Genetic analysis of an ephemeral intraspecific hybrid zone in the hypervariable tree, Metrosideros polymorpha, on Hawai'i Island.

Intraspecific hybrid zones involving long-lived woody species are rare and can provide insights into the genetic basis of early-diverging traits in speciation. Within the landscape-dominant Hawaiian tree, Metrosideros polymorpha, are morphologically distinct successional varieties, incana and glaberrima, that dominate new and old lava flows, respectively, below 1200 me on volcanically active Hawai'i Island, with var. glaberrima also extending to higher elevations and bogs. Here, we use morphological measurements on 86 adult trees to document the presence of an incana-glaberrima hybrid zone on the 1855 Mauna Loa lava flow on east Hawai'i Island and parent-offspring analysis of 1311 greenhouse seedlings from 71 crosses involving 72 adults to estimate heritabilities and genetic correlations among vegetative traits. Both the variation in adult leaf pubescence at the site and the consistency between adult and offspring phenotypes suggest the presence of two hybrid classes, F1s and var. incana backcrosses, as would be expected on a relatively young lava flow. Nine nuclear microsatellite loci failed to distinguish parental and hybrid genotypes. All four leaf traits examined showed an additive genetic basis with moderate to strong heritabilities, and genetic correlations were stronger for the more range-restricted var. incana. The differences between varieties in trait values, heritabilities and genetic correlations, coupled with high genetic variation within but low genetic variation between varieties, are consistent with a multi-million-year history of alternating periods of disruptive selection in contrasting environments and admixture in ephemeral hybrid zones. Finally, the contrasting genetic architectures suggest different evolutionary trajectories of leaf traits in these forms.

Hybrid sterility and evolution in Hawaiian Drosophila: differential gene and allele-specific expression analysis of backcross males.

The Hawaiian Drosophila are an iconic example of sequential colonization, adaptive radiation and speciation on islands. Genetic and phenotypic analysis of closely related species pairs that exhibit incomplete reproductive isolation can provide insights into the mechanisms of speciation. Drosophila silvestris from Hawai'i Island and Drosophila planitibia from Maui are two closely related allopatric Hawaiian picture-winged Drosophila that produce sterile F1 males but fertile F1 females, a pattern consistent with Haldane's rule. Backcrossing F1 hybrid females between these two species to parental species gives rise to recombinant males with three distinct sperm phenotypes despite a similar genomic background: motile sperm, no sperm (sterile), and immotile sperm. We found that these three reproductive morphologies of backcross hybrid males produce divergent gene expression profiles in testes, as measured with RNA sequencing. There were a total of 71 genes significantly differentially expressed between backcross males with no sperm compared with those backcross males with motile sperm and immotile sperm, but no significant differential gene expression between backcross males with motile sperm and backcross males with immotile sperm. All of these genes were underexpressed in males with no sperm, including a number of genes with previously known activities in adult testis. An allele-specific expression analysis showed overwhelmingly more cis-divergent than trans-divergent genes, with no significant difference in the ratio of cis- and trans-divergent genes among the sperm phenotypes. Overall, the results indicate that the regulation of gene expression involved in sperm production likely diverged relatively rapidly between these two closely related species.

Incipient radiation within the dominant Hawaiian tree Metrosideros polymorpha.

Although trees comprise a primary component of terrestrial species richness, the drivers and temporal scale of divergence in trees remain poorly understood. We examined the landscape-dominant tree, Metrosideros polymorpha, for variation at nine microsatellite loci across 23 populations on young Hawai'i Island, sampling each of the island's five varieties throughout its full geographic range. For four varieties, principal coordinate analysis revealed strong clustering of populations by variety across the 10 430 km(2) island, indicating partitioning of the species into multiple evolutionarily significant units. The single island-endemic form, riparian var. newellii, showed especially strong differentiation from other varieties despite occurring in sympatry with other varieties and likely evolved from a bog form on the oldest volcano, Kohala, within the past 500 000 years. Along with comparable riparian forms on other Pacific Islands, var. newellii appears to represent parallel incipient ecological speciation within Metrosideros. Greater genetic distance among the more common varieties on the oldest volcano and an inverse relationship between allelic diversity and substrate age appear consistent with colonization of Hawai'i Island by older, partially diverged varieties followed by increased hybridization among varieties on younger volcanoes. This study demonstrates that broad population-level sampling is required to uncover patterns of diversification within a ubiquitous and long-lived tree species. Hawaiian Metrosideros appears to be a case of incipient radiation in trees and thus should be useful for studies of divergence and the evolution of reproductive isolating barriers at the early stages of speciation.

A pharmacogenetic study of docetaxel and thalidomide in patients with castration-resistant prostate cancer using the DMET genotyping platform.

The anticancer agent docetaxel shows significant inter-individual variation in its pharmacokinetic and toxicity profile. Thalidomide is an active anticancer agent and also shows wide pharmacological variation. Past pharmacogenetic research has not explained this variation. Patients with prostate cancer enrolled in a randomized phase II trial using docetaxel and thalidomide versus docetaxel alone were genotyped using the Affymetrix DMET 1.0 platform, which tests for 1256 genetic variations in 170 drug disposition genes. Genetic polymorphisms were analyzed for associations with clinical response and toxicity. In all, 10 single-nucleotide polymorphisms (SNPs) in three genes were potentially associated with response to therapy: peroxisome proliferator-activated receptor-delta (PPAR-delta), sulfotransferase family, cytosolic, 1C, member 2 (SULT1C2) and carbohydrate (chondroitin 6) sulfotransferase 3 (CHST3). In addition, 11 SNPs in eight genes were associated with toxicities to treatment: spastic paraplegia 7 (pure and complicated autosomal recessive) (SPG7), CHST3, cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6), N-acetyltransferase 2 (arylamine N-acetyltransferase) (NAT2), ATP-binding cassette, sub-family C (CFTR/MRP), member 6 (ABCC6), ATPase, Cu++ transporting, alpha polypeptide (ATP7A), cytochrome P450, family 4, subfamily B, polypeptide 1 (CYP4B1) and solute carrier family 10 (sodium/bile acid cotransporter family), member 2 (SLC10A2). Genotyping results between drug metabolizing enzymes and transporters (DMET) and direct sequencing showed >96% of concordance. These findings highlight the role that non-CYP450 metabolizing enzymes and transporters may have in the pharmacology of docetaxel and thalidomide.

Conservation implications of hybridization in Hawaiian picture-winged Drosophila.

In this review, we discuss the importance of hybridization among species for the conservation of Hawaiian picture-winged Drosophila. Hybridization can be a positive evolutionary process that creates new species and increases the adaptation of populations and species through the spread of adaptive alleles and traits. Conversely, hybridization can disrupt the genetic integrity of species or populations and this may be most detrimental among taxa that are recently hybridizing due to recent ecological changes. The loss of biodiversity in Hawaiian Drosophila through hybridization may be facilitated by habitat alteration and introduced species that reduce population sizes and alter geographic distributions of native species. We briefly review the evidence for hybridization in the genus Drosophila and then focus on hybridization in the Hawaiian picture-winged Drosophila. We examine three general approaches for identifying hybrids and for assessing the factors that appear to contribute to hybridization and the potential ecological and evolutionary outcomes of hybrids in natural populations. Overall, the potential for hybridization among species will likely increase the risk of extinction for Hawaiian picture-winged Drosophila species. Thus, it is important to consider the potential for hybridization among species when developing plans for the conservation of Hawaiian Drosophila.

Approaches to preclinical screening of antiangiogenic agents.

Angiogenesis, or new blood vessel growth, is essential for the growth, invasion, and metastasis of solid tumors. The inhibition of this process, or antiangiogenesis, is a promising new therapeutic anticancer strategy. Several antiangiogenic compounds are currently in preclinical or clinical development for the treatment of cancer. However, the challenge for the discovery and characterization of antiangiogenic targets remains in developing efficient in vitro or in vivo preclinical angiogenesis screening assays to assess and compare antiangiogenic activity. Several semiquantitative or quantitative angiogenesis assays exist, including in vitro endothelial cell systems and ex vivo or in vivo neovascularization models utilizing mouse, rat, or human tissues. We describe the more common and cost-effective angiogenesis assays currently in use, summarizing their unique advantages and disadvantages. Since angiogenesis inhibition is a novel therapeutic modality towards controlling solid tumors, antiangiogenic drug development underlines the importance in describing, standardizing, and developing quantitative screening assays for the next generation of antiangiogenic agents.

The androgen receptor gene and its influence on the development and progression of prostate cancer.

Prostate adenocarcinoma has the highest incidence of any malignancy and is the second leading cause of cancer-related deaths in men in industrialized countries. The development and progression of prostate cancer are dependent on testosterone and dihydrotestosterone; the androgen receptor is the vehicle through which these androgens exert their regulation on prostate cellular proliferation and differentiation. As a result, much effort has been devoted to elucidating the role of the androgen receptor in prostate cancer. The CAG and GGN trinucleotide repeats in exon 1 of the androgen receptor gene have been linked to prostate cancer risk and progression in some studies. Also, androgen receptor gene amplification may be a mechanism of prostate cancer cell adaptation to hormonal therapy. In addition, androgen receptor somatic mutations can result in receptors that have altered binding specificity when compared with wild-type receptors and heightened affinity for hormones other than testosterone and dihydrotestosterone. Gene amplification and somatic mutations, coupled with the fact that various growth factors have been shown to stimulate androgen receptor activity independently of androgens, may enable prostate cancer cells to grow despite testicular-androgen ablation. Unfortunately, current medical therapy for metastatic prostate cancer is deficient, hormone-refractory prostate cancer is a major obstacle in treatment, and, as a result, prostate cancer mortality is still significant. Further study of the function of the androgen receptor will offer a better understanding of prostate cancer pathogenesis and progression, aiding the development of more effective treatments for this disease.

The phosphoprotein Op18/stathmin is differentially expressed in ovarian cancer.

To identify potential prognostic indicators of ovarian cancer and identify targets for therapeutic strategies, mRNA differential display was used to analyze gene expression differences in normal, benign, and cancerous ovarian tissue. One cDNA isolated by this technique, Op18/stathmin, is a highly conserved gene that is reported to have many different functions within a cell, including signal transduction, control of the cell cycle, and the regulation of microtubules. Quantitative Northern blot analysis of 12 malignant ovarian samples, 8 benign ovarian tumors, and 10 normal ovarian tissue samples demonstrated overexpression of Op18/stathmin mRNA in the malignant cancers. Immunohistochemistry showed an apparent overexpression of Op18/stathmin protein level and an association with proliferating cells.

Detection of Neisseria gonorrhoeae and Chlamydia trachomatis colonization of the gravid cervix.

OBJECTIVES: The aims of the study were to determine whether a Gram stain of cervical mucus can accurately rule out infection with Neisseria gonorrhoeae or Chlamydia trachomatis and to compare a diagnostic test that is based on the polymerase chain reaction with a deoxyribonucleic acid probe in the detection of these organisms. STUDY DESIGN: Gravid patients were screened for N gonorrhoeae and C trachomatis with a deoxyribonucleic acid probe, Gram stain, and analysis with the polymerase chain reaction. A normal, noninfected sample was defined by <10 polymorphonuclear leukocytes per high-power field on the Gram stain. Standard statistical methods were used to compare results of the Gram stain and the deoxyribonucleic acid probe, as well as to compare results of deoxyribonucleic acid probe hybridization and polymerase chain reaction analysis. A P value of <.05 was considered statistically significant. RESULTS: Patient enrollment totaled 519. The prevalence of infection as determined by deoxyribonucleic acid probe hybridization was 1.4% for N gonorrhoeae (7/518) and 6.8% for C trachomatis (35/518). The cervical Gram stain predicted the absence of infection in 17% (90/518) of patients, with a negative predictive value of 99% for N gonorrhoeae and 97% for C trachomatis. African American race, age <20 years, and unmarried status were all predictors of the presence of C trachomatis or N gonorrhoeae cervicitis. For the patients who lacked these risk factors (n = 74), the Gram stain had 100% negative predictive value. Analysis with the polymerase chain reaction detected 8 additional patients with C trachomatis and 105 additional patients with N gonorrhoeae, in comparison with deoxyribonucleic acid probe hybridization. CONCLUSION: The cervical Gram stain can accurately predict the absence of N gonorrhoeae and C trachomatis in gravid women. Analysis with the polymerase chain reaction indicates that N gonorrhoeae and C trachomatis are significantly more prevalent in this population than previously reported.

Effect of age on the expression of Pex (Phex) in the mouse.

Pex is a newly discovered gene (also called Phex) whose mutation is the cause of X-linked hypophosphatemia. Other members of this gene family encode endopeptidases that activate or inactivate endocrine and paracrine factors. Though embryonic bone expresses mRNA for the Pex gene at relatively high levels, we have found Pex expression to be widespread in adult organs and to be poorly expressed in adult bone. This led to the hypothesis that Pex mRNA expression changes with age. To test this, genetically normal mice of the B6C3H hybrid strain were studied at 0 (newborn), 2, 3, 10, and 72 weeks of age. Organs known to express Pex were collected, and RNA was extracted from them. Following reverse transcription, cDNA was amplified by the polymerase chain reaction with primers for Pex and G3PDH, a housekeeping gene. The amplimers were separated by electrophoresis, blotted onto nylon membranes, and hybridized with radioactively labeled internal oligonucleotide probes. The radioactivity was quantified, and the data were analyzed as the Pex/G3PDH ratio. The brain samples had high levels of Pex mRNA expression that rose slightly with age. Calvaria, kidney, and lung samples had the highest Pex mRNA expression at birth. In these organs Pex mRNA expression fell with age to undetectable or barely detectable levels. Thymus, heart, and skeletal muscle samples had low Pex mRNA expression at birth that did not change with age. Some organs showed a decline in G3PDH levels with age, but Pex expression decreased more, leading to a reduced Pex/G3PDH ratio. The widespread expression of mRNA for Pex suggests a role beyond that of phosphate homeostasis. The high level of expression in newborn animals suggests a role in growth and development. This seems to occur in addition to its role for the endocrine regulation of phosphate homeostasis by as yet unknown humoral agents that must occur throughout life. In summary, Pex mRNA expression is high in brain and bone at birth. Expression remains high in brain with age but falls with age in bone, kidney, and lung.

Age- and sex-distribution of the mutation load.

We investigate the age and sex distribution of genetic fitness under mutation-selection balance by means of simple one-locus two-allele models. We find that the extent of age and sex variation in the mutation load is very dependent on the average effect of new mutations. If the average heterozygote selective effect of new mutations is large, then age and sex differences may constitute a significant fraction of the total load, and be significant as compared to standing genetic variation. Whether the mutation load will increase or decrease with age depends on the age- and sex-specific effects of the new mutations, and on the rate of accumulation of mutations in the germ line as individuals age. We argue that the load will most likely increase with age in animals with continuous germ-cell division throughout life, and that this will occur even when mutations have unconditionally deleterious effects. We show that a male-biased mutation rate is likely to result in both a male-biased mutation load and a load that increases with male age.

Detection and clearance of prostate cells subsequent to ultrasound-guided needle biopsy as determined by multiplex nested reverse transcription polymerase chain reaction assay.

OBJECTIVES: To determine if circulating prostate cells are detectable subsequent to transrectal ultrasound (TRUS)-guided biopsy, and if so, whether cells remain in circulation for up to 4 weeks. METHODS: Blood samples were drawn from 90 patients with elevated serum prostate-specific antigen (PSA) levels and/or abnormal digital rectal examination. Two samples were drawn from all patients immediately prior to TRUS and 30 minutes postbiopsy. Blood samples were also obtained 1 week postbiopsy from 83 patients, and 1 month postbiopsy from 61 patients. Multiplex nested reverse transcription polymerase chain reaction assay (RT-PCR) for PSA and prostate-specific membrane antigen (PSM) was performed on total ribonucleic acid (RNA) from each sample. Results were reported as positive if at least one of the targets was detected. RESULTS: Of 45 patients with biopsy-proven adenocarcinoma, 22 were RT-PCR positive prebiopsy and all remained positive 30 minutes postbiopsy. Of 23 patients with adenocarcinoma who were RT-PCR negative prebiopsy, 5 (22%) converted to positive 30 minutes postbiopsy (P < 0.001). Four of these 5 patients returned to negative after 1 week or 1 month. Of 45 patients without cancer at biopsy, 32 were RT-PCR negative prebiopsy and 6 (19%) converted to positive 30 minutes postbiopsy (P < 0.001). Although four of six available samples were still positive at 1 week, all four samples available 1 month postbiopsy were negative. CONCLUSIONS: Detection of circulating prostate cells subsequent to biopsy occurred in 11 of 55 (20%) previously RT-PCR negative patients, a proportion twice that reported in the literature. We attribute this higher proportion to the simultaneous detection of PSA and PSM mRNA in our multiplex assay. Conversion rates were similar in patients regardless of biopsy result. Testing of serial postbiopsy blood demonstrates clearing of these cells by 4 weeks in most patients.

Inheritance of behavioural differences between two interfertile, sympatric species, Drosophila silvestris and D. heteroneura.

The Hawaiian fly species, Drosophila silvestris and D. heteroneura, are sympatric and interfertile but show strong behavioural isolation and major differences in male aggressive behaviour and the associated morphology. As a first step in elucidating the genetic control of the differences between these species, we examined the mating and aggressive behaviour of their reciprocal F1 hybrids. The latency to the first wing vibration and the latency to copulate did not differ significantly between the parental species. However, D. heteroneura females had a very low tendency to copulate with D. silvestris males, rarely mating during the observation period. The duration of copulation also differed significantly; same-species pairs of D. silvestris had copulations that lasted about 50% longer than those of same-species pairs of D. heteroneura. The hybrids were intermediate between the parental species for both the tendency to copulate with D. silvestris males and the duration of copulation, suggesting codominance or polygenic inheritance for those traits. The aggression traits that we scored were the leg posture and wing extension during early aggression, and the leg posture and head position during escalated aggression. The parental species showed clear differences for each of these traits. The F1 hybrids resembled one parent or the other, without showing intermediate values, suggesting single-gene dominance or threshold expression of many genes for those traits. None of the courtship or aggressive traits showed X-chromosomal effects, although the head shape of hybrids is influenced by genes on the X chromosome. It is difficult to reconcile the patterns of inheritance of aggressive behaviour and the lack of an X-chromosomal effect with the hypothesis that these traits are influenced by a coadapted gene complex.

Partial deletion of both the spermine synthase gene and the Pex gene in the X-linked hypophosphatemic, gyro (Gy) mouse.

Gy, along with Hyp, is a dominant mutation of the normal gene Pex causing X-linked hypophosphatemia in the mouse. Hemizygous Gy male mice, however, have greater defects in survival, bodily growth, skeletal mineralization, and neurological function than those found in heterozygous Gy females or in Hyp mice. Since the gene for spermine synthase is immediately upstream of the homologous human gene PEX, we compared the effects of the Gy and Hyp mutations on both the spermine synthase gene and the Pex gene. Barely detectable levels of spermine (< 5% of normal) with elevated levels of its precursor, spermidine, were found in organs of Gy male mice compared to normal male littermates. Neither Gy females nor Hyp male mice were significantly affected. Four missing introns of the spermine synthase gene were identified in Gy male mice, suggesting extensive gene disruption. A pseudogene for spermine synthase was also identified in the mouse genome. Pex mRNA was found in several but not all tissues studied in adult normal mice. Pex mRNA was altered in both Gy and Hyp mice. All male Hyp mice were lacking the 3' end of the Pex message, whereas all male Gy mice were deficient at the 5' end. In summary, the Gy mutation is associated with a recessively expressed mutation of the spermine synthase gene, leading to spermine deficiency, and a dominantly expressed mutation of the Pex gene, leading to hypophosphatemia. Alterations in two contiguous genes in Gy may explain the additional phenotypic abnormalities present in the Gy male mouse.

How does offspring quality change with age in male Drosophila melanogaster?

We examined the relationship of genetic quality to age in male Drosophila melanogaster to test two contrasting hypotheses. The traditional hypothesis is that older males have proven their viability and therefore produce offspring of superior genetic quality. This hypothesis is often evoked as an explanation for female preference for older mates. In contrast, we have recently argued that older fathers may produce offspring of inferior genetic quality. Here, we present results from an experiment designed to measure the genetic quality of offspring produced by 2 day old, 2 week old and 5 week old male D. melanogaster. We found a statistically significant small reduction in larval viability and a similar but statistically non-significant reduction in son mating ability among the offspring of the 5 week old males. Daughter fecundity showed no apparent trend for a reduction nor an increase in performance with increasing age of the fathers. There was no evidence of a difference between the 2 day old and the 2 week old males for any of these three fitness components. These results are in somewhat better accordance with our alternative hypothesis, but the relatively weak and late occurring effects indicate that mate choice based on age may not be a viable strategy in this population.

The chicken FMR1 gene is highly conserved with a CCT 5'-untranslated repeat and encodes an RNA-binding protein.

The transcriptional silencing of the human gene, fragile X mental retardation 1 (FMR1), is due to abnormal methylation in response to an expanded 5'-untranslated CGG trinucleotide repeat and accounts for most cases of fragile X syndrome, a frequent inherited form of mental retardation. Although the encoded fragile X mental retardation protein (FMRP) is known to have properties of a RNA-binding protein, the precise function of FMRP remains to be elucidated. We report the cloning of the chicken homolog of FMR1 and show strong evolutionary conservation, with nucleotide and amino acid identities of 85 and 92%, respectively, between chicken and human. In place of the mammalian CGG trinucleotide repeat, a 99-nt tripartite repetitive element containing a CCT trinucleotide repeat flanked on both sides by dinucleotide repeats was identified. Blocks of highly conserved 3'-untranslated sequence were also found. Within the coding region, two copies each of the highly conserved K homology motif and the Arg-Gly-Gly (RGG) box motif, both ribonucleotide particle family domains implicated in RNA binding, were identified. Chicken FMRP was found to bind RNA in vitro, and this activity correlated with the presence of the carboxy-terminal portion of the protein that includes the RGG motifs.

Hemobilia presenting as intermittent gastrointestinal hemorrhage with sincalide confirmation. A case report.

An 82-year-old man had his third episode of melanotic stool. Two previous workups had failed to localize the source of bleeding. A Tc-99m labeled RBC scan visualized the gallbladder early in the study. Administration of sincalide visually decreased the activity, confirming gallbladder activity. Three months later, at his second surgery, hepatic metastases were finally identified as the source of bleeding. In retrospect, the hepatic activity is inhomogeneous with at least two cold defects that could have represented hepatic metastases.

Simultaneous detection of circulating prostate specific antigen (PSA)-expressing and prostate specific membrane antigen (PSM)-expressing cells by a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) assay.

It has yet to be determined whether the detection of prostate specific antigen (PSA)-expressing or prostate specific membrane antigen (PSM)-expressing cells in the circulation of prostate cancer patients is a more accurate predictor of clinical outcome. A method of evaluating both markers simultaneously would aid in the determination of the clinical relevance of reverse transcriptase polymerase chain reaction (RT-PCR) as a staging tool for prostate cancer. We describe the development of a multiplex RT-PCR assay that simultaneously detects the presence of both PSA-expressing cells and PSM-expressing cells, as well as a ubiquitously expressed internal control all within a single reaction. Both PSA cDNA and PSM cDNA were concurrently amplified by multiplex PCR using LNCaP mRNA as the starting template. When used as part of a nested PCR system, the multiplex RT-PCR assay identified one prostate cancer cell when placed in a background of one million cultured B lymphocytes. The multiplex assay was then applied to mRNA isolated from metastatic prostate cancer patients and from healthy male and female volunteers. While all were positive for the internal control G3PDH, three of seven prostate cancer patients were positive for both PSA and PSM expression and two more were positive for either PSA or PSM. None of the male or female volunteers were positive for either PSA or PSM. Multiplex RT-PCR allows for the amplification of both PSA and PSM cDNA within a single RT-PCR reaction, and this approach should allow a consistent comparison of the clinical utility of both PSA and PSM markers as staging tools and predictors of response to therapy.