Function and regulation of the Pem homeobox gene in vivo

Pem, a member of the PEPP homeobox family, is expressed in somatic cells in male and female reproductive tissues. In the adult murine testis, Pem is specifically expressed in Sertoli cells, where it is restricted to stages IV–VIII of the seminiferous epithelial cycle. To identify Pem's function in Sertoli cells, transgenic mice were generated that express Pem in Sertoli cells during all stages of the seminiferous epithelial cycle. This resulted in an increase in double-strand DNA breaks in preleptotene spermatocytes and single-strand DNA breaks in elongating spermatids. My results suggest that Pem regulates Sertoli-cell genes that encode secreted or cell-surface proteins that serve to control premeiotic DNA replication, DNA repair, and/or chromatin remodeling in the adjacent germ cells. Three additional transgenic mouse containing varying lengths of the Pem male-specific promoter (Pp) were generated to identify the sequences responsible for regulating Pem expression in the testis and epididymis. My analysis suggests that there are at least two regulatory regions in the Pem Pp. In the testis, region II directs androgen-dependent expression specifically in Sertoli cells whereas region I fine-tunes stage-specific expression by acting as a negative regulator. In the epididymis, region II confers androgen-dependent, developmentally-regulated expression in the caput whereas region I prevents inappropriate expression in the corpus. I also report the identification and characterization of two human PEPP family members related to Pem that I have named hPEPP1 and hPEPP2. The hPEPP1 and hPEPP2 homeodomains are more closely related to PEPP subfamily homeodomains than to any other homeodomain subfamily. Both genes are localized to the specific region of the human X chromosome that shares synteny with the region on the murine X chromosome containing three PEPP homeobox genes, Pem, Psx-1, and Psx-2. hPEPP1 and hPEPP2 mRNA expression is restricted to the testis but is aberrantly expressed in tumor cells of different origins, analogous to the expression pattern of Pem but not of Psx-1 or Psx-2. Unlike all known PEPP members, neither hPEPP1 nor hPEPP2 are expressed in placenta, which suggests that the regulation of the PEPP family has undergone significant alteration since the split between hominids and rodents. ^

Treatment of solid organ transplant recipients with autologous Epstein Barr virus-specific cytotoxic T lymphocytes (CTLs).

We have investigated the in vivo safety, efficacy, and persistence of autologous Epstein Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) for the treatment of solid organ transplant (SOT) recipients at high risk for EBV-associated posttransplantation lymphoproliferative disease (PTLD). EBV-CTLs generated from 35 patients expanded with normal kinetics contained both CD8 and CD4 lymphocytes and produced significant specific killing of autologous EBV-transformed B lymphoblastoid cell lines (LCLs). Twelve SOT recipients at high risk for PTLD, or with active disease, received autologous CTL infusions without toxicity. Real-time polymerase chain reaction (PCR) monitoring of EBV-DNA showed a transient increase in plasma EBV-DNA suggestive of lysis of EBV-infected cells, although there was no consistent decrease in virus load in peripheral-blood mononuclear cells. Interferon-gamma enzyme-linked immunospot (ELISPOT) assay and tetramer analysis showed an increase in the frequency of EBV-responsive T cells, which returned to preinfusion levels after 2 to 6 months. None of the treated patients developed PTLD. One patient with liver PTLD showed a complete response, and one with ocular disease has had a partial response stable for over one year. These data are consistent with an expansion and persistence of adoptively transferred EBV-CTLs that is limited in the presence of continued immunosuppression but that nonetheless produces clinically useful antiviral activity.

Intracranial EEG reveals a time- and frequency-specific role for the right inferior frontal gyrus and primary motor cortex in stopping initiated responses.

Inappropriate response tendencies may be stopped via a specific fronto/basal ganglia/primary motor cortical network. We sought to characterize the functional role of two regions in this putative stopping network, the right inferior frontal gyrus (IFG) and the primary motor cortex (M1), using electocorticography from subdural electrodes in four patients while they performed a stop-signal task. On each trial, a motor response was initiated, and on a minority of trials a stop signal instructed the patient to try to stop the response. For each patient, there was a greater right IFG response in the beta frequency band ( approximately 16 Hz) for successful versus unsuccessful stop trials. This finding adds to evidence for a functional network for stopping because changes in beta frequency activity have also been observed in the basal ganglia in association with behavioral stopping. In addition, the right IFG response occurred 100-250 ms after the stop signal, a time range consistent with a putative inhibitory control process rather than with stop-signal processing or feedback regarding success. A downstream target of inhibitory control is M1. In each patient, there was alpha/beta band desynchronization in M1 for stop trials. However, the degree of desynchronization in M1 was less for successfully than unsuccessfully stopped trials. This reduced desynchronization on successful stop trials could relate to increased GABA inhibition in M1. Together with other findings, the results suggest that behavioral stopping is implemented via synchronized activity in the beta frequency band in a right IFG/basal ganglia network, with downstream effects on M1.

Molecular mechanisms of chondrocyte-specific expression on the mouse pro$\alpha$1(II) collagen gene

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro$\alpha1$(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a $\beta$-galactosidase reporter gene. A construction containing a 3000-bp promoter and a 3020-bp intron 1 fragment directed high levels of $\beta$-galactosidase expression specifically to chondrocytes. Successive deletions of intron 1 delineated a 48-bp fragment which targeted $\beta$-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. When the Col2a1 promoter was replaced with a minimal $\beta$-globin promoter, the 48-bp intron 1 sequence was still able to target expression of the transgene to chondrocytes, specifically. Therefore a 48-bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression to a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression. Nuclear proteins present selectively in mouse primary chondrocytes and rat chondrosarcoma cells bind to the three putative HMG (High-Mobility-Group) domain protein binding sites in this 48-bp sequence and the chondrocyte-specific proteins likely bind the DNA through minor groove. Together, my results indicate that a 48-bp sequence in Col2a1 intron 1 controls chondrocyte-specific expression in vivo and suggest that chondrocytes contain specific nuclear proteins involved in enhancer activity. ^

Project Alpha: A culturally appropriate approach to adolescent male sex education

In the United States today, adolescents face unacceptably high rates of mortality and morbidity due to the contraction of HIV/AIDS, sexually transmitted diseases and teenage pregnancy. In view of these rates, there is a need for applied preventive interventions to delay adolescent sexual behavior until adulthood. Project Alpha was a school-adopted, quasi-experimental program for adolescent male students attending Sharpstown High School in Houston, Texas. This intervention used student newsletters to provide specific role-model stories on community and student role models who have changed attitudes or improved efficacy to abstain from sexual behavior until adulthood. It was hypothesized that teenagers exposed to the intervention would show improvements in knowledge, beliefs, avoidance skills, perceived norms, intentions and self-efficacy to delay sexual behavior compared to no-treatment reference teenagers in the same school.^ In total, the Project Alpha program had a significant effect on student knowledge, beliefs (towards abstinence and having sex with multiple partners), perceived risk (HIV/STD testing), self-efficacy (could avoid sex with attractive girl who wants to have sex), perceived social norms (friends believing in sexual abstinence) and sexual intentions. However, no significant intervention effects were found in student's beliefs (that it was OK to have sex with girlfriend), perceived risk of HIV/STD, self-efficacy (to avoid sex with girlfriend) and social norms (friends believe it is OK to have sex with a girlfriend and multiple partners in the same month). ^

DIRECT EFFECT OF MELATONIN UPON THE RELEASE OF GONADOTROPINS FROM THE SUPERFUSED PITUITARY GLAND OF THE MALE GOLDEN HAMSTER

The pineal gland is known to be light sensitive and to be involved in the seasonal reproduction of male golden hamster Mesocricetus auratus. In general, the pineal gland has been demonstrated to be inhibitory to the reproductive system of the male golden hamster. Melatonin is a pineal hormone which can mimic the action of the pineal gland upon the reproductive system. However, the actual site(s) of melatonin action in the hamster has not been demonstrated. In this study a direct effect of melatonin on the release of FSH and LH from superfused hamster pituitary glands was investigated.^ The superfused pituitary glands showed a stable in vitro basal release of FSH and LH for up to 10 hours. The superfused pituitaries demonstrated reproducible responses to repeated pulses of 10('-8) M LHRH, and a dose-dependent response to stimulation with different concentrations of LHRH.^ Melatonin inhibited the basal release of FSH and LH from superfused hamster pituitary glands. This effect of melatonin was specific and not a general indolamine or catecholamine effect.^ The superfused pituitaries had a diurnal differential responsiveness to physiological concentrations of melatonin with respect to FSH and LH release which were related to the light cycle used to maintain the experimental animals. A LD 14:10 photoperiod cycle was used with light on from 5 a.m. till 7 p.m.. With pituitary glands obtained at 8:30 a.m., the basal release of FSH exhibited an initial inhibition, a gradual rebound at approximately two hours after the beginning of melatonin superfusion, and a significant overshoot of FSH release after the cessation of infusion with melatonin (Morning Response). If the pituitary glands were obtained from hamsters which were sacrificed at 3:30 p.m., the release rate of FSH exhibited an inhibition during the entire period of melatonin infusion with a rebound effect appearing only after melatonin infusion was discontinued (Afternoon Response). There was no significant difference in the responsiveness of the pituitary gland to infusion with melatonin at either 8:30 a.m. or 3:30 p.m. with respect to LH release. Also, melatonin could not inhibit the gonadotropins response to continuous superfusion with 10('-9) M LHRH in pituitaries obtained at either 8:30 a.m. or 3:30 p.m., nor inhibit the stimulatory effect of pulsatile 10('-9) M LHRH. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI^

Female-specific regulation of cytochrome P450 3As and their response to xenobiotic stress

Cytochrome P450 3As (CYP3As) are phase I enzymes responsible for metabolizing more than 50% of clinical drugs. Recent studies have revealed that expression of CYP3As is two-fold higher in women than in men leading to a faster metabolic clearance of therapeutic drugs in women. In this study, we analyzed the female specific rat CYP3A isoform, CYP3A9. We evaluated the effects of progesterone and estrogen on CYP3A9 regulation and showed a distinct role for estrogen in mediating female dominance of CYP3A9. We also observed changes in CYP3A9 expression at various stages of pregnancy which correlates well with varying physiological estradiol concentrations. In addition, by the in vitro data shows that estradiol mediated induction can be abrogated with estrogen receptor antagonist ICI182,780. We also identified three novel murine CYP3A isoforms CYP3A13, CYP3A41 and CYP3A44 and characterized their genomic structures and expression profiles. CYP3A41 and CYP3A44 show female specific expression but surprisingly this female dominance is not mediated via estrogen. Control male mice did not exhibit any CYP3A41 mRNA levels but showed minimal levels of CYP3A44. In order to gain insights into the governance ofαthe female specific genes, the hepatic regulation of CYP3A41 and CYP3A44 by the xeno-sensors PXR and CAR was examined. In female mice, pregnenolone-16α-carboxynitrile, suppressed CYP3A41 and CYP3A44 mRNA levels in PXR−/− background whereas dexamethasone-dependent suppression of CYP3A41 was mediated by PXR. In addition, phenobarbital challenge in PXR−/− revealed up-regulation of both CYP3A44, CYP3A41 levels only in males. No role for CAR was seen in the regulation of either CYP3A41 or CYP3A44 gene expression in female mice. Interestingly, PXR and CAR ligands induced male CYP3A44 levels in a receptor dependent fashion. This increase of CYP3A44 transcript in male mice is in contrast to the response seen in female mice, which clearly indicates an additional layer of regulation. Our findings suggest that gender plays a strategic role in directing the CAR/PXR mediated effects of CYP3A44/CYP3A41. This implies that differential regulation of female specific CYP3A isoforms may be the key to explain some of the gender differences observed in clearance of certain therapeutics like antidepressants and analgesics. ^

The effect of African Americans' ethnographic paradigms and cultural explanatory model of breast cancer in framing the male partner's attitudes and behaviors about the woman's breast cancer diagnosis

The disparate burden of breast cancer-related morbidity and mortality experienced by African American women compared with women of other races is a topic of intense debate in the medical and public health arenas. The anomaly is consistently attributed to the fact that at diagnosis, a large proportion of African American women have advanced-stage disease. Extensive research has documented the impacts of cultural factors and of socioeconomic factors in shaping African American women's breast-health practices; however, there is another factor of a more subtle influence that might have some role in establishing these women's vulnerability to this disease: the lack of or perceived lack of partner support. Themes expressed in the research literature reflect that many African American breast cancer patients and survivors consider their male partners as being apathetic and nonsupportive. ^ The purpose of this study was to learn how African American couples' ethnographic paradigms and cultural explanatory model of breast cancer frame the male partners' responses to the women's diagnosis and to assess his ability to cope and willingness to adapt to the subsequent challenges. The goal of the study was to determine whether these men's coping and adaptation skills positively or negatively affect the women's self-care attitudes and behaviors. ^ This study involved 4 African American couples in which the woman was a breast cancer survivor. Participants were recruited through a community-based cancer support group and a church-based cancer support group. Recruitment sessions were held at regular meetings of these organizations. Accrual took 2 months. In separate sessions, each male partner and each survivor completed a demographic survey and a questionnaire and were interviewed. Additionally, the couples were asked to participate in a communications activity (Adinkra). This activity was not done to fulfill any part of the study purpose and was not included in the data analysis; rather, it was done to assess its potential use as an intervention to promote dialogue between African American partners about the experience of breast cancer. ^ The questionnaire was analyzed on the basis of a coding schema and the interview responses were analyzed on the principles of hermeneutic phenomenology. In both cases, the instruments were used to determine whether the partner's coping skills reflected a compassionate attitude (positive response) versus an apathetic attitude (negative response) and whether his adaptation skills reflected supportive behaviors (the positive response) versus nonsupportive behaviors (the negative response). Overall, the women's responses showed that they perceived of their partners as being compassionate, yet nonsupportive, and the partner's perceived of themselves likewise. Only half of the women said that their partners' coping and adaptation abilities enabled them to relinquish traditional concepts of control and focus on their own well-being. ^ The themes that emerged indicate that African American men's attitudes and behaviors regarding his female partner's diagnosis of breast cancer and his ability to cope and willingness to adapt are influenced by their ritualistic mantras, folk beliefs, religious teachings/spiritual values, existential ideologies, socioeconomic status, and environmental factors and by their established perceptions of what causes breast cancer, what the treatments and outcomes are, and how the disease affects the entire family, particularly him. These findings imply that a culturally specific intervention might be useful in educating African American men about breast cancer and their roles in supporting their female partners, physically and psychologically, during diagnosis, treatment, and recovery. ^

The effect of specific carotenoids on lung cancer risk

A population-based case-comparison study of histologically confirmed lung cancer among white male and female residents of six Texas coastal counties was conducted from 1979-1982. Dietary information as well as information concerning smoking, alcohol consumption, occupational and residential exposures, and family history of cancer was obtained from 149 living cases and 359 comparison subjects.^ These data support the findings of previous studies that reported a protective association of total carotene intake with lung cancer (OR = 4.07, CI = 3.36-4.78), and no association for total Vitamin A or retinol. Of six specific carotenoids examined, these data reveal a statistically significant protective effect for alpha carotene (OR = 3.58, CI = 2.85-4.31), and an elevated, although non-significant effect for beta carotene (OR = 1.46, CI = 0.61-2.31). No consistent significant effect was found for cryptoxanthin, other xanthins, lutein or lycopene.^ Similar results were found for both males and females, and for both squamous cell and adenocarcinoma subtypes, although loss of power resulting from stratification may have rendered the celltype specific results more imprecise. These results should be considered with caution until confirmed by other studies, however, they suggest the importance of evaluating specific carotenoids in future diet-lung cancer investigations. ^

GENETIC INTERACTIONS OF FACTORS THAT REGULATE ALTERNATIVE RNA SPLICING IN THE MALE GERM LINE OF DROSOPHILA

Alternative RNA splicing is a critical process that contributes variety to protein functions, and further controls cell differentiation and normal development. Although it is known that most eukaryotic genes produce multiple transcripts in which splice site selection is regulated, how RNA binding proteins cooperate to activate and repress specific splice sites is still poorly understood. In addition how the regulation of alternative splicing affects germ cell development is also not well known. In this study, Drosophila Transformer 2 (Tra2) was used as a model to explore both the mechanism of its repressive function on its own pre-mRNA splicing, and the effect of the splicing regulation on spermatogenesis in testis. Half-pint (Hfp), a protein known as splicing activator, was identified in an S2 cell-based RNAi screen as a co-repressor that functions in combination with Tra2 in the splicing repression of the M1 intron. Its repressive splicing function is found to be sequence specific and is dependent on both the weak 3’ splice site and an intronic splicing silencer within the M1 intron. In addition we found that in vivo, two forms of Hfp are expressed in a cell type specific manner. These alternative forms differ at their amino terminus affecting the presence of a region with four RS dipeptides. Using assays in Drosophila S2 cells, we determined that the alternative N terminal domain is necessary in repression. This difference is probably due to differential localization of the two isoforms in the nucleus and cytoplasm. Our in vivo studies show that both Hfp and Tra2 are required for normal spermatogenesis and cooperate in repression of M1 splicing in spermatocytes. But interestingly, Tra2 and Hfp antagonize each other’s function in regulating germline specific alternative splicing of Taf1 (TBP associated factor 1). Genetic and cytological studies showed that mutants of Hfp and Taf1 both cause similar defects in meiosis and spermatogenesis. These results suggest Hfp regulates normal spermatogenesis partially through the regulation of taf1 splicing. These observations indicate that Hfp regulates tra2 and taf1 activity and play an important role in germ cell differentiation of male flies.

YKL -40, a vascular -associated serum marker for glioblastoma, correlates with chronic activation of AKT

YKL-40 is a secreted glycoprotein that has been reported to be expressed in pathologic conditions of extracellular matrix degradation and angiogenesis, such as rheumatoid arthritis, severe osteoarthritis, primary colorectal cancer, metastatic breast cancer, and recurrent ovarian cancer (Dehn, Hogdall et al. 2003). ^ We have identified YKL-40 as a serum marker for glioblastoma multiforme (GBM) using microarray analysis from samples of GBM. We compared the gene expression profile of 19 gliomas to pooled normal brain tissue using the Incyte 10,000 gene expression array. The most differentially expressed gene in this analysis was YKL-40; it was detected in GBM samples with a range of 3 to 62-fold elevation over normal brain. Western blot analysis of glioma samples for YKL-40 protein levels revealed substantial elevation in approximately 65% of GBMs, and undetectable levels in lower-grade gliomas and normal brain tissue. ELISA analysis on serum samples of glioma patients showed that YKL-40 levels were substantially elevated in many of the GBM patients. Statistical analysis indicated that in patients with glioma, serum YKL-40 levels correlate with tumor grade and potentially tumor burden in GBM. ^ Furthermore, we found that YKL-40 expression by in-situ hybridization on a brain tumor tissue array was limited to GBM's and gliosarcomas (GSA), and that YKL-40 expression was specific to the GBM component of GSA. Additional in-situ hybridization analysis, found it to be regionally associated with tumor vasculature as well as activated AKT expression in both human and mouse GBM's. Correlation of elevated YKL-40 with phospho-AKT was confirmed by Western blot analysis on a series of glioblastoma tumors, and inhibition of PI3 Kinase signaling by addition of LY294002 also decreased secretion of YKL-40 over a 7-day period in U87 glioblastoma cell tine. Lastly, YKL-40 expression was induced in response to serum starvation and altered by interaction with specific extracellular matrix (ECM) modules. In summary, we have identified the first accurate serum marker for high-grade gliomas. Furthermore, our findings indicate that YKL-40 is a highly expressed vascular-related glycoprotein in human GBM tissue and that it is affected by the AKT signaling pathway and interaction with components of brain ECM proteins. ^