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."     

After-hours telephone coverage: the application of an area-wide telephone triage and advice system for pediatric practices

BACKGROUND: After-hours telephone calls are a stressful and frustrating aspect of pediatric practice. At the request of private practice pediatricians in Denver, a metropolitan area-wide system was created to manage after-hours pediatric telephone calls and after-hours patient care. This system, the After-Hours Program (AHP), uses specially trained pediatric nurses with standardized protocols to provide after-hours telephone triage and advice for the patients of 92 Denver pediatricians, representing 56 practices. OBJECTIVES: This report describes the AHP, presents data from 4 years' experience with the program, and describes results of our evaluation of the following aspects of the program: subscribing physician satisfaction, parent satisfaction, the accuracy and appropriateness of telephone triage, and program costs. METHODS: After-Hours Program records (including quality assurance data) for all 4 years of operation were retrospectively reviewed, tabulated, and analyzed. The results of two subscribing physician surveys and one parent caller satisfaction survey are presented. A retrospective review of after-hours patient care encounter forms assessed the necessity for after-hours visits triaged by the AHP. An analysis of the total cost of this program to 10 randomly selected subscribing physicians was conducted using current AHP data and a survey of the 10 physicians. RESULTS: In 4 years, 107,938 calls have been successfully managed without an adverse clinical outcome. Minor errors in using protocols occurred in one call out of 1450 after-hours calls. After-hours phoen calls necessitated an after-hours patient visit 20% of the time and generated one after-hours hospital admission out of every 88 calls. Just over half of the patients were managed with home care advice only, and 28% were given home care advice after-hours and seen the next day in the primary physician's office. Of all patients directed by the telephone triage nurses to be seen after hours, 78% were determined to have a condition necessitating after-hours care. Data are presented regarding call volumes by time of day, day of week, patient age, and patient's initial complaint. The 6 most common complaints accounted for more than one half of the calls, and 38 complaints accounted for more than 95% of all after-hours calls. Utilization by subscribing physicians is described. Satisfaction among subscribing pediatricians was 100%, and among parents was 96% to 99% on a variety of issues. The total cost to participating Denver pediatricians (which includes revenues "given up" as a result of not seeing patients after hours) ranged from 1% to 12% of their annual net income, depending on a variety of factors. CONCLUSIONS: Large-scale after-hours telephone coverage systems can be effective and well-received by patients, parents, and primary physicians. Data presented in this report can assist in planning the training of personnel who provide after-hours telephone advice and triage. Controversies associated with this type of program are discussed. Suggestions are made regarding the direction of future programs and research.     

Working memory capacity and the top-down control of visual search: Exploring the boundaries of "executive attention".

The executive attention theory of working memory capacity (WMC) proposes that measures of WMC broadly predict higher order cognitive abilities because they tap important and general attention capabilities (R. W. Engle & M. J. Kane, 2004). Previous research demonstrated WMC-related differences in attention tasks that required restraint of habitual responses or constraint of conscious focus. To further specify the executive attention construct, the present experiments sought boundary conditions of the WMC-attention relation. Three experiments correlated individual differences in WMC, as measured by complex span tasks, and executive control of visual search. In feature-absence search, conjunction search, and spatial configuration search, WMC was unrelated to search slopes, although they were large and reliably measured. Even in a search task designed to require the volitional movement of attention (J. M. Wolfe, G. A. Alvarez, & T. S. Horowitz, 2000), WMC was irrelevant to performance. Thus, WMC is not associated with all demanding or controlled attention processes, which poses problems for some general theories of WMC. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Individual Differences in Susceptibility to False Memory in the Deese-Roediger-McDermott Paradigm.

The authors addressed whether individual differences in the working memory capacity (WMC) of young adults influence susceptibility to false memories for nonpresented critical words in the Deese-Roediger-McDermott associative list paradigm. The results of 2 experiments indicated that individuals with greater WMC recalled fewer critical words than individuals with reduced WMC when participants were forewarned about the tendency of associative lists (e.g., bed, rest, . . .) to elicit illusory memories for critical words (e.g., sleep). In contrast, both high and low WMC participants used repeated study-test trials to reduce recall of critical words. These findings suggest that individual differences in WMC influence cognitive control and the ability to actively maintain task goals in the face of interfering information or habit. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Austenite formation during intercritical annealing

A systematic experimental study has been conducted on ferrite recrystallization and intercritical austenite formation for two low-carbon steels with chemical compositions typically used for dual-phase and transformation-induced plasticity (TRIP) steels. Different initial heating rates, holding temperatures, and times were applied to the materials to examine the ferrite recrystallization and austenite formation kinetics. An Avrami model was developed to describe the isothermal ferrite recrystallization behavior and was applied successfully to the nonisothermal conditions. It was found that the initial heating rate affects the isothermal austenite formation kinetics for both the hot-rolled and cold-rolled materials albeit the effect is more pronounced for the cold-rolled material. This can be attributed to the interaction between the ferrite recrystallization and austenite formation processes. Furthermore, it was found that the distribution of austenite phase is also affected by the ferrite recrystallization process. When ferrite recrystallization is completed before the austenite formation (i.e., under sufficiently slow heating rate conditions), austenite is to a large extent randomly distributed in the ferrite matrix. On the other hand, incomplete recrystallization of ferrite due to higher heating rates leads to the formation of banded austenite grains. It is proposed that this observation is characteristic of simultaneous recrystallization and austenite formation where moving ferrite grain boundaries do not provide suitable sites for austenite nucleation.     

Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein

PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.     

Partial Reduction in BRCA1 Gene Dose Modulates DNA Replication Stress Level and Thereby Contributes to Sensitivity or Resistance

BRCA1 is a well-known breast cancer risk gene, involved in DNA damage repair via homologous recombination (HR) and replication fork protection. Therapy resistance was linked to loss and amplification of the BRCA1 gene causing inferior survival of breast cancer patients. Most studies have focused on the analysis of complete loss or mutations in functional domains of BRCA1. How mutations in non-functional domains contribute to resistance mechanisms remains elusive and was the focus of this study. Therefore, clones of the breast cancer cell line MCF7 with indels in BRCA1 exon 9 and 14 were generated using CRISPR/Cas9. Clones with successful introduced BRCA1 mutations were evaluated regarding their capacity to perform HR, how they handle DNA replication stress (RS), and the consequences on the sensitivity to MMC, PARP1 inhibition, and ionizing radiation. Unexpectedly, BRCA1 mutations resulted in both increased sensitivity and resistance to exogenous DNA damage, despite a reduction of HR capacity in all clones. Resistance was associated with improved DNA double-strand break repair and reduction in replication stress (RS). Lower RS was accompanied by increased activation and interaction of proteins essential for the S phase-specific DNA damage response consisting of HR proteins, FANCD2, and CHK1.     

Environmental air pollution and DNA adducts in Copenhagen bus drivers--effect of GSTM1 and NAT2 genotypes on adduct levels

Fecal samples from 335 dairy farm residents and 1458 cattle on 80 farms were tested for Vero cytotoxin (VT)-producing Escherichia coli (VTEC). Residents were also tested for antibodies to VT1 and O157 lipopolysaccharide (LPS). Residents and cattle on farms with VTEC-positive persons or E. coli O157:H7-positive cattle were retested. Twenty-one persons (6.3%) on 16 farms (20.8%) and 46% of cattle on 100% of the farms had VTEC in fecal samples. Human VTEC isolates included E. coli O157:H7 and 8 other serotypes, 4 of which were present in cattle on the same farms. More persons had antibodies to VT1 (41%) than to O157 LPS (12.5%). Seropositivity to O157 LPS was associated with isolation of E. coli O157:H7 on the farm (P = .022). Human VTEC infection was negatively associated with age (P < .05) and was not associated with clinical illness. Many dairy farm residents experience subclinical immunizing VTEC infections at a young age, which frequently involve non-O157 VTEC found in cattle.     

Corneal topography of photorefractive keratectomy versus laser in situ keratomileusis. Summit PRK-LASIK Study Group

OBJECTIVE: This study aimed to compare qualitative patterns of corneal topography early in the postoperative course after excimer laser photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) when used for the treatment of myopia of 6.0 to 15.0 diopters. DESIGN: The study design was a prospective, multicenter, randomized clinical trial. PARTICIPANTS: A total of 64 eyes were treated with PRK and 54 eyes were treated with LASIK. INTERVENTION: Using the Summit Apex excimer laser, patients received either PRK or LASIK using a single pass, multizone excimer laser ablation. Computer-assisted videokeratography was performed at designated postoperative examinations. MAIN OUTCOME MEASURES: Videokeratography maps at 1 and 3 months after surgery were classified using a standard classification scheme. The association of topography patterns to loss of spectacle-corrected visual acuity was tested. RESULTS: At 1 month, for the PRK (n = 60) and LASIK (n = 51) groups, respectively, 63.3% and 19.6% of eyes fell into one of the four optically irregular groups (central island, keyhole, semicircular, or irregularly irregular; P < 0.001). At 3 months, for the PRK (n = 49) and LASIK (n = 39) groups, respectively, 36.7% and 10.3% of eyes fell into one of the optically irregular groups (P = 0.004). Comparing the 1- and 3-month examination results in the PRK and LASIK groups, respectively, 19 (42%) of 45 eyes and 11 (31%) of 36 eyes had a change in topography, generally to an optically smoother pattern. The irregular groups, taken together, were associated with a greater tendency toward loss of spectacle-corrected visual acuity of two or more Snellen lines (P = 0.01). There also was greater tendency toward loss of spectacle-corrected visual acuity in the PRK group that diminished with time (P < 0.01 at 1 month, P = 0.05 at 3 months). CONCLUSIONS: After treatment for moderate-to-high myopia, LASIK topography patterns generally are more regular than are PRK patterns. This may be a result either of masking of underlying topography perturbations by the lamellar corneal flap, thus mitigating induced topography changes, or differences in surface wound healing. This study suggests that more rapid return of spectacle-corrected visual acuity found in patients treated with LASIK may be a result of more regular topography patterns early in the postoperative course.