Modulatory effects of bag cell peptides in {\it Aplysia}: Behavioral and electrophysiological studies

Reproductive hormones have effects on the nervous system not directly related to reproductive function. In the rat, for example, luteinizing hormone releasing hormone has dramatic effects on learning and memory. The present work attempts to examine the effects of reproductive hormones on non-reproductive behaviors and the neural loci and mechanisms underlying these effects in Aplysia, an animal whose behaviors, reproductive hormones and neural circuitry have been well characterized.^ In Aplysia, the neurosecretory bag cells release several peptides that are responsible for eliciting egg laying. The effects of these peptides on the defensive tail-siphon withdrawal reflex, as well as sensitization of this reflex, were examined. Sensitization, a simple form of nonassociative learning, refers to the behavioral enhancement of a response to a test stimulus after the presentation of a strong stimulus, that may last minutes (short-term) or days (long-term). An extract of the bag cells (BCE) inhibited the baseline siphon component of the tail-siphon withdrawal reflex and suppressed long-term, but not short-term, sensitization of the reflex. Preliminary experiments suggest that BCE also affects the tail component of the tail-siphon withdrawal reflex.^ To determine the neural mechanisms underlying the inhibition of the baseline reflex, electrophysiological studies were performed using an in vitro analogue of the tail-siphon withdrawal reflex to examine the ability of BCE, as well as the individual bag cell peptides (BCPs), to modulate the circuitry of the reflex. Bag cell extract attenuated the synaptic strength of the monosynaptic connections between tail sensory neurons and tail motor neurons. When individually applied only $\beta$-BCP produced a similar attenuation. This effect of $\beta$-BCP was not dependent on changes in duration of the presynaptic action potential.^ An in vitro analogue of long-term sensitization training was developed to examine the mechanisms by which the BCPs may affect long-term sensitization of the tail-siphon withdrawal reflex. This analogue exhibited both short- and long-term facilitation of the connections between the tail sensory and motor neurons.^ The results of these behavioral and electrophysiological experiments suggest that the BCPs inhibit the tail-siphon withdrawal reflex, at least in part, by modulating the synaptic strength of the connections between the sensory neurons and motor neurons underlying the reflex. One candidate for this effect is $\beta$-BCP. Thus, the peptides which elicit egg laying may also serve other functions such as the inhibition of defensive reflexes. In addition, these experiments raise the possibility that BCPs may exert a long lasting effect ($>$24 hr), suppressing long-term sensitization of the tail-siphon withdrawal reflex. ^

The prognostic significance of sialyl-Tn antigen in women treated with adjuvant chemotherapy for breast cancer

Background and purpose. Sialyl-Tn(STn) represents an aberrantly glycosylated mucin epitope which is expressed in breast cancer and other adenocarcinomas and is an important target for the development of novel immunotherapeutic approaches. It is a marker of adverse prognosis in colon and ovarian cancer, but information about its prognostic impact in breast cancer is limited. The primary aim of the present study was to investigate the influence of STn expression on outcome of invasive breast cancer in 207 women who received anthracyline-containing adjuvant chemotherapy in a prospective clinical trial.^ Methods. Expression of STn was determined by an immunohistochemical procedure using the B72.3 monoclonal antibody. The extent of staining was determined by two observers using a 0 through 4 point scale, with 0 representing $<$5% of cells staining; 1: 5-25%; 2: 26-50%; 3: 51-75%; and 4: $>$75%. Intraobserver and interobserver agreement was.78-.92 (kappa). Kaplan-Meier and Cox proportional regression survival analyses were used to compare STn-negative and STn-positive patients.^ Results. Forty-eight (23%) of the 207 specimens demonstrated positive staining of STn. With a median follow-up of five years, STn-positivity was associated with a higher 5-year recurrence-free survival time than STn-negativity (67% vs. 80%, respectively; p = 0.03). STn expression was significantly associated with menopausal status (p = 0.04) but not other conventional prognostic markers. The risk of breast cancer recurrence and death was assessed by multivariate Cox regression analyses with adjustment for lymph node status, tumor size, menopausal status, hormone receptor status, nuclear grade, S-phase fraction and ploidy. In the final multivariate model for recurrence-free survival, the three factors that showed prognostic significance were: lymph node status (hazard ratio (HR) 3.04, 95% confidence interval (CI) 1.08-8.49), STn expression (HR 2.02, 95% CI 1.09-3.73), and tumor size (HR 1.96, 95% CI 1.05-3.64). STn was also associated with worse overall survival (HR 2.16, 95% CI 0.95-4.92) in multivariate analysis.^ Conclusion. STn antigen was shown to be a predictor of poor outcome in breast cancer. This tumor-associated antigen may be a valuable marker for identifying individuals at high risk of developing recurrent disease who may benefit from adjuvant therapy targeted at STn following definitive local therapy. Further study is needed to clarify the biologic and prognostic role of STn in breast cancer. ^

Alcohol consumption and breast cancer risk among Hispanic and non-Hispanic women in New Mexico

Breast cancer incidence and mortality rates for Hispanic women are lower than for non-Hispanic white (NHW) women, but recently rates have increased more rapidly among Hispanic women. Many studies have shown a consistent increased breast cancer risk associated with modest or high alcohol intake, but few included Hispanic women. Alcohol consumption and risk of breast cancer was investigated in a New Mexico statewide population-based case-control study. The New Mexico Tumor Registry ascertained women, newly diagnosed with breast cancer (1992–1994) aged 30–74 years. Controls were identified by random digit dialing and were frequency-matched for ethnicity, age-group, and health planning district. In-person interviews of 712 cases and 844 controls were conducted. Data were collected for breast cancer risk factors, including alcohol intake. Recent alcohol intake data was collected for a four-week period, six months prior to interview. Past alcohol intake included information on alcohol consumption at ages 25, 35, and 50. History of alcohol consumption was reported by 81% of cases and 85% of controls. Of these women, 42% of cases and 48% of controls reported recent alcohol intake. Results for past alcohol intake did not show any trend with breast cancer risk, and were nonsignificant. Multivariate-adjusted odds ratios for recent alcohol intake and breast cancer suggested an increased risk at the highest level for both ethnic groups, but estimates were unstable and statistically nonsignificant. Low level of recent alcohol intake (<148 grams/week) was associated with a reduced risk for NHW women (Odds Ratio (OR) = 0.49 95% Confidence Interval (CI) 0.35–0.69). This pattern was independent of hormone-receptor status. The reduced breast cancer risk for low alcohol intake was present for premenopausal (OR = 0.29, 95% CI 0.15–0.56) and postmenopausal NHW women (OR = 0.56, 95% CI 0.35–0.90). The possibility of an increased risk associated with high alcohol intake could not be adequately addressed, because there were few drinkers with more than light to moderate intake, especially among Hispanic women. An alcohol-estrogen link is hypothesized to be the mechanism responsible for increased breast cancer risk, but has not been consistently substantiated. More studies are needed of the underlying mechanism for an association between alcohol intake and breast cancer. ^

Heterozygous GHR gene mutation in a child with idiopathic short stature.

Abstract Several monogenic defects have been reported to be associated with idiopathic short stature. Focusing on growth hormone receptor (GHR)-gene alterations, the heterozygosity of the same gene defect may be associated with a range of growth deficits. We found a heterozygous mutation (V144I) within exon 6 of the GHR gene in a patient with a low level of insulin-like growth factor I (IGF-I), normal level of GH, and severe short stature. Despite the lack of statistical difference, an overall tendency for reduced wt-GH-induction of GHR activation and Jak/Stat signalling in cells transiently expressing GHR-V144I alone or co-expressing wt-GHR compared to cells expressing only wt-GHR was found when GH doses were increased. Our results suggest that, although GHR sequence variants are responsible for some functional alterations commonly observed in children with idiopathic short stature, these changes may not explain all the height deficits observed in these subjects.

Adjuvant bevacizumab-containing therapy in triple-negative breast cancer (BEATRICE): primary results of a randomised, phase 3 trial

BACKGROUND The addition of bevacizumab to chemotherapy improves progression-free survival in metastatic breast cancer and pathological complete response rates in the neoadjuvant setting. Micrometastases are dependent on angiogenesis, suggesting that patients might benefit from anti-angiogenic strategies in the adjuvant setting. We therefore assessed the addition of bevacizumab to chemotherapy in the adjuvant setting for women with triple-negative breast cancer. METHODS For this open-label, randomised phase 3 trial we recruited patients with centrally confirmed triple-negative operable primary invasive breast cancer from 360 sites in 37 countries. We randomly allocated patients aged 18 years or older (1:1 with block randomisation; stratified by nodal status, chemotherapy [with an anthracycline, taxane, or both], hormone receptor status [negative vs low], and type of surgery) to receive a minimum of four cycles of chemotherapy either alone or with bevacizumab (equivalent of 5 mg/kg every week for 1 year). The primary endpoint was invasive disease-free survival (IDFS). Efficacy analyses were based on the intention-to-treat population, safety analyses were done on all patients who received at least one dose of study drug, and plasma biomarker analyses were done on all treated patients consenting to biomarker analyses and providing a measurable baseline plasma sample. This trial is registered with ClinicalTrials.gov, number NCT00528567. FINDINGS Between Dec 3, 2007, and March 8, 2010, we randomly assigned 1290 patients to receive chemotherapy alone and 1301 to receive bevacizumab plus chemotherapy. Most patients received anthracycline-containing therapy; 1638 (63%) of the 2591 patients had node-negative disease. At the time of analysis of IDFS, median follow-up was 31·5 months (IQR 25·6-36·8) in the chemotherapy-alone group and 32·0 months (27·5-36·9) in the bevacizumab group. At the time of the primary analysis, IDFS events had been reported in 205 patients (16%) in the chemotherapy-alone group and in 188 patients (14%) in the bevacizumab group (hazard ratio [HR] in stratified log-rank analysis 0·87, 95% CI 0·72-1·07; p=0·18). 3-year IDFS was 82·7% (95% CI 80·5-85·0) with chemotherapy alone and 83·7% (81·4-86·0) with bevacizumab and chemotherapy. After 200 deaths, no difference in overall survival was noted between the groups (HR 0·84, 95% CI 0·64-1·12; p=0·23). Exploratory biomarker assessment suggests that patients with high pre-treatment plasma VEGFR-2 might benefit from the addition of bevacizumab (Cox interaction test p=0·029). Use of bevacizumab versus chemotherapy alone was associated with increased incidences of grade 3 or worse hypertension (154 patients [12%] vs eight patients [1%]), severe cardiac events occurring at any point during the 18-month safety reporting period (19 [1%] vs two [<0·5%]), and treatment discontinuation (bevacizumab, chemotherapy, or both; 256 [20%] vs 30 [2%]); we recorded no increase in fatal adverse events with bevacizumab (four [<0·5%] vs three [<0·5%]). INTERPRETATION Bevacizumab cannot be recommended as adjuvant treatment in unselected patients with triple-negative breast cancer. Further follow-up is needed to assess the potential effect of bevacizumab on overall survival.

Adjuvant ovarian suppression in premenopausal breast cancer.

BACKGROUND Suppression of ovarian estrogen production reduces the recurrence of hormone-receptor-positive early breast cancer in premenopausal women, but its value when added to tamoxifen is uncertain. METHODS We randomly assigned 3066 premenopausal women, stratified according to prior receipt or nonreceipt of chemotherapy, to receive 5 years of tamoxifen, tamoxifen plus ovarian suppression, or exemestane plus ovarian suppression. The primary analysis tested the hypothesis that tamoxifen plus ovarian suppression would improve disease-free survival, as compared with tamoxifen alone. In the primary analysis, 46.7% of the patients had not received chemotherapy previously, and 53.3% had received chemotherapy and remained premenopausal. RESULTS After a median follow-up of 67 months, the estimated disease-free survival rate at 5 years was 86.6% in the tamoxifen-ovarian suppression group and 84.7% in the tamoxifen group (hazard ratio for disease recurrence, second invasive cancer, or death, 0.83; 95% confidence interval [CI], 0.66 to 1.04; P=0.10). Multivariable allowance for prognostic factors suggested a greater treatment effect with tamoxifen plus ovarian suppression than with tamoxifen alone (hazard ratio, 0.78; 95% CI, 0.62 to 0.98). Most recurrences occurred in patients who had received prior chemotherapy, among whom the rate of freedom from breast cancer at 5 years was 82.5% in the tamoxifen-ovarian suppression group and 78.0% in the tamoxifen group (hazard ratio for recurrence, 0.78; 95% CI, 0.60 to 1.02). At 5 years, the rate of freedom from breast cancer was 85.7% in the exemestane-ovarian suppression group (hazard ratio for recurrence vs. tamoxifen, 0.65; 95% CI, 0.49 to 0.87). CONCLUSIONS Adding ovarian suppression to tamoxifen did not provide a significant benefit in the overall study population. However, for women who were at sufficient risk for recurrence to warrant adjuvant chemotherapy and who remained premenopausal, the addition of ovarian suppression improved disease outcomes. Further improvement was seen with the use of exemestane plus ovarian suppression. (Funded by Pfizer and others; SOFT ClinicalTrials.gov number, NCT00066690.).

Adjuvant exemestane with ovarian suppression in premenopausal breast cancer.

BACKGROUND Adjuvant therapy with an aromatase inhibitor improves outcomes, as compared with tamoxifen, in postmenopausal women with hormone-receptor-positive breast cancer. METHODS In two phase 3 trials, we randomly assigned premenopausal women with hormone-receptor-positive early breast cancer to the aromatase inhibitor exemestane plus ovarian suppression or tamoxifen plus ovarian suppression for a period of 5 years. Suppression of ovarian estrogen production was achieved with the use of the gonadotropin-releasing-hormone agonist triptorelin, oophorectomy, or ovarian irradiation. The primary analysis combined data from 4690 patients in the two trials. RESULTS After a median follow-up of 68 months, disease-free survival at 5 years was 91.1% in the exemestane-ovarian suppression group and 87.3% in the tamoxifen-ovarian suppression group (hazard ratio for disease recurrence, second invasive cancer, or death, 0.72; 95% confidence interval [CI], 0.60 to 0.85; P<0.001). The rate of freedom from breast cancer at 5 years was 92.8% in the exemestane-ovarian suppression group, as compared with 88.8% in the tamoxifen-ovarian suppression group (hazard ratio for recurrence, 0.66; 95% CI, 0.55 to 0.80; P<0.001). With 194 deaths (4.1% of the patients), overall survival did not differ significantly between the two groups (hazard ratio for death in the exemestane-ovarian suppression group, 1.14; 95% CI, 0.86 to 1.51; P=0.37). Selected adverse events of grade 3 or 4 were reported for 30.6% of the patients in the exemestane-ovarian suppression group and 29.4% of those in the tamoxifen-ovarian suppression group, with profiles similar to those for postmenopausal women. CONCLUSIONS In premenopausal women with hormone-receptor-positive early breast cancer, adjuvant treatment with exemestane plus ovarian suppression, as compared with tamoxifen plus ovarian suppression, significantly reduced recurrence. (Funded by Pfizer and others; TEXT and SOFT ClinicalTrials.gov numbers, NCT00066703 and NCT00066690, respectively.).

Rebound-associated vertebral fractures after discontinuation of denosumab-from clinic and biomechanics.

Rebound-associated vertebral fractures may follow treatment discontinuation of highly potent reversible bone antiresorptives, resulting from the synergy of rapid bone resorption and accelerated microdamage accumulation in trabecular bone. INTRODUCTION The purposes of this study are to characterize rebound-associated vertebral fractures following the discontinuation of a highly potent reversible antiresorptive therapy based on clinical observation and propose a pathophysiological rationale. METHODS This study is a case report of multiple vertebral fractures early after discontinuation of denosumab therapy in a patient with hormone receptor-positive non-metastatic breast cancer treated with an aromatase inhibitor. RESULTS Discontinuation of highly potent reversible bone antiresorptives such as denosumab may expose patients to an increased fracture risk due to the joined effects of absent microdamage repair during therapy followed by synchronous excess activation of multiple bone remodelling units at the time of loss-of-effect. We suggest the term rebound-associated vertebral fractures (RVF) for this phenomenon characterized by the presence of multiple new clinical vertebral fractures, associated with either no or low trauma, in a context consistent with the presence of high bone turnover and rapid loss of lumbar spine bone mineral density (BMD) occurring within 3 to 12 months after discontinuation (loss-of-effect) of a reversible antiresorptive therapy in the absence of secondary causes of bone loss or fractures. Unlike atypical femoral fractures that emerge from failure of microdamage repair in cortical bone with long-term antiresorptive treatment, RVF originate from the synergy of rapid bone resorption and accelerated microdamage accumulation in trabecular bone triggered by the discontinuation of highly potent reversible antiresorptives. CONCLUSIONS Studies are urgently needed to i) prove the underlying pathophysiological processes suggested above, ii) establish the predictive criteria exposing patients to an increased risk of RVF, and iii) determine appropriate treatment regimens to be applied in such patients.

In vivo definition of genetic and cellular aspects of mammalian sexual differentiation

The differentiation of the reproductive organs is an essential developmental process required for the proper transmission of the genetic material. Müllerian inhibiting substance (MIS) is produced by testes and is necessary for the regression of the Müllerian ducts: the anlagen of the uterus, fallopian tubes and cervix. In vitro and standard transgenic mouse studies indicate that the nuclear hormone receptor Steroidogenic factor 1 (SF-1) and the transcription factor SOX9 play an essential role in the regulation of Mis. To test this hypothesis, mutations in the endogenous SF-1 and SOX9 binding sites in the mouse Mis promoter were introduced by gene targeting in embryonic stem (ES) cells. In disagreement with cell culture and transgenic mouse studies, male mice homozygous for the mutant SF-1 binding site correctly initiated Mis transcription in the fetal testes, although at significantly reduced levels. Surprisingly, sufficient Mis was produced for complete elimination of the Müllerian duct system. However, when the SF-1 binding site mutation was combined with an Mis -null allele, the further decrease in Mis levels led to a partial retention of uterine tissue, but only at a distance from the testes. In contrast, males homozygous for the mutant SOX9 binding site did not initiate Mis transcription, resulting in pseudohermaphrodites with a uterus and oviducts. These studies suggest an essential role for SOX9 in the initiation of Mis transcription, whereas SF-1 appears to act as a quantitative regulator of Mis transcript levels perhaps for influencing non-Müllerian duct tissues. ^ The Mis type II receptor, a member of the TGF- b superfamily, is also required for the proper regression of the Müllerian ducts. Mis type II receptor-deficient human males and their murine counterparts develop as pseudohermaphrodites. A lacZ reporter cassette was introduced into the mouse Mis type II receptor gene, by homologous recombination in ES cells. Expression studies, based on b -galactosidase activity, show marked expression of the MIS type II receptor in the postnatal Sertoli cells of the testis as well as in the prenatal and postnatal granulosa cells of the ovary. Expression is also seen in the mesenchymal cells surrounding the Müllerian duct and in the longitudinal muscle layer of the uterus. ^

Downstream targets of the Rhox5 homeobox gene

The X-linked mouse Rhox gene cluster contains over 30 homeobox genes that are candidates to regulate multiple steps in male and female gametogenesis. The founding member of the Rhox gene cluster, Rhox5, is an androgen-dependent gene expressed in Sertoli cells that promotes the survival and differentiation of the adjacent male germ cells. To decipher downstream signaling pathways of Rhox5, I used in vivo and in vitro microarray profiling to identify and characterize downstream targets of Rhox5 in the testis. This led to the identification of many Rhox5 -regulated genes, two of which I focused on in more detail. One of them, Unc5c, encodes a pro-apoptotic receptor with tumor suppressor activity that I found is negatively regulated by Rhox5 through a Rhox5-response element in the Unc5c 5' untranslated region (5' UTR). Examination of other mouse Rhox family members revealed that Rhox2 and Rhox3 also have the ability to downregulate Unc5c expression. The human RHOX protein RHOXF2 also had this ability, indicating that Unc5c repression is a conserved Rhox-dependent response. The repression of Unc5c expression by Rhox5 may, in part, mediate Rhox5's pro-survival function in the testis, as I found that Unc5c mutant mice have decreased germ cell apoptosis in the testis. This along with my other data leads me to propose a model in which Rhox5 is a negative regulator upstream of Unc5c in a Sertoli-cell pathway that promotes germ-cell survival. The other Rhox5-regulated gene that I studied in detail is insulin II (Ins2). Several lines of evidence, including electrophoretic mobility shift anaylsis, promoter mutagenesis, and chromatin immuoprecipitation analysis indicated that Ins2 is a direct target of Rhox5. Structure-function analysis identified homeodomain residues and the RHOX5 amino-terminal domain crucial for conferring Ins2 inducibility. Rhox5 regulates not only the Ins2 gene but also genes encoding other secreted proteins regulating metabolism (adiponectin and resistin), the rate-liming enzyme for monosaturated fatty acid biosynthesis (SCD-1), and transcription factors crucial for regulating metabolism (the nuclear hormone receptor PPARγ). I propose that the regulation of some or all of these molecules in Sertoli cells is responsible for the Rhox5-dependent survival of the adjacent germ cells. ^

Breast cancer risk factors among Mexican-American women

Introduction: The average age of onset of breast cancer among Hispanic women is 50 years, more than a decade earlier than non-Hispanic white women. Age at diagnosis is an important prognostic factor for breast cancer; younger age at onset is more likely to be associated with advanced disease, poorer prognosis, hormone receptor negative breast tumors, and a greater likelihood of hereditary breast cancer. Studies of breast cancer risk factors including reproductive risk factors, family history of breast cancer, and breast cancer subtype have been conducted predominately in non-Hispanic whites. Breast cancer is a heterogeneous disease with the presence of clinically, biologically, and epidemiologically distinct subtypes that also differ with respect to their risk factors. The associations between reproductive risk factors and family history of breast cancer have been well documented in the literature. However, only a few studies have assessed these associations with breast cancer subtype in Hispanic populations. Methods: To assess the associations between reproductive risk factors and family history of breast cancer we conducted three separate studies. First, we conducted a case-control study of 172 Mexican-American breast cancer cases and 344 age matched controls residing in Harris County, TX to assess reproductive and other risk factors. We conducted logistic regression analysis to assess differences in cases and controls adjusted for age at diagnosis and birthplace and then we conducted a multinomial logistic regression analysis to compare reproductive risk factors among the breast tumor subtypes. In a second study, we identified 139 breast cancer patients with a first- or second-degree family history of breast cancer and 298 without a family history from the ELLA Bi-National Breast Cancer Study. In this analysis, we also computed a multinomial logistic regression to evaluate associations between family history of breast cancer and breast cancer subtypes, and logistic regression to estimate associations between breast cancer screening practices with family history of breast cancer. In the final study, we employed a cross-sectional study design in 7279 Mexican-American women in the Mano a Mano Cohort Study. We evaluated associations with family history of breast cancer and breast cancer risk factors including body mass index (BMI), lifestyle factors, migration history, and adherence to American Cancer Society (ACS) guidelines. Results: In the results of our first analyses, reproductive risk factors differed in the magnitude and direction of associations when stratified by age and birthplace among cases and controls. In our second study, family history of breast cancer, and having at least one relative diagnosed at an early age (<50 years) was associated with triple negative breast cancer (TNBC). Mammography prior to receiving a breast cancer diagnosis was associated with family history of breast cancer. In our third study that assessed lifestyle factors, migration history and family history of breast cancer; we found that women with a first-degree family history of breast cancer were more overweight or obese compared with their counterparts without a family history. There was no indication that having a family history contributed to women practicing healthier lifestyle behaviors and/or adhering to the ACS guidelines for cancer prevention. Conclusions: We observed that among Mexican-American women, reproductive risk factors were associated with breast cancer where the woman was born (US or Mexico). Having a family history of breast cancer, especially having either a first- or second-degree relative diagnosed at a younger age, was strongly associated with TNBC subtype. These results are consistent with other published studies in this area. Further, our results indicate that women with strong family histories of breast cancer are more likely to undertake mammography but not to engage in healthier lifestyle behaviors.^

Genetic integration of a nuclear -encoded mitochondrial gene with cardiac function

Cardiovascular disease (CVD) is the leading cause of death in the United States. One manifestation of CVD known to increase mortality is an enlarged, or hypertrophic heart. Hypertrophic cardiomyocytes adapt to increased contractile demand at the genetic level with a re-emergence of the fetal gene program and a downregulation of fatty acid oxidation genes with concomitant increased reliance on glucose-based metabolism. To understand the transcriptional regulatory pathways that implement hypertrophic directives we analyzed the upstream promoter region of the muscle specific isoform of the nuclear-encoded mitochondrial gene, carnitine palmitoyltransferase-1β (CPT-1β) in cultured rat neonatal cardiac myocytes. This enzyme catalyzes the rate-limiting step of fatty acid entry into β-oxidation and is downregulated in cardiac hypertrophy and failure, making it an attractive model for the study of hypertrophic gene regulation and metabolic adaptations. We demonstrate that the muscle-enriched transcription factors GATA-4 and SRF synergistically activate CPT-1β; moreover, DNA binding to cognate sites and intact protein structure are required. This mechanism coordinates upregulation of energy generating processes with activation of the energy consuming contractile promoter for cardiac α-actin. We hypothesized that fatty acid or glucose responsive transcription factors may also regulate CPT-1β. Oleate weakly stimulates CPT-1β activity; in contrast, the glucose responsive Upstream Stimulatory Factors (USF) dramatically depresses the CPT-1β reporter. USF regulates CPT-1β through a novel physical interaction with the cofactor PGC-1 and abrogation of MEF2A/PGC-1 synergistic stimulation. In this way, USF can inversely regulate metabolic gene programs and may play a role in the shift of metabolic substrate preference seen in hypertrophy. Failing hearts have elevated expression of the nuclear hormone receptor COUP-TF. We report that COUP-TF significantly suppresses reporter transcription independent of DNA binding and specific interactions with GATA-4, Nkx2.5 or USF. In summary, CPT-1β transcriptional regulation integrates mitochondrial gene expression with two essential cardiac functions: contraction and metabolic substrate oxidation. ^

Mahogany (mg) stimulates feeding and increases basal metabolic rate independent of its suppression of agouti

The mahogany (mg) locus originally was identified as a recessive suppressor of agouti, a locus encoding a skin peptide that modifies coat color by antagonizing the melanocyte-stimulating hormone receptor or MC1-R. Certain dominant alleles of agouti cause an obesity syndrome when ectopic expression of the peptide aberrantly antagonizes the MC4-R, a related melanocyte-stimulating hormone receptor expressed in hypothalamic circuitry and involved in the regulation of feeding behavior and metabolism. Recent work has demonstrated that mg, when homozygous, blocks not only the ability of agouti to induce a yellow coat color when expressed in the skin of the lethal yellow mouse (AY), but also the obesity resulting from ectopic expression of agouti in the brain. Detailed analysis of mg/mg AY/a animals, presented here, demonstrates that mg/mg blocks the obesity, hyperinsulinemia, and increased linear growth induced by ectopic expression of the agouti peptide. Remarkably, however, mg/mg did not reduce hyperphagia in the AY/a mouse. Furthermore, mg/mg induced hyperphagia and an increase in basal metabolic rate in the C57BL/6J mouse in the absence of AY. Consequently, although mahogany is broadly required for agouti peptide action, it also appears to be involved in the control of metabolic rate and feeding behavior independent of its suppression of agouti.

Protein kinase C as a signal for exocytosis

We have studied signaling mechanisms that stimulate exocytosis and luteinizing hormone secretion in isolated male rat pituitary gonadotropes. As judged by reverse hemolytic plaque assays, phorbol-12-myristate-13-acetate (PMA) stimulates as many gonadotropes to secrete as does gonadotropin-releasing hormone (GnRH). However, PMA and GnRH use different signaling pathways. The secretagogue action of GnRH is not very sensitive to bisindolylmaleimide I, an inhibitor of protein kinase C, but is blocked by loading cells with a calcium chelator, 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. The secretagogue action of PMA is blocked by bisindolylmaleimide I and is not very sensitive to the intracellular calcium chelator. GnRH induces intracellular calcium elevations, whereas PMA does not. As judged by amperometric measurements of quantal catecholamine secretion from dopamine- or serotonin-loaded gonadotropes, the secretagogue action of PMA develops more slowly (in several minutes) than that of GnRH. We conclude that exocytosis of secretory vesicles can be stimulated independently either by calcium elevations or by activation of protein kinase C.

Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein

Multiprotein bridging factor 1 (MBF1) is a transcriptional cofactor that bridges between the TATA box-binding protein (TBP) and the Drosophila melanogaster nuclear hormone receptor FTZ-F1 or its silkworm counterpart BmFTZ-F1. A cDNA clone encoding MBF1 was isolated from the silkworm Bombyx mori whose sequence predicts a basic protein consisting of 146 amino acids. Bacterially expressed recombinant MBF1 is functional in interactions with TBP and a positive cofactor MBF2. The recombinant MBF1 also makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the FTZ-F1 binding to its recognition site. The central region of MBF1 (residues 35–113) is essential for the binding of FTZ-F1, MBF2, and TBP. When the recombinant MBF1 was added to a HeLa cell nuclear extract in the presence of MBF2 and FTZ622 bearing the FTZ-F1 DNA-binding domain, it supported selective transcriptional activation of the fushi tarazu gene as natural MBF1 did. Mutations disrupting the binding of FTZ622 to DNA or MBF1, or a MBF2 mutation disrupting the binding to MBF1, all abolished the selective activation of transcription. These results suggest that tethering of the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription. A homology search in the databases revealed that the deduced amino acid sequence of MBF1 is conserved across species from yeast to human.