Protease site-restricted selection of phage-displayed peptides on glutathione-sepharose.
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Bacteriophages↗
Biomedical subjects
T Cui
Publications and source records attributed to T Cui.
The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris.
The dengue virus envelope protein was expressed as a GST fusion protein using E. coli and P. pastoris as expression hosts. In E. coli the recombinant E protein is expressed initially as a soluble 81 kDa GST fusion protein. Treatment of the fusion protein with thrombin released a 55 kDa protein, which is the expected size for correctly processed, non-glycosylated recombinant E protein. The antiserum from animals immunised with this recombinant E protein was found to specifically recognise the dengue virus E protein in virus-infected cells, thus demonstrating the immunogenic nature of the recombinant E protein. This expression system allowed production of up to 2 mg of purified recombinant E protein from a 1 1 bacterial culture. In contrast, expression of this GST fusion protein in P. pastoris is associated with extensive proteolytic degradation of the recombinant E protein. However, this proteolytic degradation was not observed in the truncated E protein sequences which were expressed. One of these recombinant fusion proteins, GST E401 was secreted into the culture medium at levels of up to 100 microg/l of growth medium.
Animals↗
Activation of glycosylasparaginase. Formation of active N-terminal threonine by intramolecular autoproteolysis.
The activation mechanism of glycosylasparaginase of Flavobacterium meningosepticum has been analyzed by site-directed mutagenesis and activation of purified precursors in vitro. Mutation of Thr-152 to Ser or Cys leads to gene products that are not activated in vivo but are activated in vitro because processing of the mutant precursors is inhibited by certain amino acids in the cell. Kinetic studies reveal that activation is an intramolecular autoproteolytic process. The involvement of His-150 and Thr/Ser/Cys-152 in activation suggests that autoproteolysis resembles proteolysis by serine/cysteine proteases. Multiple functions of the highly conserved active threonine residue are implicated.
Amino Acid Sequence↗
Active site and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F.
Crystallographic analysis and site-directed mutagenesis have been used to identify the catalytic and oligosaccharide recognition residues of peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F (PNGase F), an amidohydrolase that removes intact asparagine-linked oligosaccharide chains from glycoproteins and glycopeptides. Mutagenesis has shown that three acidic residues, Asp-60, Glu-206, and Glu-118, that are located in a cleft at the interface between the two domains of the protein are essential for activity. The D60N mutant has no detectable activity, while E206Q and E118Q have less than 0.01 and 0.1% of the wild-type activity, respectively. Crystallographic analysis, at 2.0-A resolution, of the complex of the wild-type enzyme with the product, N,N'-diacetylchitobiose, shows that Asp-60 is in direct contact with the substrate at the cleavage site, while Glu-206 makes contact through a bridging water molecule. This indicates that Asp-60 is the primary catalytic residue, while Glu-206 probably is important for stabilization of reaction intermediates. Glu-118 forms a hydrogen bond with O6 of the second N-acetylglucosamine residue of the substrate and the low activity of the E118Q mutant results from its reduced ability to bind the oligosaccharide. This analysis also suggests that the mechanism of action of PNGase F differs from those of L-asparaginase and glycosylasparaginase, which involve a threonine residue as the nucleophile.
Amidohydrolases↗
Localization of binding site for encephalomyocarditis virus RNA polymerase in the 3'-noncoding region of the viral RNA.
We previously showed that encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) binds specifically to 3'-terminal segments of EMC virus RNA. This binding, which depends on both the 3'-noncoding region (3'-NCR) and 3'-poly (A) tail [together denoted 3'-NCR(A)], may be an important step in the initiation of virus replication. In this paper, the 3'-NCR and 3'-poly(A) were separately transcribed then mixed, but no complex with 3Dpol was obtained, showing that covalent attachment of the 3'-poly(A) to the 3'-NCR is essential for complex formation. Mutational and deletion analyses localized a critical determinant of 3Dpol binding to a U-rich sequence located 38-49 nucleotides upstream of the 3'-poly(A). Similar analyses led to the identification of a sequence of A residues between positions +10 and +15 of the 3'-poly(A) which are also critical for 3Dpol binding. As U-rich and A-rich regions are important for 3Dpol binding, a speculative model is proposed in which 3Dpol induces and stabilizes the base-pairing of the 3'-poly(A) with the adjacent U-rich sequence to form an unusual pseudoknot structure to which 3Dpol binds with high affinity.
Base Sequence↗
[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↗
Sex differences in the management and long-term outcome of acute myocardial infarction. A statewide study. MIDAS Study Group. Myocardial Infarction Data Acquisition System.
BACKGROUND: We wished to evaluate whether differences in the rate of invasive cardiac procedures between men and women with acute myocardial infarction are associated with different short- and long-term mortality. METHODS AND RESULTS: The database (Myocardial Infarction Data Acquisition System, MIDAS) included all discharges for the years 1986 and 1987 with the diagnosis of acute myocardial infarction in New Jersey, based on the New Jersey hospital discharge data system (MIDS/UB-82). Accuracy of the data was evaluated by auditing 726 randomly selected charts. The variables examined included age, sex, race, comorbidity (anemia, chronic liver disease, cancer, chronic obstructive pulmonary disease, diabetes, hypertension, prior myocardial infarction), complications (left ventricular dysfunction, arrhythmias, conduction defects), insurance status, performance of cardiac catheterization, percutaneous transluminal coronary angioplasty, or coronary artery bypass graft surgery, and survival up to 3 years. Women were older, had longer hospital stay, and were more likely than men to have anemia, diabetes, hypertension, left ventricular dysfunction, and Medicare or Medicaid insurance coverage. They were less likely than men to be admitted to a hospital equipped to perform invasive procedures or to have chronic obstructive pulmonary disease, chronic liver disease, prior myocardial infarction, or arrhythmias. After adjustment for these differences, women were less likely than men to have cardiac catheterization. Cardiac catheterization was associated with lower mortality. Women up to age 70 had higher 3-year death rates than men after adjustment for age, race, comorbidity, complications, and insurance type. This difference between men and women was somewhat diminished after the performance of cardiac catheterization and revascularization was taken into account. Unadjusted mortality was high in this study group. CONCLUSIONS: Women with acute myocardial infarction are less likely to have invasive cardiac procedures and have higher 3-year adjusted death rate up to age 70 than men.
Aged↗
The sequence of xylose isomerase gene from Streptomyces diastaticus No. 7 M1033.
The DNA sequence of the xylose isomerase gene from Streptomyces diastaticus No. 7 M1033 from Hainan Province has been determined. The structure gene of the enzyme is composed of 1161bp, corresponding to 387 amino acid residues. The G+C content of the gene is 72.1%. The probability of G or C on the third position of the codon is 98%. At the level of amino acids, this xylose isomerase displays high homology with those from other Actinomycete strains, particularly those from Streptomyces strains.
Aldose-Ketose Isomerases↗
Binding of encephalomyocarditis virus RNA polymerase to the 3'-noncoding region of the viral RNA is specific and requires the 3'-poly(A) tail.
An in vitro RNA bandshift assay has been developed to demonstrate the binding of purified recombinant encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase (3Dpol) to the 3'-noncoding region (NCR) and 30 nucleotides of the adjacent 3'-terminal poly(A) tail (3'-NCR(A)) of EMC virus RNA. The binding of 3Dpol to the 3'-NCR(A) fragment was specific since four other unrelated proteins including an RNA polymerase did not bind, and unlabeled 3'-NCR(A), but not alpha-globin mRNA, tRNA, or pure poly(A), competed with radiolabeled 3'-NCR(A) for binding. Surprisingly, 3Dpol failed to bind to the 3'-NCR of EMC virus RNA lacking the poly(A) tail. The results together show that EMC virus RNA template specificity depends only on 3Dpol, the 3'-NCR, and the poly(A) tail. This suggests that 3'-poly(A) is essential for viral RNA template selection by the EMC virus RNA polymerase.
Base Sequence↗
Arthrobacter D-xylose isomerase: protein-engineered subunit interfaces.
Mutants of Arthrobacter D-xylose isomerase were constructed in which one or two disulphide bridges or additional salt bridges were introduced at the A-A* subunit interfaces. These showed no change in enzyme activity or stability compared with the wild-type enzyme. However, a Tyr253 mutant in which a disulphide bridge was introduced at the A-B* subunit interface showed reduced thermostability that was identical in both oxidized and reduced forms, and also reduced stability in urea. X-ray-crystallographic analysis of the Mn(2+)-xylitol form of oxidized Y253C (the Tyr253-->Cys mutant) showed a changed conformation of Glu185 and also alternative conformations for Asp254, which is a ligand to the Site-[2] metal ion. With fructose, Mg(2+)-Y253C has a similar Km to that of the wild-type, and its Vmax. is also similar below pH 6.4, but declined thereafter. In the presence of Co2+, Y253C has lower activity than wild-type at all pH values, but its activity also declines at alkaline pH. These results suggest that electrostatic repulsion from the new position of Glu185 causes Asp254 to move when His219 is unprotonated, thereby preventing M2+ binding at Site [2]. These results also suggest that subunit dissociation does not lie on the pathway of thermal inactivation of D-xylose isomerase, but that movements of active-site groups are a trigger for conformational changes that initiate the unfolding process.
Aldose-Ketose Isomerases↗
The DNA-binding properties of an artificial 42-residue polypeptide derived from a natural repressor.
Bacteriophage 434 repressor recognizes the operator sequences ACAAG and ACAAT. As the same or similar sequences occur in the enhancer region of HIV-1, 434 repressor was a potential HIV enhancer-binding protein. We found that the interaction of the DNA-binding domain of 434 repressor with a 57-bp HIV enhancer DNA was very weak whereas a 42-residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly. The results of footprint and cell-free in vitro transcription studies showed that the 42-residue peptide bound preferably to the enhancer region of HIV-1 and acted as an artificial repressor. Replacement of an essential glutamine of the recognition helix by glutamic acid resulted in a partial shift of the sequence specificity of the 42-residue peptide.
Amino Acid Sequence↗
Determination of acrylamide metabolite, mercapturic acid by high performance liquid chromatography.
A HPLC Assay was developed to identify and measure the metabolite of acrylamide, mercapturic acid, N-Acetyl-s-(propionamide)-cysteine (APC) in urine. O-phthalaldehyde (OPA) was utilized as a precolumn derivatizing agent in the assay. This derivative was isolated with a good selectivity by high performance liquid chromatography (HPLC) employing reversed phase ODS columns. The quantitation of the mercapturic acid derivative was reproducible and with a detection limit of 1 pmol. The average coefficient of variation for the runs carried out on the same day was approximately 4.6% at the range of 80-160 mumol.L-1 of APC, and the mean analytical recovery from urine samples was 94%. The metabolite of urine of workers exposed to acrylamide was identified as N-acetyl-s-(propionamide)-cysteine by HPLC chromatography and fluorescence scan and HPLC-Mass spectra. All results were identical with the authentic synthesized compound.
Acetylcysteine↗
Restriction endonuclease from thermophilic bacterial species. IV. Isolation and characterization of BpuB5I.
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Bacillus↗
A nonlinear model of the arterial system incorporating a pressure-dependent compliance.
The three-element modified Windkessel model has been widely used to study the characteristics of the systemic arterial system. This model provides most of the features of the systemic input impedance, but does not describe the nonlinear effect of the pressure dependence of arterial compliance. The current investigation examines the hemodynamic consequences of such an inclusion. Simultaneous aortic pressure and flow during control and brief descending aortic occlusion were measured in open chest anesthetized experimental dogs. A numerical procedure was implemented to compute constant compliance linear and nonlinear compliance model-predicted pressure waveforms with flow as the input. Results show that the nonlinear compliance model in general can more accurately predict the measured pressure waveforms during control and during acute pressure loading. The difference between the predicted waveforms is more pronounced when blood pressure is high and when the pulse pressure is large.
Animals↗
Circular dichroism measurements show that C.C+ base pairs can coexist with A.T base pairs between antiparallel strands of an oligodeoxynucleotide double-helix.
We have studied the coil-to-helix transition of the DNA oligomer d(C4A4T4C4), using circular dichroism measurements to monitor the formation of A.T base pairs within the central self-complementary A4T4 region and the formation of protonated C.C+ base pairs at the ends of the oligomer. We found that both A.T and C.C+ base pairs formed in a coordinated fashion as the temperature and pH were lowered. The CD data of the helix form of the oligomer were consistent with the presence of paired oligomers, but not with hairpin loops. The pKa for formation of C.C+ base pairs between the C4 ends of the oligomer was higher than the pKa for formation of C.C+ base pairs in d(C8), indicating that the formation of C.C+ base pairs in the oligomer was influenced by the presence of a paired A4T4 region. We conclude that A.T and C.C+ base pairs coexist in the self-complex of the oligomer and, therefore, that C.C+ base pairs can form between antiparallel DNA strands.
Base Sequence↗
A novel mechanical cardiac assist device for reversing left ventricular failure.
Diastolic augmentation of aortic pressure is an efficacious means of improving coronary perfusion in heart failure. A novel mechanical cardiac assist device (MCAD), that has advantages over a conventional intraaortic balloon pump and left ventricular assist devices, has been developed. It consists of a high efficiency rotary solenoid, coupled to a pair of actuator plates that clamp on a shunt aortic graft section, and operates in a diastolic counterpulsation mode. The system has been evaluated in six anesthetized, thoracotomized dogs with myocardial ischemia. The MCAD was activated 30-40 min after coronary artery occlusion and synchronized with the R-wave. As illustrated by a representative sample of the data obtained from one of several trials, the preliminary experimental results demonstrated that the MCAD worked effectively to fulfill the primary functions of a counterpulsation assist device, i.e., augmentation of coronary perfusion and reduction in the vascular impedance to ventricular ejection.
Animals↗