The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics

Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the P-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins. (C) 2009 Elsevier Inc. All rights reserved.

Conservation of dual-targeted proteins in Arabidopsis and rice points to a similar pattern of gene-family evolution

Gene duplication followed by acquisition of specific targeting information and dual targeting were evolutionary strategies enabling organelles to cope with overlapping functions. We examined the evolutionary trend of dual-targeted single-gene products in Arabidopsis and rice genomes. The number of paralogous proteins encoded by gene families and the dual-targeted orthologous proteins were analysed. The number of dual-targeted proteins and the corresponding gene-family sizes were similar in Arabidopsis and rice irrespective of genome sizes. We show that dual targeting of methionine aminopeptidase, monodehydroascorbate reductase, glutamyl-tRNA synthetase, and tyrosyl-tRNA synthetase was maintained despite occurrence of whole-genome duplications in Arabidopsis and rice as well as a polyploidization followed by a diploidization event (gene loss) in the latter.

Immunohistochemical detection of polyductin and co-localization with liver progenitor cell markers during normal and abnormal development of the intrahepatic biliary system and in adult hepatobiliary carcinomas

The longest open reading frame of PKHD1 (polycystic kidney and hepatic disease 1), the autosomal recessive polycystic kidney disease (ARPKD) gene, encodes a single-pass, integral membrane protein named polyductin or fibrocystin. A fusion protein comprising its intracellular C-terminus, FP2, was previously used to raise a polyclonal antiserum shown to detect polyductin in several human tissues, including liver. In the current study, we aimed to investigate by immunohistochemistry the detailed polyductin localization pattern in normal (ductal plate [DP], remodelling ductal plate [RDP], remodelled bile ducts) and abnormal development of the primitive intrahepatic biliary system, known as ductal plate malformation (DPM). This work also included the characterization of polyductin expression profile in various histological forms of neonatal and infantile cholestasis, and in cholangiocellular carcinoma (CCC) and hepatocellular carcinoma (HCC). We detected polyductin expression in the intrahepatic biliary system during the DP and the RDP stages as well as in DPM. No specific staining was found at the stage of remodelled bile ducts. Polyductin was also detected in liver biopsies with neonatal cholestasis, including mainly biliary atresia and neonatal hepatitis with ductular reaction as well as congenital hepatic fibrosis. In addition, polyductin was present in CCC, whereas it was absent in HCC. Polyductin was also co-localized in some DP cells together with oval stem cell markers. These results represent the first systematic study of polyductin expression in human pathologies associated with abnormal development of intrahepatic biliary tree, and support the following conclusions: (i) polyductin expression mirrors developmental properties of the primitive intrahepatic biliary system; (ii) polyductin is re-expressed in pathological conditions associated with DPM and (iii) polyductin might be a potential marker to distinguish CCC from HCC.

Spectrum and Prevalence of FP/TMEM127 Gene Mutations in Pheochromocytomas and Paragangliomas

Context Pheochromocytomas and paragangliomas are genetically heterogeneous neural crest-derived neoplasms. We recently identified germline mutations of the novel transmembrane-encoding gene FP/TMEM127 in familial and sporadic pheochromocytomas consistent with a tumor suppressor effect. Objectives To examine the prevalence and spectrum of FP/TMEM127 mutations in pheochromocytomas and paragangliomas and to test the effect of mutations in vitro. Design, Setting, and Participants We sequenced the FP/TMEM127 gene in 990 individuals with pheochromocytomas and/or paragangliomas, including 898 previously unreported cases without mutations in other susceptibility genes from 8 independent worldwide referral centers between January 2009 and June 2010. A multiplex polymerase chain reaction-based method was developed to screen for large gene deletions in 545 of these samples. Confocal microscopy of 5 transfected mutant proteins was used to determine their subcellular localization. Main Outcome Measures The frequency and type of FP/TMEM127 mutation or deletion was assessed and correlated with clinical variables; the subcellular localization of 5 overexpressed mutants was compared with wild-type FP/TMEM127 protein. Results We identified 19 potentially pathogenic FP/TMEM127 germline mutations in 20 independent families, but no large deletions were detected. All mutation carriers had adrenal tumors, including 7 bilateral (P=2.7 x 10(-4)) and/or with familial disease (5 of 20 samples; P=.005). The median age at disease onset in the FP/TMEM127 mutation group was similar to that of patients without a mutation (41.5 vs 45 years, respectively; P=.54). The most common presentation was that of a single benign adrenal tumor in patients older than 40 years. Malignancy was seen in 1 mutation carrier (5%). Expression of 5 novel FP/TMEM127 mutations in cell lines revealed diffuse localization of the mutant proteins in contrast with the discrete multiorganelle distribution of wild-type TMEM127. Conclusions Germline mutations of FP/TMEM127 were associated with pheochromocytoma but not paraganglioma and occured in an age group frequently excluded from genetic screening algorithms. Disease-associated mutations disrupt intracellular distribution of the FP/TMEM127 protein. JAMA. 2010;304(23):2611-2619 www.jama.com

Identification and chromosomal localization of one locus of Leishmania (L.) major related with resistance to itraconazole

Ergosterol is an important compound responsible to maintain integrity and fluidity of Leishmania spp. membranes. Starting from an overexpression/selection method, our group has isolated and mapped nine different loci of Leishmania (L.) major related to resistance against two inhibitors of the ergosterol biosynthesis pathway, terbinafine (TBF) and itraconazole (ITZ). Individual functional analysis after overexpression induction of these loci in the presence of TBF and/or ITZ [or the ITZ analog ketoconazole (CTZ)] have shown low but significant levels of resistance after transfection into L. major wild-type parasites. In this work, we have shown the insert mapping and chromosomal identification of one of these loci (cosItz2). Functional analysis experiments associated with chromosomal localization by comparison at genomic database allowed us to identify two prospective gene-protein systems not related to the ergosterol biosynthesis and capable to confer wild-type cells resistance to ITZ-CTZ after transfection. We expected that this approach can open new insights for a better understanding of mechanisms of ITZ-CTZ action and resistance in Leishmania resulting in new strategies for the leishmaniasis treatment.

Effect of a thrombin receptor (protease-activated receptor 1, PAR-1) gene polymorphism in chronic hepatitis C liver fibrosis

Background and Aim: Tissue injury leads to activation of coagulation and generation of thrombin. Inhibition of thrombin receptor protease-activated receptor 1 (PAR-1) has been shown to reduce liver fibrosis in animals. This study aimed to evaluate the effect of PAR-1 gene polymorphism on rate of liver fibrosis (RF) in chronic hepatitis C. Methods: Polymorphisms studied: C > T transition 1426 bp upstream of translation start site (-1426C/T), 13 bp repeat of preceding -506 5`-CGGCCGCGGGAAG-3` sequence (-506I/D), and A > T transversion in intervening sequence (IVS) 14 bp upstream of exon-2 start site (IVS-14A/T). A total of 287 European and 90 Brazilian patients were studied. Results: 1426C/T polymorphism: There was a trend to higher RF in patients with the TT genotype (P = 0.06) and an association between genotype CC and slow fibrosis (P = 0.03) in Europeans. In males, RF was significantly higher in those with the TT genotype compared to CT (P = 0.003) and CC (P = 0.007). There was a significant association between TT and fast fibrosis (P = 0.04). This was confirmed in an independent cohort of Brazilians where RF was higher in TT than in CC (P = 0.03). Analysis of -506I/D showed no difference in RF and distribution of slow/fast fibrosis among different genotypes in both populations. Analysis of IVS-14A/T showed no difference between genotypes. Conclusion: In conclusion, these findings suggest that PAR-1 receptor polymorphisms influence the progression of liver fibrosis.

Detection of TGIF1 homeobox gene in oral squamous cell carcinoma according to histologic grading

Objective. TGIF1 homeobox gene involvement in oral cancer has not yet been investigated. This study analyzed the expression of TGIF1 transcripts and protein in oral squamous cell carcinoma (OSCC). Study design. Snap-frozen samples from 16 patients were taken from both OSCC and nontumoral adjacent epithelium (NT) for in situ hybridization (ISH). Forty-six paraffin-embedded samples of OSCC were submitted to immunohistochemistry (IHC). A descriptive analysis of the transcript signal detection was accomplished, and TGIF1 immunoexpression was carried out considering protein levels, localization, and cellular differentiation. Results. ISH reactions showed TGIF1 transcripts with a signal that was frequently intense in NT, and generally weak in OSCC, and that had stronger transcript signal in well-differentiated areas of OSCC when compared with poorly differentiated ones. IHC reactions had poorly differentiated cases associated with TGIF1 protein expression in both the nucleus and cytoplasm (P = .05, Fisher test). Conclusions. TGIF1 gain or loss of function might possibly play a role in oral cancer cell differentiation. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 111: 218-224)

Conceptus-mediated endometrial vascular changes during early pregnancy in mares: an anatomic, histomorphometric, and vascular endothelial growth factor receptor system immunolocalization and gene expression study

This work examined how the conceptus modulates endometrial tissue remodeling and vascular development prior to implantation in mares. A macroscopic uterine examination was completed at day 21 of pregnancy. In situ morphology revealed that the endometrium involved in encroachment is restricted to the dorsal endometrium immediately overlying the yolk sac. The amount of stromal area occupied by blood vessels and the number of endometrial glands were increased during early pregnancy. Endometrial histomorphometry as well as the endometrial mRNA abundance and immunolocalization of VEGF, VEGFR1, VEGFR2, and Ki-67 was completed at days 14 and 21 of pregnancy, at day 10 of the estrous cycle, and during estrus. No obvious differences in VEGF and VEGFR1 protein localization were detected between pregnant and cycling mares but differential staining pattern for VEGFR2 and Ki-67 was observed. VEGFR2 localized to luminal and glandular epithelium of pregnant mares, while luminal epithelium was negative in cycling mares. Ki-67 staining was weak during the luteal phase but exhibited prominent luminal epithelium staining during estrus. In pregnant mares, all endometrial layers were Ki-67 positive. Quantitative RT-PCR revealed a greater abundance of VEGF mRNA during pregnancy. VEGFR2 transcript abundance was greatest in pregnant mares on day 21. This study supports the concept that the conceptus plays an active role in directing vasculogenesis within the uterus and thereby establishing hemotrophic nutrition that supports pregnancy after implantation. Reproduction (2011) 142 593-603

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis Of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. For the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.

Gene Mapping of 18S and 5S rDNA Genes in the Karyotype of the Three-Spot Gourami Trichogaster trichopterus (Perciformes, Osphronemidae)

Ornamental fish culture is important as an economic activity and for biodiversity conservation as well. The species of the genus Trichogaster (Perciformes, Osphronemidae), popularly known as three-spot gourami, are among the several commercial species raised around the world. In the present work, eight specimens of Thrichogaster trichopterus from aquarium trade facilities were analyzed. The karyotype was composed of 23 pairs of subtelo/acrocentric chromosomes. Fluorescent in situ hybridization allowed identifying the 18S ribosomal gene at telomeric region on long arms of the largest acrocentric pair. On the other hand, the 5S rRNA gene is located at a proximal region on a pair of medium-sized chromosomes. Such information is extremely useful in face of the risks of introduction and the development of ornamental fish trade, once many fish species can be identified only by genetic studies.

Chromosomal studies in four species of genus Chaunus (Bufonidae, Anura): localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) using telomeric and ribosomal sequences was performed in four species of toad genus Chaunus: C. ictericus, C. jimi, C. rubescens and C. schneideri. Analyses based on conventional, C-banding and Ag-NOR staining were also carried out. The four species present a 2n = 22 karyotype, composed by metacentric and submetacentric chromosomes, which were indistinguishable either after conventional staining or banding techniques. Constitutive heterochromatin was predominantly located at pericentromeric regions, and telomeric sequences (TTAGGG)(n) were restricted to the end of all chromosomes. Silver staining revealed Ag-NORs located at the short arm of pair 7, and heteromorphism in size of NOR signals was also observed. By contrast, FISH with ribosomal probes clearly demonstrated absence of any heteromorphism in size of rDNA sequences, suggesting that the difference observed after Ag-staining should be attributed to differences in chromosomal condensation and/or gene activity rather than to the number of ribosomal cistrons.

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

Interaction of Mycoplasma genitalium with host cells: evidence for nuclear localization

Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.

Antimicrobial activity in the tick Rhipicephalus (Boophilus) microplus eggs: Cellular localization and temporal expression of microplusin during oogenesis and embryogenesis

Arthropods display different mechanisms to protect themselves against infections, among which antimicrobial peptides (AMPs) play an important role, acting directly against invader pathogens. We have detected several factors with inhibitory activity against Candida albicans and Micrococcus luteus on the surface and in homogenate of eggs of the tick Rhipicephalus (Boophilus) microplus. One of the anti-M. luteus factors of the egg homogenate was isolated to homogeneity. Analysis by electrospray mass spectrometry (ESI-MS) revealed that it corresponds to microplusin, an AMP previously isolated from the cell-free hemolymph of X (B.) microplus. Reverse transcription (RT) quantitative polymerase chain reactions (qPCR) showed that the levels of microplusin mRNA gradually increase along ovary development, reaching an impressive highest value three days after the adult females have dropped from the calf and start oviposition. Interestingly, the level of microplusin mRNA is very low in recently laid eggs. An enhance of microplusin gene expression in eggs is observed only nine days after the onset of oviposition, achieving the highest level just before the larva hatching, when the level of expression decreases once again. Fluorescence microscopy analysis using an anti-microplusin serum revealed that microplusin is present among yolk granules of oocytes as well as in the connecting tube of ovaries. These results, together to our previous data. suggest that microplusin may be involved not only in protection of adult female hemocele, but also in protection of the female reproductive tract and embryos, what points this AMP as a considerable target for development of new methods to control R. (B.) microplus as well as the vector-borne pathogens. (c) 2009 Elsevier Ltd. All rights reserved.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (F) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines. (c) 2009 Elsevier B.V. All rights reserved.