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Biomedical subjects

R Zhang

Publications and source records attributed to R Zhang.

At least 127 records · Page 7Linked to original sources
PubMed2000

Hepatitis C NS3 protease: restoration of NS4A cofactor activity by N-biotinylation of mutated NS4A using synthetic peptides.

The NS3 serine protase of Hepatitis C virus (HCV) requires NS4A protein as a cofactor for efficient cleavage at four sites in the nonstructural region. The cofactor activity has been mapped to the central hydrophobic region (aa 22-34) of this 54-amino-acid NS4A protein, and site-directed mutagenesis has identified alternating hydrophobic amino acids, particularly Ile25 and Ile29, as critically important. A double mutant of NS4A cofactor peptide, I25A/I29A, completely abolished the cofactor activity. We now report that the cofactor peptide activity in the I25A/I29A double mutant can be restored specifically by introducing a biotin-aminohexanoic acid fusion at the N-terminus. In addition, a similar N-terminal fusion of biotin-aminohexanoic acid with the wild-type 4A peptide significantly enhanced cofactor activity. Our data corroborate the crystal structure-based hypothesis of hydrophobic interaction between the N-terminus of NS4A and the N-terminal alpha(0) helix of NS3 protease.

Amino Acid Sequence↗
PubMed2000

Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy.

To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G(1) --> A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca(2+) sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca(2+) sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca(2+) sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca(2+) regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.

Amino Acid Sequence↗
PubMed2000

S curve, a graphic representation of protein secondary structure sequence and its applications.

A secondary structure sequence is a symbolic string composed of three kinds of letters, indicating the helix, strand, and coil (including turns), respectively. A graphic representation for this abstract symbolic sequence is proposed here, called the S curve. The S curve is the unique representation for a given secondary structure sequence in the sense that the sequence and the S curve can be uniquely determined from the other. Therefore, the S curve contains all the information that the secondary structure sequence contains. Different geometrical properties of the S curve are studied in details, which reflect the basic characteristics of the secondary structure sequences. The S curves are used to display, analyze, and compare the secondary structure sequences. Detailed application examples are presented. One advantage of the S curve methodology is that the main patterns of a given secondary structure sequence can be grasped quickly in a perceivable form. This is particularly useful in the cases in which longer sequences are involved and structures of proteins are unknown.

Amino Acid Sequence↗
PubMed2000

Determination of resibufogenin and cinobufagin in heart-protecting musk pill by HPLC.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of resibufogenin and cinobufagin in traditional Chinese medicine (heart-protecting musk pill, shexiang baoxin wan in Chinese) was developed. A reversed-phase system with a Hypersil (ODS2) C(18) column and tetrahydrofuran: methanol: water (8:31:61) mobile phase was employed for the separation of resibufogenin and cinobufagin. The detection was set at 299 nm and ethinyl estradiol was chosen as the internal standard. The limit of detection was 1.5 ng for resibufogenin and 2.0 ng for cinobufagin at a signal-to-noise ratio of 4:1. It is a rapid, simple and accurate method for quantitative analysis of resibufogenin and cinobufagin in heart-protecting musk pill.

Bufanolides↗
PubMed2000

Synthetic nano-fibrillar extracellular matrices with predesigned macroporous architectures.

Scaffolding plays a pivotal role in tissue engineering. To mimic the architecture of a natural extracellular matrix component-collagen, nona-fibrous matrices have been created with synthetic biodegradable polymers in our laboratory using a phase-separation technique. To improve the cell seeding, distribution, mass transport, and new tissue organization, three-dimensional macroporous architectures are built in the nano-fibrous matrices. Water-soluble porogen materials are first fabricated into three-dimensional negative replicas of the desired macroporous architectures. Polymer solutions are then cast over the porogen assemblies in a mold, and are thermally phase-separated to form nano-fibrous matrices. The porogen materials are leached out with water to finally form the synthetic nano-fibrous extracellular matrices with predesigned macroporous architectures. In this way, synthetic polymer matrices are created with architectural features at several levels, including the anatomical shape of the matrix, macroporous elements (100 microm to millimeters), interfiber distance (microns), and the diameter of the fibers (50-500 nm). These scaffolding materials circumvent the concerns of pathogen transmission and immuno-rejection associated with natural collagen. With the flexibility in the design of chemical structure, molecular weight, architecture, degradation rate, and mechanical properties, these novel synthetic matrices may serve as superior scaffolding for tissue engineering.

Artificial Organs↗
PubMed2000

Distinct proteins interacting with the glucocorticoid receptor.

The glucocorticoid receptor (GR) is a hormone-dependent transcription factor that modulates gene transcription by binding to its response element, interacting with other transcription factors, contacting the basal transcription machinery and by interacting with several intermediary proteins, such as coactivators. Here we discuss the most prominent examples for interacting transcription factors, interacting proteins that belong to the basal transcription apparatus and intermediary factors. Furthermore, we describe a new class of GR-interacting proteins (GRIPs) which have been identified by a blotting technique, using a radioactively labeled GR/GRE complex as a probe to identify proteins that interact with the activated GR. Two of these GRIPs, termed GRIP170 and GRIP 120, which were identified by biochemical purification and microsequencing and have turned out to play a role in modulating GR-mediated transcription, are discussed in detail.

Animals↗
PubMed2000

Coordinate expression of NGF and alpha-smooth muscle actin mRNA and protein in cutaneous wound tissue of developing and adult rats.

Nerve growth factor (NGF) is synthesized in cutaneous wound tissue, and its higher levels in the neonate may contribute to more efficient wound healing. We used in situ hybridization and immunohistochemistry to define NGF mRNA and protein expression in intact skin and following excision wounding in neonatal and adult rats. To determine whether NGF is associated with wound contractile fibroblasts (myofibroblasts), we also examined expression of alpha-smooth muscle actin (alpha-SMA) mRNA and protein, established markers for these cells. In intact skin, NGF mRNA and protein were present in vascular and arrector pili smooth muscle, hair follicle sheath cells, keratinocytes, and hypodermal fibroblasts. Neonatal adipocytes and Schwann cells also expressed NGF mRNA and protein, while adult adipocytes and Schwann cells displayed only NGF-ir. Following wounding, NGF mRNA expression was exuberant in these cell types, and increased similarly at both ages and appeared de novo in skeletal muscle cells. Additionally, both NGF mRNA and protein were present in macrophages and myofibroblasts, and expression in myofibroblasts was significantly greater in neonates. Wound myofibroblasts also expressed alpha-SMA. Surprisingly, after wounding alpha-SMA mRNA and protein were present in essentially all cells in which NGF mRNA was detected. We conclude that NGF expression is enhanced in many cell types after wounding, but greater NGF synthesis in neonates appears to be due to a more robust myofibroblast response. In addition, cell types which demonstrated NGF mRNA also expressed alpha-SMA, and staining for both markers increased following wounding, suggesting synthesis of both proteins is regulated in a coordinated fashion.

Actins↗
PubMed2000

Cross-genotypic interaction between hepatitis C virus NS3 protease domains and NS4A cofactors.

BACKGROUND/AIMS: Hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease requires NS4A as a cofactor. This cofactor activity has been mapped to the central region of NS4A which interacts with the N-terminus of NS3 protease. To investigate whether this interaction is conserved among different genotypes of HCV cross-genotypic characterization were performed to delineate the importance of NS4A cofactor function in relation to the molecular evolution of HCV METHODS: Active NS3 protease domains of genotype 1-3 (representing five subtypes: la, 1b, 2a, 2b and 3a) were produced and purified from bacterial cells. NS4A cofactor-dependent in vitro trans cleavage assays were established using the in vitro translated recombinant protein substrates. These substrates contained the junction site of NS4A/NS4B, NS4B/NS5A or NS5A/NS5B. RESULTS: Our data revealed that NS3 proteases cross-interacted with NS4A cofactors derived from different genotypes, although the genotype 2 cofactor was less efficient, which could be due to greater genetic variations in this region. Furthermore, the corresponding region in hepatitis G virus (HGV) NS4A was found to provide weak cofactor activity for HCV NS3 protease. Surprisingly, a synthetic substrate peptide from the NS4B/NS5A junction was also found to enhance HCV NS3 protease activity in a dose-dependent manner. CONCLUSION: Our study suggests that the NS4A cofactor function is well conserved among HCV It is likely that other HCV-related viruses may have developed similar strategies to regulate their protease activity.

Evolution, Molecular↗
PubMed2000

Obesity-hypertension: the effects on cardiovascular and renal systems.

Longitudinal and cross-sectional studies suggest that a large number of obese patients have a high prevalence of hypertension. This association causes the following changes: insulin and leptin resistance with a suppressed biologic activity of natriuretic peptide, which contributes to sodium retention with concomitant expanded cardiopulmonary volume and increased cardiac output. The cellular metabolism of cations may be altered in obesity and may lead to changes in vascular responsiveness and increased vascular resistance. These changes lead to structural adaptations in the heart characterized by concentric-eccentric left ventricular hypertrophy. The hypertrophic condition provides the basis for the development of congestive heart failure and cardiac arrhythmias that may explain the higher rates of cardiac sudden death in those patients. In the kidneys, obesity hypertension may initiate a derangement of renal function. The increased deposit of interstitial cells and of extracellular matrix between the tubules induces higher interstitial hydrostatic pressure and tubular sodium reabsorption. The consequent increase in renal flow and glomerular filtration enhances albuminuria excretion and the susceptibility to the development of renal damage. In summary, the hemodynamic and structural adaptations related to obesity hypertension is the cause of greater risk for adverse cardiovascular and renal events.

Animals↗
PubMed2000

Cloning and molecular characterization of calpain, a calcium-activated neutral proteinase, from different strains of Schistosoma japonicum.

cDNA coding for calpain of Schistosoma japonicum were cloned and sequenced, and serological basis of host responses to calpain were analyzed. cDNA of calpain from S. japonicum of two different isolates, Yamanashi strain (Sj-J) and Hunan strain (Sj-C), were 2, 468 bp and 2, 465 bp in length, including the same number (2, 274) of open reading frame. Nucleotide sequence and amino acid sequence between the two calpains are 99.1% and 98.8% identity, respectively. Sj-J and Sj-C calpains were considered to be translated as a preproenzyme, and a 746-amino acid mature enzyme contains eight motifs without a signal peptide at the N-terminal based on the deduced amino acid sequences. mRNA for calpain were detectable in different developmental stages, however, sera obtained from mice immunized with recombinant calpain showed enhanced binding to cercarial antigen. Human sera from S. japonicum-infected individuals recognized the large subunit of schistosomal calpain, and light-infected sera showed stronger reactivities to the recombinant calpain than moderate/high infection cases. When we tested synthetic peptides, there were four common human B cell epitopes in schistosomal calpain, all of which are shared with S. mansoni. Together with these results, calpain of S. japonicum seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.

Amino Acid Sequence↗
PubMed2000

Hyperproduction of polyesters consisting of medium-chain-length hydroxyalkanoate monomers by strain Pseudomonas stutzeri 1317.

Pseudomonas stutzeri strain 1317 was found to grow on various fatty acids, alcohols, diols, as well as glucose and gluconate for the synthesis of polyhydroxyalkanoates (PHA) with various monomer units. The PHA monomer structures were dependent on the type of fatty acids and alcohols, as well as the diols in the culture media. Only even number monomers, such as 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO) and 3-hydroxydecanoate (HD), were accumulated when even numbered fatty acids, alcohols, glucose and gluconate, as well as diol were used as carbon sources. Odd numbered fatty acids and odd numbered alcohols led to the formation of odd numbered monomers, such as 3-hydroxyvalerate (HV), 3-hydroxyheptanoate (HHp), 3-hydroxynonanoate (HN) and 3-hydroxyundecanoate (HU). The strain tolerated up to 1.5% of ethanol and made 8.3% of PHA when growth was conducted in 1.2% of ethanol. PHA formed up to 77% of cell dry weight when the strain was grown in tridecanoate. PHA synthesis was highly dependent on the nitrogen source. A depletion in nitrogen supply immediately resulted in PHA accumulation in cells grown in the glucose mineral medium.

Alcohols↗
PubMed2000

Structure of a WW domain containing fragment of dystrophin in complex with beta-dystroglycan.

Dystrophin and beta-dystroglycan are components of the dystrophin-glycoprotein complex (DGC), a multimolecular assembly that spans the cell membrane and links the actin cytoskeleton to the extracellular basal lamina. Defects in the dystrophin gene are the cause of Duchenne and Becker muscular dystrophies. The C-terminal region of dystrophin binds the cytoplasmic tail of beta-dystroglycan, in part through the interaction of its WW domain with a proline-rich motif in the tail of beta-dystroglycan. Here we report the crystal structure of this portion of dystrophin in complex with the proline-rich binding site in beta-dystroglycan. The structure shows that the dystrophin WW domain is embedded in an adjacent helical region that contains two EF-hand-like domains. The beta-dystroglycan peptide binds a composite surface formed by the WW domain and one of these EF-hands. Additionally, the structure reveals striking similarities in the mechanisms of proline recognition employed by WW domains and SH3 domains.

Amino Acid Sequence↗
PubMed2000

Genetic immunotherapy of established tumours with adenoviral vectors transducing murine interleukin-12 (mIL12) subunits in a rat medullary thyroid carcinoma model.

OBJECTIVE: Interleukin-12 (IL12) is a heterodimeric cytokine that plays an important role in the development of cellular immunity. Studies have demonstrated antitumour activity after systemic administration of recombinant IL12. As with other cytokines, with increasing dosage and longer exposure, systemic toxicity is observed. To reduce systemic toxicity and obtain local production of IL12, we developed a replication defective adenovirus transducing two subunits of the murine IL12 (AdCMVmIL12) gene. DESIGN: Two separate cassettes, expressing the p35 or p40 subunit of mIL12, under the control of human cytomeglavirus immediate early promoter, were inserted into the early1 (E1) region of adenovirus 5. Biological activity of virally expressed mIL12 was demonstrated in vitro through its ability to induce proliferation of mouse ConA blast cells. RESULTS: Rat medullary thyroid carcinoma (MTC) cells infected with AdCMVmIL12 lost their tumorigenicity in their syngenic WAG/Rij rat hosts. Efficient antitumour activity was found after direct injection of the AdCMVmIL12 vector into rMTC tumours. After intratumoural treatment with AdCMVmIL12, 86% of tumour bearing animals were apparently cured, and almost all remaining tumours were stabilized. Challenge studies showed that most animals cured after the first treatment remained tumour free after reinjection of wild type rMTC cells, indicating that long-term antitumour immunity developed. CONCLUSIONS: This study demonstrates the construction of an adenoviral vector expressing a functional heterodimeric mIL12 and its efficient antitumour activity after in vivo delivery in an animal model of medullary thyroid carcinoma.

Animals↗
PubMed2000

Protective mechanisms against the intestinal nematode Strongyloides venezuelensis in Schistosoma japonicum-infected mice.

Mice infected with Schistosoma japonicum were resistant to the intestinal nematode, Strongyloides venezuelensis. The numbers of adult S. venezuelensis recovered from mice were significantly decreased when infections were given from 6 weeks after S. japonicum infection. Larval recovery from the lungs showed that significant numbers of subcutaneously inoculated S. venezuelensis larvae were eliminated by 3 days in S. japonicum-infected mice (P < 0.0001), while histology revealed that this was associated with massive eosinophilic infiltration in the lungs. In addition, adult S. venezuelensis worms implanted in the duodenum of S. japonicum-infected mice could not establish in the intestine. This failure was associated with mucosal mastocytosis. Activation of eosinophils and intestinal mast cells was correlated with elevated expression of mRNA for interleukin (IL)-3, IL-4, and IL-5 in S. japonicum-infected mice. Sera from S. japonicum-infected mice recognized S. venezuelensis larva antigens as strongly as those from S. venezuelensis-infected mice, although transfer of sera from S. japonicum-infected mice to normal recipient mice did not protect them from S. venezuelensis challenge infection. It was concluded that the mechanisms for larval killing and adult worm expulsion of S. venezuelensis in S. japonicum-infected mice were identical to those seen in infections with S. venezuelensis only.

Animals↗
PubMed2000

A graphic approach to evaluate algorithms of secondary structure prediction.

Algorithms of secondary structure prediction have undergone the developments of nearly 30 years. However, the problem of how to appropriately evaluate and compare algorithms has not yet completely solved. A graphic method to evaluate algorithms of secondary structure prediction has been proposed here. Traditionally, the performance of an algorithm is evaluated by a number, i.e., accuracy of various definitions. Instead of a number, we use a graph to completely evaluate an algorithm, in which the mapping points are distributed in a three-dimensional space. Each point represents the predictive result of the secondary structure of a protein. Because the distribution of mapping points in the 3D space generally contains more information than a number or a set of numbers, it is expected that algorithms may be evaluated and compared by the proposed graphic method more objectively. Based on the point distribution, six evaluation parameters are proposed, which describe the overall performance of the algorithm evaluated. Furthermore, the graphic method is simple and intuitive. As an example of application, two advanced algorithms, i.e., the PHD and NNpredict methods, are evaluated and compared. It is shown that there is still much room for further improvement for both algorithms. It is pointed out that the accuracy for predicting either the alpha-helix or beta-strand in proteins with higher alpha-helix or beta-strand content, respectively, should be greatly improved for both algorithms.

Algorithms↗
PubMed2000

Predicting the standard enthalpy (deltaH0f) and entropy (S0) of alkanes by artificial neural networks.

Artificial Neural Networks (ANNs) with Extended Delta-Bar-Delta (EDBD) back propagation learning algorithm have been developed to predict the standard enthalpy and entropy of 87 acyclic alkanes. Molecular weight, boiling point and density of the compounds were used as input parameters. The network's architecture and parameters were optimized to give maximum performances. The best network was a 3-6-2 ANN, and the optimum learning epoch was about 1320. The results show that the maximum relative errors of enthalpy and entropy are less than 3%. They reveal that the performances of ANNs for predicting the enthalpy and entropy of alkanes are satisfying.

Algorithms↗
PubMed2000

Studies of the drug permeability and mechanical properties of free films prepared by cellulose acetate pseudolatex coating system.

Free films produced with cellulose acetate (CA) pseudolatex were prepared by the casting method. The effects of plasticizer concentration, drying temperature, and drying time on drug permeability and mechanical properties of free films were investigated by three-factor spherical second-order composite experimental design. The results were analyzed by the multivariable regression method. The experimental results indicated that plasticizer concentration, drying temperature, and drying time had complex effects on free film permeability and mechanical behavior. These results probably arise from the film-forming ability of CA pseudolatex particles at various conditions and the evaporation of plasticizer during the film-forming process.

Algorithms↗