Expression profiles of 50 xenobiotic transporter genes in humans and pre-clinical species: a resource for investigations into drug disposition.

Carrier-mediated transporters play a critical role in xenobiotic disposition and transporter research is complicated by species differences and their selective tissue expression. The purpose of this study was to generate a comprehensive data set of xenobiotic transporter gene expression profiles in humans and the pre-clinical species mouse, rat, beagle dog and cynomolgus monkey. mRNA expression profiles of 50 genes from the ABC, SLC and SLCO transporter superfamilies were examined in 40 human tissues by microarray analyses. Transporter genes that were identified as enriched in the liver or kidney, or that were selected for their known roles in xenobiotic disposition, were then compared in 22 tissues across the five species. Finally, as clinical variability in drug response and adverse reactions may be the result of variability in transporter gene expression, variability in the expression of selected transporter genes in 75 human liver donors were examined and compared with the highly variable drug metabolizing enzyme CYP3A4.

Updated epidemiological study of workers at two California petroleum refineries, 1950-95.

OBJECTIVES: To further assess the potential role of occupational exposures on mortality, a second update of a cohort study of workers at two petroleum refineries in California was undertaken. METHODS: Mortality analyses were based on standardised mortality ratios (SMRs) and 95% confidence intervals (95% CIs) using the general population of California as a reference. Additional analyses of lymphatic and haematopoietic cancer deaths and diseases related to asbestos were undertaken. RESULTS: The update consisted of 18,512 employees, who contributed 456,425 person-years of observation between 1950 and 1995. Both overall mortality and total cancer mortality were significantly lower than expected, as were several site specific cancers and non-malignant diseases. In particular, no significant increases were reported for leukaemia cell types or non-Hodgkin's lymphoma. Mortality excess from multiple myeloma was marginally significant. The excess was confined to employees enrolled before 1949. Furthermore, there was no significant upward trend based on duration of employment, which argues against a causal interpretation relative to employment or exposures at the refineries. No increase was found for diseases related to asbestos: pulmonary fibrosis; lung cancer; or malignant mesothelioma. There was no significant increase in mortality from any other cancers or non-malignant diseases. CONCLUSION: This second update provides additional reassurance that employment at these two refineries is not associated with increased risk of mortality.

Acoustics of a small Australian burrowing cricket: the control of low-frequency pure-tone songs.

For most insects, size determines the call frequency. This paper describes the acoustics of a small brachypterous cricket (Rufocephalus sp.; body length 9.6 mm) producing a call with a carrier frequency of approximately 3.2 kHz from a subterranean burrow. Crickets such as Gryllus campestris are approximately twice this length and produce a call frequency close to 5 kHz. The burrow of Rufocephalus opens via a small hole with a diameter of 3.2 mm. The neck of the hole at approximately 1.4 mm depth opens to a vertical two-part burrow with an upper vase-shaped chamber 16.1 mm in height with a diameter of 9.4 mm. This top chamber connects via a 6.4mm high (diameter 5.2 mm) neck to a more irregular chamber approximately 18 mm high with a width of approximately 11 mm. The walls of the top chamber neck and of the upper part of the lower chamber are smooth and appear to be sealed with saliva. The song has a mean centre frequency of 3.2 kHz and is made up of variable-length trills of pulses of mean duration 15.8 ms. Many song pulses had smooth envelopes and their frequency did not vary by more than +/-40Hz from the centre frequency, with a relative bandwidth Q-3dB of over 50. Other pulses showed considerable amplitude and frequency modulation within the pulse. When driven by external sound, burrows resonated at a mean frequency of 3.5 kHz with a mean quality factor Q of 7.4. Natural-size model burrows resonated at similar frequencies with similar Q values. One cricket, which had previously called from its own burrow at 2.95 kHz, sang at 3.27 kHz from a burrow that resonated at the same frequency. Life-size model burrows driven by external sound resonated at similar frequencies to the actual burrows; models three times life size resonated at one-third of this frequency. In all models, the sound pressure was more-or-less constant throughout the top chamber but fell rapidly in the neck of the burrow; the phase of the sound was effectively constant in the top chamber and neck and fell through approximately 180 degrees in passing from the neck into the lower chamber. A numerical model of the sound flow from region to region gave essentially similar results. A resonant electrical model fed from a high-impedance source with discrete tone bursts at different frequencies showed similar amplitude and frequency modulation to the various types of song pulses that were observed. It is suggested that the high purity of the songs results from close entrainment of the sound-producing mechanism of the insect's wings to the sharply resonant burrow.

Forensic entomology: application, education and research in Western Australia.

Forensic entomology as a science and a tool for investigation has had slow beginnings in Australia. A number of small animal decomposition trials have been recorded in the literature but mostly from an ecological rather than a forensic entomology perspective. In the last 20 years, a number of more forensically orientated field trials on small pigs and some fly developmental trials in the laboratory have been conducted but lack any replication. The following article was presented at an international seminar to detail the current research in forensic entomology, the applications of forensic entomology in scene of crime (SOC) and homicide investigations and the education of police and judiciary in the discipline of forensic entomology in Western Australia over the last 10 years.

Hearing dimorphism, trait variation and conflicts over space in the thorax of the bushcricket Requena verticalis (Listroscelidinae: Tettigoniidae: Orthoptera).

The hearing system of Requena verticalis is sexually dimorphic. Previous work has shown size of the auditory spiracle determines absolute threshold and as female spiracles are, on average, larger than males, females are more sensitive to the main energy of the male call. In all measured traits in morphology and physiology, females showed lower coefficient of variation than males. This difference was significant for bulla volume and hearing threshold. In addition, female ear size covaries with thorax dimensions but this is not so in males. Such a finding suggests stabilising selection on ear size in females, perhaps explained by the requirement of females to recognise and locate the male. As the auditory bulla is larger in females than males, so occupying thoracic space, we suggest a possible trade-off in this brachypterous species between hearing sensitivity and sound production. Finally, we examine relative growth of body structures not associated with hearing and those that influence hearing sensitivity. Scaling, where traits are under strong selection, may result in allometry. Female hearing traits show positive allometry with absolute size and while the relationship between bulla volume and spiracle area was positively allometric in females this was not the case for males.

FEMALE-FEMALE COMPETITION IN KATYDIDS: SEXUAL SELECTION FOR INCREASED SENSITIVITY TO A MALE SIGNAL?

In contrast to studies of sex-specific weaponry and other sexually selected traits, there has been no examination of Darwin's (1871, p. 418) suggestion that elaborations or enlargements of "the organs of sense" function to enhance mating success. In certain katydids the size of thoracic spiracles, which are a main input into the hearing system, determines auditory sensitivity of females. Here we present evidence that sexual dimorphism in the spiracle size of a pollen katydid, Kawanaphila nartee, is a result of sexual selection on females competing to locate nuptial-gift giving males. In field experiments in which female K. nartee were attracted to a calling male, we show a pairing advantage to females with larger auditory spiracles. The spiracle-size advantage was not a correlated result of a larger body size or mass of winners. Finally, there was no spiracle-size advantage or body-mass advantage for mating females in a later stage of competition when experimental females struggled for access to a silent male. We suggest that research on the detection of displays has lagged behind work on the displays themselves; the focus has been on the species specificity of signal perception rather than on the fitness consequences of variation in the ability to detect cues from mates or predators.

The Merck Gene Index browser: an extensible data integration system for gene finding, gene characterization and EST data mining.

MOTIVATION: To make effective use of the vast amounts of expressed sequence tag (EST) sequence data generated by the Merck-sponsored EST project and other similar efforts, sequences must be organized into gene classes, and scientists must be able to 'mine' the gene class data in the context of related genomic data. RESULTS: This paper presents the Merck Gene Index browser, an easily extensible, World Wide Web-based system for mining the Merck Gene Index (MGI) and related genomic data. The MGI is a non-redundant set of clones and sequences, each representing a distinct gene, constructed from all high-quality 3' EST sequences generated by the Merck-sponsored EST project. The MGI browser integrates data from a variety of sources and storage formats, both local and remote, using an eclectic integration strategy, including a federation of relational databases, a local data warehouse and simple hypertext links. Data currently integrated include: LENS cDNA clone and EST data, dbEST protein and non-EST nucleic acid similarity data, WashU sequence chromatograms. Entrez sequence and Medline entries, and UniGene gene clusters. Flatfile sequence data are accessed using the Bioapps server, an internally developed client-server system that supports generic sequence analysis applications. Browser data are retrieved and formatted by means of the Bioinformatics Data Integration Toolkit (B-DIT), a new suite of Perl routines.

Molecular evolution of Hox gene regulation: cloning and transgenic analysis of the lamprey HoxQ8 gene.

The mammalian Hox clusters arose by duplication of a primordial cluster. The duplication of Hox clusters created redundancy within cognate groups, allowing for change in function over time. The lamprey, Petromyzon marinus, occupies an intermediate position within the chordates, both in terms of morphologic complexity and possibly cluster number. To determine the extent of divergence among Hox genes after duplication events within vertebrates, we analyzed Hox genes belonging to cognate group 8. Here we report characterization of the HoxQ8 gene, which shows conservation with mammalian genes in its amino-terminal, homeobox and hexapeptide sequences, and in the position of its splice sites. A beta-galactosidase reporter gene was introduced in the HoxQ8 genomic region by targeted recombinational cloning using a yeast-bacteria shuttle vector, pClasper. These reporter gene constructs were tested for their ability to direct region-specific expression patterns in transgenic mouse embryos. Lamprey enhancers direct expression to posterior neural tube but not to mesoderm, suggesting conservation of neuronal enhancers. In the presence of the mouse heat shock promoter, lamprey enhancers could also direct expression to the posterior mesoderm suggesting that there has been some divergence in promoter function. Our results suggest that comparative studies on Hox gene structure and analysis of regulatory elements may provide insights into changes concomitant with Hox cluster duplications in the chordates.

Phylogenetic reconstruction of vertebrate Hox cluster duplications.

In vertebrates and the cephalochordate, amphioxus, the closest vertebrate relative, Hox genes are linked in a single cluster. Accompanying the emergence of higher vertebrates, the Hox gene cluster duplicated in either a single step or multiple steps, resulting in the four-cluster state present in teleosts and tetrapods. Mammalian Hox clusters (designated A, B, C, and D) extend over 100 kb and are located on four different chromosomes. Reconstructing the history of the duplications and its relation to vertebrate evolution has been problematic due to the lack of alignable sequence information. In this study, the problem was approached by conducting a statistical analysis of sequences from the fibrillar-type collagens (I, II, III, and IV), genes closely linked to each Hox cluster which likely share the same duplication history as the Hox genes. We find statistical support for the hypothesis that the cluster duplication occurred as multiple distinct events and that the four-cluster situation arose by a three-step sequential process.

A 50-year mortality follow-up of a large cohort of oil refinery workers in Texas.

To investigate further the possible role of occupational exposures on mortality, an update of a large Texas petroleum refinery cohort was undertaken. Between 1937 and 1987, 6799 deaths were identified among 17,844 employees. Relative to the general population of Texas, the overall standardized mortality ratio (SMR) showed a statistically significant deficit, as did nine other cause-of-death categories. Statistically significant mortality excesses were found for bone cancer (SMR = 207.8: 95% confidence interval [CI], 110.6 to 355.3), acute lymphocytic leukemia (ALL) (SMR = 259.6; 95% CI, 112.1 to 511.5), and benign/unspecified neoplasms (SMR = 194.9; 95% CI, 129.5 to 281.7). However, none of these diseases demonstrated an exposure-response relationship with length of employment. Subcohort mortality analyses by sex and race groups, length of employment, interval since hire, period of hire, and pay status were also performed. Overall, the update findings do not indicate that any excess mortality occurred as a result of employment at the refinery.

Tobacco use by Indiana children and adolescents.

Data from a five-year, longitudinal survey of cigarette smoking and smokeless tobacco use by Indiana children and adolescents are presented. A four-page, self-contained questionnaire was used to collect anonymous information from more than 240,000 students in grades six through 12. Indiana students reported prevalence levels 20% to 50% higher than levels reported in comparable national surveys. Perceived risk of physical or psychological harm was the single best statistical predictor of whether or not a student would choose to use tobacco. Tobacco use was statistically linked to increased use of illicit drugs. Physicians can play a powerful role in influencing a patient's perception of the risks associated with cigarette smoking.

Self-interest and attitudes about legislation controlling alcohol.

A stratified random sample of 505 adult Indiana residents living in households with telephones was surveyed using random-digit dialing to assess their attitudes about nine specific legislative proposals to control drunk driving or underage drinking and to assess the effects of self-interest on those attitudes. A two-stage Mitofsky-Waksberg design was used in the computer-assisted telephone-interview process. There was widespread support for all nine proposals, but self-interest, related to personal vulnerability for enforcement of those measures, reduced the support of regular drinkers for drunk-driving controls compared with nondrinkers' support. Parents of children aged 18 and younger were less likely than nonparents to support imposing parental liability for the consequences of children's underage drinking.

A national survey of public support for restrictions on youth access to tobacco.

A national telephone survey was conducted to measure public support for seven proposals to restrict youth access to tobacco products, including increases in the cigarette excise tax. A random digit dialing survey, using computer-assisted telephone interviews and a two-stage Mitofsky-Waksberg design, was used to generate and replace telephone numbers and to select individuals from within households. More than 94% of respondents believed cigarette smoking by children and adolescents to be a "very serious" or "somewhat serious" problem. Most respondents expressed support for all the proposed measures to restrict youth access to tobacco products (fines for sellers, fines for youthful violators, licensing of all tobacco vendors, restrictions on cigarette vending machines, ban on sponsorship of youth-oriented events, and ban on all tobacco advertising), and for increases in the cigarette excise tax.

Molecular evidence on primate phylogeny from DNA sequences.

Evidence from DNA sequences on the phylogenetic systematics of primates is congruent with the evidence from morphology in grouping Cercopithecoidea (Old World monkeys) and Hominoidea (apes and humans) into Catarrhini, Catarrhini and Platyrrhini (ceboids or New World monkeys) into Anthropoidea, Lemuriformes and Lorisiformes into Strepsirhini, and Anthropoidea, Tarsioidea, and Strepsirhini into Primates. With regard to the problematic relationships of Tarsioidea, DNA sequences group it with Anthropoidea into Haplorhini. In addition, the DNA evidence favors retaining Cheirogaleidae within Lemuriformes in contrast to some morphological studies that favor placing Cheirogaleids in Lorisiformes. While parsimony analysis of the present DNA sequence data provides only modest support for Haplorhini as a monophyletic taxon, it provides very strong support for Hominoidea, Catarrhini, Anthropoidea, and Strepsirhini as monophyletic taxa. The parsimony DNA evidence also rejects the hypothesis that megabats are the sister group of either Primates or Dermoptera (flying lemur) or a Primate-Dermoptera clade and instead strongly supports the monophyly of Chiroptera, with megabats grouping with microbats at considerable distance from Primates. In contrast to the confused morphological picture of sister group relationships within Hominoidea, orthologous noncoding DNA sequences (spanning alignments involving as many as 20,000 base positions) now provide by the parsimony criterion highly significant evidence for the sister group relationships defined by a cladistic classification that groups the lineages to all extant hominoids into family Hominidae, divides this ape family into subfamilies Hylobatinae (gibbons) and Homininae, divides Homininae into tribes Pongini (orangutans) and Hominini, and divides Hominini into subtribes Gorillina (gorillas) and Hominina (humans and chimpanzees). A likelihood analysis of the largest body of these noncoding orthologues and counts of putative synapomorphies using the full range of sequence data from mitochondrial and nuclear genomes also find that humans and chimpanzees share the longest common ancestry.

Cigarette smoking as a predictor of alcohol and other drug use by children and adolescents: evidence of the "gateway drug effect".

Data from a statewide survey, conducted by the Indiana Prevention Resource Center, of 20,629 Indiana students in grades 5-12 were analyzed to determine the extent to which cigarette smoking predicted use of alcohol and other drugs and acted as a so-called "gateway drug." A three-stage purposive/quota cluster sampling strategy yielded a representative sample of Indiana students, stratified by grade. Cross-tabulated data revealed a strong, dose-dependent relationship between smoking behavior and binge drinking, as well as use of alcohol and illicit drugs. Daily pack-a-day smokers were three times more likely to drink alcohol, seven times more likely to use smokeless tobacco, and 10-30 times more likely to use illicit drugs than nonsmokers. A stepwise multiple regression analyzed the role that the student's perceptions of the risk of using drugs and of peer approval/disapproval of the student's drug use, gender, grade in school, and ethnic background played in predicting alcohol and other drug use.

Phylogenetic footprinting reveals unexpected complexity in trans factor binding upstream from the epsilon-globin gene.

The human epsilon-globin gene undergoes dramatic changes in transcriptional activity during development, but the molecular factors that control its high expression in the embryo and its complete repression at 6-8 weeks of gestation are unknown. Although a putative silencer has been identified, the action of this silencer appears to be necessary but not sufficient for complete repression of epsilon gene expression, suggesting that multiple control elements may be required. Phylogenetic footprinting is a strategy that uses evolution to aid in the elucidation of these multiple control points. The strategy is based on the observation that the characteristic developmental expression pattern of the epsilon gene is conserved in all placental mammals. By aligning epsilon genomic sequences (from -2.0 kb upstream to the epsilon polyadenylylation signal), conserved sequence elements that are likely binding sites for trans factors can be identified against the background of neutral DNA. Twenty-one such conserved elements (phylogenetic footprints) were found upstream of the epsilon gene. Oligonucleotides spanning these conserved elements were used in a gel-shift assay to reveal 47 nuclear binding sites. Among these were 8 binding sites for YY1 (yin and yang 1), a protein with dual (activator or repressor) activity; 5 binding sites for the putative stage selector protein, SSP; and 7 binding sites for an as yet unidentified protein. The large number of high-affinity interactions detected in this analysis further supports the notion that the epsilon gene is regulated by multiple redundant elements.

Reexamination of the African hominoid trichotomy with additional sequences from the primate beta-globin gene cluster.

Additional DNA sequence information from a range of primates, including 13.7 kb from pygmy chimpanzee (Pan paniscus), was added to data sets of beta-globin gene cluster sequence alignments that span the gamma 1, gamma 2, and psi eta loci and their flanking and intergenic regions. This enlarged body of data was used to address the issue of whether the ancestral separations of gorilla, chimpanzee, and human lineages resulted from only one trichotomous branching or from two dichotomous branching events. The degree of divergence, corrected for superimposed substitutions, seen in the beta-globin gene cluster between human alleles is about a third to a half that observed between two species of chimpanzee and about a fourth that between human and chimpanzee. The divergence either between chimpanzee and gorilla or between human and gorilla is slightly greater than that between human and chimpanzee, suggesting that the ancestral separations resulted from two closely spaced dichotomous branchings. Maximum parsimony analysis further strengthened the evidence that humans and chimpanzees share the longest common ancestry. Support for this human-chimpanzee clade is statistically significant at P = 0.002 over a human-gorilla clade or a chimpanzee-gorilla clade. An analysis of expected and observed homoplasy revealed that the number of sequence changes uniquely shared by human and chimpanzee lineages is too large to be attributed to homoplasy. Molecular clock calculations that accommodated lineage variations in rates of molecular evolution yielded hominoid branching times that ranged from 17-19 million years ago (MYA) for the separation of gibbon from the other hominoids to 5-7 MYA for the separation of chimpanzees from humans. Based on the relatively late dates and mounting corroborative evidence from unlinked nuclear genes and mitochondrial DNA for the close sister grouping of humans and chimpanzees, a cladistic classification would place all apes and humans in the same family. Within this family, gibbons would be placed in one subfamily and all other extant hominoids in another subfamily. The later subfamily would be divided into a tribe for orangutans and another tribe for gorillas, chimpanzees, and humans. Finally, gorillas would be placed in one subtribe with chimpanzees and humans in another, although this last division is not as strongly supported as the other divisions.