Effect of Menopause on Melatonin and Alertness Rhythms Investigated in Constant Routine Conditions

Although studies have reported the effects of the menstrual cycle on melatonin rhythmicity, none has investigated the effects of menopause on the melatonin rhythm. The circadian rhythm in melatonin and its relationship to subjective alertness was investigated in pre‐ and postmenopausal women under constant routine conditions (controlled posture, dim lighting, calorie intake, temperature, and prolonged wakefulness). Eleven healthy pre‐menopausal (42±4 yr) and 10 postmenopausal women (55±2 yr) participated in the study. Salivary melatonin samples and subjective measures of alertness and sleepiness were assessed hourly during the 22 h constant routine protocol. Postmenopausal women had a significantly earlier melatonin acrophase (1.1±0.5 h clock time in decimal h; mean±SEM, p<0.05) compared to the pre‐menopausal women (2.3±0.3 h). There was no significant difference between melatonin onset and amplitude between the pre‐menopausal and postmenopausal women. Self‐rated alertness declined in both study groups as the length of sleep deprivation increased. Melatonin onset preceded the onset of self‐rated sleepiness in both groups. The time interval between melatonin onset and the onset of sleepiness and alertness offset was significantly greater in the postmenopausal women compared to the pre‐menopausal women. In conclusion, under controlled experimental conditions the timing of the melatonin rhythm was advanced in postmenopausal women altering its phase relationship to subjective alertness and sleepiness.     

Risk-related standard inevitable in assessing competence

In a preceding article, Mark Wicclair suggested that where patient preferences clash with the doctor's recommendations, a minimal understanding of the task at hand, rather than the risks involved, should be the basis for standards of patient competence in decision-making (Bioethics 5(2):91-104, April 1991). Skene, Principal Law Reform Officer of the Victoria, Australia, Law Reform Commission, questions Wicclair's minimum standard from a legal standpoint, pointing out contradictions in the effects of such a standard. Skene concludes that legal protection and the education of both doctors and patients are the most suitable safeguards of patient autonomy.     

Structural basis for the autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase

The activity of the c-Kit receptor protein-tyrosine kinase is tightly regulated in normal cells, whereas deregulated c-Kit kinase activity is implicated in the pathogenesis of human cancers. The c-Kit juxtamembrane region is known to have an autoinhibitory function; however the precise mechanism by which c-Kit is maintained in an autoinhibited state is not known. We report the 1.9-A resolution crystal structure of native c-Kit kinase in an autoinhibited conformation and compare it with active c-Kit kinase. Autoinhibited c-Kit is stabilized by the juxtamembrane domain, which inserts into the kinase-active site and disrupts formation of the activated structure. A 1.6-A crystal structure of c-Kit in complex with STI-571 (Imatinib or Gleevec) demonstrates that inhibitor binding disrupts this natural mechanism for maintaining c-Kit in an autoinhibited state. Together, these results provide a structural basis for understanding c-Kit kinase autoinhibition and will facilitate the structure-guided design of specific inhibitors that target the activated and autoinhibited conformations of c-Kit kinase.     

Crystal structure of human dipeptidyl peptidase IV in complex with a decapeptide reveals details on substrate specificity and tetrahedral intermediate formation

Dipeptidyl peptidase IV (DPPIV) is a member of the prolyl oligopeptidase family of serine proteases. DPPIV removes dipeptides from the N terminus of substrates, including many chemokines, neuropeptides, and peptide hormones. Specific inhibition of DPPIV is being investigated in human trials for the treatment of type II diabetes. To understand better the molecular determinants that underlie enzyme catalysis and substrate specificity, we report the crystal structures of DPPIV in the free form and in complex with the first 10 residues of the physiological substrate, Neuropeptide Y (residues 1-10; tNPY). The crystal structure of the free form of the enzyme reveals two potential channels through which substrates could access the active site-a so-called propeller opening, and side opening. The crystal structure of the DPPIV/tNPY complex suggests that bioactive peptides utilize the side opening unique to DPPIV to access the active site. Other structural features in the active site such as the presence of a Glu motif, a well-defined hydrophobic S1 subsite, and minimal long-range interactions explain the substrate recognition and binding properties of DPPIV. Moreover, in the DPPIV/tNPY complex structure, the peptide is not cleaved but trapped in a tetrahedral intermediate that occurs during catalysis. Conformational changes of S630 and H740 between DPPIV in its free form and in complex with tNPY were observed and contribute to the stabilization of the tetrahedral intermediate. Our results facilitate the design of potent, selective small molecule inhibitors of DPPIV that may yield compounds for the development of novel drugs to treat type II diabetes.     

Rapid arrest of axon elongation by brefeldin A: a role for the small GTP-binding protein ARF in neuronal growth cones

Members of the ADP-ribosylation factor (ARF) family of small guanosine triphosphate-binding proteins play an essential role in membrane trafficking which subserves constitutive protein transport along exocytic and endocytic pathways within eukaryotic cell bodies. In growing neurons, membrane trafficking within motile growth cones distant from the cell body underlies the rapid plasmalemmal expansion which subserves axon elongation. We report here that ARF is a constituent of axonal growth cones, and that application of brefeldin A to neurons in culture produces a rapid arrest of axon extension that can be ascribed to inhibition of ARF function in growth cones. Our findings demonstrate a role for ARF in growth cones that is coupled tightly to the rapid growth of neuronal processes characteristic of developmental and regenerative axon elongation, and indicate that ARF participates not only in constitutive membrane traffic within the cell body, but also in membrane dynamics within growing axon endings.     

Presence of ferric hydroxide clusters in mutants of Haemophilus influenzae ferric ion-binding protein A

The periplasmic iron binding protein plays an essential role in the iron uptake pathway of Gram-negative pathogenic bacteria from the Pasteurellaceae and Neisseriaceae families and is critical for survival of these pathogens within the host. In this study, we report the crystal structures of two mutant forms of ferric ion-binding protein A (FbpA) from Haemophilus influenzae with bound multinuclear oxo-metal clusters. Crystals of site-directed mutants in the metal or anion binding ligands contain protein in the open conformation, and two mutant FbpAs, H9A and N175L, contain different cluster arrangements in the iron-binding pocket. The iron clusters are anchored by binding to the two tyrosine ligands (Tyr195 and Tyr196) positioned at the vertex of the iron-binding pocket but are not coordinated by the other metal binding ligands. Our results suggest that the metal clusters may have formed in situ, suggesting that the mutant FbpAs may serve as a simple model for protein-mediated mineralization.