Influence of variable salinity and low light on copper accumulation in the potential seagrass bioindicator, Zostera muelleri.

Utilising a potential coastal trace element bioindicator requires understanding its accumulation patterns under varying environmental scenarios. The present study aimed to understand, from two experiments, the influence and effect of low light (15.3 µmol photons m-2 s-1) and variable salinity (normal 36 and reduced 29) on Zostera muelleri accumulating variable Cu concentrations (control, low 5 µg L-1 and high 50 µg L-1) in order to determine its capability as a potential trace element bioindicator. Initial (24 h) leaf Cu concentration was in proportion to exposure Cu concentrations, irrespective of manipulated environmental conditions, suggesting passive accumulation. Final below-ground Cu concentrations, during the low light experiment, significantly increased over time, suggesting active Cu accumulation. Zostera muelleri leaves could act as a Cu bioindicator at times of reduced light and salinity while further interpretation is required of below-ground Cu concentrations. It is recommended that Z. muelleri could be utilised as a Cu bioindicator.

Monitoring Cyp2b10 mRNA expression at cessation of 2-year carcinogenesis bioassay in mouse liver provides evidence for a carcinogenic mechanism devoid of human relevance: the dalcetrapib experience.

INTRODUCTION: Dalcetrapib is a cholesteryl ester transfer protein (CETP) modulator in clinical assessment for cardiovascular outcome benefits. In compliance with regulatory requirements, dalcetrapib was evaluated in rodent 2-year carcinogenesis bioassays. In the mouse bioassay, male mice demonstrated increased liver weight and statistically increased incidences of hepatocellular adenoma/carcinoma. Hepatic cytochrome p450 (Cyp) 2b10 mRNA induction and increased Cyp2b10 enzyme activity signify activation of hepatic nuclear receptor constitutive androstane receptor (CAR), a widely established promoter of rodent-specific hepatic tumors. We therefore monitored hepatic Cyp2b10 mRNA and its enzyme activity in a subset of dalcetrapib-treated male mice from the bioassay. METHODS: Liver samples were obtained from ~1/3 of male mice from each dose group including vehicle-controls (mean and earliest study day of death 678 and 459 respectively). Quantitative real time PCR (qRT-PCR) was performed to determine Cyp2b10 mRNA expression and Cyp1a-, Cyp2b10- and Cyp3a-selective activities were monitored. RESULTS: Cyp2b10 mRNA was strongly induced by dalcetrapib with an expected wide inter-individual variation (5-1421-fold). Group average fold-induction versus vehicle-controls showed a dose-related increase from 48-fold (250mg/kg/day) to 160-fold (750mg/kg/day), which declined slightly at 2000mg/kg/day (97-fold). Cyp enzyme activities showed approximate doubling of total Cyp P450 content per milligram protein and a 9-fold increase in Cyp2b10-selective pentoxyresorufin O-dealkylase activity (750mg/kg/day). DISCUSSION: These data from hepatic Cyp2b10 monitoring are strongly suggestive of CAR activation by dalcetrapib, a mechanism devoid of relevance towards hepatocarcinogenesis in humans; results show feasibility of Cyp2b10 as a surrogate marker for this mechanism at cessation of a carcinogenesis bioassay.

Comparison of different methods of ventilation via cannula cricothyroidotomy in a trachea-lung model.

BACKGROUND: Cannula cricothyroidotomy is recommended in recent guidelines as a rescue intervention in the 'cannot-intubate cannot-ventilate' scenario. Several methods of providing ventilation via a cannula cricothyroidotomy have been described, but there are no data comparing these methods and using cannulae of differing diameters. METHODS: Using a bench-top trachea-lung model (comprising a Siemens test lung attached to commercially available breathing system tubing), we compared delivered minute volumes (MVs) for five methods of ventilation administered through cannulae of diameters 20, 16, 14, and 13 G. The ventilation methods were: an ENK oxygen flow modulator, a Manujet, a self-inflating resuscitation bag, the oxygen flush of an anaesthetic machine, and oxygen from a wall-mounted flow meter attached via a three-way tap to the cannula. All experiments were performed with and without a proximal 2.5 mm diameter constriction to simulate partial upper airway obstruction. RESULTS: MVs increased with increasing cannula diameter. In the absence of a proximal constriction, MVs delivered via a 20 G cannula were <1 litre min(-1) with all devices; only the Manujet delivered MVs >2 litre min(-1), at cannula sizes of >or=16 G. MVs were greater in the presence of a proximal constriction, but did not exceed 4 litre min(-1) using the low-pressure devices. CONCLUSIONS: Extrapolated to the clinical situation, these data suggest that low-pressure devices will not deliver adequate MVs via a cannula cricothroidotomy and should no longer be advocated. Purpose-made devices should be available in all areas where anaesthesia is administered or airway interventions are performed.

Microarray-based identification of a novel Streptococcus pneumoniae regulon controlled by an autoinduced peptide.

We have identified in the Streptococcus pneumoniae genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of blpC, a small gene located downstream of blpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the blp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended -10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.

Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae.

In bacteria, adaptive responses to environmental stimuli are often initiated by two-component signal transduction systems (TCS). The prototypical TCS comprises two proteins: a histidine kinase (HK, hk) and a response regulator (RR rr). Recent research has suggested that compounds that inhibit two-component systems might have good antibacterial activity. In order to identify TCS that are crucial for growth or virulence of Streptococcus pneumoniae, we have examined the genomic sequence of a virulent S. pneumoniae strain for genes that are related to known histidine kinases or response regulators. Altogether 13 histidine kinases and 13 response regulators have been identified. The protein sequences encoded by these genes were compared with sequences deposited in public databases. This analysis revealed that two of the 13 pneumococcal TCSs have been described before (ciaRH and comDE) and two are homologous to the yycFG and the phoRP genes of Bacillus subtilis. All the pneumococcal response regulators contain putative DNA binding motifs within the C-terminal output domain, implying that they are involved in transcriptional control. Two of these response regulators are obviously the first representatives of a new subfamily containing an AraC-type DNA-binding effector domain. To assess the regulatory role of these transcription factors, we disrupted each of the 13 response regulator genes by insertional mutagenesis. All the viable mutant strains with disrupted response regulator genes were further characterized with regard to growth in vitro, competence, and experimental virulence. Two response regulator genes could not be inactivated, indicating that they may regulate essential cellular functions. The possibility of using these systems as targets for the development of novel antibacterials will be discussed.

Noncompliance with drug therapy for chronic obstructive pulmonary disease: a risk factor for hospitalization?

OBJECTIVE: To determine if patients who were noncompliant with prescribed medications for chronic obstructive pulmonary disease (COPD) had higher rates of hospitalization. METHODS: A retrospective case-control study was performed in a tertiary-care university-affiliated Veterans Administration Health Care System setting. Subjects included 93 patients hospitalized for exacerbation of COPD and 93 controls with a diagnosis of COPD who did not require hospitalization. Utilizing pharmacy prescription fill records, medication noncompliance rates of patients who required hospitalization for exacerbation of COPD were compared with patients who did not require such hospitalization. RESULTS: The mean noncompliance ratio for the hospitalized patients was lower than the ratio for the controls (0.19 vs. 0.20) although the difference was not statistically significant (P = .95). There was no statistically significant difference between the demographics of the two groups. However, the patients who were hospitalized had a significantly greater number of COPD and nonCOPD medications (P < .0001, P < .0001) prescribed. They also had significantly more nonCOPD admissions and lengths of stay (P = .02, P = .01). CONCLUSION: At the levels of medication noncompliance observed in this population, there was no difference in rates of hospitalization. Hospitalization could be attributed to other causes such as severity of illness and existence of other comorbid conditions. The effects of environmental pollution and cigarette smoking were not studied. KEYWORDS: chronic obstructive pulmonary disease; hospitalization, noncompliance; risk factors.

Csk-mediated phosphorylation of substrates is regulated by substrate tyrosine phosphorylation.

Csk is a cellular protein tyrosine kinase (PTK) that has been shown to specifically regulate the activity of Src kinase family members by phosphorylation of a carboxy-terminal tyrosine residue. The molecular mechanisms controlling Csk regulation and its substrate specificity have not been elucidated. Here we report a novel type of overlay kinase assay that allows to probe for Csk-mediated phosphorylation of cellular substrates separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. Most of the cell lines analyzed with this method revealed only a few potential Csk substrates. However, an increased number of Csk substrates was detected in NIH3T3 cells expressing a constitutively activated form of the Src kinase Lck or in PC12 and NIH3T3 cells that had been treated with pervanadate. These cells all display an increased level of cellular protein tyrosine phosphorylation which led to the conclusion that Csk preferentially phosphorylates tyrosine-phosphorylated proteins. To verify this hypothesis we analyzed Csk-mediated phosphorylation of recombinant Lck, a known Csk substrate. Results demonstrated that autophosphorylation of Lck (at Tyr394) facilitates Csk-mediated phosphorylation of Lck at its regulatory site (Tyr505). Subsequent peptide binding studies revealed that Csk can bind to a peptide corresponding to the Lck-autophosphorylation site only when it is phosphorylated. These findings suggest that autophosphorylation of Lck at Tyr394 triggers an interaction with Csk and thereby facilitates subsequent phosphorylation and inactivation of Lck. The phosphorylation of other cellular Csk substrates may be regulated by a similar mechanism.

Post-infection fatigue syndrome following Q fever.

In 1989, 147 individuals in the West Midlands, UK, were infected with Q fever. Five years later, following anecdotal reports of fatigue, we used a questionnaire-based case-control study to determine the prevalence of chronic fatigue syndrome symptoms in this group. Replies from 71 patients were compared with those from 142 age- and sex-matched controls. Increased sweating (52.9% vs. 31.6%, p = 0.006), breathlessness (50.7% vs. 30.6%, p = 0.006), blurred vision (34.3% vs. 17.8%, p = 0.016) and undue tiredness (68.7% vs. 51.5%, p = 0.03) were found in controls compared to cases. These findings were similar to those in Australian abbatoir workers occupationally exposed to Q fever. CDC criteria for chronic fatigue syndrome were fulfilled by 42.3% of cases and 26% of controls. Using visual analogue scores, symptoms were more severe in cases than in controls. Our findings support the existence of a chronic fatigue state following acute Q fever, in a group of patients exposed just once to the organism, and in circumstances free of such confounding factors as lawsuits over compensation.

Residency training--the profession's forge for leadership development.

The concept of leadership may be the most widely studied and least understood topic in the domain of social services. Leadership has been described as a major force in the profession's transition to pharmaceutical care and effective program development. Pharmacy practice residency programs are a major means of developing and sustaining leadership for the growth of the profession. Three concepts that are explored include: (1) some insight into noncognitive elements of residency training that are critical to the development of professional practice, (2) analysis of leadership perceived to be critical for pharmacists, and (3) a definition of how residency training can develop professional leaders to meet the challenges for pharmacy.

Purification and characterization of recombinant human p50csk protein-tyrosine kinase from an Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL.

An Escherichia coli expression system overproducing the bacterial chaperones GroES and GroEL was engineered and has been successfully used to produce large quantities of the recombinant human protein-tyrosine kinase p50csk. The co-overproduction of the two chaperones with p50csk results in increased solubility of the kinase and allows purification of milligram amounts of active enzyme. Analysis of the purified protein by SDS/polyacrylamide gel electrophoresis reveals a single band with an apparent molecular mass of 50 kDa, indicating that recombinant human p50csk has been purified to near homogeneity. The purified enzyme displays tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties, including in vitro substrate specificity and enzymatic characteristics of the enzyme, have been assessed and compared with those of members of the Src family of protein-tyrosine kinases. Results indicate that p50csk and p56lck have different substrate specificities and that p50csk and p60c-src have similar kinetic parameters. The successful production and purification of an enzymatically active form of p50csk will enable further characterization of this important kinase and allow clarification of its physiological role. In addition, the results suggest that the approach described may be generally applicable to improve the solubility of recombinant proteins which otherwise are produced in an insoluble form in E. coli.

Mapping of the p56lck-mediated phosphorylation of GAP and analysis of its influence on p21ras-GTPase activity in vitro.

The protein tyrosine kinase p56lck and other members of the src family can transduce signals from activated cell-surface receptors. As we showed earlier the GTPase-activating protein (GAP), a regulator of p21ras, is a substrate of p56lck. Here, tryptic peptides of p56lck-phosphorylated GAP were generated and analyzed by two-dimensional thin layer chromatography and mass spectroscopy. Results revealed that p56lck phosphorylates GAP specifically on Tyr-460 in vitro and in vivo. The effect of tyrosine phosphorylation of GAP on its GTPase-activating activity versus p21ras was then tested using a p21ras-dependent GTPase assay system. Our results demonstrate that p56lck-mediated tyrosine phosphorylation of GAP is not sufficient to change directly its effect on the intrinsic GTPase activity of p21ras.

Purification and characterization of an activated form of the protein tyrosine kinase Lck from an Escherichia coli expression system.

The lymphocyte-specific, nonreceptor protein tyrosine kinase Lck has been purified from an Escherichia coli expression system using a monoclonal antibody column followed by dye-affinity chromatography. Polyacrylamide gel electrophoretic analysis of purified protein revealed a single 56 kDa band, indicating that recombinant Lck was purified to near-homogeneity. The purified enzyme displayed tyrosine kinase activity as measured by both autophosphorylation and phosphorylation of exogenous substrates. Biochemical properties including protein phosphorylation and kinetic characteristics of the enzyme have been assessed. Peptide map analysis revealed that bacterially expressed Lck is phosphorylated predominantly on the autophosphorylation site (tyrosine-394), which is characteristic for activated protein tyrosine kinases. Indeed, we found that the recombinant enzyme is approximately fivefold more active than Lck from resting T cells, which is extensively phosphorylated at the regulatory carboxy-terminal tyrosine residue (tyrosine-505). Thus, we have overproduced recombinant human Lck in E. coli and developed a simple two-step purification procedure which yields highly active enzyme. This will enable the identification and characterization of potential regulators and targets of Lck and thereby greatly facilitate studies which will clarify its role in T cell signal transduction.

Generation of neomucosa in vivo by transplantation of dissociated rat postnatal small intestinal epithelium.

A novel method to study the generation of rat small intestinal mucosa, by transplantation of disaggregated postnatal rat small intestinal epithelium is described. Cellular aggregates, comprised of epithelium with attached proliferative cells and closely associated stromal tissue, were isolated from postnatal rat small intestine by enzymatic digestion, then grafted immediately to the subcutaneous plane of adult recipients. On graft retrieval after 14 days, 39% of cellular transplants to nude mice, and 84% of cellular transplants to inbred rats had developed into small intestine-like structures. These structures were comprised of a circumferential layer of epithelium surrounding a central mucin filled lumen. This neomucosal layer exhibited well formed crypts and villi, and contained all epithelial stem cell lineages i.e. absorptive enterocytes, goblet cells, Paneth's cells and entero-endocrine cells. Proliferative activity within this neomucosa was confined to crypt regions as in normal postnatal small intestine. Developmental maturation within the regenerated neomucosa was demonstrated by organotypic morphogenesis, i.e. formation of mature crypts and villi, and progressive cytodifferentiation with increased numbers of goblet cells, entero-endocrine cells and Paneth's cells. Altered patterns of brush border enzyme expression further confirmed a temporal progression of development within neomucosal enterocytes. It is concluded that after "extensive" mucosal disaggregation, postnatal small intestinal epithelial progenitor cells retain the capacity for organotypic regeneration of neomucosa when transplanted to ectopic sites in adult recipients. These small aggregates of epithelium and stroma are capable of generating the topographical signals necessary for the three dimensional regeneration of this tissue. Furthermore, the multipotent generative potential of the stem cells within these cellular aggregates is maintained with production of all progeny.

Heparin stimulates the proliferation of intestinal epithelial cells in primary culture.

Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminoglycans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysaccharides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration.

Identification of a signaling complex involving CD2, zeta chain and p59fyn in T lymphocytes.

CD2 is a cell surface receptor molecule which has been implicated in cell-cell adhesion and signaling functions in T lymphocytes and natural killer cells. The mechanism by which extracellular stimuli induce CD2-regulated signal transduction events is largely unknown. However, there is increasing evidence that in cells of hematopoietic origin several receptor-mediated signaling mechanisms involve transmembrane polypeptides related to the CD3 zeta chain and the activation of protein tyrosine kinases. We have therefore investigated the potential involvement of zeta chain and src family protein tyrosine kinases in signal transduction pathways initiated by CD2. Using in vitro kinase assays on CD2 immunoprecipitates from detergent lysates of T lymphocytes, we identified a complex consisting of CD2, zeta chain and the src family kinases p59fyn and p56lck. Furthermore, using double indirect immunofluorescence combined with capping techniques, we have revealed such complexes in viable T lymphocytes. These findings provide evidence for a multimolecular signaling complex consisting of at least CD2, zeta chain and p59fyn in T lymphocytes and suggest a critical role for this complex in the initiation of CD2-mediated cellular activation by regulating the activation of intracellular signaling molecules.

Cytokeratin expression in epithelial cells isolated from the crypt and villus regions of the rodent small intestine.

Many physiological and structural features of epithelium in the small intestine are regulated during their transit from the crypt base to the villus tip. This crypt-villus axis is an important model for the study of the regulation of cell proliferation and differentiation. We have investigated the expression of cytokeratins in purified epithelial cells from the proliferative (crypt) and differentiated (villus) regions of this tissue. Three polypeptides were identified (cytokeratins 8, 18 and 19) as well as a fourth, 46 kDa polypeptide with similar electrophoretic characteristics to the recently identified cytokeratin 20. The distribution of these molecules was found to vary along the crypt-villus axis, with cytokeratin 18 being restricted to the proliferative crypt and cytokeratins 8 and 19 demonstrating more uniform distributions. The 46 kDa component was found to be expressed predominantly within the villus epithelium. Although there is no substantial evidence of a direct role for cytokeratins in the process of epithelial differentiation, these data suggest that differential expression of cytokeratins is associated with changes in intestinal epithelial differentiation.

Progressive morphogenesis in vivo after transplantation of cultured small bowel epithelium.

An experimental model for the primary culture and transplantation of late foetal rat small intestinal epithelium is described. Multicellular aggregates of mucosal epithelium containing pre-crypt proliferative cells were isolated from 20-day foetal rat intestine by enzymatic disaggregation. Cellular aggregates, which we refer to as "epithelial organoids," attached readily in culture, proliferated, and spread to produce coalescing colonies within 10 days. Enterocytes were maintained in culture for 3 days, removed as cell sheets, and incubated overnight with foetal mesenchyme. Fourteen recombinant preparations were then grafted to the renal subcapsular space of adult nude mice. Four of six grafts retrieved after 1 wk had developed. Histology demonstrated the formation of simple tubular structures lined by a polarized columnar epithelium. At 14 days, two of eight grafts had developed and demonstrated temporal progression of morphogenesis. Histology showed rudimentary crypts and villi lined by different epithelial cell types, including enterocytes and goblet cells. Small bowel proliferative cells within "epithelial organoids" from 20-day foetal intestine, may be maintained in primary culture for up to four days. After short term primary culture, these proliferative cells retain the capacity for progressive organotypic morphogenesis and pluripotent cytodifferentiation, after transplantation to adult recipients.