONS: The Forskolin had the effect on lowering the IOP of rabbits. The mechanism of IOP reduction by Forskolin is related to its AC activation.

Adenylyl Cyclases↗

PubMed1995

[Analysis of human DNA fingerprints by using fluorescein labelled probe JH12.6].

A method of analysis of human DNA fingerprints has been established by using fluorescein (Fl-11-dUTP) labelled probe JH12.6. The clear and high distinctive patterns of human DNA fingerprints were obtained with this method. The DNA fingerprints of 78 unrelated individuals living in Yunnan province of China were detected, and their probability of chance association between random individuals was calculated as 7.4 x 10(-11), and their mean allelic frequency as 0.09. Comparing of DNA fingerprints with labelling probe JH12.6 by using between radioactive 32P and fluorescein showed that the method by using fluorescein labelled probe JH12.6 was simple, fast, safe and economical. It can totally replace the method by using radioactive 32P labelled probe JH12.6, and it can be broadly applied in forensic medicine and other fields.

DNA Fingerprinting↗

PubMed1994

Triple hydroxylation of tetracenomycin A2 to tetracenomycin C in Streptomyces glaucescens. Overexpression of the tcmG gene in Streptomyces lividans and characterization of the tetracenomycin A2 oxygenase.

Nucleotide sequence analysis of the tcmG gene has suggested that the TcmG protein is responsible for the triple-hydroxylation of tetracenomycin (Tcm) A2 to Tcm C in Streptomyces glaucescens (Decker, H., Motamedi, H., and Hutchinson, C.R. (1993) J. Bacteriol. 175, 3876-3886). The heterologous expression of the tcmG gene in Streptomyces lividans and the purification and characterization of TcmG protein, which we have named Tcm A2 oxygenase, are described here. NH2-terminal amino acid analysis of the purified enzyme led to the revision of the translational start site of tcmG to a TTG, 33 base pairs downstream of the GTG site assigned initially on the basis of nucleotide sequence analysis. Tcm A2 oxygenase is a monomeric protein in solution and contains 1 mol of non-covalently bound FAD; the apoenzyme can be partially reconstituted in vitro by addition of FAD. Tcm A2 oxygenase exhibits an optimal pH of 9.0-9.5 and prefers NADPH over NADH as an electron donor. The apparent K'm of the enzyme for Tcm A2, NADH, and NADPH are 1.81 +/- 0.38, 260 +/- 19, and 82.1 +/- 17 microM, respectively, and the apparent V'max for the reaction is 14.7 +/- 1.1 nmol Tcm C/min.mg. Purification and characterization of Tcm A2 oxygenase provide direct evidence to support the notion that the angular hydroxy groups of naphthacenequinones like Tcm C are introduced from 18O2 via a mono- or dioxygenase process.

Amino Acid Sequence↗

PubMed1994

Azotobacter vinelandii ferredoxin I. Alteration of individual surface charges and the [4FE-4S]2+/+ cluster reduction potential.

The structures of Azotobacter vinelandii ferredoxin I (AvFdI) and Peptococcus aerogenes ferredoxin (PaFd), near their analogous [4e-4S]2+/+ clusters, are highly conserved (Backes, G., Mino, Y., Loehr, T.M., Meyer, T.E., Cusanovich, M.A., Sweeney, W.V., Adman, E.T., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 11, 2055-2064). Despite these similarities, the reduction potential (E0') of the AvFdI [4Fe-4S]2+/+ cluster is more than 200 mV more negative than that of PaFd. We have tested the contribution that individual amino acid residues make to the control of E0' by converting residues in AvFdI into the corresponding residue in PaFd. Four mutations involved substitutions of negatively charged surface residues with neutral residues and two involved substitution of buried hydrophobic residues. All AvFdI variants were characterized by x-ray crystallography, absorption, CD, EPR, and 1H NMR spectroscopies and by electrochemical methods. For the F25I mutation, significant structural changes occurred that affected the EPR and 1H NMR spectroscopic properties of AvFdI and had a minor influence on E0'. For all other mutations there were no changes in reduction potential. Thus we conclude, that variations in charged surface residues do not account for the observed differences in E0' between the analogous [4Fe-4S]2+/+ cluster of PaFd and AvFdI. These differences are therefore most likely to be due to differences in solvent accessibility.

Amino Acid Sequence↗

PubMed1994

Long-lasting effects of partial hippocampal kindling on hippocampal physiology and function.

The objective of this project was to study the behavioral and physiological effects at 6-9 weeks after evoking 15 afterdischarges (ADs) in hippocampal CA1 (partial hippocampal kindling). Rats were trained on the open radial arm maze (RAM) with all eight arms baited, kindled, and then tested again on the RAM, followed by in vitro recordings at 8-9 weeks after kindling. Partial kindling was manifested by an increase in hippocampal AD duration. Enhancement of the commissural basal dendritic excitatory postsynaptic potential (EPSP) was observed for at least 1 day after the ADs. Kindled rats performed worse than control rats during the 1st but not during the 7th or 8th week after kindling. Rats that were slow in acquiring the RAM showed more RAM errors after kindling than those that showed fast acquisition. At 8-9 weeks after kindling, as shown by field potential recording in the hippocampal slice in vitro, kindled rats showed an increase in paired-pulse facilitation (PPF) of the EPSP in CA1 but a decreased PPF of the perforant path to dentate gyrus EPSP; no change in the PPF of the population spike was found in CA1 or DG. In a second group of rats that were not run on the RAM, at 6 weeks after kindling, PPF of the population EPSP and population spike were enhanced in the kindled rats compared to the control rats in CA1, but not in DG or CA3 in vitro (at 1.5, 2, or 4 times threshold intensity).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Overexpression of ferredoxin I in Azotobacter vinelandii.

Azotobacter vinelandii has recently been used for a variety of genetic experiments which take advantage of its facile transformation system and its high-frequency homologous recombination. One gene that has been cloned and sequenced is the fdxA gene that encodes a small Fe-S protein called A. vinelandii ferredoxin I (AvFdI). Because this protein has been extensively characterized by X-ray crystallography and spectroscopic methods, we are using it as a model to address some general questions in Fe-S biochemistry. AvFdI is not a very abundant protein in wild-type cells, so to facilitate our biochemical studies we have developed the overexpression system described herein. The results show that AvFdI can be easily overproduced ca. 50-fold in its native background, by introducing multiple copies of the fdxA gene into A. vinelandii, on the broad-host-range multicopy plasmid, pKT230. The protein can be expressed from its own constitutive promoter or from the controlled nifH promoter. The overproduced protein has no deleterious effects on the organism and is identical to the protein produced by wild-type cells. This A. vinelandii-based system should be generally useful for the overproduction of other A vinelandii proteins or for the expression of genes from thermophilic or other organisms with similarly high G-C contents, or for the expression of O2-sensitive metalloproteins that are unstable in other systems.

Azotobacter vinelandii↗

PubMed1994

Partial sequencing and mapping of clones from two maize cDNA libraries.

As one component of a maize genome project, we report the analysis of a number of randomly selected cDNAs, by a combination of measuring mRNA expression, 'single-pass' sequencing (SPS), and genome mapping. Etiolated seedling (490) and membrane-free polysomal endosperm cDNA clones (576) were evaluated for their transcription levels by hybridizing with a probe prepared from total mRNA and categorized as corresponding to abundantly or rarely expressed mRNAs and as either constitutive or tissue-specific. A total 313 clones from the two libraries were submitted to 'single-pass' sequencing from the presumed 5' end of the mRNA and the nucleotide sequence compared with the GenBank database. About 61% of the clones showed no significant similarities within GenBank, 14% of the clones exhibited a high degree of similarity, while the remaining 25% exhibited a lesser degree of similarity. The chromosomal location of more than 300 clones was determined by RFLP mapping using standard populations. The results demonstrate that a combination of analyses provides synergistic information in eventually deducing the actual function of these types of clones.

Chromosomes↗