The role of calcium and other ions in sorting and delivery in the late endocytic pathway.

The passage of endocytosed receptor-bound ligands and membrane proteins through the endocytic pathway of mammalian cells to lysosomes occurs via early and late endosomes. The latter contain many luminal vesicles and are often referred to as MVBs (multivesicular bodies). The overall morphology of endosomal compartments is, in major part, a consequence of the many fusion events occurring in the endocytic pathway. Kissing events and direct fusion between late endosomes and lysosomes provide a means of delivery to lysosomes. The luminal ionic composition of organelles in the endocytic pathway is of considerable importance both in the trafficking of endocytosed ligands and in the membrane fusion events. In particular, H(+) ions play a role in sorting processes and providing an appropriate environment for the action of lysosomal acid hydrolases. Na(+)/H(+) exchangers in the endosomal membrane have been implicated in the formation of MVBs and sorting into luminal vesicles. Ca(2+) ions are required for fusion events and luminal content condensation in the lysosome. Consistent with an important role for luminal Ca(2+) in traffic through the late endocytic pathway, mutations in the gene encoding mucolipin-1, a lysosomal non-specific cation channel, result in abnormalities in lipid traffic and are associated with the autosomal recessive lysosomal storage disease MLIV (mucolipidosis type IV).

Relationship between endosomes and lysosomes.

Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing assay using late endosomes and lysosomes from rat liver. There is evidence from electron microscopy studies that the same process occurs in intact cells. The fusion process results in the formation of hybrid organelles from which lysosomes are re-formed. The discovery of the hybrid organelle has opened up three areas of investigation: (i) the mechanism of direct fusion of late endosomes and lysosomes, (ii) the mechanism of re-formation of lysosomes from the hybrid organelle, and (iii) the function of the hybrid organelle. Fusion has analogies with homotypic vacuole fusion in yeast. It requires syntaxin 7 as part of the functional trans-SNARE [SNAP receptor, where SNAP is soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein] complex and the release of lumenal calcium to achieve membrane fusion. Re-formation of lysosomes from the hybrid organelle occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular traffic. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a 'cell stomach', acting as a major site of hydrolysis of endocytosed macromolecules.

An outbreak of scabies in a teaching hospital: lessons learned.

OBJECTIVE: To investigate an outbreak of scabies in an inner-city teaching hospital, identify pathways of transmission, institute effective control measures to end the outbreak, and prevent future occurrences. DESIGN: Outbreak investigation, case-control study, and chart review. SETTING: Large tertiary acute-care hospital. RESULTS: A patient with unrecognized Norwegian (crusted) scabies was admitted to the acquired immunodeficiency syndrome (AIDS) service of a 940-bed acute-care hospital. Over 4 months, 773 healthcare workers (HCWs) and 204 patients were exposed to scabies. Of the exposed HCWs, 147 (19%) worked on the AIDS service. Risk factors for being infested with scabies among HCWs included working on the AIDS service (odds ratio [OR], 5.3; 95% confidence interval [CI95], 2.17-13.15) and being a nurse, physical therapist, or HCW with extensive physical contact with infected patients (OR, 4.5; CI95, 1.26-17.45). Aggressive infection control precautions beyond Centers for Disease Control and Prevention barrier and isolation recommendations were instituted, including the following: (1) early identification of infected patients; (2) prophylactic treatment with topical applications for all exposed HCWs; (3) use of two treatments 1 week apart for all cases of Norwegian scabies; (4) maintaining isolation for 8 days and barrier precautions for 24 hours after completing second treatment for a diagnosis of Norwegian scabies; and (5) oral ivermectin for treatment of patients who failed conventional therapy. CONCLUSIONS: HCWs with the most patient contact are at highest risk of acquiring scabies. Because HCWs who used traditionally accepted barriers while caring for patients with Norwegian scabies continued to develop scabies, we found additional measures were required in the acute-care hospital. HCWs with skin exposure to patients with scabies should receive prophylactic treatment. We recommend (1) using heightened barrier precautions for care of patients with scabies and (2) extending the isolation period for 8 days or 24 hours after the second treatment with a scabicide for those patients with Norwegian scabies. Oral ivermectin was well tolerated for treating patients and HCWs who failed conventional treatment. Finally, we developed a surveillance system that provides a "barometric measure" of the infection rate in the community. If scabies increases in the community, a tiered triage system is activated to protect against transmission among HCWs or hospital patients.

Causes of urine marking in cats and effects of environmental management on frequency of marking.

OBJECTIVE: To evaluate effects of environmental management alone on marking frequency in cats with urine marking and to obtain demographic data on cats with urine marking and data on owner-perceived factors that contributed to urine marking behavior. DESIGN: Single-intervention study. ANIMALS: 40 neutered male and 7 spayed female cats. PROCEDURE: During a 2-week baseline phase, owners maintained a daily record of the number of urine marks. This phase was followed by a 2-week environmental management phase during which owners cleaned recently deposited urine marks daily, scooped waste from the litter box daily, and changed the litter and cleaned the litter box weekly while continuing to record urine marks. RESULTS: Male cats and cats from multicat households were significantly overrepresented, compared with the general pet cat population in California. The most commonly mentioned causative factors for urine marking were agonistic interactions with other cats outside or inside the home. Environmental management procedures resulted in an overall reduction in urine marking frequency. Among cats that marked > or = 6 times during the baseline phase, females were significantly more likely to respond to treatment (> or = 50% reduction in marking frequency) than were males. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that male cats and cats from multicat households are more likely to exhibit urine marking behavior than females and cats from single-cat households. Results also suggest that attention to environmental and litter box hygiene can reduce marking frequency in cats, regardless of sex or household status of the cats, and may come close to resolving the marking problem in some cats.

Effects of a selective serotonin reuptake inhibitor on urine spraying behavior in cats.

OBJECTIVE: To determine the effectiveness of a readily available selective serotonin reuptake inhibitor (SSRI), fluoxetine hydrochloride, on reducing problem urine spraying in cats. DESIGN: Randomized placebo-controlled double-blind clinical trial. ANIMALS: 17 neutered cats > 1 year old with objectionable urine spraying behavior. Procedure-Owners recorded urine-spraying events for 2 weeks (baseline). Cats that vertically marked a mean of > or = 3 times per week were treated for 8 weeks with fluoxetine or fish-flavored liquid placebo. If urine spraying was not reduced by 70% by weeks 4 through 5, the dosage was increased by 50% for weeks 7 and 8. After discontinuation of treatment at the end of 8 weeks, owners recorded daily urine marks for another 4 weeks. RESULTS: The mean (+/- SE) weekly rate of spraying episodes in treated cats was 8.6 (+/- 2.0) at baseline, decreased significantly by week 2 (1.7 +/- 0.6), and continued to decrease by weeks 7 and 8 (0.4 +/- 0.2). The mean weekly spraying rate of cats receiving placebo was 7.8 (+/- 1.5) at baseline, decreased only slightly during week 1 (5.5 +/- 1.8), and did not decline further. When treatment was discontinued after 8 weeks, the spraying rate of cats that had received treatment varied. The main adverse reaction to the drug was a reduction in food intake, which was observed in 4 of 9 treated cats. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of fluoxetine hydrochloride for treatment of urine spraying in cats can be expected to considerably reduce the rate of urine marking. The frequency of spraying before treatment is predictive of the spraying rate when the drug is discontinued.

A preliminary study of the effect of imaginary sexual stimulation on the perceived covariation between freedom and responsibility.

The authors evaluated subjective estimates of the relationship between freedom and responsibility under predictions made in accordance with cognitive-experiential self-theory (CEST; V. Denes-Raj & S. Epstein, 1994; S. Epstein, A. Lipson, C. Holstein, & E. Huh, 1992; S. Epstein, R. Pacini, V. Denes-Raj, & H. Meier, 1996; L. A. Kirkpatrick & S. Epstein, 1992). Half of the participants viewed sexually stimulating primes before making judgments. The other participants viewed neutral stimuli before making judgments. Two dependent measures were used: A set of alternate-forms propositions measured perceived relationships between the variables, and response latencies were used to evaluate the hypothesis that persons operating experientially would make judgments faster than persons operating rationally. Results indicated a significant effect for the priming condition with respect to the within-subject dependent variable. In accordance with predictions, further analysis indicated that positive contingency items were endorsed less often by primed participants, and negative contingency items were endorsed more often by primed participants. Results are in line with predictions afforded by the CEST model. Implications are discussed.

The equivalence of contingency structure for intuitive covariation judgments about height, weight, and body fat.

In a complete between-subjects design, 576 respondents judged covariations of problems framed around height, weight, and body fat. A verbal measure was used, consisting of propositional statements representing relationships between two of the variables, as well as two types of response options: true or false, and a 7-point, bipolar, Likert-type scale. Consistent with previous findings, weight and body fat were unambiguously judged as if positively correlated; there were indications of a perceived negative correlation of height and body fat; and there was no evidence that participants regarded height and weight as positively correlated. Furthermore, there was no reliable evidence in support of framing effects. However, there were indications of influence for conditional format. These findings are discussed relative to previous and ongoing research efforts, and it is concluded that it may be inappropriate to regard variations in conditional format as parallel items. Measures involving all possible variations are considered fundamentally unbiased and consistent with I. Ajzen's (1988) principle of aggregation.

The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles.

We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

Chronic insulin effects on insulin signalling and GLUT4 endocytosis are reversed by metformin.

Decreases in insulin-responsive glucose transport and associated levels of cell surface GLUT4 occur in rat adipocytes maintained in culture for 20 h under hyperinsulinaemic and hyperglycaemic conditions. We have investigated whether this defect is due to reduced signalling from the insulin receptor, GLUT4 expression or impaired GLUT4 trafficking. The effects of chronic insulin treatment on glucose transport and GLUT4 trafficking were ameliorated by inclusion of metformin in the culture medium. In comparison with the ic insulin treatment attenuated changes in signalling processes leading to glucose transport. These included insulin receptor tyrosine phosphorylation, phosphoinositide 3-kinase activity and Akt activity, which were all reduced by 60-70%. Inclusion of metformin in the culture medium prevented the effects of the chronic insulin treatment on these signalling processes. In comparison with cells maintained in culture without insulin, the total expression of GLUT4 protein was not significantly altered by chronic insulin treatment, although the level of GLUT1 expression was increased. Trafficking rate constants for wortmannin-induced cell-surface loss of GLUT4 and GLUT1 were assessed by 2-N-4-(1-azi-2, 2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannose-4-yloxy)-2-propyla min e (ATB-BMPA) photolabelling. In comparison with cells acutely treated with insulin, chronic insulin treatment resulted in a doubling of the rate constants for GLUT4 endocytosis. These results suggest that the GLUT4 endocytosis process is very sensitive to the perturbations in signalling that occur under hyperinsulinaemic and hyperglycaemic conditions, and that the resulting elevation of endocytosis accounts for the reduced levels of net GLUT4 translocation observed.

Lysosome-endosome fusion and lysosome biogenesis.

Recent data both from cell-free experiments and from cultured cells have shown that lysosomes can fuse directly with late endosomes to form a hybrid organelle. This has a led to a hypothesis that dense core lysosomes are in essence storage granules for acid hydrolases and that, when the former fuse with late endosomes, a hybrid organelle for digestion of endocytosed macromolecules is created. Lysosomes are then re-formed from hybrid organelles by a process involving condensation of contents. In this Commentary we review the evidence for formation of the hybrid organelles and discuss the current status of our understanding of the mechanisms of fusion and lysosome re-formation. We also review lysosome biosynthesis, showing how recent studies of lysosome-like organelles including the yeast vacuole, Drosophila eye pigment granules and mammalian secretory lysosomes have identified novel proteins involved in this process.

Association of need for cognition with judgments of height, weight, and body fat covariation.

559 college students, assessed for Need for Cognition, judged whether height, weight, and body fat were correlated using judgment probes that controlled for framing and conditional format. A principal components analysis of Need for Cognition scores identified two factors underlying the scale, which may be important in judgment outcome. In addition, judgments about correlations among height, weight, and body fat were similar to those of previous studies. Furthermore, the hypothesis that Need for Cognition would be related to the tendency to judge a negative correlation between height and body fat, i.e., a possible illusory correlation, was confirmed. Results are discussed relative to the Elaboration Likelihood Model of Persuasion.

Association of AP1 adaptor complexes with GLUT4 vesicles.

Nycodenz gradients have been used to examine the in vitro effects of GTP-(gamma)-S on adaptor complex association with GLUT4 vesicles. On addition of GTP-(gamma)-S, GLUT4 fractionates as a heavier population of vesicles, which we suggest is due to a budding or coating reaction. Under these conditions there is an increase in co-sedimentation of GLUT4 with AP1, but not with AP3. Western blotting of proteins associated with isolated GLUT4 vesicles shows the presence of high levels of AP1 and some AP3 but very little AP2 adaptor complexes. Cell free, in vitro association of the AP1 complex with GLUT4 vesicles is increased approximately 4-fold by the addition of GTP-(gamma)-S and an ATP regenerating system. Following GTP-(gamma)-S treatment in vitro, ARF is also recruited to GLUT4 vesicles, and the temperature dependence of ARF recruitment closely parallels that of AP1. The recruitment of both AP1 and ARF are partially blocked by brefeldin A. These data demonstrate that the coating of GLUT4 vesicles can be studied in isolated cell-free fractions. Furthermore, at least two distinct adaptor complexes can associate with the GLUT4 vesicles and it is likely that these adaptors are involved in mediating distinct intracellular sorting events at the level of TGN and endosomes.

Evidence against protein kinase B as a mediator of contraction-induced glucose transport and GLUT4 translocation in rat skeletal muscle.

Both insulin and muscle contraction stimulate glucose transport activity. However, contraction stimulation does not involve the insulin signalling intermediate phosphatidylinositol 3-kinase (PI 3-kinase). Protein kinase B (PKB) has recently been identified as a direct downstream target of PI 3-kinase in the insulin signalling pathway. We have examined here whether the two stimuli share PKB as a convergent step in separate signalling pathways. Insulin stimulates both glucose transport, GLUT4 cell-surface content and PKB activity (by 4-6-fold above basal) in a wortmannin-sensitive manner in in vitro incubated rat soleus muscles. By contrast, muscle contraction, which stimulates glucose transport and the cell surface content of GLUT4 by 3-fold above basal levels, had no effect on PKB activity. These data demonstrate that PKB is not a mediator of contraction-induced glucose transport and GLUT4 translocation.

Radiographic standing cervical segmental alignment in adult volunteers without neck symptoms.

STUDY DESIGN: A radiographic evaluation of standing cervical and global sagittal alignment in 100 adult volunteers with no neck or radicular arm symptoms was undertaken. OBJECTIVES: To document and define cervical standing lateral alignment and balance in a volunteer population without cervical problems. SUMMARY OF BACKGROUND DATA: Minimal data exist on cervical segmental sagittal alignment distributions in stance. METHODS: Radiographic measures of spinal alignment were collected and statistically analyzed. RESULTS: Data were categorized into two age- and sex-matched groups. Group 1 had no cervical or lumbar symptoms. Group 2 had no cervical symptoms, but had lumbar symptoms. Mean total and segmental cervical alignment distributions were not statistically different between groups. Mean total cervical lordosis in stance for the entire group was -40.0 +/- 9.7 degrees. Thoracic and lumbar alignment were within previously reported measures and not statistically different between groups. Plumb line markers at C7 and sacral reference points were not statistically different between groups. Pearson correlations revealed that cervical sagittal plane alignment vaired inversely with thoracic alignment. Independent orthopedists noted cervical osteoarthritis was present in 17% of each group of volunteers with substantial kappa agreement (0.73) between observers. Intraobserver and interobserver reliability of sagittal alignment measures of 30 randomly remeasured radiographs revealed strong correlation between observations. CONCLUSIONS: No differences in total cervical lordosis were noted between cervical asymptomatic volunteers with or without low back pain. Most cervical lordosis occurred at the C1-C2 level in stance, whereas only 6 degrees (15%) occurred at the lowest three cervical levels (C4-C7). Changes in cervical lordosis correlated inversely with changes in thoracic alignment.

IL-4 enhances IL-3 and IL-8 gene expression in a human leukemic mast cell line.

We examined the capacity of interleukin (IL)-4 to induce or enhance the expression of certain cytokines in resting and activated cells of the HMC-1 human leukemic mast cell line. The HMC-1 mast cells were cultured with or without recombinant human IL-4 and then activated with the calcium ionophore ionomycin. Stimulation of non-IL-4-treated cells with ionomycin (10 microM) for periods of 30 min to 8 hr induced expression of mRNA encoding IL-3, IL-4 and IL-8 but was without effect on levels of mRNA for tumour necrosis factor (TNF)-alpha or beta-actin. Culture of the cells with IL-4 (100 ng/ml) for 24 hr led to a small increase in resting levels of mRNA for IL-3 and IL-8 but not for IL-4, TNF-alpha or beta-actin. More notably, the IL-4 treatment produced a pronounced elevation of mRNA for IL-3 and IL-8 when the cells were subsequently activated with ionomycin. The IL-4 treatment produced a negligible effect on IL-4 mRNA, and no effect on TNF-alpha or beta-actin mRNA levels in ionomycin-activated cells. Quantitation of cDNA by competitive polymerase chain reaction (PCR) revealed that the IL-4 treatment produced a sixfold increase in ionomycin-induced levels of cellular IL-3 mRNA, a fourfold increase in induced IL-8 mRNA and less than a twofold increase in induced IL-4 mRNA. The IL-4 treatment led to a 15- to 20-fold increase in ionomycin-induced secretion of IL-3 product and a doubling of induced IL-8 product. These effects of IL-4 were not associated with increased mast cell numbers. We conclude that IL-4 alone is a weak activator of IL-3 and IL-8 gene expression in mast cells, but is able to enhance activation signals in stimulated mast cells leading to transcription and secretion of these two cytokines.

Lidocaine for the prevention of pain due to injection of propofol.

Propofol has a high incidence of pain with injection, particularly into small veins. We sought to determine whether concomitant administration of lidocaine could prevent this pain. In a randomized double-blind trial, 368 women were allocated to one of four groups to receive 19 mL of propofol mixed with either 1 mL of 0.9% saline, 1 mL of 0.5% (5 mg) lidocaine, 1 mL of 1% (10 mg) lidocaine, or 1 mL of 2% (20 mg) lidocaine. The pain of injection was scored as none, mild, moderate, or severe. There was a significant reduction in the overall incidence of pain from 73% with saline to 32% with 20 mg lidocaine. A highly significant negative dose-response relationship between the dose of lidocaine and the severity of pain was demonstrable, both at induction of anesthesia and as recalled in the recovery room (P less than 0.001 for both). Lidocaine (20 mg IV) will significantly reduce the incidence and severity of pain with propofol injection, but about 6% of patients will still suffer unpleasant pain if the dorsum of the hand is used.

Pharmacokinetics and bioavailability of ticarcillin and clavulanate in foals after intravenous and intramuscular administration.

The pharmacokinetics and bioavailability of ticarcillin and clavulanate were determined after intravenous (i.v.) or intramuscular (i.m.) administration of ticarcillin disodium (50 mg/kg) combined with clavulanate potassium (1.67 mg/kg) to groups of healthy foals at 3 days and 28 days of age. After i.v. administration of the combination to five foals, the disposition kinetics of ticarcillin and clavulanate were best described using a two-compartment open model. Mean plasma elimination-rate constant (beta) and clearance (ClB) for ticarcillin were significantly less (P less than 0.01), and volume of distribution at steady state (Vd(ss)) was significantly larger (P less than 0.05), in the foals at 3 days compared with 28 days of age. This indicated that renal excretion mechanisms were immature and ticarcillin was more widely distributed in 3-day-old foals. The mean elimination rate constant for clavulanate was significantly less (P less than 0.01) at 3 days than at 28 days of age. Values of the major kinetic terms describing the disposition of ticarcillin after i.m. administration to five 3-day-old foals were not significantly different from values of these parameters in the same foals at 28 days of age. After i.m. administration of the drug combination, plasma clavulanate concentrations peaked significantly later (P less than 0.01), and the elimination-rate constant (kd) for clavulanate was significantly less (P less than 0.01), in 3-day-old foals than in 28-day-old foals. The bioavailabilities of ticarcillin and clavulanate after i.m. administration in 3-day-old foals were 100% and 88.3%, respectively, and in 28-day-old foals were 100% and 27.4%, respectively. Mean plasma ticarcillin concentrations exceeded 16 micrograms/ml for a longer period after i.m. administration of the drug combination than after i.v. administration to foals of both age groups. By virtue of the frequency of administration required and the painful response elicited by i.m. injection, it is recommended that when the combination of ticarcillin disodium (50 mg/kg) and clavulanate potassium (1.67 mg/kg) is used in foals to treat infections caused by susceptible organisms (MIC less than or equal to 16 micrograms/ml), it should be administered i.v. four times daily.