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Biomedical subjects

Z Lin

Publications and source records attributed to Z Lin.

At least 289 records · Page 16Linked to original sources
PubMed1995

Roa307, a protein encoded on Coxiella burnetii plasmid QpH1, shows homology to proteins encoded in the replication origin region of bacterial chromosomes.

TnphoA mutagenesis identified an open reading frame, roa307, immediately upstream of the partition locus qsopAB on the Coxiella burnetii plasmid QpH1. The protein sequence deduced from roa307 displayed homology to Orf290 of Pseudomonas putida, Orf283 and Orf282 (SpoOJ) of Bacillus subtilis-hypothetical products of genes in the chromosomal replication origin region. Expression of roa307 was demonstrated by PhoA activity of an Roa307-PhoA fusion.

Amino Acid Sequence↗
PubMed1995

Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES.

In an effort to clarify the role of GroES in chaperonin-facilitated protein folding, a plasmid-encoding expression system for GroES incorporating a histidine-tagged, thrombin-cleavable, N-terminal sequence was constructed. This approach facilitated the rapid purification of native-like, histidine-cleaved GroES (HC-GroES). The addition of NaSCN to purification buffers to mildly promote subunit dissociation enabled the complete separation of chromosomally encoded, wild-type GroES chains from recombinant chains, allowing the production of homogeneous mutant variants of GroES. A substitution of histidine-7 to tryptophan in GroES was used to demonstrate the concentration-dependent modulation of the heptameric quaternary structure of the chaperonin. Fluorescence and light scattering studies of this mutant suggest that GroES heptamers dissociate to monomers upon dilution with half-times of 2-4 min. Sedimentation equilibrium experiments using either wild-type or HC-GroES can best be described by a monomer--heptamer equilibrium, yielding dissociation constants of 1 x 10(-38) M6 for native GroES and 2 x 10(-32) M6 for HC-GroES. These results are supported by subunit exchange experiments using mixtures of native or HC-GroES and GroES containing the complete N-terminal histidine tail. Native polyacrylamide gel electrophoresis demonstrates that these mixtures form an eight-membered hybrid set within minutes. The studies described here suggest a dynamic equilibrium for the quaternary structure of GroES, which may be an important feature for its role in GroEL-mediated protein folding reactions.

Amino Acid Sequence↗
PubMed1995

Proposed cation-pi mediated binding by factor Xa: a novel enzymatic mechanism for molecular recognition.

Factor Xa (FXa) is an important serine protease in the blood coagulation cascade. Small synthetic competitive inhibitors of FXa are under development as potential anticoagulants. To better understand FXa structural features and molecular recognition mechanisms, we have constructed three dimensional models of FXa-inhibitor complex structures via a new search approach that samples conformational space and binding space simultaneously for DABE and DX-9065a, two bis amidinoaryl derivatives that are among the most potent and selective FXa inhibitors reported to date. We find the most probable binding modes for the two inhibitors to be a folded conformation, with one distal amidino group extending into the S1 pocket, forming a salt-bridge with FXa Asp-189, and the other positively charged group fitting into the S4 subsite, and stabilized by a cation-pi interaction. We propose as a hypothesis that the cavity-like S4 subsite formed by the three pi-faces of the aromatic residues Tyr-99, Phe-174 and Trp-215 is sufficiently rich in pi electrons that it is not only a hydrophobic pocket, but also forms a cation recognition site. This proposed cation-pi binding mechanism is one of the first proposed for enzymatic molecular recognition, and for which experimental verification can be obtained without any complicating charge compensation mechanism. Our models provide plausible explanations of the structure-activity relationships observed for these inhibitors, and suggest that cation-pi interactions may provide a novel mechanism for molecular recognition.

Amino Acid Sequence↗
PubMed1995

A role for calcineurin (protein phosphatase-2B) in the regulation of glutamate release.

Previous studies have shown that 4-aminopyridine (4AP) induced Ca-influx effects the release of glutamate from nerve terminals (synaptosomes) isolated from rat cerebral cortex. We now show that the Ca-dependent component of this release is potentiated by preincubation of the synaptosomes with the immunosuppressant, FK506, an inhibitor of protein phosphatase-2B (calcineurin). FK506 did not inhibit the Ca-independent release of glutamate from a cytosolic pool. Examination of the effect of FK506 on the influx of Ca elicited by 4AP indicated that inhibition of calcineurin activity resulted in an increase of voltage-dependent Ca-influx. Based on these results, we suggest that protein dephosphorylation effected by calcineurin may suppress voltage-dependent Ca-channel activity and in so doing inhibits evoked glutamate release. Activation of calcineurin produced by initial Ca-entry may represent a negative feedback to limit the activity of Ca-channels coupled to the release of glutamate.

Aminopyrine↗
PubMed1995

The partition region of plasmid QpH1 is a member of a family of two trans-acting factors as implied by sequence analysis.

Sequencing analysis revealed that the partition region of the Coxiella burnetii plasmid QpH1 contains a putative operon, designated qsopAB. The two open reading frames (ORFs), qsopA and qsopB, specify the QsopA and QsopB proteins, with deduced molecular masses of 45.7 and 37.6 kDa, respectively. Maxicell analysis demonstrated that although qsopB was located downstream from qsopA, it had its own promoter that was active in Escherichia coli. Several direct or inverted repeats were found around this operon. The most distinct was a 20-bp long imperfect palindrome in the promoter region of qsopA, with homology to a palindrome in the promoter region of P1 parA. Structurally qsopAB was similar to parAB of the P1 plasmid. However, at the amino acid (aa) sequence level, QsopA and QsopB were closest to the F plasmid SopA and SopB proteins, respectively. QsopA shared 58.0% homology and 32.7% identity with SopA, but only 45-50% homology and 22-26% identity with other members of the protein A partition family. QsoB had even lower (41-45%) homology to other members of the protein B partition family, with the highest homology and identity to SopB. Despite lower homologies, both QsopA and QsopB did share conserved aa sequence regions and invariant residues with other members within each family.

Amino Acid Sequence↗
PubMed1995

Activation of the B-cell surface receptor CD40 induces A20, a novel zinc finger protein that inhibits apoptosis.

CD40 activation is critical for B-cell function, leading to activation and expression of cell surface markers, proliferation, immunoglobulin class switching and inhibition of programmed cell death (PCD). Germinal center B-cells, for example, can be prevented from undergoing PCD by CD40 activation. The mechanism by which PCD is inhibited has been an enigma. A potential role for A20, a novel zinc finger protein, in inhibiting B-cell apoptosis was suggested by our previous finding that it is induced by the Epstein-Barr virus LMP-1 gene product, a potent cell death inhibitor. We now show that CD40 activation induces A20 and that expression of A20 renders B-cell lines resistant to PCD. Additionally, we show that CD40 activation of A20 expression is mediated by inducible binding of NF-kappa B complexes to the A20 promoter and provide evidence for a critical role for Thr234 (in the CD40 cytoplasmic domain) in activating NF-kappa B.

Antigens, CD↗
PubMed1995

Photoaffinity labelling of cyanomethaemoglobin with derivatives of tryptophan and 5-bromotryptophan.

Tryptophan and 5-bromotryptophan (5-BrTrp) are relatively potent inhibitors of sickle-haemoglobin polymerization. The binding sites of these compounds to normal and sickle haemoglobin (HBA and HBS) have been suggested, but not firmly established, through the use of spin-labelled derivatives and/or computer modeling. In the present study we approached the problem by utilizing the technique of photoaffinity labelling. The cyanomet forms of HBA and HBS were subjected to photoaffinity labelling with N alpha-(4-azidotetrafluorobenzoyl)tryptophan and N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan respectively. Both irradiated samples of HBA and HBS were denatured, digested with trypsin, and then separated by reversed-phase HPLC. A labelled tryptic peptide was isolated from the photolabelling of HBS with N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan. The peptide was identified to be Val1(alpha)-Lys7(alpha), with the label attached to Val1(alpha), by virtue of amino acid analysis and sequencing, in conjunction with fast-atom-bombardment MS. The binding mode of N alpha-(1-ethyl-2-diazomalonyl)-5-bromotryptophan is proposed and its relevance to the potency of the 5-BrTrp-based anti-sickling agents is discussed.

Affinity Labels↗
PubMed1995

The hydrophobic nature of GroEL-substrate binding.

The molecular chaperone GroEl from Escherichia coli is a member of the highly conserved Hsp60 family of proteins that facilitates protein folding. A central question regarding the mechanism of GroEL-assisted refolding of proteins concerns its broad substrate specificity. The nature of GroEL-polypeptide chain interaction was investigated by isothermal titration calorimetry using proteins that maintain a non-native conformation in neutral buffer solutions. A single molecule of an unfolded variant of subtilisin BPN' binds non-cooperatively to GroEL with micromolar affinity and a positive enthalpy change. Additional calorimetric titrations of this chain with GroEL show that the positive enthalpy change decreases with increasing temperature between 6 and 25 degrees C, yielding a delta CP of -0.85 kcal mol-1 degree-1. alpha-Casein similarly binds to GroEL with micromolar affinity and a positive enthalpy change in the range of 15-20 degrees C, yielding a delta CP of -0.44 kcal mol-1 degree-1. The negative heat capacity change provides strong evidence for the role of hydrophobic interactions as the driving force for the association of these substrates with the GroEL chaperonin.

Amino Acid Sequence↗
PubMed1995

In vivo oxidation of [13C]galactose in patients with galactose-1-phosphate uridyltransferase deficiency.

We developed an intravenous and oral [13C]galactose breath test for the in vivo study of galactose metabolism. Following an intravenous bolus of 7 mg/kg of [1-13C]galactose in the fasting state, normal children and adults eliminated 3-6% and 21-47% of the bolus as 13CO2 in expired air collected over 1 and 5 h, respectively. Comparable fractional elimination was seen when the dose was given orally. Patients with galactosemia who have barely detectable or absent galactose-1-phosphate uridyltransferase (GALT) activity in erythrocytes and are homoallelic for the Q188R gene mutation, when given a 7 mg/kg intravenous bolus had barely detectable 13CO2 in air samples in the first hour, but eventually eliminated as much as 3.6% of the dose in 5 h. A galactosemia/Duarte (Q188R/N314D) compound heterozygote and a homozygous Duarte subject, as well as a subject with one normal allele and one Q188R allele, showed normal in vivo oxidation. An assessment of whole body galactose metabolism can be made with this procedure. Further use of this in vivo modality in patients with different genetic backgrounds should increase our understanding of genotype-phenotype relationships in hereditary galactosemia.

Adolescent↗
PubMed1995

The chemical and mineralogical behaviour of Pb in shooting range soils from central Sweden.

Recently investigations have shown that the annual flux of lead from shotgun pellets to shooting range soils is significant in some countries. This paper presents the data of chemical and mineralogical analyses of soils and Pb-pellet crusts from five shooting ranges in Sweden and, based on these results, evaluates the retention of lead in these shooting range soils. In the soils, Pb-pellets and bullets are readily decomposed and transformed to crust materials composed of Pb-bearing minerals. The transformation products in the crust materials, identified by X-ray diffraction, are predominantly hydrocerussite [Pb(3)(CO(3))(2) (OH)(2)], associated with cerussite (PbCO(3)) and anglesite (PbSO(4)). In a period of 20-25 years, an average of 4.8% metallic lead in the pellets has been transformed to lead carbonate and lead sulphate, where the former is the more stable mineral in the surface environment. However, in soils relatively rich in humus an average of 15.6% metallic lead in the pellets was transformed to secondary lead compounds in the same period. The results of the chemical analyses indicate that Pb is rather immobile in the soil profile. The surficial horizon contains higher concentrations of lead (52-3400 mg kg(-1)), while lower concentrations of lead were found in the E and B horizons where the total Pb concentrations (8-37 mg kg(-1)) are within about one standard deviation of the mean reference sample concentration. An inverse relationship is revealed between the aluminium hydroxide content of the soil fraction and EDTA-extractable Pb, which suggests that these compounds have affected the retention of lead.

Journal Article↗
PubMed1995

Non-invasive identification of gastric contractions from surface electrogastrogram using back-propagation neural networks.

Gastric contractions play an important role in the digestive process of the stomach. The established method for the measurement of gastric contractions is invasive and involves the insertion through the nose of a manometric probe into the stomach. A non-invasive method is introduced in this paper for the identification of gastric contractions using the surface electrogastrogram. The electrogastrogram (EGG) was measured by placing surface electrodes on the abdominal skin over the stomach in ten subjects. Gastric contractions were simultaneously monitored using an intraluminal manometric probe. The back-propagation neural network was applied to identify gastric contractions from the EGG. The input of the neural network was composed of spectral data points of the EGG which was computed using the exponential distribution method. Experiments were conducted to optimize network structures and parameters. Using the EGG data in five subjects as the training set and the EGG data in another five subjects as the testing set, an overall accuracy of 92% was achieved in the identification of gastric contractions with an optimized three-layer back-propagation neural network (number of nodes for input:hidden:output layers being 64:10:2).

Adult↗
PubMed1995

Screening of volatile substances and determination of toluene (a thinner component) in the blood and urine in emergency medical care and autopsy cases by the pulse heating method.

Screening of volatile substances was performed by pulse heating gas chromatography - mass spectrometry (GC-MS) using a GS-Q column in 211 emergency medical care and 342 autopsy cases. At least 36 standard substances could be separately detected. Six kinds of volatile substances were screened in a total of 553 cases. Toluene and/or hippuric acid were detected in the blood and/or urine in respectively, 4 emergency medical care and 8 autopsy cases. There were 11 abusers (9 males and 2 females) in these 12 positive cases. The ages of the abusers ranged from 13-26 years. There was no particular pattern to the monthly frequency distribution of identification of thinner (toluene) abuse cases, which occurred throughout the year. It is believed that these data at least partly reflect the present status of thinner/glue abuse in Japan. We conclude that pulse heating GC-MS is useful in the screening and quantitative determination of volatile substances including toluene and other thinner/glue components.

Journal Article↗