A comparison of genes involved in sphingan biosynthesis brought up to date

Microbial polysaccharides have a wide range of functional properties and show high relevance in industrial applications. The possibility to create tailor-made polysaccharides by genetic engineering will further enhance the product portfolio and may open new fields of application. Here, we have examined in detail the recently sequenced genome of the welan-producing strain Sphingomonas sp. ATCC 31555 to identify the complete welan cluster and further genes involved in EPS production. The corresponding genes were compared on the nucleotide and amino acid sequence level to the EPS clusters of the described gellan-producing Sphingomonas elodea ATCC 31461, diutan-producing Sphingomonas sp. ATCC 53159, and the S-88-producing Sphingomonas sp. ATCC 31554 strains. We also compared the previously mentioned strains to each other and included the genes upstream of the main cluster in gellan and welan cluster. The cluster organization of Sphingomonas strain S-7 was also compared based on previous hybridization experiments, without nucleotide sequences. We have found that the occurrence of genes in all biosynthesis clusters is connected to the structures of the various produced sphingans. Along these lines, homologous genes responsible for the assembly of the identical repeating unit generally show high sequence identity, whereas genes for putative side chain attachment urf31, urf31.4, and urf34 vary more in distinct areas. Moreover, gene clusters for biosynthesis of diutan, welan, gellan, and S-88 as well as S-7 are similar in general organization but differ in location and arrangement of some genes. Finally, we summarized genetic and mutational engineering approaches toward modified sphingan variants as described in literature.     

Anticancer Activities of Antimicrobial BmKn2 Peptides Against Oral and Colon Cancer Cells

There is considerable current interest in developing antimicrobial and anticancer agents with a new mode of action. The antimicrobial peptides are regarded as a potential solution for treating cancer cells. The antimicrobial effect of 6 synthetic peptides against 7 bacterial species was evaluated. The result showed that IsCT, BmKn2 and BMAP-28 exhibited broad range of action against Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6538, methicillin resistant S. aureus DMST 20651, Staphylococcus epidermidis ATCC 12228, Acinetobacter baumanii ATCC 19066, Escherichia coli ATCC 25922 and Salmonella typhi DMST 562 at minimal inhibitory concentrations (MIC) of 2.97–24.28 μM. Neither AMP induced significant hemolysis, or showed cytotoxic on dental pulp stem cells and smooth muscle cells at their MICs. In addition, BmKn2 inhibited growth of human oral squamous carcinoma HSC4 cells and human colon cancer SW620 cells with IC₅₀ of 17.26 and 40 µM, respectively. Taken together, BmKn2 peptide from scorpion venom may offer a novel therapeutic strategy for development of cationic antimicrobial and anticancer peptides as potential new therapeutic agents.     

Recent advances in biochemistry and biotechnological synthesis of avermectins and their derivatives

Avermectins (AVMs), produced by Streptomyces avermitilis MA-4680 (or ATCC 31267, NRRL 8165, NCBIM 12804), are 16-member macrocylic lactones that play very important functions as bactericidal and antiparasitic agents against nematodes and anthropods, as well as Mycobacterium tuberculosis H37Rv. Since its discovery in 1975, use of AVM has been widely spreading around the globe. To date, the whole genome sequence of S. avermitilis K139 has been acquired, in which the AVM biosynthetic gene cluster was the most highly investigated to mine the genes responsible for functional as well as regulatory roles. Therefore, significant progress has been achieved for understanding and manipulating the biosynthesis, improved production, regulation mechanism, side effects, as well as the resistance of AVMs and their derivatives. These findings will facilitate further strain improvement and biosynthesis of novel derivatives bearing stable and improved biological activities, as well as overcoming the resistance mechanism to open up a bright period for these compounds. In this review, we have summarized and analyzed the update in advanced progress in biochemistry and biotechnological approaches used for the production of AVMs and their derivatives.     

Dietary l-glutamine supplementation modulates microbial community and activates innate immunity in the mouse intestine

This study was conducted to determine effects of dietary supplementation with 1 % l-glutamine for 14 days on the abundance of intestinal bacteria and the activation of intestinal innate immunity in mice. The measured variables included (1) the abundance of Bacteroidetes, Firmicutes, Lactobacillus, Streptococcus and Bifidobacterium in the lumen of the small intestine; (2) the expression of toll-like receptors (TLRs), pro-inflammatory cytokines, and antibacterial substances secreted by Paneth cells and goblet cells in the jejunum, ileum and colon; and (3) the activation of TLR4-nuclear factor kappa B (NF-κB), mitogen-activated protein kinases (MAPK), and phosphoinositide-3-kinases (PI3K)/PI3K-protein kinase B (Akt) signaling pathways in the jejunum and ileum. In the jejunum, glutamine supplementation decreased the abundance of Firmicutes, while increased mRNA levels for antibacterial substances in association with the activation of NF-κB and PI3K-Akt pathways. In the ileum, glutamine supplementation induced a shift in the Firmicutes:Bacteroidetes ratio in favor of Bacteroidetes, and enhanced mRNA levels for Tlr4, pro-inflammatory cytokines, and antibacterial substances participating in NF-κB and JNK signaling pathways. These results indicate that the effects of glutamine on the intestine vary with its segments and compartments. Collectively, dietary glutamine supplementation of mice beneficially alters intestinal bacterial community and activates the innate immunity in the small intestine through NF-κB, MAPK and PI3K-Akt signaling pathways.     

Deep-sea water inhibits metastatic potential in HT-29 human colorectal adenocarcinomas via MAPK/NF-κB signaling pathway

A previous investigation showed that deep-sea water (DSW) can affect the expression of genes that regulate metastasis, including cyclooxygenase-2 (COX-2), matrix metalloproteinase-2 (MMP-2), urokinase plasminogen activator (uPA) and uPA receptor (uPAR), in HT-29 human colorectal adenocarcinomas. In the present study, we investigated the effects of DSW on inducible nitric oxide synthase (iNOS) expression and cell migration and also explored the mechanism of DSW-induced anti-metastatic potential in HT-29 human colorectal adenocarcinomas. Cytokine-induced expression of iNOS, which is highly expressed in colon cancer and enhances cancer growth and metastasis, was decreased in a hardness-dependent manner by DSW. Also, the wound healing assay revealed that DSW inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration in a hardness-dependent manner. DSW also decreased the phosphorylation of various MAPKs, including p38, ERK and JNK, and suppressed the nuclear translocation of NF-κB but not c-Jun. The results suggest that DSW may inhibit cancer cell growth related to iNOS overexpression and PKC-mediated cell migration in HT-29 human colorectal adenocarcinomas and the antimetastatic potential of DSW may be regulated by prevention of NF-κB nuclear translocation via inhibition of p38, ERK and JNK phosphorylation. In conclusion, the present investigation demonstrates that DSW inhibits cancer growth and metastasis via down-regulation of iNOS expression and the MAPK/NF-κB signaling pathway.     

Basic amino acids and dimethylarginines targeted metabolomics discriminates primary hepatocarcinoma from hepatic colorectal metastases

Hepatocellular carcinoma (HCC) is a very aggressive neoplasia requiring early and accurate diagnosis to improve patient outcomes with timely treatment. The liver is also very frequently colonized by metastases, and the most frequent differential diagnosis is HCC against intrahepatic cholangiocarcinoma or metastatic adenocarcinoma. Metabolomics is a powerful tool for identification of altered biomarkers in cancer, and to evaluate the efficacy of drug treatments. Here we analyzed by HILIC-MS/MS methylated arginines, basic amino acids (Arg, Cit, Orn), and their ratios in the extracts of primary HCC tissues, liver metastases from colorectal carcinoma (MET), cirrhotic related hepatitis-C-virus (CIR), and non-cirrhotic normal liver (NT) adjacent tissues. We found high levels of Arg (p < 0.0001) and Arg/Orn (p < 0.01) in MET compared to other tissues. In MET, compared to NT and CIR, Arg concentration was fivefold higher, while in HCC it was twofold higher. ADMA increased twofold compared to NT and CIR, while in HCC it was 50 % higher. Arg/Cit and ADMA/SDMA ratios were significantly higher in MET compared to NT and CIR (p < 0.005). Arg/Orn, Arg/Cit, and ADMA/SDMA ratios increased progressively from NT, CIR, HCC, to MET tissues. Arg/Cit correlated significantly with Arg/Orn ratios (r = 0.77; p < 0.0001), and discriminates tumor from non-tumor samples. In addition, the discriminant lactate/glucose ratio we previously found by NMR, also correlated significantly with the Arg levels (r = 0.64; p < 0.0001), and discriminated MET from all other tissues. The results indicated that Arg in MET is higher than other tissue classes, suggesting that, together with the lactate/glucose ratio, it can be considered a further biomarker for HCC-metastases differentiation.     

Expression status of let-7a and miR-335 among breast tumors in patients with and without germ-line BRCA mutations

The genetic factors of cancer predisposition remain elusive in the majority of familial and/or early-onset cases of breast cancer (BC). This type of BC is promoted by germ-line mutations that inactivate BRCA1 or BRCA2. On the other hand, recent studies have indicated that alterations in the levels of miRNA expression are linked to this disease. Although BRCA1 and BRCA2 gene mutations have been reported to commonly lead to alterations in genes that encode cancer-related proteins, little is known regarding the putative impact of these mutations on noncoding miRNAs. In the present study, we aimed to determine whether miRNA dysregulation is involved in the pathogenesis of BRCA-mutated BC. An expression analysis of 14 human miRNAs previously shown to be related to BC diagnosis, prognosis, and drug resistance was conducted using tissues from 60 familial and/or early-onset patients whose peripheral blood samples had been screened for BRCA1 and BRCA2 mutations through sequence analysis. Let-7a and miR-335 expression levels were significantly downregulated in the tumors of patients with a BRCA mutation compared with those of patients without a BRCA mutation (P = 0.04 and P = 0.02, respectively). Our results defined the associations between the expression status of let-7a and miR-335 and BRCA mutations. The expression analysis of these miRNAs might be used as biomarkers of the BRCA mutation status of early-onset and/or familial BC.     

Isolation and characterization of flagellar filaments from Bacillus cereus ATCC 14579

Isolated flagellar filaments from the type strain of Bacillus cereus, ATCC 14579, were shown to consist of 34, 32 and 31 kDa proteins in similar proportions as judged by band intensities on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of these three proteins of strain ATCC 14579 were identical with the deduced sequences of three flagellin genes BC1657, BC1658 and BC1659 in the whole genome sequence. Strain ATCC 14579 was classified into serotype T2 by a flagellar serotyping scheme for B. cereus strains that are untypeable into known flagellar serotypes H1 to H23. Flagellar filaments from a reference strain of serotype T2 contained two protein bands at 34 and 32 kDa, but a single protein band at 39 kDa was detected in flagellar filaments of a reference strain of serotype H1. Two murine monoclonal antibodies, 1A5 and 2A5, which recognize both the 34 and 32 kDa flagellins and a single flagellin of 32 kDa, respectively, were specifically reactive with B. cereus strains ATCC 14579 and serotype T2 in whole-cell ELISA and bacterial motility inhibition tests. In immunoelectron microscopy with monoclonal antibodies 1A5 and 2A5, colloidal gold spheres were shown to localize almost evenly over the entire part of flagellar filaments. Since strain ATCC 14579, and presumably strain serotype T2, are unusual among B. cereus strains in possessing multiple genes that encode flagellin subunits, a possible unique mechanism may contribute to assembly of multiple flagellin subunits into the filament over its entire length.     

Increased Expression of Angiogenic Factors in Cultured Human Brain Arteriovenous Malformation Endothelial Cells

To compare the mRNA level of angiogenic factor vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMP)-2, and MMP-9 in cultured human brain arteriovenous malformation (AVM) endothelial cells (ECs) and normal brain endothelial cells (BECs). Tissue explants both from deformed vessels of AVM and normal microvessel were put into culture for endothelial cells. After the monolayer adherent ECs reached confluence, they were tested with endothelial specific marker CD34 and von Willebrand factor (vWF) by immunochemical assay. mRNA levels of VEGF-A, MMP-2, and MMP-9 in AVM endothelial cells (AVMECs) and BECs were measured by PCR. Immunostaining confirmed that more than 95 % of the cultured cells were CD34 (Fig. 1b) and/or vWF positive. Expression levels of VEGF-A and MMP-2 mRNAs were significantly higher in AVMECs than in BECs. The MMP-9 level was also increased in AVMECs, but the difference was not statistically significant. Vascular tissue explants adherent method is a better approach for isolation and culture of AVMECs. Cultured AVMECs expressed higher angiogenic factors (VEGF, MMP-2) than the controlled BECs, implicating angiogenesis plays an important role in the pathogenesis of AVM.     

The Neuroprotective Effects of α-Iso-cubebene on Dopaminergic Cell Death: Involvement of CREB/Nrf2 Signaling

As a part of ongoing studies to elucidate pharmacologically active components of Schisandra chinensis, we isolated and studied α-iso-cubebene. The neuroprotective mechanisms of α-iso-cubebene in human neuroblastoma SH-SY5Y cells were investigated. α-Iso-cubebene significantly inhibited cytotoxicity and apoptosis due to 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in dopaminergic SH-SY5Y cells. Pretreatment of cells with α-iso-cubebene reduced intracellular accumulation of ROS and calcium in response to 6-OHDA. The neuroprotective effects of α-iso-cubebene were found to result from protecting the mitochondrial membrane potential. Notably, α-iso-cubebene inhibited the release of apoptosis-inducing factor from the mitochondria into the cytosol and nucleus after 6-OHDA treatment. α-Iso-cubebene also induced the activation of PKA/PKB/CREB/Nrf2 and suppressed 6-OHDA-induced neurotoxicity. α-Iso-cubebene was found to induce phosphorylation of PKA and PKB and activate Nrf2 and CREB signaling pathways in a dose-dependent manner. Additionally, α-iso-cubebene stimulated the expression of the antioxidant response genes NQO1 and HO-1. Finally, α-iso-cubebene-mediated neuroprotective effects were found to be reversible after transfection with CREB and Nrf2 small interfering RNAs.     

Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae

Strain improvement is a powerful tool in commercial development of microbial fermentation processes. Strains of Aspergillus sojae which were previously identified as polygalacturonase producers were subjected to the cost-effective mutagenesis and selection method, the so-called random screening. Physical (ultraviolet irradiation at 254 nm) and chemical mutagens (N-methyl-N′-nitro-N-nitrosoguanidine) were used in the development and implementation of a classical mutation and selection strategy for the improved production of pectic acid-degrading enzymes. Three mutation cycles of both mutagenic treatments and also the combination of them were performed to generate mutants descending from A. sojae ATCC 20235 and mutants of A. sojae CBS 100928. Pectinolytic enzyme production of the mutants was compared to their wild types in submerged and solid-state fermentation. Comparing both strains, higher pectinase activity was obtained by A. sojae ATCC 20235 and mutants thereof. The highest polygalacturonase activity (1,087.2?±?151.9 U/g) in solid-state culture was obtained by mutant M3, which was 1.7 times increased in comparison to the wild strain, A. sojae ATCC 20235. Additional, further mutation of mutant M3 for two more cycles of treatment by UV irradiation generated mutant DH56 with the highest polygalacturonase activity (98.8?±?8.7 U/mL) in submerged culture. This corresponded to 2.4-fold enhanced polygalacturonase production in comparison to the wild strain. The results of this study indicated the development of a classical mutation and selection strategy as a promising tool to improve pectinolytic enzyme production by both fungal strains.     

The neurobiological link between OCD and ADHD

Obsessive compulsive disorder (OCD) and attention deficit hyperactivity disorder (ADHD) are two of the most common neuropsychiatric diseases in paediatric populations. The high comorbidity of ADHD and OCD with each other, especially of ADHD in paediatric OCD, is well described. OCD and ADHD often follow a chronic course with persistent rates of at least 40–50 %. Family studies showed high heritability in ADHD and OCD, and some genetic findings showed similar variants for both disorders of the same pathogenetic mechanisms, whereas other genetic findings may differentiate between ADHD and OCD. Neuropsychological and neuroimaging studies suggest that partly similar executive functions are affected in both disorders. The deficits in the corresponding brain networks may be responsible for the perseverative, compulsive symptoms in OCD but also for the disinhibited and impulsive symptoms characterizing ADHD. This article reviews the current literature of neuroimaging, neurochemical circuitry, neuropsychological and genetic findings considering similarities as well as differences between OCD and ADHD.     

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells

Cystic fibrosis transmembrane conductance regulator (CFTR) acts as a cAMP-dependent chloride channel, has been studied in various types of cells. CFTR is abundantly expressed in vascular smooth muscle cells and closely linked to vascular tone regulation. However, the functional significance of CFTR in basilar vascular smooth muscle cells (BASMCs) remains elusive. Accumulating evidence has shown the direct role of CFTR in cell apoptosis that contributes to several main pathological events in CF, such as inflammation, lung injury and pancreatic insufficiency. We therefore investigated the role of CFTR in BASMC apoptotic process induced by hydrogen peroxide (H₂O₂). We found that H₂O₂-induced cell apoptosis was parallel to a significant decrease in endogenous CFTR protein expression. Silencing CFTR with adenovirus-mediated CFTR specific siRNA further enhanced H₂O₂-induced BASMC injury, mitochondrial cytochrome c release into cytoplasm, cleaved caspase-3 and -9 protein expression and oxidized glutathione levels; while decreased cell viability, the Bcl-2/Bax ratio, mitochondrial membrane potential, total glutathione levels, activities of superoxide dismutase and catalase. The pharmacological activation of CFTR with forskolin produced the opposite effects. These results strongly suggest that CFTR may modulate oxidative stress-related BASMC apoptosis through the cAMP- and mitochondria-dependent pathway and regulating endogenous antioxidant defense system.     

Inhibition of MMP-2 but not MMP-9 Influences Inner Ear Spiral Ganglion Neurons In Vitro

Matrix metalloproteinases (MMPs) play an important role in modeling of the extracellular matrix. There is increasing evidence that these proteases are important in neurite elongation and axonal guidance during development in the central nervous system and retina. Moreover, they are also expressed after acute injury and can be the key mediators of pathogenesis. However, the role of MMPs in the inner ear is largely unknown. Our group recently demonstrated that general inhibition of MMPs resulted in auditory hair cell loss in vitro. In the present study, we investigated the role of MMPs in inner ear spiral ganglion neuron (SGN) survival, neuritogenesis and neurite extension by blocking MMPs known to be involved in axonal guidance, neurite elongation, and apoptosis in other neuronal systems. Spiral ganglion (SG) explants from 5-day-old Wistar rats were treated with different concentrations of the general MMP inhibitor GM6001, a specific MMP-2 inhibitor, and a specific MMP-9 inhibitor, in vitro. The general inhibitor of MMPs and the specific inhibition of MMP-2 significantly reduced both the number of neurites that extended from SG explants, as well as the length of individual neurites. However, neither the general inhibitor of MMPs nor the specific inhibition of MMP-2 influenced SGN survival. Inhibition of MMP-9 had no influence on SGNs. The data suggest that MMPs, and more specifically MMP-2, influence the growth of developing afferent neurites in the mammalian inner ear in vivo.     

Tumor necrosis factor-α-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase

An elevated level of tumor necrosis factor (TNF)-α is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-α activates nuclear factor (NF)-kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-α and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-α-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-α with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-α-induced ROS production was measured using 5-(and-6)-chloromethyl-2’, 7’-dichlorodihydrofluorescein diacetate assay. TNF-α-induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-α-induced upregulation of interleukin (IL)-1β and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-α-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-α/β and p65, degradation of IkappaBα, binding of NF-kappaB to its binding motif, and upregulation of IL-1β and VCAM-1 induced by TNF-α were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-α-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1β and VCAM-1, in human cardiomyocytes.