Caveolae and sarcoplasmic reticular coupling in smooth muscle cells of pressurised arteries: the relevance for Ca2+ oscillations and tone

OBJECTIVE: A close association of caveolae and sarcoplasmic reticulum (SR) has been suggested to be important for contractile activation of smooth muscle. Here, we investigate the presence of such arrangements in pressurised resistance arteries and examine the influence of two agents purported to disrupt caveolae and/or SR conformations by different mechanisms of action. METHODS: Rat mesenteric small arteries (RMSA) were mounted on a pressure myograph and the functional (lumen diameter and Ca(2+) oscillations) and ultrastructural effects of the phosphatase inhibitor calyculin-A (cal-A), or the cholesterol binding agent methyl-beta-cyclodextrin (mbetacd), examined by light and electron microscopy. RESULTS: Smooth muscle cells of RMSA exhibited a prominent peripheral SR that often encircled individual caveolae. The peripheral SR on occasion was observed to make contact with centrally located SR allowing for a structural association of caveoale-SR-myofilaments. Cal-A maximally constricted RMSA and disrupted the regular SR-caveolae appearance such that concentrated swirls of SR not enveloping caveolae were evident. Mbetacd treatment, in contrast, inhibited agonist contractility and reduced the appearance of caveolae whilst peripheral SR apposition to the plasmalemma could still be observed. Treatment with either agent inhibited agonist-mediated smooth muscle Ca(2+) oscillations. CONCLUSION: We present data that supports a structural arrangement of caveolae and underlying peripheral SR in smooth muscle cells of pressurised resistance arteries that serves to regulate Ca(2+) oscillations and contractile activation.     

Problem-based learning of research skills

PURPOSE: To determine whether a short-term, problem-based educational intervention leads to increased research activity among health care practitioners. SUBJECTS AND METHODS: Participant's success was evaluated as a composite of 2 outcomes. These were (1) reporting results for the project designed during the practicum and (2) conducting subsequent research activities. The study population included 36 clinical research outcomes projects developed by clinical practitioners, postgraduate trainees, and medical students during 6 separate practicums. All project teams received the same educational intervention, an "outcomes research practicum" that was divided into 4 primary learning modules administered over a 1 to 4 month period. Each module included a preparatory videotape lecture, supplemental readings, and a 90-minute interactive laboratory session during which faculty members worked with participants to develop answers to a series of predefined questions relating to the design of clinical outcomes research projects. Follow-up continued for a minimum of 12 months and a maximum of 36 months. RESULTS: Eighty-three percent of project teams completed all 4 practicum modules, and 69% completed one of the study outcomes (50% completed their research project and 47% completing a subsequent research activity). Practitioners were more likely to complete subsequent research activities, whereas trainees were more likely to complete their study project. DISCUSSION: This short-term, problem-based educational intervention was successful in increasing the collective research activities of participants. Further, more rigorous structured research is needed to determine the ultimate impact on practice change and patient outcomes.     

Clinical nurse specialists as entrepreneurs: constrained or liberated

Aims. This qualitative study explored the experiences of two groups of clinical nurse specialists – continence advisors and tissue viability nurses – working in primary care in the UK. In particular, the study focused on how clinical nurse specialists’ relationships with other health‐care professionals had an impact on their role. Background. Clinical nurse specialists are recognized worldwide as having expertise in a given field, which they use to develop the practice of others. Additionally, clinical nurse specialists share many of the characteristics of entrepreneurs, which they use to develop services related to their speciality. However, little research has been conducted in relation to clinical nurse specialists’ experiences as they attempt to diversify nursing practice. Design/methods. An ethnographic approach was adopted comprising many elements of Glaserian grounded theory. Data were collected via participant observation and face‐to‐face interviews with 22 clinical nurse specialists. Findings. Services provided by clinical nurse specialists were not static, clinical nurse specialists being the main drivers for service developments. However, clinical nurse specialists encountered difficulties when introducing new ideas. Given their role as advisors, clinical nurse specialists lacked authority to bring about change and were dependent on a number of mechanisms to bring about change, including ‘cultivating relationships’ with more powerful others, most notably the speciality consultant. Conclusions. The UK government has pledged to ‘liberate the talents of nurses’ so that their skills can be used to progress patient services. This study highlights the fact that a lack of collaborative working practices between health‐care professionals led to clinical nurse specialists being constrained. Relevance to clinical practice. Health‐care organizations need to provide an environment in which the entrepreneurial skills of clinical nurse specialists may be capitalized on. In the absence of an outlet for their ideas regarding service developments, clinical nurse specialists may remain dependent on the mechanisms witnessed in this study for some time.     

Robust expansion of viral antigen-specific CD4+ and CD8+ T cells for adoptive T cell therapy using gene-modified activated T cells as antigen presenting cells

Cytomegalovirus (CMV) reactivation after stem cell transplantation can be treated with CMV-specific T cells, but current in vitro techniques using dendritic cells as antigen-presenting cells are time-consuming and expensive. To simplify the production of clinical grade CMV-specific T cells, we evaluated gene-modified activated T cells [antigen presenting T cells (T-APCs)] as a reliable and easily produced source of APCs to boost CD4+ and CD8+ T-cell responses against the immunodominant CMV antigen pp65. T-APCs expressing the full-length immunodominant CMV pp65 gene were used to stimulate the expansion of autologous T cells. After 10 to 14 days, the T cell lines were tested for antigen specificity by using the flow cytometric intracellular detection of interferon-gamma after stimulation for 6 hours with a pp65 peptide library of 15-mers, overlapping by 11 amino acids. Under optimal conditions, this technique induced a median 766-fold and a 652-fold expansion of pp65-specific CD4+ and CD8+ responder cells, respectively, in 15 T cell lines. In 13 of 15 T cell lines, over 10 antigen-specific CD4+ plus CD8+ T cells were generated starting with only 5x10 peripheral blood mononuclear cells, representing an over 3-log increase. These data indicate that T-APCs efficiently boost pp65-specific CD4+ and CD8+ T cell numbers to clinically useful levels. The approach has the advantage of using a single leukocyte collection from the donor to generate large numbers of CMV-specific T cells within a total 3-week culture period using only one stimulation of antigen.     

Acceptability of routine screening for perinatal depression

BACKGROUND: To assess the acceptability of routine screening for perinatal depression. METHOD: Postnatal women (n=860) and health professionals (n=916) were surveyed after 3 years of routine perinatal (antenatal and postnatal) use of the Edinburgh Postnatal Depression Scale (EPDS). RESULTS: Over 90% of women had the screening explained to them and found the EPDS easy to complete; 85% had no difficulties completing it. Discomfort with screening was significantly related to having a higher EPDS score. A majority of health professionals using the EPDS was comfortable and found it useful. LIMITATIONS: The sample involved only maternity services supporting depression screening. In addition, the response rate from GPs was low. CONCLUSIONS: Routine screening with the EPDS is acceptable to most women and health professionals. Sensitive explanation, along with staff training and support, is essential in implementing depression screening.     

Patterns of sequence evolution at epitopes for host antibodies and cytotoxic T-lymphocytes in human immunodeficiency virus type 1

Analysis of published sequence data from the nine protein-coding genes of human immunodeficiency virus type 1 (HIV-1) showed striking differences in evolutionary pattern between epitopes for host neutralizing antibodies (Ab) and epitopes for cytotoxic T cells (CTL). In all sequences analyzed, the greatest median amino acid residue diversity was seen at sites that formed part of Ab epitopes, but not of CTL epitopes. By contrast, sites belonging to CTL epitopes but not to Ab epitopes showed reduced median amino acid sequence diversity not only in comparison to sites in Ab epitopes but also in comparison to non-epitope sites. Ab epitopes that did not overlap CTL epitopes showed the highest frequency of comparisons in which the rate of nonsynonymous (amino acid-altering) nucleotide substitution exceeded that of synonymous nucleotide substitution, supporting the hypothesis that much of the diversity at Ab epitopes results from positive selection exerted by the host immune system. Though less frequent than that at Ab epitopes, there was evidence of such selection at certain CTL epitopes as well; and amino acid differences between sister pairs of sequences in CTL epitopes were more likely to be convergent than those in Ab epitopes. The pattern seen at CTL epitopes may represent the result of conflicting pressures favoring conservation of the amino acid sequence for functional reasons and amino acid replacements for reasons of CTL escape.     

Previously unidentified complex cytogenetic changes found in a pediatric case of solid-pseudopapillary neoplasm of the pancreas

Solid pseudopapillary neoplasm of the pancreas (SPNP) is a rare tumor with low malignant potential found in adolescent girls and young women. The pathogenesis of SPNP remains uncertain and its management is controversial. Genetic changes associated with SPNP have seldom been reported. We describe here the cytogenetic investigation of a case of SPNP in a 13-year-old girl whose tumor cells revealed two unrelated clones: one clone characterized by complex karyotypic changes, including breakpoints in two common fragile sites at chromosome 2, band q33, and chromosome 4, band q31, and the second clone defined by partial monosomy for chromosome X. Loss of heterozygosity for HRAS was also identified by array comparative genomic hybridization (a-CGH). These cumulative changes seem insufficient for activation of cell transformation, but could possibly play a role in priming the cell for future mutagenic events.     

Listeria monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features

A collection of Listeria monocytogenes serotype 4b strains belonging to lineages I and III were examined by PCR and Southern blot analysis using species-, virulence-, and serotype-specific primers and probes. Whereas four serotype 4b lineage I strains reacted in PCR with the serotype 4b-, 4d-, and 4e-specific ORF2110 and virulence-specific lmo1134 and lmo2821 primers, all nine serotype 4b lineage III strains were negative by ORF2110 and lmo1134 primers. In addition, the nine serotype 4b lineage III strains formed two separate groups through their reactions in PCR with virulence-specific lmo2821 primers. Southern blot analysis using species-specific lmo0733 and virulence-specific lmo2821 gene probes largely confirmed the PCR results. These findings indicate that L. monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.     

Paclitaxel induces calcium oscillations via an inositol 1,4,5-trisphosphate receptor and neuronal calcium sensor 1-dependent mechanism

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Despite a well documented tubulin-stabilizing effect, many side effects of taxol therapy cannot be explained by cytoskeletal mechanisms. In the present study submicromolar concentrations of taxol, mimicking concentrations found in patients, induced cytosolic calcium (Ca(2+)) oscillations in a human neuronal cell line. These oscillations were independent of extracellular and mitochondrial Ca(2+) but dependent on intact signaling via the phosphoinositide signaling pathway. We identified a taxol binding protein, neuronal Ca(2+) sensor 1 (NCS-1), a Ca(2+) binding protein that interacts with the inositol 1,4,5-trisphosphate receptor from a human brain cDNA phage display library. Taxol increased binding of NCS-1 to the inositol 1,4,5-trisphosphate receptor. Short hairpin RNA-mediated knockdown of NCS-1 in the same cell line abrogated the response to taxol but not to other agonists stimulating the phosphoinositide signaling pathway. These findings are important for studies involving taxol as a research tool in cell biology and may help to devise new strategies for the management of side effects induced by taxol therapy.     

Effect of methylphenidate on Stroop Color-Word task performance in children with attention deficit hyperactivity disorder

Attention deficit hyperactivity disorder (ADHD) is a neuropsychiatric syndrome common in the pediatric population. It is associated with multiple nonspecific deficits on neuropsychological tests of executive function, and a beneficial response to pharmacotherapy with methylphenidate (MPH) and other psychostimulants. The Stroop Color-Word task is used empirically as an aid in diagnosis and treatment monitoring of ADHD; however, data on the sensitivity of the Stroop interference score to the effects of MPH are limited. To address this issue, we studied Stroop performance in a cohort of 18 MPH-treated prepubescent boys with ADHD and six healthy controls on and off MPH treatment conditions. MPH significantly improved performance in both groups, with the ADHD participants consistently displaying worse scores than those of controls both on and off MPH. These results suggest that though the diagnostic value of the Stroop task in ADHD remains controversial, it has heuristic value for monitoring clinical responses to MPH treatment. More research is needed to ascertain the clinical significance of our findings and to replicate this relatively small effect in a larger cohort, to determine whether MPH effects on Stroop performance are specific to ADHD symptoms or they generalize to other forms of symptomatology.