ABSTRACT. The serotoninergic system has been implicated in the etiology of schizophrenia and other behavioral disorders. Association studies have focused on the tryptophan hydroxylase 2 gene (TPH2) and the 5-hydroxytryptamine receptor 2A gene (5-HTR2A). We genotyped two single-nucleotide polymorphisms, A1438G of 5-HTR2A and intronic rs1386494 of TPH2 in the Malay population, using a sample size of 289 schizophrenic patients and 130 healthy controls. We found a significant association of A1438G of 5-HTR2A with schizophrenia in Malays. On the other hand, TPH2 polymorphism was not associated with schizophrenia. This is the first genetic association study concerning schizophrenia in the Malay population.

ABSTRACT. Cacao (Theobroma cacao) is one of the most important tropical crops; however, production is threatened by numerous pathogens, including the hemibiotrophic fungus Moniliophthora perniciosa, which causes witches’ broom disease. To understand the mechanisms that lead to the development of this disease in cacao, we focused our attention on cacao transcription factors (TFs), which act as master regulators of cellular processes and are important for the fine-tuning of plant defense responses. We developed a macroarray with 88 TF cDNA from previously obtained cacao-M. perniciosa interaction libraries. Seventy-two TFs were found differentially expressed between the susceptible (Catongo) and resistant (TSH1188) genotypes and/or during the disease time course - from 24 h to 30 days after infection. Most of the differentially expressed TFs belonged to the bZIP, MYB and WRKY families and presented opposite expression patterns in susceptible and resistant cacao-M. perniciosa interactions (i.e., up-regulated in Catongo and down-regulated in TSH1188). The results of the macroarray were confirmed for bZIP and WRKY TFs by real-time PCR. These differentially expressed TFs are good candidates for subsequent functional analysis as well as for plant engineering. Some of these TFs could also be localized on the cacao reference map related to witches’ broom resistance, facilitating the breeding and selection of resistant cacao trees.

ABSTRACT. The pathogenic fungus Fusarium graminearum is an ongoing threat to agriculture, causing losses in grain yield and quality in diverse crops. Substantial progress has been made in the identification of genes involved in the suppression of phytopathogens by antagonistic microorganisms; however, limited information regarding responses of plant pathogens to these biocontrol agents is available. Gene expression analysis was used to identify differentially expressed transcripts of the fungal plant pathogen F. graminearum under antagonistic effect of the bacterium Pantoea agglomerans. A macroarray was constructed, using 1014 transcripts from an F. graminearum cDNA library. Probes consisted of the cDNA of F. graminearum grown in the presence and in the absence of P. agglomerans. Twenty-nine genes were either up (19) or down (10) regulated during interaction with the antagonist bacterium. Genes encoding proteins associated with fungal defense and/or virulence or with nutritional and oxidative stress responses were induced. The repressed genes coded for a zinc finger protein associated with cell division, proteins containing cellular signaling domains, respiratory chain proteins, and chaperone-type proteins. These data give molecular and biochemical evidence of response of F. graminearum to an antagonist and could help develop effective biocontrol procedures for pathogenic plant fungi.

ABSTRACT. In order to determine whether Pro12Ala polymorphism of the peroxisome proliferator-activated receptor γ2 (PPARγ2) gene contributes to susceptibility to primary hypertension and metabolic lipid disorders, 482 unrelated subjects from Inner Mongolia were studied, including 137 healthy normotensive (controls) and 345 hypertensive subjects. PCR-RFLP was used to determine the genotypes of Pro12Ala variants of the PPARγ2 gene, and direct sequencing was used to check the results. The frequency of the Ala allele was lower in patients with hypertension (1.3%) than in controls (3.6%). The incidence of the Ala allele was significantly lower in patients with hypertension (P = 0.018) and in those with elevated blood lipids (P = 0.040), compared to the control group. Total plasma cholesterol, triglycerides and high-density lipoprotein cholesterol were significantly higher (P < 0.05), and low-density lipoprotein cholesterol was significantly lower (P < 0.05) in primary hypertension patients than in the control group. We conclude that the Ala allele is involved in genetic susceptibility to hypertension and metabolic lipid disorders in the Han population of Inner Mongolia.

ABSTRACT. cDNA fragments of lea3 genes with a high GC content (from 68 to 77%) were found in several Poaceae, including Sorghum vulgare, Saccharum officinarum, Oryza officinalis, Oryza meyeriana, Ampelocalamus calcareus, Cynodon dactylon, and Zizania latifoli. They were successfully isolated by means of optimal experimental parameters, which included dimethyl sulfoxide as additive and degenerate primers “AGETKAS” and “AGKDKTG”, and their sequences were analyzed. Compared to the method of isolating genes by screening of a cDNA library using abscisic acid- and other stress-responsive cDNA clones, which is time-consuming and costly, this method is relatively easy and inexpensive. Using this new method, many new homologue lea3 genes were rapidly determined.

ABSTRACT. Peroxisome proliferator-activated receptor delta (PPAR-δ) is a transcription factor implicated in metabolism and inflammation. The +294T/C polymorphism in the PPAR-δ gene is associated with risk of coronary artery disease (CAD) in dyslipidemic women and hypercholesterolemic men. Whether this polymorphism influences the risk of CAD in the absence of dyslipidemia was not known, so we investigated a possible association of this polymorphism with plasma lipid and lipoprotein levels and with risk and outcome of CAD in a normolipidemic Tunisian population. Genotyping was performed by PCR-RFLP in 112 CAD patients and 113 healthy volunteers. The C-allele was significantly more frequent in patients than in controls (0.320 vs 0.189, P = 0.001). This association remained significant after adjustment for age, gender, body mass index, smoking, hypertension, and high-density lipoprotein cholesterol. Subjects carrying either one or two copies of the C-allele had a 2.7-fold higher risk of CAD than subjects homozygous for the T-allele. PPAR-δ genotypes were not associated with lipoprotein concentrations or outcome of CAD. We conclude that PPAR-δ +294T/C polymorphism is an independent risk factor of CAD in normolipidemic Tunisian subjects. The lack of association with lipoprotein concentrations suggests that the effect of the polymorphism on CAD is not mediated through lipoprotein levels in this population and that it may influence the atherosclerotic process through mechanisms involving inflammation.

ABSTRACT. Three DNA extraction protocols were compared for their ability to yield DNA from the leaves of herbarium specimens of nine species from nine genera of the Papilionoideae. We tested two protocols that use classic procedures for lysis and purification with cetyl trimethylammonium bromide (CTAB); a third protocol used a Nucleospin Plant kit. DNA obtained from all three procedures was quantified and tested by PCR. Test results indicated the superiority of one of the CTAB protocols. We made some modifications, developing a protocol that produced high-quality DNA from all nine species. The modification involved the use of a lower EDTA concentration (20 mM instead of 50 mM) and a higher β-mercaptoethanol concentration (1% instead of 0.4%) in the extraction buffer. The modified protocol avoids the necessity for a second DNA precipitation step. This new CTAB protocol includes the use of 1.4 M NaCl, 20 mM EDTA and 1% β-mercaptoethanol in the extraction; DNA precipitation time is reduced. A reduction in contaminating metabolites (such as PCR inhibitors) in the sample mixtures and lower costs for reagents are characteristics of this modified protocol; the cost of analysis per sample was lowered, compared to previous options. The quality of DNA was suitable for PCR amplification. This is a practical alternative to more difficult, time-consuming and expensive protocols.

ABSTRACT. Although they are of economic importance, there have been few cytogenetic studies of the Gerridae (Heteroptera) in Brazil. We examined spermatogenesis (meiosis and spermiogenesis) and nucleolar behavior in three species of the family Gerridae. Brachymetra albinerva and Halobatopsis platensis were found to have a chromosome complement of 2n = 25 (24A + X0) and Cylindrostethus palmaris 2n = 29 (28A + X0) chromosomes. Fifteen individuals of these species were collected from the reservoir of São José do Rio Preto, SP, using screens and were transported in pots containing water to the laboratory, where cytogenetic preparations were made. The polyploidy nuclei are formed by several heteropyknotic regions; cells in meiotic prophase have a heteropyknotic region that is probably the sex chromosome, and the chromosomes from chiasmata. The spermatids are rounded and have a heteropyknotic region at the periphery of the nucleus; the sperm head is small, with a long tail. Silver impregnation of meiotic cells showed one or more disorganized bodies around the perichromosomal sheath. The round spermatids had two bodies next to each other, but these were elongated; one of the bodies remained in the head and the other migrated to the initial part of the tail at the end of spermagenesis, when the staining was no longer evident. The meiotic cells appear during spermatogenesis and have very similar silver-impregnation patterns in different species of Heteroptera.

ABSTRACT. Some factors complicate comparisons between linkage maps from different studies. This problem can be resolved if measures of precision, such as confidence intervals and frequency distributions, are associated with markers. We examined the precision of distances and ordering of microsatellite markers in the consensus linkage maps of chromosomes 1, 3 and 4 from two F₂ reciprocal Brazilian chicken populations, using bootstrap sampling. Single and consensus maps were constructed. The consensus map was compared with the International Consensus Linkage Map and with the whole genome sequence. Some loci showed segregation distortion and missing data, but this did not affect the analyses negatively. Several inversions and position shifts were detected, based on 95% confidence intervals and frequency distributions of loci. Some discrepancies in distances between loci and in ordering were due to chance, whereas others could be attributed to other effects, including reciprocal crosses, sampling error of the founder animals from the two populations, F₂ population structure, number of and distance between microsatellite markers, number of informative meioses, loci segregation patterns, and sex. In the Brazilian consensus GGA1, locus LEI1038 was in a position closer to the true genome sequence than in the International Consensus Map, whereas for GGA3 and GGA4, no such differences were found. Extending these analyses to the remaining chromosomes should facilitate comparisons and the integration of several available genetic maps, allowing meta-analyses for map construction and quantitative trait loci (QTL) mapping. The precision of the estimates of QTL positions and their effects would be increased with such information.

ABSTRACT. The family Heliconiaceae contains a single genus, Heliconia, with approximately 180 species of Neotropical origin. This genus was formerly allocated to the family Musaceae, but today forms its own family, in the order Zingiberales. The combination of inverted flowers, a single staminode and drupe fruits is an exclusive characteristic of Heliconia. Heliconias are cultivated as ornamental garden plants, and are of increasing importance as cut flowers. However, there are taxonomic confusions and uncertainties about the number of species and the relationships among them. Molecular studies are therefore necessary for better understanding of the species boundaries of these plants. We examined the genetic variability and the phylogenetic relationships of 124 accessions of the genus Heliconia based on RAPD markers. Phenetic and cladistic analyses, using 231 polymorphic RAPD markers, demonstrated that the genus Heliconia is monophyletic. Groupings corresponding to currently recognized species and some subgenera were found, and cultivars and hybrids were found to cluster with their parents. RAPD analysis generally agreed with morphological species classification, except for the position of the subgenus Stenochlamys, which was found to be polyphyletic.

ABSTRACT. Mytilus coruscus is one of the most important cultured species of marine shellfish in China. Using an expressed sequence tag-library and two microsatellite-enriched genomic libraries of M. coruscus, we isolated and characterized 12 polymorphic microsatellites in a test population; the number of alleles ranged from three to seven, and the observed and expected heterozygosities ranged from 0.0333 to 0.8571 and from 0.3452 to 0.8267, respectively. Four loci deviated from Hardy-Weinberg equilibrium. These polymorphic microsatellite loci can be employed in genetic diversity analysis and molecular marker-assisted breeding of M. coruscus.

ABSTRACT. We determined whether ADRβ3 polymorphism is associated with obesity-related traits in 140 obese patients. Molecular analysis was performed by PCR and RFLP. Individuals carrying the Arg64 allele had a lower waist-to-hip ratio, higher adiponectin and high-density lipoprotein cholesterol levels, and a tendency towards lower blood pressure. In contrast, Trp64/Trp64 carriers were at greater risk for dysmetabolic phenotypes (odds ratio = 2.88, P = 0.03).

ABSTRACT. Crossbreeding is an efficient means to increase production and quality in plants; however, hybridization is seldom reported in bamboo. We crossbred two bamboo species Phyllostachys kwangsiensis (female parent) and Phyllostachys bambusoides (male parent) for the first time, and obtained suspected bamboo hybrids. We identified two bamboo hybrids from the above parents using PCR/ISSR. We concluded that ISSR markers are useful to identify bamboo hybrids, and that breeding between bamboo species is possible and useful.

ABSTRACT. Testicular sperm extraction (TESE) associated with intracytoplasmic sperm injection has allowed many men presenting non-obstructive azoospermia to achieve fatherhood. Microdissection TESE (microTESE) was proposed as a method to improve sperm retrieval rates in these patients; however, there have been failures. Little is known about whether microTESE leads to spermatogenic alterations in the contralateral testis. We assessed histological outcomes of experimental microTESE in the contralateral testis of adult male rabbits. Nine adult male rabbits were divided into three groups: control (testicular biopsy to observe normal histological and morphometric values), sham (incision of the tunica vaginalis, and a contralateral testicular biopsy to observe histological and morphometric patterns, 45 days later), and study (left testicular microTESE, and a right testicular biopsy to observe histological and morphometric patterns, 45 days later). Sections were assessed by calculating Johnsen-like scores, and measuring total tubule diameter, lumen diameter and epithelial height. The results were compared using ANOVA and Bonferroni’s statistical analysis. Morphometric evaluation of the seminiferous tubules did not demonstrate differences between the three groups. However, microTESE caused spermatogenic alterations, leading to maturation arrest in the contralateral testis.

ABSTRACT. Genetic diversity analysis of chickpea germplasm can provide practical information for the selection of parental material and thus assist in planning breeding strategies. Chickpea seed is a good source of carbohydrates and proteins, constituting 80% of the total dry seed weight. Released cultivars and advanced lines of 30 chickpea genotypes were subjected to RAPD analysis for assessment of genetic diversity. We used 16 RAPD primers. Amplification of genomic DNA of the 30 genotypes yielded 62 fragments that could be scored. The number of amplification products produced per primer varied from two to four, with a mean of three bands. The total number of bands amplified by 16 anchored primers varied from 16 to 34. The primer GLK-15 produced the largest number (N = 4) of fragments, whereas primers GLK-19 and GLD-19 produced the smallest number (N = 1) of fragments. The single band produced by the GTGTGCCCCA primer in the PB-2000 and 07005 genotypes may be attributed to temperature tolerance phenotypes.

ABSTRACT. Pathogenicity of strains of the entomopathogenic fungus Beauveria bassiana and endophytic strains of Beauveria sp against the bovine tick Rhipicephalus (Boophilus) microplus was tested in laboratory bioassays and under field conditions. Suspensions containing 10⁵, 10⁷ and 10⁹ conidia/mL were prepared of each fungal strain for laboratory bioassays. The ticks were maintained at 28ºC, 90 ± 5% relative humidity, and the following variables were evaluated: initial female weight, egg weight, hatching percentage, reproductive efficiency, and percentage control. For tests under field conditions, a Beauveria suspension containing 10⁶ conidia/mL was sprayed on tick-infested cows. After 72 h, the ticks were collected to estimate mortality under field conditions. Laboratory bioassays showed a mortality of 20 to 50% of the ticks seven days after inoculation with 10⁷ Beauveria conidia/mL. Under field conditions 10⁶ Beauveria conidia/mL induced 18-32% mortality. All Beauveria strains were effective in biological control of R. (Boophilus) microplus under laboratory and field test conditions. This is the first demonstration that endophytic fungi can be used for biological control of the cattle tick; this could help reduce environmental contamination by diminishing the need for chemical acaricides. Two endophytic strains were isolated from maize leaves and characterized by molecular sequencing of 5.8S rDNA ITS1 and ITS2 and morphological analyses of conidia. We found that these two endophytic Beauveria isolates, designated B95 and B157, are close to Beauveria amorpha.

ABSTRACT. We examined whether single-nucleotide polymorphisms (SNPs) in the calpain (CAPN) and calpastatin (CAST) genes, described from Bos primigenius taurus, are polymorphic in Nellore cattle. We also looked for a possible association of linkage disequilibrium of this polymorphism with tenderness of the longissimus dorsi muscle after 7, 14 and 21 days of postmortem aging in 638 purebred Nellore bulls. Meat tenderness was measured as Warner-Bratzler shear force. Additive and dominance effects were tested for SNPs of the three genotypic classes; the substitution effect was tested for SNPs with missing genotypic classes. Genotypic and gene frequencies were also calculated for the different SNPs. An increase in tenderness was observed from 7 to 21 days; the average values for shear force at 7, 14 and 21 days of aging were 5.92 ± 0.06, 4.92 ± 0.05, and 4.38 ± 0.04 kg, respectively. All markers showed polymorphism, but there was no CC genotype for CAPN316, and few animals showed the AA genotype for CAPN530. The alleles CAPN4751, UOGCAST1, and WSUCAST were found to have additive and dominance effects for shear force at 7, 14 and 21 days, while CAPN316 showed a substitution effect for shear force at 7 and 21 days. An additive-by-additive epistatic interaction was observed between CAPN4751 and markers on the CAST gene. In conclusion, these markers should be considered for use in breeding programs.

ABSTRACT. We investigated the ABO genotypes and heterogeneity of the O alleles in Plasmodium falciparum-infected and non-infected individuals from the Brazilian Amazon region. Sample collection took place from May 2003 to August 2005, from P. falciparum malaria patients from four endemic regions of the Brazilian Amazon. The control group consisted of donors from four blood banks in the same areas. DNA was extracted using the Easy-DNA^TM extraction kit. ABO genotyping was performed using PCR/RFLP. There was a high frequency of ABO*O01O01. ABO*AO01 was the second most frequent genotype, and the third most frequent genotype was ABO*BO01. There were low frequencies of the ABO*O01O02, ABO*AA, ABO*AB, ABO*BB, and ABO*O02O02 genotypes. We analyzed the alleles of the O phenotype; the O^1variant allele was the most frequent, both in malaria and non-malaria groups; consequently, the homozygous genotype O¹^vO¹^v was the most frequently observed. There was no evidence of the homozygous O² allele. Significant differences were not detected in the frequency of individuals with the various alleles in the comparison of the malaria patients and the general population (blood donors).

ABSTRACT. Despite the implementation control programs, schistosomiasis continues to spread throughout the world. Among modern control strategies, vector control is currently being emphasized. Within this context, analysis of the genetic variability of intermediate host snails (Biomphalaria spp) is important because it allows identification of specific sequences of the genome of this mollusk related to susceptibility/resistance to Schistosoma mansoni infection. We investigated Brazilian albino (non-pigmented) and pigmented (wild type) strains of Biomphalaria glabrata; these strains differ in their susceptibility to S. mansoni infection. Genetic variability was studied by RAPD-PCR using different random primers. The electrophoretic patterns resulting from amplification showed specific polymorphic markers for the albino and pigmented strains of B. glabrata. This information will help in the identification and isolation of genes specifically related to susceptibility, demonstrating that RAPD-PCR is an appropriate and efficient methodological approach for analysis of the genetic variability of schistosomiasis vectors.

ABSTRACT. High polyphenol and polysaccharide levels in plant tissues such as banana fruit and leaves constitute a significant challenge to the extraction of sufficient amounts of high-quality RNA required for cDNA library synthesis and molecular analysis. To determine their comparative effectiveness at eliminating polyphenols, polysaccharides and proteins, three protocols for RNA extraction from in vitro banana plantlet leaves were tested: Concert^TM Plant RNA isolation kit, a small-scale protocol based on Valderrama-Cháirez, and a modified version of the Valderrama-Cháirez protocol. RNA quantity and purity were evaluated by UV-spectrophotometry using DEPC-treated water and Tris-HCl, pH 7.5. Purity was greater using Tris-HCl. The Concert^TM Plant protocol produced the poorest quality RNA. Reverse transcription into cDNAs from RNA isolated from in vitro banana plantlet leaves infected with Mycosphaerella fijiensis using the modified Valderrama-Cháirez protocol, followed by PCR using primers designed against γ-actin from banana and M. fijiensis, yielded products of the anticipated size. In addition, this protocol reduced the processing time, lowered costs, used less expensive equipment, and could be used for other plants that have the same problems with high polyphenol and polysaccharide levels.

ABSTRACT. Transgenic animals are used extensively in the study of in vivo gene function, as models for human diseases and in the production of biopharmaceuticals. The technology behind obtaining these animals involves molecular biology techniques, cell culture and embryo manipulation; the mouse is the species most widely used as an experimental model. In scientific research, diverse models are available as tools for the elucidation of gene function, such as transgenic animals, knockout and conditional knockout animals, knock-in animals, humanized animals, and knockdown animals. We examined the evolution of the science for the development of these animals, as well as the techniques currently used in obtaining these animal models. We review the phenotypic techniques used for elucidation of alterations caused by genetic modification. We also investigated the role of genetically modified animals in the biotechnology industry, where they promise a revolution in obtaining heterologous proteins through natural secretions, such as milk, increasing the scale of production and facilitating purification, thereby lowering the cost of production of hormones, growth factors and enzymes.