Interactions between domains of apo calmodulin alter calcium binding and stability

Calmodulin (CaM) is an essential protein that exerts exquisite spatial and temporal control over diverse eukaryotic processes. Although the two half-molecule domains of CaM each have two EF-hands and bind two calcium ions cooperatively, they have distinct roles in activation of some targets. Interdomain interactions may mediate coordination of their actions. Proteolytic footprinting titrations of CaM [Pedigo and Shea (1995) Biochemistry 34, 1179-1196; Shea, Verhoeven, and Pedigo (1996) Biochemistry 35, 2943-2957] showed that calcium binding to the high-affinity sites (III and IV in the C-domain) alters the conformation of helix B in the N-domain despite sites I and II being vacant. This may arise from calcium-induced disruption of interactions between the apo domains. In this study, comparing the cloned domains (residues 1-75, 76-148) to whole CaM, the proteolytic susceptibility of helix B in the apo isolated N-domain was higher than in apo CaM. The isolated N-domain was monotonically protected by calcium binding and had a higher calcium affinity than when part of whole CaM. The change in affinity was small (1-1.5 kcal/mol) but acted to separate the domain saturation curves of whole CaM. Unfolding enthalpies and melting temperatures of the apo isolated domains did not correspond to the two transitions resolved for apo CaM. In summary, the interactions between domains of apo CaM protected the N-domain from proteolysis and raised its Tm by 10 degrees C, demonstrating that CaM is not the sum of its parts.     

A haplotype and linkage disequilibrium analysis of the hereditary hemochromatosis gene region

Monoclonal antibodies were generated against serotonin (5-HT) and the C-terminal portion of the neuronal form of nitric oxide synthase (nNOS), the enzyme producing nitric oxide in neurons. These antibodies were used to compare the distribution of 5-HT- and nNOS-containing neurons in the raphe nuclei of four animal species (rat, mouse, guinea pig, and cat). It was found that the rat was the only species in which the raphe nuclei contain a substantial number of nNOS-immunoreactive (IR) cell bodies. In this species and as observed by other authors, all mesencephalic raphe nuclei contained nNOS-IR cells, the largest group being located in the nucleus raphe dorsalis. The coexistence of nNOS and 5-HT immunoreactivities in these nuclei was visualized by double labeling. In the medulla, the nuclei raphe magnus and obscurus displayed a rather low number of nNOS-IR neurons. In the other species, nNOS-IR cell bodies were found in very low numbers, whatever raphe nucleus was considered. The rostral pole of the nucleus raphe dorsalis and the nuclei raphe magnus and obscurus contained a few nNOS-IR neurons which did not show any coincidence with the 5-HT neurons. In addition, nNOS-IR axons were rare. It is concluded that in the mouse, guinea pig, and cat the involvement of nitric oxide in functions subserved by 5-HT within the raphe nuclei might be minimal.     

New trends in the development of cancer vaccines

Recent advances in understanding of the molecular mechanisms of antigen processing and presentation, and the identification of tumor-associated antigens in melanoma and other cancers, have stimulated the development of a new generation cancer vaccines. This review summarizes the most recent approaches for the design of safe and more effective vaccines for cancer. Peptide-based vaccines are safe and can be synthesized with high purity and reproducibility. Recombinant viruses encoding tumor-associated antigens allow efficient delivery and precise control over the form and the quantity of the delivered antigens. DNA-based vaccines induce long-lasting immune responses and are considered very safe. Antigen-loaded dendritic cells, and the use of newly developed adjuvants are also very promising new approaches. In this review, we also discuss the possible clinical applications and future directions for vaccine development.     

Effects of postural change on aspiration in head and neck surgical patients

This study was designed to define the effects of postural change on liquid aspiration during videofluorographic examination of oropharyngeal swallow in head and neck surgical patients. Thirty-two patients were given two swallows of five different amounts of liquid barium as tolerated. When aspiration occurred, the patient's head and/or body position was changed, new posture being determined by the swallowing disorder identified as the cause of the aspiration. Postural techniques were successful in eliminating aspiration on at least one volume of liquid in 81% of these patients. Patients in all surgical groups were able to use postures with equal success. A variety of positions were used in each type of surgical patient, indicating that these patients exhibited a variety of swallowing problems. Results emphasize the importance of introducing postural techniques during the radiographic study of oropharyngeal swallow to facilitate safe oral intake of liquids.     

Illicit substance use among adolescents: a matrix of prospective predictors

This paper reviews findings from 58 prospective studies of illicit substance use (ISU) among adolescents. It arranges 384 findings according to three types of influence (viz., social, attitudinal, and intrapersonal) and four levels of influence (viz., ultimate, distal, proximal, and immediate). The bulk of evidence reconfirms the importance of several predictors of ISU (e.g., intentions and prior substance-related behavior, friendship patterns and peer behaviors, absence of supportive parents, psychological temperament), reveals that a few variables thought to be well-established predictors may not be (e.g., parental behaviors, parental permissiveness, depression, low self-esteem), and uncovers several variables where findings were either sparse or inconsistent (e.g., the role of public policies concerning ISU, mass media depictions of ISU, certain parenting styles, affective states, perceptions of parental disapproval for ISU, and substance-specific refusal skills). Directions for future research are discussed.     

Adrenarche is associated with decreased 3 beta-hydroxysteroid dehydrogenase expression in the adrenal reticularis

The increased production of adrenal dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) which occurs during the prepubertal period is known as adrenarche. One hypothesis for adrenarche is that alterations in intra-adrenal expression of steroidogenic enzymes within the inner reticularis zone leads to the increased production of 19-carbon steroids. We tested the hypothesis that at the time of adrenarche there is decreased expression of 3 beta HSD in the reticularis. Immunohistochemical localization of 3 beta HSD was performed and staining intensities compared between adrenal glands from children ages 4 months to 7 years (N = 11) and ages 8 to 11 years (N = 6). No difference was observed between the levels of staining in the glomerulosa and fasciculata from either age group. However, the reticularis from the older children exhibited diminished 3 beta HSD immunoreactivity. These findings suggest that as children mature there is a decreased level of 3 beta HSD in the adrenal reticularis which may contribute to the increased production of DHEA and DHEAS seen during adrenarche.     

Anandamide- and delta9-tetrahydrocannabinol-evoked arachidonic acid mobilization and blockade by SR141716A [N-(Piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4 -methyl-1H-pyrazole-3-carboximide hydrochloride]

The purpose of this study was to investigate whether anandamide induces cannabimimetic responses, mainly mobilization of arachidonic acid, in primary cultures of rat brain cortical astrocytes. Confluent monolayer cultures of astrocytes, prelabeled with [3H]arachidonic acid, were incubated with anandamide or delta9-tetrahydrocannabinol (delta9-THC) in the presence or absence of thimerosal, a fatty acid acyl CoA transferase inhibitor and phenylmethylsulfonyl fluoride, an amidohydrolase inhibitor. Anandamide and delta9-THC induced a time- and concentration-dependent release of arachidonic acid in the presence, but not in the absence, of thimerosal. Anandamide- and delta9-THC-stimulated arachidonic acid release was pertussis toxin-sensitive, indicating a receptor/G-protein involvement. A novel and selective cannabinoid receptor antagonist, SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4- methyl-1H-pyrazole-3-carboximide hydrochloride], blocked the arachidonic acid release, suggesting a cannabinoid receptor-mediated pathway. In astrocytes, the magnitude of anandamide-induced arachidonic acid release was equal to that released by equimolar concentrations of delta9-THC. Furthermore, direct assay of amidohydrolase activity indicated that degradation of anandamide into arachidonic acid and ethanolamine was negligible in cortical astrocytes. Our results suggest that anandamide stimulates receptor-mediated release of arachidonic acid, and the receptor may be the cannabinoid receptor. Astrocytes, containing a cannabinoid receptor and lower or negligible amidohydrolase activity, may be an important brain cell model in which to study the cannabimimetic effects of anandamide at a cellular and molecular level.