Faculty Bibliography

Few studies have examined the influence of the age and the strain of mouse on the pharmacokinetics of psychostimulants, or the role of pharmacokinetics in age-related differences in drug responses. The present study compared concentrations of cocaine, and its metabolite, benzoylecgonine (BZE), in the blood and brain of early (P35) and later (P42) periadolescent and adult (P63) CD-1 and C57BL/6 male mice 15 min after acute intraperitoneal injection of cocaine (20 mg/kg). Brain levels of cocaine and BZE after seven daily cocaine injections in CD-1 and C57BL/6 mice beginning on P35 and on P63 were also measured. P35 periadolescents of both strains had lower blood cocaine levels than did the adults, but only C57BL/6 periadolescents had lower brain cocaine levels than the adults. C57BL/6 mice of both ages had higher blood cocaine levels than did the corresponding CD-1 mice. Concomitant with lower cocaine levels, periadolescent CD-1 mice had higher blood BZE levels than the adults, suggesting that periadolescents may metabolize cocaine faster. Brain cocaine levels in P42 C57BL/6 mice were similar to those of adults. Cocaine-induced activity did not differ between periadolescent and adult CD-1 mice after a single injection of cocaine, whereas periadolescent C57BL/6 mice had lower activity levels than did the adults after a single cocaine injection. Periadolescent CD-1 mice exhibited higher levels of locomotor activity following cocaine injection than did periadolescent C57BL/6 mice. Following chronic cocaine administration, cocaine and BZE levels in the brains of periadolescent and adult mice did not differ from each other in either strain. However, brain cocaine levels at both ages were lower in CD-1 mice than in C57BL/6 mice. In conclusion, the age and the strain of mouse significantly affect the levels of cocaine obtained in brain and blood following acute administration. Our data are consistent with the notion that CD-1 and C57BL/6 mice metabolize cocaine faster during the early periadolescent period than as adults. Furthermore, potentially important strain differences between CD-1 and C57BL/6 mice were noted in cocaine levels following acute and chronic cocaine administration, and in locomotor activity following acute cocaine administration

Effective therapies for stroke must interdict multiple parallel and sequential pathophysiological events. A paradigm which offers insight into multivalent but thoughtfully coordinated protective programs is ischemic preconditioning. A central hypothesis of our group and others is that pharmacological agents that activate programs of gene expression normally induced by ischemic preconditioning will be effective agents for the prevention and treatment of stroke. Inhibitors of a class of enzymes, the hypoxia inducible factor-1 (HIF-1) prolyl hydroxylases stabilize the transcriptional activator HIF-1 and activate target genes involved in compensation for ischemia, including erythropoeitin (Epo) and vascular endothelial growth factor (VEGF). Here, we review evidence suggesting that the HIF-1 prolyl hyroxylases are inhibited during ischemic preconditioning and that pharmacological inhibitors of these enzymes are viable targets for stroke therapy.

BACKGROUND: Heat shock proteins (HSPs) are a family of highly conserved proteins found ubiquitously in mammalian cells, believed to be regulators of normal cell physiology and the cellular stress response. In addition, the small 27-kDa heat shock protein (HSP27) has previously been found to be a differentiation marker for keratinocytes and a prognostic marker associated with increased survival in certain cancerous tumors. METHODS: Using immunohistochemistry on routinely processed paraffin sections, we examined skin biopsies from 15 invasive melanomas, 13 intradermal nevi, and two compound nevi immunostained with a mouse monoclonal antibody to HSP27. In addition, cultured melanocytes were heat stressed at 45 degrees C for 1 h and then fixed and immunostained in order to localize HSP27 expression intracellularly. RESULTS: We found cytoplasmic and strong perinuclear staining of HSP27 in melanocytes in normal skin, in melanomas, and in nevi. Nuclear reactivity was absent. In addition, in cultured non-malignant melanocytes, HSP27 expression relocated from the cytoplasm to the nucleus with heat stress. CONCLUSIONS: To our knowledge, this investigation is the first to demonstrate that HSP27 is expressed in melanocytes in normal skin, in nevi, and in non-malignant cultured melanocytes

BACKGROUND: We have developed a novel, microsphere-based universal array platform referred to as the Tag-It platform. This platform is suitable for high-throughput clinical genotyping applications and was used for multiplex analysis of a panel of thrombophilia-associated single-nucleotide polymorphisms (SNPs). METHODS: Genomic DNA from 132 patients was amplified by multiplex PCR using 6 primer sets, followed by multiplex allele-specific primer extension using 12 universally tagged genotyping primers. The products were then sorted on the Tag-It array and detected by use of the Luminex xMAP system. Genotypes were also determined by sequencing. RESULTS: Empirical validation of the universal array showed that the highest nonspecific signal was 3.7% of the specific signal. Patient genotypes showed 100% concordance with direct DNA sequencing data for 736 SNP determinations. CONCLUSIONS: The Tag-It microsphere-based universal array platform is a highly accurate, multiplexed, high-throughput SNP-detection platform.

The cerebellum consists of a highly organized set of folia that are largely generated postnatally during expansion of the granule cell precursor (GCP) pool. Since the secreted factor sonic hedgehog (Shh) is expressed in Purkinje cells and functions as a GCP mitogen in vitro, it is possible that Shh influences foliation during cerebellum development by regulating the position and/or size of lobes. We studied how Shh and its transcriptional mediators, the Gli proteins, regulate GCP proliferation in vivo, and tested whether they influence foliation. We demonstrate that Shh expression correlates spatially and temporally with foliation. Expression of the Shh target gene Gli1 is also highest in the anterior medial cerebellum, but is restricted to proliferating GCPs and Bergmann glia. By contrast, Gli2 is expressed uniformly in all cells in the developing cerebellum except Purkinje cells and Gli3 is broadly expressed along the anteroposterior axis. Whereas Gli mutants have a normal cerebellum, Gli2 mutants have greatly reduced foliation at birth and a decrease in GCPs. In a complementary study using transgenic mice, we show that overexpressing Shh in the normal domain does not grossly alter the basic foliation pattern, but does lead to prolonged proliferation of GCPs and an increase in the overall size of the cerebellum. Taken together, these studies demonstrate that positive Shh signaling through Gli2 is required to generate a sufficient number of GCPs for proper lobe growth

The effect of light on neuroendocrine functions is thought to be mediated through retinal inputs to the circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). The present studies were conducted to provide experimental evidence for this signaling modality in non-human primates. In the St. Kitts vervet monkey, anterograde tracing of SCN efferents revealed a monosynaptic pathway between the circadian clock and hypothalamic neurons producing luteinizing hormone-releasing hormone (LHRH). Using a variety of tracing techniques, direct retinal input was found to be abundant in the SCN and in other hypothalamic sites. Strikingly, in hypothalamic areas other than the SCN, primary visual afferents established direct contacts with neuroendocrine cells including those producing LHRH and dopamine, neurons that are the hypothalamic regulators of pituitary gonadotrops and prolactin. Thus, our data reveal for the first time in primates that light stimuli can reach the hypothalamo-pituitary-gonadal axis, directly providing a pathway independent of but parallel to that of the circadian clock for the photic modulation of hormone release

OBJECTIVE: To assess the impact of cannulation of a resistant cervical os with the outer malleable sheath of a double-lumen, soft ET catheter on IVF-ET outcomes. DESIGN: Retrospective cohort study. SETTING: University-based IVF center. PATIENT(S): One hundred forty-two patients undergoing 142 ETs. INTERVENTION(S): Trial ultrasound-guided ET at all transfers, leaving the malleable outer sheath in situ when the soft inner catheter could not negotiate the internal os. MAIN OUTCOME MEASURE(S): Implantation rate and clinical pregnancy rate. RESULT(S): In 102 ETs (71.8%), the soft inner sheath easily negotiated the internal os (group 1). Forty ETs (28.2%) required cannulation of resistant internal ora with the outer sheath of the trial catheter (group 2). Implantation rates (35% vs. 32% in groups 1 and 2, respectively) and clinical pregnancy rates (50% vs. 45%) were not significantly different between groups. Blood was present on the transfer catheter after ET more frequently in group 2 than in group 1 (55% vs. 15%); however, neither the implantation rate nor the clinical pregnancy rate were affected by the presence of blood. CONCLUSION(S): Cannulation of a resistant internal os by the malleable outer sheath and blood on the transfer catheter after ET do not have an adverse effect on implantation rate or clinical pregnancy rate

Factors of both cytoplasmic and nuclear origin regulate metaphase chromosome alignment and spindle checkpoint during mitosis. Most aneuploidies associated with maternal aging are believed to derive from nondisjunction and meiotic errors, such as aberrations in spindle formation and chromosome alignment at meiosis I. Senescence-accelerated mice (SAM) exhibit aging-associated meiotic defects, specifically chromosome misalignments at meiosis I and II that resemble those found in human female aging. How maternal aging disrupts meiosis remains largely unexplained. Using germinal vesicle nuclear transfer, we found that aging-associated misalignment of metaphase chromosomes is predominately associated with the nuclear factors in the SAM model. Cytoplasm of young hybrid B6C3F1 mouse oocytes could partly rescue aging-associated meiotic chromosome misalignment, whereas cytoplasm of young SAM was ineffective in preventing the meiotic defects of old SAM oocytes, which is indicative of a deficiency of SAM oocyte cytoplasm. Our results demonstrate that both nuclear and cytoplasmic factors contribute to the meiotic defects of the old SAM oocytes and that the nuclear compartment plays the predominant role in the etiology of aging-related meiotic defects

Sexual dimorphism requires the integration of positional information in the embryo with the sex determination pathway. Homeotic genes are a major source of positional information responsible for patterning along the anterior-posterior axis in embryonic development, and are likely to play a critical role in sexual dimorphism. Here, we investigate the role of homeotic genes in the sexually dimorphic development of the gonad in Drosophila. We have found that Abdominal-B (ABD-B) is expressed in a sexually dimorphic manner in the embryonic gonad. Furthermore, Abd-B is necessary and sufficient for specification of a sexually dimorphic cell type, the male-specific somatic gonadal precursors (msSGPs). In Abd-B mutants, the msSGPs are not specified and male gonads now resemble female gonads with respect to these cells. Ectopic expression of Abd-B is sufficient to induce formation of extra msSGPs in additional segments of the embryo. Abd-B works together with abdominal-A to pattern the non-sexually dimorphic somatic gonad in both sexes, while Abd-B alone specifies the msSGPs. Our results indicate that Abd-B acts at multiple levels to regulate gonad development and that Abd-B class homeotic genes are conserved factors in establishing gonad sexual dimorphism in diverse species.