Suppressors of clathrin deficiency: overexpression of ubiquitin rescues lethal strains of clathrin-deficient Saccharomyces cerevisiae

Clathrin-mediated vesicular transport is important for normal growth of the yeast Saccharomyces cerevisiae. Previously, we identified a genetic locus (SCD1) that influences the ability of clathrin heavy-chain-deficient (Chc-) yeast cells to survive. With the scd1-v allele, Chc- yeast cells are viable but grow poorly; with the scd1-i allele, Chc- cells are inviable. To identify the SCD1 locus and other genes that can rescue chc1 delta scd1-i cells to viability, a multicopy suppressor selection strategy was developed. A strain of scd1-i genotype carrying the clathrin heavy-chain gene under GAL1 control (GAL1:CHC1) was transformed with a YEp24 yeast genomic library, and colonies that could grow on glucose were selected. Plasmids from six distinct genetic loci, none of which encoded CHC1, were recovered. One of the suppressor loci was shown to be UBI4, the polyubiquitin gene. UBI4 rescues only in high copy number and is not allelic to SCD1. The conjugation of ubiquitin to intracellular proteins can mediate their selective degradation. Since UBI4 is required for survival of yeast cells under stress and is induced during starvation, ubiquitin expression in GAL1:CHC1 cells was examined. After a shift to growth on glucose to repress synthesis of clathrin heavy chains, UBI4 mRNA levels were elevated > 10-fold, whereas the quantity of free ubiquitin declined severalfold relative to that of Chc+ cells. In addition, novel higher-molecular-weight ubiquitin conjugates appeared in clathrin-deficient cells. We suggest that higher levels of ubiquitin are required for turnover of mislocalized or improperly processed proteins that accumulate in the absence of clathrin and that ubiquitin may play a general role in turnover of proteins in the secretory or endocytic pathway.     

Invasive electrophysiologic evaluation in octogenarians: is age a limiting factor?

To assess the indications, diagnostic yield, and incidence of complications of electrophysiologic testing in the elderly we reviewed our experience with 60 procedures in 45 patients aged > or = 80 years (range 80 to 92 years, mean age 83) undergoing full electrophysiologic evaluation in our laboratory over the past 7 years. The yield of inducible ventricular tachycardia (31%), supraventricular tachycardia (4%), and previously unsuspected conduction abnormalities significant enough to warrant permanent pacemaker implantation (9%), together with the low incidence of complications (1 patient had a deep venous thrombosis and femoral artery pseudoaneurysm, representing an incidence of 2.2% of patients undergoing studies or 3.3% incidence of complications per procedure), suggest that invasive electrophysiologic procedures in the elderly can provide useful information at a complication rate comparable with that of younger patients.     

Perimenstrual complaints in women complaining of PMS, menorrhagia, and dysmenorrhea: toward a dismantling of the premenstrual syndrome

Four groups of women were compared in terms of their perimenstrual symptoms, reported menstrual blood loss and period pain, and neuroticism scores: three patient groups were referred to a Gynaecology Outpatient Clinic because of menorrhagia (N = 101), PMS (N = 104), dysmenorrhea (N = 56), and a control group (N = 105). The three patient groups showed considerable overlap in a number of symptoms. This has led us to postulate three factors contributing to perimenstrual complaints: a) a 'timing factor' linked to the ovarian cycle; b) a 'menstruation factor,' associated with the buildup of the endometrium and its shedding; and c) a 'vulnerability factor,' one aspect of which, 'neuroticism,' was measured in this study. Depressive symptoms, which were the most important in leading women to seek help for their PMS, were related to all three factors. Depressive mood changes seemed to be linked to the 'timing factor' but were noticeably worse and more prolonged in women with high neuroticism, heavy bleeding, or severe pain. One premenstrual symptom, food craving, was of considerable interest. This was weakly related to neuroticism, not apparently affected by the 'menstruation factor' and differed in severity between those in the PMS group and the other three groups. It is potentially relevant that both carbohydrate craving and depression are linked to serotonergic changes in the brain, which may prove to be particularly marked in the late luteal phase.     

Expression in animal cells and characterization of the hepatitis E virus structural proteins

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to obtain fertilization of the oocyte in vivo and in vitro. Although most of the biochemical/ biophysical events that occur during capacitation in vitro have been characterized, the molecular mechanisms underlying these complex events are still obscure. Increases of intracellular free Ca2+ concentrations ([Ca2+]i) and protein tyrosine phosphorylation have previously been demonstrated during in vitro capacitation of human spermatozoa. In the present study we investigated the relationship between extracellular/intracellular Ca2+, protein tyrosine phosphorylation, and tyrosine kinase and phosphatase activities during sperm capacitation. We report that the increase in tyrosine phosphorylation of several protein bands that occurs during sperm capacitation is independent of the presence of Ca2+ in the external medium and, at least partially, of the increase in [Ca2+]i occurring during the process. Indeed, the spontaneous increase in phosphorylation was still present in Ca(2+)-free/EGTA-containing-medium and in the presence of the intracellular Ca2+ chelator BAPTA/AM. Moreover, phosphorylation of proteins and protein tyrosine kinase (PTK) activity was enhanced if spermatozoa were incubated in Ca(2+)-free medium, suggesting the presence of Ca(2+)-inhibited tyrosine kinase(s) in human sperm. This hypothesis is further substantiated by the lower tyrosine phosphorylation observed after incubation with the ionophore A23187 and the endoplasmic Ca(2+)-ATPase inhibitor thapsigargin, which promote Ca2+ influx in human sperm. The ability of the cells to undergo acrosome reaction in response to progesterone, which can be considered a functional endpoint of capacitation, was highly compromised when spermatozoa were incubated in Ca(2+)-free medium or in the presence of EGTA, confirming that Ca2+ is required for sperm capacitation. Conversely, in the presence of erbstatin, a inhibitor of tyrosine kinase activity, which blunts tyrosine phosphorylation during capacitation, response to progesterone was maintained, suggesting that tyrosine phosphorylation must be kept at a low level (physiologically by the presence of Ca2+ in the external medium, or pharmacologically by the presence of erbstatin) in order to obtain response to progesterone. This mechanism may be important in vivo during sperm transit in the female genital tract to ensure appropriate timing of full capacitation in the proximity of the oocyte.     

Guidance of locomotion on foot uses perceived target location rather than optic flow

What visual information do we use to guide movement through our environment? Self-movement produces a pattern of motion on the retina, called optic flow. During translation, the direction of movement (locomotor direction) is specified by the point in the flow field from which the motion vectors radiate - the focus of expansion (FoE) [1-3]. If an eye movement is made, however, the FoE no longer specifies locomotor direction [4], but the 'heading' direction can still be judged accurately [5]. Models have been proposed that remove confounding rotational motion due to eye movements by decomposing the retinal flow into its separable translational and rotational components ([6-7] are early examples). An alternative theory is based upon the use of invariants in the retinal flow field [8]. The assumption underpinning all these models (see also [9-11]), and associated psychophysical [5,12,13] and neurophysiological studies [14-16], is that locomotive heading is guided by optic flow. In this paper we challenge that assumption for the control of direction of locomotion on foot. Here we have explored the role of perceived location by recording the walking trajectories of people wearing displacing prism glasses. The results suggest that perceived location, rather than optic or retinal flow, is the predominant cue that guides locomotion on foot.     

Association of periampullary diverticula with primary choledocholithiasis but not with secondary choledocholithiasis

The precision and the diagnostic performance of the Boehringer Mannheim CEDIA DAU LSD assay was evaluated. The assay was performed in the semi-quantitative mode on a Hitachi 917 analyzer. Within-run coefficients of variation (CVs) of the semiquantitative values for 0.25 and 1.0 ng/mL were 11.2 and 6.2%, respectively. Day-to-day CVs for the same concentrations were 12.6 and 8.6%. We analyzed 318 urine samples by CEDIA, DPC Coat-A-Count RIA and Behring EMIT II. Confirmation was performed by GC-MS, after extraction on Bond Elut Certify columns. Two hundred sixty-three samples were negative by all methods. Twenty-five samples were positive by all immunoassays, 19 of which were confirmed by gas chromatography-mass spectrometry (GC-MS). One sample was falsely negative by CEDIA. Three samples were positive by EMIT and CEDIA, but negative by RIA and GC-MS. Twenty-six samples were positive by EMIT alone, but they were not confirmed by GC-MS. The LSD CEDIA assay seems to be less specific than DPC RIA but more specific than the EMIT LSD assay.