Cytotoxicity and Glycan-Binding Profile of a <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (<Emphasis Type="Italic">Aplysia kurodai</Emphasis>)

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k _ass and k _diss values are 2.4 × 10³ M^?1 s^?1 and 3.8 × 10^?3 s^?1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.     

Dimethylfumarate induces cell cycle arrest and apoptosis via regulating intracellular redox systems in HeLa cells

Dimethylfumarate (DMF) is cytotoxic to several kinds of cells and serves as an anti-tumor drug. This study was designed to investigate the effects and underlying mechanism of DMF on cervical cancer cells. HeLa cells were cultured and treated with 0, 50, 100, 150, and 200 μM DMF, respectively. After 24 h, cell growth was evaluated using Cell Counting Kit-8 (CCK-8) assay and the cell cycle was examined using flow cytometry. In addition, cell apoptosis was detected by Annexin V/propidium iodide (PI) staining and the expressions of caspase-3 and poly-ADP-ribose polymerase (PARP) were detected using western blotting. The redox-related factors were then assessed. Furthermore, all of the indicators were detected in HeLa cells after combined treatment of DMF and N-acetyl-l-cysteine (NAC, an oxygen-free radical scavenger). The cell number and cell growth of HeLa were obviously inhibited by DMF in a dose-dependent manner, as the cell cycle was arrested at G0/G1 phase (P?<?0.05). The apoptotic HeLa cells were markedly increased, and the expression levels of caspase-3 and PARP were significantly increased in a DMF concentration-dependent way (P?<?0.05). Meanwhile, loss of △Ψm, increase in reactive oxygen species and O₂ ^·?, and the decrease in catalase activity and glutathione (GSH) level were found after DMF treatment (P?<?0.05). All these changes were significantly attenuated and even completely disappeared by adding NAC (P?<?0.05). In conclusion, the cytotoxicity of DMF on cell proliferation and apoptosis of HeLa cells was mainly related to the intracellular redox systems by depletion of intracellular GSH.     

Obtaining and study of monosaccharide derivatives of low-molecular-weight chitosan

The possibility of obtaining monosaccharide derivatives of low-molecular-weight chitosan with the use of the Maillard reaction was studied. Chitosan derivatives (molecular weight, 24 and 5 kDa) obtained with glucosamine, N-acetyl galactosamine, galactose, and mannose with a substitution degree of 4–14% and a yield of 60–80% were obtained. Some physicochemical and biological properties of these derivatives were studied. We showed that monosaccharide derivatives of low-molecular-weight chitosan exhibited antibacterial activity. Chitosan at a concentration of 0.01% caused 100% death of bacteria B. subtilis and E. coli. The strongest antibacterial effect was exhibited by 24-kDa derivatives: only 0.02–0.08% of cells survived. These derivatives were two orders of magnitude more effective than the 5-kDa chitosan modified with galactose.     

Synthesis of a tetra- and a trisaccharide related to an anti-tumor saponin “Julibroside J<Subscript>28</Subscript>” from <Emphasis Type="Italic">Albizia julibrissin</Emphasis>

Simple and convergent synthesis of a tetra- and a trisaccharide portions of an antitumor compound Julibroside J₂₈, isolated from Albizia julibrissin, that showed significant in vitro antitumor activity against HeLa, Bel-7402 and PC-3M-1E8 cancer cell lines is reported. The tetrasaccharide has been synthesized as its p-methoxyphenyl glycoside starting from commercially available d-glucose, l-rhamnose and l-arabinose. The trisaccharide part has been synthesized from commercially available N-acetyl d-glucosamine, d-fucose and d-xylose using simple protecting group manipulations. Sulfuric acid immobilized on silica has been used successfully as a Brönsted acid catalyst for the crucial glycosylation steps.     

Phosphorylation regulates interaction of 210-kDa myosin light chain kinase <Emphasis Type="Italic">N</Emphasis>-terminal domain with actin cytoskeleton

High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303–314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Cationic Antimicrobial Peptides Derived from <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> Leukocyte Extract,Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC₅₀ values of KT2 and RT2 for HeLa and CaSki cells showed 28.7–53.4 and 17.3–30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.     

An acidic,thermostable exochitinase with β-<Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase activity from <Emphasis Type="Italic">Paenibacillus barengoltzii</Emphasis> converting chitin to <Emphasis Type="Italic">N</Emphasis>-acetyl glucosamine

Background

N-acetyl-β-D-glucosamine (GlcNAc) is widely used as a valuable pharmacological agent and a functional food additive. The traditional chemical process for GlcNAc production has some problems such as high production cost, low yield, and acidic pollution. Hence, to identify a novel chitinase that is suitable for bioconversion of chitin to GlcNAc is of great value.

Results

A novel chitinase gene (PbChi74) from Paenibacillus barengoltzii was cloned and heterologously expressed in Escherichia coli as an intracellular soluble protein. The gene has an open reading frame (ORF) of 2,163 bp encoding 720 amino acids. The recombinant chitinase (PbChi74) was purified to apparent homogeneity with a purification fold of 2.2 and a recovery yield of 57.9%. The molecular mass of the purified enzyme was estimated to be 74.6 kDa and 74.3 kDa by SDS-PAGE and gel filtration, respectively. PbChi74 displayed an acidic pH optimum of 4.5 and a temperature optimum of 65°C. The enzyme showed high activity toward colloidal chitin, glycol chitin, N-acetyl chitooligosaccharides, and p-nitrophenyl N-acetyl β-glucosaminide. PbChi74 hydrolyzed colloidal chitin to yield N- acetyl chitobiose [(GlcNAc)₂] at the initial stage, which was further converted to its monomer N-acetyl glucosamine (GlcNAc), suggesting that it is an exochitinase with β-N-acetylglucosaminidase activity. The purified PbChi74 coupled with RmNAG (β-N-acetylglucosaminidase from Rhizomucor miehei) was used to convert colloidal chitin to GlcNAc, and GlcNAc was the sole end product at a concentration of 27.8 mg mL^-1 with a conversion yield of 92.6%. These results suggest that PbChi74 may have great potential in chitin conversion.

Conclusions

The excellent thermostability and hydrolytic properties may give the exochitinase great potential in GlcNAc production from chitin. This is the first report on an exochitinase with N-acetyl-β-D-glucosaminidase activity from Paenibacillus species.

     

1,6-anhydro-<Emphasis Type="Italic">N</Emphasis>-acetyl-β-<Emphasis Type="Italic">D</Emphasis>-glucosamine in oligosaccharide synthesis: II. The synthesis of the spacered Le<Superscript>y</Superscript> tetrasaccharide

3-Aminopropyl glycoside of 3,2′-di-O-α-L-fucosyl-N-acetyllactosamine (Le^y tetrasaccharide) was synthesized. The glycosyl donor, 2-O-acetyl-2,4,6-tri-O-benzoyl-α-D-galactopyranosyl bromide, was coupled with glycosyl acceptor, 1,6-anhydro-2-acetamido-2-deoxy-β-D-glucopyranose or its 3-O-acetyl derivative, to give the corresponding N-acetyllactosamine derivatives in 20 and 71% yields, respectively. The glycosyl donor was synthesized from 1,2-di-O-acetyl-3,4,6-triO-benzoyl-D-galactopyranose, which was obtained by the treatment of benzobromogalactose with sodium borohydride to yield 1,2-O-benzylidene derivative and subsequent removal of benzylidene group and acetylation. Acidic methanolysis of the disaccharide derivatives resulted in the selective removal of one or both acetyl groups to give the disaccharide acceptor bearing hydroxy groups at C3 of the glucosamine residue and C2 of the galactose residue. The introduction of fucose residues in these positions by the treatment with tetrabenzylfucopyranosyl bromide resulted in a tetrasaccharide derivative, which was converted into 3,2′-di-O-α-L-fucopuranosyl-1,6-anhydro-N-acetyllactosamine peracetate after substitution of acetyl groups for benzoyl and benzyl groups. Opening of the anhydro ring by acetolysis resulted in peracetate, which was then converted into the corresponding oxazoline derivative by two steps. Glycosydation of the oxazoline derivative with 3-trifluoroacetamidopropan-1-ol and removal of O-acetyl and N-trifluoroacetyl protective groups resulted in a free spacered Le^y tetrasaccharide.     

Expression of gene for <Emphasis Type="Italic">N</Emphasis>-acyl-homoserine lactonase <Emphasis Type="Italic">AiiA</Emphasis> affects properties of rhizospheric strain <Emphasis Type="Italic">Pseudomonas chlororaphis</Emphasis> 449

The introduction into strain Pseudomonas chlororaphis 449 of plasmid pME6863 that contains the cloned gene for N-acyl-homoserine lactonase, AiiA, leads to the degradation of all three types of N-acylhomoserine lactones produced by this strain (N-butanoyl-homoserine lactone, N-hexanoyl-homoserine lactone, and N-3-oxo-hexanoyl-homoserine lactone). This causes a drastic reduction in the synthesis of phenazine pigment and decreases the ability of cells to migrate on the surface of nutrient medium. However, the antagonistic activity of P. chlororaphis 449 toward phytopathogenic fungi Sclerotinia sclerotiorum and Rhizoctonia solani is not only decreased, but is even slightly increased; no essential changes in the exoprotease activity were observed. It is assumed that one of the QS systems of P. chlororaphis 449 may exert the repression effect on the expression of genes, which determine the two latter cell activities.     

Site-specific and linkage analyses of fucosylated <Emphasis Type="Italic">N</Emphasis>-glycans on haptoglobin in sera of patients with various types of cancer: possible implication for the differential diagnosis of cancer

Fucosylation is an important type of glycosylation involved in cancer, and fucosylated proteins could be employed as cancer biomarkers. Previously, we reported that fucosylated N-glycans on haptoglobin in the sera of patients with pancreatic cancer were increased by lectin-ELISA and mass spectrometry analyses. However, an increase in fucosylated haptoglobin has been reported in various types of cancer. To ascertain if characteristic fucosylation is observed in each cancer type, we undertook site-specific analyses of N-glycans on haptoglobin in the sera of patients with five types of operable gastroenterological cancer (esophageal, gastric, colon, gallbladder, pancreatic), a non-gastroenterological cancer (prostate cancer) and normal controls using ODS column LC-ESI MS. Haptoglobin has four potential glycosylation sites (Asn184, Asn207, Asn211, Asn241). In all cancer samples, monofucosylated N-glycans were significantly increased at all glycosylation sites. Moreover, difucosylated N-glycans were detected at Asn 184, Asn207 and Asn241 only in cancer samples. Remarkable differences in N-glycan structure among cancer types were not observed. We next analyzed N-glycan alditols released from haptoglobin using graphitized carbon column LC-ESI MS to identify the linkage of fucosylation. Lewis-type and core-type fucosylated N-glycans were increased in gastroenterological cancer samples, but only core-type fucosylated N-glycan was relatively increased in prostate cancer samples. In metastatic prostate cancer, Lewis-type fucosylated N-glycan was also increased. These data suggest that the original tissue/cell producing fucosylated haptoglobin is different in each cancer type and linkage of fucosylation might be a clue of primary lesion, thereby enabling a differential diagnosis between gastroenterological cancers and non-gastroenterological cancers.     

Structural and quantitative evidence of α2–6-sialylated <Emphasis Type="Italic">N</Emphasis>-glycans as markers of the differentiation potential of human mesenchymal stem cells

Human somatic stem cells such as mesenchymal stem cells (hMSCs) have the capacity to differentiate into mesenchymal tissue lineages and to alter immune regulatory functions. As such, they hold promise for use in stem cell-based therapies. However, no method is currently available to evaluate the actual differentiation capacity of hMSCs prior to cell transplantation. Previously, we performed a comprehensive glycan profiling of adipose-derived hMSCs using high-density lectin microarray and demonstrated that α2–6-sialylation is a marker of the differentiation potential of these cells. Nevertheless, no information was available about the structural details of these of α2–6-sialylated glycans. Here we used high performance liquid chromatography (HPLC) analysis combined with mass spectrometry (MS) to perform a structural and quantitative glycome analysis targeting both N- and O-glycans derived from early (with differentiation ability) and late (without differentiation ability) passages of adipose tissue-derived hMSCs. Findings in these cells were compared with those from human induced pluripotent stem cells (hiPSCs), human dermal fibroblasts (hFibs) and cartilage tissue-derived chondrocytes. A higher percentage of α2–6-sialylated N-glycans was detected in early passage cells (24–28 % of sialylated N-glycans) compared with late passage cells (13–15 %). A major α2–6-sialylated N-glycan structure detected in adipose-derived hMSCs was that of mono-sialylated biantennary N-glycan. Similar results were obtained for the cartilage tissue-derived chondrocytes, Yub621c (28 % for passage 7 and 5 % for passage 28). In contrast, no significant differences were observed between early and late passage hMSCs with respect to α2–6-sialylated O-glycan percentages. These results demonstrate that levels of α2–6-sialylated N-glycans, but not O-glycans, could be used as markers of the differential potential of hMSCs.     

<Emphasis Type="Italic">Camellia sinensis</Emphasis> increased apoptosis on U2OS osteosarcoma cells and wound healing potential on NIH3T3 fibroblast cells

Camellia sinensis (Cs) is a plant which is rich in polyphenols and has antioxidant, antiinflammatory, antimutagenic, anticarcinogenic and antibacterial activities. In this study, two different methanol extracts (Cs-I and Cs-II) were prepared from the leaf of C. sinensis in order to investigate the wound healing and anticancer activities. Total phenolic content and antioxidant activity of the extracts were determined. Wound healing effects of Cs extracts were evaluated by using Masson’s Trichrome Tecnique on NIH3T3 fibroblast cells. Cytotoxic and apoptotic effects of the extracts were determined by MTT and AnnexinV-PI assays on U2OS osteosarcoma cells. Total phenolic contents and antioxidant activities of the extracts were almost the same. The highest concentration (60 µg/mL) of the extracts showed significant cytotoxic and apoptotic effects on U2OS cells. Especially, the highest apoptotic effect was determined with 60 µg/mL Cs-I extract. Significant wound healing potential on NIH3T3 fibroblast cells were determined especially with low extract concentrations (0.5, 1 and 5 µg/mL), while high extract concentrations showed significant anticancer effects. As a result, two Cs leaf extracts exhibited important apoptotic properties and both have wound healing potential. However, the Cs-I extract was found more effective on apoptotic osteosarcoma cell death and has an increased wound healing potential than the Cs-II extract.     

Monosaccharide profiling of silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis> L.) nervous system during development and aging

Glycoconjugates have various functions in differentiation, development, aging and in all aspects of normal functioning of organisms. The reason for increased research on this topic is that glycoconjugates locate mostly on the cell surface and play crucial biological roles in the nervous system including brain development, synaptic plasticity, learning, and memory. Considering their roles in the nervous system, information about their existence in the insect nervous system is rather sparse. Therefore, in order to detect monosaccharide content of N- and O-glycans, we carried out capLC–ESI–MS/MS analysis to determine the concentration changes of glucose, mannose, galactose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose, xylose, arabinose, and ribose monosaccharides in the nervous system of Bombyx mori during development and aging processes. In addition to LC–MS, lectin blotting was done to detect quantitative changes in N- and O-glycans. Developmental stages were selected as 3rd (the youngest sample), 5th (young) larval instar, motionless prepupa (the oldest sample), and pupa (adult development). Derivatization of monosaccharides was performed with a solution of PMP agent and analyzed with capLC–ESI–MS/MS. For lectin blotting, determination of glycan types was carried out with Galanthus nivalis agglutinin and Peanut agglutinin lectins. In all stages, the most abundant monosaccharide was glucose. Although all monosaccharides were present most abundantly in the youngest stage (3rd instar), they are generally reduced gradually during the aging process. It was observed that amounts of monosaccharides increased again in the pupa stage. According to lectin blotting, N- and O-linked glycoproteins expressions were different and there were some specific glycoprotein expression differences between stages. These findings suggest that the glycosylation state of proteins in the nervous system changes during development and aging in insects in a similar fashion to that reported for vertebrates.     

In vitro virulence characteristics of rare serovars of <Emphasis Type="Italic">Salmonella enterica</Emphasis> isolated from sand lizards (<Emphasis Type="Italic">Lacerta agilis</Emphasis> L.)

The aim of this study was to estimate virulence potential of Salmonella enterica strains colonizing the gut of free-living sand lizards (Lacerta agilis L.). The strains belonged to three Salmonella serovars: Abony, Schleissheim, and Telhashomer. Adhesion and invasion abilities of the strains were determined in quantitative assays using the gentamicin protection method. Induction of apoptosis was assessed using HeLa cell monolayers. PCR assays were used for detection of 26 virulence genes localised within mobile elements: pathogenicity islands, virulence plasmids, and prophage sequences. In vitro studies revealed that all strains had adhesion and invasion abilities to human epithelial cells. The isolates were cytotoxic and induced apoptosis of the cells. The serovars differed in the number of virulence-associated genes: up to 18 genes were present in Salmonella Schleissheim, 17 in Salmonella Abony, whereas as few as six genes were found in Salmonella Telhashomer. Generally, Salmonella Abony and Salmonella Schleissheim did not differ much in gene content connected with the presence SPI-1 to -5. All of the strains lacked genes localised within bacteriophages and plasmids. The presence of virulence-associated genes and in vitro pathogenicity assays suggest that Salmonella sp. strains originating from autochthonous, free-living lizards can potentially infect and cause disease in humans.     

Alteration of a recombinant protein <Emphasis Type="Italic">N</Emphasis>-glycan structure in silkworms by partial suppression of <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase gene expression

Objective

To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae.

Results

Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man₃GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins.

Conclusions

Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

     

Characterization of a <Emphasis Type="Italic">Pinus sylvestris</Emphasis> thaumatin-like protein gene and determination of antimicrobial activity of the in vitro expressed protein

Thaumatin-like proteins (TLPs) are pathogenesis-related proteins, which are involved in plant defense responses to pathogen infection. Expression of the Pinus sylvestris L. TLP gene is up-regulated by methyl jasmonate treatment and inoculation with Heterobasidion annosum. A full-length Pinus taeda TLP gene sequence was used to design PCR primers for amplification of the full-length TLP gene from P. sylvestris. A putative 705-bp open reading frame of TLP gene was cloned into Escherichia coli cells, and then subcloned into the overexpression vector pET100 using BL21 Star expression bacteria. Optimization of the expression of recombinant TLP was achieved by decreasing both expression temperature and IPTG concentration. The purified 24.6-kDa TLP shows antimicrobial activity against 12 fungal species.     

Molecular analysis and bioactivity of luteinizing hormone from Japanese eel, <Emphasis Type="Italic">Anguilla japonica</Emphasis>, produced in silkworm pupae

Luteinizing hormone (LH), a gonadotropin hormone (GTH) of the pituitary glycoprotein family, is important in oocyte maturation, ovulation, and spermiation. In this study, we generated a Japanese eel LH (JeLH) with and without the equine chorionic gonadotropin (eCG) carboxyl-terminal peptides under the control of the polyhedron in the silkworm pupae BES to determine their expression levels, glycosylation profile, and in vitro bioactivity. The target proteins were highly expressed in the pupae hemolymph. Recombinant JeLH·eCG and JeLH were N- or O-glycosylated, as shown by periodic-acid Schiff staining, deglycosidase enzyme treatment, and lectin blot analyses, and showed no significant difference in the in vitro bioactivity. Both single-chain hormones and salmon pituitary extract induced maturation of Japanese eel oocytes in vitro. Recombinant LHs produced in silkworm pupae might be suitable candidates for in vivo experiments, because they can be produced in sufficient amount and can undergo N-glycosylation.     

Structure and antitumor activity of the extracellular polysaccharides from <Emphasis Type="Italic">Aspergillus aculeatus</Emphasis> via apoptosis and cell cycle arrest

Two extracellular polysaccharides, designated as WPA and WPB, were isolated from the fungus Aspergillus aculeatus using Q-Sepharose fast flow and Sephacryl S-300 column chromatography. WPA composed of mannose and galactose in a molar ratio of 3.9:1.0, and WPB mainly contained mannose. The molecular weight of WPA and WPB was about 28.1 kDa and 21.0 kDa, respectively. On the basis of methylation and NMR analysis, the possible main chain of WPA was [→5)-β-D-Galf-(1 → 2,6)-α-D-Manp(1→], and WPB was mainly [→2,6)-α-D-Manp(1→], both with [α-D-Manp(1 → 2)-α-D-Manp(1 → 2)-α-D-Manp(1→] substituted at C-2 of [→2,6)-α-D-Manp(1→]. Meanwhile, WPA displayed a stronger anti-proliferative effect than WPB on HeLa, MCF-7 and MGC-803 cells in vitro. WPA and WPB could arrest HeLa cells in G2/M phase and induce HeLa cells apoptosis. Thus, our study provides evidence that WPA and WPB may be taken as potential candidates for treating cervical carcinoma.