Occurrence of sibling species of Lutzomyia longipalpis (Diptera : Psychodidae) in Venezuela: First evidence from reproductively isolated sympatric populations

The delimitation of cryptic species within the main vector of the American visceral leishmaniasis, Lutzomyia longipalpis, remains a topic of controversy. An analysis of generic variability based on 8 enzymatic loci revealed fixed differences in 2 diagnostic loci, adenylate kinase (Ak) and hexokinase (Hk), between sympatric and allopatric populations at 4 localities in Venezuela. The absence of heterozygotes for these 2 loci within 1 locality indicates, for the first time, the presence of 2 sympatric reproductively isolated populations or cryptic species within L. longipalpis. Significant differences were also detected between these cryptic species in the allele frequencies of glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase, decarboxylating (Me). One species showed mean heterozygosities that ranged between 6.6% and 6.7%, with 1.6-1.9 alleles detected per locus, while the other had mean heterozygosities that ranged from 4.3% to 6.3%, with 1.3-1.6 alleles per locus. Comparisons of isozyme profiles with published data suggests that 1 species is similar to the L. longipalpis described in Colombian and Brazilian populations, whereas the other has not been previously reported.

Estimation of outcrossing rate in a breeding population of Eucalyptus urophylla with dominant RAPD and AFLP markers

Eucalyptus breeding is typically conducted by selection in open-pollinated progenies. As mating is controlled only on the female side of the cross, knowledge of outcrossing versus selling rates is essential for maintaining adequate levels of genetic variability for continuous gains. Outcrossing rate in an open-pollinated breeding population of Eucalyptus urophylla was estimated by two PCR-based dominant marker technologies, RAPD and AFLP, using 11 open-pollinated progeny arrays of 24 individuals. Estimated outcrossing rates indicate predominant outcrossing and suggest maintenance of adequate genetic variability within families. The multilcous outcrossing rate (t(m)) estimated from RAPD markers (0.93 +/- 0.027), although in the same range, was higher (alpha > 0.01) than the estimate based on AFLP (0.89 +/- 0.033). Both estimates were of similar magnitude to those estimated for natural populations using isozymes. The estimated Wright's fixation index was lower than expected based on t, possibly resulting from selection against selfed seedlings when sampling plants for the study. An empirical analysis suggests that 18 is the minimum number of dominant marker loci necessary to achieve robust estimates of t,. This study demonstrates the usefulness of dominant markers, both RAPD and AFLP, for estimating the outcrossing rate in breeding and natural populations of forest trees. We anticipate an increasing use of such PCR-based technologies in mating-system studies, in view of their high throughput and universality of the reagents, particularly for species where isozyme systems have not yet been optimized.

Structure, genomic DNA typing, and kinetic characterization of the D allozyme of placental alkaline phosphatase (PLAP/ALPP)

The D allozyme of placental alkaline phosphatase (PLAP) displays enzymatic properties at variance with those of the common PLAP allozymes. We have deduced the amino acid sequence of the PLAP D allele by PCR cloning of its gene, ALPP Two coding substitutions were found in comparison With the cDNA of the common PLAP F allele, i.e., 692C>G and 1352A>G, which translate into a P209R and E429G substitution. A single nucleotide primer extension (SNuPE) assay was developed using PCR primers that enable the amplification of a 1.9 kb PLAP fragment. Extension primers were then used on this PCR fragment to detect the 692C>G and 1352A>G substitution. The SNuPE assay on these two nucleotide substitutions enabled us to distinguish the PLAP F and D alleles from the PLAP S/I alleles. Functional studies on the D allozyme were made possible by constructing and expressing a PLAP D cDNA, i.e., [Arg209, Gly429] PLAP, into wildtype Chinese hamster ovary cells. We determined the k(cat) and K-m, of the PLAP S, F. and D allozymes using the non,physiological substrate p-nitrophenylphosphate at an optimal pH (9.8) as well as two physiological substrates, i.e., pyridoxal-5'-phosphate and inorganic pyrophosphate at physiological pH (7.5). We found that the biochemical properties of the D allozyme of PLAP are significantly different from those of the common PLAP allozymes. These biochemical findings suggest that a suboptimal enzymatic function by the PLAP D allozyme may be the basis for the apparent negative selective pressure of the PLAP D allele. The development of the SNuPE assay will enable us to test the hypothesis that the PLAP D allele is subjected to intrauterine selection by examining genomic DNA from statistically informative population samples. Hum Mutat 19:258-267, 2002. (C) 2002 Wiley-Liss, Inc.

Triiodothyronine mimics the effects of estrogen in breast cancer cell lines

MCF-7 (estrogen receptor positive - ER(+)) and MDA-MB-231 (estrogen receptor negative - ER(-)) are human breast cancer cell lines which express functional thyroid hormone receptors (c-erb A alpha 1 and c-erb beta 1) as indicated by stimulation of mitochondrial alpha-glycerophosphate dehydrogenase. In MCF-7, mimicking E(2), T-3 stimulated growth in a dose-dependent (10(10) M-10(-8) M) manner, induced the expression of progesterone receptor and growth factor TGF alpha mRNAs and inhibited that of TGF beta mRNA; T-3 also increased progesterone binding and LDH5 isozyme activities. None of these effects were observed in (ER(-)) MDA-MB-231 cells. 10(-6) M tamoxifen (TAM) reverted growth stimulation, suppressed progesterone receptor and TGF alpha mRNA induction and restored TGF beta mRNA to control levels in T-3-treated MCF-7 cells. That T-3 is acting in MCF-7 cells via its binding to ER is suggested by the immunoprecipitation of pre-bound I-125-T-3 from MCF-7 nuclear extracts by an ER-specific monoclonal antibody and by the displacement of H-3-estradiol binding to ER by radioinert T-3. Copyright (C) 1996 Elsevier B.V. Ltd.

Chemical composition of flavonoids and styrylpyrones and the genetic variability of isozymes in natural populations of Cryptocarya mandioccana Meisner (Lauraceae)

The chemical composition of leaves of 57 trees of Cryptocarya mandioccana from three populations of southeastern Brazil was investigated through HPLC, assaying six flavonoids, seven styrylpyrones, and seven unidentified compounds. From 51 of the former trees, genotypes were obtained from 40 polymorphic loci of 19 isozymes. Cluster analyses of the phytochemical and genetical variation revealed that trees exhibited four chemotypes and five clusters from isozymes, respectively. Discriminant analyses from selected variables of the isozymic and chemical data sets were performed, respectively, in relation to the four chemotypes and the five isozyme clusters. The classification of individuals presented respective error estimates of 9.16% and 13.57%, indicating that the genetic data could explain the clusters from chernotypes and vice versa at acceptable error levels. Linear regressions with Dummy variable showed significant association of locus Skdh-2 with quercetin-3-O-beta-D-glucopyranoside and cryptofolione, indicating that its alleles would be responsible for the chemotype variation between individuals. (c) 2006 Elsevier Ltd. All rights reserved.

Purification and properties of buffalo (Bubalus bubalis) erythrocyte hexokinase

Buffalo erythrocytes contain one isozyme of hexokinase that apparently lacks microheterogeneity as shown by chromatographic properties. A single protein band was detected by means of Western blotting using an antibody raised in rabbits against homogeneous rat brain hexokinase I. The native protein has a molecular weight of 200,000 +/- 2880 by gel filtration. Partial purification of erythrocyte hexokinase by a combination of several procedures, including affinity chromatography, which was previously applied successfully to the purifica tion of other mammalian type I hexokinases, produced a partially purified enzyme that showed several contami nants after SDS-polyacrylamide gel electrophoresis. The affinity of buffalo erythrocyte hexokinase for glucose (K-m = 0.012 +/- 0.001 mM) is lower than most other mammal hexokinases type I. It phosphorylates other sugars, with considerably higher K-m values. This isozyme is able to use MgATP but does not use MgGTP, MgCTP or MgUTP. We used inhibition patterns, obtained with products to elucidate enzyme sequential mechanisms. Our results are clearly in agreement with a random sequential mechanism and in disagreement with an ordered sequential mechanism with either glucose or ATP as the obligatory first substrates. The ADP inhibition was of mixed type with both ATP and glucose as substrates. (C) 1997 Elsevier B.V.

Thyroid State and Tolerance of Mammalian Myocardium to Hypoxia

Thyroid hormone is known to affect myocardial glycogen stores and thereby possibly limit anaerobic performance of mammalian cardiac muscle. Thyroid hormone administration (3,5,T-triiodo-L-thyroxine, 300 mu g/kg/day, sc) for 10 days decreased left ventricle (LV) glycogen concentration relative to euthyroid animals (2.78 +/- 0.46 vs. 4.28 +/- 0.29 mg/g of LV (mean +/- SEM)) while increasing the percent of V(1) myosin isozyi-ne, contractile activity and cardiac mass. In contrast, thyroidectomy increased myocardial glycogen stores (8.50 +/- 0.56 mg/g of LV) and shifted the myosin isozyme toward V(3), prolonged contractile activity and decreased LV mass. Thyroxine administration for 3, 7 and 10 days to thyroidectomized animals progressively decreased contractile duration and increased LV mass. Thyroxine administration for 3 or 7 days to thyroidectomized rats did not reduce glycogen stores (7.75 +/- 1.02 and 9.62 +/- 1.16 mg/g of LV, respectively), whereas myocardial glycogen declined to 3.30 +/- 0.58 mg/g of LV after 10 days of treatment. During hypoxia, cardiac muscle from thyroidectomized rats maintained greater active force and developed less contracture relative to euthyroid and, to a greater extent, than hyperthyroid rats. Removal of glucose from the bath decreased anaerobic performance and impaired recovery; however, myocardium from thyroidectomized rats remained more tolerant to hypoxia than the euthyroid group. Overall, the intrinsic LV glycogen content was positively correlated to anaerobic performance. These data demonstrate that the thyroid state profoundly affects myocardial growth, contractility and anaerobic performance of rat myocardium. Although energy demand may affect function during hypoxia, anaerobic substrate reserve (cardiac glycogen concentration) appears to be the primary factor determining tolerance to hypoxic stress. J. Exp. Zool. 311A:399-407, 2009. (C) 2009 Wiley-Liss, Inc.

Novos Registros sobre Meloidogyne mayaguensis no Brasil e Estudo Morfológico Comparativo com M. incognita

Almeida, E.J., P.L.M. Soares, A.R. Silva & J.M. Santos. 2008. New records on Meloidogyne mayaguensis in Brazil and comparative study with M. incognita. Meloidogyne mayaguensis is the plant-parasitic nematode responsible for a great impact on guava production in Brazil, since it has been the cause for eradication of thousand of hectares of guava plantations in the Northeastern region. In the present study, this species was detected in soybean fields of Ituverava municipality, São Paulo state, and in different vegetable crops (lettuce, cucumber, pepper and cherry tomato) in Chapada dos Guimaraes municipality, Mato Grosso state, causing root galls and other symptoms. The species was identified on the basis of the perineal pattern of females, and on the morphology and morphometry of anterior region of males. Isozyme phenotype for esterase was used for confirmation. This constitutes the first report on the occurrence of M. mayaguensis on lettuce, cucumber, pepper and cherry tomato cultures in Mato Grosso state and the first one in soybean in São Paulo state. It was found that morphological features of male anterior region and female perineal pattern are enough for the safe distinction between M. mayaguensis and M. incognita.

Evaluation of microsatellite markers in cultivars of sweet orange (Citrus sinensis [L.] Osbeck)

Sweet orange is considered a very important species in the citrus world market and presents wide morphological variability. However, its characterization at the molecular level by random amplified polymorphic DNA (RAPD) and isozyme markers is not appropriate. Microsatellite or simple sequence repeats (SSRs) have been suggested as ideal for studies in cultures of vegetative propagation and as value markers for mapping in several species. However, information on microsatellite polymorphism in citrus species is scarce. In this work, microsatellite markers (AG-repeats) were developed from an enrichment library of genomic DNA of sweet orange cv. Pera (Citrus sinensis [L.] Osbeck), and 31 cultivars of sweet orange were evaluated. Evaluation of 18 microsatellite primers did not permit differentiation of the varieties studied. New microsatellite primers are being evaluated with the aim of detecting polymorphisms among the cultivars and closely related species to be used in genetic mapping programs.

Genetic diversity of Rhizoctonia solani AG-3 from potato and tobacco in North Carolina

Anastomosis group 3 (AG-3) of Rhizoctonia solani (teleomorph = Thanatephorus cucumeris) is frequently associated with diseases of potato (AG-3 PT) and tobacco (AG-3 TB). Although isolates of R. solani AG-3 from these two Solanaceous hosts are somatically related based on anastomosis reaction and taxonomically related based on fatty acid, isozyme and DNA characters, considerable differences are evident in their biology, ecology, and epidemiology. However, genetic diversity among field populations of R. solani AG-3 PT and TB has not been documented. In this study, the genetic diversity of field populations of R. solani AG-3 PT and AG-3 TB in North Carolina was examined using somatic compatibility and amplified fragment length polymorphism (AFLP) criteria. A sample of 32 isolates from potato and 36 isolates from tobacco were paired in all possible combinations on PDA plus activated charcoal and examined for their resulting somatic interactions. Twenty-eight and eight distinct somatic compatibility groups (SCG) were identified in the AG-3 PT and AG-3 TB samples, respectively. AFLP analyses indicated that each of the 32 AG-3 PT isolates had a distinct AFLP phenotype, whereas 28 AFLP phenotypes were found among the 36 isolates of AG-3 TB. None of the AG-3 PT isolates were somatically compatible or shared a common AFLP phenotype with any AG-3 TB isolate. Clones (i.e., cases where two or more isolates were somatically compatible and shared the same AFLP phenotype) were identified only in the AG-3 TB population. Four clones from tobacco represented 22% of the total population. All eight SCG from tobacco were associated with more than one AFLP phenotype. Compatible somatic interactions between AG-3 PT isolates occurred only between certain isolates from the same field (two isolates in each of four different fields), and when this occurred AFLP phenotypes were similar but not identical.

Characterization of peroxidase changes in tolerant and susceptible soybeans challenged by soybean aphid (Hemiptera: Aphididae)

Changes in protein content, peroxidase activity, and isozyme profiles in response to soybean aphid feeding were documented at V1 (fully developed leaves at unifoliate node, first trifoliate leaf unrolled) and V3 (fully developed leaf at second trifoliate node, third trifoliate leaf unrolled) stages of soybean aphid-tolerant (KS4202) and -susceptible (SD76R) soybeans. Protein content was similar between infested and control V1 and V3 stage plants for both KS4202 and SD76R at 6, 16, and 22 d after aphid introduction. Enzyme kinetics studies documented that control and aphid-infested KS4202 V1 stage and SD76R V1 and V3 stages had similar levels of peroxidase activity at the three time points evaluated. In contrast, KS4202 aphid-infested plants at the V3 stage had significantly higher peroxidase activity levels than control plants at 6 and 22 d after aphid introduction. The differences in peroxidase activity observed between infested and control V3 stage KS4202 plants at these two time points suggest that peroxidases may be playing multiple roles in the tolerant plant. Native gels stained for peroxidase were able to detect differences in the isozyme profiles of aphid-infested and control plants for both KS4202 and SD76R.

Enzyme replacement prevents enamel defects in hypophosphatasia mice

Hypophosphatasia (HPP) is the inborn error of metabolism characterized by deficiency of alkaline phosphatase activity, leading to rickets or osteomalacia and to dental defects. HPP occurs from loss-of-function mutations within the gene that encodes the tissue-nonspecific isozyme of alkaline phosphatase (TNAP). TNAP knockout (Alpl-/-, aka Akp2-/-) mice closely phenocopy infantile HPP, including the rickets, vitamin B6-responsive seizures, improper dentin mineralization, and lack of acellular cementum. Here, we report that lack of TNAP in Alpl-/- mice also causes severe enamel defects, which are preventable by enzyme replacement with mineral-targeted TNAP (ENB-0040). Immunohistochemistry was used to map the spatiotemporal expression of TNAP in the tissues of the developing enamel organ of healthy mouse molars and incisors. We found strong, stage-specific expression of TNAP in ameloblasts. In the Alpl-/- mice, histological, mu CT, and scanning electron microscopy analysis showed reduced mineralization and disrupted organization of the rods and inter-rod structures in enamel of both the molars and incisors. All of these abnormalities were prevented in mice receiving from birth daily subcutaneous injections of mineral-targeting, human TNAP at 8.2?mg/kg/day for up to 44 days. These data reveal an important role for TNAP in enamel mineralization and demonstrate the efficacy of mineral-targeted TNAP to prevent enamel defects in HPP. (C) 2012 American Society for Bone and Mineral Research.

Mitochondrial localization and structure-based phosphate activation mechanism of Glutaminase C with implications for cancer metabolism

Glutamine is an essential nutrient for cancer cell proliferation, especially in the context of citric acid cycle anaplerosis. In this manuscript we present results that collectively demonstrate that, of the three major mammalian glutaminases identified to date, the lesser studied splice variant of the gene gls, known as Glutaminase C (GAC), is important for tumor metabolism. We show that, although levels of both the kidney-type isoforms are elevated in tumor vs. normal tissues, GAC is distinctly mitochondrial. GAC is also most responsive to the activator inorganic phosphate, the content of which is supposedly higher in mitochondria subject to hypoxia. Analysis of X-ray crystal structures of GAC in different bound states suggests a mechanism that introduces the tetramerization-induced lifting of a "gating loop" as essential for the phosphate-dependent activation process. Surprisingly, phosphate binds inside the catalytic pocket rather than at the oligomerization interface. Phosphate also mediates substrate entry by competing with glutamate. A greater tendency to oligomerize differentiates GAC from its alternatively spliced isoform and the cycling of phosphate in and out of the active site distinguishes it from the liver-type isozyme, which is known to be less dependent on this ion.

Comparison of microsatellites and isozymes in genetic diversity studies of Oryza glumaepatula (Poaceae) populations

The study of the genetic structure of wild plant populations is essential for their management and conservation. Several DNA markers have been used in such studies, as well as isozyme markers. In order to provide a better comprehension of the results obtained and a comparison between markers which will help choose tools for future studies in natural populations of Oryza glumaepatula, a predominantly autogamous species, this study used both isozymes and microsatellites to assess the genetic diversity and genetic structure of 13 populations, pointing to similarities and divergences of each marker, and evaluating the relative importance of the results for studies of population genetics and conservation. A bulk sample for each population was obtained, by sampling two to three seeds of each plant, up to a set of 50 seeds. Amplified products of eight SSR loci were electrophoresed on non-denaturing polyacrylamide gels, and the fragments were visualized using silver staining procedure. Isozyme analyses were conducted in polyacrylamide gels, under a discontinuous system, using six enzymatic loci. SSR loci showed higher mean levels of genetic diversity (A=2.83, p=0.71, A(P)=3.17, H-o=0.081, H-e=0.351) than isozyme loci (A=1.20, p=0.20, A(P)=1.38, H-o=0.006, H-e=0.056). Interpopulation genetic differentiation detected by SSR loci (R-ST=0.631, equivalent to F-ST=0.533) was lower than that obtained with isozymes (F-ST=0.772). However, both markers showed high deviation from Hardy-Weinberg expectations (F-IS=0.744 and 0.899, respectively for SSR and isozymes). The mean apparent outcrossing rate for SSR ((t) over bar (a)=0.14) was higher than that obtained using isozymes ((t) over bar (a)=0.043), although both markers detected lower levels of outcrossing in Amazonia compared to the Pantanal. The migrant number estimation was also higher for SSR (Nm=0.219) than isozymes (Nm=0.074), although a small number for both markers was expected due to the mode of reproduction of this species, defined as mixed with predominance of self fertilization. No correlation was obtained between genetic and geographic distances with SSR, but a positive correlation was found between genetic and geographic distances with isozymes. We conclude that these markers are divergent in detecting genetic diversity parameters in O. glumaepatula and that microsatellites are powerful for detecting information at the intra-population level, while isozymes are more powerful for inter-population diversity, since clustering of populations agreed with the expectations based on the geographic distribution of the populations using this marker. Rev. Biol. Trop. 60 (4): 1463-1478. Epub 2012 December 01.

Diversity and genetic structure in natural populations of Hancornia speciosa var. speciosa Gomes in northeastern Brazil

Hancornia speciosa Gomes is a fruit tree native from Brazil that belongs to Apocinaceae family, and is popularly known as Mangabeira. Its fruits are widely consumed raw or processed as fruit jam, juices and ice creams, which have made it a target of intense exploitation. The extractive activities and intense human activity on the environment of natural occurrence of H. speciosa has caused genetic erosion in the species and little is known about the ecology or genetic structure of natural populations. The objective of this research was the evaluation of the genetic diversity and genetic structure of H. speciosa var. speciosa. The genetic variability was assessed using 11 allozyme loci with a sample of 164 individuals distributed in six natural populations located in the States of Pernambuco and Alagoas, Northeastern Brazil. The results showed a high level of genetic diversity within the species (e= 0.36) seeing that the most of the genetic variability of H. speciosa var. speciosa is within its natural populations with low difference among populations ( or = 0.081). The inbreeding values within ( = -0.555) and among populations ( =-0.428) were low showing lacking of endogamy and a surplus of heterozygotes. The estimated gene flow ( m ) was high, ranging from 2.20 to 13.18, indicating to be enough to prevent the effects of genetic drift and genetic differentiation among populations. The multivariate analyses indicated that there is a relationship between genetic and geographical distances, which was confirmed by a spatial pattern analysis using Mantel test (r = 0.3598; p = 0.0920) with 1000 random permutations. The high genetic diversity index in these populations indicates potential for in situ genetic conservation.