Genetic and environmental influences on sleep problems: a study of preadolescent and adolescent twins.

BACKGROUND/AIM: The aim of this study was to examine the extent to which additive genetic, shared environmental and non-shared environmental factors contribute to adolescent and preadolescent sleep problems. METHODS: The sample consisted of a cohort of 270 monozygotic and 246 dizygotic twins from a university-based twin registry. RESULTS: Results demonstrated that genetic and environmental influences each appear to be important to adolescent sleep problems. CONCLUSIONS: While the magnitude of genetic influence on sleep problems was consistent with findings from the adult literature, it was smaller than in studies with younger children, suggesting genetic effects may be less influential in adolescence and adulthood.

Sleep problems and temperament in adolescents.

The aim of this study was to determine the association between temperament and sleep in adolescents. Participants included 516 adolescents and their mothers drawn from the community. Findings indicated that as with younger children, sleep and dimensions of temperament (sociability, impulsivity and negative affect) are related in adolescents.

Nightmares and anxiety in elementary-aged children: is there a relationship.

OBJECTIVE: To determine whether a relationship exists between children's anxiety level and nightmare occurrence. METHOD: A total of 60 kindergarten, second and fourth grade school children and their parents completed questionnaires assessing nightmare occurrence and anxiety. RESULTS: According to parental report, children who experience nightmares have significantly higher levels of anxiety than children who do not experience nightmares. The results also indicate a relationship between nightmare distress and trait anxiety. CONCLUSION: These findings suggest that anxiety issues should be considered in children who are experiencing nightmares.

ClC chloride channels.

SUMMARY: Chloride-conducting ion channels of the ClC family are emerging as critical contributors to a host of biological processes. These polytopic membrane proteins form aqueous pathways through which anions are selectively allowed to pass down their concentration gradients. The ClCs are found in nearly all organisms, with members in every mammalian tissue, yet relatively little is known about their mechanism or regulation. It is clear, however, that they are fundamentally different in molecular construction and mechanism from the well-known potassium-, sodium-, and calcium-selective channels. The medical importance of ClC channels - four inherited diseases have been blamed on familial ClC dysfunction to date - highlights their diverse physiological functions and provides strong motivation for further study.

Projection structure of a ClC-type chloride channel at 6.5 A resolution.

Virtually all cells in all eukaryotic organisms express ion channels of the ClC type, the only known molecular family of chloride-ion-selective channels. The diversity of ClC channels highlights the multitude and range of functions served by gated chloride-ion conduction in biological membranes, such as controlling electrical excitability in skeletal muscle, maintaining systemic blood pressure, acidifying endosomal compartments, and regulating electrical responses of GABA (gamma-aminobutyric acid)-containing interneurons in the central nervous system. Previously, we expressed and purified a prokaryotic ClC channel homologue. Here we report the formation of two-dimensional crystals of this ClC channel protein reconstituted into phospholipid bilayer membranes. Cryo-electron microscopic analysis of these crystals yields a projection structure at 6.5 A resolution, which shows off-axis water-filled pores within the dimeric channel complex.

Sleep disturbances during pregnancy.

OBJECTIVE: To assess sleep patterns and prevalence of sleep disturbances during pregnancy. DESIGN: Cross-sectional design; prospective questionnaire. SETTING: Outpatient, private obstetric clinic. PARTICIPANTS: 127 consecutive patients, with women evaluated at one of four points in pregnancy, 8-12 weeks (n = 37), 18-22 weeks (n = 28), 25-28 weeks (n = 24), and 35-38 weeks (n = 38). MAIN OUTCOME MEASURE: Questionnaire of sleep habits and sleep disturbances. RESULTS: A large percentage of the women experienced sleep disturbances during pregnancy, These problems included frequent night wakings, difficulty falling asleep, and symptoms of sleep apnea. Few differences in sleep patterns were found across pregnancy, although women were found to sleep more and nap more by the end of pregnancy. CONCLUSION: Sleep disturbances are common during pregnancy, especially late in pregnancy.

A decade of CLC chloride channels: structure, mechanism, and many unsettled questions.

ClC-type chloride channels are ubiquitous throughout the biological world. Expressed in nearly every cell type, these proteins have a host of biological functions. With nine distinct homologues known in eukaryotes, the ClCs represent the only molecularly defined family of chloride channels. ClC channels exhibit features of molecular architecture and gating mechanisms unprecedented in other types of ion channels. They form two-pore homodimers, and their voltage-dependence arises not from charged residues in the protein, but rather via coupling of gating to the movement of chloride ions within the pore. Because the functional characteristics of only a few ClC channels have been studied in detail, we are still learning which properties are general to the whole family. New approaches, including structural analyses, will be crucial to an understanding of ClC architecture and function.

Developmental features of sleep.

In sum, sleep disorders are common problems for children and adolescents, with estimates indicating that approximately 20% to 25% of the pediatric population experiences some type of sleep disturbance. Furthermore, clinicians should be aware that sleep disturbances may not only exist in isolation, but can be related to psychiatric or medical issues. Although much appears to be known about sleep disorders in the pediatric population, our knowledge of this area is still in its infancy. Additional research is still needed to investigate differences in clinical presentation of specific sleep disturbances among different age groups (i.e., children, adolescents, adults, and elderly), to develop the most appropriate treatments for given populations, and to study the effects of sleep disturbances on functioning. Given the prevalence of these problems in the child and adolescent population and its likely impact on cognitive and behavioral functioning, health professionals need to become increasingly aware of and knowledgeable about sleep and sleep disorders. We all spend about one third of our lives sleeping, or trying to sleep; thus, we should understand as much as we can about it.

Empirically supported treatments in pediatric psychology: bedtime refusal and night wakings in young children.

OBJECTIVE: To review the literature for empirically supported treatments for bedtime refusal and night wakings in young children. METHODS: An extensive review of the literature resulted in the inclusion of 41 studies that were evaluated according to the criteria established by the Task Force on Promotion and Dissemination of Psychological Procedures (1995). RESULTS: Evidence exists indicating that extinction and parent education on the prevention of sleep problems can be considered well-established treatments. Furthermore, graduated extinction and scheduled awakenings are probably efficacious treatments, with positive routines a promising intervention. CONCLUSIONS: A discussion of effectiveness, treatment feasibility, cost-effectiveness, and methodological limitations of the studies is provided. Recommendations for future directions for research in the treatment of these two common sleep disorders are presented.

A practical approach to acute renal failure.

Acute renal failure represents a wide variety of renal diseases, which may be challenging to diagnose and even more challenging to treat. As understanding of these diseases improves, so perhaps will clinicians' ability to treat them.

Mutational analysis of the helical hairpin region of diphtheria toxin transmembrane domain.

Entry of the catalytic domain of diphtheria toxin into the cytoplasma of eukaryotic cells depends on insertion of the T (transmembrane) domain into the endosomal membrane, a process triggered by low pH. To probe the mechanism of insertion, we mutated ionizable residues within the helical hairpin region of the T domain. Only three mutations caused significant effects on cytotoxicity, D295K, E349K, and D352K. Each of these represents a substitution of a basic for an acidic residue at the tip of a helical hairpin. Substitution of Lys for Glu349 or Asp352, in the TH8/9 hairpin, reduced toxicity for Vero cells > 100-fold, whereas a Lys substitution for Asp295, one of 3 acidic residues in the TH5/6/7 hairpin, caused a less marked reduction. All three mutations also altered the pH-dependent formation, and/or ion conductance, of channels formed by the toxin in artificial bilayers or the plasma membrane. E349K or D352K did not alter the pH dependence of conformational changes in the toxin occurring near pH 5. Our findings support the hypothesis that the TH8/9 hairpin inserts into the endosomal membrane after low pH-mediated partial unfolding of the T domain. A positive residue at the tip of this hairpin apparently inhibits insertion and blocks toxin action. The ion-conducting properties of channels formed by selected mutants, described elsewhere, are consistent with this model. The status of the TH5/6/7 hairpin in the integral membrane form of the T domain remains uncertain.

Pediatricians and sleep disorders: training and practice.

OBJECTIVE: A series of studies were conducted to investigate pediatricians' training, knowledge, and practices regarding sleep and sleep disorders in children and adolescents. METHOD AND RESULTS: Study 1, a national survey of 156 pediatric residency programs, found that pediatricians receive a mean of 4.8 hours of instruction on sleep and sleep disorders, although the mode and median hours of instruction is 0 hours. In study 2, 88 pediatricians completing a questionnaire concerning general knowledge about sleep disorders in children and adolescents received a mean score of 71.8% (range, 40% to 93%). Pediatricians appear to know the most about developmental issues and sleep hygiene and the least about specific disorders such as narcolepsy and parasomnias. In the third study, 183 pediatricians were surveyed about their actual beliefs and practices regarding young children's sleep problems. Together, those surveyed reported that approximately 25% of their patients experience some type of sleep problem. Most pediatricians recommend behavioral interventions, although 14.8% of pediatricians report prescribing pharmacological treatments, and 48.9% inform parents that their child is likely to outgrow the problem. CONCLUSIONS: The results of these studies support the need for more education in sleep and sleep disorders in children and adolescents within medical schools, pediatric residency programs, and the practicing pediatric community.

Reaction of diphtheria toxin channels with sulfhydryl-specific reagents: observation of chemical reactions at the single molecule level.

The diphtheria toxin channel is believed to be a homooligomer of its T domain in which each subunit consists of two alpha-helices, lying within the membrane, connected by a short interhelical loop of four amino acids (residues 349-352). To investigate the validity and implications of this model, we singly mutated each of these amino acids to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and added to the trans compartment sulfhydryl-specific reagents [methanethiosulfonate derivatives (MTS-ER)] that introduce a positive or negative charge to reacted cysteines. The introduction of a positive charge at residue 351 or 352 (through the MTS-ER reactions) resulted in a step decrease in single-channel conductance, whereas the introduction of a negative charge resulted in a step increase. The opposite sign of these effects indicates the predominantly electrostatic nature of the phenomenon and implies that residues 351 and 352 lie close to the channel entrance. The same reactions at residue 350 resulted in very little change in channel conductance but instead changed the character of the natural rapid flickering of the channel between open and closed states to one in which the channel spent more time in the closed state; this may have resulted from the group introduced at position 350 acting as a tethered channel blocker. The MTS derivatives had no effect on channels containing a cysteine at position 349, suggesting that this residue faces away from the channel entrance. We propose that the step changes in conductance or flickering pattern result from the chemical reaction of one MTS-ER molecule with one cysteine, and thus a bimolecular chemical reaction is being witnessed at the single molecule level. From the distribution of waiting times between the appearance (i.e., the opening) of a channel and the step change in its conductance or flickering pattern, we can calculate a pseudo-first-order rate constant, which can then be converted to a second-order rate constant, for the chemical reaction.

Global social skill ratings: measures of social behavior or physical attractiveness?

Calvert reviewed the literature on social skills and physical attractiveness and concluded that many ratings of social skill may be confounded by the physical attractiveness of the target individual, possibly due to a general perception that physical attractiveness and social competence are positively correlated. In order to examine the influence of physical attractiveness on social skill ratings, Ss made global ratings of social skill and attractiveness for a confederate whose appearance and behavior had been altered to appear attractive or unattractive and socially skilled or unskilled in an assertiveness and heterosocial vignette. The results indicated that the same skilled behavior was viewed as more competent when performed by an attractive person compared to an unattractive person. Attractiveness had no influence on ratings of generally incompetent behavior. Thus, it appears that physical attractiveness does not compensate for poor interpersonal skills, but a skilled, attractive individual may be judged to have particularly good skills. Implications for the assessment of social skills are discussed.

Structure-function relationships in diphtheria toxin channels: I. Determining a minimal channel-forming domain.

Diphtheria Toxin (DT) is a 535 amino acid exotoxin, whose active form consists of two polypeptide chains linked by an interchain disulphide bond. DT's N-terminal A fragment kills cells by enzymatically inactivating their protein synthetic machinery; its C-terminal B chain is required for the binding of toxin to sensitive cells and for the translocation of the A fragment into the cytosol. This B fragment, consisting of its N-terminal T domain (amino acids 191-386) and its C-terminal R domain (amino acids 387-535) is responsible for the ion-conducting channels formed by DT in lipid bilayers and cellular plasma membranes. To further delineate the channel-forming region of DT, we studied channels formed by deletion mutants of DT in lipid bilayer membranes under several pH conditions. Channels formed by mutants containing only the T domain (i.e., lacking the A fragment and/or the R domain), as well as those formed by mutants replacing the R domain with Interleukin-2 (IL-2), have single channel conductances and selectivities essentially identical to those of channels formed by wild-type DT. Furthermore, deleting the N-terminal 118 amino acids of the T domain also has minimal effect on the single channel conductance and selectivity of the mutant channels. Together, these data identify a 61 amino acid stretch of the T domain, corresponding to the region which includes alpha-helices TH8 and TH9 in the crystal structure of DT, as the channel-forming region of the toxin.

Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH.

Ion-conducting channels formed in lipid bilayers by diphtheria toxin are highly pH dependent. Among other properties, the channel's single channel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH-dependent single channel properties as that of the intact toxin. This region corresponds to an alpha-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two alpha-helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysine has no effect on the DT channel's single channel conductance or selectivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3 cis/7.2 trans (in 1 M KCl), consistent with this group interacting electrostatically with ions in the channel. The cation selectivity of these mutant channels is also reduced from that of wild-type channels, a direction consistent with residue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 (in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0 cis/7.2 trans (with a high concentration of permeant buffer in the cis compartment) imply that residue 352 is on or near the trans side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. The trans location of position 352 severely constrains the number of possible membrane topologies for this region.

Structure-function relationships in diphtheria toxin channels: III. Residues which affect the cis pH dependence of channel conductance.

The conductance of channels formed by diphtheria toxin (DT) in lipid bilayer membrane depends strongly on pH. We have previously shown that a 61 amino acid region of the protein, denoted TH8-9, is sufficient to form channels having the same pH-dependent conductance properties as those of whole toxin channels. One residue in this region, Aspartate 352, is responsible for all the dependence of single channel conductance on trans pH, whereas another, Glutamate 349, has no effect. Here, we report that of the seven remaining charged residues in the TH8-9 region, mutations altering the charge on H322, H323, H372, and R377 have minimal effects on single channel conductance; mutations of Glutamates 326, 327, or 362, however, significantly affect single channel conductance as well as its dependence on cis pH. Moreover, Glutamate 362 is titratable from both the cis and trans sides of the membrane, suggesting that this residue lies within the channel; it is more accessible, however, to cis than to trans protons. These results are consistent with the membrane-spanning topology previously proposed for the TH8-9 region, and suggest a geometric model for the DT channel.

Treatment of childhood sleep disorders: generalization across disorders and effects on family members.

Investigated the treatment of bedtime problems and its generalization to night wakings. Six children (M age = 35 months) and their parents participated in this study. A multiple-baseline design across subjects was employed and found that treatment instituted at bedtime was successful in relieving both bedtime disturbances and night wakings. Furthermore, significant positive changes in parental sleep and family satisfaction occurred following amelioration of the children's sleep problems. Data support recent work suggesting that chronic sleep problems in children are amenable to behavioral interventions. In addition, this method appears to be more cost-effective and less stressful for parents to implement than behavioral interventions that directly target night wakings.

Structure/function analysis of the transmembrane domain of DAB389-interleukin-2, an interleukin-2 receptor-targeted fusion toxin. The amphipathic helical region of the transmembrane domain is essential for the efficient delivery of the catalytic domain to the cytosol of target cells.

Cassette and deletion mutagenesis were used to analyze the function of the amphipathic alpha-helices in the transmembrane domain of DAB389-interleukin-2 (IL-2), a fusion protein which is targeted to the interleukin-2 receptor. We demonstrate that the in-frame deletion of 60 amino acids, from Asn204 to Glu263 in DAB389-IL-2, results in complete loss of cytotoxic activity, whereas when the amphipathic regions from Asp208 to Ser220 and Ala244 to His258 are replaced with idealized amphipathic helices composed of repeating Glu, Lys, and Leu residues, the mutant fusion toxin has low but detectable activity. DAB389-IL-2 and both variants form channels in artificial phospholipid bilayers with conductances identical to those formed by diphtheria toxin. Both mutant fusion toxins bind to the high affinity IL-2 receptor with affinities similar to that of DAB389-IL-2. The fact that these mutants have markedly reduced or absent cytotoxic activity, but possess "wild type" catalytic activity, binding affinities, and channel conductances, suggests the existence of a step in the intoxication pathway, defective in the mutants, which occurs after DAB389-IL-2 binds to the IL-2 receptor. It is unknown whether this step occurs prior or subsequent to channel formation, but it is essential for the efficient delivery of the ADP-ribosyltransferase from DAB389-IL-2 to the cytosol of target cells.