Bursera fagaroides, effect of an ethanolic extract on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica

The effect of an ethanolic extract from the stem bark of Bursera fagaroides on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica was evaluated. For this purpose, increasing concentrations of the extract, up to 8.0 mg/mL, were added to amoeba cultures or ODC reaction mixtures, which were incubated at 37 °C. Metronidazole and G418 were added as controls. After 1.5 and 72 h, the ODC activity in vitro and growth, respectively, were determined. Results revealed a strong inhibition of growth with IC₅₀ values on the order of 0.05 mg/mL. ODC activity, on the other hand, was inhibited by 12% and 50% at concentrations of 4.0 and 8.0 mg/mL, respectively.     

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.     

Differentially enhanced gene expression in hemocytes from Macrobrachium rosenbergii challenged in vivo with lipopolysaccharide

In order to better understand the immune response in prawns after treatment with the immunostimulant lipopolysaccharide (LPS), in this study, the differential gene expression of the hemocytes from LPS-injected versus non-injected prawns (Macrobrachium rosenbergii) were isolated and identified using suppression subtractive hybridization (SSH). The hemocytes were extracted after treatment for 1, 6, and 12 h. The upregulated genes (i.e., where gene expression was elevated) were identified and could be divided into four classes on the basis of physiological function: genes concerning defense-related molecules, genes involved in energy-production (respiration), genes related to protein synthesis and folding, and genes with unknown function. The time-course for gene expression indicated that, except for expression of the gene anti-microbial peptide (amp), which was increased at 12 h after LPS treatment, the expression of the other two immune-related genes was much earlier (at 1 h), including alpha-2-macroglobulin (α2-M) and Mas-like protein (mas). These results suggest that in the early phase of LPS stimulation some immune reactions regulated by α-2M and Mas may be induced, such as the activation of prophenoloxidase activating system, opsonization, and anti-microbial activity. In addition, six unigenes with unproven function were particularly interesting and worthy of further study because their expression in LPS-treated hemocytes was clearly enhanced.     

Semaphorin-plexin signalling genes associated with human breast tumourigenesis

Introduction

Gene expression profiling has enabled us to demonstrate the heterogeneity of breast cancers. The potential of a tumour to grow and metastasise is partly dependant on its ability to initiate angiogenesis or growth and remodelling of new blood vessels, usually from a pre-existing vascular network, to ensure delivery of oxygen, nutrients, and growth factors to rapidly dividing transformed cells along with access to the systemic circulation. Cell-cell signalling of semaphorin ligands through interaction with their plexin receptors is important for the homeostasis and morphogenesis of many tissues and has been widely studied for a role in neural connectivity, cancer, cell migration and immune responses. This study investigated the role of four semaphorin/plexin signalling genes in human breast cancers in vivo and in vitro.

Materials and methods

mRNA was extracted from formalin fixed paraffin embedded archival breast invasive ductal carcinoma tissue samples of progressive grades (grades I-III) and compared to tissue from benign tumours. Gene expression profiles were determined by microarray using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays and validated by Q-PCR using a Corbett RotorGene 6000. Following validation, the gene expression profile of the identified targets was correlated with those of the human breast cancer cell lines MCF-7 and MDA-MD-231.

Results

The array data revealed that 888 genes were found to be significantly (p ≤ 0.05) differentially expressed between grades I and II tumours and 563 genes between grade III and benign tumours. From these genes, we identified four genes involved in semaphorin-plexin signalling including SEMA4D which has previously been identified as being involved in increased angiogenesis in breast cancers, and three other genes, SEMA4F, PLXNA2 and PLXNA3, which in the literature were associated with tumourigenesis, but not directly in breast tumourigenesis. The microarray analysis revealed that SEMA4D was significantly (P = 0.0347) down-regulated in the grade III tumours compared to benign tumours; SEMA4F, was significantly (P = 0.0159) down-regulated between grades I and II tumours; PLXNA2 was significantly (P = 0.036) down-regulated between grade III and benign tumours and PLXNA3 significantly (P = 0.042) up-regulated between grades I and II tumours. Gene expression of SEMA4D was validated using Q-PCR, demonstrating the same expression profile in both data sets. When the sample set was increased to incorporate more cases, SEMA4D continued to follow the same expression profile, including statistical significance for the differences observed and small standard deviations. In vitro the same pattern was present where expression for SEMA4D was significantly higher in MDA-MB-231 cells when compared to MCF-7 cells. The expression of SEMA4F, PLXNA2 and PLXNA3 could not be validated using Q-PCR, however in vitro analysis of these three genes revealed that both SEMA4F and PLXNA3 followed the microarray trend in expression, although they did not reach significance. In contrast, PLXNA2 demonstrated statistical significance and was in concordance with the literature.

Discussion

We, and others, have proposed SEMA4D to be a gene with a potentially protective effect in benign tumours that contributes to tumour growth and metastatic suppression. Previous data supports a role for SEMA4F as a tumour suppressor in the peripheral nervous system but our data seems to indicate that the gene is involved in tumour progression in breast cancer. Our in vitro analysis of PLXNA2 revealed that the gene has higher expression in more aggressive breast cancer cell types. Finally, our in vitro analysis on PLXNA3 also suggest that this gene may have some form of growth suppressive role in breast cancer, in addition to a similar role for the gene previously reported in ovarian cancer. From the data obtained in this study, SEMA4D may have a role in more aggressive and potentially metastatic breast tumours.

Conclusions

Semaphorins and their receptors, the plexins, have been implicated in numerous aspects of neural development, however their expression in many other epithelial tissues suggests that the semaphorin-plexin signalling system also contributes to blood vessel growth and development. These findings warrant further investigation of the role of semaphorins and plexins and their role in normal and tumour-induced angiogenesis in vivo and in vitro. This may represent a new front of attack in anti-angiogenic therapies of breast and other cancers.     

Small RNAs with 5′-Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica

Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5′-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5′-polyphosphate siRNAs extends to single-celled eukaryotic organisms.     

Effect of finishing period length with α-tocopherol supplementation on the expression of vitamin E-related genes in the muscle and subcutaneous fat of light lambs

The aim of this study was to investigate how different finishing period lengths with α-tocopherol supplementation or alfalfa grazing affect mRNA expression levels of genes related to vitamin E metabolism in L. thoracis (LT) muscle and subcutaneous fat (SF) from lambs of the Rasa Aragonesa breed. Indoors, concentrate-fed light lambs (n = 48) were supplemented with 500 dl-α-tocopheryl acetate/kg concentrate for an average finishing period length of 0 (C), 10.7 (VE10d), 21.2 (VE20d) and, 32.3 (VE30d) days before slaughtering. Simultaneously, 8 lambs with their dams were alfalfa-grazed. The α-tocopherol affected in a short-term the expression of genes in LT muscle (ABCA1, LPL, APOE, and SREBP1) and SF (ABCA1, SCARB1, LPL, and PPARG). On the contrary, PPARA gene expression showed a long-term α-tocopherol effect because the highest levels of PPARA mRNA were found in the VE30d.     

Adipose triglyceride lipase regulates lipid metabolism in dairy goat mammary epithelial cells

Adipose triglyceride lipase (ATGL) catalyzes the initial step in the lipid lipolysis process, hydrolyzing triglyceride (TG) to produce diacylglycerol (DG) and free fatty acids (FFA). In addition, ATGL regulates lipid storage and release in adipocyte cells. However, its role in mammary gland tissue remains unclear. To assess the role of the ATGL gene in the goat mammary gland, this study analyzed the tissue distribution and expression of key genes together with lipid accumulation after knockdown of the ATGL gene. The mRNA of ATGL was highly expressed in subcutaneous adipose tissue, the lung and the mammary gland with a significant increase in expression during the lactation period compared with the dry period of the mammary gland. Knockdown of the ATGL gene in goat mammary epithelial cells (GMECs) using siRNA resulted in a significant decrease in both ATGL mRNA and protein levels. Silencing of the ATGL gene markedly increased lipid droplet accumulation and intracellular TG concentration (P < 0.05), while it reduced FFA levels in GMECs (P < 0.05). Additionally, the expression of HSL for lipolysis, FABP3 for fatty acid transport, PPARα for fatty acid oxidation, ADFP, BTN1A1, and XDH for milk fat formation and secretion was down-regulated (P < 0.05) after knockdown of the ATGL gene, with increased expression of CD36 for fatty acid uptake (P < 0.05). In conclusion, these data suggest that the ATGL gene plays an important role in triglyceride lipolysis in GMECs and provides the first experimental evidence that ATGL may be involved in lipid metabolism during lactation.     

Construction of theta-type shuttle vector for Leuconostoc and other lactic acid bacteria using pCB42 isolated from kimchi

The pCB42 plasmid from Leuconostoc citreum CB2567, a strain isolated from kimchi, was characterized, and a shuttle vector for Escherichia coli and lactic acid bacteria (LAB) was constructed. The pCB42 plasmid has a circular structure of 4312 bp, a low G + C content, and no single-stranded DNA intermediates during replication, which indicates that pCB42 replicates via the theta-type replication mechanism. In silico analysis of this plasmid revealed 6 open reading frames: 1 transposase gene, 1 DNA-binding gene, 2 putative replication genes, and 2 unknown genes. The fragment encompassing ORF5 contains a functional plasmid replicon. This plasmid was capable of replicating in various LAB, including L. citreum, L. mesenteroides, Lactobacillus plantarum, Lb. reuteri, Lactococcus lactis, Streptococcus thermophilus, Weissella confusa, and Oenococcus oeni. The LAB-E. coli shuttle vector was constructed by ligating pCB42 and pEK104, and the resulting shuttle vector, pLeuCM42, showed a high segregational stability in L. citreum CB2567 after 100 generations of cell division. By using this shuttle vector, the β-gal gene from Lb. plantarum was successfully expressed in the host strain, L. citreum CB2567. The pLeuCM42 shuttle vector can serve as a useful gene-delivery and expression tool for the genetic study or metabolic engineering of various strains of LAB.     

The complete mitochondrial genome of Taeniogonalos taihorina (Bischoff) (Hymenoptera: Trigonalyidae) reveals a novel gene rearrangement pattern in the Hymenoptera

The family Trigonalyidae is considered to be one of the most basal lineages in the suborder Apocrita of Hymenoptera. Here, we determine the first complete mitochondrial genome of the Trigonalyidae, from the species Taeniogonalos taihorina (Bischoff, 1914). This mitochondrial genome is 15,927 bp long, with a high A + T-content of 84.60%. It contains all of the 37 typical animal mitochondrial genes and an A + T-rich region. The orders and directions of all genes are different from those of previously reported hymenopteran mitochondrial genomes. Eight tRNA genes, three protein-coding genes and the A + T-rich region were rearranged, with the dominant gene rearrangement events being translocation and local inversion. The arrangements of three tRNA clusters, trnY–trnM–trnI–trnQ, trnW–trnL2–trnC, and trnH–trnA–trnR–trnN–trnS–trnE–trnF, and the position of the cox1 gene, are novel to the Hymenoptera, even the insects. Six long intergenic spacers are present in the genome. The secondary structures of the RNA genes are normal, except for trnS2, in which the D-stem pairing is absent.     

Evidence of expression variation and allelic imbalance in Crohn's disease susceptibility genes NOD2 and ATG16L1 in human dendritic cells

Human dendritic cells (DCs) play an important role in induction and progression of Crohn's disease (CD). Accumulating evidence suggests that viral infection is required to trigger CD pathogenesis in genetically predisposed individuals. NOD2 and ATG16L1 are among the major CD susceptibility genes implicated in impaired immune response to bacterial infection. In this study, we investigated gene expression and allelic imbalance (AI) of NOD2 and ATG16L1 using common variants in human monocyte-derived DCs. Significant AI was observed in ~ 40% and ~ 70% of NOD2 and ATG16L1 heterozygotes, respectively (p < 0.05). AI of NOD2 was inversely associated with its expression level (p = 0.015). No correlation was detected between gene expression and AI for ATG16L1. When infected with Newcastle Disease Virus (NDV), NOD2 expression in DCs was induced about four-fold (p < 0.001), whereas ATG16L1 expression was not affected (p = 0.88). In addition, NDV infection tended to lower the variance in AI among DC populations for the NOD2 gene (p = 0.05), but not the ATG16L1 gene (p = 0.32). Findings of a simulation study, aimed to verify whether the observed variation in gene expression and AI is a result of sample-to-sample variability or experimental measurement error, suggested that NOD2 AI is likely to result from a deterministic event at a single cell level. Overall, our results present initial evidence that AI of the NOD2 and ATG16L1 genes exists in populations of human DCs. In addition, our findings suggest that viral infection may regulate NOD2 expression.     

The effect of the introduction of exogenous strain Acidithiobacillus thiooxidans A01 on functional gene expression, structure and function of indigenous consortium during pyrite bioleaching

Acidithiobacillus thiooxidans A01 was added to a consortium of bioleaching bacteria including Acidithiobacilluscaldus, Leptospirillumferriphilum, Acidithiobacillus ferrooxidans, Sulfobacillus thermosulfidooxidans, Acidiphilium spp., and Ferroplasma thermophilum cultured in modified 9 K medium containing 0.5% (w/v) pyrite, and 10.7% increase of bioleaching rate was observed. Changes in community structure and gene expression were monitored with real-time PCR and functional gene arrays (FGAs). Real-time PCR showed that addition of At. thiooxidans caused increased numbers of all consortium members except At. caldus, and At. caldus, L. ferriphilum, and F. thermophilum remained dominant in this community. FGAs results showed that after addition of At. thiooxidans, most genes involved in iron, sulfur, carbon, and nitrogen metabolisms, metal resistance, electron transport, and extracellular polymeric substances of L. ferriphilum, F. thermophilum, and Acidiphilium spp., were up-regulated while most of these genes were down-regulated at 70-78 h in At. caldus and up-regulated in At. ferrooxidans, then down-regulated at 82-86 h.     

Enhanced resistance of Saccharomyces cerevisiae to vanillin by expression of lacA from Trametes sp. AH28-2

Saccharomyces cerevisiae is affected by the presence of certain phenolic compounds such as vanillin during fermentation of pretreated lignocellulosic hydrolysates. Since vanillin can be polymerized in the presence of laccase into compounds with lower toxicity, the laccase gene, lacA, from Trametes sp. AH28-2 was fused to the α-factor signal sequence and transferred into S. cerevisiae CEN.PK strains for secretory expression. Furthermore, the chaperone gene, KAR2, was overexpressed to promote the translocation of laccase. In the presence of 8 mmol/L vanillin, a shorter lag phase was observed in the lacA gene expressing strains. The vanillin-specific conversion rate of the lacA-expressing strain BSJX0A2 was 0.069 g g⁻¹ biomass h⁻¹, while it was 0.065 g g⁻¹ biomass h⁻¹ in the reference strain.     

Entamoeba histolytica uses ferritin as an iron source and internalises this protein by means of clathrin-coated vesicles

Entamoeba histolytica is a parasitic protozoan that produces dysentery and often reaches the liver, leading to abscess formation. Ferritin is an iron-storage protein that is mainly found in liver and spleen in mammals. The liver contains a plentiful source of iron for amoebae multiplying in that organ, making it a prime target for infection since iron is essential for the growth of this parasite. The aim of this study was to determine whether trophozoites are able to take up ferritin and internalise this protein for their growth in axenic culture. Interaction between the amoebae and ferritin was studied by flow cytometry, confocal laser-scanning microscopy and transmission electron microscopy. Amoebae were viable in iron supplied by ferritin. Trophozoites quickly internalised ferritin via clathrin-coated vesicles, a process that was initiated within the first 2 min of incubation. In 30 min, ferritin was found colocalizing with the LAMP-2 protein at vesicles in the cytosol. The uptake of ferritin was time- temperature- and concentration-dependent, specific and saturated at 46 nM of ferritin. Haemoglobin and holo-transferrin did not compete with ferritin for binding to amoebae. Amoebae cleaved ferritin leading to the production of several different sized fragments. Cysteine proteases of 100, 75 and 50 kDa from amoeba extracts were observed in gels copolymerised with ferritin. For a pathogen such as E. histolytica, the capacity to utilise ferritin as an iron source may well explain its high pathogenic potential in the liver.     

Early Trichinella spiralis and Trichinella nativa infections induce similar gene expression profiles in rat jejunal mucosa

Trichinella spiralis causes a significantly higher parasite burden in rat muscle than Trichinella nativa. To assess whether the difference in infectivity is due to the early intestinal response, we analyzed gene expression changes in the rat jejunum during Trichinella infection with a whole-genome microarray. The rats were euthanized on day five of infection, and their jejunal mucosa was sampled for microarray analysis. In addition, intestinal histology and hematology were examined. Against our expectations, the gene expression changes were similar in both T.nativa- and T. spiralis-infected groups. The two groups were hence pooled, and in the combined Trichinella-infected group, 551 genes were overexpressed and 427 underexpressed when compared to controls (false discovery rate ?0.001 and fold change at least 2 in either direction). Pathway analysis identified seven pathways significantly associated with Trichinella infection (p < 0.05). The microarray data suggested nonspecific damage and an inflammatory response in the jejunal mucosa. Histological findings, including hyperemia, hemorrhage and a marked infiltration of inflammatory cells, supported the microarray data. Trichinella infection caused complex gene expression changes that indicate a host response to tissue damage in the mucosa of the jejunum, but the changes were not notably dependent on the studied species of Trichinella.