An endogenous 'hypertensive factor' enhances the voltage-dependent calcium current

The effects of an immunoaffinity-purified putative endogenous hypertensive factor (HF) on voltage-dependent calcium current in frog cardiac myocytes were assessed. In 9 out of 10 cells, HF reversibly increased the peak amplitude of the calcium current. HF increased peak calcium current density at -5 mV from a control level of 1.8 +/- 1.3 pA/pF (mean +/- SD) to 4.4 +/- 2.0 pA/pF. HF shifted the peak of the calcium current-voltage relationship in the hyperpolarizing direction. HF shifted the voltage dependence of the inactivation of the calcium current to more negative potentials with prepulses from -80 to 0 mV, but the inactivation was not affected with prepulses more positive than 0 mV. Modulation of the voltage-dependent calcium current by HF may be the mechanism underlying its pressor effects.     

Conformational distributions of melittin in water/methanol mixtures from frequency-domain measurements of nonradiative energy transfer

We used fluorescence energy transfer to examine the effects of solvent composition on the distribution of distances between the single tryptophan residue of melittin (residue 19) to the N-terminal alpha-amino group, which was labeled with a dansyl residue. The tryptophan intensity decays, with and without the dansyl acceptor, were measured by the frequency-domain method. The data were analyzed by a least-squares algorithm which accounts for correlation between the parameters. A wide distribution of tryptophan to dansyl distances was found for the random-coil state, with a Gaussian half-width of 25 A. Increasing concentrations of methanol, which were shown to induce and alpha-helical conformation, resulted in a progressive decrease in the width of the distribution, reaching a limiting half-width of 3 A at 80% (v/v) methanol. The distance from the indole moiety of Trp-19 to the dansyl group in 80% (v/v) methanol/water was found to be 25 A, as assessed from the center of the distance distribution. A distance of 24-25 A was recovered from the X-ray crystal structure of the tetramer, which is largely alpha-helical. At low ionic strength (less than 0.01) the CD spectra revealed a small fraction or amount of alpha-helix for melittin in water, which implies a small fraction of residual structure. This residual structure is apparently lost in guanidine hydrochloride as demonstrated by a further broadening in the distribution of distances. These results demonstrate the usefulness of frequency-domain measurements of resonance transfer for resolution of conformational distributions of proteins.     

A novel RNA helicase gene tightly linked to the Triplo-lethal locus of Drosophila.

The Triplo-lethal (Tpl) locus of Drosophila is the only known locus which is lethal when present in three copies rather than the normal two. After recovering a hybrid-dysgenesis-induced mutation of Tpl we used a rapid combination of transposon tagging, chromosome microdissection and PCR to clone the P element that had transposed into the Tpl region. That P element is located within the gene for a new and unique member of the RNA helicase family. This new helicase differs from all others known by having glycine-rich repeats at both the amino and carboxyl termini. Curiously, genetic analysis shows that the P element inserted into this gene is not responsible for the Tpl mutant phenotype. We present possible explanations for these findings.     

The occurrence of polyenoic very long chain fatty acids with greater than 32 carbon atoms in molecular species of phosphatidylcholine in normal and peroxisome-deficient (Zellweger''s syndrome) brain.

The n-6 tetra- and pentaenoic fatty acids with carbon chain lengths greater than 32 found in normal brain are located predominantly in a separable species of phosphatidylcholine. A similar phospholipid is found in increased amounts in the brain of peroxisome-deficient (Zellweger's syndrome) patients, but the fatty acid composition differs in that penta- and hexaenoic derivatives predominate. Our data strongly suggest that the polyenoic very long chain fatty acids are confined to the sn-1 position of the glycerol moiety, while the sn-2 position is enriched in saturated, monounsaturated and polyunsaturated fatty acids with less than 24 carbon atoms. It is postulated that these unusual molecular species of phosphatidylcholine may play some, as yet undefined, role in brain physiology.     

Transmission electron microscopy of small numbers of sorted cells

A new method that allows the transmission electron microscopic examination of as few as 1 x 10(4) cells obtained by flow cytometric sorting is described. The approach involves "sandwiching" fixed cells in an agarose case by a microcentrifugation system consisting of small-diameter cell-centrifugation tubes and subsequent processing of the cells by conventional techniques. The advantages offered by this method are discussed.     

Subcutaneous narcotic infusions for cancer pain: treatment outcome and guidelines for use.

OBJECTIVE: To provide guidelines for the institution and maintenance of a continuous subcutaneous narcotic infusion program for cancer patients with chronic pain through an analysis of the narcotic requirements and treatment outcomes of patients who underwent such therapy and a comparison of the costs of two commonly used infusion systems. DESIGN: Retrospective study. SETTING: Tertiary care facilities and patients'' homes. PATIENTS: Of 481 patients seen in consultation for cancer pain between July 1987 and April 1990, 60 (12%) met the eligibility criteria (i.e., standard medical management had failed, and they had adequate supervision at home). INTERVENTION: Continuous subcutaneous infusion with hydromorphone hydrochloride or morphine started on an inpatient basis and continued at home whenever possible. OUTCOME MEASURES: Patient selectivity, narcotic dosing requirements, discharge rate, patient preference for analgesic regimen, side effects, complications and cost-effectiveness. RESULTS: The mean initial maintenance infusion dose after dose titration was almost three times higher than the dose required before infusion (hydromorphone or equivalent 6.2 v. 2.1 mg/h). Eighteen patients died, and the remaining 42 were discharged home for a mean of 94.4 (standard deviation 128.3) days (extremes 12 and 741 days). The mean maximum infusion rate was 24.1 mg/h (extremes 0.5 and 180 mg/h). All but one of the patients preferred the infusion system to their previous oral analgesic regimen. Despite major dose escalations nausea and vomiting were well controlled in all cases. Twelve patients (20%) experienced serious systemic toxic effects or complications; six became encephalopathic, which necessitated dose reduction, five had a subcutaneous infection necessitating antibiotic treatment, and one had respiratory depression. The programmable computerized infusion pump was found to be more cost-effective than the disposable infusion device after a break-even point of 8 months. CONCLUSIONS: Continuous subcutaneous infusion of opioid drugs with the use of a portable programmable pump is safe and effective in selected patients who have failed to respond to standard medical treatment of their cancer pain. Dose titration may require rapid dose escalation, but this is usually well tolerated. For most communities embarking on such a program a programmable infusion system will be more cost-effective than a disposable system.     

Glycogen phosphorylase: control by phosphorylation and allosteric effectors.

Structural studies of muscle glycogen phosphorylase during the last two decades have provided a detailed mechanism for the molecular basis of the control by phosphorylation and by allosteric effectors and the catalytic mechanism. Control by phosphorylation is effected by a disorder to order transition of the NH2-terminal residues that promotes localized changes in the structure of the protein at the region of subunit-subunit contacts and larger changes in the quaternary structure. The covalently attached phosphate group acts like an allosteric effector but the full manifestation of the response is also dependent on the NH2-terminal tail residues. The noncovalently bound allosteric effectors produce similar shifts in the structural states although these are bound at sites that are remote from the serine-phosphate site. The communication from these sites to the catalytic site is through long-range interactions that result in activation of the enzyme through opening access to the buried catalytic site and through creation of the substrate phosphate recognition site by an interchange of an acidic group with a basic group. Recent advances in expression systems have opened the way to a study of properties both for the muscle and other isozymes and other species that should illuminate the different regulatory roles of the enzyme in different tissues and organisms. The allosteric mechanism of activation of phosphorylase by phosphorylation may be relevant to other enzymes although it is now known that other mechanisms such as electrostatic steric blocking mechanisms also exist.