Thomas W. Seale

Establishment of chronic intravenous drug self-administration in the C57BL/6J mouse.

A wide variety of drugs that have significant human abuse potential have been demonstrated to function as positive reinforcers in animals. The present study was designed to characterize a new mouse model of chronic intravenous drug self-administration. Adult male C57BL/6J mice, implanted with external jugular infusion catheters, were given access to response-contingent injections. They did not initiate responding for saline delivery, whereas the C57BL/6J mice initiated morphine, cocaine, methamphetamine and pentobarbital self-administration. Drug-maintained responding was consistently and significantly higher for each compound than for saline responding. In contrast to C57BL/6J mice, DBA/2J mice failed to initiate cocaine self-administration. Thus, chronic intravenous drug self-administration procedures can be adapted to the inbred mouse.

Systematic comparison of apomorphine-induced behavioral changes in two mouse strains with inherited differences in brain dopamine receptors

Dosage and time dependencies of apomorphine-induced changes in stereotyped behaviors (climbing, gnawing and sniffing), locomotor activity and rearing activity were compared in young adult male mice of two inbred strains, DBA/2 and C57BL/6. These two strains are known to differ in their genetically specified brain dopamine receptor number. Apomorphine administered intraperitoneally at dosages of 0.5-20 mg/kg failed to induced stereotyped climbing in DBA/2 at any of the doses tested but markedly increased climbing in C57BL/6 at higher dosages. Apomorphine-induced stereotyped gnawing occurred in both strains at higher dosages although the latency was shorter and maximal effect greater in C57BL/6. Stereotyped sniffing was induced in both strains to a comparable degree at doses greater than or equal to 2.0 mg/kg, and the duration of this stereotypy was indistinguishable between strains. Locomotor activity was inhibited maximally in DBA/2 at an apomorphine dosage of 2 mg/kg and was inhibited to a greater extent than was C57BL/6. Rearing was inhibited in both strains by doses of apomorphine greater than or equal to 0.5 mg/kg; however the duration of the effect was considerably greater in DBA/2 than in C57BL/6. These data suggest that genetically determined differences in central dopamine receptors may have profound and selective effects on behaviors mediated by dopamine pathways; that complex behavioral patterns, e.g., apomorphine-induced stereotypy, may be dissected in to individual components by identifying neuropharmaco genetic differences between strains; that marked strain-specific, inherited differences in dopamine agonist-induced behavioral changes do occur among inbred, non-mutant mouse strains and that their occurrence in other mammalian species including man should be considered.

The iron/heme regulated genes of Haemophilus influenzae: comparative transcriptional profiling as a tool to define the species core modulon

BackgroundHaemophilus influenzae requires heme for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. Although an understanding of the heme acquisition mechanisms of H. influenzae is emerging, significant gaps in our knowledge remain. Unresolved issues include the identities of all genes exhibiting altered transcription in response to iron and heme availability, the fraction of such genes functioning in iron/heme acquisition, and the heterogeneity of this gene set among clinical isolates. Previously we utilized H. influenzae strain Rd KW20 to demonstrate the utility of transcriptional profiling in defining the genes exhibiting altered transcription in response to environmental iron and heme levels. The current study expands upon those observations by determining the iron/heme modulons of two clinical isolates, the type b isolate 10810 and the nontypeable isolate R2866. These data are used to begin to define the core iron/heme modulon of the species.ResultsMicroarray studies were performed to compare gene expression on transition from iron/heme-restricted to iron/heme-replete conditions for each isolate. Of 1820 ORFs on the array corresponding to R2866 genes, 363 were significantly differentially expressed: 233 were maximally transcribed under iron/heme-replete conditions and 130 under iron/heme-restricted conditions. Of the 1883 ORFs representing genes of strain 10810, 353 were significantly differentially transcribed: 150 were preferentially transcribed under iron/heme-replete conditions and 203 under iron/heme-restricted conditions. Comparison of the data sets indicated that 163 genes exhibited similar regulation in both isolates and that 74 of these exhibited similar patterns of regulation in Rd KW20. These comprise the putative core iron/heme modulon.ConclusionThis study provides evidence for a conserved core of H. influenzae genes the transcription of which is altered by the availability of iron and/or heme in the growth environment. Elucidation of this modulon provides a means to identify genes with unrecognized roles in iron/heme acquisition or homeostasis, unanticipated responsiveness to environmental levels of the micronutrients or potential roles in virulence. Defining these core genes is also of potential importance in identifying targets for therapeutic and vaccine designs since products of these genes are likely to be preferentially expressed during growth in iron/heme restricted sites of the human body.

Inherent differences in sensitivity to methylxanthines among inbred mice

The behavioral effects of caffeine, theophylline, paraxanthine, and theobromine on locomotor activity were analyzed in four strains of inbred mice that were previously shown to differ in their acute toxic responses to caffeine administered at high dosages. Dose response curves for the effects of caffeine, theophylline, paraxanthine and theobromine on locomotor activity were established in CBA/J, C57BL/6J, DBA/2J and SWR/J strains of inbred mice. Paraxanthine was the maximally effective methylxanthine in the CBA/J, DBA/2J and SWR/J strains, while in the C57BL/6J strain, caffeine was the maximally effective methylxanthine. Theophylline failed to stimulate locomotor activity in the C57BL/6J strain and theobromine failed to stimulate activity in all of the strains tested. Decreases in locomotor activity were seen at the 100 mg/kg dose of caffeine in the C57BL/6J mice and at the 100 mg/kg dose of theophylline in the C57BL/6J, DBA/2J and SWR/J strains. Theobromine produced decreases in locomotor activity in the C57BL/6J, DBA/2J and SWR/J strains of mice. In contrast to the other methylxanthines, paraxanthine failed to decrease activity across the range of doses tested (1.0-150 mg/kg). These data suggest that the methylxanthines have genetically specified multiple modes of action upon locomotor activity and that the use of genetically distinct strains of mice may have important value in the neurochemical and pharmacological dissection of methylxanthine-induced behavioral effects.

The heme-binding protein (HbpA) of Haemophilus influenzae as a virulence determinant

Haemophilus influenzae has an absolute growth requirement for heme and the heme-binding lipoprotein (HbpA) and has been implicated in the utilization of this essential nutrient. We constructed an insertional mutation of hbpA in a type b and a nontypeable H. influenzae strain. In the type b strain, the hbpA mutant was impaired in utilization of heme complexed to either hemopexin or to albumin and in the utilization of low levels of heme but not in the utilization of heme at high levels or of hemoglobin or hemoglobin-haptoglobin complexes. In contrast, the hbpA mutant derivative of the nontypeable strain was impaired in utilization of all tested heme sources. We further examined the impact of the hbpA mutation in animal models of H. influenzae disease. The hbpA mutant of the nontypeable strain was indistinguishable from the wild-type strain in the chinchilla model of otitis media. The hbpA mutant derivative of the type b strain caused bacteremia as well as the wild-type strain in 5-day old infant rats. However, in 30-day old rats the hbpA caused significantly lower rates of bacteremia than the wild-type strain indicating a role for hbpA and heme acquisition in virulence in this model of H. influenzae disease. In conclusion, HbpA is important for heme utilization by multiple H. influenzae strains and is a virulence determinant in a model of H. influenzae invasive disease.

Complex Role of Hemoglobin and Hemoglobin-Haptoglobin Binding Proteins in Haemophilus influenzae Virulence in the Infant Rat Model of Invasive Infection

ABSTRACT Haemophilus influenzae requires an exogenous heme source for aerobic growth in vitro. Hemoglobin or hemoglobin-haptoglobin satisfies this requirement. Heme acquisition from hemoglobin-haptoglobin is mediated by proteins encoded by hgp genes. Both Hgps and additional proteins, including those encoded by the hxu operon, provide independent pathways for hemoglobin utilization. Recently we showed that deletion of the set of three hgp genes from a nontypeable strain (86-028NP) of H. influenzae attenuated virulence in the chinchilla otitis media model of noninvasive disease. The present study was undertaken to investigate the role of the hgp genes in virulence of the wild-type serotype b clinical isolate HI689 in the infant rat model of hematogenous meningitis, an established model of invasive disease requiring aerobic growth. Bacteremia of high titer and long duration (>14 days) and histopathologically confirmed meningitis occurred in >95% of infant rats challenged at 5 days of age with strain HI689. While mutations disrupting either the Hgp- or Hxu-mediated pathway of heme acquisition had no effect on virulence in infant rats, an isogenic mutant deficient for both pathways was unable to sustain bacteremia or produce meningitis. In contrast, mutations disrupting either pathway decreased the limited ability of H. influenzae to initiate and sustain bacteremia in weanling rats. Biochemical and growth studies also indicated that infant rat plasma contains multiple heme sources that change with age. Taken together, these data indicate that both the hgp genes and the hxuC gene are virulence determinants in the rat model of human invasive disease.

Transcriptional Profile of Haemophilus influenzae: Effects of Iron and Heme

Haemophilus influenzae requires either heme or a porphyrin and iron source for growth. Microarray studies of H. influenzae strain Rd KW20 identified 162 iron/heme-regulated genes, representing approximately 10% of the genome, with > or =1.5-fold changes in transcription in response to iron/heme availability in vitro. Eighty genes were preferentially expressed under iron/heme restriction; 82 genes were preferentially expressed under iron/heme-replete conditions.

Characterization of Three New Competence-Regulated Operons in Haemophilus influenzae

Haemophilus influenzae is one of a growing number of bacteria in which the natural ability to uptake exogenous DNA for potential genomic transformation has been recognized. To date, several operons involved in transformation in this organism have been described. These operons are characterized by a conserved 22-bp regulatory element upstream of the first gene and are induced coincident with transfer from rich to nutrient-depleted media. The previously identified operons comprised genes encoding proteins that include members of the type II secretion system and type IV pili, shown to be essential for transformation in other bacteria, and other proteins previously identified as required for transformation in H. influenzae. In the present study, three novel competence operons were identified by comparative genomics and transcriptional analysis. These operons have been further characterized by construction of null mutants and examination of the resulting transformation phenotypes. The putative protein encoded by the HI0366 gene was shown to be essential for DNA uptake, but not binding, and is homologous to a protein shown to be required for pilus biogenesis and twitching motility in Pseudomonas aeruginosa. An insertion in HI0939 abolished both DNA binding and uptake. The predicted product of this gene shares characteristics with PulJ, a pseudopilin involved in pullulanase export in Klebsiella oxytoca.

Genotypic Differences Between C57BL/6 and A Inbred Mice in Anxiolytic and Sedative Actions of Diazepam

The role of genotype in susceptibility to the behavioral actions of benzodiazepines is not well characterized. To develop a model for such studies, we have characterized the anxiolytic and sedative activities of diazepam in C57BL/6J and A/J inbred mice. C57BL/6J mice were more responsive than A/J mice to diazepam-induced anxiolytic-like activity in the mirrored chamber aversion assay and the elevated plus-maze assay. Basal activity of the two strains did not differ in either assay. In contrast, the two strains were equally responsive to the anxiolytic effects of the 5-HT1Areceptor partial agonist, buspirone. C57BL/6J mice were also more susceptible to the sedative effects of diazepam than were A/J mice. Flumazenil blocked the effects of diazepam in these behavioral assays. Measurement of diazepam and nordiazepam in blood and brain suggested that the response differences are of a pharmacodynamic rather than a pharmacokinetic nature. Taken together, these findings indicate that C57BL/6J and A/J mice provide a valuable tool for behavioral genetic studies of the mechanisms underlying the pharmacological actions of benzodiazepines.

Genetic Differences in Response to Cocaine and Stimulant Drugs

The term psychomotor stimulant generally refers to pharmacologically active compounds recognized by their prominent ability to enhance motor activity through their activation of the central nervous system. This class of agents includes cocaine, amphetamine, and caffeine. These and other compounds included in this pharmacological class differ from one another in their chemical structures and in their mechanisms of direct action on brain cells (Ritz et al., 1987; Miller et al., 1989; Snyder, 1985; Frantz, 1985; Johanson and Fischman, 1989; Pitts and Marwah, 1988). These compounds share the common property of selectively activating neural firing rates in specific regions of the brain (Wechsler et al., 1979; London et al., 1986; Porrino and Kornetsky, 1988; Nehlig et al., 1988), and they induce similar but nonidentical behavioral changes (Balster, 1988; Snyder, 1985; Gold et al., 1989).