Biomedical subjects

Z Jia

Publications and source records attributed to Z Jia.

At least 91 records · Page 5Linked to original sources

PubMed1999

Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding.

The crystallographic study of chimeric B72.3 antibody illustrated that there are three FR side-chain interactions with either CDR residue's side chain or main chain. For example, hydrogen bonds are formed between the hydroxyl group of threonine at L5 in FR1 and the guanidinal nitrogen group of arginine at L24 in CDR1, between the hydroxyl group of tyrosine at L36 in FR2 and the amide nitrogen group of glutamine at L89 in CDR3 and between the hydroxyl group of tyrosine at L71 in FR3 and the carbonyl group of isoleucine at L29 as well as the amide nitrogen group of serine at L31 in CDR1. Elimination of these hydrogen bonds at these FR positions may affect CDR loop conformations. To confirm these assumptions, we altered these FR residues by site-directed mutagenesis and determined binding affinities of these mutant chimeric antibodies for the TAG72 antigen. We found that the substitution of tyrosine by phenylalanine at L71, altering main-chain hydrogen bonds, significantly reduced the binding affinity for the TAG72 antigen by 23-fold, whereas the substitution of threonine and tyrosine by alanine and phenylalanine at L5 and L36, eliminating hydrogen bonds to side-chain atoms, did not affect the binding affinity for the TAG72 antigen. Our results indicate that the light-chain FR residue tyrosine at L71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding. Our results will thus be of importance when the humanized B72.3 antibody is constructed, since this important mouse FR residue tyrosine at L71 must be maintained.

Amino Acid Sequence↗

PubMed1999

Blockade of glutamate receptors and barbiturate anesthesia: increased sensitivity to pentobarbital-induced anesthesia despite reduced inhibition of AMPA receptors in GluR2 null mutant mice.

BACKGROUND: Barbiturates enhance gamma-aminobutyric acid type A (GABA(A)) receptor function and also inhibit the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype of glutamate receptor. The relative contribution of these actions to the behavioral properties of barbiturates is not certain. Because AMPA receptor complexes that lack the GluR2 subunit are relatively insensitive to pentobarbital inhibition, GluR2 null mutant mice provide a novel tool to investigate the importance of AMPA receptor inhibition to the anesthetic effects of barbiturates. METHODS: GluR2 null allele (-/-), heterozygous (+/-), and wild-type (+/+) mice were injected with pentobarbital (30 and 35 mg/kg intraperitoneally). Sensitivity to anesthetics was assessed by measuring the latency to loss of righting reflex, sleep time, and the loss of corneal, pineal, and toe-pinch withdrawal reflexes. In addition, patch-clamp recordings of acutely dissociated CA1 hippocampal pyramidal neurons from (-/-) and (+/+) mice were undertaken to investigate the effects of barbiturates on kainate-activated AMPA receptors and GABA-activated GABA(A) receptors. RESULTS: Behavioral tests indicate that sensitivity to pentobarbital was increased in (-/-) mice. In contrast, AMPA receptors from (-/-) neurons were less sensitive to inhibition by pentobarbital (concentrations that produced 50% of the maximal inhibition [IC50], 301 vs. 51 microM), thiopental (IC50, 153 vs. 34 microM), and phenobarbital (IC50, 930 vs. 205 microM) compared with wild-type controls, respectively. In addition, the potency of kainate was greater in (-/-) neurons, whereas no differences were observed for the potentiation of GABA(A) receptors by pentobarbital. CONCLUSIONS: The GluR2 null mutant mice were more sensitive to pentobarbital anesthesia despite a reduced sensitivity of GluR2-deficient AMPA receptors to barbiturate blockade. Our results indicate that the inhibition of AMPA receptors does not correlate with the anesthetic effects of barbiturates in this animal model. We postulate that the increase in the sensitivity to anesthetics results from a global suppression of excitatory neurotransmission in GluR2-deficient mice.

Alleles↗

PubMed1999

Crystallization and X-ray crystallographic analysis of m-calpain, a Ca2+-dependent protease.

The absolute requirement of Ca(2+) for proteolytic activity is a feature unique to the calpains, a family of heterodimeric cysteine proteases. Conditions are described which give rise to diffraction-quality crystals of m-calpain in two crystal forms, P1 and P2(1). Data have been collected from native crystals of m-calpain in both P1 and P2(1) forms, to 2.6 and 2.15 A, respectively. Selenomethionine-containing crystals have been grown in both forms, and anomalous data from the P2(1) selenomethionine enzyme provided the location of 17 of the 19 Se atoms in the protein.

Animals↗

PubMed1999

New saponins from the seeds of Aesculus chinensis.

Eight new acylated polyhydroxyoleanene triterpenoidal saponins, aesculiosides A-H (1-8), along with four known ones, have been isolated from the seeds of Aesculus chinensis. On the basis of extensive NMR studies, the structures of the new compounds were determined to be 21-O-tigloylprotoaescigenin 3-O-[beta-D-glucopyranosyl-(1-->2)][beta-D-glucopyranosyl-(1-->4)]- beta-D-glucuronopyranosyl acid (1), 21-O-angeloylprotoaescigenin 3-O-[beta-D-glucopyranosyl- (1-->2)][beta-D-glucopyranosyl-(1-->4)]-beta-D-glucuronopyranosyl acid (2), 21,22-O-ditigloylprotoaescigenin 3-O-[beta-D-glucopyranosyl-(1-->2)][beta-d-glucopyranosyl- (1-->4)]-beta-D-glucuronopyranosyl acid (3), 21-O-tigloyl-22-O-angeloylprotoaescigenin 3-O-[beta-D-glucopyranosyl- (1-->2)][beta-D-glucopyranosyl-(1-->4)]-beta-D-glucuronopyranosyl acid (4), 21,22-O-ditigloylprotoaescigenin 3-O-[beta-D-glucopyranosyl- (1-->2)][beta-D-glucopyranosyl-(1-->4)]-methyl beta-d-glucuronopyranosate (5), 21-O-tigloyl-22-O-angeloylprotoaescigenin 3-O-[beta-D-glucopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)]-methy l beta-D-glucuronopyranosate (6), 21-O-tigloyl-28-O-acetylprotoaescigenin 3-O-[beta-D-glucopyranosyl-(1-->2)][beta-D-glucopyranosyl-(1-->4)]-methy l beta-D-glucuronopyranosate (7) and 21-O-angeloyl-28-O-acetylprotoaescigenin 3-O-[beta-D-glucopyranosyl-(1-->2)][beta-D-glucopyranosyl-(1-->4)]-methy l beta-D-glucuronopyranosate (8).

Carbohydrate Conformation↗

PubMed1999

Triterpenoidal saponins acylated with two monoterpenic acids from Gleditsia sinensis.

The isolation and characterization of three new oleanane-type triterpenoidal saponins, called gleditsiosides E-G, along with two known ones, from the anomalous fruits of Gleditsia sinensis LAM. are described. Their structural details were unambiguously determined by using a combination of modern NMR techniques, including distortionless enhancement by polarization transfer (DEPT), double-quantum filtered 1H-1H correlated spectroscopy (DQF-COSY), homonuclear hartmann-hahn (HOHAHA), 1H-13C heteronuclear correlation (HET-COR), heteronuclear multiple-bond connectivity (HMBC) and rotating-frame overhauser enhancement spectroscopy (ROESY) experiments as well as some chemical methods. The five bisdesmosidic triterpenoidal saponins consisting of the same sugar sequence were all acylated with two different or identical monoterpenic acids to the C-2 and C-3 positions of rhamnose moiety.

Carbohydrate Sequence↗

PubMed1999

Minor triterpenoid saponins from Ardisia crenata.

Two minor triterpenoid saponins, ardisicrenoside G [3 beta-O-{alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->4)- [beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl}-16 alpha,28-dihydroxyolean-12-en-30-oic acid] and ardisicrenoside H [3 beta-O-{beta-D-xylopyranosyl-(1-->2)-beta-D- glucopyranosyl-(1-->4)-[beta-D-glucopyranosyl-(1-->2)]-alpha-L- arabinopyranosyl}-16 alpha,28-dihydroxyolean-12-en-30-oic acid] were isolated from the roots of Ardisia crenata. Structural assignments are based on NMR, MS and chemical reactions.

Carbohydrate Sequence↗

PubMed1999

[Orbicularis oculi muscle suspension and orbital septum fixation in blepharoplasty].

OBJECTIVE: The study is to correct all the deformities of the lower eyelid. There is a regressive keletonization of the orbit as the soft-tissue covering of the lower eyelid skin and orbicularis oculi descend downward due to loss of tone, and the orbital fat herniates through a weakened septum orbital, resulting in an infraorbital crease. METHODS: We suspend the orbicularis oculi muscle and fix the orbital septum in blepharoplasty. RESULTS: This method has been used in 32 patients with satisfactory results in recent years. CONCLUSION: The technique can improve all the deformities of the baggy eyelids and achieve long-lasting youthful countenance.

Adult↗

PubMed1999

[Effect of tongxinluo capsule in treating variant angina pectoris patients and its influence on endothelial function].

OBJECTIVE: To assess the efficacy of Tongxinluo capsule (TXLC) in treating variant angina pectoris and its effect on endothelial function. METHODS: Sixty-four patients with variant angina pectoris were enrolled in the study for four weeks by a randomized clinical trial treatment with TXLC or isosorbide mononitrate. RESULTS: (1) The symptoms of both groups were significantly improved, the total effective rate of TXLC and isosorbide were 86.67% and 87.10% respectively; (2) The level of serum nitric oxide was increased, and the serum endothelin was decreased after treatment, there was no significant difference between these two groups. CONCLUSION: TXLC could effectively improve the symptoms of variant angina pectoris, the mechanism of which may likely be mediated by nitric oxide and endothelin.

Adult↗

PubMed1999

[Immune responses to recombinant expression plasmid encoding hepatitis C virus core antigen].

OBJECTIVE: To study the feasibility of DNA immunization with recombinant expression plasmid containing hepatitis C virus (HCV) core gene against HCV infection. METHODS: The recombinant plasmid containing HCV C gene was constructed and expressed transiently by using lipofectamine in the mouse SP2/0 cells. After appraisal and purification, these plasmid and control DNA were directly injected intramuscularly into BALB/c mice, HCV C specific antibody in the sera was checked with ELISA. Proliferative responses of spleen cells from experimental and control mice were determined by (3)H thymidine incorporation and cytotoxicity T lymphocyte (CTL) activity was measured by standard 4-h (51)Cr release assays. RESULTS: All immunized mice developed anti-HCV core antibodies and this antibody was dose and immune number dependent. Lymphoproliferative responses for HCV core antigen were higher in immunized mice than in control mice (P < 0.05), stimulation indices (SI) were 2.42 - 6.10. CTLs might induce approximately 63.13% lysis of pcDNA HCV C transfected SP2/0 target cells. CONCLUSION: It is suggested that HCV C gene immunization is a promising method of vaccination against HCV infection.

Animals↗

PubMed1999

[Efficacy of native carvedilol in patients with essential hypertension].

To compare the efficacy of native carvedilol(Cv) with native atenolol(At), we treated 80 cases of essential hypertension(stage I-II) for 4 weeks, using both open-test and double blind randomized imitative methods. The daily dose of carvedilol was 10 mg to 40 mg, and that of atenolol was 50 mg to 100 mg. Both drugs were given once daily. Out of 30 cases of Cv group, carvedilol showed significant efficiency in 20 cases, and was effective in 6 cases. The total therapeutic efficiency was 86.2% (26/30). In the double blind groups(25 patients in each group), those treated with carvedilol(Group A) had 21 effective cases(84.0%) while those treated with atenolol(Group B) had 18 effective cases(72.0%). A lower incidence of adverse effects was observed in carvedilol group. Dizziness occurred similarly in both drugs, but it lasted only a short time and could be tolerated. The recommended dose of 10-30 mg once daily would be appropriate. After treatment there was a significant reduction in blood pressure in both groups(P < 0.01). It suggests that native carvedilol is a safe and effective anti-hypertensive agent.

Adolescent↗

PubMed1999

[Determination of lignan glycosides in the roots of Gaultheria yunnanensis Rehd. by HPLC].

OBJECTIVE: To determine the contents of four lignan glycosides in the root of Gaultheria yunnanensis, namely, D1: gaultheroside A, D2:(-)-8'-methoxyisolariciresinol-2a-O-beta-D-xylopyranoside, D3:(-)-isolariciresinol-2a-O-beta-D-xylopyranside, D4:(+)-lyoniresinol-2a-O-beta-D-glucoxylopyranoside. METHOD: Rp-HPLC was used to separate and analyze the lignan glycosides in the root of the title plant. RESULT: The method for sample preparation was set up; the gradient program for the mobile phase was established; and the retention times of four lignan glycosides were determined. CONCLUSION: The method has been proved to be accurate, reproducible and of higher recovery rate.

Chromatography, High Pressure Liquid↗

PubMed1998

A fluorescence study of ligand-induced conformational changes in cytosolic fructose-1,6-bisphosphatase from germinating castor oil seeds.

The intrinsic fluorescence of homogeneous castor oil seed cytosolic fructose-1,6-bisphosphatase (FBPasec) was used as an indicator of conformational changes due to ligand binding. Binding of the substrate and the inhibitor fructose-2,6-bisphosphate (F-2,6-P2) was quantitatively compared to their respective kinetic effects on enzymatic activity. There are two distinct types of substrate interaction with FBPasec, corresponding to catalytic and inhibitory binding, respectively. Inhibitory substrate binding shares several characteristics with F-2,6-P2 binding which indicates that both ligands bind at the same site. However, F-2,6-P2 does not prevent fluorescence transitions attributed to catalytic substrate binding. The marked synergistic inhibition of FBPasec by AMP and F-2,6-P2 appears to arise via AMP's promotion of F-2,6-P2 binding. Based on the X-ray crystal structure of porcine kidney FBPase our modelling studies suggest the existence of a distinct F-1,6-P2/F-2,6-P2 inhibitory binding site which partially overlaps with the enzyme's catalytic site. We propose that a pronounced allosteric transition mediated by AMP binding increases access of F-1,6-P2 and F-2,6-P2 to this common inhibitory binding site.

Adenosine Monophosphate↗

PubMed1998

Aldehyde reductase: the role of C-terminal residues in defining substrate and cofactor specificities.

The only major structural difference between aldehyde reductase, a primarily NADPH-dependent aldo-keto reductase, and aldose reductase, a dually coenzyme-specific (NADPH/NADH) member of the same superfamily, is an additional eight amino acid residues in the substrate/inhibitor binding site (C-terminal region) of aldehyde reductase. On the premise that this segment defines the substrate specificity of the enzyme, a mutant of aldehyde reductase lacking residues 306-313 was constructed. In contrast to wild-type enzyme, the mutant enzyme reduced a narrower range of aldehydes and the new substrate specificity was not similar to aldose reductase as might have been predicted. A major change in coenzyme specificity was observed, however, the mutant enzyme being distinctly NADH preferring(Km, NADH = 35 microM, compared to <5 mM for wild-type and Km, NADPH = 670 microM, compared to 35 microM for wild type). Upon analyzing coordinates of aldehyde and aldose reductase, we found that deletion of residues 306-313 may have created a truncated enzyme that retained the three-dimensional structural features of the enzyme's C-terminal segment. The change in substrate specificity could be explained by the new alignment of amino acids. The reversal of coenzyme specificity appeared to be due to a significant backbone shift initiated by the formation of a strong hydrogen bond between Tyr319 and Val300. A similar bond exists in aldose reductase (Tyr309-Ala299). It appears, therefore, that as far as coenzyme specificity is concerned, deletion of residues 306-313 has converted aldehyde reductase into an aldose reductase-like enzyme.

Aldehyde Reductase↗

PubMed1998

Purification, crystallization and preliminary X-ray analysis of the Escherichia coli phytase.

A recombinant form of Escherichia coli phytase, which hydrolyzes phytic acid into phosphate and myo-inositol, has been expressed, purified and crystallized. Crystals have been obtained by the method of bulk crystallization in 10 mM sodium acetate buffer (pH 4.5) without using a conventional precipitant. The enzyme crystallized in space group P21, with unit-cell dimensions a = 74.9, b = 72.2, c = 82.4 A, and beta = 92.0 degrees. Crystals diffract to at least 2.2 A at a rotating-anode X-ray source and a 2.3 A resolution data set has been collected, giving completeness of 98.0% and an Rsym of 0.072. Assuming there are two phytase molecules in the asymmetric unit, the solvent content is calculated to be 42.1%. A self-rotation function shows a clear twofold non-crystallographic symmetry relating two molecules of E. coli phytase in the asymmetric unit.

6-Phytase↗

PubMed1998

Structure of type III antifreeze protein at 277 K.

Fish antifreeze proteins (AFP's) depress the freezing point of blood and other body fluids below that of the surrounding seawater by binding to and inhibiting the growth of seed ice crystals. The high-resolution crystal structure of type III AFP, determined at room temperature, reveals a remarkably flat surface containing most of the ice-binding residues [Jia et al. (1996). Nature (London), 384, 285-288]. Since AFP's function at temperatures close to 273 K, it is important to know whether the structure determined at room temperature undergoes any change at much lower temperature. Therefore, type III AFP has been crystallized at 277 K and its structure determined. Although crystallization conditions at 277 K were similar to those at approximately 295 K, crystal growth took much longer at the lower temperature. Crystals grown at the two temperatures were isomorphous. Initial crystals appeared within 40-50 d and grew to their final size in about 8-12 months, instead of a couple of days at approximately 295 K. The type III antifreeze protein structure from crystals grown at 277 K was essentially the same as that determined at approximately 295 K, with the exception of some minor changes in side-chain conformation. The result is an indication that temperature has a minimal effect on the structure of type III AFP, thus lending increased physiological validity to the room-temperature structure which was used for the initial ice-binding modelling.

Animals↗

PubMed1998

Inhibition of protein tyrosine phosphatases PTP1B and CD45 by sulfotyrosyl peptides.

Sulfotyrosyl peptides corresponding to the known high-affinity substrate phosphotyrosyl peptide sequences in casein and the autophosphorylation sites of insulin receptor and EGF receptor were investigated as inhibitors of protein tyrosine phosphatases PTP1B and CD45. These peptides inhibit both PTP1B and CD45 in the micromolar range competitively and reversibly. The elements required for inhibition were investigated by truncation and substitution of these peptides. Acidic residues N-terminal to the sulfotyrosyl residues are essential for high-affinity binding to PTP1B. The recognition elements required for inhibition of PTP1B and CD45 are different and this suggests the possibility of identifying selective active-site-directed inhibitors for these enzymes.

Binding Sites↗

PubMed1998

Affinity selection from peptide libraries to determine substrate specificity of protein tyrosine phosphatases.

Affinity selection from peptide libraries is a powerful tool that has been used for determining the sequence specificities of a number of enzymes and protein binding domains, including protein kinases, src homology 2 domains, and PDZ domains. We have extended this approach to protein tyrosine phosphatases using peptide libraries containing a nonhydrolyzable phosphotyrosine analog, difluorophosphonomethylphenylalanine. A size-exclusion method is used to separate enzyme-peptide complexes from free peptide, providing several advantages over the traditional immobilized protein affinity column approach. In addition, the feasibility of using mass spectrometric detection to quantitate peptides rapidly and reproducibly is demonstrated as an alternative to quantitation by peptide sequencing. The validity of this analysis is demonstrated by synthesizing individual peptides and comparing their affinity for enzyme with the predictions from the affinity selection process. As a model for these studies the protein tyrosine phosphatase PTP1B is used, providing additional insights into the sequence specificity of this enzyme. In particular, a selection for aromatic amino acids at the pY - 1 position (immediately N-terminal to the phosphotyrosine), as well as a broad pY + 1 selectivity, is observed in addition to the general preference for acidic residues N-terminal to the phosphotyrosine. The approach described here should prove applicable to protein tyrosine phosphatases in general as well as for the study of nonpeptidyl combinatorial libraries.

Amino Acid Sequence↗

PubMed1998

The effects of steric mutations on the structure of type III antifreeze protein and its interaction with ice.

The interaction of proteins with ice is poorly understood and difficult to study, partly because ice is transitory and can present many binding surfaces, and partly because structures have been determined for only two ice-binding proteins. This paper focuses on one of these, a 66-residue antifreeze protein (AFP) from eel pout. The high resolution X-ray structure of this fish AFP demonstrated that the proposed ice-binding surface is remarkably flat for such a small protein. The residues on the planar surface thought to be involved in ice binding are restrained by hydrogen bonds or by tight packing of their side-chains. To probe the requirement for a flat binding surface, a conserved alanine in the center of the AFP planar surface was substituted with larger residues. Six alanine replacement mutants (Ala16 > Cys, Thr, Met, Arg, His and Tyr), designed to disrupt the planarity of the surface and sterically block binding to ice, were characterized by X-ray crystallography and compared with the wild-type AFP. In each case, the detail provided by these crystal structures has helped explain the effects of the mutation on antifreeze activity. The substitutions, Ala16 > His and Ala16 > Tyr, were large enough to shield Gln44, one of the putative ice-binding residues, contributing to their very low thermal hysteresis activity. In addition to sterically hindering the putative ice-binding site, the bulkier residues also caused shifts in the putative ice-binding residues owing to the tight packing of side-chains on the planar surface. This unexpected consequence of the mutations helps account for the severely reduced antifreeze activity. One explanation for residual antifreeze activity in some of the mutants lies in the possibility that AFPs have a role in shaping the site on the ice to which they bind. Thus, side-chain dislocations might be partially accommodated by ice that can freeze around them. It is evident that the disruption of the planarity, by introducing larger residues at the center of the proposed ice-binding site, is not the only factor responsible for the loss of antifreeze activity. There are multiple causes including positional change and steric blockage of some putative ice-binding residues.

Alanine↗