Enhancement of keratinolytic activity of a thermophilic subtilase by improving its autolysis resistance and thermostability under reducing conditions.

WF146 protease, a thermophilic subtilase from thermophile Bacillus sp. WF146, suffers excessive autolysis in the presence of reducing agents. In this report, two autolytic sites of WF146 protease were modified by site-directed mutagenesis.

Electrochemical determination of L-Tryptophan, L-Tyrosine and L-Cysteine using electrospun carbon nanofibers modified electrode.

A novel and simple method for the direct and quantitative determination of L-tryptophan (Trp), L-tyrosine (Tyr) and L-cysteine (Cys) was proposed in this work. Carbon nanofibers (CNFs), made by electrospinning technique, were used to modify carbon paste electrode (CPE) without any treatment to study the electrochemical behaviors of the three amino acids using cyclic voltammetry (CV) and constant potential amperometric method. The results demonstrated that the CNFs modified carbon paste electrode (CNF-CPE) exhibited high electrocatalytic activity and good analytical performance towards the oxidation of the three amino acids.

A threshold photoelectron-photoion coincidence spectrometer with double velocity imaging using synchrotron radiation.

A novel threshold photoelectron-photoion coincidence (TPEPICO) imaging spectrometer at the U14-A beamline of the Hefei National Synchrotron Radiation Laboratory is presented. A set of open electron and ion lenses are utilized to map velocity imaging of photoelectrons and photoions simultaneously, in which a repelling electric field using an extra lens is applied to magnify images of photoelectrons instead of traditional accelerating electric field in order to suppress the contribution of energetic electrons in the threshold photoelectron spectroscopy (TPES) and the mass-selected TPEPICO spectroscopy. The typical energy resolution of TPES is measured to be 9 meV (full width at half maximum), as shown on the (2)P(1/2) ionization of argon.

Effect of hirulog-like peptide on balloon catheter injury-induced neointimal formation in femoral arteries of minipigs and relationship with inflammatory mediators.

Vascular intervention-induced neointimal formation is a major drawback for managing atherosclerotic cardiovascular diseases using invasive vascular procedures. Our previous studies demonstrated that hirulog-like peptide (HLP) reduced balloon catheter dilation-induced neointimal formation or restenosis in carotid arteries of rats or atherosclerotic rabbits with less interruption in coagulation or bleeding than heparin or hirulog-1. The present study examined the effect of HLP on balloon catheter injury-induced neointimal formation in femoral arteries of minipigs.

Improvement of extracellular production of a thermophilic subtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide.

Limited secretion capacity remains a drawback of using Escherichia coli as the host for the production of recombinant proteins. In this report, random mutagenesis was performed within the N-terminal propeptide of thermostable WF146 protease, a subtilase from thermophilic Bacillus sp. WF146, generating a variant named WBM(MT) with improved capacity for extracellular production when expressed in E.

Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF.

A gene encoding a spore-associated subtilase, designated protease CDF, was cloned from Thermoactinomyces sp. CDF and expressed in Escherichia coli. The enzyme gene is translated as a proform consisting of a 94 aa propeptide and a 283 aa mature protease domain.

Improvement of low-temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site-directed mutagenesis.

By directed evolution and subsequent site-directed mutagenesis, cold-adapted variants of WF146 protease, a thermophilic subtilase, have been successfully engineered. A four-amino acid substitution variant RTN29 displayed a sixfold increase in caseinolytic activity in the temperature range of 15-25 degrees C, a down-shift of optimum temperature by approximately 15 degrees C, as well as a decrease in thermostability, indicating it follows the general principle of trade-off between activity and stability. Nevertheless, to some extent RTN29 remained its thermophilic nature, and no loss of activity was observed after heat-treatment at 60 degrees C for 2 h.

Characterization of the Haloarcula hispanica amyH gene promoter, an archaeal promoter that confers promoter activity in Escherichia coli.

Archaea form a third domain of life that is distinct from Bacteria and Eukarya. According to the current knowledge, the basal transcription machinery of Archaea (including the core promoter architecture, the RNA polymerase, and the basal transcription factors) closely resembles that of Eukarya in structure and function, while differing considerably from the bacterial paradigm. In the present study, the promoter region of the halophilic archaeon Haloarcula hispanica's amyH gene was isolated and characterized, and it was surprisingly revealed that the amyH gene promoter could confer promoter activity (i.

Biochemical and molecular characterization of plant MYB transcription factor family.

MYB genes are widely distributed in higher plants and comprise one of the largest transcription factors, which are characterized by the presence of a highly conserved MYB domain at their N-termini. Over recent decades, biochemical and molecular characterizations of MYB have been extensively studied and reported to be involved in many physiological and biochemical processes. This review describes current knowledge of their structure characteristic, classification, multi-functionality, mechanism of combinational control, evolution, and function redundancy.

[The roles of MYB transcription factors on plant defense responses and its molecular mechanism.].

Transcriptional regulation of defense gene expression is a crucial part of plant defense responses in plant defense environment stresses. As one of the largest plant transcription factor families, MYB (v-myb avian myeloblastosis viral on-cogene homolog) transcription factors play an important role in plant stress tolerance. In this paper, we review the structural features, functional characterization and molecular mechanism of MYB transcription factor family, and discuss the regula-tory roles of transcription factors in plant defense responses.

Cold-adapted maturation of thermophilic WF146 protease by mimicking the propeptide binding interactions of psychrophilic subtilisin S41.

Thermophilic WF146 protease matures efficiently at 60 degrees C, but quite slowly at low temperatures. In this report, seven amino acid residues involved in interactions between the mature domain and the propeptide of the enzyme were substituted by corresponding residues of psychrophilic subtilisin S41 to generate mutant Mut7 (S105G/G107D/Y117E/S136N/V143G/K144E/D145S). Mut3 (S105G/G107D/Y117E) and Mut4 (S136N/V143G/K144E/D145S) were also constructed.

Pharmacokinetics and pharmacology of hirulog-like peptide.

Hirulog-like peptide (HLP), a new thrombin peptide inhibitor, effectively reduces neointimal formation or restenosis in rat and rabbit vascular injury models. The present study investigated the pharmacokinetics and pharmacology of HLP in Sprague-Dawley (SD) rats. The input response of HLP in rats was studied using radioisotopic tracing method.

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease.

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+).

[Research progress of graft-versus-host disease after liver transplantation].

Graft-versus-host disease (GVHD) is an uncommon but fatal complication of orthotopic liver transplantation (OLT). The pathogenesis and mechanism of GVHD after OLT remains unclear and the treatment is therefore largely empirical. The prevention of GVHD including avoidance of closely matched human leukocyte antigen donors and old-age recipients is of particular importance because effective treatment is lacking.

An extracellular halophilic protease SptA from a halophilic archaeon Natrinema sp. J7: gene cloning, expression and characterization.

A gene encoding an extracellular protease, sptA, was cloned from the halophilic archaeon Natrinema sp. J7. It encoded a polypeptide of 565 amino acids containing a putative 49-amino acid signal peptide, a 103-amino acid propeptide, as well as a mature region and C-terminal extension, with a high proportion of acidic amino acid residues.

Therapeutic angiogenesis by intramuscular injection of fibrin particles into ischaemic hindlimbs.

1. Fibrin gel has been used as a carrier of angiogenic molecules to promote neovascularization in animal models of limb ischaemia. However, little is known about the effects of fibrin itself under such pathological conditions.

[Insulin secretion and insulin sensitivity in 1193 inpatients with essential hypertension].

Objective: To evaluate the metabolic profiles of insulin secretion and insulin sensitivity in different categories of nondiabetic patients with essential hypertension, we made a retrospecting study in our hospital.

Methods: According to the criteria of WHO and ADA, we calculated the distribution of plasma glucose levels during a 75 g oral glucose tolerance test (OGTT). Nondiabetic subjects with hypertension were classified into the following four groups: (1) normal glucose tolerance (NGT) with fasting plasma glucose (FPG) < 6.

Domain topology of the DNA polymerase D complex from a hyperthermophilic archaeon Pyrococcus horikoshii.

Family D DNA polymerase (PolD) is a recently found DNA polymerase extensively existing in Euryarchaeota of Archaea. Here, we report the domain function of PolD in oligomerization and interaction with other proteins, which were characterized with the yeast two-hybrid (Y2H) and surface plasmon resonance (SPR) assays. A proliferating cell nuclear antigen, PhoPCNA, interacted with the N-terminus of the small subunit, DP1(1-200).

[Graft versus host disease after liver transplantation: a report of 3 cases].

Objective: To document the clinical experience in the diagnosis and treatment of graft-versus-host disease(GVHD) after liver transplantation.

Methods: Clinical course was followed up and laboratory examinations were done in 3 patients with orthotopic liver transplantation (OLT) who developed acute GVHE. The diagnosis depended on clinical manifestations, skin biopsy, HLA typing and PCR short tandem repeat (PCR-STR).

A 21-amino acid peptide from the cysteine cluster II of the family D DNA polymerase from Pyrococcus horikoshii stimulates its nuclease activity which is Mre11-like and prefers manganese ion as the cofactor.

Family D DNA polymerase (PolD) is a new type of DNA polymerase possessing polymerization and 3'-5' exonuclease activities. Here we report the characterization of the nuclease activity of PolD from Pyrococcus horikoshii. By site-directed mutagenesis, we verified that the putative Mre11-like nuclease domain in the small subunit (DP1), predicted according to computer analysis and structure inference reported previously, is the catalytic domain.

Subunit interaction and regulation of activity through terminal domains of the family D DNA polymerase from Pyrococcus horikoshii.

Functions of the terminal domains of the family D DNA polymerase from Pyrococcus horikoshii (PolDPho) were analyzed by making and characterizing various truncated proteins. Based on a co-expression vector developed previously (Shen, Y., Musti, K.