Cul4A is an oncogene in malignant pleural mesothelioma

Cullin 4A (Cul4A) is important in cell survival, development, growth and the cell cycle, but its role in mesothelioma has not been studied. For the first time, we identified amplification of the Cul4A gene in four of five mesothelioma cell lines. Consistent with increased Cul4A gene copy number, we found that Cul4A protein was overexpressed in mesothelioma cells as well. Cul4A protein was also overexpressed in 64% of primary malignant pleural mesothelioma (MPM) tumours. Furthermore, knockdown of Cul4A with shRNA in mesothelioma cells resulted in up‐regulation of p21 and p27 tumour suppressor proteins in a p53‐independent manner in H290, H28 and MS‐1 mesothelioma cell lines. Knockdown of Cul4A also resulted in G0/G1 cell cycle arrest and decreased colony formation in H290, H28 and MS‐1 mesothelioma cell lines. Moreover, G0/G1 cell cycle arrest was partially reversed by siRNA down‐regulation of p21 and/or p27 in Cul4A knockdown H290 cell line. In the contrary, overexpression of Cul4A resulted in down‐regulation of p21 and p27 proteins and increased colony formation in H28 mesothelioma cell line. Both p21 and p27 showed faster degradation rates in Cul4A overexpressed H28 cell line and slower degradation rates in Cul4A knockdown H28 cell line. Our study indicates that Cul4A amplification and overexpression play an oncogenic role in the pathogenesis of mesothelioma. Thus, Cul4A may be a potential therapeutic target for MPM.     

Ribosomal Proteins S27E, P2, and L37A from Marine Invertebrates

Single complementary DNAs encoding sequences for 40S ribosomal proteins related to S27E from the American lobster Homarus americanus and mussel Mytilus galloprovincialis were characterized. Single genes for ribosomal proteins L37A and P2 from the gumboot chiton Cryptochiton stellerii are similarly described. The lobster S27E protein contains the highly conserved cysteine residues, suggesting its likely designation in the C₄ protein family containing zinc finger motifs. The lobster S27E protein also appears to have an intermediate gene copy number between lower and higher euckaryotes. Expression of the S27E protein in lobster hepatopancreas was slightly elevated during several postmolt and premolt stages. Chlorinated pesticide treatment significantly reduced S27E expression in hepatopancreas, indicating that this gene is responsive to endogenous and exogenous cues. Received March 6, 1998; accepted October 2, 1998.     

Genetic polymorphisms and preliminary association analysis with production traits of the porcine <Emphasis Type="Italic">SLC27A4</Emphasis> gene

Solute carrier family 27 (fatty acid transporter), member 4 (SLC27A4) is a fatty acyl-CoA synthetase producing very long chain fatty acid-CoA for lipid metabolic pathways, suggesting that the SLC27A4 gene is a potential candidate gene for traits related to fat deposition in animals. This study was conducted to sequence the genomic region from exon 6 to 12 of porcine SLC27A4 and detect polymorphisms by comparative sequencing. In silico mapping assigned SLC27A4 gene between gene COQ4 (coenzyme Q4 homolog) and URM1 (ubiquitin related modifier 1 homolog) on pig chromosome 1q24-q2.12 where significant QTL affecting backfat depth had previously been identified. Thirty six putative sites of variation were detected, of which 31 polymorphisms including 28 SNPs and 3 indels were located in the intronic region, and 5 in the exonic regions. The g.1777G>A (EU703769) in intron 8 was confirmed by PCR-RFLP using HpaII restriction enzyme and further genotyped in four Chinese native pig breeds (Meishan, Erhualian, Tongcheng and Qingping) and three western meat-type pig breeds (Duroc, Large White and Landrace). Allele G was exclusively present in Tongcheng and Qingping pigs and predominant in the other pig populations analyzed. Significant differences of backfat at rump, body weight at birth and average daily gain on weaning between the AG and GG genotype were observed in Landrace pig population (P < 0.05).     

Identification of an antilymphocyte transformation substance from Pasteurella multocida

Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai‐1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS‐PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT‐2 cells, which are an IL‐2‐dependent T cell line, nor did it modify IL‐2 production by Con A‐stimulated mouse spleen cells. The N‐terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai‐1 was cloned into expression vector pQE‐60 in Escherichia coli XL‐1 Blue. Recombinant UPase (rUPase) tagged with His at the C‐terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A‐stimulated mouse spleen cells and may be a virulence factor.     

Hessian fly-resistance gene transferred from chromosome 4Mv of Aegilops ventricosa to Triticum aestivum

 A new Hessian fly (Mayetiola destructor) resistance gene from Aegilops ventricosa and its transfer to hexaploid wheat is described. The 4D(4M^v) substitution line H-93-33 derived from the cross [(Triticum turgidum H-1-1×Aegilops ventricosa no. 11)×Triticum aestivum H-10-15] was highly resistant to the Spanish population tested. Resistance seemed to be inherited as a single dominant factor in the F₂ generation resulting from a cross of H-93-33 with its susceptible parent (H-10-15). Resistance in Ae. venticosa no. 10 was located on chromosome 4M^v using M^v wheat/Ae. ventricosa addition lines. The resistance gene transferred from Ae. ventricosa no. 11 to H-93-33 (H27) is allelic with respect to that of Ae. ventricosa no. 10 and is non-allelic with respect to the genes H3 and H6 from Monon and Caldwell respectively. The assignment of H27 gene to chromosome 4M^v is further supported by its linkage to a gene encoding isozyme Acph-M^v1, previously located on chromosome 4M^v in the line H-93-33. A new marker from homoeologous chromosome group 4 (Amp-M^v2) present in H-93-33 and the 4M^v addition line is described. Received: 12 October 1996 / Accepted: 22 November 1996     

Atg27 Tyrosine Sorting Motif is Important for Its Trafficking and Atg9 Localization

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9‐positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP‐3, which mediates direct transport from the trans‐Golgi to the vacuole. The four C‐terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP‐3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.      

Serum levels of interleukin (IL)-27 in patients with ischemic heart disease

Objective

Cytokines, the key mediators of immune responses, play an important role in the pathogenesis of cardiovascular diseases. The aim of this study was to evaluate the serum levels of IL-27 in patients with ischemic heart disease (IHD) and also to clarify its association with traditional risk factors of the disease.

Methods

A total of 120 patients with IHD as having acute myocardial infarction (AMI; n = 60) or unstable angina (UA; n = 60) and 60 sex- and age-matched healthy subjects as a control group were enrolled in this cross-sectional, case-controlled study. Serum samples were collected from all participants (for AMI patients at 3–5 days after events and for UA at admission time) and tested for the levels of IL-27 by use of ELISA method.

Results

The mean serum levels of IL-27 in AMI group (38.00 ± 14.38 Pg/ml) and UA group (35.77 ± 18.93 Pg/ml) were significantly higher than those observed in the control group (24.91 ± 14.96 Pg/ml; P < 0.0001 and 0.001, respectively). The mean serum levels of IL-27 in IHD patients with or without a certain traditional risk factor including hypertension, dyslipidemia, diabetes smoking were significantly higher as compared to those in the control group.

Conclusions

These results showed that the higher serum levels of IL-27 were associated with IHD. The presence or absence of certain traditional risk factors of IHD did not influence the serum levels of cytokine.     

Age-related expression profile of the <Emphasis Type="Italic">SLC27A1</Emphasis> gene in chicken tissues

The solute carrier family 27 (SLC27, also known as fatty acid transport proteins [FATPs]) plays important biological roles in cells. However, there is no report about the expression profile of SLC27 member in chicken. In this study, we quantified the expression of SLC27A1 (FATP1) mRNA in a mountainous black-boned chicken breed (MB) and a commercial meat type chicken breed (S01), to discern the tissue and age-related specific expression pattern and their potential involvement in fat deposition and muscle fatty acid metabolism. Real-time quantitative PCR assays were developed for accurate measurement of SLC27A1 mRNA levels in different tissues from chicken with different ages (0–12 weeks). Expression of SLC27A1 mRNA was detected in all tissues examined. There was a significantly age-related change of the SLC27A1 mRNAs in heart, breast muscle (BMW), leg muscle (LMW), liver, and abdominal fat (AF) tissues (P < 0.05). The breast muscle and leg muscle tissues had the highest expression of SLC27A1 mRNA than the other tissues from the same individual at 0, 2 and 4 weeks. The overall SLC27A1 mRNA level exhibited a “rise-decline” developmental change in all tissues except for breast muscle, subcutaneous fat, and brain. The S01 chicken had a higher expression of the SLC27A1 mRNA in breast muscle, subcutaneous fat, and heart tissues than the MB chicken. Our results showed that the expression of SLC27A1 mRNA in chicken tissues exhibits specific developmental changes and age-related patterns.     

Partial deacetylation of 1,6-anhydro-2,3,4-tri-O-acetyl-β-d-glucopyranose byAureobasidium pullulans cells

The ability of 58 yeasts and yeast-like organisms representing 44 genera to deacetylate 1,6-anhydro-2,3,4-tri-0-acetyl-β-d-glucopyranose was investigated.Aureobasidium pullulans strains can perform this deacetylation selectively.A. pullulans CCY 27-1-11 and 27-1-14 release preferentially the acetyl group in position C(4), whereas the subspeciesA. pullulans CCY 27-1-14a (with no pigmentation) preferentially releases the acetyl group in position C₍₂₎.A. pullulans cells catalyzing the selective deacetylation of 1,6-anhydro-2,3,4-tri-O-acetyl-β-D-glucopyranose were permeabilized and immobilized on a matrix of polyethyleneimine cross-linked with 2-chloromethyloxirane.     

Functional and structural characteristics of anticancer peptide Pep27 analogues

Background  

A secreted peptide Pep27 initiates the cell death program in S. pneumoniae through signal transduction. This study was undertaken to evaluate the relation between the structure and cytotoxic activity of Pep27 and its analogues on cancer cells.     

A new powdery mildew resistance allele at the Pm4 wheat locus transferred from einkorn (Triticum monococcum)

Powdery mildew is one of the most destructive foliar diseases of wheat. A set of differential Blumeria graminis f.sp. tritici (Bgt) isolates was used to test the powdery mildew response of a Triticum monococcum-derived resistant hexaploid line, Tm27d2. Segregation analysis of 95 F_2:3 lines from a Chinese Spring/Tm27d2 cross revealed that the resistance of Tm27d2 is controlled by a single dominant gene. Using monosomic analysis and a molecular mapping approach, the resistance gene was localized to the terminal end of chromosome 2AL. The linkage map of chromosome 2AL consisted of nine simple sequence repeat markers and one sequence-tagged site (STS) marker (ResPm4) indicative for the Pm4 locus. According to the differential reactions of 19 wheat cultivars/lines with known powdery mildew resistance genes to 13 Bgt isolates, Tm27d2 carried a new resistance specificity. The complete association of the resistance allele with STS marker ResPm4 indicated that it represented a new allele at the Pm4 locus. This new allele was designated Pm4d. The two flanking markers Xgwm526 and Xbarc122 closely linked to Pm4d at genetic distances of 3.4 and 1.0 cM, respectively, are present in chromosome bin 2AL1-0.85-1.00.     

Expression and Purification of Recombinant lysostaphin in Escherichia coli

Summary A 1.5 kb plasmid-encoded lysostaphin gene fragment of Staphylococcus staphylolyticus was amplified by polymerase chain reaction (PCR) and cloned in Escherichia coli by using plasmid pET29b(+) as an expression vector. By optimizing culture conditions, the activities of lysostaphin were expressed as 66 %, 30 %, and 4 % in extracellular, intracellular, and periplasmic fractions of recombinant E. coli, respectively. The enzyme was purified to homogeneity by using a simple one-step fractionation on bacterial cells of lysostaphin-resistant Staphylococcus aureus mutant. The recombinant enzyme had an Mr of approximate 27 kDa, and its bacteriolytic activity was indistinguishable to the authentic lysostaphin purified from Staphylococcus staphylolyticus.     

Degradation of naphthalene by thermophilic bacteria <Emphasis Type="Italic">via</Emphasis> a pathway,through protocatechuic acid

A number of thermophilic bacteria capable of utilizing naphthalene as a sole source of carbon were isolated from a high-temperature oilfield in Lithuania. These isolates were able to utilize several other aromatic compounds, such as anthracene, benzene, phenol, benzene-1, 3-diol, protocatechuic acid as well. Thermophilic isolate G27 ascribed to Geobacillus genus was found to have a high aromatic compound degrading capacity. Spectrophotometric determination of enzyme activities in cell-free extracts revealed that the last aromatic ring fission enzyme in naphthalene biotransformation by Geobacillus sp. G27 was inducible via protocatechuate 3, 4-dioxygenase; no protocatechuate 4, 5-dioxygenase, protocatechuate 2, 3-dioxygenase activities were detected. Intermediates such as o-phthalic and protocatechuic acids detected in culture supernatant confirmed that the metabolism of naphthalene by Geobacillus sp. G27 can proceed through protocatechuic acid via ortho-cleavage pathway and thus differs from the pathways known for mesophilic bacteria.     

The effect of low temperatures on fatty acid composition of crops with different cold resistance

The study was focused on fatty acid (FA) composition of lipids from the seedlings and roots of crops having different cold resistance and grown at 27°C or 4°C. Biosynthesis of FA in the lipids of seedlings and roots of cold-susceptible maize (Zea mays L.) at both growth temperatures was controlled by chloroplast ω6 desaturase and microsomal ω6 desaturase, respectively. The content of linoleic acid was 56.2% and 43.3% in the coleoptiles of maize seedlings grown at 4 and 27°C, respectively, and in the roots it was 52.2% and 38.5%, respectively. The content of α-linolenoic acid in the coleoptiles was 6.7–6.8% at both temperatures, while in the root lipids it was higher at low temperature (3.15% at 27°C vs. 4.7% at 4°C). FA biosynthesis in the seedling coleoptiles of wheat (Triticum aestivum L.) and Siberian wild rye (Elymus sibiricus L.) grown at low temperature was controlled by the chloroplast ω3 desaturase. A minor increase in the content of α-linolenoic acid was observed at low temperature: 29.7% to 30.2% in wheat and 22.8% to 25.8% in wild rye. In the root tissues of these species, the biosynthesis of α-linolenoic acid was controlled by the microsomal ω3 desaturase. The content of α-linolenoic acid was higher at low temperature: in wheat it was 6.1% at 27°C and 17.1%, and 4°C, while in Siberian wild rye, 7.1% and 12.0% at 4 and 27°C, respectively.     

Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2

Human Griscelli syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2-a/melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2-a/melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The Rab27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a.     

Characterization of brevicin 27, a bacteriocin synthetized byLactobacillus brevis SB27

Lactobacillus brevis SB27, isolated from sausages, produced an antimicrobial substance active against numerous strains of heterofermentative lactobacilli and against some strains of pediococci andBacillus. The antibacterial agent was shown to be heat stable, resistant over a wide pH range, and sensitive to proteolytic enzymes. It was identified as a bacteriocin and termed brevicin 27. Dialysis and ultrafiltration suggested an apparent molecular weight between 10 and 30 kDa for the crude inhibitory molecule. Brevicin 27 exhibited a hydrophobic character. A partially purified preparation, resulting from ammonium sulfate precipitation and cation exchange chromatography, permitted confirmation of some characteristics of the bacteriocin, previously established with the crude extract. After treatment of the original brevicin 27-producing strain with novobiocin, a nonproducing mutant was obtained. This mutant was sensitive to brevicin 27, and its plasmid profile revealed the loss of a plasmid of about 3 MDa.     

Regulation of H3K27me3 and H3K4me3 during early porcine embryonic development

The epigenetic marks H3K27me3 and H3K4me3 are important repressive and permissive histone modifications, respectively, which are involved in gene regulation such as Hox gene expression during embryonic development. In this study, we investigated the global levels of these two histone modifications. We also investigated the expression of H3K27me3's methyltransferase (EZH2), EZH2 co‐factors (EED and SUZ12) and demethylases (JMJD3 and UTX), as well as H3K4me3's methylases (ASH1L and MLL1) and demethylase (RBP2) in porcine pre‐implantation embryos. In addition, the expression of Hox genes, HOXA2, HOXA3, HOXA7, HOXA10, HOXB4, HOXB7, HOXC8, HOXD8, and HOXD10 was investigated. We found that global levels of H3K27me3 decreased from the 1‐ to the 4‐cell stage, corresponding to the time of major embryonic genome activation. Subsequently, the levels increased in hatched blastocysts, particularly in the trophectoderm. The expression levels of EZH2, EED, SUZ12, JMJD3, and UTX correlated well with these findings. The global levels of H3K4me3 decreased from the 1‐cell to the morula stage and increased in hatched blastocysts, especially in trophectoderm. A peak in expression of ASH1L was seen at the 4‐cell stage, but overall, expression of ASH1L, MLL1, and RBP2 correlated poorly with H3K4me3. HOXA3, A7, and B4 were expressed in 4‐cell embryos, and HOXA7, A10, B4, and D8 were expressed in hatched blastocysts, and did not correlate well to global methylation of H3K27me3 or H3K4me3. Thus, H3K4me3 may play a role in early porcine embryonic genome activation, whereas, H3K27me3 may be involved in initial cell lineage segregation in the blastocyst. Mol. Reprod. Dev. 77: 540–549, 2010. © 2010 Wiley‐Liss, Inc.     

Antifungal Activities of N-Arylbenzenesulfonamides against Phytopathogens and Control Efficacy on Wheat Leaf Rust and Cabbage Club Root Diseases

A set of N-arylbenzenesulfonamides with various substituents at the arylamine and benzenesulfonyl positions were prepared, and their antifungal properties were measured in vitro against such plant pathogenic fungi as Pythium ultimum, Phytophthora capsici, Rhizoctonia solani, and Botrytis cinerea. Compounds 3, 4, 8, 9, 10, 14, 16, 18, 20, 21, 24 and 27 had antifungal activity over a broad spectrum of the phytopathogenic fungi tested, where 50% of inhibition (ED₅₀) was in the range of 3-15 μg/ml. Based on the in vitro activity, six derivatives (3, 4, 10, 18, 21 and 27) were selected and tested further for their fungicidal efficacy in vivo. The fungicidal efficacy of 10, 21 and 27 had a disease control value of over 85% at 50 μg/ml against wheat leaf rust, while that of 4 was selective against cabbage club root disease.