Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion

The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate the biochemistry and physiology of membrane fusion. Homologues of vesicle-associated membrane protein (VAMP), syntaxin, and SNAP-25 were identified in CV membranes. A VAMP and syntaxin immunoreactive band at a higher apparent molecular mass (approximately 70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, and syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolysis by tetanus toxin. All criteria identify the SNARE complex as that described in other secretory systems. Complexes exist pre-formed on individual CV membranes and form between contacting CV. Most notably, CV SNARE complexes are disrupted in response to [Ca2+]free that trigger maximal fusion. N-Ethylmaleimide, which blocks fusion at or before the Ca2+-triggering step, blocks complex disruption by Ca2+. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca2+-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca2+ disrupts rather than stabilizes the complex, the presumably coiled-coil SNARE interactions are not needed at the time of fusion. These findings rule out models of fusion in which SNARE complex formation goes to completion ("zippers-up") after Ca2+ binding removes a "fusion-clamp."     

The Internet & Healthcare Education: HELIX

With the advent of the World Wide Web (WWW), we are now on the cusp of a revolution in computer technology that will dramatically enhance medical education. An historical analogy might be Johann Gutenberg's invention of movable type in the 1400's-radically decreasing the cost, time, and expertise required to reproduce printed materials. Now, the WWW can decrease the cost of disseminating medical educational materials. When an educational module is authored for the Web, it can be placed on a computer "server" which in turn, distributes the program on the WWW to anyone with a computer and Internet access. Rapidly emerging standards are being developed to allow increasingly rich educational experiences on the Internet. With the introduction of HTML (hypertext markup language), a standardized method of placing text and graphics, as well as the connections between them, was created.     

Why talk about mental states? The significance of children's conversations with friends, siblings, and mothers

Natural language data from 38 47-month-olds recorded at home in unstructured observations were analyzed and comparisons made of characteristics of mental state term use in child-friend, child-sibling, and child-mother dyads. Significantly more references to mental states were made by the children in conversations with siblings and friends than with mothers. Frequent use of mental state terms by both partners was related to cooperative interaction in both child-friend and child-sibling dyads and several associations were found with measures of language fluency, gender, and maternal education, although these varied across the 2 dyads. Children's use of mental state terms in conversations with siblings and friends was correlated with their performance on two false belief measures. Results highlight the importance of extending investigations into the social implications of the development of children's "theories of mind."     

Ultrarapid metabolizers of debrisoquine: characterization and PCR-based detection of alleles with duplication of the CYP2D6 gene

During a six-month period 2,221 haemocultures obtained from patients hospitalized in the Faculty Hospital Olomouc were examined. In all 304 isolated bacteria the sensitivity was assessed by the standard dilution micromethod and moreover all positive haemocultures were examined the "direct" disc method. Agreement between the results of the two methods was proved in 84% of pairs of tests and within a range from 67 to 100%, depending on the type of antimicrobial preparation. Based on these findings it may be stated that assessment of the sensitivity by the "direct" method agrees significantly with assessment of the sensitivity according to minimal inhibitory concentrations (MIC). In patients with septicaemia this procedure makes it possible to change empirical antibiotherapy by 24 hours sooner to aimed therapy.     

Coping, drinking motives, goal attainment expectancies and family models in relation to alcohol use among college students

Associations between coping responses, drinking motivations, expectations of meeting social and academic goals, and family of origin problem drinking and measures of college students' quantity/frequency of alcohol use and social complications of alcohol use were investigated in a sample of 218 college students. Positive associations were found between "emotion-focused" forms of coping such as detachment and the criterion measures, whereas "problem-focused coping" was not significantly associated with quantity/frequency of alcohol use or drinking complications. Positive correlations were also found between drinking motives, goal attainment expectancies and family models measures and the criterion measures. Regression models constructed for alcohol quantity/frequency and drinking complications implicated the total number of drinking motives, family models of problem drinking and the coping strategy of self-blame as strongly related to criterion measures. Positive social drinking motives and coping by seeking social support were implicated as possible protective factors.     

The gradual threshold model of ambivalence: relating the positive and negative bases of attitudes to subjective ambivalence

Eighty subjects viewed and visually imagined upright or rotated alphanumeric characters and later judged whether test characters were previously seen or imagined (reality monitoring). Identification and test characters were presented verbally or visually. When characters were identified and tested verbally, source confusions (misjudging a seen character as "imagined" and vice-versa) were infrequent and were comparable for rotated and upright characters. When characters were identified and tested visually, source confusions were more frequent and were influenced by character rotation. Memories for imagined characters were especially susceptible to source confusion. Also source confusions for seen characters increased when characters were rotated. These results are consistent with the proposal that increasing sensory similarity between perceived and imagined items increases source confusion and that perceived rotation generates cognitive operations similar to those generated when the subject imagines a character rotated.     

Loss of heterozygosity in tuberous sclerosis hamartomas

We have previously described in tuberous sclerosis (TSC) hamartomas the phenomenon of loss of heterozygosity (LOH) for DNA markers in the region of both the TSC2 gene on chromosome 16p13.3 and the TSC1 gene on 9q34. We now describe the spectrum of LOH in 51 TSC hamartomas from 34 cases of TSC. DNA was extracted from leucocytes or normal paraffin embedded tissue, and from frozen paraffin embedded hamartoma tissue from the same patient. The samples were analysed for 11 markers spanning the TSC1 locus and nine markers spanning the TSC2 locus. Twenty-one of 51 hamartomas showed LOH (41%). There was significantly more LOH on 16p13.3, with 16 hamartomas showing LOH around TSC2, and five in the vicinity of TSC1. No hamartoma showed LOH for markers around both loci. All the areas of LOH on chromosome 9 were large, but the smallest region of overlap lay between the markers D9S149 and D9S114, providing independent evidence for the localisation of the TSC1 gene. These data show that LOH is a common finding in a wide range of hamartomas, affecting the same TSC locus in different lesions from the same patient but not affecting both loci. These data support the hypothesis that both the TSC genes act as tumour suppressors and that the manifestations of TSC in patients with germline TSC mutations rise from "second hit" somatic mutations inactivating the remaining normal copy of the TSC gene.     

Measurement of activin in biological fluids by radioimmunoassay, utilizing dissociating agents to remove the interference of follistatin

BACKGROUND: The aim of this morphological investigation was to obtain more information about the structural and cellular mechanisms of interalveolar pore formation in postnatal lung development. Assuming that alveolar pore formation is related to the general thinning of interalveolar walls observed in the postnatal period, we have focused our attention on the topographical relationship between epithelial cells and connective tissue in the septum. Thereby we tried to formulate a uniform concept of pore formation. METHODS: After fixation with glutaraldehyde and osmiumtetroxide, tissue blocks of rat lungs aged 44 days were embedded in Epon. Serial sections were obtained in order to analyse precisely pores and supposed sites of pore formation (type II cells and thin spots in transsections of interalveolar walls). RESULTS: We made the following observations: there are pores with or without type II cells in the neighbourhood, and "pre-pores" with either fully transseptal granular pneumocytes, or thin spots in the interalveolar wall consisting of one or two layers of type I cell epithelium or of type II and type I cells without intervening connective tissue. CONCLUSIONS: From these findings we deduce that there is a general principle of interalveolar pore formation which consists in the formation of transseptal interepithelial cell contacts (i.e., between cells of type II and type I or type I and type I), promoted by the thinning of interalveolar walls in the stage of microvascular maturation. Within the zone of contact the cells thin out and give way to form an interalveolar opening.     

Cell surface chondroitin sulfate proteoglycans in tumor cell adhesion, motility and invasion

Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors. Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells. In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene. A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L). This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L. In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6). This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites. In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice. In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells. These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG.     

Nonsanction seat belt law enforcement: a modern day tale of two cities

This paper describes the implementation and evaluation of a nonsanction seat belt law enforcement program in two experimental communities of contrasting size in a state with a mandatory belt law. The main ingredients of the program were seat belt "salutes," public information and education, and limited use of inexpensive economic incentives. Driver shoulder belt use data collected before, during, and after the experimental programs, compared to similar data collected in a comparison community, showed the approach to be effective. While standard seat belt enforcement activities without incentives have been shown to be effective, many police departments, especially in smaller communities, are reluctant to make wholesale increases in seat belt citations. Although requiring some additional level of manpower and resources, a nonsanction approach to seat belt law enforcement can provide an alternate way of increasing belt use in these communities.