Mutations in RIT1 cause Noonan syndrome - additional functional evidence and expanding the clinical phenotype.

RASopathies are a clinically heterogeneous group of conditions caused by mutations in 1 of 16 proteins in the RAS-mitogen activated protein kinase (RAS-MAPK) pathway. Recently, mutations in RIT1 were identified as a novel cause for Noonan syndrome. Here we provide additional functional evidence for a causal role of RIT1 mutations and expand the associated phenotypic spectrum. We identified two de novo missense variants p.Met90Ile and p.Ala57Gly. Both variants resulted in increased MEK-ERK signaling compared to wild-type, underscoring gain-of-function as the primary functional mechanism. Introduction of p.Met90Ile and p.Ala57Gly into zebrafish embryos reproduced not only aspects of the human phenotype but also revealed abnormalities of eye development, emphasizing the importance of RIT1 for spatial and temporal organization of the growing organism. In addition, we observed severe lymphedema of the lower extremity and genitalia in one patient. We provide additional evidence for a causal relationship between pathogenic mutations in RIT1, increased RAS-MAPK/MEK-ERK signaling and the clinical phenotype. The mutant RIT1 protein may possess reduced GTPase activity or a diminished ability to interact with cellular GTPase activating proteins; however the precise mechanism remains unknown. The phenotypic spectrum is likely to expand and includes lymphedema of the lower extremities in addition to nuchal hygroma.

Comparing the safety, efficacy and recovery of intranasal midazolam vs. oral chloral hydrate and promethazine.

PURPOSE: The purpose of this study was to compare the safety, efficacy and recovery time of intranasal midazolam spray administered using an atomizer to orally administered chloral hydrate and promethazine for the sedation of pediatric dental patients. METHODS: A randomized double-blind crossover study design was utilized in which 31 patients (mean age 41.8 months, range 26-58 months) underwent two restorative dental appointments. At one appointment, subjects received 0.2 mg/kg intranasal midazolam; at the other appointment subjects received 62.5 mg/kg chloral hydrate with 12.5 mg promethazine. Administered at each appointment was 25%-50% N(2)0/0(2). Physiologic parameters (heart rate, blood pressure, respiratory rate, oxygen saturation) and behavior assessments (crying, movement, sleep) using the Houpt Sedation Rating Scale were recorded at baseline and every five minutes during treatment. Overall behavior was assessed at baseline and at the end of treatment. Following treatment, a modified Vancouver Recovery Scale was used to determine the length of time it took each subject to meet established discharge criteria. RESULTS: There were no clinically significant differences in physiologic parameters, however a statistically significant decrease in systolic and diastolic blood pressure was observed in patients sedated with chloral hydrate/promethazine. There were no significant differences in behavior between groups. Patients sedated with intranasal midazolam slept less and recovered quicker than patients sedated with oral chloral hydrate/promethazine. CONCLUSIONS: Intranasal midazolam administered using an atomizer is as safe (as assessed by physiologic parameters) and effective (as assessed by behavior ratings) as oral chloral hydrate/promethazine for conscious sedation of pediatric dental patients.

Action potential waveform voltage clamp shows significance of different Ca2+ channel types in developing ascidian muscle.

1. Early in development, ascidian muscle cells generate spontaneous, long-duration action potentials that are mediated by a high-threshold, inactivating Ca2+ current. This spontaneous activity is required for appropriate physiological development. 2. Mature muscle cells generate brief action potentials only in response to motor neuron input. The mature action potential is mediated by a high-threshold sustained Ca2+ current. 3. Action potentials recorded from these two stages were imposed as voltage-clamp commands on cells of the same and different stages from which they were recorded. This strategy allowed us to study how immature and mature Ca2+ currents are optimized to their particular functions. 4. Total Ca2+ entry during an action potential did not change during development. The developmental increase in Ca2+ current density exactly compensated for decreased spike duration. This compensation was a function purely of Ca2+ current density, not of the transition from immature to mature Ca2+ current types. 5. In immature cells, Ca2+ entry was spread out over the entire waveform of spontaneous activity, including the interspike voltage trajectory. This almost continuous Ca2+ entry may be important in triggering Ca2+-dependent developmental programmes, and is a function of the slightly more negative voltage dependence of the immature Ca2+ current. 6. In contrast, Ca2+ entry in mature cells was confined to the action potential itself, because of the slightly more positive voltage dependence of the mature Ca2+ current. This may be important in permitting rapid contraction-relaxation cycles during larval swimming. 7. The inactivation of the immature Ca2+ current serves to limit the frequency and burst duration of spontaneous activity. The sustained kinetics of the mature Ca2+ current permit high-frequency firing during larval swimming.

CoREST: a functional corepressor required for regulation of neural-specific gene expression.

Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for CoREST interaction. Mutations of the motif that disrupt binding also abrogate repression. When fused to a Gal4 DNA-binding domain, CoREST functions as a repressor. CoREST is present in cell lines that express REST, and the proteins are found in the same immunocomplex. CoREST contains two SANT (SW13/ADA2/NCoR/TFIIIB B) domains, a structural feature of the nuclear receptor and silencing mediator for retinoid and thyroid human receptors (SMRT)-extended corepressors that mediate inducible repression by steroid hormone receptors. Together, REST and CoREST mediate repression of the type II sodium channel promoter in nonneural cells, and the REST/CoREST complex may mediate long-term repression essential to maintenance of cell identity.

Spontaneous activity regulates calcium-dependent K+ current expression in developing ascidian muscle.

1. In embryonic ascidian muscle, outward K+ currents develop in two stages: the initial expression of a slowly activating, voltage-gated K+ current (IKv) near the time of neurulation is followed about 6 h later by a rapidly activating calcium-activated K+ current (IK(Ca)). During this 6 h interval, inward Ca2+ currents (ICa) appear and the inward rectifier (IK(IR)), the sole resting conductance, is transiently downregulated. These events predict a period of spontaneous activity. The following experiments were designed to test this prediction and to examine the relevance of spontaneous activity for muscle cell development. 2. By recording activity in cell-attached patches, we have found that muscle cells generate spontaneous action potentials during this 6 h window of time when IK(IR) is downregulated and outward K+ currents are slow. Action potentials occur at a mean frequency of 13.9 min-1.3. When activity is blocked by the transient application of the Ca2+ channel blocker Cd2+, IK(Ca) fails to develop. This disruption is specific for IK(Ca): IK(IR) and ICa develop normally in activity-blocked cells. Application of Cd2+ either before or after the window of activity has no effect. 4. The reappearance of IK(IR) and the development of IK(Ca) and the mature form of ICa are all prevented by transcription blockers, with a sensitive period corresponding to the period of activity. 5. These data show that, although the expression of three channel types depends on transcription during the period of spontaneous activity, only the development of IK(Ca) depends on activity.

Co-ordinated modulation of Ca2+ and K+ currents during ascidian muscle development.

1. The development of Ca2+ and K+ currents was studied in ascidian muscle cells at twelve embryonic stages from gastrulation to the mature cell, a period of 24 h. A high degree of co-ordination occurs between the development of the inwardly rectifying K+ current (IK(IR)), which sets the resting potential, and Ca2+ and outward K+ currents, which determine action potential waveform. 2. At neurulation IK(IR), which had been present since fertilization, begins to decrease, reaching 12% of its previous density in 6 h. IK(IR) then immediately begins to increase again, reaching its previous density in another 6 h. 3. When IK(IR) begins to decrease, a high-threshold inactivating Ca2+ current and a slowly activating voltage-gated K+ current appear. 4. When IK(IR) returns to its previous density, two new currents appear: a sustained Ca2+ current with the same voltage dependence, but different conotoxin sensitivity than the inactivating Ca2+ current; and a Ca(2+)-dependent K+ current, which activates 8-10 times faster and at potentials 20-30 mV more negative than the voltage-dependent K+ current. 5. The transient downregulation of IK(IR) destabilizes the resting potential and causes spontaneous action potentials to occur. Because IK(IR) is absent when only a slowly activating high-threshold outward K+ current is present, these action potentials are long in duration. 6. The return of IK(IR) and the appearance of the rapidly activating Ca(2+)-dependent K+ current eventually terminate this activity. The action potentials of the mature cell occur only on stimulation, and are 10 times shorter in duration than those in the immature cell.

Comparison of ionic currents expressed in immature and mature muscle cells of an ascidian larva.

We have compared the voltage-gated ion channels present in larval ascidian muscle at two developmental stages: muscle precursor cells just after the terminal cell division and mature contractile muscle, 7-11 hr later. All precursor cells express a high-threshold transient Ca current and a slowly activating delayed K current, and about half the cells express a low-threshold transient Ca current. An inwardly rectifying K current, which had been present from fertilization until just before the terminal cell division, is absent. Mature muscle retains two of the tailbud currents: the low-threshold transient Ca current and the slow delayed K current, although at larger densities, and also expresses a high-threshold Ca current that is similar in most respects to the precursor cell current but that lacks inactivation. In addition, mature muscle expresses two rapidly activating outward K currents, one voltage and one Ca dependent, that generate a composite outward K current that is eight times larger and activates eight times faster than the tailbud K current. Mature muscle also reexpresses the inward rectifier. We propose that the transient absence of the inward rectifier and the slow activation of the delayed K current early in development create a window of developmental time when spontaneous electrical activity is likely.

Essential areas for behavioral science training: a needs assessment approach.

The design of effective behavioral science curricula for graduate medical education is an important link in the delivery of high quality medical care. Despite the work that has gone into developing methods of teaching and evaluating behavioral science training, as shown by numerous published articles, practicing primary care physicians still show deficits in handling patients' psychosocial problems. In this study, the authors propose the use of a behavioral science needs assessment questionnaire to ascertain the needs of residents for content areas based on the residents' medical school backgrounds and preferences for training. The questionnaire can also be used to monitor the effectiveness of existing curricula. The questionnaire was administered to 40 family practice residents in four programs to determine essential content areas. It was then used for curriculum planning in one program with 19 residents. Results from this program identified areas not covered in medical school and reflected the resident's comfort/confidence in practice in areas emphasized in the residency curriculum.

Influence of cimetidine on oral diazepam elimination with measurement of subsequent cognitive change.

In a double-blind crossover design, the effect of cimetidine on oral diazepam pharmacokinetics and an evaluation of cognitive function change was studied in seven healthy volunteers. Subjects randomly received placebo or cimetidine 300 mg orally every 6 h for a total of five doses prior to diazepam 10 mg. A significant increase in diazepam elimination half-life from (mean +/- s.e. mean) 66.9 +/- 21.4 to 89.6 +/- 22.5 h (P = 0.006) and area under the plasma concentration-time curve from 5.06 +/- 1.47 to 8.93 +/- 1.88 micrograms ml-1 h (P = 0.028) was observed indicating a probable cimetidine induced inhibition of diazepam hepatic metabolism. Digit symbol substitution and visual analogue scales were used to measure the effect of an alteration of diazepam kinetics on cognitive function. An enhancement of pharmacological effect as reflected in digit symbol substitution scores and visual analogue scale measurements was not observed.