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This study examines how retirees' formal and informal social participation change over time and investigates gendered differences. Seven waves of the Korean Longitudinal Study of Ageing track the frequency of social participation. A comparison group of employed individuals, weighted with coarsened exact matching, controls for age and time trends, and mixed model regressions estimate changes over time. Retirees show a gradual decline in the frequency of meeting friends and an abrupt decrease in the frequency of attending a social gathering, compared to their working peers. These trends are much stronger for men than women, and compound pre-existing gender differences in social participation. The more severe decline in the social participation of men is of great concern and points to the persistence of gendered employment structures into the retirement transition, putting Korean retirees at risk of social isolation and related health deterioration.

This article presents a new method for understanding and visualizing convolutional neural networks (CNNs). Most existing approaches to this problem focus on a global score and evaluate the pixelwise contribution of inputs to the score. The analysis of CNNs for multilabeled outputs or regression has not yet been considered in the literature, despite their success on image classification tasks with well-defined global scores. To address this problem, we propose a new inverse-based approach that computes the inverse of a feedforward pass to identify activations of interest in lower layers. We developed a layerwise inverse procedure based on two observations: 1) inverse results should have consistent internal activations to the original forward pass and 2) a small amount of activation in inverse results is desirable for human interpretability. Experimental results show that the proposed method allows us to analyze CNNs for classification and regression in the same framework. We demonstrated that our method successfully finds attributions in the inputs for image classification with comparable performance to state-of-the-art methods. To visualize the tradeoff between various methods, we developed a novel plot that shows the tradeoff between the amount of activations and the rate of class reidentification. In the case of regression, our method showed that conventional CNNs for single image super-resolution overlook a portion of frequency bands that may result in performance degradation.

Objectives: Social participation is important to the quality of life of older adults, especially widows. This is the first study to test whether older widows' formal and informal social participation rises or declines using multiple panel observations pre- and postbereavement. This article also tests the moderating effects of economic and marital satisfaction, depression, and husband's illness before death on these trends., Methods: Seven waves of the Korean Longitudinal Study of Ageing are used to track changes in 4 measures of social participation-meeting and contacting a child, meeting friends, and attending a formal group. A comparison group of married individuals, weighted with coarsened exact matching, controls for age and time trends. Mixed model regressions estimate the effects of widowhood over time., Results: Social participation shows little change before bereavement and rises significantly after bereavement for all measures. However, frequencies of meeting and contacting a child peak and decline early postbereavement, while meeting friends and attending a group show delayed but long-lasting effects. With regard to moderators, economic and marital satisfaction are positively associated with overall social participation levels but negatively associated with social participation postbereavement., Discussion: Increased social participation after bereavement underscores the resilience of widows and the social support they receive. However, differences in timing suggest that contact with children is gradually substituted with extrafamilial relationships in the long run. The negative moderating roles of economic and marital satisfaction point to a paradox where seemingly well-off individuals may be more vulnerable to widowhood., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Calcium ions play a pivotal role in cell proliferation, differentiation, and migration. Under basal conditions, the calcium level is tightly regulated; however, cellular activation by growth factors increase the ion level through calcium pumps in the plasma membrane and endoplasmic reticulum for calcium signaling. Orai1 is a major calcium channel in the cell membrane of non-excitable cells, and its activity depends on the stromal interaction molecule 1 (Stim1). Several groups reported that the store-operated calcium entry (SOCE) can be modulated through phosphorylation of Stim1 by protein kinases such as extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and p21-activated kinase (PAK). PKC is a protein kinase that is activated by calcium and diacylglycerol (DAG), but it remains unclear what role activated PKC plays in controlling the intracellular calcium pool., Objectives: Here, we investigated whether PKC-β controls intracellular calcium dynamics through Stim1., Methods: Several biochemical methods such as immune-precipitation, site directed mutagenesis, in vitro kinase assay were employed to investigate PKC interaction with and phosphorylation of Stim1. Intracellular calcium mobilization, via Stim1 mediated SOCE channel, were studied using in the presence of PKC activator or inhibitor under a confocal microscope., Results: Our data demonstrate that PKC interacts with and phosphorylates Stim1 in vitro. phosphorylation of Stim1 at its C-terminal end appears to be important in the regulation of SOCE activity in HEK293 and HeLa cells. Additionally, transient intracellular calcium mobilization assays demonstrate that the SOCE activity was inhibited by PKC activators or activated by PKC inhibitors., Conclusion: In sum, our data suggest a repressive role of PKC in regulating calcium entry through SOCE., (© 2022. The Author(s).)

This paper presents a new variational inference framework for image restoration and a convolutional neural network (CNN) structure that can solve the restoration problems described by the proposed framework. Earlier CNN-based image restoration methods primarily focused on network architecture design or training strategy with non-blind scenarios where the degradation models are known or assumed. For a step closer to real-world applications, CNNs are also blindly trained with the whole dataset, including diverse degradations. However, the conditional distribution of a high-quality image given a diversely degraded one is too complicated to be learned by a single CNN. Therefore, there have also been some methods that provide additional prior information to train a CNN. Unlike previous approaches, we focus more on the objective of restoration based on the Bayesian perspective and how to reformulate the objective. Specifically, our method relaxes the original posterior inference problem to better manageable sub-problems and thus behaves like a divide-and-conquer scheme. As a result, the proposed framework boosts the performance of several restoration problems compared to the previous ones. Specifically, our method delivers state-of-the-art performance on Gaussian denoising, real-world noise reduction, blind image super-resolution, and JPEG compression artifacts reduction. Our code and more details are available on our project page, https://github.com/JWSoh/VDIR.

Objectives: Ageism has increased over 200 years and costs the U.S. health care system $63 billion a year. While scholars agree on the consequences of ageism, there are disagreements on whether it is related to the demographics of aging, or society's cultural values. We test both hypotheses across 20 countries., Method: To circumvent the sampling limitations of survey studies, we used an 8-billion-word corpus, identified 3 synonyms with the highest prevalence-aged, elderly, old people-and compiled the top 300 words (collocates) that were used most frequently with these synonyms for each of the 20 countries. The resulting 6,000 collocates were rated on an ageism scale by 2 raters to create an ageism score per country. Cultural dimension scores-Power Distance, Individualism, Masculinity, Uncertainty Avoidance, and Long-term Orientation-were taken from Hofstede, and demographics-size and speed of population aging-came from the World Development Indicators., Results: Of the 20 countries, UK topped the ageism table, while Sri Lanka had the lowest ageism score. Multiple regression models showed that higher levels of masculinity and long-term orientation are associated with ageism, controlling for other cultural dimensions, demographics (size and speed of aging), and economics (GDP-per-capita)., Discussion: Our findings blunt the deterministic nature of ageism at the societal level. Demographics is only one side of the ageism coin, and the cultural side is equally, if not more important. This study lays the groundwork to tackle societal ageism-one of our generation's most pernicious threats., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Gerontological Society of America.)

Transmembrane Bax Inhibitor Motif-containing 6 (TMBIM6) is upregulated in several cancer types and involved in the metastasis. Specific downregulation of TMBIM6 results in cancer cell death. However, the TMBIM6 gene transcriptional regulation in normal and cancer cells is least studied. Here, we identified the core promoter region (-133/+30 bp) sufficient for promoter activity of TMBIM6 gene. Reporter gene expression with mutations at transcription factor binding sites, EMSA, supershift, and ChIP assays demonstrated that Sp1 is an essential transcription factor for basal promoter activity of TMBIM6. The TMBIM6 mRNA expression was increased with Sp1 levels in a concentration dependent manner. Ablation of Sp1 through siRNA or inhibition with mithramycin-A reduced the TMBIM6 mRNA expression. We also found that the protein kinase-C activation stimulates promoter activity and endogenous TMBIM6 mRNA by 2- to 2.5-fold. Additionally, overexpression of active mutants of PKCι, PKCε, and PKCδ increased TMBIM6 expression by enhancing nuclear translocation of Sp1. Immunohistochemistry analyses confirmed that the expression levels of PKCι, Sp1, and TMBIM6 were correlated with one another in samples from human breast, prostate, and liver cancer patients. Altogether, this study suggests the involvement of Sp1 in basal transcription and PKC in the enhanced expression of TMBIM6 in cancer.

Rationale: Degenerative spondylolisthesis is defined as forward slippage of a vertebra with respect to the underlying vertebra and is associated with the induction of lumbar canal stenosis. The use of anterior column support for degenerative lumbar conditions has been well documented. Direct lateral interbody fusion (DLIF) gains access via a lateral approach through the retroperitoneal fat and psoas muscle. It avoids many of the access-related complications yet comes with its own risks and limitations. The location of the iliac wing precludes exposure of the L5-S1 disc space and may make L4-5 surgery difficult. Therefore, accurate preoperative patient positioning is essential., Patient Concerns: A 71-year-old female with a body mass index (BMI) of 39.2 kg/m (height 155.9 cm, weight 79.5 kg) presented with lumbar pain radiating to the left lower limb. She complained of neurologic claudication with more than 100 m ambulation., Diagnosis: Plain standing view on spine radiography revealed L4-5 spondylolisthesis and disc-space narrowing. Magnetic resonance imaging (MRI) revealed severe L4-5 bilateral foraminal stenosis. After the first surgery, simple rib cage radiography was performed to examine the source of her right-sided flank pain and it revealed acute fracture of the right ninth and tenth ribs., Interventions: The patient was laterally positioned on the table in an extreme bending position. The intervertebral cage was inserted in the L4-5 disc space and disc height was restored. With respect to the rib fracture, treatment was conservative., Outcomes: The patient's radiating pain was immediately relieved and her lower back pain disappeared at 3 months after surgery. The patient reported right-sided flank pain after the first surgery. Simple rib cage radiography was performed and revealed fracture of the right ninth and tenth ribs. Follow-up assessments conducted 2 months later revealed complete bony union., Lessons: DLIF avoids many access-related complications. However, it is associated with other intraoperative complications, including injury to the lumbar nerve root and plexus. In addition, there are preoperative complications associated with improper patient placement on the table. In this case, the patient's obesity and strict positional requirements resulted in rib fracture. We suggest that surgeons consider this complication and exercise care in preoperative positioning.

Conjugated linoleic acid (CLA) constitutes a group of isomers derived from linoleic acid. Diverse studies have suggested that these unsaturated fatty acids have beneficial effects on human health. However, it has also been reported that their consumption can generate alterations in hepatic tissue. Thus, in the present study, we evaluated the effect of two of the major isomers of CLA, cis-9, trans-11-CLA and trans-10, cis-12-CLA, in the regulation of insulin signaling in a hepatic cell model, clone 9 (C9). We found that the two isomers decrease insulin-stimulated phosphorylation of the main proteins involved in insulin signaling, such as Akt at Ser 473 and Thr 308 , the insulin receptor at Tyr 1158 , IRS-1 at Tyr 632 , and GSK-3 at Ser 9/21 . Protein expression, however, was unaffected. Interestingly, both isomers of CLA promoted phosphorylation and activation of PKCε. Inhibition of PKCε activity by a dominant-negative form or knockdown of endogenous PKCε prevented the adverse effects of CLA isomers on insulin-induced Akt phosphorylation. Additionally, we also found that both isomers of CLA increase phosphorylation of IRS-1 at Ser 612 , a mechanism that probably underlies the inhibition of IRS-1 signaling by PKCε. Using confocal microscopy, we found that both isomers of CLA induced lipid accumulation in C9 cells with the presence of spherical cytosolic vesicles, suggesting their identity as neutral lipid droplets. These findings indicate that cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers could have a significant role in the development of insulin resistance in hepatic C9 cells through IRS-1 serine phosphorylation, PKCε activation, and hepatic lipid accumulation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background Context: In the posterior instrumented fusion surgery for thoracolumbar (T-L) burst fracture, early postoperative re-collapse of well-reduced vertebral body fracture could induce critical complications such as correction loss, posttraumatic kyphosis, and metal failure, often leading to revision surgery. Furthermore, re-collapse is quite difficult to predict because of the variety of risk factors, and no widely accepted accurate prediction systems exist. Although load-sharing classification has been known to help to decide the need for additional anterior column support, this radiographic scoring system has several critical limitations., Purpose: (1) To evaluate risk factors and predictors for postoperative re-collapse in T-L burst fractures. (2) Through the decision-making model, we aimed to predict re-collapse and prevent unnecessary additional anterior spinal surgery., Study Design: Retrospective comparative study., Patient Sample: Two-hundred and eight (104 men and 104 women) consecutive patients with T-L burst fracture who underwent posterior instrumented fusion were reviewed retrospectively. Burst fractures caused by high-energy trauma (fall from a height and motor vehicle accident) with a minimum 1-year follow-up were included. The average age at the time of surgery was 45.9 years (range, 15-79). With respect to the involved spinal level, 95 cases (45.6%) involved L1, 51 involved T12, 54 involved L2, and 8 involved T11. Mean fixation segments were 3.5 (range, 2-5). Pedicle screw instrumentation including fractured vertebra had been performed in 129 patients (62.3%)., Outcome Measures: Clinical data using self-report measures (visual analog scale score), radiographic measurements (plain radiograph, computed tomography, and magnetic resonance image), and functional measures using the Oswestry Disability Index were evaluated., Methods: Body height loss of fractured vertebra, body wedge angle, and Cobb angle were measured in serial plain radiographs. We assigned patients to the re-collapse group if their body height loss progressed greater than 20% at any follow-up time compared with immediate postoperative body height loss; we assigned the remaining patients to the well-maintained group. The chi-square test and t test of SPSS were used for comparison of differences between two groups and multiple logistic regression analysis for risk factor evaluation. Through the decision tree analysis of statistical package R, a decision-making model was composed, and a cutoff value of revealed risk factors and re-collapse rate of each subgroup were identified. The present study wassupported by the University College of Medicine Research Fund (university to which authors belong). There was no external funding source for this study. The authors have no conflict of interest to declare., Results: Re-collapse occurred in 31 of 208 patients (14.9%). In this group, age, the proportion of male gender, preoperative height loss, and preoperative wedge angle were significantly greater than the well-maintained group. Multivariable logistic regression analysis identified two independent risk factors: age (adjusted odds ratio 1.084, p=.002) and body height loss (adjusted odds ratio 1.065, p=.003). According to the decision-making tree, age (>43 years) was the most discriminating variable, andpreoperative body height loss (>54%) was the second. In this model, the re-collapse rate was zero in ages less than 43 years, and among those remaining, nearly 80% patients with greater than 54% of body height loss belonged to the re-collapse group., Conclusions: The independent predictors of re-collapse after posterior instrumented fusion for T-L burst fracture were the age at operation (>43 years old) and preoperative body height loss (>54%). Careful assessment using our decision-making model could help to predict re-collapse and prevent unnecessary additional spinal surgery for anterior column support, especially in young patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study is to present the effective design of N-plasty of the iliotibial band and surgical results of its use as a treatment for refractory external snapping hip., Materials and Methods: We evaluated 17 patients (24 cases) with external snapping hip who underwent N-plasty between October 2013 and May 2016 and who were followed up for at least 12 months. All patients were male and the mean age was 20.8 years. The mean duration of symptoms prior to surgical intervention was 28.5 months with an average follow up of 24.5 months. Surgery was defined as being successful when patients could carry out their daily activities and exercise without a clicking sensation or pain 6 months after surgery until their last follow-up. Failure was defined when either a clicking sensation or pain was present. The visual analog scale (VAS) and modified Harris hip score (mHHS) were measured and compared preoperatively and at last follow-up., Results: All patients had complete resolution of pain and snapping. The VAS decreased from 6.77 preoperatively to 0.09 postoperatively and mHHS improved from 69.5 to 97.8 after surgery., Conclusion: Modified designed N-plasty is considered to be an excellent treatment method facilitating operation reproducibility with maximum elongation effect of the iliotibial band., Competing Interests: CONFLICT OF INTEREST: The authors declare that there is no potential conflict of interest relevant to this article.

KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRAS(G12V) into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRAS(G12V) overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.

Background: There have been few randomized control trials comparing the incidence of stent fracture and primary patency among different self-expanding nitinol stents to date. The SMART™ CONTROL stent (Cordis Corp, Miami Lakes, Florida, United States) has a peak-to-valley bridge and inline interconnection, whereas the COMPLETE™-SE stent (Medtronic Vascular, Santa Rosa, California, United States) crowns have been configured to minimize crown-to-crown interaction, increasing the stent's flexibility without compromising radial strength. Further, the 2011 ESC (European society of cardiology) guidelines recommend that dual antiplatelet therapy with aspirin and a thienopyridine such as clopidogrel should be administered for at least one month after infrainguinal bare metal stent implantation. Cilostazol has been reported to reduce intimal hyperplasia and subsequent repeat revascularization. To date, there has been no randomized study comparing the safety and efficacy of two different antiplatelet regimens, clopidogrel and cilostazol, following successful femoropopliteal stenting., Methods/design: The primary purpose of our study is to examine the incidence of stent fracture and primary patency between two different major representative self-expanding nitinol stents (SMART™ CONTROL versus COMPLETE™-SE) in stenotic or occlusive femoropopliteal arterial lesion. The secondary purpose is to examine whether there is any difference in efficacy and safety between aspirin plus clopidogrel versus aspirin plus cilostazol for one month following stent implantation in femoropopliteal lesions. This is a prospective, randomized, multicenter trial to assess the efficacy of the COMPLETE™-SE versus SMART™ CONTROL stent for provisional stenting after balloon angioplasty in femoropopliteal arterial lesions. The study design is a 2x2 randomization design and a total of 346 patients will be enrolled. The primary endpoint of this study is the rate of binary restenosis in the treated segment at 12 months after intervention as determined by catheter angiography or duplex ultrasound., Discussion: This trial will provide powerful insight into whether the design of the COMPLETE™-SE stent is more fracture-resistant or effective in preventing restenosis compared with the SMART™ CONTROL stent. Also, it will determine the efficacy and safety of aspirin plus clopidogrel versus aspirin plus cilostazol in patients undergoing stent implantation in femoropopliteal lesions., Trial Registration: Registered on 2 April 2012 with the National Institutes of Health Clinical Trials Registry (ClinicalTrials.gov identifier# NCT01570803).

Study Design: A retrospective study., Objective: To determine the incidence and risk factors of adjacent segment disease (ASD) requiring surgery among patients previously treated with spinal fusion for degenerative lumbar disease and to compare the survivorship of adjacent segment according to various risk factors including comparison of fusion methods: posterior lumbar interbody fusion (PLIF) versus posterolateral fusion (PLF)., Summary of Background Data: One of the major issues after lumbar spinal fusion is the development of adjacent segment disease. Biomechanically, PLIF has been reported to be more rigid than PLF, and therefore, patients who undergo PLIF are suspected to experience a higher incidence of ASD than those who underwent PLF. There have been many studies analyzing the risk factors of ASD, but we are not aware of any study comparing PLIF with PLF in incidence of ASD requiring surgery., Methods: A consecutive series of 490 patients who had undergone lumbar spinal fusion of 3 or fewer segments to treat degenerative lumbar disease was identified. The mean age at index operation was 53 years, and the mean follow-up period was 51 months (12-236 mo). The number of patients treated by PLF and PLIF were 103 and 387, respectively. The incidence and prevalence of revision surgery for ASD were calculated by Kaplan-Meier method. For risk factor analysis, we used log-rank test and Cox regression analysis with fusion methods, sex, age, number of fused segments, and presence of laminectomy adjacent to index fusion., Results: After index spinal fusion, 23 patients (4.7%) had undergone additional surgery for ASD. Kaplan-Meier analysis predicted a disease-free survival rate of adjacent segments in 94.2% of patients at 5 years and 89.6% at 10 years after the index operation. In the analysis of risk factors, PLIF was associated with 3.4 times higher incidence of ASD requiring surgery than PLF (P = 0.037). Patients older than 60 years at the time of index operation were 2.5 times more likely to undergo revision operation than those younger than 60 years (P = 0.038). There were no significant differences in survival rates of the adjacent segment according to sex, preoperative diagnosis, number of fused segments, and concomitant laminectomy to adjacent segment., Conclusion: It was predicted that 10% of patients would undergo additional surgery for treating ASD within 10 years after index lumbar fusion. In this study, PLIF showed higher incidence of ASD than did PLF. Patient age greater than 60 years was another independent risk factor. Surgeons should carefully consider these factors at the time of surgical planning of lumbar fusion., Level of Evidence: 3.

Larval feeding with curcumin induces an extended health span with significantly increased median and maximum longevities in the adult fly. This phenotype is diet insensitive and shows no additive effect on longevity when combined with an adult dietary restriction (DR) diet, suggesting that curcumin and DR operate via the same or overlapping pathways for this trait. This treatment significantly slows the aging rate so that it is comparable with that of genetically selected long lived animals. The larval treatment also enhances the adult animal's geotactic activity in an additive manner with DR, suggesting that curcumin and DR may use different pathways for different traits. Feeding the drug to adults during only the health span also results in a significantly extended health span with increased median and maximum life span. This extended longevity phenotype is induced only during these stage-specific periods. Feeding adults with the drug over their whole life results in a weakly negative effect on median longevity with no increase in maximum life span. There are no negative effects on reproduction, although larval curcumin feeding increases development time, and also apparently accelerates the normal late-life neuromuscular degeneration seen in the legs. Gene expression data from curcumin-fed larvae shows that the TOR pathway is inhibited in the larvae and the young to midlife adults, although several other genes involved in longevity extension are also affected. These data support the hypothesis that curcumin acts as if it is a DR mimetic nutraceutical. These data also suggest that the search for DR mimetics may be enhanced by the use of stage-specific screening of candidate molecules., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Sclerotium rot was found on Cymbidium orchids at Seosan-si, Chungcheongnam-do, Korea, in July, 2010. Symptoms occurred on low leaves, which turned yellowish, after which the entire plant wilted. Severely infected plants were blighted and eventually died. White mycelial mats and sclerotia appeared on pseudobulbs. Based on the mycological characteristics and pathogenicity, the causal fungus was identified as Sclerotium rolfsii. This is the first report of new Sclerotium rot on Cymbidium spp. caused by S. rolfsii in Korea.

Objectives: This study compared rate of cell proliferation, viability, cell size, expression patterns of genes related to pluripotency and epigenetic modification between canine foetal fibroblasts (cFF) and canine adipose tissue-derived mesenchymal stem cells (cAd-MSC)., Materials and Methods: Proliferation pattern, cell viability as well as cell size at each passage of cFF and cAd-MSC were measured when cultures reached confluence. In addition, real-time PCR was performed to investigate expression of Dnmt1, HDAC1, OCT4, SOX2, BAX, BCL2 genes with reference to β-actin gene expression as an endogenous control in both cell lines., Results: cFF and cAd-MSC differed in number of generations, but not in doubling times, at all passages. Mean cell size of cAd-MSC was significantly smaller than that of cFF. Cell viability was significantly lower in cFFs and apoptotic level was significantly lower in cAd-MSC compared to passage-matched cFF. In the expression of genes related to pluripotency and epigenetic modification, level of HDAC1 in cAd-MSC was significantly higher than in cFF, but expression of Dnmt1 did not differ between the two groups. OCT4 and SOX2 were significantly more highly expressed in cAd-MSC compared to cFF., Conclusions: cAd-MSC have higher stem-cell potential than cFF in terms of proliferation patterns, epigenetic modification and pluripotency, thus cAd-MSC could be more appropriate than cFF as donors of nuclei in somatic cell nuclear transfer for transgenesis., (© 2012 Blackwell Publishing Ltd.)

Protein kinases and protein phosphatases constitute about 2-4% of the genes in a typical eukaryotic genome. Protein phosphatases are important players in many cellular processes such as proliferation, differentiation, cell adhesion, and motility. In this study, we identified, classified, and analyzed protein phosphatase complement of the dog genome. In this article, we report the identification of at least 178 putative protein phosphatases in dog which include 51 PSTPs, 112 PTPs, and 15 Asp-based protein phosphatases. Interestingly, we found at least five novel protein phosphatases in dog, namely DUSP5L, DUSP18L, MTMR9L, MTMR12L, and PPP6CL which are not present in human, mouse, rat, and cow. In addition, we found PTP4A1-rt, a retro-transposed copy of the PTP4A1 gene, in chromosome 27. Furthermore, we modeled three-dimensional structures of the catalytic domains of these putative protein phosphatases and aligned them to see the structural similarities between them. We docked PPP2CA with okadaic acid and calculated the value of affinity energy as -8.8 kcal/mol. Our nucleotide substitution rate study revealed that apparently none of the phosphatase family is under significantly higher evolutionary pressure.

Induction of terminal differentiation represents a potentially less toxic cancer therapy. Treatment of HO-1 human metastatic melanoma cells with IFN-β plus mezerein (MEZ) promotes terminal differentiation with an irreversible loss of growth potential. During this process, the transcription factor FOXM1 is down-regulated potentially inhibiting transactivation of target genes including those involved in G(2)/M progression and cell proliferation. We investigated the mechanism of FOXM1 down-regulation and its physiological role in terminal differentiation. Genetic and pharmacological studies revealed that FOXM1 down-regulation was primarily caused by MEZ activation of PKCα and co-treatment with IFN-β plus MEZ augmented the effect of PKCα. Promoter analysis with a mutated E-box on the FOXM1 promoter, and in vitro and in vivo binding assays confirm a direct role of c-Myc on FOXM1 expression. Reduction of c-Myc and overexpression of Mad1 by IFN-β plus MEZ treatment should cause potent and persistent reduction of FOXM1 expression during terminal differentiation. Overexpression of FOXM1 restored expression of cell cycle-associated genes and increased the proportion of cells in the S phase. Our experiments support a model for terminal differentiation in which FOXM1 down-regulation via activation of PKCα followed by suppression of c-Myc expression, are causal events in promoting growth inhibition during terminal differentiation.

Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKCα, -δ, -ε, and -μ/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKCα, δ, and ε and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a -83/-67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the steroidogenic machinery in mouse Leydig cells.

Nitric oxide (NO) production in endothelial cells (EC) is regulated by multisite phosphorylation of specific serine and threonine residues in endothelial NO synthase (eNOS). Among these, eNOS-Ser116 is phosphorylated in the basal state, and its phosphorylation contributes to basal NO production. Here, we investigated the mechanism by which eNOS-Ser116 is phosphorylated during the basal state using bovine aortic EC. Although a previous study suggested that protein kinase C was involved in eNOS-Ser116 phosphorylation, overexpression of various protein kinase C isoforms did not affect eNOS-Ser116 phosphorylation. An in silico analysis using a motif scan revealed that the eNOS-Ser116 residue might be a substrate for proline-directed protein kinases. Roscovitine, a specific inhibitor of cyclin-dependent kinase (CDK), 1, 2, and 5, but not an inhibitor of mitogen-activated protein kinase kinase or glycogen synthase kinase 3beta, inhibited eNOS-Ser116 phosphorylation dose dependently. Furthermore, purified CDK1, 2, or 5 directly phosphorylated eNOS-Ser116 in vitro. Ectopic expression of the dominant-negative CDK5 but not dominant-negative CDK1 or dominant-negative CDK2 repressed eNOS-Ser116 phosphorylation and increased NO production. In addition, CDK5 activity was detected in bovine aortic EC, and coimmunoprecipitation and confocal microscopy studies revealed a colocalization of eNOS and CDK5. Cotransfection of CDK5 and p25, the specific CDK5 activator, increased eNOS-Ser116 phosphorylation and decreased NO production, but its parent molecule, p35, and p39, another activator, were not detected in bovine aortic EC, which suggests the existence of a novel CDK5 activator. Overall, this is the first study to find that CDK5 is a physiological kinase responsible for eNOS-Ser116 phosphorylation and regulation of NO production.

Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. To provide a more practical method of visualizing melanosomes in melanocytes as well as in keratinocytes, we attempted to use murine cell lines instead of human primary cells. We generated various fluorescent fusion proteins of tyrosinase, a melanin synthesis enzyme located in the melanosome, by using green fluorescent protein and red fluorescent protein. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy. Co-culture of murine melanocytes and keratinocytes was optimized and melanosome transfer was either stimulated with alphaMSH or partially inhibited by niacinamide. To the best of our knowledge, this is the first study showing that a murine co-culture model, in addition to human primary cell co-culture, can be a good tool for depigmenting agent screening by monitoring melanosome transfer.

Protein kinase C delta (PKCdelta) is one of the important isoforms of PKCs that regulate various cellular processes, including cell survival and apoptosis. Studies have shown that activation of PKCdelta is correlated with apoptosis in various cell types, depending upon various stimuli. Phosphorylation of Thr505, Ser643 and Ser662 is crucial in activation of PKCdelta. Furthermore, phosphorylation of tyrosine residues, in particular that of Tyr311, is associated with PKCdelta activation and induction of apoptosis. Here, we generated a hydrophobic motif phosphorylation-deficient mutant of PKCdelta (PKCdelta-S662A) by mutating Ser662 to Ala, and studied the effect of this mutation in inducing apoptosis in L929 murine fibroblasts. We report that this mutation renders PKCdelta apoptotically more active. Furthermore, we found that the mutant PKCdelta-S662A is tyrosine-phosphorylated and translocated to the membrane faster than its wild-type counterpart., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Activation of V1 vasopressin (VP) receptors prevents serum deprivation-induced apoptosis in neuronal H32 cells, partially through mitogen-activated protein kinase (MAPK) mediated Bad phosphorylation. In this study, we investigated the role of protein kinases C (PKC) and B (PKB) mediating VP-induced antiapoptosis in H32 cells. Serum deprivation increased PKCdelta but not PKCalpha or PKCbeta activity, while VP increased PKCalpha and PKCbeta without affecting PKCdelta activity. Inhibition of PKCdelta prevented caspase 3 activation, indicating that PKCdelta mediates the pro-apoptotic actions of serum deprivation. Simultaneous inhibition of PKCalpha and beta and MAPK abolished VP-induced Bad phosphorylation, but it only partially prevented caspase 3 inhibition. Complete abolition of the protective effect of VP on serum deprivation-induced caspase 3 activity required additional blockade of phosphoinositide 3 kinase (PI3K)/protein kinase B. The data demonstrate that VP exerts antiapoptosis through multiple pathways; while PKCalpha and beta together with extracellular signal-regulated kinases/MAPK activation mediates Bad phosphorylation (inactivation), the full protective action of VP requires additional activation of PKB (PI3K/protein kinase B) pathway.

TPA is known to cooperate with an activated Ras oncogene in the transformation of rodent fibroblasts, but the biochemical mechanisms responsible for this effect have not been established. In the present study we used c-fos promoter-luciferase constructs as reporters, in transient transfection assays, in NIH3T3 cells to assess the mechanism of this cooperation. We found a marked synergistic interaction between TPA and a transfected v-Ha-ras oncogene in the activation of c-fos promoter and SRE. SRE has binding sites for TCF and SRF. A dominant-negative Ras (ras-N17) inhibited the TPA-Ras synergy by blocking the PKC-MAPK-TCF pathway. Dominant-negative RhoA and Rac1 (but not Cdc42Hs) inhibited the TPA-Ras synergy by blocking the Ras-Rho-SRF signaling pathway. Constitutively active PKCalpha and PKCepsilon showed synergy with v-Ras. These results suggest that the activation of two distinct pathways such as Ras-Raf-ERK-TCF pathway and Rho-SRF pathway are responsible for the induction of c-fos by TPA and Ras in mitogenic signaling pathways.

Although it is often assumed that the antitumor effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are due to inhibition of cyclooxgenase (COX) activity, specifically COX-2, there is accumulating evidence that COX-2 independent mechanisms can also play an important role. Studies with sulindac sulfone (Aptosyn) and related derivatives have revealed a novel pathway of tumor growth inhibition and apoptosis mediated by activation of the guanosine 3',5' monophosphate (cGMP)-dependent enzyme protein kinase G (PKG). The present study indicates that concentrations of the NSAIDs celecoxib, indomethacin, and meclofenamic acid that inhibit growth of SW480 human colon cancer cells inhibit subcellular cGMP-phosphodiesterase (PDE) enzymatic activity and in intact cells induce a two- to threefold increase in intracellular levels of cGMP. This is associated with phosphorylation of the protein VASP, a marker of PKG activation, activation of JNK1 and a decrease in cellular levels of cyclin D1; effects seen with other agents that cause activation of PKG in these cells. On the other hand even a high concentration of the COX-2 specific inhibitor rofecoxib (500 microM) did not inhibit growth of SW480 cells. Nor did rofecoxib inhibit cGMP-PDE activity or cause other changes related to PKG activation in these cells. Since activation of the PKG pathways by celecoxib, indomethacin, and meclofenamic acid in this cell culture system required high concentrations of these compounds, it remains to be determined whether activation of this pathway contributes to the in vivo antitumor effects of specific NSAIDs., (Copyright 2007 Wiley-Liss, Inc.)

It has been reported previously that cyclin G1 enables cells to overcome radiation-induced G(2) arrest and increased cell death and that these effects are mediated by transcriptional activation of cyclin B1. In this study, we further investigated the mechanism by which cyclin G1 transcriptionally activates cyclin B1. Deletion or point mutations within the cyclin B1 promoter region revealed that the c-Myc binding site (E-box) is necessary for cyclin G1-mediated transcriptional activation of cyclin B1 to occur. In addition, the kinase activity of Cdk5 was increased by cyclin G1 overexpression, and Cdk5 directly phosphorylated c-Myc on Ser-62. Furthermore, cyclin G1 mediated increased radiosensitivity, and radiation-induced M phase arrest was attenuated when RNA interference of Cdk5 was treated. Taken together, the results of this study indicate that Cdk5 activation in cells that overexpress cyclin G1 leads to c-Myc phosphorylation on Ser-62, which is responsible for cyclin G1-mediated transcriptional activation of cyclin B1.

Activation-induced upregulation of inhibitory killer Ig-like receptor (KIR) is regulated by protein kinase Cs (PKCs). Conventional PKCs increase KIR expression on the post-transcriptional level by increasing the recycling of surface molecules and endoplasmic reticulum (ER)-Golgi processing. PKCdelta plays a role in the secretion of cytoplasmic KIR through lytic granules. In this study, we identified amino acid sequence motifs associated with PKC-mediated KIR membrane trafficking by systematic mutagenesis. Mutations of Y(398) and HLWC(364) completely inhibited the PMA-induced increase of KIR molecules at surface as well as total protein levels, indicating that these are associated with ER-Golgi processing and sorting to plasma membrane through lytic granules. Mutations of Y-based motif, including Y(398), acidic region (PE(394)), dileucine motif-like region (IL(423)) and PKC-phosphorylatable S(415) caused a blockade of surface KIR endocytosis after PKC stimulation. Mutation of T(145) caused an accumulation of mutant proteins in late endosomes and lysosomes after PKC activation, suggesting that T(145) might be related to the recovery of endocytosed KIR to the surface membrane. We also demonstrated that PKCs could directly phosphorylate the KIR cytoplasmic tail by means of western blot and in vitro kinase assay, implying that phosphorylation status of KIR cytoplasmic tail can direct the fate of surface KIR molecules. Taken together, various sequence motifs are implicated in the PKC-mediated post-transcriptional upregulation of KIR, and each of these motifs work in different steps after PKC activation.

Eukaryotic protein kinases, containing a conserved catalytic domain, represent one of the largest superfamilies of the eukaryotic proteins and play distinct roles in cell signaling and diseases. Near completion of rat genome sequencing project enables the evaluation of a near complete set of rat protein kinases. Publicly accessible genetic sequence databases were searched for rat protein kinases, and 515 eukaryotic protein kinases, 40 atypical protein kinases and 45 kinase pseudogenes were identified. The rat has 509 putative protein kinases orthologous to human kinases. Unlike microtubule affinity-regulating kinases, the rat has a few more kinases, in addition to the orthologous pairs of mouse kinases. The comparison of 11 different eukaryotic species revealed the evolutionary conservation of this diverse family of proteins. The evolutionary rate studies of human disease and non-disease associated kinases suggested that relatively uniform selective pressures have been applied to these kinase classes. This bioinformatic study of the rat protein kinases provides a suitable framework for further characterization of the functional and structural properties of these protein kinases.

Protein kinase C (PKC), a multi-gene family of enzymes, plays key roles in the pathways of signal transduction, growth control and tumorigenesis. Variations in the intracellular localization of the individual isoforms are thought to be an important mechanism for the isoform-specific regulation of enzyme activity and substrate specificity. To provide a dynamic method of analyzing the localization of the specific isoforms of PKC in living cells, we generated fluorescent fusion proteins of the various PKC isoforms by using the green fluorescent protein (GFP) as a fluorescent marker at the carboxyl termini of these enzymes. The intracellular localization of the specific PKC isoforms was then examined by fluorescence microscopy after transient transfection of the respective PKC-GFP expression vector into NIH3T3 cells and subsequent TPA stimulation. We found that the specific isoforms of PKC display distinct localization patterns in untreated NIH3T3 cells. For example, PKCalpha is localized mainly in the cytoplasm while PKCepsilon is localized mainly in the Golgi apparatus. We also observed that PKCalpha, beta1, beta2, gamma, delta, epsilon, and eta translocate to the plasma membrane within 10 min of the start of TPA treatment, while the cellular localizations of PKCzeta and iota were not affected by TPA. Using a protein kinase inhibitor, we also showed that the kinase activity was not important for the translocation of PKC. These results suggest that specific PKC isoforms exert spatially distinct biological effects by virtue of their directed translocation to different intracellular sites.

We tested the effects of dietary restriction (DR) on the standard w(1118) strain as well as on our previously described Ra and La strains and their reciprocal isogenic 'cybrid' lines containing heterologous nuclear-mitochondrial combinations. The w(1118) and Ra strains, but neither the La strains nor their 'cybrid' derivatives, are DR-inducible. The Ra and La animals are more robust than the w(1118) animals, and the Ra strain shows an upward shift in its DR threshold. The non-inducibility of the La strain suggests that it expresses a constituitive version of the inducible DR phenotype of its predecessor Ra strain. The difference in mitochondrial efficiency observed between the Ra and La mitochondria (Ross, 2000) has real effects on longevity in the adults: the presence of the more efficient La mitochondria enhances the longevity of an otherwise Ra animal; while the maximum longevity inherent in the La nuclear information cannot be fully expressed in the presence of the Ra mitochondria. Despite the absence of any demographic mathematical parameters robustly and uniquely associated with extended longevity, there are some common trends in that longer lived cohorts have a longer period of low and often constant early life mortality rates (comprising the "health span"), leading to a delayed onset of senescence as noted by the late life increase in age-specific mortality rates (comprising the "senescence span"). There is a genetic basis to this phenotype, but the context-dependence of the demographic data suggests that there is not likely to be only one mechanism or pathway involved in the DR response. In addition, the presence of live yeast had systemic strain specific effects such that it increased longevity in the w(1118) animals but decreased it in the Ra and derived lines. Higher density (4-10x) foods yielded a decreased longevity in all strains at the highest level, showing that malnutrition occurs at both low and high caloric levels.

Heat shock protein 27 (HSP27), which is highly expressed in human lung and breast cancer tissues, induced resistance to cell death against various stimuli. Treatment of NCI-H1299 cells, which express a high level of HSP27, with small interference RNA specifically targeting HSP27 resulted in inhibition of their resistance to radiation or cisplatin, suggesting that HSP27 contributed to cellular resistance in these lung cancer cells. Furthermore, because HSP27 interacts directly with the COOH terminus of the protein kinase C delta (PKC delta)-V5 region with ensuing inhibition of PKC delta activity and PKC delta-mediated cell death, we wished to determine amino acid residues in the V5 region that mediate its interaction with HSP27. Investigation with various deletion mutants of the region revealed that amino acid residues 668 to 674 of the V5 region mediate its interaction with HSP27. When NCI-H1299 cells were treated with biotin or with FITC-tagged heptapeptide of the residues 668 to 674 (E-F-Q-F-L-D-I), the cells exhibited dramatically increased cisplatin or radiation-induced cell death with the heptapeptide having efficient interaction with HSP27, which in turn restored the PKC delta activity that had been inhibited by HSP27. In vivo nude mice grafting data also suggested that NCI-H1299 cells were sensitized by this heptapeptide. The above data strongly show that the heptapeptide of the PKC delta-V5 region sensitized human cancer cells through its interaction with HSP27, thereby sequestering HSP27. The heptapeptide may provide a novel strategy for selective neutralization of HSP27.

Inhibitory killer Ig-like receptor (KIR) expression was upregulated by protein kinase C (PKC) activation in stable Jurkat clones that express KIR or CD8KIR fusion proteins. PKC-induced KIR upregulation was mediated by the cytoplasmic tail of KIR and regulated at the post-transcriptional level. PKC inhibition, metabolic labeling and colocalization studies demonstrated that the activation of the conventional PKCs upregulated surface and cellular KIR levels by stimulating the maturation processes in endoplasmic reticulum-Golgi and by promoting the recycling of surface KIR through sorting endosomes. Similar studies also revealed that KIR was secreted to plasma membrane through lytic granules in a PKCdelta-dependent manner. Consequently, PKCdelta inhibition caused the formation of giant perinuclear granules, which trapped KIR and FasL as well as CPE and Lamp1.

GnRH regulates the reproductive system by stimulating synthesis and release of gonadotropins. GnRH acts through a receptor coupled to multiple intracellular events including a rapid phosphoinositide turnover. Although the cAMP pathway is essential for gonadotrope function, the ability of GnRH to induce cAMP, as well as the coupling mechanisms involved, remain controversial. In this study, we established that GnRH increases intracellular cAMP levels in a concentration-dependent manner in LbetaT2 gonadotrope cells (maximal increase, 2.5-fold; EC(50), 0.30 nm), and this was further evidenced by GnRH activation of a cAMP-sensitive reporter gene. The GnRH effect was Ca(2+) independent, mimicked by the phorbol ester phorbol 12-myristate 13-acetate, and blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, indicating that the GnRH effect was mediated by PKC. Pharmacological inhibition of conventional PKC isoforms with Gö6976 did not prevent GnRH-induced cAMP production, whereas down-regulation of novel PKCdelta, -epsilon, and -theta by a long-term treatment with GnRH markedly reduced it. Expression of dominant-negative (DN) mutants of PKCdelta or -epsilon but not PKCtheta impaired GnRH activation of a cAMP-sensitive promoter, demonstrating that PKCdelta and -epsilon are the two endogenous isoforms mediating GnRH activation of the adenylyl cyclase (AC) pathway in LbetaT2 cells. Accordingly, we identified by RT-PCR and immunocytochemical analysis, two PKC-sensitive AC isoforms, i.e. AC5 and AC7 as potential targets for GnRH. Lastly, we showed that only sustained stimulation of GnRH receptor significantly increased cAMP, suggesting that in vivo, the cAMP signaling pathway may be selectively recruited under intense GnRH release such as the preovulatory GnRH surge.

Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by beta-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCepsilon (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, beta-phorbol myristate acetate (PMA), but not 4alpha-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events.

Despite the involvement in diverse physiological process and pleiotropic expression profile, the molecular functions of Nur77 are not likely to be fully elucidated. From the effort to find a novel function of Nur77, we detected molecular interaction between Nur77 and PKC. Details of interaction revealed that C-terminal ligand binding domain (LBD) of Nur77 specifically interacted with highly conserved glycine-rich loop of PKC required for catalytic activity. This molecular interaction resulted in inhibition of catalytic activity of PKCtheta by Nur77. C-terminal LBD of Nur77 is sufficient for inhibiting the phosphorylation of substrate by PKCtheta. Ultimately, inhibition of catalytic activity by Nur77 is deeply associated with repression of PKC-mediated activation of AP-1 and NF-kappaB. Therefore, these findings demonstrate a novel function of Nur77 as a PKC inhibitor and give insights into molecular mechanisms of various Nur77-mediated physiological phenomena.

Although cyclin G1 has been implicated in certain p53-related biological phenomena, other aspects of its function remain unclear. Here we report hitherto unknown mechanism by which cyclin G1 increases radiation sensitivity by regulating the level of cyclin B1. Overexpression of cyclin G1 was observable in lung carcinoma tissues. Irradiation of human lung cells with cyclin G1 overexpression resulted in increased cell death and gamma-H2AX foci suggesting that cyclin G1 rendered the cells more susceptible to DNA damage. Enhanced radiosensitivity by cyclin G1 was correlated with increased cyclin B1, CDC2/cyclin B1 complex, and MPM2. Cell cycle synchronization clearly showed coexpression of cyclin G1 and cyclin B1 in G2/M phase. Depletion of cyclin G1 by interference RNA revealed that cyclin G1 regulated transcription of cyclin B1 in a p53-independent manner, and confirmed that the increased mitotic cells and cell death by cyclin G1 were dependent upon cyclin B1. Therefore, our data suggest that cyclin G1 enhanced radiation sensitivity by overriding radiation-induced G2 arrest through transcriptional upregulation of cyclin B1.

Expression of MHC class II genes requires CIITA. Although the transactivation function of CIITA is well characterized, the signaling events that regulate CIITA expression are less understood. In this study, we report that CIITA expression in B cells depends on protein kinase Cdelta (PKCdelta). PKCdelta controls CIITA gene transcription mainly via modulating CREB recruitment to the CIITA promoter without affecting CIITA mRNA stability. Inhibition of PKCdelta by a pharmacological inhibitor or knocking down of endogenous PKCdelta expression by small interfering RNA reduced CREB binding to the CIITA promoter. The decrease of CIITA gene expression in the presence of the PKCdelta inhibitor was prevented by ectopically expressing a constitutively active form of CREB. In addition, histone acetylation of the CIITA promoter is regulated by PKCdelta since the PKCdelta inhibitor treatment or PKCdelta small interfering RNA resulted in decreased histone acetylation. Taken together, our study reveals that PKCdelta is an important signaling molecule necessary to maintain CIITA and MHC class II expression in B cells.

Intracellular signaling molecules and apoptotic factors seem to play an important role in determining the radiation response of tumor cells. However, the basis for the link between signaling pathway and apoptotic cell death machinery after ionizing irradiation remains still largely unclear. In this study, we showed that c-Abl-PKCdelta-Rac1-p38 MAPK signaling is required for the conformational changes of Bak and Bax during ionizing radiation-induced apoptotic cell death in human non-small cell lung cancer cells. Ionizing radiation induced conformational changes and subsequent oligomerizations of Bak and Bax, dissipation of mitochondrial membrane potential, and cytochrome c release from mitochondria. Small interference (siRNA) targeting of Bak and Bax effectively protected cells from radiation-induced mitochondrial membrane potential loss and apoptotic cell death. p38 MAPK was found to be selectively activated in response to radiation treatment. Inhibition of p38 MAPK completely suppressed radiation-induced Bak and Bax activations, dissipation of mitochondrial membrane potential, and cell death. Moreover, expression of a dominant negative form of protein kinase Cdelta (PKCdelta) or siRNA targeting of PKCdelta attenuated p38 MAPK activation and conformational changes of Bak and Bax. In addition, ectopic expression of RacN17, a dominant negative form of Rac1, markedly inhibited p38 MAPK activation but did not affect PKCdelta activation. Upon stimulation of cells with radiation, PKCdelta was phosphorylated dramatically on tyrosine. c-Abl-PKCdelta complex formation was also increased in response to radiation. Moreover, siRNA targeting of c-Abl attenuated radiation-induced PKCdelta and p38 MAPK activations, and Bak and Bax modulations. These data support a notion that activation of the c-Abl-PKCdelta-Rac1-p38 MAPK pathway in response to ionizing radiation signals conformational changes of Bak and Bax, resulting in mitochondrial activation-mediated apoptotic cell death in human non-small cell lung cancer cells.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived mature B cells with the distinctive phenotype CD19(hi) CD5+ CD23+ IgM(lo), which are refractory to apoptosis. An increased level of apoptosis has been observed on treatment of human B-CLL cells with protein kinase C (PKC) inhibitors, suggesting that this family of protein kinases mediate survival signals within B-CLL cells. Therefore, to investigate the ability of individual PKC isoforms to transform developing B cells, we stably expressed plasmids encoding PKC mutants in fetal liver-derived hematopoietic progenitor cells (HPC) from wild-type mice and then cultured them in B-cell generation systems in vitro and in vivo. Surprisingly, we noted that expression of a plasmid-encoding dominant-negative PKC alpha (PKC alpha-KR) in HPCs and subsequent culture both in vitro and in vivo resulted in the generation of a population of cells that displayed an enhanced proliferative capacity over untransfected cells and phenotypically resemble human B-CLL cells. In the absence of growth factors and stroma, these B-CLL-like cells undergo cell cycle arrest and, consistent with their ability to escape growth factor withdrawal-induced apoptosis, exhibited elevated levels of Bcl-2 expression. These studies therefore identify a unique oncogenic trigger for the development of a B-CLL-like disease resulting from the subversion of PKC alpha signaling. Our findings uncover novel avenues not only for the study of the induction of leukemic B cells but also for the development of therapeutic drugs to combat PKC alpha-regulated transformation events.

Since radiation-induced caspase-dependent apoptosis and ROS generation were partially prevented by HSP25 overexpression, similar to the treatment of control cells with antioxidant agents such as DPI and tiron, questions arise whether radiation-mediated ROS generation contributes to the apoptotic cell death, and also whether HSP25 overexpression can reduce ROS mediated apoptotic cell death. In the present study, radiation-induced cytochrome c release from mitochondria and activation of caspases accompanied by a decrease of mitochondrial membrane potential in Jurkat T cells were shown to be inhibited by mitochondrial complex I inhibitor rotenone, suggesting that mitochondrial ROS might be important in radiation-induced caspase-dependent apoptosis. When HSP25 was overexpressed, effects similar to the treatment of cells with the antioxidants were obtained, indicating that HSP25 suppressed radiation-induced mitochondrial alteration that resulted in apoptosis. Furthermore, activation of p38 MAP kinase by radiation was associated with radiation-induced cell death and ROS production and PKCdelta was an upstream molecule for p38 MAP kinase activation, ROS generation and subsequent caspase-dependent apoptotic events. However, in the HSP25 overexpressed cells, the above-described effects were blocked. In fact, radiation-induced membrane translocation of PKCdelta and tyrosine phosphorylation were inhibited by HSP25. Based on the above data, we suggest that HSP25 downregulates PKCdelta, which is a key molecule for radiation-induced ROS generation and mitochondrial-mediated caspase-dependent apoptotic events.

Heat shock protein 25 (HSP25) interferes negatively with apoptosis through several pathways that involve its direct interaction with cytochrome c or Akt. Here we show that HSP25 inhibits protein kinase C (PKC) delta-mediated cell death through direct interaction. HSP25 binds to kinase-active PKCdelta to inhibit its kinase activity and translocation to the membrane, which results in reduced cell death. Deletion constructs of HSP25 and PKCdelta identified amino acids 90-103 of HSP25 and the C-terminal V5 region of PKCdelta as binding sites. In addition, the interaction between HSP25 and PKCdelta induced HSP25 phosphorylation at Ser-15 and Ser-86, and these phosphorylations permitted HSP25 release from PKCdelta. Based on these observations, we propose that after PKCdelta activation, HSP25 binds to the exposed V5 region of PKCdelta. This novel function of HSP25 accounts for its cytoprotective properties via the inhibition of PKCdelta and the enhancement of HSP25 phosphorylation.

We previously demonstrated that protein kinase C-eta (PKC-eta) mediates a phorbol 12-myristate-13-acetate (PMA)-induced proliferative response in human glioblastoma (GBM) cells. In this report, we show that PMA-stimulated activation of PKC-eta in U-251 GBM cells resulted in activation of both Akt and the mammalian target of rapamycin (mTOR) signaling pathways and an increase in cell proliferation. Expression of a kinase dead PKC-eta (PKC-etaKR) construct reduced the basal and PMA-evoked proliferation of PKC-eta-expressing U-251 GBM cells, as well as abrogated the PMA-induced activation of Akt, mTOR, and the mTOR targets 4E-BP1 and STAT-3. Treatment of cells with the PI-3 kinase inhibitor LY294002 (10 muM) or the mTOR inhibitor rapamycin (10 nM) also reduced PMA-induced proliferation and cell-cycle progression. Expression of a constitutively active PKC-eta (PKC-etaDeltaNPS) construct in a GBM cell line with no endogenous PKC-eta (U-1242) also provided evidence that PKC-eta targets the Akt and mTOR signaling pathways. Moreover, activation of 4E-BP1 and STAT-3 in both PMA-treated U-251 and PKC-etaDeltaNPS-expressing U-1242 GBM cells was inhibited by rapamycin. However, activation of Akt, but not mTOR was inhibited by the PI-3 kinase inhibitor LY294002. This study identifies Akt and mTOR as downstream targets of PKC-eta that are involved in GBM cell proliferation.

Epidermal keratinocyte differentiation is a tightly regulated stepwise process that requires protein kinase C (PKC) activation. Studies on cultured mouse keraitnocytes induced to differentiate with Ca2+ have indirectly implicated the involvement of PKCa isoform. When PKCalpha was overexpressed in undifferentiated keratinocytes using adenoviral system, expressions of differentiation markers such as loricrin, filaggrin, keratin 1 (MK1) and keratin 10 (MK10) were increased, and ERK1/2 phosphorylation was concurrently induced without change of other MAPK such as p38 MAPK and JNK1/2. Similarly, transfection of PKCalpha kinase active mutant (PKCalpha- CAT) in the undifferentiated keratinocyte, but not PKCbeta-CAT, also increased differentiation marker expressions. On the other hand, PKCalpha dominant negative mutant (PKCbeta-KR) reduced Ca2+ -mediated differentiation marker expressions, while PKCbeta-KR did not, suggesting that PKCalpha is responsible for keratinocyte differentiation. When downstream pathway of PKCalpha in Ca2+ -mediated differentiation was examined, ERK1/2, p38 MAPK and JNK1/2 phosphorylations were increased by Ca2+ shift. Treatment of keratinocytes with PD98059, MEK inhibitor, and SB20358, p38 MAPK inhibitor, before Ca2+ shift induced morphological changes and reduced expressions of differentiation markers, but treatment with SP60012, JNK1/2 inhibitor, did not change at all. Dominant negative mutants of ERK1/2 and p38 MAPK also inhibited the expressions of differentiation marker expressions in Ca2+ shifted cells. The above results indicate that both ERK1/2 and p38 MAPK may be involved in Ca2+ -mediated differentiation, and that only ERK1/2 pathway is specific for PKCalpha-mediated differentiation in mouse keratinocytes.

Protein kinase C (PKC) family, which functions through serine/threonine kinase activity, is involved in signal transduction pathways necessary for cell proliferation, differentiation, and apoptosis. Its critical role in neoplastic transformation and tumor invasion renders PKC a potential target for anticancer therapy. In this study, we investigated the effect of targeting individual PKCs on gastric carcinogenesis. We established gastric cancer cell lines stably expressing antisense PKCalpha, PKCbeta1, and PKCbeta2 cDNA. These stable transfectants were characterized by cell morphology, cell growth, apoptosis, and tumorigenicity in vitro and in vivo. PKCalpha-AS and PKCbeta1-AS transfectants showed a different morphology with flattened, long processes and decreased nuclear:cytoplasmic ratio compared with the control cells. Cell growth was markedly inhibited in PKCalpha-AS and PKCbeta1-AS transfectants. PKCalpha-AS and PKCbeta1-AS cells were more responsive to mitomycin C- or 5-fluorouracil-induced apoptosis. However, antisense targeting of PKCbeta2 did not have any significant effect on cell morphology, cell growth, or apoptosis. Furthermore, antisense inhibition of PKCalpha and PKCbeta1 markedly suppressed colony-forming efficiency in soft agar and in nude mice xenografts. Inhibition of PKCalpha or PKCbeta1 significantly suppressed transcriptional and DNA binding activity of activator protein in gastric cancer cells, suggesting that PKCalpha or PKCbeta1 exerts their effects on cell growth through regulation of activator protein activity. These data provide evidence that targeting PKCalpha and PKCbeta1 by antisense method is a promising therapy for gastric cancer.

HSP25 has been shown to induce resistance to radiation and oxidative stress; however, its exact mechanisms remain unclear. In the present study, a high concentration of H2O2 was found to induce DNA fragmentation in L929 mouse fibroblast cells, and HSP25 overexpression attenuated this phenomenon. To elucidate the mechanisms of H2O2-mediated cell death, ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in the cells after treatment with H2O2 were examined. ERK1/2 and JNK1/2 were activated by H2O2; ERK1/2 activation was inhibited in HSP25-overexpressed cells, while JNK1/2 was indifferent. Inhibition of ERK1/2 activation by treatment of the cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced cell death; similarly treated HSP25-overexpressed cells were not at all affected. Moreover, inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 transfection did not affect H2O2-mediated cell death in control cells. Dominant-negative Ras or Raf transfection inhibited H2O2-mediated ERK1/2 activation and cell death in control cells. On the contrary, HSP25-overexpressed cells did not show any differences. Upstream pathways of H2O2-mediated ERK1/2 activation and cell death involved both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta, while in HSP25-overexpressed cells these kinases did not respond to H2O2 treatment. Since HSP25 overexpression reduced reactive oxygen species (ROS), increased manganese superoxide dismutase (MnSOD) gene expression, and increased enzyme activity, involvement of MnSOD in HSP25-mediated attenuation of H2O2-mediated ERK1/2 activation and cell death was examined. Blockage of MnSOD with antisense oligonucleotides prevented DNA fragmentation and returned the ERK1/2 activation to the control level. Indeed, when MnSOD was overexpressed in L929 cells, similar to in HSP25-overexpressed cells, DNA fragmentation and ERK1/2 activation were reduced. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated downregulation of ERK1/2.

Oxidative stress is known to induce apoptosis in a wide variety of cell types, apparently by modulating intracellular signaling pathways. High concentrations of H2O2 have been found to induce apoptosis in L929 mouse fibroblast cells. To elucidate the mechanisms of H2O2-mediated apoptosis, ERK1/2, p38-MAPK, and JNK1/2 phosphorylation was examined, and ERK1/2 and JNK1/2 were found to be activated by H2O2. Inhibition of ERK1/2 activation by treatment of L929 cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced apoptosis, while inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 or MKK4 or MKK7 transfection did not affect H2O2-mediated apoptosis. H2O2-mediated ERK1/2 activation was not only Ras-Raf dependent, but also both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta dependent. H2O2-mediated PKCdelta-dependent and tyrosine kinase-dependent ERK1/2 activations were independent from each other. Based on the above results, we suggest for the first time that oxidative damage-induced apoptosis is mediated by ERK1/2 phosphorylation which is not only Ras-Raf dependent, but also both tyrosine kinase and PKCdelta dependent.