Mutations in the yeast Hsp40 chaperone protein Ydj1 cause defects in Axl1 biogenesis and pro-a-factor processing.

The heat shock protein (Hsp) 70/Hsp40 chaperone system plays an essential role in cell physiology, but few of its in vivo functions are known. We report that biogenesis of Axl1p, an insulinase-like endoprotease from yeast, is dependent upon the cytosolic Hsp40 protein Ydj1p. Axl1 is responsible for cleavage of the P2 processing intermediate of pro-a-factor, a mating pheromone, to its mature form. Mutant ydj1 strains exhibited a severe mating defect, which correlated with a 90% reduction in a-factor secretion. Reduced levels of a-factor export were caused by defects in the endoproteolytic processing of P2, which led to its intracellular accumulation. Defective P2 processing correlated with the reduction in the steady state level of active Axl1p. Two mechanisms were uncovered to explain why Axl1p activity was diminished in ydj1 strains. First, AXL1 mRNA levels were reduced ydj1 strains. Second, the half-life of newly synthesized Axl1p was greatly diminished in ydj1 strains. Collectively, these data indicate Ydj1p functions to promote AXL1 mRNA accumulation and in addition appears to facilitate the proper folding of nascent Axl1p. This study is the first to suggest a role for Ydj1p in RNA metabolism and identifies Axl1p as an in vivo substrate of the Hsp70/Ydj1p chaperone system.

The bits and bytes of computer/Internet addiction: a factor analytic approach.

A factor analytic approach was used to explore possible constructs underlying so-called computer/Internet addiction. A 94-item survey was developed, from which two major and two minor factors were derived. Factor 1 focused on problematic computer-related behaviors in heavy users of the Internet, whereas Factor 2 focused on the usefulness and general purpose nature of computers and the Internet. Factor 3 isolated a combination of use of the Internet for sexual gratification and shyness/introversion, and Factor 4 focused on an absence of problems related to Internet use that were coupled with a mild aversion or a disinterest in this technology. These data support the notion that some individuals have a mixture of obsessive-like characteristics related specifically to their computer/Internet use but that, not surprisingly, they also exhibit a preference for on-line, rather than in-person, interactions.

Mutations within the first LSGGQ motif of Ste6p cause defects in a-factor transport and mating in Saccharomyces cerevisiae.

Mating between the two haploid cell types (a and alpha) of the yeast Saccharomyces cerevisiae depends upon the efficient secretion and delivery of the a- and alpha-factor pheromones to their respective target cells. However, a quantitative correlation between the level of transported a-factor and mating efficiency has never been determined. a-Factor is transported by Ste6p, a member of the ATP-binding cassette (ABC) family of transporter proteins. In this study, several missense mutations were introduced in or near the conserved LSGGQ motif within the first nucleotide-binding domain of Ste6p. Quantitation of extracellular a-factor levels indicated that these mutations caused a broad range of a-factor transport defects, and those directly within the LSGGQ motif caused the most severe defects. Overall, we observed a strong correlation between the level of transported a-factor and the mating efficiency of these strains, consistent with the role of Ste6p as the a-factor transporter. The LSGGQ mutations did not cause either a significant alteration in the steady-state level of Ste6p or a detectable change in its subcellular localization. Thus, it appears that these mutations interfere with the ability of Ste6p to transport a-factor out of the MATa cell. The possible involvement of the LSGGQ motif in transporter function is consistent with the strong conservation of this sequence motif throughout the ABC transporter superfamily.

Intrathecal fentanyl for post-thoracotomy pain.

This double-blind, placebo-controlled study investigated the efficacy of intermittent doses of intrathecal fentanyl in 30 patients undergoing thoracotomy. They were allocated randomly to three groups, two of which had microspinal catheters inserted into the lumbar subarachnoid space at the end of surgery; the third group acted as a control. Intrathecal fentanyl or 0.9% saline was administered through the catheters and all patients received morphine using a patient-controlled analgesia (PCA) system. Pain scores, morphine consumption and peak expiratory flow rates (PEFR) were recorded on an hourly basis. Intrathecal fentanyl resulted in a faster onset of analgesia (mean visual analogue scale (VAS) score at 1 h = 0.9 compared with 6.3 (95% confidence intervals for the difference -6.8, -4.0) for the other groups; P < 0.001) and significantly lower pain scores at rest, on cough and on movement. PEFR values were consistently higher in the intrathecal fentanyl group. There were no cases of early or delayed respiratory depression.

The posttranslational modification of phosphoglucomutase is regulated by galactose induction and glucose repression in Saccharomyces cerevisiae.

The enzyme phosphoglucomutase functions at a key point in carbohydrate metabolism. In this paper, we show that the synthesis of the major isoform of yeast phosphoglucomutase, encoded by the GAL5 (PGM2) gene, is regulated in a manner that is distinct from that previously described for other enzymes involved in galactose metabolism in the yeast Saccharomyces cerevisiae. Accumulation of this isoform increased four- to sixfold when the culture experienced either glucose depletion or heat shock. However, heat shock induction did not occur unless the cells were under glucose repression. This nonadditive increase in expression suggests that the regulatory mechanisms controlling the heat shock induction and glucose repression of the GAL5 gene are functionally related. We previously demonstrated that phosphoglucomutase is modified by a posttranslational Glc-phosphorylation reaction. We now show that this posttranslational modification, like phosphoglucomutase expression itself, is also regulated by galactose induction and glucose repression. Finally, no evidence was found to indicate that the Glc-phosphorylation of phosphoglucomutase alters its enzymatic activity under the conditions examined.

Propofol infusion and the suppression of consciousness: dose requirements to induce loss of consciousness and to suppress response to noxious and non-noxious stimuli.

We have defined the infusion dose requirements of propofol to suppress consciousness and response to a variety of graded non-noxious and noxious stimuli in 52 unpremedicated patients aged 16-40 yr and 32 patients aged 41-65 yr. They were allocated to receive one of five loading dose-infusion schemes designed to establish stable conditions covering the range from wakefulness, through sedation, to loss of consciousness and anaesthesia. At 10 and 20 min after the loading dose, each patient's response to a graded series of stimuli was recorded. Probit analysis was used to derive mean values (95% confidence interval) for the ED50 and ED95 (as final infusion rate) for loss of response to verbal command at 4.9 (4.7-5.1) mg kg-1 h-1 and 7.9 (7.3-8.8) mg kg-1 h-1, respectively, in the young group and 4.2 (4.0-4.4) mg kg-1 h-1 and 5.8 (5.4-6.4) mg kg-1 h-1, respectively, in the older group. In both groups the dose-response curves for suppression of proprioception, finger counting and perception of light touch in conscious patients were shifted to the left of the curves for loss of consciousness and eyelash reflex. Dose-response curves for noxious stimuli were shifted to the right of those for loss of consciousness.

Propofol and alfentanil in children: infusion technique and dose requirement for total i.v. anaesthesia.

We estimated the dose of propofol (initial dose followed by a stepped infusion) when given with two different infusion rates of alfentanil for total i.v. anaesthesia in 59 children aged 3-12 yr. Patients in series 1 (four groups) received an alfentanil loading dose of 85 micrograms kg-1 and an infusion of 65 micrograms kg-1 h-1. Patients in series 2 (groups 5 and 6) received an alfentanil loading dose of 65 micrograms kg-1 and infusion of 50 micrograms kg-1 h-1. Parents gave their informed consent. Premedication comprised temazepam 0.3 mg kg-1. Glycopyrronium 5 micrograms kg-1 was administered and anaesthesia induced and maintained with alfentanil (loading dose and infusion) followed by propofol (loading dose and three-stage manual infusion scheme). Suxamethonium 1 mg kg-1 was used to facilitate tracheal intubation and the lungs were ventilated artificially to normocapnia with 30% oxygen in air. Probit analysis was used to determine the dose requirement of propofol. In series 1, the ED50 was 6.0 mg kg-1 h-1 (95% confidence limits 5.5-6.2 mg kg-1 h-1) and ED95 8.6 (6.8-7.8) mg kg-1 h-1. Corresponding values for series 2 were ED50 7.5 (8.0-9.8) mg kg-1 h-1 and ED95 10.5 (9.6-13.1) mg kg-1 h-1.