Molecular cloning, expression and characterization of a novel geneβ-N-acetylglucosaminidase from Bombyxmori

Previously, we have reported that a gene encoding Bombyxmoriβ-N-acetylglucosaminidase 2 (BmGlcNAcase2) has been identified differentially expressed in the midgut of Bombyxmori strain NB resistant to nucleopolyhedrovirus (BmNPV), strain 306 susceptible to NPV and a near isogenic line BC9 with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). To get more knowledge about the relationship between β-N-acetylglucosaminidase and the resistance of NPV, in this study, the 1542 bp open reading frame of a putative bombyxmoriβ-N-acetylglucosaminidase 2 gene (BmGlcNAcase2) was amplified from a pool of bombyxmoricDNAs and inserted into the prokaryotic expression plasmid pET-30a(+).Western blotting analysis showed that BmGlcNAcase2 was expressed in hemolymph, ovary, testis, fat body, trachea, midgut and silk gland of fifth instar larvae respectively . Immunofluoresence analysis indicated that BmGlcNAcase2 was mainly located to the cytoplasm or some structure in cytoplasm.

Cloning of Endo-β-Glucanase Ⅲ and Expression in Eerevisiae Fermentum

Objective The aim was to construct bioengineering strains that could degrade the cellulosic solid waste. Method The cDNA of endo-β-glucanase III of Trichoderma vi ride AS313711 was cloned by RT-PCR method. After sequenced, this gene was constructed to expression vector pESP-2, and then the plasmid was transformed into competent cell of cerevisiae fermentum by electric shock, the transformant was then obtained. The enzyme activity of this transformant at the different temperatures and pH was measured by DNS method. Result The length of ORF of EG III was 1 257 bp, encoding 418 amino acids, while the deduced molecular weight was 44.1 × 103 kD. Conclusion The enzyme activity of EG III was the highest when it was at PH 4.9 and tempeture was of 60℃. Then the corresponding enzyme activity was about 100%.

Improved extracellular endo-1,4-β-mannosidase activity of recombinant Pichia pastoris by optimizing signal peptide

In order to improve the extracellular endo-1,4-β-mannosidase(MAN) activity of recombinant Pichia pastoris, optimization of signal peptides was investigated. At first, five potential signal peptides(W1, MF4 I, INU1 A, αpre, HFBI) were chosen to be analyzed by Signal P 4.0, among which W1 was designed. Then, the widely used signal peptide α-factor in expression vector p GAPZαA was replaced by those five signal peptides to reconstruct five new expression vectors. MAN activity was assayed after expression vectors were transformed into Pichia pastoris. The data show that the relative efficiencies of W1, MF4 I, INU1 A, αpre, and HFBI signal peptides are 23.5%, 203.5%, 0, 79.7%, and 120.3% compared with α-factor, respectively. The further gene copy number determination by the quantitative real-time PCR reveals that the MAN activities mediated by α-factor from 1 to 6 gene copy number levels are 12.95, 43.33, 126.63, 173.53, 103.23 and 88.63 U/m L, while those mediated by MF4 I are 79.22, 133.89, 260.14, 347.5, 206.15 and 181.89 U/m L, respectively. The maximum MAN activity reached 347.5 U/m L with 4 gene copies mediated by MF4 I. These results indicate that replacing the signal peptide α-factor with MF4 I and increasing MAN gene copies to a proper number can greatly improve the secretory expression of MAN.

Cloning and Expression of Extremely Heat-Resistant Endo-β-1,4-Glucanase Gene from Thermotoga maritima in Bacillus subtilis

Gene encoding endo-β-1,4-glucanase(TM1525)is derived from Thermotoga maritima(T.maritima),which has an open reading frame of 825 bp and encodes a 274 amino acid endo-β-1,4-glucanase.This enzyme has the same high temperature resistance as thermophilic bacteria,which is an ideal property for industrial applications.By molecular biological means,TM1525 was cloned into pHT43 vector and introduced into Bacillus subtilis(B.subtilis)WB800N by electroporation.The results showed that the WB800N expression system was successfully constructed,and extracellular expression of the recombinant gene was achieved.Cellulose hydrolyzed activity of the protein was exhibited.

Syntheses and Olfactory Characters of Some Endo-α-Substituted-Bicyclo[2. 2. 2]-Octenyl (or Octanyl) Methanols

Sixteen title compounds were synthesized, twelve of which are new ones. Their structures were determined by 1H NMR, IR and MS, the refractive indices or melting points were measured. Odors of all the title compounds were evaluated and the structure-odor relationship was briefly discussed.

Purification and Some Properties of Endo-1,4-β-Glucanases of Trichoderma harzianum UzCF-28

This work aimed at isolation, purification and study of biochemical features of cellulolytic enzymes synthesized by Trichoderma harzianum UzCF-28 strain. Strain UzCF-28 revealed a high cellulolytic activity during submerged cultivation in the liquid culture on modified Mandels nutrient medium, where wheat straw was used as a source of carbon. As a result of purification by precipitation with ammonium sulfate and further ion exchange chromatography, two isoforms of endo- 1,4-β-glucanase-EG II and EG III with molecular weight of 135 and 75 kDa respectively were revealed. The pH optimum for EG I and EG III was 4.5, while for EG II—4.7, irrespective of the applied substrates—either CMC or “Whatman filter” paper. Heating up to 40°C of EG III did not lead to its inactivation, and on the contrary, its activity increased by more than three times comparing to the initial activity of the enzyme, i.e. thermostability of EG III among tested enzymes significantly varied.

RNA interference to reveal roles of β-N-acetylglucosaminidase gene during molting process in Locusta migratoria

β-N-acetylglucosaminidases are crucial enzymes involved in chitin degrada- tion in insects. We identified a β-N-acetylglucosaminidase gene （LmNAG1） from Locusta migratoria. The full-length complementary DNA （cDNA） of LmNAG1 consists of 2 667 nucleotides, including an open reading frame （ORF） of 1 845 nucleotides encoding 614 amino acid residues, and 233- and 589-nucleotide non-coding regions at the 5＇- and 3＇- ends, respectively. Phylogenetic analysis grouped the cDNA-deduced LmNAG1 protein with the enzymatically characterized β-N-acetylglucosaminidases in group I. Analyses of stage- and tissue-dependent expression patterns of LmNAG1 were carried out by real- time quantitative polymerase chain reaction. Our results showed that LmNAG1 transcript level in the integument was significantly high in the last 2 days of the fourth and fifth instar nymphs. LmNAG1 was highly expressed in foregut and hindgut. RNA interference of LmNAG1 resulted in an effective silence of the gene and a significantly reduced total LmNAG enzyme activity at 48 and 72 h after the injection of LmNAG1 double-stranded RNA （dsRNA）. As compared with the control nymphs injected with GFP dsRNA, 50% of the dsLmNAGl-injected nymphs were not able to molt successfully and eventually died. Our results suggest that LmNAG1 plays an essential role in molting process ofL. migratoria.

Neuroprotective Effect of Escitalopram Oxalate in Rats with Chronic Hypoperfusion

Summary： The neuroproteetive effects of escitalopram oxalate in rats with chronic hypoperfusion and the possible mechanism were explored. Chronic hypoperfusion （2-VO） model was prepared and given escitalopram oxalate （experimental group） or PBS （control group） after 6 weeks. Eight weeks after the operation, Morris water maze test was carried out to evaluate the learning and memory ability of the rats. The cell proliferation, three-dimensional vascular distribution, cell morphological changes in ischemic area and the plasma vascular endothelial growth factor （VEGF） were detected to explore the possible mechanisms. （1） Morris water maze test showed that the escape latency in the experimental group was significantly shorter than in the control group, while the first quadrant swimming time in the experi- mental group was significantly longer than the control group （both P〈0.01）. （2） Cerebrovascular confo- cal detection results showed that the inside diameter of capillaries was significantly less in the experi- mental group than in the control group; the vascular density was significantly increased in the experi- mental group and the total area of capillaries was also significantly increased in the experimental group as compared with the control group. （3） There was statistically significant difference in BrdU-positive cells in the ischemic brain tissue between the experimental group and the control group （P=0.003〈0.01）. （4） VEGF concentrations in the plasma and the ischemic area were higher in the experimental group than in the control group （P〈0.05）. It was concluded that escitalopram oxalate could significantly im- prove the learning and memory ability of the rats with chronic cerebral ischemia probably by the VEGF-mediated angiogenesis.

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Gastric cancer:Where is the place for the surgeon,the oncologist and the endoscopist today?

Gastric cancer remains a major health issue and a leading cause of death worldwide.While the incidence is decreasing in western countries,there has been a shift to more proximal cancers of the diffuse type,which are usually more aggressive and associated with a worse prognosis.Radical surgery still offers the only chance of long term survival,but surgery has reached a plateau of effectiveness and more aggressive approaches like"ultraradical" lymphadenectomy have not improved prognosis.There are three options to improve the situation:Earlier detection,neoadjuvant chemotherapy and adjuvant therapy.Whilst systematic gastroscopic screening makes sense in countries with a high incidence of gastric cancer,in other regions targeted investigation of risk groups including first-degree relatives of cancer patients,patients with a chronic corpusdominant gastritis or with defined genetic abnormalities may help to detect cancer at an earlier stage.Neoadjuvant chemotherapy has meanwhile proved to significantly improve the prognosis not only in patients with a locally advanced cancer who cannot be resected for cure but but also in those who are potentially amenable to curative resection.In the largest randomised study so far reported,perioperative chemotherapy raised overall survival after 5 years from 23%to 36%.The role of adjuvant chemotherapy has been discussed for over 30 years.Meta-analyses demonstrate a small but significant effect which,however,seems to be restricted to Asian patients.In a large USstudy,adjuvant radiochemotherapy appeared to significantly improve outcomes.However,less than 50%of the study patients underwent a systematic lymphadenectomy and so the results of the therapy group were not better to those of"only resected"patients in two large European studies.Thus,the indication of adjuvant(radio-)chemotherapy in gastric cancer currently remains uncertain.Endoscopists have found a therapeutic role through endoscopic resection of early cancers,introduced mainly by Japanese authors.With the development of high resolution endoscopy,endosonography and adequate equipment,the endoscopic curative resection of T1a-tumors(restricted to the mucosal layer) has been established.

Update of cholangioscopy and biliary strictures

Cholangioscopy remains another modality in the investigation of biliary strictures. At cholangioscopy, the"tumour vessel" sign is considered a specific sign formalignancy. Through its ability to not only visualisemucosa, but to take targeted biopsies, it has a greater accuracy, sensitivity and specificity for malignant strictures than endoscopic retrograde cholangiopancreatography guided cytopathological acquisition. Cholangioscopy however, is time consuming and costly, requires greater technical expertise, and should be reserved for the investigation of undifferentiated strictures after standard investigations have failed.

The New Approach of Expansion Baldwin-Aibassov＇s Rules for Ring-Closing Reaction for d- and f-elements Periodic Table of Elements

The authors have studied the effect of a magnetic field on Baldwin＇s rules. The authors have proposed a new mechanism that takes into account the effect of the angle and energy endo- or exo-cyclization. The authors propose to extend the rule Bouldwin not only for sp^3-, sp^2- and sp- orbits, but and for d^1 - d^10 and f^1 - f^14 elements of I-VIII of the Periodic table.

Xyloglucans of Monocotyledons Have Diverse Structures

Except in the Poaceae, little is known about the structures of the xyloglucans in the primary walls of monocotyledons. Xyloglucan structures in a range of monocotyledon species were examined. Wall preparations were isolated, extracted with 6 M sodium hydroxide, and the extracts treated with a xyloglucan-specific endo-（1→4）-β-glucanase preparation. The oligosaccharides released were analyzed by high-performance anion-exchange chromatography and by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. Oligosaccharide profiles of the non-commelinid monocotyledons were similar to those of most eudicotyledons, indicating the xyloglucans were fucogalactoxyloglucans, with a XXXG a core motif and the fucosylated units XXFG and XLFG. An exception was Lemna minor （Araceae）, which yielded no fucosylated oligosaccharides and had both XXXG and XXGn core motifs. Except for the Arecales （palms） and the Dasypogonaceae, which had fucogalactoxyloglucans, the xyloglucans of the commelinid monocotyledons were structurally different. The Zingiberales and Commelinales had xyloglucans with both XXGn and XXXG core motifs; small proportions of XXFG units, but no XLFG units, were present. In the Poales, the Poaceae had xyloglucans with a XXGn core motif and no fucosylated units. In the other Poales families, some had both XXXG and XXGn core motifs, others had only XXXG; XXFG units were present, but XLFG units were not.