Characterization and identification of Lactobacillus fermentum 4,6-α-glucosyltransferase and its products / 生物工程学报

4,6-α-glucosyltransferases (4,6-α-GTs), which converts amylose into α(1-6) bonds-containing α-glucan, possesses great application potential in enzymatic synthesis of dietary fiber. Primers were designed according to the conserved motifs existing in the amino acid sequence of 4,6-α-GTs, and used to amplify a putative GTFB-Like 4,6-α-GTs gene (named as gtf16) from the genomic DNA of Lactobacillus. The gtf16 gene was cloned into the plasmid pET15b, expressed in Escherichia coli BL21(DE3), followed by purification and characterization. The optimum pH and the optimum temperature of the purified enzyme were 5.0 and 40 °C, respectively. The biotransformation product of this enzyme was systematically characterized by thin-layer chromatography, NMR spectroscopy, and hydrolysis reaction. The Gtf16-catalyzed product shows a similar structure to that of the isomalto/malto-polysaccharide (IMMP), which is the amylose-derived product catalyzed by GtfB from Lactobacillus reuteri 121. Moreover, The Gtf16-catalyzed product contains up to 75% of α(1-6) bonds and has an average molecular weight of 23 793 Da. Furthermore, the content of the anti-digestive components was 88.22% upon hydrolysis with digestive enzymes.

unusual case of Peters Plus syndrome with sexual ambiguity and absence of mutations in the B3GALTL gene

Peters Plus syndrome [MIM 261540] is a rare autosomal recessive condition characterized by ocular defects [typically Peters anomaly] and other systemic major/minor abnormalities. Mutations in the B3GALTL gene encoding the beta -1,3-glucosyltransferase have been found in virtually all patients with typical Peters Plus syndrome. We report here a female patient with severe manifestations of Peters Plus syndrome including facial dysmorphism and bilateral corneal opacity associated with left renal pyelo-calicial dilatation and sexual ambiguity. Total sequencing of the B3GALTL gene revealed no mutation in the patient. To our knowledge, sexual ambiguity has not previously been reported in Peters Plus syndrome so far, and renal malformation is also apparently rare in the syndrome

Short-Cut Pathway to Synthesize Cellulose of Encysting Acanthamoeba

The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.

The roles of glycosphingolipids in the proliferation and neural differentiation of mouse embryonic stem cells

Glycosphingolipids including gangliosides play important regulatory roles in cell proliferation and differentiation. UDP-glucose:ceramide glucosyltransferase (Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs (shRNAs) to evaluate the roles of glycosphingolipids in proliferation and neural differentiation of mouse embryonic stem cells (mESCs). HPTLC/immunofluorescence analyses of shRNA-transfected mESCs revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM3 and GD3. Furthermore, MTT and Western blot/immunofluorescence analyses demonstrated that inhibition of the Ugcg expression in mESCs resulted in decrease of cell proliferation (P < 0.05) and decrease of activation of the ERK1/2 (P < 0.05), respectively. To further investigate the role of glycosphingolipids in neural differentiation, the embryoid bodies formed from Ugcg-shRNA transfected mESCs were differentiated into neural cells by treatment with retinoic acid. We found that inhibition of Ugcg expression did not affect embryoid body (EB) differentiation, as judged by morphological comparison and expression of early neural precursor cell marker, nestin, in differentiated EBs. However, RT-PCR/immunofluorescence analyses showed that expression of microtubule- associated protein 2 (MAP-2) for neurons and glial fibrillary acidic protein (GFAP) for glial cells was decreased in neural cells differentiated from the shRNA-transfected mESCs. These results suggest that glycosphingolipids are involved in the proliferation of mESCs through ERK1/2 activation, and that glycosphingolipids play roles in differentiation of neural precursor cells derived from mESCs.

Comparative analyses of the structure of the 1, 3-beta-glucan synthase gene in Paracoccidioides brasiliensis isolates

The evolutionary origin and significance of spliceosomal introns have been the subject of many investigations. Two theories, [quot ]introns-early[quot ] theory and [quot ]introns-late[quot ] theory, have been proposed to explain the evolution of introns in eukaryotic genes. Intron position is generally conserved in paralogue and orthologue genes. Some introns occur at similar but not necessarily identical positions in homologous genes, which were separated by great evolutionary distances. This event can be explained by insertion, loss or movement of the intron over short distances. Intron loss and gain events are unique in evolution and can be useful as markers for phylogenetic analyses. The insertion of introns at an identical position suggests a common ancestor gene. Here we analyzed, using PCR and RT-PCR, the structure of the 1,3-beta-glucan synthase gene (FKS) in several clinical isolates of Paracoccidioides brasiliensis (Pb): isolates Pb 01, Pb 4940, Pb 8515, Pb 8311, Pb 8334, Pb 4268, Pb 1668, and Pb E. Our results showed that seven of the isolates examined showed identical structures concerning the position of introns in PbFKS1. PbFKS4940 showed the intron described at the 3' end and had lost that one at the 5' end. The presence of the PbFKS4940 transcript suggests that it could be a functional gene. These data suggest a divergent evolution for introns with regard to the 1,3-beta-glucan synthase gene in P. brasiliensis isolates.

Epigallocatechin-3-gallate Suppresses Galactose-alpha1,4-galactose-beta1,4-glucose Ceramide Expression in TNF-alpha Stimulated Human Intestinal Epithelial Cells Through Inhibition of MAPKs and NF-kappa B

Intestinal epithelial cells (IECs) have been known to produce galactose-alpha1,4-galactose-beta1,4-glucose ceramide (Gb3) that play an important role in the mucosal immune response. The regulation of Gb3 is important to prevent tissue damage causing shiga like toxin. Epigallocatechin-3-gallate (EGCG) has been studied as anti-carcinogenic, anti-oxidant, anti-angiogenic, and anti-viral activities, and anti-diabetic. However, little is known between the expressions of Gb3 on IECs. The aim of this study was to examine the inhibitory effect of EGCG, a major ingredient of green tea, on Gb3 production via mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-kappa B) in the TNF-alpha stimulated human colon epithelial cells, HT29. To investigate how Gb3 is regulated, ceramide glucosyltransferase (CGT), lactosylceramide synthase (GalT2), and Gb3 synthase (GalT6) were analyzed by RT-PCR in HT 29 cells exposed to TNF-alpha in the presence or absence of EGCG. EGCG dose-dependently manner, inhibits TNF-alpha induced Gb3 expression by blocking in both the MAPKs and NF-kappaB pathways in HT29 cells. TNF-alpha enhanced CGT, GalT2 and GalT6 mRNA levels and EGCG suppressed the level of these enzymes enhanced by TNF-alpha treatment.