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Biomedical subjects

L Niu

Publications and source records attributed to L Niu.

At least 37 records · Page 2Linked to original sources
PubMed1998

Crystal structures of acutolysin A, a three-disulfide hemorrhagic zinc metalloproteinase from the snake venom of Agkistrodon acutus.

Acutolysin A alias AaHI, a 22 kDa hemorrhagic toxin isolated from the snake venom of Agkistrodon acutus, is a member of the adamalysin subfamily of the metzincin family and is a snake venom zinc metalloproteinase possessing only one catalytic domain. Acutolysin A was found to have a high-activity and a low-activity under weakly alkaline and acidic conditions, respectively. With the adamalysin II structure as the initial trial-and-error model, the crystal structures were solved to the final crystallographic R-factors of 0. 168 and 0.171, against the diffraction data of crystals grown under pH 5.0 and pH 7.5 conditions to 1.9 A and 1.95 A resolution, respectively. One zinc ion, binding in the active-site, one structural calcium ion and some water molecules were localized in both of the structures. The catalytic zinc ion is coordinated in a tetrahedral manner with one catalytic water molecule anchoring to an intermediate glutamic acid residue (Glu143) and three imidazole Nepsilon2 atoms of His142, His146 and His152 in the highly conserved sequence H142E143XXH146XXGXXH152. There are two new disulfide bridges (Cys157-Cys181 and Cys159-Cys164) in acutolysin A in addition to the highly conserved disulfide bridge Cys117-Cys197. The calcium ion occurs on the molecular surface. The superposition showed that there was no significant conformational changes between the two structures except for a few slight changes of some flexible residue side-chains on the molecular surface, terminal residues and the active-site cleft. The average contact distance between the catalytic water molecule and oxygen atoms of the Glu143 carboxylate group in the weakly alkaline structure was also found to be closer than that in the weakly acidic structure. By comparing the available structural information of the members of the adamalysin subfamily, it seems that, when lowering the pH value, the polarization capability of the Glu143 carboxylate group to the catalytic water molecule become weaker, which might be the structural reason why the snake venom metalloproteinases are inactive or have a low activity under acidic conditions.

Acids↗
PubMed1998

A Tetrahymena thermophila G4-DNA binding protein with dihydrolipoamide dehydrogenase activity.

G4-DNA is a four-stranded structure that is formed by guanine-rich sequences. We report here the purification and characterization of a novel G4-DNA binding protein from Tetrahymena thermophila, designated TGP2. TGP2 was found to preferentially bind to G4-DNA oligonucleotides with adjacent single-stranded domains containing phosphorylated 5' ends and the sequence element, 5'-ACTG-3'. The amino acid sequence of TGP2 has high similarity to dihydrolipoamide dehydrogenase (DLDH) from a variety of species, and TGP2 was shown to have DLDH activity. Purified DLDH from porcine heart and bovine intestinal mucosa were shown to bind specifically to G4-DNA oligonucleotides. On the basis of these results we conclude that TGP2 is DLDH in T. thermophila and suggest that the G4-DNA binding capability of TGP2/DLDH may be biologically relevant.

Amino Acid Sequence↗
PubMed1998

Tetrahymena mutants with short telomeres.

Telomere length is dynamic in many organisms. Genetic screens that identify mutants with altered telomere lengths are essential if we are to understand how telomere length is regulated in vivo. In Tetrahymena thermophila, telomeres become long at 30 degrees, and growth rate slows. A slow-growing culture with long telomeres is often overgrown by a variant cell type with short telomeres and a rapid-doubling rate. Here we show that this variant cell type with short telomeres is in fact a mutant with a genetic defect in telomere length regulation. One of these telomere growth inhibited forever (tgi) mutants was heterozygous for a telomerase RNA mutation, and this mutant telomerase RNA caused telomere shortening when overexpressed in wild-type cells. Several other tgi mutants were also likely to be heterozygous at their mutant loci, since they reverted to wild type when selective pressure for short telomeres was removed. These results illustrate that telomere length can regulate growth rate in Tetrahymena and that this phenomenon can be exploited to identify genes involved in telomere length regulation.

Animals↗
PubMed1998

[The clinical value of intraoperative ultrasound of the liver].

OBJECTIVE: To evaluate usefulness of intraoperative ultrasound of the liver. METHODS: A 5.0 MHz ultrasound transducer was used to detect liver lesions, their number, location and relation to the intrahepatic blood vessels. RESULTS: Forty two patients with primary liver tumors received intraoperative ultrasonographic examination. Liver tumor was resected in 29 patients(69.0%). The tumors in 4 patients were unresectable due to their close relationship to the hepatic vasculature as judged by the ultrasound image. Multiple lesions were found in 6 patients during operation. CONCLUSIONS: Intraoperative ultrasound of the liver is sensitive enough to detect most liver lesions and to locate them by liver segment which makes radical tumor resection possible. It is useful to define the tumor in relation to hepatic vasculature. It can compensate for missing lesions in preoperative imaging studies. Fenestration operation for liver abscess and intervention treatment for liver cysts can be performed during operation.

Adult↗
PubMed1997

Nucleoprotein-based nanoscale assembly.

A system for addressing in the construction of macromolecular assemblies can be based on the biospecificity of DNA (cytosine-5) methyltransferases and the capacity of these enzymes to form abortive covalent complexes at targeted 5-fluorocytosine residues in DNA. Using this system, macromolecular assemblies have been created using two representative methyltransferases: M-HhaI and M x MspI. When 5-fluorocytosine (F) is placed at the targeted cytosine in each recognition sequence in a synthetic oligodeoxynucleotide (GFGC for M x HhaI or FCGG for M x MspI), we show that the first recognition sequence becomes an address for M x HhaI, while the second sequence becomes an address for M x MspI. A chimeric enzyme containing a dodecapeptide antigen linked to the C terminus of M-HhaI retained its recognition specificity. That specificity served to address the linked peptide to the GFGC recognition site in DNA. With this assembly system components can be placed in a preselected order on the DNA helix. Axial spacing for adjacent addresses can be guided by the observed kinetic footprint of each methyltransferase. Axial rotation of the addressable protein can be guided by the screw axis of the DNA helix. The system has significant potential in the general construction of macromolecular assemblies. We anticipate that these assemblies will be useful in the construction of regular protein arrays for structural analysis, in the construction of protein-DNA systems as models of chromatin and the synaptonemal complex, and in the construction of macromolecular devices.

Base Sequence↗
PubMed1997

Purification, characterization and conformational analysis of a haemorrhagin from the venom of Agkistrodon acutus.

AaHIV, a medium-sized toxin with a mol. wt of 44,000, a pI of 5.0 and a low cysteine content, was isolated from the venom of Agkistrodon acutus by ion-exchange and gel filtration chromatography. It had haemorrhagic, lethal and caseinolytic activities, the last of which was inhibited by EDTA, Zn(CH3COO)2 or CuSO4. The circular dichroism spectrum at pH 7.0 showed two negative bands at 210 nm and 219 nm, corresponding to secondary structure contents of 18.2% alpha-helix, 31.0% beta-sheet, 17.2% beta-turn and 33.6% random-coil. The effect of various pH values or the addition of EDTA or Zn(CH3COO)2 on the circular dichroism spectrum was also investigated.

Agkistrodon↗
PubMed1996

Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes.

Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-microseconds time resolution, respectively, for studying neurotransmitter receptors in giant (approximately 20-microns diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.

Animals↗
PubMed1996

Extraction of molecular edges on the average difference map by a Monte-Carlo method.

A method giving a low-resolution image of the molecules in the unit cell has been described, which was based only on the observed structure factors. An operator, called the average difference operator (ADO), was introduced in reciprocal space to flatten the electron densities everywhere but the regions on either side of the molecular envelope in real space. The observed structure factors were first modified by ADO, then a Monte-Carlo condensing protocol [Subbiah (1991). Science, 252, 128-133; (1993). Acta Cryst. D49, 108-119] was employed to stimulate the modified electron-density map at low resolution. It was found that molecular edges could be extracted, especially when there was relatively large solvent content in the unit cell.

Journal Article↗
PubMed1996

Synthesis and characterization of a caged receptor ligand suitable for chemical kinetic investigations of the glycine receptor in the 3-microseconds time domain.

Here we report the development and characterization of a new photolabile protecting group for the carboxyl group of neurotransmitters, 2-methoxy-5-nitrophenyl. The synthesis and characterization of a photolabile derivative of beta-alanine, caged beta-alanine, are described. beta-Alanine can activate the glycine receptor, a major inhibitory receptor in the mammalian central nervous system; the 2-methoxy-5-nitrophenyl derivative of beta-alanine combined with a laser-pulse photolysis method makes it possible to investigate the chemical kinetic mechanism of the receptor in the 3-microseconds time domain. The derivative is photolyzed by a laser pulse to release free beta-alanine within 3 microseconds and with a product quantum yield of 0.2. In aqueous solution in the dark and at neutral pH, the compound is more stable, by a factor of approximately of 25, than the analogous derivative of glycine [Ramesh, D., Wieboldt, R., Niu, L., Carpenter, B. K., & Hess, G. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11074-11078]. 2-Methoxy-5-nitrophenyl-beta-alanine hydrolyzes in aqueous solution at neutral pH with a t1/2 of approximately 1.5 h. Neither the 2-methoxy-5-nitrophenyl-beta-alanine nor the 2-methoxy-5-nitrophenol photolysis side product activates, inhibits, or potentiates the response of glycine receptors in rat hippocampal neurons to glycine. Photolysis of 2-methoxy-5-nitrophenyl-beta-alanine by irradiation with a 600-ns laser pulse at 333 nm releases beta-alanine, which then activates glycine receptor-channels on neurons equilibrated with the caged compound, as detected by whole-cell current recording. Compared with the analogous derivative of glycine, in terms of quantum yield, photolysis rate, and stability, this new compound is not only a better candidate for use in chemical kinetic investigations of the glycine receptor, but can also be used in determining the location of glycine receptors in neuronal cells.

Animals↗
PubMed1996

Purification, crystallization and preliminary X-ray diffraction analysis of haemorrhagin IV from the snake venom of Agkistrodon acutus.

Haemorrhagin IV, a medium molecular weight haemorrhagin from the snake venom of Agkistrodon acutus (AaHIV), has been purified and crystallized. The molecular weight and isoelectric point of AaHIV are 44 kDa and pI 5.0, respectively. The crystal belongs to space group C222(1) with unit-cell dimensions of a = 124.2, b = 114.5, c = 98.4 A, and could o diffract X-rays to 3.0 A, resolution. There are one or two molecules in the crystallographic asymmetric unit.

Journal Article↗
PubMed1996

Synthesis and photochemical properties of a kainate precursor and activation of kainate and AMPA receptor channels on a microsecond time scale.

Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolylpropionate (AMPA) receptors are transmembrane proteins that can form ion channels upon binding a specific ligand. The receptors are located at major excitatory synapses in the mammalian central nervous system. Kainate and AMPA receptors participate in many physiological activities of the brain, including learning and memory, and are involved in many neurological disorders. Elucidation of the mechanisms of receptor transmembrane channel formation, inhibition, and regulation is important in understanding fundamental central nervous system function and in designing potential therapeutic agents. Kainate can activate both kainate and AMPA receptors, leading to channel opening in the microsecond to millisecond time region. A newly developed laser pulse photolysis technique, with a microsecond time resolution, has been successfully used to study the chemical reactions of receptor proteins in the microsecond to millisecond time region. To apply the technique to kainate and AMPA receptors, a photolabile kainate precursor in which a caging group, the alpha-carboxy-2-nitrobenzyl group, is attached to the gamma-carboxyl group of kainic acid has been synthesized. The photolytic release of free kainate from the caged kainate on the microsecond time scale, initiated by a pulse of laser light at 308 nm, was measured. The quantum yield is 0.34 at pH 6.8 and room temperature. The half-life of the major component (approximately 86%) of the photolytic reaction is 45 microseconds, while that of the minor component (approximately 14%) is 0.7 ms. The effects of the caged kainate on kainate and AMPA receptors endogenously expressed in rat hippocampal neurons were also evaluated. Caged kainate (750 microM) did not activate the receptor channels, nor did it potentiate or inhibit the kainate response. Photolysis of the caged kainate by a pulse of 333-nm laser light resulted in a rapid rise (with a t1/2 of 0.4 ms) in the whole-cell current due to the opening of kainate-activated receptor channels. The results presented demonstrate that this kainate precursor is suitable for rapid chemical kinetic investigations of the kainate and AMPA receptors in the microsecond to millisecond time region.

Animals↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of haemorrhagin I from the snake venom of Agkistrodon acutus.

Haemorrhagin I from the snake venom of Agkistrodon acutus (AaHI) has been crystallized using the hanging-drop vapour diffusion method. The crystals belong to space group P4(1)2(1)2 or P4(3)2(1)2 with unit-cell dimensions a = b = 63.61 and c = 95.69 A. There is one molecule in the asymmetric unit. Data to 2.35 A resolution have been collected using a single-crystal.

Journal Article↗
PubMed1996

Steady flow in an aneurysm model: correlation between fluid dynamics and blood platelet deposition.

Laminar and turbulent numerical simulations of steady flow in an aneurysm model were carried out over Reynolds numbers ranging from 300 to 3600. The numerical simulations are validated with Digital particle Image Velocimetry (DPIV) measurements, and used to study the fluid dynamic mechanisms that characterize aneurysm deterioration, by correlating them to in vitro blood platelet deposition results. It is shown that the recirculation zone formed inside the aneurysm cavity creates conditions that promote thrombus formation and the viability of rupture. Wall shear stress values in the recirculation zone are around one order of magnitude less than in the entrance zone. The point of reattachment at the distal end of the aneurysm is characterized by a pronounced wall shear stress peak. As the Reynolds number increases in laminar flow, the center of the recirculation region migrates toward the distal end of the aneurysm, increasing the pressure at the reattachment point. Under fully turbulent flow conditions (Re = 3600) the recirculation zone inside the aneurysm shrinks considerably. The wall shear stress values are almost one order of magnitude larger than those for the laminar cases. The fluid dynamics mechanisms inferred from the numerical simulation were correlated with measurements of blood platelet deposition, offering useful explanations for the different morphologies of the platelet deposition curves.

Aortic Aneurysm, Abdominal↗
PubMed1995

Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by a laser-pulse photolysis technique.

Effects of cocaine on the muscle nicotinic acetylcholine receptor were investigated by using a chemical kinetic technique with a microsecond time resolution. This membrane-bound receptor regulates signal transmission between nerve and muscle cells, initiates muscle contraction, and is inhibited by cocaine, an abused drug. The inhibition mechanism is not well understood because of the lack of chemical kinetic techniques with the appropriate (microsecond) time resolution. Such a technique, utilizing laser-pulse photolysis, was recently developed; by using it the following results were obtained. (i) The apparent cocaine dissociation constant of the closed-channel receptor form is approximately 50 microM. High carbamoylcholine concentration and, therefore, increased concentrations of the open-channel receptor form, decrease receptor affinity for cocaine approximately 6-fold. (ii) The rate of the receptor reaction with cocaine is at least approximately 30-fold slower than the channel-opening rate, resulting in a cocaine-induced decrease in the concentration of open receptor channels without a concomitant decrease in the channel-opening or -closing rates. (iii) The channel-closing rate increases approximately 1.5-fold as the cocaine concentration is increased from 20 to 60 microM but then remains constant as the concentration is increased further. The results are consistent with a mechanism in which cocaine first binds rapidly to a regulatory site of the receptor, which can still form transmembrane channels. Subsequently, a slow step (t1/2 approximately 70 ms) leads to a receptor form that cannot form transmembrane channels, and acetylcholine receptor-mediated signal transmission is, therefore, blocked. Implications for the search for therapeutic agents that alleviate cocaine poisoning are mentioned.

Animals↗
PubMed1995

[Promoter activity detection and transcription initiation site determination of the D-xylose isomerase gene of Streptomyces diastaticus strain No.7 M1033].

The DNA segment of the Streptomyces diastaticus strain No.7 M1033 D-xylose Isomerase gene, from -192 to +581 bp according to the translation start site (tss), was cloned in Streptomyces promoter-probe plasmid pIJ4083 from recombination plasmid pUB1 and its M13 subclone S1 respectively. The protoplasts of S. lividans TK24 were transformed with the recombinant, and then detected the promoter activity by measuring the catethol dioxygenase expression. Results of S1 mapping of total RNA of S. di. M1033 indicatedthat: the transcription initiation site of D-XI gene was 40bp upstream the coding region; there was another gene encoded on the antisense strand upstream D-XI gene, with a 114-nucleotide sequence separating their coding regions. According to its DNA sequence, we believed it was the xylulose kinase gene. Data from quantitive S1 mapping also suggested that transcription of the two genes was induced by D-xylose and repressed by glucose.

Aldose-Ketose Isomerases↗
PubMed1994

Characterization of the mouse Rep-3 gene: sequence similarities to bacterial and yeast mismatch-repair proteins.

The mouse Rep-3 gene is transcribed divergently from the same promoter region as the dihydrofolate reductase-encoding gene and has a deduced amino-acid sequence that shares identity with the bacterial protein, MutS, which is involved in DNA mismatch repair. We have cloned Rep-3, mapped it and sequenced all of the known exons and their intron junction sequences. We find that the open reading frame is considerably larger than initially reported and that the most abundant form of Rep-3 mRNA encodes a protein of 123 kDa. The gene spans at least 134 kb and consists of 26 exons, including several alternatively spliced exons. All of the exon/intron junctions match the expected consensus sequences with the exception of the splice junctions for intron 6, which has AT and AC dinucleotides instead of the usual GT and AG bordering the exon sequences. The junction sequences for this intron share consensus sequences with three intron sequences from other genes, thereby helping to establish an alternative consensus sequence.

Amino Acid Sequence↗