Keiko Ishino

Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C.

ABSTRACT Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the α, δ, η, and ζ isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the α, δ, and η isoforms. Overexpression of the η isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The η-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the η-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the η isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative η isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1α,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the δ isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the α and ζ isoforms did not. These findings indicate that the η and δ isoforms of PKC are involved crucially in squamous cell differentiation.

PKCη associates with cyclin E/cdk2/p21 complex, phosphorylates p21 and inhibits cdk2 kinase in keratinocytes

PKC is activated on the cell membrane by phospholipids, thereby transducing signals to intracellular pathways. We provide here another function of PKC, namely, regulating cell cycle by interaction with the cyclin E/cdk2/p21 complex. Among the 10 isoforms of PKC, PKCη is predominantly expressed in squamous cell epithelia and induces terminal differentiation of keratinocytes. PKCη that is endogenously expressed or overexpressed was found to associate with the cyclin E/cdk2/p21 complex in keratinocytes of mice and humans. Requirement of a possible adaptor protein to the binding was suggested by the reconstitution of PKCη and the cyclin E/cdk2/p21 complex which were prepared from human keratinocytes or Sf9 insect cells. Colocalization of PKCη with cdk2 and cyclin E was observed in the cytoplasm, particularly in the perinuclear region. p21 was phosphorylated in the complex in a PKC-activator dependent manner. Association of PKCη with cdk2 resulted in marked inhibition of cdk2-kinase activity when measured by phosphorylation of Rb. Dominant negative PKCη associated with the cyclin E/cdk2/p21 complex, but caused a little inhibition of cdk2 kinase activity. Among the known regulatory mechanisms of cdk2 activity, dephosphorylation of Thr160 was demonstrated.

Specific decrease in the level of Hic-5, a focal adhesion protein, during immortalization of mouse embryonic fibroblasts, and its association with focal adhesion kinase

Hic‐5 is a paxillin homologue with four LIM domains in its C‐terminal region, localized mainly in focal adhesions in normal fibroblasts. Hic‐5 is also known to associate with focal adhesion kinase (FAK) or the related CAKβ, and with vinculin. In the present study, we examined changes in Hic‐5 and paxillin protein levels in primary mouse embryo fibroblasts (MEF) during mortal and immortal stages. The Hic‐5 level was markedly decreased when cells became immortalized, whereas that of paxillin was increased. The vinculin level was not changed significantly. Hic‐5 was mainly localized in focal adhesion plaques of mortal MEF but was localized in the nuclear periphery in the immortalized MEF; the number of focal adhesion plaques was decreased in these cells. Mouse Hic‐5 contains three LD domains in its N‐terminal half, and the first LD domain (LD1) appears to be involved in interaction with FAK. However, this interaction was not essential for recruitment of Hic‐5 to focal adhesions, since its subcellular localization was similar in FAK−/− cells. Forced expression of Hic‐5 decreased colony forming ability of MEF from FAK+/+ mice, but not of FAK−/− cells. These observations suggested the involvement of Hic‐5 in determination of cellular proliferative capacity in collaboration with other cytoskeletal components. J. Cell. Biochem. 76:411–419, 2000.

Phorbol Ester‐induced G1 Arrest in BALB/MK‐2 Mouse Keratinocytes Is Mediated by δ and η Isoforms of Protein Kinase C

We investigated the possible negative regulation of the cell cycle by protein kinase C (PKC) isoforms in synchronously grown BALB/MK‐2 mouse keratinocytes, in which PKC isoforms were overexpressed by using the adenovirus vector Ax. Cells at the G1/S boundary of the cell cycle were the most sensitive to the inhibitory effect of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a PKC agonist, resulting in G1 arrest. TPA‐induced inhibition of DNA synthesis was augmented by overexpression of the η and δ isoforms, but rescued by the dominant‐negative and antisense η isoforms. In contrast, the α and ζ isoforms showed no effect on DNA synthesis with or without TPA treatment. Immunoblotting indicated cell cycle‐dependent expression of the η isoform, being highest in cells at the G1/S boundary. The present study provides evidence that the η and δ isoforms of PKC are involved in negative regulation of cell cycle at the G1/S boundary in mouse keratinocytes.

Effect of interventions by an antimicrobial stewardship team on clinical course and economic outcome in patients with bloodstream infection.

BACKGROUND Bloodstream infections (BSIs) represent one of the most severe and clinically important conditions in the hospital setting. We have organized an interdisciplinary antimicrobial stewardship team (AST) at our hospital and performed consultations focusing on BSI patients since 2013. This study aimed to evaluate the impact of AST interventions on the diagnosis, treatment, and clinical outcomes of BSI patients. METHODS We conducted a retrospective quasi-experimental study of BSI patients at a single Japanese university hospital. AST provided recommendations to attending physicians regarding appropriate diagnosis, therapy, and management of BSI patients after reviewing medical charts. RESULTS We identified a total of 308 cases of BSI from January to December, 2012 (pre-intervention group) and 324 cases of BSI from April, 2013 to March, 2014 (post-intervention group). No significant differences in the in-hospital mortality or 30-day mortality rates were observed between both the groups. Inappropriate therapy was initiated in a significantly lower proportion of patients in the post-intervention group (18.5% vs. 11.4%; P = 0.012). Multivariate analysis confirmed that inappropriate therapy was significantly associated with in-hospital mortality (odds ratio, 2.62; 95% confidence interval, 1.42-4.82; P = 0.002). CONCLUSIONS An interdisciplinary AST intervention approach decreases the use of inappropriate therapy and may improve clinical outcomes in BSI patients.

More accurate measurement of vancomycin minimum inhibitory concentration indicates poor outcomes in meticillin-resistant Staphylococcus aureus bacteraemia

Meticillin-resistant Staphylococcus aureus (MRSA) is an important pathogen associated with community-acquired and nosocomial infections. The aim of this study was to validate the vancomycin (VAN) minimum inhibitory concentration (MIC) and administration of VAN that may affect the prognosis of patients with MRSA bacteraemia. In total, 140 clinical MRSA strains from blood cultures were collected from January 2009 to December 2013 at a university hospital in Tokyo (Japan). Patient background, their clinical situation and the susceptibility of isolates to anti-MRSA agents in all cases were reviewed, and factors contributing to 30-day mortality were analysed. Susceptibility to anti-MRSA agents was measured by a microdilution susceptibility testing method. The VAN MIC was further evaluated at 0.25 μg/mL intervals from 0.5 μg/mL to 2.0 μg/mL. Multiple logistic regression analysis revealed a 4-fold increase in mortality of patients with a VAN MIC ≥1.5 μg/mL [odds ratio (OR)=3.952, 95% confidence interval (CI) 1.471-10.614; P=0.006]. A one-score increase in the Charlson co-morbidity index resulted in a 1.2-fold increase in the risk of death (OR=1.199, 95% CI 1.054-1.364; P=0.006). However, no significant difference was found in the ratio of the VAN 24-h area under the concentration-time curve to MIC between VAN MIC ≥1.5 μg/mL and <1.5 μg/mL. A significant increase in the MICs of teicoplanin and daptomycin was observed in strains with high VAN MICs. For patients with high VAN MICs, administration of these anti-MRSA antibiotics may have a poor outcome owing to cross-resistance.

Epidemiology and risk factors for mortality in bloodstream infections: A single-center retrospective study in Japan

Background: Few published data are available on the morbidity and mortality of bloodstream infections (BSIs) in Japan. We sought to investigate the epidemiology of BSIs, the involvement of antimicrobial resistance, and the factors that influence patient prognosis. Methods: This single‐center study retrospectively evaluated patients who were found to have positive blood cultures at a tertiary teaching hospital between January 2012 and December 2016. Results: A total of 2,105 patients with BSIs were included; 1,786 survived and 319 died, and the 30‐day mortality rate was 15.2% over the 5‐year study period. BSIs caused by yeasts were independently associated with 30‐day mortality. The 30‐day mortality rate of BSIs caused by extended‐spectrum beta lactamase–producing gram‐negative bacteria was significantly higher than that of BSIs caused by nonproducing bacteria. Discussion: The differences in mortality may be caused by differences in the distribution of pathogens and in the delivery of health care. Conclusions: This study reported epidemiology and antimicrobial resistance data of BSIs in Japan and identified several risk factors associated with 30‐day mortality. National surveillance of BSIs is required in Japan for comparison with other countries.