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.

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.

Requirements for the membrane insertion of signal-anchor type proteins.

Proteins which are inserted and anchored in the membrane of the ER by an uncleaved signal-anchor sequence can assume two final orientations. Type I signal-anchor proteins translocate the NH2 terminus across the membrane while type II signal-anchor proteins translocate the COOH terminus. We investigated the requirements for cytosolic protein components and nucleotides for the membrane targeting and insertion of single-spanning type I signal-anchor proteins. Besides the ribosome, signal recognition particle (SRP), GTP, and rough microsomes (RMs) no other components were found to be required. The GTP analogue GMPPNP could substitute for GTP in supporting the membrane insertion of IMC-CAT. By using a photocrosslinking assay we show that for secreted, type I and type II signal-anchor proteins the presence of both GTP and RMs is required for the release of the nascent chain from the 54-kD subunit of SRP. For two of the proteins studied the release of the nascent chain from SRP54 was accompanied by a new interaction with components of the ER. We conclude that the GTP-dependent release of the nascent chain from SRP54 occurs in an identical manner for each of the proteins studied.

Structural requirements for membrane assembly of proteins spanning the membrane several times.

We have investigated the structural requirements for the biogenesis of proteins spanning the membrane several times. Proteins containing various combinations of topological signals (signal anchor and stop transfer sequences) were synthesized in a cell-free translation system and their membrane topology was determined. Proteins spanning the membrane twice were obtained when a signal anchor sequence was followed by either a stop transfer sequence or a second signal anchor sequence. Thus, a signal anchor sequence in the second position can function as a stop transfer sequence, spanning the membrane in the opposite orientation to that of the first signal anchor sequence. A signal anchor sequence in the third position was able to insert amino acid sequences located COOH terminal to it. We conclude that proteins spanning the membrane several times can be generated by stringing together signal anchor and stop transfer sequences. However, not all proteins with three topological signals were found to span the membrane three times. A certain segment located between the first and second topological signal could prevent stable membrane integration of a third signal anchor segment.

A tripartite structure of the signals that determine protein insertion into the endoplasmic reticulum membrane.

Multilineage colony stimulating factor is a secretory protein with a cleavable signal sequence that is unusually long and hydrophobic. Using molecular cloning techniques we exchanged sequences NH2- or COOH-terminally flanking the hydrophobic signal sequence. Such modified fusion proteins still inserted into the membrane but their signal sequence was not cleaved. Instead the proteins were now anchored in the membrane by the formerly cleaved signal sequence (signal-anchor sequence). They exposed the NH2 terminus on the exoplasmic and the COOH terminus on the cytoplasmic side of the membrane. We conclude from our results that hydrophilic sequences flanking the hydrophobic core of a signal sequence can determine cleavage by signal peptidase and insertion into the membrane. It appears that negatively charged amino acid residues close to the NH2 terminal side of the hydrophobic segment are compatible with translocation of this segment across the membrane. A tripartite structure is proposed for signal-anchor sequences: a hydrophobic core region that mediates targeting to and insertion into the ER membrane and flanking hydrophilic segments that determine the orientation of the protein in the membrane.

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.

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.

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.

Mouse monoclonal antibodies that specifically recognize an amino-terminal epitope of p56lck protein tyrosine kinase.

p56lck is a T cell-specific protein tyrosine kinase of the src family of proto-oncogenes which has been implicated in T cell signal transduction. Here we describe the production of mouse monoclonal antibodies directed against recombinant human p56lck purified from an E. coli expression system. The antibodies were characterized, by ELISA. RIA and immunoprecipitation of p56lck from T cell lysates. A specific epitope was revealed at the aminoterminus of the p56lck molecule by using Western blotting of deletion mutants and distinct domains of p56lck expressed in E. coli. Potential applications of the results obtained are discussed.

The 68 kDa protein of signal recognition particle contains a glycine-rich region also found in certain RNA-binding proteins.

Signal recognition particle (SRP) interacts with the signal sequence in nascent secretory and membrane proteins and directs them to the membrane of the endoplasmic reticulum. Membrane targeting is mediated by the 68 and the 72 kDa proteins of SRP. We have cloned and sequenced cDNA encoding the 68 kDa protein of canine signal recognition particle (SRP68). SRP68 is a basic protein comprised of 622 amino acid residues. Close to the amino terminus there is a glycine-rich region which SRP68 has in common with some RNA-binding proteins. SRP68 shares no detectable similarity to any of the proteins in data libraries.

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.

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.

Colonic mucosal replacement by syngeneic small intestinal stem cell transplantation.

A novel method of colonic mucosal replacement by transplantation of disaggregated small intestinal epithelium is described. Thirty-one inbred rats had the ascending colon isolated, and surgical mucosectomy was performed on the "free" loop. Epithelial cell aggregates were isolated from postnatal small intestine using collagenase and dispase digestion, then 20 microL of the cell suspension was "seeded" over the denuded colonic muscle of 25 recipient rats. Six control rats had surgical mucosectomy only. All loops were retrieved after 14 days for histologic examination. Stem cell lineage studies were used with selective staining protocols to identify enterocytes, goblet cells, entero-endocrine cells, and Paneth cells. A neomucosa with typical small bowel morphology including crypts and villi and all four stem cell lineages was regenerated by transplanted cells on the colonic muscle in 19 of 25 (76%) recipients. Control loops showed no epithelial regrowth confirming total mucosectomy. With appropriate stromal support, transplanted small intestinal stem cells have the capacity to re-epithelialize denuded colonic muscle with small bowel neomucosa.

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.

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.

Subfractionation of hepatic endosomes in Nycodenz gradients and by free-flow electrophoresis. Separation of ligand-transporting and receptor-enriched membranes.

The complexity of rat liver endosome fractions containing internalized radioiodinated asialotransferrin, asialo-(alkaline phosphatase), insulin and prolactin was investigated by using free-flow electrophoresis and isopycnic centrifugation in Nycodenz gradients. Two subfractions were separated by free-flow electrophoresis. Both subfractions contained receptors for asialoglycoprotein and insulin. Glycosyltransferase activities were associated with the more electronegative vesicles, whereas 5'-nucleotidase and alkaline phosphodiesterase activities were associated with the less electronegative vesicles. Three subfractions were separated on Nycodenz gradients. Two subfractions, previously shown to become acidified in vitro, contained the ligands. At short intervals after uptake (1-2 min), ligands were mainly in subfraction DN-2 (density 1.115 g/cm3), but movement into subfraction DN-1 (density 1.090 g/cm3) had occurred 10-15 min after internalization. Low amounts of glycosyltransferase activities were associated with subfraction DN-2, and 5'-nucleotidase and alkaline phosphodiesterase activities were mainly located in subfraction DN-1. The binding sites for asialoglycoproteins and insulin were distributed towards the higher density range in the Nycodenz gradients, thus indicating a segregation of receptor-enriched vesicles and those vesicles containing the various ligands 10-15 min after internalization. Electron microscopy of the subfractions separated on Nycodenz gradients indicated that whereas the ligand-transporting fractions consisted mainly of empty vesicles (average diameter 100-150 nm), the receptor-enriched component was more granular and smaller (average diameter 70-95 nm). The properties of the endosome subfraction are used to assign their origin to the regions of the endocytic compartment where ligand-receptor dissociation and separation occur.