A new polymorphism in the Growth and Differentiation Factor 9 (GDF9) gene is associated with increased ovulation rate and prolificacy in homozygous sheep

P>Brazilian Santa Ines (SI) sheep are very well-adapted to the tropical conditions of Brazil and are an important source of animal protein. A high rate of twin births was reported in some SI flocks. Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are the first two genes expressed by the oocyte to be associated with an increased ovulation rate in sheep. All GDF9 and BMP15 variants characterized, until now, present the same phenotype: the heterozygote ewes have an increased ovulation rate and the mutated homozygotes are sterile. In this study, we have found a new allele of GDF9, named FecGE (Embrapa), which leads to a substitution of a phenylalanine with a cysteine in a conservative position of the mature peptide. Homozygote ewes presenting the FecGE allele have shown an increase in their ovulation rate (82%) and prolificacy (58%). This new phenotype can be very useful in better understanding the genetic control of follicular development; the mechanisms involved in the control of ovulation rate in mammals; and for the improvement of sheep production.

A new set of microsatellite markers for the genetic characterization of Ceratocystis fimbriata, an economically important plant pathogen

Ceratocystis fimbriata is a fungal pathogen which attacks several economically important plants, but occurs in host-associated, morphologically indistinguishable forms. In Brazil, this fungus seriously attacks mango trees (Mangifera indica), causing severe loss of yield. This work aimed to develop and characterize a novel set of microsatellite markers for this important pathogen, providing researchers with new molecular tools for the characterization of isolates. Twenty polymorphic primer pairs were designed from a microsatellite-enriched library. We tested the usefulness of these markers through genotyping thirteen isolates of the fungus. On average, 6.65 alleles per locus were detected, revealing the ability of this set of markers to characterize C. fimbriata isolates associated to mango and to other plant species.

Development and characterization of microsatellite markers for castor (Ricinus communis L.), an important oleaginous species for biodiesel production

Castor (Ricinus communis L.) is an important oleaginous plant from both economic and social points of view. The seeds contain an oil with excellent properties for industrial uses. This paper presents the main results of a study aiming to develop microsatellite markers for castor. Twelve new polymorphic microsatellite markers were isolated and characterized in 38 genotypes accessions from the castor germplasm of the Brazilian Agricultural Research Company (EMBRAPA). Knowledge on the genetic diversity of castor can be used to gain a better understanding on genetic diversity conservation, and germplasm management, guiding breeding programs and conservation strategies.

Growth-active gibberellins overcome the very slow shoot growth of Hancornia speciosa, an important fruit tree from the Brazilian ""Cerrado""

Shoot elongation of Hancornia speciosa, an endangered tree from the Brazilian savannah ""Cerrado"", is very slow, thus limiting nursery production of plants. Gibberellins (GAs) A(1), A(3), and A(5), and two inhibitors of GA biosynthesis, trinexapac-ethyl and ancymidol were applied to shoots of Hancornia seedlings. GA(1) and GA(3) significantly stimulated shoot elongation, while GA(5) had no significant effect. Trinexapac-ethyl and ancymidol, both at 100 A mu g per seedling, inhibited shoot elongation up to 45 days after treatment, though the effect was statistically significant only for ancymidol. Somewhat surprisingly, exogenous GA(3) more effectively stimulated shoot elongation in SD-grown plants, than in LD-grown plants. The results from exogenous application of GAs and inhibitors of GA biosynthesis imply that Hancornia shoot growth is controlled by GAs, and that level of endogenous growth-active GAs is likely to be the limiting factor for shoot elongation in Hancornia. Application of GAs thus offer a practical method for nursery production of Hancornia seedlings for outplanting into the field.

Hereditary hemochromatosis: Mutations in genes involved in iron homeostasis in Brazilian patients

Background: p.C282Y mutation and rare variants in the HFE gene have been associated with hereditary hemochromatosis (HH). HH is also caused by mutations in other genes, such as the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1). The low rate homozygous p.C282Y mutation in Brazil is suggestive that mutations in non-HFE genes may be linked to HH phenotype. Aim: To screen exon-by-exon DNA sequences of HFE, HJV, HAMP, TFR2 and SLC40A1 genes to characterize the molecular basis of HH in a sample of the Brazilian population. Materials and methods: Fifty-one patients with primary iron overload (transferrin saturation >= 50% in females and >= 60% in males) were selected. Subsequent bidirectional DNA sequencing of HFE, HJV, HAMP, TFR2 and SLC40A1 exons was performed. Results: Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n = 11, 21.6%). In addition, heterozygous HFE p.S65C mutation was found in combination with p.H63D in two patients and homozygous HFE p.H63D was found in two patients as well. Sequencing in the HJV and HAMP genes revealed HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Molecular and clinical diagnosis of juvenile hemochromatosis (homozygous form for the HJV p.G320V) was described for the first time in Brazil. Three TFR2 polymorphisms (p.A75V, p.A617A and p.R752H) and six SLC40A1 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704, rs11568346) and the novel mutation SLC40A1 p.G204S were also found. Conclusions: The HE p.C282Y in homozygosity or in heterozygosity with p.H63D was the most frequent mutation associated with HH in this sample. The HJV p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G2045 mutation may also be linked to primary iron overload but their role in the pathophysiology of HH remain to be elucidated. (C) 2011 Elsevier Inc. All rights reserved.

Hemojuvelin and Hepcidin Genes Sequencing in Brazilian Patients with Primary Iron Overload

Background: Most hereditary hemochromatosis (HH) patients are homozygous for the p. C282Y mutation in the HFE gene. Some studies reported that HH phenotypic expression could be modulated by genetic factors such as HJV and HAMP gene mutations. Aims: The aims of this study were to identify HJV and HAMP mutations and to analyze their impact on HH phenotype in non-p. C282Y homozygous individuals. Methods: Twenty-four Brazilian patients with primary iron overload and non-p. C282Y homozygous genotype (transferrin saturation >50% in women and >60% in men and absence of secondary causes) were selected. Subsequent bidirectional sequencing of the HJV and HAMP exons was performed. Results: Sequencing revealed a substitution in heterozygosis, c. 929C>G, which corresponds to p.A310G polymorphism in HJV exon 4 (rs7540883). In the same gene, in another individual, an IVS1-36C>G intronic variant was detected in heterozygosis. In the HAMP gene, an IVS3 + 42G>A intronic variant was identified. There were six (25.0%) patients carrying a heterozygous genotype for the HFE p. C282Y and nine (37.5%) patients carrying a heterozygous genotype for the HFE p. H63D. Conclusion: HJV p.A310G polymorphism and two intronic variants were found, but none of these alterations were associated with digenic inheritance with the HFE gene. Our data indicate that HJV and HAMP functional mutations are not frequent in these patients.

Effects of Protein-Energy Malnutrition on NF-KappaB Signalling in Murine Peritoneal Macrophages

Protein-energy malnutrition (PEM) is an important public health problem affecting millions of people worldwide. PEM decreases resistance to infection, impairing a number of physiological processes. In unstimulated cells, NF-kappa B is kept from binding to its consensus sequence by the inhibitor I kappa B alpha, which retains NF-kappa B in the cytoplasm. Upon various signals, such as lipopolysaccharide (LPS), I kappa B alpha is rapidly degraded and NF-kappa B is induced to translocate into the nucleus, where it activates expression of various genes that participate in the inflammatory response, including those involved in the synthesis of TNF-alpha. TRAF-6 is a cytoplasmic adapter protein that links the stimulatory signal from Toll like receptor-4 to NF-kappa B. The aim of this study was to evaluate the effect of malnutrition on induction of TNF-a by LPS in murine peritoneal macrophages. We evaluated peritoneal cellularity, the expression of MyD88, TRAF-6, IKK, I kappa B alpha and NF-kappa B, NF-kappa B activation and TNF-alpha mRNA and protein synthesis inmacrophages. Two-month-old male BALB/Cmice were submitted to PEM with a low-protein diet that contained 2% protein, compared to 12% protein in the control diet. When the experimental group had lost about 20% of the original body weight, it was used in the subsequent experiments. Malnourished animals presented anemia, leucopenia and severe reduction in peritoneal cavity cellularity. TNF-a mRNA and protein levels of macrophages stimulated with LPS were significantly lower in malnourished animals. PEM also decreased TRAF-6 expression and NF-kappa B activation after LPS stimulation. These results led us to conclude that PEM changes NF-kappa B signalling pathway in macrophages to LPS stimulus.

Two physically, functionally, and developmentally distinct peritoneal macrophage subsets

The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (Mempty set) are commonly drawn for functional studies. Here we define two Mempty set subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal Mempty set (LPM), contains approximately 90% of the PerC Mempty set in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical Mempty set surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal Mempty set (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of Mempty set heterogeneity and shed new light on PerC Mempty set diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC Mempty set.

Enteroinvasive Escherichia coli vs. Shigella flexneri : how different patterns of gene expression affect virulence

Important features of the enteroinvasive Escherichia coli (EIEC) phenotype and gene expression likely to confer EIEC with a lower ability to cause disease than Shigella flexneri were described here for the first time. To confirm the lower pathogenicity of EIEC, we have analyzed the keratoconjunctivitis developed in guinea-pigs with EIEC or S. flexneri. Shigella flexneri induced a more pronounced proinflammatory response, whereas EIEC induced a mild form of the disease. EIEC showed a significantly less efficient cell-to-cell Caco-2 dissemination when compared with S. flexneri. Plaques formed by EIEC during intercellular spreading were four times smaller than those formed by S. flexneri. At the molecular level, the lower expression of virulence genes by EIEC during infection of Caco-2 cells highlighted the importance of effective gene transcription for bacterial pathogenicity.

Plasmodium vivax recombinant vaccine candidate AMA-1 plays an important role in adaptive immune response eliciting differentiation of dendritic cells

The Apical Membrane Antigen-1 (AMA-1) is a well-characterized and functionally important merozoite protein and is currently considered a major candidate antigen for a malaria vaccine. Previously, we showed that AMA-1 has an influence on cellular immune responses of malaria-naive subjects, resulting in an alternative activation of monocyte-derived dendritic cells and induction of a pro-inflammatory response by stimulated PBMCs. Although there is evidence, from human and animal malaria model systems that cell-mediated immunity may contribute to both protection and pathogenesis, the knowledge on cellular immune responses in vivax malaria and the factors that may regulate this immunity are poorly understood. In the current work, we describe the maturation of monocyte-derived dendritic cells of P. vivax naturally infected individuals and the effect of P. vivax vaccine candidate Pv-AMA-1 on the immune responses of the same donors. We show that malaria-infected subjects present modulation of DC maturation, demonstrated by a significant decrease in expression of antigen-presenting molecules (CD1a, HLA-ABC and HLA-DR), accessory molecules (CD40, CD80 and CD86) and Fc gamma RI (CD64) receptor (P <= 0.05). Furthermore, Pv-AMA-1 elicits an upregulation of CD1a and HLA-DR molecules on the surface of monocyte-derived dendritic cells (P=0.0356 and P=0.0196, respectively), and it is presented by AMA-1-stimulated DCs. A significant pro-inflammatory response elicited by Pv-AMA-1-pulsed PBMCs is also demonstrated, as determined by significant production of TNF-alpha, IL-12p40 and IFN-gamma (P <= 0.05). Our results suggest that Pv-AMA-1 may partially revert DC down-modulation observed in infected subjects, and exert an important role in the initiation of pro-inflammatory immunity that might contribute substantially to protection. (c) 2009 Elsevier Ltd. All rights reserved.

Transcript profiling of papaya fruit reveals differentially expressed genes associated with fruit ripening

Papaya (Carica papaya L) fruit has a short shelf life due to fast ripening induced by ethylene, but little is known about the genetic control of ripening and attributes of fruit quality. Therefore, we identified ripening-related genes affected by ethylene using cDNA-AFLP (Amplified Fragment Length Polymorphism of cDNA). Transcript profiling of non-induced and ethylene-induced fruit samples was performed, and 71 differentially expressed genes were identified. Among those genes some involved in ethylene biosynthesis, regulation of transcription, and stress responses or plant defence were found (heat shock proteins, polygalacturonase-inhibiting protein, and acyl-CoA oxidases). Several transcription factors were isolated, and except for a 14-3-3 protein, an AP2 domain-containing factor, a salt-tolerant zinc finger protein, and a suppressor of PhyA-105 1, most of them were negatively affected by ethylene, including fragments of transcripts similar to VRN1, and ethylene responsive factors (ERF). With respect to fruit quality, genes related to cell wall structure or metabolism, volatiles or pigment precursors, and vitamin biosynthesis were also found. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Expression patterns of cell wall-modifying genes from banana during fruit ripening and in relationship with finger drop

Few molecular studies have been devoted to the finger drop process that occurs during banana fruit ripening. Recent studies revealed the involvement of changes in the properties of cell wall polysaccharides in the pedicel rupture area. In this study, the expression of cell-wall modifying genes was monitored in peel tissue during post-harvest ripening of Cavendish banana fruit, at median area (control zone) and compared with that in the pedicel rupture area (drop zone). To this end, three pectin methylesterase (PME) and seven xyloglucan endotransglycosylase/hydrolase (XTH) genes were isolated. The accumulation of their mRNAs and those of polygalaturonase, expansin, and pectate lyase genes already isolated from banana were examined. During post-harvest ripening, transcripts of all genes were detected in both zones, but accumulated differentially. MaPME1, MaPG1, and MaXTH4 mRNA levels did not change in either zone. Levels of MaPME3 and MaPG3 mRNAs increased greatly only in the control zone and at the late ripening stages. For other genes, the main molecular changes occurred 1-4 d after ripening induction. MaPME2, MaPEL1, MaPEL2, MaPG4, MaXTH6, MaXTH8, MaXTH9, MaEXP1, MaEXP4, and MaEXP5 accumulated highly in the drop zone, contrary to MaXTH3 and MaXTH5, and MaEXP2 throughout ripening. For MaPG2, MaXET1, and MaXET2 genes, high accumulation in the drop zone was transient. The transcriptional data obtained from all genes examined suggested that finger drop and peel softening involved similar mechanisms. These findings also led to the proposal of a sequence of molecular events leading to finger drop and to suggest some candidates.

Farnesol induces the transcriptional accumulation of the Aspergillus nidulans Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase

Farnesol (FOH) is a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, a catabolite of the cholesterol biosynthetic pathway. These isoprenoids inhibit proliferation and induce apoptosis. It has been shown previously that FOH triggers morphological features characteristic of apoptosis in the filamentous fungus Aspergillus nidulans. Here, we investigate which pathways are influenced through FOH by examining the transcriptional profile of A. nidulans exposed to this isoprenoid. We observed decreased mRNA abundance of several genes involved in RNA processing and modification, transcription, translation, ribosomal structure and biogenesis, amino acid transport and metabolism, and ergosterol biosynthesis. We also observed increased mRNA expression of genes encoding a number of mitochondrial proteins and characterized in detail one of them, the aifA, encoding the Apoptosis-Inducing Factor (AIF)-like mitochondrial oxidoreductase. The Delta aifA mutant is more sensitive to FOH (about 8.0% and 0% survival when exposed to 10 and 100 mu M FOH respectively) than the wild type (about 97% and 3% survival when exposed to 10 and 100 mu M FOH respectively). These results suggest that AifA is possibly important for decreasing the effects of FOH and reactive oxygen species. Furthermore, we showed an involvement of autophagy and protein kinase C in A. nidulans FOH-induced apoptosis.

Functional characterization of the Aspergillus fumigatus CRZ1 homologue, CrzA

The protein phosphatase calcineurin is an important mediator connecting calcium-dependent signalling to various cellular responses in multiple organisms. In fungi calcineurin acts largely through regulating Crz1p-like transcription factors. Here we characterize an Aspergillus fumigatus CRZ1 homologue, CrzA and demonstrate its mediation of cellular tolerance to increased concentrations of calcium and manganese. In addition to acute sensitivitiy to these ions, and decreased conidiation, the crzA null mutant suffers altered expression of calcium transporter mRNAs under high concentrations of calcium, and loss of virulence when compared with the corresponding complemented and wild-type strains. We use multiple expression analyses to probe the transcriptional basis of A. fumigatus calcium tolerance identifying several genes having calA and/or crzA dependent mRNA accumulation patterns. We also demonstrate that contrary to previous findings, the gene encoding the Aspergillus nidulans calcineurin subunit homologue, cnaA, is not essential and that the cnaA deletion mutant shares the morphological phenotypes observed in the corresponding A. fumigatus mutant, Delta calA. Exploiting the A. nidulans model system, we have linked calcineurin activity with asexual developmental induction, finding that CrzA supports appropriate developmental induction in a calcineurin and brlA-dependent manner in both species.

Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA

Nucleotide excision repair (NER) eliminates helix-distorting DNA base lesions. Seven XP-deficient genetic complementation groups (XPA to XPG) have already been identified in mammals, and their corresponding genes have been cloned. Hereditary defects in NER are associated with several diseases, including xeroderma pigmentosum (XP). UV-DDB (XPE) is formed by two associated subunits, DDB1 and DDB2. UV-DDB was identified biochemically as a protein factor that exhibits very strong and specific binding to ultraviolet (UV)-treated DNA. As a preliminary step to characterize the components of the NER in the filamentous fungus Aspergillus nidulans, here we identified a putative DDB1 homologue, DdbA. Deletion and expression analysis indicated that A. nidulans ddbA gene is involved in the DNA damage response, more specifically in the UV light response and 4-nitroquinoline oxide (4-NQO) sensitivity. Furthermore, the Delta ddbA strain cannot self-cross and expression analysis showed that ddbA can be induced by oxidative stress and is developmentally regulated in both asexual and sexual processes. The Delta ddbA mutation can genetically interact with uvsB(ATR), atmA(ATM), nkuA(KU70), H2AX-S129A (a replacement of the conserved serine in the C-terminal of H2AX with alanine), and cshB (a mutation in CSB Cockayne`s syndrome protein involved in the transcription-coupled repair subpathway of NER) mutations. Finally, to determine the DdbA cellular localization, we constructed a GFP:DdbA strain. In the presence and absence of DNA damage, DdbA was mostly detected in the nuclei, indicating that DdbA localizes to nuclei and its cellular localization is not affected by the cellular response to DNA damage induced by 4-NQO and UV light.