Emotional intelligence and coping strategies as determinants of quality of life in depressed patient-caregiver dyads: An actor-partner interdependence analysis.

OBJECTIVE: Patients with major depressive disorder (MDD) and their natural caregivers experience major lifestyle difficulties. Little is known concerning dyadic (i.e., patient and natural caregiver) characteristics' impact on quality of life. In a sample of depressed patient-caregiver dyads, we examined quality of life (QoL) levels compared with the general population and whether QoL is influenced by emotional intelligence (EI) and coping strategies using the actor-partner interdependence model (APIM). METHODS: This cross-sectional study involved 79 patient-caregiver dyads. The self-reported data, completed by patients and their primary caregivers, included QoL (SF-36), EI (TEIQue-SF) and coping strategies (BriefCope). The QoL of patients and caregivers was compared with 158 French age-sex-matched healthy controls. The dyadic interactions were analyzed using structural equation modeling. RESULTS: Patients and their caregivers experienced lower QoL levels than French age-sex-matched controls. The EI findings showed actor (degree to which the person's EI was associated with his/her own QoL) and partner (degree to which the person's EI was associated with QoL of the other member of the dyad) effects for patients and caregivers. The coping strategies (i.e., problem solving, positive thinking, avoidance and social support) revealed only actor effects. CONCLUSION: QoL is seriously impaired in depressed patients and their primary caregivers and is associated with EI and coping strategies. Targeted interventions focusing on EI and coping strategies could be offered to improve QoL in dyads.

[Multidisciplinary approach as a model for detection and monitoring of psychiatric morbidity in patients treated with interferon and ribavirin]. / Abordaje multidisciplinar como modelo de detección y seguimiento de la moribilidad psiquiátrica en pacientes en tratamiento con interferón de la moribilidad psiquiátrica en pacientes en tratamiento con interferón y ribavirina.

PURPOSE: We aimed to describe the incidence of psychiatric disorders in a cohort of HCV infected patients treated with interferon and ribavirin, and their impact on treatment adherence and viral response rate (SVR). MATERIALS AND METHODS: Retrospective analysis of a cohort of HCV patients visited at an outpatient pharmacy service (OPS). We included all adult patients monoinfected with HCV who had initiated treatment in 2010. Monitoring of psychiatric disorders was assessed at weeks 0, 4, 12, 24, 48, and 72 through the self-administered questionnaires Hospital Anxiety and Depression Scale (HADS) and General Health Questionnaire (Goldberg).Adherence to treatment was assessed by counting of drug dispensations and patient reporting and drug exposure with the 80/80/80 rule. Virologic response was determined by the physician according to standard definitions. RESULTS: Among 76 included patients, 19 (25%) had a preexisting psychiatric disorder. The incidence of confirmed psychiatric disorders was 33% (n=25),with a peak of abnormal results in the tests by week 12. Overall, 43% of patients achieved an SVR. There were not significant differences between strict adherence and SVR in patients with or without medically confirmed disorders(96.0% vs 96,8%; p = NS) and SVR (39% vs 52%; p = NS], respectively. CONCLUSIONS: Psychiatric side effects had no effect on adherence to treatment nor on attainment of SVR. Multidisciplinary monitoring provided during the treatment of hepatitis C can contribute to early detection and management of psychiatric disorders and to improve integrated patient care.

Objetivo: Describir la experiencia recogida durante el programa multidisciplinar,y en particular describir la incidencia de los trastornos psiquiátricosen pacientes con hepatitis C crónica (HCC) durante el tratamiento coninterferón y ribavirina, y determinar la adherencia al tratamiento antiviraly la respuesta viral sobtenida (RVS).Material y métodos: Estudio observacional, descriptivo y retrospectivo realizadoa partir de los datos recogidos durante el programa de dispensaciónambulatoria de tratamiento antiviral.Se incluyó a todos los pacientes monoinfectados por el virus hepatitis C(VHC) que iniciaron tratamiento durante el 2010. El cribaje de lostrastornos psiquiátricos se realizó mediante el Hospital Anxiety-DepressionScale (HADS) y el General Health Questionnaire (Goldberg) las semanas 0,4, 12, 24, 48 y 72. La adherencia se evaluó mediante el recuento de dispensacionesy de la medicación sobrante del paciente y la exposición al fármacosegún la regla 80/80/80. La respuesta virológica se determinó por elmédico responsable de acuerdo a las definiciones estándar.Resultados: Se incluyeron 76 pacientes, 19 (25%) de los cuales teníanantecedentes psiquiátricos. La incidencia de trastornos psiquiátricos fue del33% (n = 25). El pico de resultados anormales en los test fue en la semana12. El 43% alcanzó RVS, sin diferencias entre ambos grupos (p > 0,05). Laadherencia (96,0% y 96,8%, p > 0,05) y RVS (39% y 52%, p > 0,05)fueron similares en ambos subgrupos con y sin trastornos.Conclusiones: Los trastornos psiquiátricos no tuvieron impacto en la adherenciay la RVS. El seguimiento multidisciplinar durante el tratamiento de la hepatitisC crónica (HCC) puede contribuir a la detección precoz y manejo de lostrastornos psiquiátricos y a mejorar la atención integral del paciente.

Abordaje multidisciplinar como modelo de detección y seguimiento de la morbilidad psiquiátrica en pacientes en tratamiento con interferón y ribavirina / Multidisciplinary approach as a model for detection and monitoring of psichiatric morbidity in patients treated with interferon and ribavirin

Objetivo: Describir la experiencia recogida durante el programa multidiscplinar, y en particular describir la incidencia de los trastornos psiquiátricos en pacientes con hepatitis C crónica (HCC) durante el tratamiento con interferón y ribavirina, y determinar la adherencia al tratamiento antiviraly la respuesta viral sobtenida (RVS). Material y métodos: Estudio observacional, descriptivo y retrospectivo realizado a partir de los datos recogidos durante el programa de dispensación ambulatoria de tratamiento antiviral. Se incluyó a todos los pacientes monoinfectados por el virus hepatitis C(VHC) que iniciaron tratamiento durante el 2010. El cribaje de los trastornos psiquiátricos se realizó mediante el Hospital Anxiety-Depression Scale (HADS) y el General Health Questionnaire (Goldberg) las semanas 0,4, 12, 24, 48 y 72. La adherencia se evaluó mediante el recuento de dispensaciones y de la medicación sobrante del paciente y la exposición al fármaco según la regla 80/80/80. La respuesta virológica se determinó por el médico responsable de acuerdo a las definiciones estándar. Resultados: Se incluyeron 76 pacientes, 19 (25%) de los cuales tenían antecedentes psiquiátricos. La incidencia de trastornos psiquiátricos fue del33% (n = 25). El pico de resultados anormales en los test fue en la semana12. El 43% alcanzó RVS, sin diferencias entre ambos grupos (p > 0,05). La adherencia (96,0% y 96,8%, p > 0,05) y RVS (39% y 52%, p > 0,05)fueron similares en ambos subgrupos con y sin trastornos. Conclusiones: Los trastornos psiquiátricos no tuvieron impacto en la adherencia y la RVS. El seguimiento multidisciplinar durante el tratamiento de la hepatitis C crónica (HCC) puede contribuir a la detección precoz y manejo de los trastornos psiquiátricos y a mejorar la atención integral del paciente

Purpose: We aimed to describe the incidence of psychiatric disorders in a cohort of HCV infected patients treated with interferon and ribavirin, and their impact on treatment adherence and viral response rate (SVR).Materials and methods: Retrospective analysis of a cohort of HCV patients visited at an outpatient pharmacy service (OPS). We included all adult patients monoinfected with HCV who had initiated treatment in 2010. Monitoring of psychiatric disorders was assessed at weeks 0, 4, 12, 24, 48, and72 through the self-administered questionnaires Hospital Anxiety and Depression Scale (HADS) and General Health Questionnaire (Goldberg).Adherence to treatment was assessed by counting of drug dispensations and patient reporting and drug exposure with the 80/80/80 rule. Virologic response was determined by the physician according to standard definitions. Results: Among 76 included patients, 19 (25%) had a preexisting psychiatric disorder. The incidence of confirmed psychiatric disorders was 33% (n=25),with a peak of abnormal results in the tests by week 12. Overall, 43% of patients achieved an SVR. There were not significant differences between strictad herence and SVR in patients with or without medically confirmed disorders(96.0% vs 96,8%; p = NS) and SVR (39% vs 52%; p = NS], respectively. Conclusions: Psychiatric side effects had no effect on adherence to treatment nor on attainment of SVR. Multidisciplinary monitoring provided during the treatment of hepatitis C can contribute to early detection and man-agement of psychiatric disorders and to improve integrated patient care

Safety, tolerability, pharmacokinetics, and antiviral activity of GSK2336805, an inhibitor of hepatitis C virus (HCV) NS5A, in healthy subjects and subjects chronically infected with HCV genotype 1.

GSK2336805 is an orally bioavailable hepatitis C virus (HCV) inhibitor working through an NS5A-mediated mechanism. This first-time-in-human study was conducted to assess the safety, tolerability, pharmacokinetics, metabolism, and efficacy of GSK2336805 in healthy subjects and subjects infected with HCV genotype 1. We performed a three-part, randomized, double-blind, placebo-controlled study in 46 healthy subjects and 23 HCV-infected subjects. After an overnight fast, healthy subjects received GSK2336805 as 10 mg, 30 mg, 30 mg plus food, and 60 mg in a single dose and 10 mg (7 days), 30 mg (7 days), and 75 mg (14 days) in a once-daily multiple dose. Subjects with HCV received GSK2336805 as a 1- to 120-mg single dose. In subjects with HCV, reductions in HCV RNA were observed within 4 h and a single dose of GSK2336805 of ≥10 mg resulted in a statistically significant ≥2-log reduction in HCV RNA compared with placebo at 24 h postdose. GSK2336805 was readily absorbed in all subjects, and the half-life (t1/2) was suitable for once-daily dosing. Administration of GSK2336805 with food had no effect on plasma GSK2336805 exposure; however, absorption was delayed, with a median tmax (time to maximum concentration of drug in serum) of 4.5 versus 2.0 h. Twenty subjects who received GSK2336805 experienced mild to moderate adverse events; none were serious. GSK2336805 was well tolerated and exhibited rapid, significant antiviral activity after a single dose in HCV-infected subjects. These results support the conduct of further studies evaluating GSK2336805 administered once daily for longer durations in combination with peginterferon, ribavirin, and other direct-acting antivirals. (This study has been registered at ClinicalTrials.gov under registration no. NCT01277692.).

Cytoplasmic p27 is oncogenic and cooperates with Ras both in vivo and in vitro.

p27(Kip1) (p27) can have opposing roles during malignant transformation depending on cellular context: on one hand it functions as a tumor suppressor by inhibiting cyclin-cyclin-dependent kinase (CDK) activity in the nucleus and on the other it may adopt an oncogenic role that is less well understood. To gain further insight into the roles played by p27 during tumorigenesis, we compared the susceptibility with urethane-induced tumorigenesis of two p27 mouse models, p27(-/-) and p27(CK-) knockin, in which p27 cannot bind or inhibit cyclin-CDKs. In this K-Ras-driven tumorigenesis model, p27(CK-) mice had an increase in both tumor number and aggressiveness compared with p27(-/-), indicating a cooperation between p27(CK-) and activated Ras. In the lung, increased tumorigenesis was associated with cytoplasmic localization of p27(CK-) and bronchiolaveolar stem cell amplification. The ability of p27(CK-) to cooperate with other oncogenes was not universal. When c-Myc was used as a transforming agent, p27 status became irrelevant and c-Myc was equally potent in transforming p27(+/+), p27(-/-) and p27(CK-) cells. In fact, c-Myc induced the degradation of wild-type p27 via the Skp-Cullin-F-box (SCF)-Skp2 pathway. In contrast, p27(CK-) levels were not affected by c-Myc expression, as p27(CK-) is insensitive to Skp2-mediated degradation because of its inability to bind cyclin E/CDK2. However, in presence of c-Myc, p27(CK-) remained mostly nuclear, providing an explanation for its inability to cooperate with Myc during transformation. Thus, we propose that the p27(CK-) protein needs to be localized in the cytoplasm in order to function as an oncogene, otherwise it just behaves similar to a null allele.

Responsiveness of human monocyte-derived dendritic cells to thimerosal and mercury derivatives.

Several cases of skin sensitization have been reported following the application of thimerosal, which is composed of ethyl mercury and thiosalicylic acid (TSA). However, few in vitro studies have been carried out on human dendritic cells (DCs) which play an essential role in the initiation of allergic contact dermatitis. The aim of the present study was to identify the effect of thimerosal and other mercury compounds on human DCs. To address this purpose, DCs derived from monocytes (mono-DCs) were used. Data show that thimerosal and mercury derivatives induced DC activation, as monitored by CD86 and HLA-DR overexpression associated with the secretion of tumor necrosis factor alpha and interleukin 8, similarly to lipopolysaccharide and the sensitizers, 1-chloro-2,4-dinitrobenzene (DNCB) and nickel sulfate, which were used as positive controls. In contrast, TSA, the non-mercury part of thimerosal, as well as dichloronitrobenzene, a DNCB negative control, and the irritant, sodium dodecyl sulfate, had no effect. Moreover, oxidative stress, monitored by ROS induction and depolarization of the mitochondrial membrane potential, was induced by thimerosal and mercury compounds, as well as DNCB, in comparison with hydrogen peroxide, used as a positive control. The role of thiol oxidation in the initiation of mono-DC activation was confirmed by a pre-treatment with N-acetyl-l-cysteine which strongly decreased chemical-induced CD86 overexpression. These data are in agreement with several clinical observations of the high relevance of thimerosal in patch-test reactions and prove that human mono-DCs are useful in vitro tools for determining the allergenic potency of chemicals.

Withdrawal of TNF-alpha after the fifth day of differentiation of CD34+ cord blood progenitors generates a homogeneous population of Langerhans cells and delays their maturation.

Human cord blood CD34+ progenitors cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) generate a heterogeneous population of dendritic cells (DC), including Langerhans cells (LC). This combination of cytokines has been shown to be crucial for differentiation into LC. After day 5 of culture, TNF-alpha has been maintained in the medium in most studies despite the observation of spontaneous maturation of LC after day 12. Five-day samples of in vitro differentiated LC were cultured in parallel with or without TNF-alpha. The absence of TNF-alpha was shown to: (1) slow down proliferation without triggering apoptotic cell death, (2) enhance the percentage of LC, (3) delay or abrogate the expression of CD83, CD86, HLA-DR and CD208 molecules, and (4) maintain endocytosis by receptor and macropinocytosis. The withdrawal of TNF-alpha abrogated the spontaneous synthesis of matrix metalloproteinases. At day 12, TNF-alpha-deprived LC were less efficient in allogeneic T cell activation than LC cultivated with TNF-alpha. These data indicate that the suppression of TNF-alpha after day 5 maintains cells in an immature state and provides a population with 80% of LC at day 12.

Comparative studies on the secretion and activation of MMPs in two reconstructed human skin models using HaCaT- and HaCaT-ras-transfected cell lines.

Matrix metalloproteinases play an important role in tissue regeneration, wound healing and tumor invasion. Our previous studies have shown a higher motility of HaCaT-ras-transfected cells compared with HaCaT or normal human keratinocytes (NHK) in correlation with a higher secretion of MMP-2 (72 kDa) or MMP-9 (92 kDa), according to the medium used for cell cultures. Presently, the expression and activity of MMPs were investigated in two reconstructed skin models, using a dead de-epidermized dermis (DED) or a dermal substitute including living fibroblasts. In all experiments, MMP-9 was essentially secreted by NHK and to a greater extent by HaCaT cells. Its active form (86 kDa) was only detected in both reconstructed skin models according to keratinocyte differentiation. MMP-2 was mainly secreted by living fibroblasts included in the dermal substitute skin model. In this case, its activation was up-regulated when HaCaT cell lines were seeded onto the dermal substitute according to their culture at air/liquid interface as shown for MMP-9. The collagenase MMP-1 and stromelysin-1 (MMP-3), susceptible to activate pro-MMP-2 and -9, respectively, were detected in their inactive form by ELISA. MMP-1 was expressed in both models but MMP-3 required the presence of living fibroblasts. Their activities were not detected using specific fluorogenic substrates. In the skin equivalent model using HaCaT, the extensive secretion and activation of MMP-2 and MMP-9 could explain the defect observed in basal membrane reconstruction, suggesting a direct interaction of HaCaT with fibroblasts.

A functional update of the Escherichia coli K-12 genome.

BACKGROUND: Since the genome of Escherichia coli K-12 was initially annotated in 1997, additional functional information based on biological characterization and functions of sequence-similar proteins has become available. On the basis of this new information, an updated version of the annotated chromosome has been generated. RESULTS: The E. coli K-12 chromosome is currently represented by 4,401 genes encoding 116 RNAs and 4,285 proteins. The boundaries of the genes identified in the GenBank Accession U00096 were used. Some protein-coding sequences are compound and encode multimodular proteins. The coding sequences (CDSs) are represented by modules (protein elements of at least 100 amino acids with biological activity and independent evolutionary history). There are 4,616 identified modules in the 4,285 proteins. Of these, 48.9% have been characterized, 29.5% have an imputed function, 2.1% have a phenotype and 19.5% have no function assignment. Only 7% of the modules appear unique to E. coli, and this number is expected to be reduced as more genome data becomes available. The imputed functions were assigned on the basis of manual evaluation of functions predicted by BLAST and DARWIN analyses and by the MAGPIE genome annotation system. CONCLUSIONS: Much knowledge has been gained about functions encoded by the E. coli K-12 genome since the 1997 annotation was published. The data presented here should be useful for analysis of E. coli gene products as well as gene products encoded by other genomes.

Interim report on genomics of Escherichia coli.

We present a summary of recent progress in understanding Escherichia coli K-12 gene and protein functions. New information has come both from classical biological experimentation and from using the analytical tools of functional genomics. The content of the E. coli genome can clearly be seen to contain elements acquired by horizontal transfer. Nevertheless, there is probably a large, stable core of >3500 genes that are shared among all E. coli strains. The gene-enzyme relationship is examined, and, in many cases, it exhibits complexity beyond a simple one-to-one relationship. Also, the E. coli genome can now be seen to contain many multiple enzymes that carry out the same or closely similar reactions. Some are similar in sequence and may share common ancestry; some are not. We discuss the concept of a minimal genome as being variable among organisms and obligatorily linked to their life styles and defined environmental conditions. We also address classification of functions of gene products and avenues of insight into the history of protein evolution.

The pharmacokinetics and pharmacodynamics of GW395058, a peptide agonist of the thrombopoietin receptor, in the dog, a large-animal model of chemotherapy-induced thrombocytopenia.

GW395058, a PEGylated peptide agonist of the thrombopoietin receptor, stimulates megakaryocytopoiesis and has previously been shown to increase platelet counts in vivo. The pharmacokinetics and pharmacodynamics of GW395058 were characterized using a randomized, crossover study in a large-animal model (dog) of chemotherapy-induced thrombocytopenia. Nine beagle dogs received i.v. carboplatin (350 mg/m(2)) on day 0 and day 28. GW395058 (1.31 mg/kg) (n = 6) or vehicle control (n = 3) was administered on day 1 and day 29 either as an i.v. bolus or s.c. injection. After i.v. administration, peak concentrations of GW395058 occurred rapidly, while the half-life averaged approximately 56 h. Bioavailability (+/- standard deviation) of GW395058 given s.c. was 78.2% (20.9%). GW395058 (i.v. and s.c.) ameliorated the platelet nadir (p = 0.0086) and resulted in a shorter time to recovery compared to the control group. The mean nadir platelet counts following carboplatin administration were 197,000 cells/microl (80,000) for the i.v. GW395058-dose group, 183,000 cells/microl (72,000) for the s.c.-dose group and 71,000 cells/microl (38,000) for the vehicle-alone group. GW395058 reduced the thrombocytopenic effects of carboplatin in dogs. No GW395058-related adverse side effects were observed.

Expression of E-, P-, n-cadherins and catenins in human bladder carcinoma cell lines.

PURPOSE: Cadherins are cell surface glycoproteins that mediate Ca2+-dependent, homophilic cell-cell adhesion. The classical cadherins, E-, P- and N-cadherins, are known to self-associate from their extracellular domain, while their cytoplasmic domain interacts with either beta-catenin or plakoglobin (gamma-catenin), which in turn is bound to alpha-catenin that links the complex to the actin cytoskeleton. The aim of the present study was to analyze the expression of E-, P- and N-cadherins and catenins in human bladder carcinoma cells. MATERIALS AND METHODS: Five human bladder carcinoma cell lines, representing a variety of differentiation states, were grown in cell culture. We performed a cell aggregation assay, specific for biological cadherin activity. The expression of cadherins and catenins was analyzed by immunocytochemistry, Western blotting and RT-PCR. The interactions between cadherins and catenins were assessed by immunoprecipitation. RESULTS: We observed a reduced E-cadherin expression in the poorly differentiated and invasive-tumor derived cells. Interestingly, immunofluorescence study reveals the persistent localization of catenins at intercellular contacts in two E-cadherin deficient cell lines (T24 and TCCSUP) which yet exhibit an epithelial-like morphology and a calcium-dependent adhesive capacity. This suggests that other cadherin(s) are expressed in these both cell lines. P-cadherin, another epithelial cadherin, is expressed only in E-cadherin positive cells. On the other hand, N-cadherin is present at cell-cell borders in the very anaplastic cell lines, T24 and TCCSUP, and is able to link beta-catenin or plakoglobin. CONCLUSION: These results indicate that N-cadherin may participate in intercellular adhesion, while facilitating bladder tumorigenesis.

The disruption of adherens junctions is associated with a decrease of E-cadherin phosphorylation by protein kinase CK2.

The down-regulation of E-cadherin is a common event in carcinogenesis. Phosphorylation/dephosphorylation is one posttranscriptional process which may regulate intercellular junctions. Here we show that in okadaic acid-treated keratinocytes, E-cadherin expression is shifted from the membrane to the cytoplasm, preventing cells from forming aggregates. These changes of E-cadherin localization and function are associated with a decrease in its phosphorylation state. The decrease in E-cadherin phosphorylation was essentially detected in okadaic acid-treated cell lysates isolated from 0.5% Triton-soluble fraction and not in the Triton-insoluble fraction linked to the cytoskeleton, suggesting a role of E-cadherin phosphorylation in cell-cell interactions. E-cadherin was markedly phosphorylated by CK2, either the purified recombinant enzyme or the endogenous enzyme. Using specific CK2 inhibitors such as heparin and 5, 6-dichloro-1-beta-d-ribofuranosylbenzimidazole, endogenous CK2 was confirmed as the main enzyme phosphorylating E-cadherin. The decrease in E-cadherin phosphorylation by endogenous CK2 was not restored by the addition of purified CK2, confirming that it is not due to a defect in CK2 expression or to its reduced activity, but rather to the incapacity of CK2 to phosphorylate E-cadherin. The co-immunoprecipitation and colocalization of E-cadherin and CK2 suggests that CK2 may play a critical role in the maintenance of epidermis cohesion.

An ultrasensitive competitive binding assay for the detection of toxins affecting protein phosphatases.

An ultrasensitive assay is described for microcystin-LR and other substances (microcystins, nodularin, okadaic acid, calyculin A, tautomycin) which block the active site of protein phosphatases (PP) 1 and 2A. The assay is based on competition between the unknown sample and [125I]microcystin-YR for binding to the catalytic subunit of PP2A. The PP2A-bound [125I]microcystin-YR was stable (half-time of dissociation = 1.8 h), allowing non-bound [125I]microcystin-YR to be removed by Sephadex G-50 size-exclusion chromatography. Compared to current assays based on inhibition of protein phosphatase activity the present assay was more robust against interference (from fluoride, ATP, histone, and casein), and had an even better sensitivity. The detection limit was below 50 pM (2.5 fmol) for nodularin and microcystin-LR, and below 200 pM (10 fmol) for okadaic acid. The method was used successfully to detect extremely low concentrations of either microcystin or nodularin in drinking water or seawater, and okadaic acid in shellfish extract.

MultiFun, a multifunctional classification scheme for Escherichia coli K-12 gene products.

An enriched classification system for cellular functions of gene products of Escherichia coli K-12 was developed based on the initial classification by Riley. In the new classification scheme, MultiFun, cellular functions are divided into 10 major categories: Metabolism, Information Transfer, Regulation, Transport, Cell Processes, Cell Structure, Location, Extra-chromosomal Origin, DNA Site, and Cryptic Gene. These major categories are further sub-divided into a hierarchical scheme. Two thousand nine hundred twenty-two gene products of E. coli K-12 were assigned to one or more functions depending on the role they play in the cell. Functional assignments were made to 66% of E. coli gene products, ranging from 1 to 16 assignments per gene product. The expansion of cellular function categories and the assignment to more than one category (multifunction) provides a more complete description of the gene products and their roles and hence better reflects the functional complexity of organisms. We believe this classification system will be useful in the field of genome analysis, both for annotation purposes and for comparative studies. The functional classification scheme and the cellular function assignments made to E. coli gene products can be accessed from the web at the databases GenProtEC (http://genprotec.mbl.edu) and EcoCyc (http://www.ecocyc.org).

Pharmacokinetics and hematological effects of the PEGylated thrombopoietin peptide mimetic GW395058 in rats and monkeys after intravenous or subcutaneous administration.

GW395058, a potent PEGylated peptide human thrombopoietin receptor (HuTPOr) agonist in vitro, is being evaluated for the treatment of thrombocytopenia. GW395058 shares no sequence homology with TPO. In this report the pharmacokinetics and hematological effects of GW395058 in rats and monkeys are described. Doses eliciting thrombocytosis in rodents (2 or 10 microg/kg s.c.) produced insufficient plasma concentration data for pharmacokinetic parameter estimate calculations. At higher i.v. doses in rats (500, 1,000 or 2,000 microg/kg) serum t1/2 (half-life) values were >20 h, and the area under the concentration time curve increased proportionally with dose. In cynomolgus monkeys GW395058 plasma t1/2 values ranged from 37 to 68 h after s.c. or i.v. dosing, and similar values were observed in rhesus monkeys following s.c. dosing. Rat platelet counts increased following 2 (1.6-fold) or 10 microg/kg (fourfold) s.c. doses. Cynomolgus and rhesus monkey platelet counts did not change significantly at comparable s.c. doses, but did increase slightly (

Immunogenicity of thrombopoietin mimetic peptide GW395058 in BALB/c mice and New Zealand white rabbits: evaluation of the potential for thrombopoietin neutralizing antibody production in man.

Administration of exogenous proteins and peptides as therapeutics carries with it the potential for immune system recognition and the development of neutralizing antibodies to endogenous regulatory proteins. PEGylation of proteins typically reduces their immunogenicity in vivo. GW395058 is a PEGylated peptide thrombopoietin receptor (TPOr) agonist being evaluated for the treatment of chemotherapy-induced thrombocytopenia. Although GW395058 shares no homology with TPO, it does compete with TPO for binding to a common receptor, and a similarity in local structure could result in shared epitopes. Thus GW395058 could elicit TPO-neutralizing antibodies. In this study, we evaluated the immunogenicity of GW395058 in mice, the potential of rabbit antibodies elicited by immunizations with the non-PEGylated parent peptide AF15705 to cross-react with recombinant human (rHu) TPO, and the potential of mouse anti-rHuTPO antibodies elicited by repeated dosing with rHuTPO to cross-react with AF15705. GW395058-dosed mice failed to produce antibodies to AF15705 or rHuTPO. Mouse anti-rHuTPO did not cross-react with AF15705 and rabbit anti-AF15705 antibodies failed to cross-react with rHuTPO. GW395058 caused no immune-mediated lesions in mice, but rHuTPO suppressed megakaryocytopoiesis and caused B-lymphocyte hyperplasia in lymphoid tissues consistent with antigenic stimulation. These data suggest that the potential for an immune response to GW395058 in man would be low.

The up-regulation of vascular endothelial growth factor in mutated Ha-ras HaCaT cell lines is reduced by a farnesyl transferase inhibitor.

The induction of tumor angiogenesis is mediated in particular by an increased production of VEGF. As ras oncogene is implicated in tumorigenesis, the inhibition of farnesyl transferase activity has recently been developed. The purpose of this study was to evaluate whether expression of mutated Ha-ras oncogene is associated with an altered expression of VEGF in an in vitro model of human skin carcinogenesis and to appreciate the effect of a new farnesyl transferase inhibitor on this VEGF expression. The amounts of VEGF secreted by an HaCaT cell line and two cell clones (metastatic or not) obtained after mutated c-Ha-ras transfection were compared. Our findings showed that the release of VEGF is greater for HaCaT-ras than for HaCaT cells and could be down-regulated using a protein farnesyl transferase inhibitor, in a reversible and dose-dependent manner. These results confirm that the Ha-ras oncogene can contribute to tumor development and progression of epidermal tumors through neoangiogenesis and that farnesyl transferase inhibitors as anticancer drugs may be efficient for the reduction of skin tumor growth.

Ras-transfection up-regulated HaCaT cell migration: inhibition by Marimastat.

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-nis clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines.

Cell migration and MMP-9 secretion are increased by epidermal growth factor in HaCaT-ras transfected cells.

Mutated RAS oncoproteins and epidermal growth factor (EGF) are thought to contribute to the proliferative, invasive and metastatic properties of transformed cells. In the present study, we investigated the role of EGF in two H-ras transfected clones and compared it to that in the parental cell line, HaCaT and primary cultured keratinocytes. Our findings show that the motility on type I collagen, measured by the migration index, was similar for both the HaCaT cell line and normal human keratinocytes, whereas it was higher for the HaCaT-ras clones. These results suggest an involvement of the ras oncogene in the stimulation of cell migration. EGF in cell pretreatment or during the migration assay also caused an increase in migration of all the cells, but preserved the difference between HaCaT and HaCaT-ras. However, no significant difference in EGF-R expression was detected between normal cultured keratinocytes, HaCaT and HaCaT-ras cell lines with or without EGF pretreatment. Moreover, when the cells were stimulated with EGF, the MMP-9 activity was greatly increased in a dose-dependent manner in all the cells, and EGF stimulation particularly highlights the increased amount of MMP-9 in HaCaT-ras cells compared to HaCaT cells. In conclusion, EGF is able to enhance motility and to up-regulate MMP-9 activity in all cells, but with a higher impact in HaCaT-ras cells without an overexpression of EGF-R. As EGF acts in synergy with the H-ras mutation, they could be implicated in the local invasion by the HaCaT-ras clones.