Karyotypical Confirmation of Natural Hybridization between Two Manatee Species, Trichechus manatus and Trichechus inunguis.

Two species of manatees are found in Northern Brazil-the Antillean manatee (Trichechus manatus), which is found along the coast from Florida to Northeastern Brazil, and the Amazonian manatee (Trichechus inunguis), endemic to the Amazon drainage basin. These species show a sympatric distribution in the region of the Marajó Archipelago, an estuarine area surrounding the Amazon River mouth. There is evidence of the occurrence of interspecific hybrids in this area, based on mitochondrial DNA analyses, although the use of nuclear markers has not corroborated this proposal. Considering that these species show very distinct karyotypes, despite being closely related (2n = 48 in T. manatus and 2n = 56 in T. inunguis), hybrids would present distinct chromosome numbers. Based on this, we conducted cytogenetic analyses using classic and molecular techniques in three calves found stranded in the Marajó Island and Amapá coast. The results showed that one of them, morphologically classified as T. inunguis, presented the correspondent karyotype, with 2n = 56. However, the other two, which were phenotypically similar to T. manatus, showed 2n = 49. Despite the same diploid number, their G-banding patterns revealed some differences. The results of the distribution of some microsatellite sequences have also confirmed the heterozygosity of some chromosomal pairs in these two individuals. These results are the first indubitable confirmation of the occurrence of natural hybrids between T. manatus and T. inunguis, and also brings about some issues concerning the viability of hybrids, considering that these two individuals do not correspond to an F1 hybrid, but instead, both presented a possible F2 karyotype.

Recurrent chromosome reshuffling and the evolution of neo-sex chromosomes in parrots.

The karyotype of most birds has remained considerably stable during more than 100 million years' evolution, except for some groups, such as parrots. The evolutionary processes and underlying genetic mechanism of chromosomal rearrangements in parrots, however, are poorly understood. Here, using chromosome-level assemblies of four parrot genomes, we uncover frequent chromosome fusions and fissions, with most of them occurring independently among lineages. The increased activities of chromosomal rearrangements in parrots are likely associated with parrot-specific loss of two genes, ALC1 and PARP3, that have known functions in the repair of double-strand breaks and maintenance of genome stability. We further find that the fusion of the ZW sex chromosomes and chromosome 11 has created a pair of neo-sex chromosomes in the ancestor of parrots, and the chromosome 25 has been further added to the sex chromosomes in monk parakeet. Together, the combination of our genomic and cytogenetic analyses characterizes the complex evolutionary history of chromosomal rearrangements and sex chromosomes in parrots.

Comparative Cytogenetics in Four Leptodactylus Species (Amphibia, Anura, Leptodactylidae): Evidence of Inner Chromosomal Diversification in Highly Conserved Karyotypes.

With 82 species currently described, the genus Leptodactylus is the most diverse and representative one in the family Leptodactylidae. Concerning chromosomal organization, this genus represents an interesting and underexplored group since data from molecular cytogenetics are incipient, and little is known about the organization and distribution of repetitive DNA elements in the karyotypes. In this sense, this study aimed at providing a comparative analysis in 4 Leptodactylus species (L. macrosternum, L. pentadactylus, L. fuscus, and Leptodactylus cf. podicipinus), combining conventional cytogenetics (Giemsa staining, C-banding, and AgNOR staining) and mapping of molecular markers (18S rDNA, telomeric and microsatellite probes), to investigate mechanisms underlying their karyotype differentiation process. The results showed that all species had karyotypes with 2n = 22 and FN = 44, except for Leptodactylus cf. podicipinus which presented FN = 36. The 18S rDNA was observed in pair 8 of all analyzed species (corresponding to pair 4 in L. pentadactylus), coinciding with the secondary constrictions and AgNOR staining. FISH with microsatellite DNA probes demonstrated species-specific patterns, as well as an association of these repetitive sequences with constitutive heterochromatin blocks and ribosomal DNA clusters, revealing the dynamics of microsatellites in the genome of the analyzed species. In summary, our data demonstrate an ongoing process of genomic divergence inside species with almost similar karyotype, driven most likely by a series of pericentric inversions, followed by differential accumulation of repetitive sequences.

Two new cytotypes reinforce that Micronycteris hirsuta Peters, 1869 does not represent a monotypic taxon.

BACKGROUND: The genus Micronycteris is a diverse group of phyllostomid bats currently comprising 11 species, with diploid number (2n) ranging from 26 to 40 chromosomes. The karyotypic relationships within Micronycteris and between Micronycteris and other phyllostomids remain poorly understood. The karyotype of Micronycteris hirsuta is of particular interest: three different diploid numbers were reported for this species in South and Central Americas with 2n = 26, 28 and 30 chromosomes. Although current evidence suggests some geographic differentiation among populations of M. hirsuta based on chromosomal, morphological, and nuclear and mitochondrial DNA markers, the recognition of new species or subspecies has been avoided due to the need for additional data, mainly chromosomal data. RESULTS: We describe two new cytotypes for Micronycteris hirsuta (MHI) (2n = 26 and 25, NF = 32), whose differences in diploid number are interpreted as the products of Robertsonian rearrangements. C-banding revealed a small amount of constitutive heterochromatin at the centromere and the NOR was located in the interstitial portion of the short arm of a second pair, confirmed by FISH. Telomeric probes hybridized to the centromeric regions and weakly to telomeric regions of most chromosomes. The G-banding analysis and chromosome painting with whole chromosome probes from Carollia brevicauda (CBR) and Phyllostomus hastatus (PHA) enabled the establishment of genome-wide homologies between MHI, CBR and PHA. CONCLUSIONS: The karyotypes of Brazilian specimens of Micronycteris hirsuta described here are new to Micronycteris and reinforce that M. hirsuta does not represent a monotypic taxon. Our results corroborate the hypothesis of karyotypic megaevolution within Micronycteris, and strong evidence for this is that the entire chromosome complement of M. hirsuta was shown to be derivative with respect to species compared in this study.

Intervention strategies and agents mediating the prevention of xenorejection.

Xenotransplantation, the transplantation of cells, tissues, and/or organs across species, has proven to be an enormous challenge, resulting in only limited achievements over the last century. Unlike allotransplantation, the immunologic barriers involved in xenotransplant rejection are aggressive and usually occur within minutes in a hyperacute fashion. The use of organs from phylogenetically related concordant species may not be practical. Discordant xenotransplantation is characterized by hyperacute graft rejection, and to use nonprimate discordant organs for human benefit will require manipulation of the taxonomic differences. The hyperacute rejection process is primarily due to the attachment of preformed xenoreactive antibodies to the donor vascular endothelium, which results in hyperactivation of the complement system beyond the control of the natural complement regulatory proteins. Understanding the complex and diverse immune components involved in hyperacute, acute, and accelerated rejections has resulted in the development of different hematologic and molecular strategies. Plasmapheresis has been used to remove xenoantibodies, and xenoperfusion techniques are used to create a suitable and familiar environment for the xenograft. Various molecular approaches, such as the development of transgenic animals expressing human complement regulatory proteins such as CD59 or decay accelerating factor (DAF), to downregulate complement activation or the production of pigs lacking the xenoreactive antigen by knockout of the Gal alpha-1,3-galactosyl transferase gene have also been attempted. A combination of these techniques together with the administration of soluble complement inhibitors such as the vaccinia virus complement control protein (VCP) may well contribute to prolong graft survival. However, various issues including the possible emergence of new viral infections have confounded the topic of xenotransplantation. Here the different modulatory approaches and agents mediating interventions in xenorejection are discussed.

Broadcasts of wing-fanning vibrations recorded from calling male Ceratitis capitata (Diptera: Tephritidae) increase captures of females in traps.

Female Mediterranean fruit flies, Ceratitis capitata (Wiedemann), from the sterile-male rearing facility in El Pino, Guatemala, were exposed to broadcasts of wing-fanning vibrations recorded from males engaged in calling behavior to investigate the feasibility of developing a female-selective acoustic trap. The recorded signals had frequent amplitude fluctuations and peak frequencies approximately 350 Hz, typical of signals observed in previous studies of Mediterranean fruit fly acoustic behavior. Females did not exhibit long-distance phonotaxis, but remained near a speaker significantly longer when the sounds were broadcast at 103-107 dB than when the speaker was silent. In addition, significantly higher percentages of females were captured by yellow adhesive traps next to a broadcasting speaker than by traps next to a silent mimic. Additional bioassays were conducted with synthetic, 350-Hz tones produced by a thermoacoustic tube as well as with silent mimics of the different sound sources to examine the relative responsiveness of female Mediterranean fruit flies to traps with different acoustic and visual features. The visual attributes of the different sound source assemblies significantly affected capture rates. The range over which the broadcast significantly increased the percentage of female captures was <0.5 m, which may limit the utility of these acoustic cues in large-scale trapping programs. However, the findings of this study do justify further testing of whether optimized short-range acoustic signals could be used to augment longer range pheromonal and visual cues to improve the efficacy of female-selective traps.

Acoustic detection of termite infestations in urban trees.

A portable, low-frequency acoustic system was used to detect termite infestations in urban trees. The likelihood of infestation was rated independently by a computer program and an experienced listener that distinguished insect sounds from background noises. Because soil is a good insulator, termite sounds could be detected easily underneath infested trees, despite the presence of high urban background noise. Termite sounds could be detected also in trunks, but background noise often made it difficult to identify termite signals unambiguously. High likelihoods of termite infestation were predicted at four live oak (Quercus virginiana Mill, Fagacae), two loblolly pine (Pinus taeda L., Pinacae), and two baldcypress (Taxodium distichum Rich. Pinacae) trees that wood-baited traps had identified as infested with Coptotermes formosanus Shiraki. Infestations also were predicted at two pine trees with confirmed recoveries of Reticulitermes flavipes (Kollar). Low likelihoods of infestation were predicted in four oak trees where no termites were found. Additional tests were conducted in anechoic environments to determine the range of acoustic detectability and the feasibility of acoustically estimating termite population levels. There was a significant regression between the activity rate and the number of termites present in a wood trap block, with a minimum detectable number of approximately 50 workers per liter of wood. The success of these field tests suggests that currently available acoustic systems have considerable potential to detect and monitor hidden infestations of termites in urban trees and around building perimeters in addition to their present uses to detect and monitor termite infestations in buildings.

The progression of benign prostatic hyperplasia: examining the evidence and determining the risk.

BACKGROUND: Benign prostatic hyperplasia (BPH) is often associated with enlargement of the prostate gland, lower urinary tract symptoms, decreased urinary flow and a reduced quality of life. Furthermore, if the symptoms associated with BPH are left untreated, serious complications, such as acute urinary retention, may ensue. Evidence is emerging from long-term clinical studies to suggest that BPH is a progressive disease, with some patients progressing much more rapidly than others. OBJECTIVE: This article aims to explore the natural history of BPH progression from a molecular, pathological and clinical perspective, with emphasis on the key clinical evidence to support the progressive nature of this disease. How our increased understanding of the disease and of the risk factors for BPH progression might be applied to improve current management practices are also discussed. CONCLUSION: Strategies to identify patients most at risk and guidelines directed towards long-term management, in addition to short-term treatment, may be useful in helping to prevent BPH progression.

Multilocus genotyping indicates that the ability to invade the bloodstream is widespread among Candida albicans isolates.

Multilocus genotyping was used to compare populations of Candida albicans from oral mucosa and blood. No significant differences in allele frequencies between the two samples were detected, and in a dendrogram of genotypic similarities, genotypes from both types of samples were finely interspersed. This is evidence for widespread distribution of invasive potential.

Divergence in fitness and evolution of drug resistance in experimental populations of Candida albicans.

The dissemination and persistence of drug-resistant organisms in nature depends on the relative fitness of sensitive and resistant genotypes. While resistant genotypes are expected to be at an advantage compared to less resistant genotypes in the presence of drug, resistance may incur a cost; resistant genotypes may be at a disadvantage in the absence of drug. We measured the fitness of replicate experimental populations of the pathogenic yeast Candida albicans founded from a single progenitor cell in a previous study (L. E. Cowen, D. Sanglard, D. Calabrese, C. Sirjusingh, J. B. Anderson, and L. M. Kohn, J. Bacteriol. 182:1515-1522, 2000) and evolved in the presence, and in the absence, of the antifungal agent fluconazole. Fitness was measured both in the presence and in the absence of fluconazole by placing each evolved population in direct competition with the drug-sensitive ancestor and measuring the reproductive output of each competitor in the mixture. Populations evolved in the presence of drug diverged in fitness. Any significant cost of resistance, indicated by reduced fitness in the absence of drug, was eliminated with further evolution. Populations evolved in the absence of drug showed more uniform increases in fitness under both conditions. Fitness in the competition assays was not predicted by measurements of the MICs, doubling times, or stationary-phase cell densities of the competitors in isolation, suggesting the importance of interactions between mixed genotypes in competitions.

Infrequent genetic exchange and recombination in the mitochondrial genome of Candida albicans.

Previous analyses of diploid nuclear genotypes have concluded that recombination has occurred in populations of the yeast Candida albicans. To address the possibilities of clonality and recombination in an effectively haploid genome, we sequenced seven regions of mitochondrial DNA (mtDNA) in 45 strains of C. albicans from human immunodeficiency virus-positive patients in Toronto, Canada, and 3 standard reference isolates of C. albicans, CA, CAI4, and WO-1. Among a total of 2,553 nucleotides in the seven regions, 62 polymorphic nucleotide sites and seven indels defined nine distinct mtDNA haplotypes among the 48 strains. Five of these haplotypes occurred in more than one strain, indicating clonal proliferation of mtDNA. Phylogenetic analysis of mtDNA haplotypes resulted in one most-parsimonious tree. Most of the nucleotide sites undergoing parallel change in this tree were clustered in blocks that corresponded to sequenced regions. Because of the existence of these blocks, the apparent homoplasy can be attributed to infrequent, past genetic exchange and recombination between individuals and cannot be attributed to parallel mutation. Among strains sharing the same mtDNA haplotypes, multilocus nuclear genotypes were more similar than expected from a random comparison of nuclear DNA genotypes, suggesting that clonal proliferation of the mitochondrial genome was accompanied by clonal proliferation of the nuclear genome.

Flow cytometric analysis of hematologic neoplasia.

Flow cytometry (FCM) is a powerful technology that allows the rapid analysis of cellular components such as surface and intracellular antigens, or DNA content. The measurements are fast and are based on optical signals emitted by cells labeled with fluorochromes as they flow suspended in a liquid medium through an intense laser beam. The signals include scattered light, which provides information on cell size and granularity, and fluorescence derived from dyes or fluorochrome-labeled antibodies bound to specific cell components.

Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice.

Obesity is a disorder of energy balance. Hormone-sensitive lipase (HSL) mediates the hydrolysis of triacylglycerol, the major form of stored energy in the body. Perilipin (encoded by the gene Plin), an adipocyte protein, has been postulated to modulate HSL activity. We show here that targeted disruption of Plin results in healthy mice that have constitutively activated fat-cell HSL. Plin -/- mice consume more food than control mice, but have normal body weight. They are much leaner and more muscular than controls, have 62% smaller white adipocytes, show elevated basal lipolysis that is resistant to beta-adrenergic agonist stimulation, and are cold-sensitive except when fed. They are also resistant to diet-induced obesity. Breeding the Plin -/- alleles into Leprdb/db mice reverses the obesity by ncreasing the metabolic rate of the mice. Our results demonstrate a role for perilipin in reining in basal HSL activity and regulating lipolysis and energy balance; thus, agents that inactivate perilipin may prove useful as anti-obesity medications.

Bicalutamide monotherapy compared with castration in patients with nonmetastatic locally advanced prostate cancer: 6.3 years of followup.

PURPOSE: Nonsteroidal antiandrogen monotherapy may be a treatment option for some patients with advanced prostate cancer. We report a survival and safety update from an analysis of 2 studies in which patients with nonmetastatic (M0) locally advanced disease were treated with either 150 mg. bicalutamide monotherapy or castration. MATERIALS AND METHODS: Data from 2 open label, multicenter studies of identical design were pooled according to protocol. Patients with stage T3/4 prostate cancer were randomized to receive 150 mg. bicalutamide daily or castration (orchiectomy or 3.6 mg. goserelin acetate every 28 days) in a 2:1 ratio. RESULTS: A total of 480 patients with locally advanced prostate cancer were randomized to treatment. After a median followup of 6.3 years mortality was 56%. There was no statistically significant difference between the 2 groups in overall survival (hazard ratio 1.05, upper 1-sided 95% confidence limit 1.31, p = 0.70) or time to progression (1.20, 1.45, p = 0.11). There were statistically significant benefits in the bicalutamide monotherapy group in the 2 quality of life parameters of sexual interest (p = 0.029) and physical capacity (p = 0.046). The highest incidences of adverse events were the pharmacological side effects of hot flashes in the castration group, and breast pain and gynecomastia in the bicalutamide group. The incidences of other types of adverse events were low. Bicalutamide was well tolerated, with few drug related withdrawals from study, and no new safety issues were identified during this longer followup. CONCLUSIONS: Monotherapy with 150 mg. bicalutamide is an attractive alternative to castration in patients with locally advanced prostate cancer for whom immediate hormone therapy is indicated.

Monte Carlo methods in electronic structures for large systems.

Quantum Monte Carlo methods have recently made it possible to calculate the electronic structure of relatively large molecular systems with very high accuracy. These large systems range from positron complexes [NH(2),Ps] with approximately 10 electrons to C(20) isomers with 120 electrons, to silicon crystal structures of 250 atoms and 1000 valence electrons. The techniques for such calculations and a sampling of applications are reviewed.

Follicular lymphoma can be distinguished from benign follicular hyperplasia by flow cytometry using simultaneous staining of cytoplasmic bcl-2 and cell surface CD20.

The distinction between benign follicular hyperplasia (FH) and follicular lymphoma (FL) is sometimes problematic. We wanted to determine whether the expression of bcl-2 of FH was quantitatively different from that of FL, using surface CD20 expression as a discriminator of the various lymphoid compartments. Lymph node cell suspensions from 12 cases of FH and 17 cases of FL were analyzed by flow cytometry using a combined surface CD20 and intracellular bcl-2 staining. CD20- T cells in FH demonstrated the same bcl-2 expression as the CD20+ mantle cells, but the bright CD20+ germinal center cells showed near absence of bcl-2 expression. In contrast, the neoplastic cells of FL showed greater bcl-2 expression than the T cells of the same tumors and all cell populations of FH. This difference was particularly significant between the neoplastic B cells of FL and the germinal center cells of FH. The combined analysis of CD20 and bcl-2 should be useful for the differential diagnosis between FH and FL and particularly applicable to limited samples or when B-cell clonality is in question. Whether the quantitation of bcl-2 expression can be of further discriminatory value in malignant lymphomas remains to be determined.

Numerical studies of blood shear and washing in a continuous flow ventricular assist device.

The third prototype of a continuous flow ventricular assist device (CF3) is being developed and tested for implantation in humans. The blood in the pump flows through a fully shrouded four bladed impeller (supported by magnetic bearings) and through small clearance regions on either side of the impeller. Computational fluid dynamics (CFD) solutions for this flow have been obtained by using TascFlow, a software package available from AEA Technology, UK. These flow solutions have been used to estimate the shear stresses on the blood in the pump and, hence, to minimize hemolysis. In addition, the solutions are informative for achieving a design that will provide good washing of the blood to minimize the possibility of stagnation points that can lead to thrombosis. This study presents numerical studies of these phenomena in the CF3. The calculated shear rate results are compared with values published in the open literature. The comparisons indicate that hemolysis will not be a problem with CF3, which is in agreement with preliminary experimental measurements. Flow studies are being conducted to determine the optimal size of the clearance regions.