KSR and CNK: two scaffolds regulating RAS-mediated RAF activation.

The RAS-RAF-MEK-extracellular-regulated kinase (RAS/ERK) pathway is a major intracellular route used by metazoan cells to channel to downstream targets a diverse array of signals, including those controlling cell proliferation and survival. Recent findings suggest that the pathway is assembled by specific scaffolding proteins that in turn regulate the efficiency, the location and/or the duration of signal transmission. Here, through the angle of studies conducted in Drosophila and C. elegans, we present two such proteins, the kinase suppressor of RAS (KSR) and connector enhancer of KSR (CNK) scaffolds, and highlight their implication in a novel mechanism regulating RAS-mediated RAF activation. Based on recent findings, we discuss the possibility that KSR, a RAF-like protein, does not solely act as a scaffold, but directly induces RAF catalytic function by a kinase-independent mechanism apparently shared by RAF-like proteins.

The Ras signaling pathway in Drosophila.

During Drosophila eye development, a Ras cascade mediates the decision between neuronal and non-neuronal differentiation of the R7 photoreceptor precursor. Recent genetic and molecular studies have identified a set of protein kinases as components of the Ras cascade and nuclear targets of the cascade, including Yan, Pointed, Jun, and Phyllopod. The Ras cascade functions in other Drosophila signal transduction pathways, eliciting a distinct response in each case, presumably through phosphorylation of specific transcription factors.

Pituitary pro-opiomelanocortin gene expression requires synergistic interactions of several regulatory elements.

The pro-opiomelanocortin (POMC) gene is expressed very early during pituitary development, before expression of the other pituitary hormone genes, growth hormone and prolactin, and before expression of the Pit-1/GHF-1 transcription factor which activates those genes. Thus, analysis of the POMC promoter should provide markers of the early stages of pituitary development at the time when cells are being committed to expression of one or the other pituitary hormone. We have previously localized the rat POMC promoter to a 543-bp 5'-flanking DNA fragment of the gene using transfection and transgenic mice experiments. We have now used mutagenesis and in vitro protein-DNA binding studies to define three domains of the promoter which have distinct and complementary activities. Within these domains which require each other for full activity, at least nine regulatory elements were defined by in vitro footprinting and replacement mutagenesis. Each element appeared equally important for promoter activity, as mutagenesis of any element had similar effect on promoter activity. Most of the elements bound different AtT-20 nuclear proteins in gel mobility shift experiments. Whereas only two elements appeared to be binding sites for the known transcription factors AP-1 and chicken ovalbumin upstream promoter, the seven other elements appeared to bind nuclear proteins with novel properties. Thus, in contrast to the predominant role of Pit-1/GHF-1 in transcription of the growth hormone and prolactin genes, the control of an early pituitary gene, POMC, appears to depend on the synergistic interaction of several regulatory elements which bind different nuclear proteins.

Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene.

Pro-opiomelanocortin (POMC)-expressing cells appear to be the first pituitary cells committed to hormone production. In this work, we have identified an element of the POMC promoter which confers cell-specific activity. This element did not exhibit any activity on its own and required at least one other element of the promoter to manifest its cell-specific activity. Fine mutagenesis of this element indicated that a CANNTG motif is responsible for activity. This E-box motif is typical of binding sites for helix-loop-helix (HLH) transcription factors; however, the POMC cell-specific E box cannot be replaced by other E boxes like the kappa E2 site of the immunoglobulin gene or a muscle-specific E box. Similar E boxes which are present in the insulin gene promoter were shown to contribute to the pancreatic specificity of the insulin promoter. However, E-box-binding proteins found in nuclear extracts from POMC-expressing AtT-20 cells and from insulin-expressing cells have different electrophoretic mobilities. The AtT-20 proteins were named CUTE (for corticotroph upstream transcription element-binding) proteins, and they were not found in any other cells. CUTE proteins have DNA-binding properties characteristic of HLH transcription factors. Overexpression of the dominant negative HLH protein Id or of the ubiquitous positive HLH factor rat Pan-2 decreased or augmented POMC promoter activity, respectively. These observations are consistent with the hypothesis that CUTE factors might be heterodimers. This hypothesis was further supported by antibody shift experiments and by abrogation of DNA binding in the presence of bacterially expressed Id protein. Thus, the cell-specific CUTE proteins and their binding site in the POMC promoter appear to be important determinants for cell specificity of this promoter. The requirement for HLH factors in POMC transcription also presents the possibility that these factors are involved in differentiation of pituitary cells, in analogy with the role of HLH factors in muscle development.

Identification of constitutive and ras-inducible phosphorylation sites of KSR: implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression.

Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, we have initiated studies investigating the effect of phosphorylation and protein interactions on KSR function. Here, we report the identification of five in vivo phosphorylation sites of KSR. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Further, we find that treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and that activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites did not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, we found that the biological effect of KSR varied dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functioned as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocked Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocked R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels.

Trends in the quality of care for Medicare beneficiaries admitted to the hospital with unstable angina.

OBJECTIVES: We sought to 1) determine the proportion of appropriate elderly patients admitted to the hospital with unstable angina who are treated with aspirin and heparin; 2) identify patient factors associated with the Agency for Health Care Policy and Research (AHCPR) guideline-based use of aspirin and heparin; and 3) compare practice patterns and patient outcomes before and after publication of the AHCPR guidelines. BACKGROUND: Improving the care of patients with unstable angina may provide immediate opportunities to mitigate the adverse consequences of unstable angina. However, despite the importance of this diagnosis, there is a paucity of information on the patterns of treatment and outcomes across diverse sites and recent trends in practice that have occurred, especially since the publication of the AHCPR practice guidelines. METHOD: We performed a retrospective cohort study using data created from medical charts and administrative files. The sample included 300 consecutive patients admitted to one of three Connecticut hospitals in the period 1993 to 1994 and 150 consecutive patients admitted in 1995 with a principal discharge diagnosis of unstable angina or chest pain. RESULTS: Of the 384 patients > or =65 years old who had no contraindications to aspirin on hospital admission, 276 (72%) received it. Of the 369 patients > or =65 years old who had no contraindications to heparin on admission, 88 (24%) received it. Among the 321 patients > or =65 years old who had no contraindications to aspirin at hospital discharge, 208 (65%) were prescribed it. When 1995 was compared with 1993 to 1994, the use of aspirin (odds ratio [OR] 2.3, 95% confidence interval [CI] 1.3 to 4.0) and heparin (OR 2.8, 95% CI 1.6 to 4.9) on hospital admission significantly increased, and the use of aspirin at discharge (OR 1.4, 95% CI 0.8 to 2.4) increased. Concomitantly, there was a significant reduction in 30-day readmission (OR 0.52, 95% CI 0.27 to 0.99). CONCLUSIONS: Our results indicate an improvement in the care and outcomes of elderly patients with unstable angina, but there remain opportunities for further improvement.

A genetic screen for modifiers of a kinase suppressor of Ras-dependent rough eye phenotype in Drosophila.

kinase suppressor of Ras (ksr) encodes a putative protein kinase that by genetic criteria appears to function downstream of RAS in multiple receptor tyrosine kinase (RTK) pathways. While biochemical evidence suggests that the role of KSR is closely linked to the signal transduction mechanism of the MAPK cascade, the precise molecular function of KSR remains unresolved. To further elucidate the role of KSR and to identify proteins that may be required for KSR function, we conducted a dominant modifier screen in Drosophila based on a KSR-dependent phenotype. Overexpression of the KSR kinase domain in a subset of cells during Drosophila eye development blocks photoreceptor cell differentiation and results in the external roughening of the adult eye. Therefore, mutations in genes functioning with KSR might modify the KSR-dependent phenotype. We screened approximately 185,000 mutagenized progeny for dominant modifiers of the KSR-dependent rough eye phenotype. A total of 15 complementation groups of Enhancers and four complementation groups of Suppressors were derived. Ten of these complementation groups correspond to mutations in known components of the Ras1 pathway, demonstrating the ability of the screen to specifically identify loci critical for Ras1 signaling and further confirming a role for KSR in Ras1 signaling. In addition, we have identified 4 additional complementation groups. One of them corresponds to the kismet locus, which encodes a putative chromatin remodeling factor. The relevance of these loci with respect to the function of KSR and the Ras1 pathway in general is discussed.

A screen for genes that function downstream of Ras1 during Drosophila eye development.

Cell-fate specification of the R7 photoreceptor cell is controlled by the sevenless receptor tyrosine kinase (SevRTK) and Ras1, the Drosophila homologue of mammalian H-ras, K-ras and N-ras oncogenes. An activated form of Ras1 expressed under control of the sevenless enhancer/promoter (sev-Ras1V12) induces production of supernumerary R7 photoreceptor cells, which causes the eye to become rough in appearance. To isolate mutations in genes functioning downstream of Ras1, we carried out a screen for dominant suppressors and enhancers of this rough eye phenotype. Approximately 850,000 mutagenized flies were screened, and 282 dominant suppressors and 577 dominant enhancers were isolated. Mutations in the Drosophila homologues of Raf, MEK, MAPK, type I Geranylgeranyl Transferase and Protein Phosphatase 2A were isolated, as were mutations in several novel signaling genes. Some of these mutant genes appear to be general signaling factors that function in other Ras1 pathways, while one seems to be more specific for photoreceptor development. At least two suppressors appear to function either between Ras1 and Raf or in parallel to Raf.

Plasmapheresis in a case of eosinophilia-myalgia syndrome with ascending polyneuropathy.

Eosinophilia-myalgia syndrome complicated by ascending polyneuropathy in a 40-year-old woman is described. High-dose intravenous steroids had no beneficial effect on the clinical course. Dramatic and rapid clinical improvement occurred with the use of plasmapheresis. The use of this therapeutic modality should be considered in patients with a similar clinical presentation.

Hydrophobic and fibrillar microporous polyetherurethane urea prosthesis: an ESCA study on the internal and external surfaces of explanted grafts.

The ESCA study gives a good qualitative and quantitative elemental analysis of internal and external surfaces of foreign materials. Microporous hydrophobic Mitrathane (a polyetherurethane urea) grafts were implanted as blood conduits in dogs for up to 6 months. Surface analysis of explanted grafts demonstrated the presence of different contaminants: sodium, chlorine, silicon, in patent grafts, i.e. those implanted for 1 month and less. The sulphur probably comes from the presence of proteins on the surface of the polymer and the high level of nitrogen is also protein-related. At 6 month implantation, the grafts were occluded and a decrease of proteins on the surface was observed. The values of N/C and O/C ratios are also reported. For the virgin material, these ratios correspond to the quantity of hard and soft segments; but, for the explanted grafts, these parameters are also influenced by the presence of proteins due to the Versaclean washing which did not wash away all the proteins on the surface of the polymer. The SEM photographs showed a certain degradation of polyurethane after 6 month of implantation. However, by ESCA study, it is difficult to compare the surface of virgin and explanted grafts because it is masked by the presence of proteins.

In vitro exposure of a novel polyesterurethane graft to enzymes: a study of the biostability of the Vascugraft arterial prosthesis.

The biostability of the Vascugraft arterial prosthesis, a porous synthetic graft made by a novel spinning process from a unique poly(ester urethane) polymer, has been studied by means of an in vitro enzyme incubation technique. Samples of the Vascugraft were exposed to buffered solutions of collagenase and pancreatin, as well as the buffer solutions alone, for periods of up to 100 days at 37 +/- 1 degrees C. On removal and after cleaning, a number of different analytic methods, including X-ray photoelectron spectroscopy for chemical analysis (ESCA), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), scanning electron microscopy (SEM), interference microscopy, moisture content and contact angle measurements, were used to examine the changes in chemical structure and surface morphology of the samples. During incubation in both enzymes the molecular weight of the polyurethane appeared to decrease in the presence of enzyme but increase in the presence of buffer. Further microphase separation in the polyurethane material developed during incubation in buffer solutions. Such changes in microstructure were associated with increased surface hydrophilicity, increased moisture content and a significant improvement in the extent of order and preferred orientation of the hard segment domains within the fibres. In the sampling depth of about 5 nm, both enzymes decreased the carbonate group content at the surface of the prosthesis to as little as 40% of their original values. The results from ATR-FTIR and DSC demonstrated that this phenomenon was limited primarily to the soft segment phase. While the Vascugraft prosthesis did exhibit some limited chemical modifications on exposure to concentrated enzyme solutions, nevertheless such changes were confined to the surface layer of the polyurethane microfibres. The importance and significance of those results will be more adequately determined by in vivo investigation.

Expanded polytetrafluoroethylene arterial prostheses in humans: chemical analysis of 79 explanted specimens.

The expanded polytetrafluoroethylene (ePTFE) vascular prostheses are widely used as small and medium diameter blood conduits when an autologous venous material is not available or is not suitable. The long-term performance of a prosthesis is dependent on several factors, including its healing characteristics and its stability in vivo. This study was undertaken to assess whether chemical degradation of ePTFE occurs when such arterial substitutes are implanted in humans. Seventy-nine ePTFE grafts excised for complications were analysed using the following techniques: measurement of the contact angle (theta), electron spectroscopy for chemical analysis (ESCA or XPS), Fourier transform infra-red spectroscopy (FTIR) and differential scanning calorimetry (DSC). The results were compared with those obtained from virgin ePTFE and virgin ePTFE washed prostheses. The measurement of the contact angle (theta) permits the comparison of the level of hydrophobicity of material after in vivo residency. The contact angles of explanted ePTFE grafts are greater than those of virgin ones but remain close to those of washed virgin prostheses. The ESCA method allowed investigation of the chemical changes which occur on the surface of ePTFE prostheses after implantation because of the low penetration of the X-ray (about 50 A). This study did not reveal any chemical degradation of the ePTFE with time of implantation for periods up to 6.5 yr. Changes in the surface composition were probably related to lipid and/or protein uptake. The FTIR spectroscopy provides information about the chemical composition of material. Compared with the virgin ePTFE prostheses, the FTIR spectra of explanted prostheses showed specific bands which are characteristic of lipid and/or protein absorptions. The bulk properties of ePTFE studied by DSC did not show any significant changes with time of implantation. It is concluded that ePTFE grafts remain stable in vivo for periods up to 6.5 yr.

Morphological, physical and chemical evaluation of the Vascugraft arterial prosthesis: comparison of a novel polyurethane device with other microporous structures.

In this study the morphology, physical properties, surface chemical characteristics and microstructure of the Vascugraft arterial prosthesis have been investigated. This is a novel microporous polyurethane device, recently developed by the company Braun-Melsungen AG in Germany for use as a small calibre arterial substitute. This comparative study included two other synthetic grafts: the Mitrathane prosthesis, a hydrophilic prototype polyetherurethane urea graft with closed internal pores, and the commercially successful expanded polytetrafluoroethylene reinforced Goretex prosthesis with an open microporous structure. The Vascugraft prosthesis contains a network of fused microfibres of varying thickness and orientation which provide open and communicating pores similar in size to those in the Goretex material. In addition, they extend from one side of the graft wall to the other. As well as having superior longitudinal and radial compliance to the reinforced Goretex device, the Vascugraft prosthesis has more than adequate bursting and suture retention strengths. Through the use of contact angle measurements, electron spectroscopy for chemical analysis, Fourier transform infrared spectroscopy, differential scanning calorimetry and molecular weight analysis by size exclusion chromatography, the surface of the Vascugraft prosthesis has been shown to be uniquely hydrophobic, as well as containing carbonate groups within an aliphatic polyesterurethane polymer. In addition, variations in micro-phase separation structure of hard and soft segment domains between different sizes and batches of product are marginal. Because of the interesting physical and chemical properties, it is recommended that in vitro biocompatibility and biostability studies be undertaken prior to using the prosthesis in animal or clinical trials.

Maximum likelihood positioning in the scintillation camera using depth of interaction.

Specific effects of the depth of interaction (DOI) on the photomultiplier (PM) response in an Auger gamma camera were quantified. The method was implemented and tested on a Monte Carlo simulator with special care to the noise modeling. Two models were developed, one considering only the geometric aspects of the camera and used for comparison, and one describing a more realistic camera environment. In a typical camera configuration and 140-keV photons, the DOI alone can account for a 6.4-mm discrepancy in position and 12% in energy between two scintillations. Variation of the DOI can still bring additional distortions when photons do not enter the crystal perpendicularly such as in slant hole, cone beam and other focusing collimators. With a 0.95-cm crystal and a 30 degrees slant angle, the obliquity factor can be responsible for a 5.5-mm variation in the event position. Results indicate that both geometrical and stochastic effects of the DOI are definitely reducing the camera performances and should be included in the image formation process.

CP-225,917 and CP-263,114, novel Ras farnesylation inhibitors from an unidentified fungus. I. Taxonomy, fermentation, isolation, and biochemical properties.

During the course of our screening for squalene synthase inhibitors and Ras farnesylation inhibitors, a novel fungal culture was discovered to produce two structurally unique compounds, CP-225,917 and CP-263,114, as well as zaragozic acid A (squalestatin I). The two compounds are characterized by a bicyclo[4.3.1]dec-1,6-diene core plus two extended alkyl chains. CP-225,917 and CP-263,114 inhibit Ras farnesyl transferase from rat brain with IC50 values of 6 microM and 20 microM, respectively. CP-225,917 inhibits squalene synthase with an IC50 value of 43 microM and CP-263,114 with an IC50 of 160 microM. The producing organism, though not fully classified, exhibits the characteristics of a sterile Phoma species.

Community mental health myths and the fate of former hospitalized patients.

This paper addresses one of these important emerging problems--the fate of former hospitalized patients--and the policy issues that the problem raises. The discussion is derived from observations of the experience of the mental health system in the State of Hawaii, but the issues raised are of relevance to other states as well.

A collaborative project in Connecticut to improve the care of patients with acute myocardial infarction.

BACKGROUND: State-based peer review organizations (PROs) and individual hospitals are challenged to achieve their quality improvement (QI) goals with shrinking resources. In 1993-1994 the Connecticut PRO and 15 local hospitals generated a comparative QI database on acute myocardial infarction (AMI) care for 1,202 Medicare and non-Medicare patients discharged in 1992 and 1993. METHODS: A steering committee composed of hospital and PRO representatives was assembled to provide oversight. PRO staff developed a chart abstraction tool and trained hospital abstracters who collected and submitted data to the PRO for comparative analyses. Written feedback was provided to all hospitals and supplemented with onsite presentations when requested. Each hospital prepared a written QI plan based on its unique data profile. RESULTS: Opportunities for improvement were identified at all hospitals. The most commonly targeted areas for improvement included the use of thrombolytics at presentation, aspirin at presentation and at discharge, and beta blockers at discharge. Improvement interventions included staff education sessions, development of AMI critical paths and standing orders, and storage of appropriate medications in emergency departments. Self-report data from the hospitals indicate improvements in care. DISCUSSION: PROs and hospitals can augment their individual QI activities by working together to share data, resources, and lessons learned. Twenty-three hospitals are now collaborating with the Connecticut PRO on a similarly designed QI project aimed at improving the care of patients hospitalized with atrial fibrillation. This project includes a more formal means of communicating QI interventions.