Cloning and Bioinformatics Analysis of Rosa rugosa β-1,3-Glucanase Gene (RrGlu)

In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong” with RT-PCR and RACE methods and named as RrGlu. The full-length cDNA is 1380 bp with an open reading frame of 1041 bp, encoding 346 amino acids. The derived protein has a molecular weight of 37.85 kD, a calculated pI of 9.12, a pfam00332 conserved domain at position 36 - 345, and belongs to glycosyl hydrolase family 17. The derived protein is a hydrophilic protein secreted into the vacuole. There is a signal peptide cleavage site at position 34 - 35, a transmembrane domain at position 13 - 32, six Ser phosphorylation sites, three Thr phosphorylation sites, three Tyr phosphorylation sites, one N-glycosylation site, and five O-glycosylation sites. There are 31.50% α-helixes, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner structure. This protein and the Glu protein from eight other species, including Prunus persica, share a sequence homology of greater than 72%;all of the proteins contain a pfam00332 conserved domain and a β-1,3-glucanase active center sequence (LIVM)-X-(LIVMFYW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of R. rugosa pollination incompatibility and improve the theory and techniques of breeding ornamental R. rugosa.

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.

Improving the Heat Resistance ofβ-1,4 Glucanase by Introducing Disulfide Bonds

Each possible pair of residues inβ-1,4 glucanase for disulfide formation was assessed using online websites,and four pairs L28C-S256C,Q41C-P278C,S122C-N163C and A184C-A215C were selected.Accordingly,four recombinant plasmids pET28a(+)EccslH28,pET28a(+)EccslH41,pET28a(+)EccslH122 and pET28a(+)EccslH184 were prepared and transformed into E.coli to express the recombinant enzymes.Then analysis on enzymatic properties showed that T50 of the recombinant enzymes was increased from 10 min for EccslHt2 to 90 min for EccslH28 and 40 min for EccslH41 at 70℃,while their optimum pH value and pH stability were not affected,which proved that the introduction of disulfide bond improved the thermal stability ofβ-1,4 glucanase.

Screening of NKX2-5,GATA4,ZIC3 gene mutations in sporadic congenital simple heart disease in Hainan Province

Objective:Congenital heart disease(CHD)is caused by abnormal cardiac development,which is the most common congenital malformation at home and abroad.NKX2-5,GATA4 and ZIC3 have been shown to be associated with CHD.This experiment explored the relationship between NKX2-5,GATA4 and ZIC3 gene mutations and sporadic CHD in Hainan Province.Methods:To collect 210 sporadic CHD patients in Hainan,the DNA of patients was extracted from blood,and the target gene fragments were amplified.Using high-resolution melting(HRM)and DNA sequencing technology,and we analyzed the sequences of NKX2-5,GATA4 and ZIC3 genes.Results:NKX2-5,GATA4 and ZIC3 genes were sequenced in 210 CHD patients,and seven gene mutations were found,including NKX2-5 heterozygous missense mutation(c.178G>T)and three heterozygous mutations in GATA4(c.677C>T,c.928A>G,c.1123G>A),three heterozygous mutations in ZIC3(c.19G>C,c.1255C>G,c.1348C>T),in which NKX2-5(c.178G>T),GATA4(c.1123G>A),and ZIC3(c.1255C>G,c.1348C>T)are new mutation sites.These gene mutations were predicted to be pathogenic mutations by bioinformatics software.Conclusion:Conclusion:Seven gene mutations were found in 210 patients,and it was the first report that the gene mutations of NKX2-5,GATA4 and ZIC3 in Hainan Province associated with the pathogenesis of CHD.

Anti-tumor effects induced by gene vaccines co-expressing truncated human prostate specific membrane antigen gene and mouse 4-1BBL

Objective To investigate the influence of m4-1BBL on anti-tumor effects induced by truncated human prostate specific membrane antigen ( tPSMA ) gene in mice. Methods A eukaryotic expression plasmid encoding tPSMA and m4-1BBL ( pDC316-tPSMA-IRES m4-1BBL) ,pDC316-tPSMA and pDC316 were constructed.

PtrCel9A6, an Endo-l,4-β-Glucanase, Is Required for Cell Wall Formation during Xylem Differentiation in Populus

ABSTRACT Endo-l,4-β-glucanases （EGases） are involved in many aspects of plant growth. Our previous study found that an EGase, PtrCel9A6, is specifically expressed in differentiating xylem cells during Populus secondary growth. In this study, the xylem-specific PtrCel9A6 was characterized for its role in xylem differentiation. The EGase is localized on the plasma membrane with catalytic domain toward the outside cell wall, hydrolyzing amorphous cellulose. Suppression of PtrCel9A6 expression caused secondary cell wall defects in xylem cells and significant cellulose reduction in Populus. Heterologous expression of PtrCelgA6 in Arabidopsis enhanced plant growth as well as increased fiber cell length. In addition, introduction of PtrCel9A6 into Arabidopsis resulted in male sterility due to defects in anther dehiscence. Together, these results demonstrate that PtrCel9A6 plays a critical role in remodeling the 1,4-β-glucan chains in the wall matrix and is required for cell wall thickening during Populus xylem differentiation.

Association of ALOX5AP and PDE4D with the risk of lacunar infarct in people from Jiangsu Province,China

The genes for 5-1ipoxygenase activating protein （ALOX5AP） and phosphodiesterase 4D （PDE4D） have been demonstrated as susceptibility genes for lacunar in the Icelandic and Pakistani populations, but little is known about the role of these genes in Chinese populations. The present study utilized polymerase chain reaction and ligase detection reaction to detect single nucleotide polymorphisms （SNPs） in 280 consecutive stroke patients and 258 unrelated population-based controls from Nanjing, Jiangsu Province, China. The allele frequency, genotypes, and haplotypes of the two SNPs （rs456009 and rs966221） in PDE4D were similar between the two groups. However, A allele frequency of rs4073259 （A/G） and rs4769055 （A/C） in the ALOX5AP gene exhibited differences in two groups, and especially the haplotype of the SNP was significantly different between the two groups. Results suggested that the ALOX5AP gene might be involved in lacunar infarct, while PDE4D gene was not a risk factor for lacunar infarct in individuals from Jiangsu Province, China.

Steroidal Plant Growth Promoters vs. Phytopathogens, via Enzymatic Regulation;An in <i>Silico</i>Approach

Steroidal plant growth promoters (SPGP) have been continuously studied due to their high activity increasing biomass and resistance to diverse stress factors. In our hands, a new SPGP family of 22-oxocholestanic compounds stands out at a comparative level to brassinosteroids (BSs). The potential activity of new SPGP against phytopathogens was studied through in silico molecular docking, these assays were performed with relevant ensymes of phytopatogens Chitinase B and 1,3-β-Glucanase. Nine Chitinase B inhibitors and two 1,3-β-Glucanase inhibitors were proposed. The launched study analyzed the interactional and spatial level, determining the presence of interactions with key amino acids in receptors in comparison to reference inhibitors. Even more, the AVR4 and ECP6 effectors were also examined. No compound that blocks ECP6 was found;due to, probably, the influence of its highly hydrophilic environment. In the case of AVR4, two SPGP showed a better docking score (DS) than a chitin fragment (endogenous ligand);this fact demonstrates the latent potential of the 22-oxocholestanic derivatives against phytopathogens, with a specific regulation via proliferation inhibition. Moreover, this SPGP does not affect the symbiotic fungi that are beneficial for the natural plant system.

Distinct regulatory mechanism of immunoglobulin gene transcription in epithelial cancer cells

The restriction of immunoglobulin(Ig)expression to B lymphocytes is well established.However,several reports have confirmed that the Ig gene can be expressed in many non-B cancer cells and/or some normal cells.Our aim is to determine whether the Ig gene promoter can be activated in non-B cancer cells and to identify the regulatory mechanism for Ig gene expression.Our results show that the Ig promoter of VH4-59 was activated in several non-B cancer cell lines.Moreover,two novel positive regulatory elements,an enhancer-like element at 2800 to 2610 bp and a copromoter-like element at 2610 to 2300 bp,were identified in two epithelial cancer cell lines,HeLa S3 and HT-29.The octamer element(59-ATGCAAAT-39)located in the Ig promoter,a crucial element for B-cell-derived Ig gene transcription,was also very important for non-B-cell-derived Ig gene transcription.More importantly,we confirmed that octamer-related protein-1(Oct-1),but not Oct-2,was a crucial transcriptional factor for Ig gene transcription due to its ability to bind to the octamer element of the Ig promoter in epithelial cancer cells.These results suggested the presence of a distinct regulatory mechanism for Ig gene expression in non-B cancer cells.

The RNA binding proteins TIA1 and TIAL1 promote Mcl1 mRNA translation to protect germinal center responses from apoptosis

Germinal centers(GCs)are essential for the establishment of long-lasting antibody responses.GC B cells rely on post-transcriptional RNA mechanisms to translate activation-associated transcriptional programs into functional changes in the cell proteome.However,the critical proteins driving these key mechanisms are still unknown.Here,we show that the RNA binding proteins TIA1 and TIAL1 are required for the generation of long-lasting GC responses.TIA1-and TIAL1-deficient GC B cells fail to undergo antigen-mediated positive selection,expansion and differentiation into B-cell clones producing high-affinity antibodies.Mechanistically,TIA1 and TIAL1 control the transcriptional identity of dark-and light-zone GC B cells and enable timely expression of the prosurvival molecule MCL1.Thus,we demonstrate here that TIA1 and TIAL1 are key players in the post-transcriptional program that selects high-affinity antigen-specific GC B cells.

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.