Biomedical subjects

X Huang

Publications and source records attributed to X Huang.

At least 379 records · Page 21Linked to original sources

PubMed1998

Expression of soluble VEGF receptor 2 and characterization of its binding by surface plasmon resonance.

Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that induces angiogenesis in several pathological conditions. To block angiogenesis, soluble VEGF receptor can be used. In this study, we describe a method for high yield expression of soluble VEGF receptor 2 (sFlk-1) in a baculovirus expression system (30 mg purified sFlk-1 per L of insect cell supernatant). We also determined the binding constants for both human and mouse VEGF to the recombinant receptor by surface plasmon resonance. In this cell-free assay, under the given experimental conditions, the on-rate ka was 0.5-2.2 x 10(6) M-1s-1 and the off-rate kd was 2-4 x 10(-4) s-1 (KD = 2-6 x 10(-10) M). To our knowledge this is the first study to report on- and off-rates for the VEGF:sFlk-1 interaction. Heparin was not required for the binding of VEGF to sFlk-1 in this assay. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptor.

Animals↗

PubMed1998

Inhibition of intrathymic T cell development by expression of a transgenic antagonist peptide.

The mature T cell receptor (TCR) repertoire is shaped by positive- and negative-selection events taking place during T cell development. These events are regulated by interactions between the TCR and major histocompatibility complex molecules presenting self-peptides. It has been shown that many antagonist peptides are efficient at mediating positive selection. In this study we analyzed the effects of a transgene encoding an antagonist peptide (influenza NP34) that is presented by H-2Db in a Tap-1-independent fashion in mice expressing the influenza NP68-specific TCR F5. We find that the transgenic peptide does not mediate positive or negative selection in F5(+)Tap-1(-/-) mice, but inhibits maturation of CD8(+) single positive thymocytes in F5(+)Tap-1(+) mice without inducing signs of negative selection. We conclude that antagonism of antigen recognition occurs not only at the level of mature T cells but also in T cell development.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Direct observations of structural phase transitions in planar crystallized ion plasmas

Laser-cooled 9Be+ ions confined in two-dimensionally extended lattice planes were directly observed, and the images were used to characterize the structural phases of the ions. Five different stable crystalline phases were observed, and the energetically favored structure could be sensitively tuned by changing the areal density of the confined ions. The experimental results are in good agreement with theoretical predictions for the planar (infinite in two dimensions) one-component plasma. Qualitatively similar structural phase transitions occur, or are predicted to occur, in other experimentally realizable planar systems.

Journal Article↗

PubMed1998

Selective recognition and cleavage of RNA loop structures by Ni(II).Xaa-Gly-His metallopeptides.

The recognition and cleavage of tRNAPhe and the TAR RNA of HIV-1 by metallopeptides of the general form Ni(II).Xaa-Gly-His (where Xaa is Gly, Lys, or Arg) were investigated. The results of RNA cleavage analyses suggest that KHSO5- or magnesium monoperoxyphthalate-activated metallopeptides (1) induce nucleobase damage which requires aniline acetate for complete RNA strand scission and (2) selectively target the loops of stem-loop structures of the above-named substrates. In targeting RNA loop regions, the metallopeptides may be sensitive to intraloop structural features, including the overall structural environment of the loop itself and possibly the presence of intraloop hydrogen bonding. Overall, these results suggest that the metallopeptides interact selectively within a loop, in a fashion reminiscent of many RNA binding proteins, instead of targeting RNA single-stranded character alone. These observations further suggest a possible metallopeptide-based strategy for the molecular recognition of native RNA structures and insight with regard to the general features available for ligand binding site discrimination.

Base Sequence↗

PubMed1998

NMR identification of epitopes of Lyme disease antigen OspA to monoclonal antibodies.

Outer surface protein A (OspA) from the Lyme disease spirochete Borrelia burgdorferi has been a focus of vaccine development. We have identified epitopes of OspA to two monoclonal antibodies (mAbs) by comparing NMR chemical shifts of free OspA and those in Fab complexes. Deuteration of non-labile protons in OspA extended the size limit of this technique so that it was applicable to the 78 kDa complexes of OspA and the Fab fragment. The epitope identified by NMR to an mAb, 184.1, agrees well with that previously defined by the crystal structure of the same complex, indicating the ability of the NMR method to accurately map an epitope in a large protein complex. The technique mapped the epitope to mAb 336, a mAb of clinical interest, to a region centered at the C-terminal alpha-helix. The results provides a basis for rational design of OspA-based Lyme disease vaccines.

Amino Acid Sequence↗

PubMed1998

Identification of target promoters for the Bacillus subtilis sigma X factor using a consensus-directed search.

The promoter selectivity of RNA polymerase (RNAP) can be altered by the association with alternative sigma subunits. Bacillus subtilis hosts a multitude of sigma factors, several of which coordinate the complex developmental program culminating in endospore formation. Genome sequencing has revealed an unanticipated seven new sigma factors of the highly divergent extracytoplasmic function (ECF) sub-family. Virtually nothing is known regarding either the promoter selectivity or the target genes for these newly identified sigma factors. We have used saturation mutagenesis to define a promoter consensus for recognition by one such ECF sigma factor, sigma X. The resulting consensus sequence was used to identify candidate sigma X target sites. Three newly identified sigma X-dependent promoters precede genes encoding regulatory proteins: an AbrB homolog (Abh), a putative response regulator aspartate phosphatase (RapD), and a regulator of autolysin expression (LytR). sigma X also contributes to the expression of CsbB, a putative membrane-bound glucosyl transferase that is partially controlled by the sigma B stress response sigma factor. Since LytR modulates the expression of the major autolytic amidase and CsbB may function in peptidoglycan synthesis or modification, we suggest that sigma X participates in the regulation of peptidoglycan synthesis and turnover.

Bacillus subtilis↗

PubMed1998

Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis.

The cortical deposition of Abeta is an event that occurs in Alzheimer's disease, Down's syndrome, head injury, and normal aging. Previously, in appraising the effects of different neurochemical factors that impact upon the solubility of Abeta, we observed that Zn2+ was the predominant bioessential metal to induce the aggregation of soluble Abeta at pH 7.4 in vitro and that this reaction is totally reversible with chelation. We now report that unlike other biometals tested at maximal biological concentrations, marked Cu2+-induced aggregation of Abeta1-40 emerged as the solution pH was lowered from 7.4 to 6.8 and that the reaction was completely reversible with either chelation or alkalinization. This interaction was comparable to the pH-dependent effect of Cu2+ on insulin aggregation but was not seen for aprotinin or albumin. Abeta1-40 bound three to four Cu2+ ions when precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low nanomolar concentrations of both Abeta1-40 and Abeta1-42 with submicromolar concentrations of Cu2+. Unlike Abeta1-40, Abeta1-42 was precipitated by submicromolar Cu2+ concentrations at pH 7.4. Rat Abeta1-40 and histidine-modified human Abeta1-40 were not aggregated by Zn2+, Cu2+, or Fe3+, indicating that histidine residues are essential for metal-mediated Abeta assembly. These results indicate that H+-induced conformational changes unmask a metal-binding site on Abeta that mediates reversible assembly of the peptide. Since a mildly acidic environment together with increased Zn2+ and Cu2+ are common features of inflammation, we propose that Abeta aggregation by these factors may be a response to local injury. Cu2+, Zn2+, and Fe3+ association with Abeta explains the recently reported enrichment of these metal ions in amyloid plaques in Alzheimer's disease.

Acidosis↗

PubMed1998

Sintered octadecylsilica as monolithic column packing in capillary electrochromatography and micro high-performance liquid chromatography.

Fused-silica capillaries were packed with porous 6-microns octadecylated silica microspheres and subjected to thermal treatment in order to obtain a column with porous silica based monolithic packing. After sintering, the monolithic packing was reoctadecylated in situ with dimethyloctadecylchlorosilane. The mechanical strength and stability of the monolithic column were significantly greater than those of conventional columns packed with particulate stationary phase. The performance of the columns was evaluated in both mu-HPLC and capillary electrochromatography (CEC) of small aromatic compounds and polycyclic aromatic hydrocarbons with 10 mM borate, pH 8.0, in acetonitrile-water mixtures as the mobile phase. Since no untoward bubble formation was observed, CEC could be performed in a CZE unit without the need for pressurizing the monolithic column. The plate efficiency of the monolithic column was similar to that of columns freshly packed with the same particles and was always higher in the CEC than in the mu-HPLC under otherwise identical conditions. The electrosmotic flow (EOF) velocity increased with the acetonitrile content of the eluent in open fused-silica capillaries with octadecylated innerwall and in columns packed with sintered octadecylsilica. In contradistinction, the EOF velocity decreased with the organic strength of the eluent in raw fused-silica capillaries. The opposite trend is attributed to the different effect of changing organic modifier concentration on the accessibility of silanol groups at the raw and octadecylated silica surfaces.

Chromatography, High Pressure Liquid↗

PubMed1998

Molecular cloning, functional expression, and characterization of pyruvate dehydrogenase kinase from anaerobic muscle of the parasitic nematode Ascaris suum.

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic mitochondrial metabolism of the parasitic nematode Ascaris suum. A cDNA coding for an A. suum pyruvate dehydrogenase kinase (APDK) has been cloned and sequenced from poly(A)+ RNA isolated from adult A. suum muscle.2 APDK exhibited significant sequence identity to mammalian PDKs. Nucleotide sequence analysis of the APDK cDNA revealed a 22-nucleotide spliced leader, characteristic of many nematode mRNAs, a 5'-UTR of 6 nucleotides, an open reading frame of 1197 nucleotides, and a 3'-UTR of 101 nucleotides that included a putative polyadenylation signal. The open reading frame predicted a protein of 399 amino acids with a molecular weight of 45,402 that included a putative 18-aminoacid leader peptide. Recombinant APDK (rAPDK) was functionally expressed in Escherichia coli with a his tag at its N-terminus and purified to apparent homogeneity on Ni-NTA-agarose. Recombinant APDK was a dimer and was not autophosphorylated and its activity was stimulated in the presence of APDK-deficient adult A. suum muscle PDC presumably by the binding of APDK to the dihydrolipoyl transacetylase (E2) core of the complex. After binding to the core, rAPDK activity was stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios within the same ranges as observed for the native APDK. Immunoblotting suggested that native APDK focused as a series of 43-kDa spots (pI 6.1-6.8) on two-dimensional gels of the purified adult A. suum muscle PDC.

Amino Acid Sequence↗

PubMed1998

The effect of two antipodal fluorine-induced sugar puckers on the conformation and stability of the Dickerson-Drew dodecamer duplex [d(CGCGAATTCGCG)]2.

UV thermal melting studies, CD and NMR spectroscopies were employed to assess the contribution of antipodal sugar conformations on the stability of the canonical B-DNA conformation of the Dickerson-Drew dodecamer duplex [[d(CGCGAATTCGCG)]2, (ODN 1)]. Different oligodeoxynucleotide versions of ODN 1 were synthesized with modified thymidine units favoring distinct sugar conformations by using a 3'- endo (north) 2'-fluoro-2'-deoxyribofuranosyl thymine (1) or a 2'- endo (south) 2'-fluoro-2'-deoxyarabinofuranosyl thymine (2). The results showed that two south thymidines greatly stabilized the double helix, whereas two north thymidines destabilized it by inducing a more A-like conformation in the middle of the duplex. Use of combinations of north and south thymidine conformers in the same oligo destabilized the double helix even further, but without inducing a conformational change. The critical length for establishing a detectable A-like conformation in the middle of a B-DNA ODN appears to be 4 bp. Our results suggest that manipulation of the conformation of DNA in a sequence-independent manner is possible.

DNA↗

PubMed1998

Vascular endothelial growth factor as a marker of tumor endothelium.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that is a primary stimulant of the vascularization of solid tumors. VEGF production is induced by oncogenic gene mutations in the tumor cells and by hypoxic conditions inside the tumor mass. Hypoxia and the locally increased concentration of VEGF lead to an up-regulation of VEGF receptor expression on tumor endothelial cells. Therefore, in the tumor microenvironment, there is an up-regulation of both VEGF and its receptor, leading to a high concentration of occupied receptor on tumor vascular endothelium. The VEGF:receptor complex presents an attractive target for the specific delivery of drugs or other effectors to tumor endothelium. In the present study, several hybridomas that secrete monoclonal antibodies against the VEGF:receptor (Flk-1) complex or against VEGF itself have been raised. Three of the antibodies (3E7, GV39M, and 11B5) bind with high affinity to the VEGF:Flk-1 complex in ELISA and to tumor endothelium in frozen sections of human tumors, rodent tumors, and human tumor xenografts. 3E7 and GV39M localize selectively to tumor endothelium after i.v. injection into mice bearing human tumor xenografts. Additionally, one antibody (2C3) was raised that blocks the interaction between VEGF and KDR/Flk-1. 2C3 inhibits VEGF-mediated growth of endothelial cells in vitro and localizes strongly to connective tissue in tumors after injection into mice bearing human tumor xenografts. These findings suggest that 3E7, GV39M, and 2C3 are candidates for targeting and imaging the vasculature or connective tissue of tumors.

Animals↗

PubMed1998

[Expression of human ciliary neurotrophic factor gene in Escherichia coli].

OBJECTIVE: To express biologically active human ciliary neurotrophic factor(hCNTF) gene in Escherichia coli. METHODS: Total RNA was extracted from human peripheral nerves and cDNA was synthesized by superscript reverse-transcriptase, a polymerase chain reaction(PCR) was conducted to obtain a full length cDNA fragment encode for hCNTF gene. After recovery from gel and purification, the PCR product was cloned into the pGEM-5Zf(+) vector and DNA sequence analysis was performed to verify hCNTF gene. The hCNTF gene was then placed under control of T7 promoter in the expression vector pET-11d and transformed into Escherichia coli strain BL21(DE3). Cultures of this transformat were induced by IPTG for the expression of recombinant protein. The bioactivity of recombinant protein was evaluated by its ability to support the survival of embryonic chick dorsal root ganglion neurons in culture. RESULTS: The human CNTF gene was cloned and biologically active hCNTF was expressed efficiently. Based on densitometry of stained gel,the recombinant hCNTF accounted for more than 25% of the total bacterial protein. CONCLUSION: The cloning and expression at high level of hCNTF gene in E.coli provides a basis for understanding the structure-activity relationship of CNTF and its potential clinical application.

Cells, Cultured↗

PubMed1998

Site-specific phosphorylation of Lys-Ser-Pro repeat peptides from neurofilament H by cyclin-dependent kinase 5: structural basis for substrate recognition.

Recent work has shown that high molecular weight neurofilament (NF) proteins are phosphorylated in their carboxy-terminal tail portion by the enzyme cyclin-dependent kinase 5 (CDK-5). The tail domain of neurofilaments contains 52 tripeptide repeats, viz. Lys-Ser-Pro, which mainly exist as KSPXK and KSPXXX motifs (X = amino acid). CDK-5 specifically phosphorylates the serine residues within the KSPXK sites. We probed the structural basis for this type of substrate selectivity by studying the conformation of synthetic peptides containing either KSPXK or KSPXXX repeats designed from native neurofilament sequences. Synthetic peptides with KSPXK repeats were phosphorylated on serine with a recombinant CDK-5/p25 complex whereas those with KSPXXX repeats were unreactive in this system. Circular dichroism (CD) studies in 50% TFE/H2O revealed a predominantly helical conformation for the KSPXXX-containing peptides, whereas the CD spectra for KSPXK-containing peptides indicated the presence of a high population of extended structures in water and 50% TFE solutions. However, detailed NMR analysis of one such peptide which included two such KSPXK repeats suggested a turn-like conformation encompassing the first KSPXK repeat. Restrained molecular dynamics calculations yielded an unusually stable, folded structure with a double "S"-like bend incorporating the central residues of the peptide. The data suggest that a transient reverse turn or loop-type structure may be a requirement for CDK-5-promoted phosphate transfer to neurofilament-specific peptide segments.

Amino Acid Sequence↗

PubMed1998

Electrochemical oxidation of N-acyldopamines and regioselective reactions of their quinones with N-acetylcysteine and thiourea.

The metabolism of catechols often involves their oxidation to quinones and subsequent nucleophilic addition reactions with sulfur-containing compounds. Adducts formed during these reactions may play important roles in many biological systems. We have studied the electrochemical oxidation of N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD) in the presence of two sulfur-centered nucleophiles, N-acetylcysteine (NACySH) and thiourea (TU), and have characterized the adducts and reaction pathways. NADA and NBAD react similarly, but their adducts with NACySH and TU were formed regioselectively. NACySH yields mainly 5-adducts and TU only 6-adducts. The NACySH adducts are oxidized more easily than the parent N-acyldopamine, and their oxidations are chemically reversible. However, the TU adducts are more difficult to oxidize, and their oxidation products undergo further chemical reactions. An intramolecular base catalysis mechanism for adduct formation with NACySH is proposed, which facilitates removal of the proton from the sulfhydryl group of NACySH and directs formation of the 5-adduct via a 1,6-Michael addition reaction. The absence of a proton on the thioureylene sulfur atom leads to formation of the 6-thioureylene adduct via a 1,4-Michael addition reaction of TU. This mechanism is consistent with the formation of other sulfur-centered adducts of catechols previously reported in the literature.

Acetylcysteine↗

PubMed1998

[DNA sequencing of human ciliary neurotropic factor gene by silver staining method].

OBJECTIVE: To introduce a non-radioactive protocol for DNA sequence analysis which employs a silver staining procedure METHODS: DNA template is amplified by Taq DNA polymerase to generate sequence ladder, No labeled nucleotide or primer is involved in the thermal-cycling sequencing reaction. A sensitive silver staining procedure is employed to visualize bands in sequencing gel. RESULTS: This silver staining method is used for DNA sequence analysis of human cilliary neurotrophic factor (hCNTF) gene. The resulted band resolution is comparable to radioactive sequencing method, and data can be obtain within less than 2 hours after sequencing reaction. CONCLUSION: Silver staining is a rapid, inexpensive and reliable method for DNA sequence analysis.

Ciliary Neurotrophic Factor↗

PubMed1998

Bragg diffraction from crystallized ion plasmas

Single crystals of a one-component plasma were observed by optical Bragg diffraction. The plasmas contained 10(5) to 10(6) single-positive beryllium-9 ions (9Be+) at particle densities of 10(8) to 10(9) per cubic centimeter. In approximately spherical plasmas, single body-centered cubic (bcc) crystals or, in some cases, two or more bcc crystals having fixed orientations with respect to each other were observed. In some oblate plasmas, a mixture of bcc and face-centered cubic ordering was seen. Knowledge of the properties of one-component plasma crystals is required for models of white dwarfs and neutron stars, which are believed to contain matter in that form.

Journal Article↗

PubMed1998

Overexpression, purification, and biophysical characterization of the heterodimerization domain of the core-binding factor beta subunit.

Core-binding factors (CBF) are heteromeric transcription factors essential for several developmental processes, including hematopoiesis. CBFs contain a DNA-binding CBF alpha subunit and a non-DNA binding CBF beta subunit that increases the affinity of CBF alpha for DNA. We have developed a procedure for overexpressing and purifying full-length CBF beta as well as a truncated form containing the N-terminal 141 amino acids using a novel glutaredoxin fusion expression system. Substantial quantities of the CBF beta proteins can be produced in this manner allowing for their biophysical characterization. We show that the full-length and truncated forms of CBF beta bind to a CBF alpha DNA complex with very similar affinities. Sedimentation equilibrium measurements show these proteins to be monomeric. Circular dichroism spectroscopy demonstrates that CBF beta is a mixed alpha/beta protein and NMR spectroscopy shows that the truncated and full-length proteins are structurally similar and suitable for structure determination by NMR spectroscopy.

Circular Dichroism↗

PubMed1998

Molecular cloning, expression, and characterization of a novel human serine/threonine protein phosphatase, PP7, that is homologous to Drosophila retinal degeneration C gene product (rdgC).

A novel serine/threonine protein phosphatase (PPase) designated PP7 was identified from cDNA produced from human retina RNA. PP7 has a molecular mass of approximately 75 kDa, and the deduced amino acid sequence of PP7 contains a phosphatase catalytic core domain that possesses all of the invariant motifs of the PP1, PP2A, PP2B, PP4, PP5, and PP6 gene family. However, PP7 has unique N- and C-terminal regions and shares < 35% identity with the other known PPases. The unique C-terminal region of PP7 contains multiple Ca2+ binding sites (i.e. EF-hand motifs). This region of PP7 is similar to the Drosophila retinal degeneration C gene product (rdgC), and PP7 and rdgC share 42.1% identity. Unlike the other known PPases, the expression of PP7 is not ubiquitous; PP7 was only detected in retina and retinal-derived Y-79 retinoblastoma cells. Expression of recombinant human PP7 in baculovirus-infected SF21 insect cells produces an active soluble enzyme that is capable of utilizing phosphohistone and p-nitrophenyl phosphate as substrates. The activity of recombinant PP7 is dependent on Mg2+ and is activated by calcium (IC50 approximately equal to 250 microM). PP7 is not affected by calmodulin and is insensitive to inhibition by okadaic acid, microcystin-LR, calyculin A, and cantharidin.

Amino Acid Sequence↗