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Biomedical subjects

Y L Zhang

Publications and source records attributed to Y L Zhang.

At least 37 records · Page 2Linked to original sources
PubMed2001

[Genetic transformation of hairy roots in Trichosanthes kirilowii Maxim. by Ti and Ri plasmids].

OBJECTIVE: To establish a hairy root culture system by double transformation for Trichosanthes kirilowii. METHOD: 1. Crown galls were induced by direct infection of sterile seedlings of T. kirilonii with Agrobacterium tumefaciens C58, and then the hairy roots were obtained from the regenerated plants by infection with A. rhzogenes 15834; 2. Transformation of Ti and Ri plasmids was inspected by high-pressure-paper electrophoresis; 3. The protein contents in the tissues of T. kirilowii were inspected by spectrophotometer and SDS-PAGE. RESULT: A hairy root culture system has been established successfully by double transformation with Ti and Ri plasmids in T. kirilowii. CONCLUSION: Compared with the ordinary hairy roots, the double transformed hairy roots grow faster but retain similar protein contents.

Arginine↗
PubMed2000

Thermodynamic study of ligand binding to protein-tyrosine phosphatase 1B and its substrate-trapping mutants.

The binding of several phosphonodifluoromethyl phenylalanine (F(2)Pmp)-containing peptides to protein-tyrosine phosphatase 1B (PTP1B) and its substrate-trapping mutants (C215S and D181A) has been studied using isothermal titration calorimetry. The binding of a high affinity ligand, Ac-Asp-Ala-Asp-Glu-F(2)Pmp-Leu-NH(2), to PTP1B (K(d) = 0.24 microm) is favored by both enthalpic and entropic contributions. Disruption of ionic interactions between the side chain of Arg-47 and the N-terminal acidic residues reduces the binding affinity primarily through the reduction of the TDeltaS term. The role of Arg-47 may be to maximize surface contact between PTP1B and the peptide, which contributes to high affinity binding. The active site Cys-215 --> Ser mutant PTP1B binds ligands with the same affinity as the wild-type enzyme. However, unlike wild-type PTP1B, peptide binding to C215S is predominantly driven by enthalpy change, which likely results from the elimination of the electrostatic repulsion between the thiolate anion and the phosphonate group. The increased enthalpic contribution is offset by reduction in the binding entropy, which may be the result of increased entropy of the unbound protein caused by this mutation. The general acid-deficient mutant D181A binds the peptide 5-fold tighter than the C215S mutant, consistent with the observation that the Asp to Ala mutant is a better "substrate-trapping" reagent than C215S. The increased binding affinity for D181A as compared with the wild-type PTP1B results primarily from an increase in the DeltaH of binding in the mutant, which may be related to decreased electrostatic repulsion between the phosphate moiety and PTP1B. These results have important implications for the design of high affinity PTP1B inhibitors.

Amino Acid Sequence↗
PubMed2000

1-Aminomethylisoquinoline-4-carboxylates as novel dipeptidylpeptidase IV inhibitors.

Structure-activity relationship within a series of 1-aminoalkylisoquinoline-4-carboxylates as inhibitors of DPP-IV is described. A primary aminomethyl group is required to maintain biological activity. Substitution of the isoquinoline at the 6- and 8-positions with methoxy groups increases potency to 53 times that of the lead compound SDZ 029-576.

Carboxylic Acids↗
PubMed2000

Transcriptional suppression of synuclein gamma (SNCG) expression in human breast cancer cells by the growth inhibitory cytokine oncostatin M.

Previously, we have shown that synuclein gamma (SNCG), a member of the brain protein synuclein family, is highly expressed in human infiltrating breast carcinomas but not expressed in normal or benign breast tissues. The SNCG mRNA was also detected in several human breast cancer cell lines with the highest expression found in H3922, a cell line derived from an infiltrating ductal carcinoma. In this study, we show that expression of SNCG mRNA in H3922 cells is significantly decreased by treating cells with the cytokine oncostatin M (OM) who has a growth-inhibitory effect on these cells. A decrease in SNCG mRNA level can be detected as early as 30 min after OM addition. By 4 h OM treatment, the level of SNCG mRNA was decreased to 70% of control, and by 24 h the mRNA was below detectable level. Western blot analysis further demonstrated the suppression of SNCG protein expression by OM. The level of SNCG protein in H3922 cells was reduced more than 90% by OM after 2 days. Since OM-induced growth inhibition occurs after 3-4 days, the down-regulation of SNCG expression appears to proceed the effect of OM on cell growth. Additional experiments to measure the transcriptional rates of SNCG indicate that the observed OM-induced down-regulation of SNCG mRNA occurs mainly at the transcriptional level. In an attempt to examine the role of SNCG gene in the proliferation of breast cancer cells, SNCG cDNA was stably transfected into MCF-7 cells that do not express endogenous SNCG gene. Examination of cell growth under anchorage-dependent and anchorage-independent conditions demonstrates that over expression of SNCG gene significantly stimulated the growth of MCF-7 cells both in monolayer culture and in soft agar. These data together suggest that SNCG may play a role in cell proliferation.

Blotting, Northern↗
PubMed2000

Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish.

Aeromonas hydrophila, a normal inhabitant of aquatic environments, is an opportunistic pathogen of a variety of aquatic and terrestrial animals, including humans. A. hydrophila PPD134/91 is defined as virulent whereas PPD35/85 is defined as avirulent on the basis of their different LD50 values in fish. Suppression subtractive hybridization (SSH) was used to identify genetic differences between these two strains. Sixty-nine genomic regions of differences were absent in PPD35/85, and the DNA sequences of these regions were determined. Sixteen ORFs encoded by 23 fragments showed high homology to known proteins of other bacteria. ORFs encoded by the remaining 46 fragments were identified as new proteins of A. hydrophila, showing no significant homology to any known proteins. Among these PPD134/91-specific genes, 22 DNA fragments (21 ORFs) were present in most of the eight virulent strains studied but mostly absent in the seven avirulent strains, suggesting that they are universal virulence genes in A. hydrophila. The PPD134/91-specific genes included five known virulence factors of A. hydrophila: haemolysin (hlyA), protease (oligopeptidase A), outer-membrane protein (Omp), multidrug-resistance protein and histone-like protein (HU-2). Another 47 DNA fragments (44 ORFs) were mainly present in PPD134/91, indicating the heterogeneity among motile aeromonads. Some of these fragments encoded virulence determinants. These included genes for the synthesis of O-antigen and type II restriction/modification system. The results indicated that SSH is successful in identifying genetic differences and virulence genes among different strains of A. hydrophila.

Aeromonas hydrophila↗
PubMed2000

Morphology of luminal and glandular epithelial cells from pig endometrium grown on plastic or extracellular matrices.

Luminal (LE) and glandular epithelial (GE) cells from d-13 pregnant pigs were cultured on plastic, matrix secreted from endometrial stroma, and EHS matrix (Matrigel) in culture medium (RPMI-1640) supplemented with 20 and 10% fetal bovine serum, respectively. After culture for 7 and 14 d, GE and LE cells were prepared for transmission and GE cells for scanning electron microscopy. The two types of endometrial epithelial cells displayed different morphological characteristics when grown on different culture substrates. On plastic, the GE and LE cells formed flattened monolayers. However, stroma-secreted matrix directed the polarization of endometrial epithelia. The GE and LE cells reacted differently to thick Matrigel coatings; LE cells formed a colony after 7 d of culture and then proliferated further to form a colony with a cavity, but GE cells organized to form a colony with a shallow depression in the center at 7 d and developed duct-like structures after 14 d in vitro. Luminal epithelial cells grown on either diluted or thin-coated Matrigel and grown on stroma-secreted matrix formed a monolayer but no three-dimensional structures.

Animals↗
PubMed2000

[Morphometric study on the effects of estrone on bone in vitro].

AIM: To study the effects of estrone on the bone resorption and bone formation in vitro. METHODS: Long bones from 16-day-old female mouse fetuses were cultured in BGJb medium for 48 h. The bones were harvested, then bones length and length of their diaphyses were measured under stereo microscope. The histomorphormetric analyses on midlongitudinal sections of bones were performed. The numbers of osteoclasts and hypertrophic chondrocytes were observed under biological microscope. RESULTS: Compared with control, there were significant increase in bones length and length of their diaphyses after treated with 10(-7) mol/L estrone. Under this condition, decreased osteoclasts and increased hypertrophic chondrocytes were observed, too. CONCLUSION: Estrone stimulates bone formation and inhibites bone resorption in vitro.

Animals↗
PubMed2000

[Dynamics of growth and total tanshinones accumulation in crown gall cultures of salvia miltiorrhiza].

AIM: To determine the dynamics of growth and total tanshinones accumulation in crown gall cultures of Salvia miltiorrhiza in MS and 67-V liquid media. METHODS: Fresh, dry weight and total tanshinones yields in the cultures and in the medium were determined every 5 days in crown gall suspension cultures. RESULTS: In MS medium, the logarithmic growth phase of crown gall cultures in S. miltiorrhiza was from the 5th to 30th days, and the stationary growth phase was from the 30th to 35th days. From the 25th to 30th days, physiological activity of crown gall cultures was higher and their growth was better. However, in 67-V medium, the logarithmic growth phase of crown gall cultures was from the 10th to 25th days, and the stationary growth phase was from the 25th to 35th days. Total tanshinones were largely accumulated in the cultures and in the medium after 25 days. The total tanshinones yield (60 mg.L-1) was reached at the 35th day. CONCLUSION: Knowing the regularity of the growth and total tanshinones accumulation in crown gall cultures of S. miltiorrhiza will be helpful to take proper regulative measures in order to obtain the maximum total tanshinones yield.

Abietanes↗
PubMed2000

[A survey of the changes of serum inorganic salts and alkaline phosphatase after jaw fractures].

OBJECTIVE: To study the changes of serum salts and alkaline phosphatase in patients with jaw fractures and its roles. METHODS: The serum salts and alkaline phosphatase were determined in 40 cases of jaw fracture and 14 health persons. RESULTS: The results showed that there was no significant difference in Na(+), K(+), Cl(-), Ca(2+) contents between jaw fracture group and normal group (P>0.05), but P(3-) Mg(2+), ALP levels were higher in jaw fracture group than that in normal group (P<0.05). Furthermore the Ca(2+), P(3-), Mg(2+), ALP were higher in multiple or communition fracture of jaw than in single or double line fracture (P<0.05) except Ca content. The level of Ca(2+) was higher in single jaw fracture than in jaw damage with whole body injury (P>0.05) but the levels of P(3-),Mg(2+), ALP were no difference (P>0.05). As the same all the Ca(2+), P(3-), Mg(2+), ALP levels were higher in early stage of fracture than in 4 weeks after fracture (P<0.05). CONCLUSION: The contents of Ca(2+) P(3-), Mg(2+) ALP were increased after the axilla mandible were fractured and it has an important regulation role in the activity of osteoblast and osteoclast in healing of fractured jaw.

English Abstract↗
PubMed1999

Impaired transition state complementarity in the hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases.

The hydrolysis of O-arylphosphorothioates by protein-tyrosine phosphatases (PTPases) was studied with the aim of providing a mechanistic framework for the reactions of this important class of substrate analogues. O-arylphosphorothioates are hydrolyzed 2 to 3 orders of magnitude slower than O-aryl phosphates by PTPases. This is in contrast to the solution reaction where phosphorothioates display 10-60-fold higher reactivity than the corresponding oxygen analogues. Kinetic analyses suggest that PTPases utilize the same active site and similar kinetic and chemical mechanisms for the hydrolysis of O-arylphosphorothioates and O-aryl phosphates. Thio substitution has no effect on the affinity of substrate or product for the PTPases. Bronsted analyses suggest that like the PTPase-catalyzed phosphoryl transfer reaction the transition state for the PTPase-catalyzed thiophosphoryl transfer is highly dissociative, similar to that of the corresponding solution reaction. The side chain of the active-site Arg residue forms a bidentate hydrogen bond with two of the terminal phosphate oxygens in the ground state and two of the equatorial oxygens in a transition state analog complex with vanadate [Denu et al. (1996) Proc. Natl. Acad. Sci. USA 93, 2493-2498; Zhang, M. et al. (1997) Biochemistry 36, 15-23; Pannifer et al. (1998) J. Biol. Chem. 273, 10454-10462]. Replacement of the active-site Arg409 in the Yersinia PTPase by a Lys reduces the thio effect by 54-fold, consistent with direct interaction and demonstrating strong energetic coupling between Arg409 and the phosphoryl oxygens in the transition state. These results suggest that the large thio effect observed in the PTPase reaction is the result of inability to achieve precise transition state complementarity in the enzyme active site with the larger sulfur substitution.

Binding Sites↗
PubMed1999

Distinct contributions of residue 192 to the specificity of coagulation and fibrinolytic serine proteases.

Archetypal members of the chymotrypsin family of serine proteases, such as trypsin, chymotrypsin, and elastase, exhibit relatively broad substrate specificity. However, the successful development of efficient proteolytic cascades, such as the blood coagulation and fibrinolytic systems, required the evolution of proteases that displayed restricted specificity. Tissue-type plasminogen activator (t-PA), for example, possesses exquisitely stringent substrate specificity, and the molecular basis of this important biochemical property of t-PA remains obscure. Previous investigations of related serine proteases, which participate in the blood coagulation cascade, have focused attention on the residue that occupies position 192 (chymotrypsin numbering system), which plays a pivotal role in determining both the inhibitor and substrate specificity of these enzymes. Consequently, we created and characterized the kinetic properties of new variants of t-PA that contained point mutations at position 192. These studies demonstrated that, unlike in coagulation serine proteases, Gln-192 does not contribute significantly to the substrate or inhibitor specificity of t-PA in physiologically relevant reactions. Replacement of Gln-192 with a glutamic acid residue did, however, decrease the catalytic efficiency of mature, two-chain t-PA toward plasminogen in the absence of a fibrin co-factor.

Amino Acid Substitution↗
PubMed1999

Restoration of potent protein-tyrosine phosphatase activity into the membrane-distal domain of receptor protein-tyrosine phosphatase alpha.

Most transmembrane, receptor-like protein-tyrosine phosphatases (RPTPs) contain two cytoplasmic catalytic protein-tyrosine phosphatase (PTP) domains, of which the membrane-proximal domain, D1, contains the majority of the activity, while the membrane-distal domain, D2, exhibits little or no activity. We have investigated the structural basis for reduced activity in RPTP-D2s, using RPTPalpha as a model system. Sequence alignment of PTP domains indicated that two motifs, the KNRY motif and the WpD motif, are highly conserved in all PTP domains, but not in RPTP-D2s. In RPTPalpha-D2, the Tyr in the KNRY motif is substituted by Val (position 555) and the Asp in the WpD motif by Glu (position 690). Mutation of Val555 and Glu690 had synergistic effects on RPTPalpha-D2 activity, in that the PTP activity of RPTPalpha-D2-V555Y/E690D was greatly enhanced to levels that were similar to or approaching those of RPTPalpha-D1. Therefore, Val555 and Glu690 are responsible in large part for reduced RPTPalpha-D2 activity. In addition, we established that the increased PTP activity is due to restoration of effective transition-state stabilization in RPTPalpha-D2-V555Y/E690D. Since the KNRY motif and the WpD motif are mutated in all RPTP-D2s, it is highly unlikely, due to lack of transition-state stabilization, that the residual RPTP-D2 catalytic activity plays a role in the function of RPTPs.

Amino Acid Sequence↗
PubMed1999

On the spontaneous mutability of CpG sites in cultured S49 mouse lymphoma cells.

The frequent occurrence of human mutations at CpG dinucleotide sites has been attributed to cytosine methylation and hydrolytic deamination of the resulting 5-methylcytosine residue. Previously, we reported an unusually strong hotspot for spontaneous transitions at a CpG site in the gene for regulatory (R) subunit of protein kinase A in S49 mouse lymphoma cells. Now, using polymerase chain reaction-based methods to screen mutant populations for mutations at particular CpG sites, we show that two methylated CpG sites in the gene for hypoxanthine phosphoribosyltransferase are much less mutable than the R subunit hotspot site, suggesting that different methylated CpG sites are differentially susceptible to spontaneous mutation. We also present data on spontaneous R subunit mutations in cloned populations of 5-azacytidine-treated S49 cells that had been demethylated at the hotspot site in both R subunit alleles. Of 13 independent mutants isolated from populations grown from fully demethylated cells, seven had the hotspot mutation. We conclude that CG-->TA mutations at strong CpG hotspots do not require prior methylation of CpG sites.

5-Methylcytosine↗
PubMed1998

Conformational and dynamic changes of Yersinia protein tyrosine phosphatase induced by ligand binding and active site mutation and revealed by H/D exchange and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Protein tyrosine phosphatases (PTPase) play important roles in the intracellular signal transduction pathways that regulate cell transformation, growth, and proliferation. Here, solvent accessibility is determined for backbone amide protons from various segments of wild-type Yersinia PTPase in the presence or absence of 220 microM vanadate, a competitive inhibitor, as well as an active site mutant in which the essential cysteine 403 has been replaced by serine (C403S). The method consists of solution-phase H/D exchange, followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform ion cyclotron resonance mass spectrometry. Proteolytic segments spanning approximately 93.5% of the primary sequence are analyzed. Binding of vanadate reduces the H/D exchange rate throughout the protein, both for the WpD loop and for numerous other residues that are shielded when that loop is pulled down over the active site on binding of the inhibitor. The single active site C403S mutation reduces solvent access to the WpD loop itself, but opens up the structure in several other segments. Although the 3D structure of the ligand-bound C403S mutant is similar to that of the wild-type PTPase, and the C403S mutant and the wild-type enzyme display similar affinities for vanadate, the thermodynamics for binding of vanadate is different for the two proteins. Collectively, these results establish the flexibility of the WpD loop (previously inferred by comparing PTPase X-ray single-cyrstal diffraction structures in the presence and absence of a tungstate inhibitor), as well as several other signficant changes in segment exposure and/or flexibility that are not evident from X-ray structures.

Amino Acid Sequence↗
PubMed1998

Synthesis, biological activity, and conformational analysis of peptidomimetic analogues of the Saccharomyces cerevisiae alpha-factor tridecapeptide.

Biochemical and biophysical investigations on the Saccharomyces cerevisiae alpha-factor indicate that this tridecapeptide mating pheromone (WHWLQLKPGQPMY) might adopt a type II beta-turn in the center of the peptide when it binds to its G protein-coupled receptor. To test this hypothesis we synthesized analogues of alpha-factor incorporating a (R or S)-gamma-lactam conformational constraint [3-(R or S)-amino-2-oxo-1-pyrrolidineacetamido] in place of the Pro-Gly at residues 8 and 9 of the peptide and tested their biological activities and receptor binding. Analogues were purified to >99% homogeneity as evidenced by high-performance liquid chromatography and capillary electrophoresis and characterized by amino acid analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The restricted alpha-factor analogue WHWLQLK[(R)-gamma-lactam]QP[Nle]Y was more active than its lactam-containing diastereomeric homologue WHWLQLK[(S)-gamma-lactam]QP[Nle]Y and about equally active with the [Nle12]-alpha-factor in growth arrest and FUS1-lacZ gene induction assays. Both lactam analogues competed with tritiated [Nle12]-alpha-factor for binding to the alpha-factor receptor (Ste2p) with the (R)-gamma-lactam-containing peptide having 7-fold higher affinity than the (S)-gamma-lactam-containing homologue. Two-dimensional NMR spectroscopy and modeling analysis gave evidence that the (R)-gamma-analogue is a flexible peptide that assumes a transient gamma-turn structure around the lactam moiety. The results represent the first example of an alpha-factor analogue containing a peptidomimetic constraint that is as active as the native pheromone. The correlation between activity and structure provides further evidence that the biologically active conformation of the molecule contains a turn in the middle of the pheromone. This study provides new insights into the structural basis of alpha-factor activity and adds to the repertoire of conformationally biasing constraints that can be used to maintain and even enhance biological activity in peptide hormones.

Amides↗
PubMed1998

Low-affinity binding determined by titration calorimetry using a high-affinity coupling ligand: a thermodynamic study of ligand binding to protein tyrosine phosphatase 1B.

A competition-based method is used for the determination of the thermodynamic parameters for a low-affinity ligand binding reaction by isothermal titration calorimetry. This method is based on the coupling of a high-affinity ligand to the binding of the low-affinity ligand. Results are presented for the binding of a nonhydrolyzable phosphotyrosine analog phosphonodifluoromethyl phenylalanine (F2Pmp)-containing peptide (Ac-Asp-Ala-Asp-Glu-F2Pmp-Leu-NH2), arsenate, and inorganic phosphate to the intracellular human protein tyrosine phosphatase 1B(PTP1B). The binding constants are 3.3 x 10(6), 4.3 x 10(3), and 48 M-1 for the F2Pmp-containing peptide, arsenate, and inorganic phosphate, respectively. The binding of arsenate and inorganic phosphate to PTP1B is enthalpy driven. This is in contrast to the binding of the F2Pmp-containing peptide which is mainly driven by entropy. The calorimetrically determined binding constants are in agreement with the Ki values determined by enzyme inhibition studies. This demonstrates that isothermal titration calorimetry can be used to quantitatively determine the thermodynamic parameters for the interactions between proteins and low-affinity ligands if a proper coupling ligand can be identified.

Amino Acid Sequence↗
PubMed1998

Equilibrium intermediates in the unfolding pathway of creatine kinase.

The unfolding of creatine kinase in various concentrations of guanidine hydrochloride of increasing concentrations has been investigated by combination of size-exclusion chromatography (SEC) with other methods. There are two peaks in the profiles of SEC in GuHCl at moderate concentrations, showing that unfolding of creatine kinase goes through dimeric and monomeric intermediates with increasing guanidine hydrochloride concentrations. Both intermediates have relatively compact structure and retain considerable ordered structure.

Chromatography, Gel↗