Genomic checkpoints for exon 10 usage in the luteinizing hormone receptor type 1 and type 2

Alternative splicing is a hallmark of glycoprotein hormone receptor gene regulation, but its molecular mechanism is unknown. The LH receptor (LHR) gene possesses 11 exons, but exon 10 is constitutively skipped in the New World monkey lineage (LHR type 2), whereas it is constitutively spliced in the human (LHR type 1). This study identifies the regulatory elements of exon 10 usage. Sequencing of genomic marmoset DNA revealed that the cryptic LHR exon 10 is highly homologous to exon 10 from other species and displays intact splice sites. Functional studies using a minigene approach excluded the contribution of intronic, marmoset-specific long interspersed nucleotide-1 elements to exon 10 skipping. Sequencing of the genomic regions surrounding exon 10 from several primate lineages, sequence comparisons including the human and mouse LHR gene, revealed the presence of unique nucleotides at 3'-intronic position -19 and -10 and at position +26 within exon 10 of the marmoset LHR. Exon trap experiments and in vitro mutagenesis of these nucleotides resulted in the identification of a composite regulatory element of splicing consisting of cis-acting elements represented by two polypyrimidine tracts and a trans-acting element within exon 10, which affect the secondary RNA structure. Changes within this complex resulted either in constitutive exon inclusion, constitutive skipping, or alternative splicing of exon 10. This work delineates the molecular pathway leading to intronization of exon 10 in the LHR type 2 and reveals, for the first time, the essential function of regulatory and structural elements involved in glycoprotein hormone receptor splicing.     

First report of Phytopythium vexans causing the “Avocado sadness” in Michoacan,Mexico

Mexico is the main producer, consumer and exporter of avocado in the world, being Michoacan the main producer state contributing more than 80% of the national production. There are phytopathogens that decimate the production causing the death of the tree. Root samples were collected in avocado trees that showed the characteristic symptomatology of the disease known as avocado sadness, the sampling was carried out in four of the main avocado producing towns, in the state of Michoacan, Mexico. The isolation consisted in sowing root tissue in Petri dishes with V8^®-PARPH culture medium, subsequently they were identified morphologically and for species level it was determined by molecular biology, with the PCR-ITS technique. Pathogenicity tests were performed in triplicate with avocado seedlings with more than six leaves. After 24 hours, the inoculated plants expressed decay in the apical part, after 120 hours the leaves showed yellowing and after 15 days there was a generalized wilt on the stem and leaves, re-isolating the phytopathogen Phytopythium vexans. This study confirms the first report of the oomycete P. vexans affecting avocado trees in the most important producing region of the Mexican Republic.     

Human gene copy number spectra analysis in congenital heart malformations

The clinical significance of copy number variants (CNVs) in congenital heart disease (CHD) continues to be a challenge. Although CNVs including genes can confer disease risk, relationships between gene dosage and phenotype are still being defined. Our goal was to perform a quantitative analysis of CNVs involving 100 well-defined CHD risk genes identified through previously published human association studies in subjects with anatomically defined cardiac malformations. A novel analytical approach permitting CNV gene frequency "spectra" to be computed over prespecified regions to determine phenotype-gene dosage relationships was employed. CNVs in subjects with CHD (n = 945), subphenotyped into 40 groups and verified in accordance with the European Paediatric Cardiac Code, were compared with two control groups, a disease-free cohort (n = 2,026) and a population with coronary artery disease (n = 880). Gains (≥200 kb) and losses (≥100 kb) were determined over 100 CHD risk genes and compared using a Barnard exact test. Six subphenotypes showed significant enrichment (P ≤ 0.05), including aortic stenosis (valvar), atrioventricular canal (partial), atrioventricular septal defect with tetralogy of Fallot, subaortic stenosis, tetralogy of Fallot, and truncus arteriosus. Furthermore, CNV gene frequency spectra were enriched (P ≤ 0.05) for losses at: FKBP6, ELN, GTF2IRD1, GATA4, CRKL, TBX1, ATRX, GPC3, BCOR, ZIC3, FLNA and MID1; and gains at: PRKAB2, FMO5, CHD1L, BCL9, ACP6, GJA5, HRAS, GATA6 and RUNX1. Of CHD subjects, 14% had causal chromosomal abnormalities, and 4.3% had likely causal (significantly enriched), large, rare CNVs. CNV frequency spectra combined with precision phenotyping may lead to increased molecular understanding of etiologic pathways.     

C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.     

A hydrogen peroxide electrode assay to measure thiol peroxidase activity for organoselenium and organotellurium drugs

Molecular mimics of the enzyme glutathione peroxidase (GPx) are increasingly being evaluated as redox active drugs. Their molecular mechanism of action parallels that of the native enzyme; however, a major distinction is that GPx mimics can use alternative thiol substrates to glutathione. This generic thiol peroxidase activity implies that it is necessary to assess a GPx mimic's recognition of a range of cellular thiols in order to determine its potential therapeutic effects. We report an electrochemical assay that, by measuring the rate of decrease of the peroxide substrate, allows the activity of GPx mimics to be directly compared against an array of thiols. The derived pseudo zero-order rate constants, k(obs), for representative GPx mimics range between 0 and 6.6min(-1) and can vary by more than an order of magnitude depending on the thiol electron donor. An additional advantage of the assay is that it enables synergistic interactions between GPx mimics and cellular proteins to be evaluated. Here we report that glutathione disulfide reductase, which is commonly used to evaluate GPx mimic activity, recognizes the GPx mimic ebselen as a substrate, increasing its apparent k(obs). Therefore, reports relying on glutathione disulfide reductase to evaluate GPx mimic activity may exaggerate drug antioxidant action.     

Nonoverlapping expression of IL10, IL12p40, and IFNgamma mRNA in the marginal zone and T cell zone of the spleen after antigenic stimulation

The differentiation of CD4(+) T cells is regulated by cytokines locally within the compartments of secondary lymphoid organs during adaptive immune responses. Quantitative data about the expression of cytokine mRNAs within the T and B cell zones of lymphoid organs are lacking. In this study, we assessed the expression of multiple cytokine genes within the lymphoid compartments of the spleen of rats after two types of stimulation. First, the spleen was stimulated directly by a blood-derived Ag. Second, the spleen was stimulated indirectly by incoming lymphocytes that had been activated and released during a proceeding immune response at a distant tissue site. Using laser microdissection, we show that the expression of cytokine mRNAs was compartment specific, transient, and preceded cell proliferation after the direct antigenic stimulation. Surprisingly, the indirect stimulation by incoming activated lymphocytes induced similar cytokines in the T cell zone. However, the nonoverlapping expression was lost and IL10 appeared as the major cytokine in all compartments. Thus, tracking two types of immune activation without disturbing the integrity of structures reveals distinct and overlapping events in the compartments of the spleen. This information adds a new dimension to the understanding of immune responses in vivo.     

The human neonatal small intestine has the potential for arginine synthesis; developmental changes in the expression of arginine-synthesizing and -catabolizing enzymes

Background  

Milk contains too little arginine for normal growth, but its precursors proline and glutamine are abundant; the small intestine of rodents and piglets produces arginine from proline during the suckling period; and parenterally fed premature human neonates frequently suffer from hypoargininemia. These findings raise the question whether the neonatal human small intestine also expresses the enzymes that enable the synthesis of arginine from proline and/or glutamine. Carbamoylphosphate synthetase (CPS), ornithine aminotransferase (OAT), argininosuccinate synthetase (ASS), arginase-1 (ARG1), arginase-2 (ARG2), and nitric-oxide synthase (NOS) were visualized by semiquantitative immunohistochemistry in 89 small-intestinal specimens.     

Fermentation characterization and flux analysis of recombinant strains of Clostridium acetobutylicum with an inactivated solR gene

The effect of solR inactivation on the metabolism of Clostridium acetobutylicum was examined using fermentation characterization and metabolic flux analysis. The solR-inactivated strain (SolRH) of this study had a higher rate of glucose utilization and produced higher solvent concentrations (by 25%, 14%, and 81%, respectively, for butanol, acetone, and ethanol) compared to the wild type. Strain SolRH(pTAAD), carrying a plasmid-encoded copy of the bifunctional alcohol/aldehyde dehydrogenase gene (aad) used in butanol production, produced even higher concentrations of solvents (by 21%, 45%, and 62%, respectively, for butanol, acetone, and ethanol) than strain SolRH. Clarithromycin used for strain SolRH maintenance during SolRH(pTAAD) fermentations did not alter product formation; however, tetracycline used for pTAAD maintenance resulted in 90% lower solvent production. Journal of Industrial Microbiology & Biotechnology (2001) 27, 322–328. Received 12 September 2000/ Accepted in revised form 21 July 2001