Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by a mutation in either the TSC1 or TSC2 tumour suppressor gene. The disease is characterized by a broad phenotypic spectrum that can include seizures, mental retardation, renal dysfunction and dermatological abnormalities. TSC2 encodes tuberin, a putative GTPase activating protein for rap1 and rab5. The TSC1 gene was recently identified and codes for hamartin, a novel protein with no significant homology to tuberin or any other known vertebrate protein. Here, we show that hamartin and tuberin associate physically in vivo and that the interaction is mediated by predicted coiled-coil domains. Our data suggest that hamartin and tuberin function in the same complex rather than in separate pathways.      

Synthesis and acid ionization constants of cyclic cystine peptides

Cyclic peptide disulfides of the general formula were synthesized from the corresponding peptide derivatives [Boc-Cys(Trt)(Gly)-_n-Cys(Trt)-OBu^t] by oxidation with iodine in methanol and by subsequent removal of the terminal groups with trifluoroacetic acid. Acid ionization constants of the obtained peptides were determined by potentiometric titration in aqueous KCl (0.1 mol/L) medium. All compounds have two dissociable hydrogens, corresponding to carboxyl (pK¹= 2.35–2.84) and to terminal amino group (pK₂= 5.61–6.93); pK₁, values show first an upward and then a downward trend with the increase in ring size; the opposite is true for pK₂, values. These trends could be tentatively attributed to the intramolecular salt bridge (-COO——-NH⁺₃-) formation.     

Leukotriene B4 Receptor (BLT-1) Modulates Neutrophil Influx into the Peritoneum but Not the Lung and Liver during Surgically Induced Bacterial Peritonitis in Mice

Leukotriene B₄ (LTB₄) is a rapidly synthesized, early neutrophil chemoattractant that signals via its cell surface receptor, BLT-1, to attract and activate neutrophils during peritonitis. BLT-1-deficient (BLT-1^−/−) mice were used to determine the effects of LTB₄ on neutrophil migration and activation, bacterial levels, and survival after cecal ligation and puncture (CLP). Male BLT-1^−/− or wild-type (WT) BALB/c mice underwent CLP. Tissues were harvested for determination of levels of bacteria, myeloperoxidase (MPO), LTB₄, macrophage inflammatory protein 2 (MIP-2), and neutrophil (polymorphonuclear leukocyte [PMN]) numbers at 4 and 18 h after CLP. PMN activation was determined by an assessment of phagocytosis ability and CD11b expression. Survival was also determined. BLT-1^−/− mice had decreased numbers of PMNs in the peritoneum at both 4 and 18 h after CLP but increased numbers of PMNs in the blood at 18 h compared with WT mice. Liver and lung MPO levels were significantly higher in BLT-1^−/− mice at both 4 and 18 h after CLP, with increased bacterial levels in the blood, the liver, and peritoneal fluid at 4 h. Bacterial levels remained higher in peritoneal fluid at 18 h, but blood and liver bacterial levels at 18 h were not different from levels at 4 h. PMN phagocytosis and CD11b levels were decreased in BLT-1^−/− mice. LTB₄ levels were similar between the groups before and after CLP, but MIP-2 levels were decreased both locally and systemically in BLT-1^−/− mice. Survival was significantly improved in BLT-1^−/− mice (71%) compared with WT mice (14%) at 48 h post-CLP. Thus, LTB₄ modulates neutrophil migration into the mouse peritoneum, but not the lung or liver, after CLP. Despite higher bacterial and PMN levels at remote sites, there was increased survival in BLT-1^−/− mice compared to WT mice. Decreased PMN activation may result in less remote organ dysfunction and improved survival.     

Mutation analysis of 184 cystic fibrosis families in Wales.

We describe a molecular analysis of 184 cystic fibrosis (CF) families in Wales. To determine accurate frequency data for the CF mutations in the Welsh population, families with at least three Welsh grandparents were strictly regarded as Welsh. Of these 74 families, we have identified approximately 90% of mutations causing CF, with delta F508 accounting for 71.8% and 621 + 1G greater than T 6.7%. We observed a significant difference between the Welsh and Scottish frequencies of 621 + 1G greater than T. To allow the rapid and efficient screening for the more common mutations we modified a multiplex used by Watson et al enabling the detection of delta F508, G551D, and R553X simultaneously with 621 + 1G greater than T. In parallel to this system we ran the Cellmark Diagnostics ARMS multiplex kit, which detects delta F508, 621 + 1G greater than T, G551D, and G542X. RFLP analysis of the 184 families shows that the delta F508 chromosomes are almost exclusively found on the B haplotype (XV2c 1, KM19 2); the other CF mutations have more heterogeneous backgrounds. Strong haplotype correlations exist between the markers XV2c, KM19, D9, and G2 and the other CF mutations. Haplotype data suggest that there are at least seven mutations that remain to be identified in these families.     

The GAP-related domain of tuberin, the product of the TSC2 gene, is a target for missense mutations in tuberous sclerosis

Tuberous sclerosis is an autosomal dominant trait in which the dysregulation of cellular proliferation and differentiation results in the development of hamartomatous growths in many organs. The TSC2 gene is one of two genes determining tuberous sclerosis. Inactivating germline mutations of TSC2 in patients with tuberous sclerosis and somatic loss of heterozygosity at the TSC2 locus in the associated hamartomas indicate that TSC2 functions as a tumour suppressor gene and that loss of function is critical to expression of the tuberous sclerosis phenotype. The TSC2 product, tuberin, has a region of homology with the GTPase activating protein rap1GAP and stimulates the GTPase activity of rap1a and rab5a in vitro. Here we show that the region of homology between tuberin and human rap1GAP and the murine GAP mSpa1 is more extensive than previously reported and spans approximately 160 amino acid residues encoded within exons 34-38 of the TSC2 gene. Single strand conformation polymorphism analysis of these exons in 173 unrelated patients with tuberous sclerosis and direct sequencing of variant conformers together with study of additional family members enabled characterisation of disease associated mutations in 14 cases. Missense mutations, which occurred in exons 36, 37 and 38 were identified in eight cases, four of whom shared the same recurrent change P1675L. Each of the five different missense mutations identified was shown to occur de novo in at least one sporadic case of tuberous sclerosis. The high proportion of missense mutations detected in the region of the TSC2 gene encoding the GAP-related domain supports its key role in the regulation of cellular growth.      

Organ specific apoptosis following polymicrobial intraperitoneal infection

Background Leukocyte apoptosis allows safe removal of potentially harmful cells and facilitates resolution of inflammation. We hypothesized that the number of apoptotic cells changes in a disproportionate fashion in parenchymal organs in response to intra-abdominal infection. Materials and methods The percentage of apoptotic cells in the liver, spleen, lung, and peripheral blood was evaluated following cecal ligation and puncture (CLP) in mice. Tissue myeloperoxidase (MPO) levels were measured as an index of neutrophil extravasation. Results Liver & spleen MPO continually increased, while lung MPO remained low after CLP. In parallel to the increase in MPO, liver & spleen apoptosis continually increased throughout the 9-day follow-up period, whereas lung apoptosis remained unchanged. Conclusions The distribution of apoptotic cells during intraperitoneal infection occurs in an organ specific manner, with significant increases in the spleen and liver. This distribution likely reflected the clearance of apoptotic cells as the inflammatory focus became contained. Supported by the American Association for the Surgery of Trauma, John H. Davis Research Scholarship, and by the Veterans Administration Merit Review Project 0005. Received 7 October 2005; returned for revision 22 November 2005; accepted by G. Wallace 23 December 2005     

Assessing the validity of a survey of the restaurant health promotion environment

Our primary question was whether a telephone survey of restaurant personnel could provide accurate community-level measures of the restaurant health promotion environment. An obvious concern was that restaurant personnel might exaggerate the extent to which their establishment had a positive health promotion environment. Comparison with the most obvious "gold standard"--direct observation--showed fairly accurate reporting about nonsmoking seating arrangements, but restaurant personnel exaggerated the extent to which menu items were designated as low in fat. We also compared the restaurant-survey measures of nonsmoking seating availability at the community level with measures of the restrictiveness of local no-smoking ordinances. We found a positive relationship, as expected, between measures of the restrictiveness of ordinances and the amount of nonsmoking seating indicated by the restaurant survey.     

Counseling Fathers (2010) by C.Z. Oren and D.C. Oren

No abstract available for this article.     

Race and ethnic variation in college students’ allostatic regulation of racism-related stress

Racism-related stress is thought to contribute to widespread race/ethnic health inequities via negative emotion and allostatic stress process up-regulation. Although prior studies document race-related stress and health correlations, due to methodological and technical limitations, they have been unable to directly test the stress-reactivity hypothesis in situ. Guided by theories of constructed emotion and allostasis, we developed a protocol using wearable sensors and daily surveys that allowed us to operationalize and time-couple self-reported racism-related experiences, negative emotions, and an independent biosignal of emotional arousal. We used data from 100 diverse young adults at a predominantly White college campus to assess racism-related stress reactivity using electrodermal activity (EDA), a biosignal of sympathetic nervous system activity. We find that racism-related experiences predict both increased negative emotion risk and heightened EDA, consistent with the proposed allostatic model of health and disease. Specific patterns varied across race/ethnic groups. For example, discrimination and rumination were associated with negative emotion for African American students, but only interpersonal discrimination predicted increased arousal via EDA. The pattern of results was more general for Latinx students, for whom interpersonal discrimination, vicarious racism exposure, and rumination significantly modulated arousal. As with Latinx students, African students were particularly responsive to vicarious racism while 1.5 generation Black students were generally not responsive to racism-related experiences. Overall, these findings provide support for allostasis-based theories of mental and physical health via a naturalistic assessment of the emotional and sympathetic nervous system responding to real-life social experiences.

Racism in the United States shapes interracial social interactions (1) and, mirroring society-wide trends in the racialization of American politics (2, 3), the situation may be worsening with rising racial animosity (4, 5). Consequently, racism-related stress is hypothesized to contribute to widespread minority health disparities (6) and Black/White mortality differentials on a scale sufficient to influence election outcomes (7). One possible explanation for these disparities is that race-related stress increases negative emotions and physiological wear and tear via modulation of the sympathetic-adrenal-medullary (SAM) axis, hypothalamic-pituitary-adrenal (HPA) axis, and immune system (8). Up-regulation of these systems, often referred to generically as “the stress process,” is the outcome of the brain’s predictive modeling and regulation of the body’s energetic needs, or allostasis (9). Here, race-related stress is thought to increase energy demands as the brain prepares the body to deal with threats by marshaling oxygen, glucose, and other energetic mediators (10). Consequently, researchers have argued that ongoing allostatic up-regulation of the autonomic and endocrine systems, combined with immune activity modulation, in response to racism-related threats is physiologically taxing and over time increases vulnerability to chronic disease (11, 12). For example, downstream physiological adaptions to a body energized on high alert are thought to increase risks for excess adiposity, hypertension, diabetes, and cardiovascular disease (13–15), traditionally the leading cause of death in the United States.Despite the theoretical importance of regulatory allostatic processes for understanding racial health inequities, racism-related physiological responding has not been directly measured in real time in the real world. Conducting these assessments is challenging because the timing of many socially mediated experiences, such as racism-related incidents, occur in “social time”; that is, they are dependent upon actions beyond the control of the individual, are largely unobserved, and are highly variable and stochastic. Acute physiological changes are thus responses to temporally variable stimuli and are modulated on precise time scales in the order of seconds (i.e., SAM) and minutes (i.e., HPA). Although systematic and metaanalytic reviews find evidence that discriminatory experiences are correlated with mental and physical health both early (6) and later in life (16), most studies are cross-sectional, retrospective, and rely on behavioral and psychological self-reports, even when employing temporally sensitive methodologies (17). Although a few recent studies in natural settings incorporating biomarkers consistent with models of allostasis have begun filtering into the literature (18), their measurement strategies are not time-synchronized with race-related experiences. These studies provide post hoc evidence of allostatic process modulation, but do not measure allostatic regulation concurrently as it transpires in situ.Our study was designed to address these limitations and to directly test allostatic modulation of the sympathetic component of the SAM response to racism-related stress. To these ends, we developed a prospective protocol capturing events throughout the day, including the timing of racism-related experiences of perceived interpersonal discrimination, rumination on racism, and vicarious racism exposure, as well as negative emotions. Our design also included a wearable device that continuously tracked SAM activity using an electrodermal activity (EDA) sensor, a direct measure of the sympathetic nervous system (SNS) division of the autonomic nervous system that indexes affective arousal. This approach facilitated approximate time-synchronization of racism-related experiences with the SNS-mediated first-stage allostatic stress-response pathway. We were therefore able to operationalize two key aspects of stress-response dynamics temporally coupled with racism-related stressors: Negative emotion and SNS arousal. To our knowledge, this study provides a temporally coregistered and ecologically embedded assessment of the dynamic links between race-related stressors, negative emotions, and the SAM-mediated SNS component of a stress response among individuals of diverse ethnic backgrounds.