Bioinformatic analysis of responsive genes in two-dimension and three-dimension cultured human periodontal ligament cells subjected to compressive stress.

BACKGROUND AND OBJECTIVE: Analyzing responses of human periodontal ligament cells to mechanical stress and mechanotransduction is important for understanding periodontal tissue physiology and remodeling. It has been shown that the cellular response to mechanical stress can vary according to the type and duration of force and to extracellular attachment conditions. This study investigated the gene-expression profile of human periodontal ligament cells cultured in two-dimension (2D) and three-dimension (3D) conditions after application of compressive stress for 2 and 48 h. MATERIAL AND METHODS: Human primary periodontal ligament cells were obtained from premolars extracted for orthodontic purposes. Cells were cultured in a conventional 2D culture dish or in 3D collagen gel and compressive stress was applied for 2 and 48 h. Control cells were cultured under identical conditions but without the application of compressive stress. After the application of compressive stress, total RNA was extracted and a cDNA microarray was performed. Microarray data were analyzed using statistical methods, including david and gene set enrichment analysis to identify significant signaling pathways. Real-time PCR was performed for five mRNAs in order to confirm the cDNA microarray results. RESULTS: The cDNA microarray analysis revealed that after application of compressive stress for 2 h, 191 and 553 genes showed changes in their expression levels in 2D and 3D cultured cells, respectively. After application of compressive stress for 48 h, 280 and 519 genes showed changes in their expression levels in 2D and 3D cultured cells, respectively. Euclidean clustering method was used to demonstrate the gene-expression kinetics. CONCLUSION: Analysis of the results showed that several signaling pathways, including the MAPK pathway and the focal adhesion kinase pathway are relevant to the compressive force-induced cellular response. 2D and 3D cultured cells showed significantly different gene-expression profiles, suggesting that cellular attachment to extracellular matrix influences cellular responses to mechanical stresses.

Increased procoagulant cell-derived microparticles (C-MP) in splenectomized patients with ITP.

BACKGROUND: Splenectomy is frequently employed for therapeutic and diagnostic purposes in various clinical disorders. However its long-term safety is not well elucidated. Although risk of infection by encapsulated organisms is widely recognized, less well-known are risks of thrombosis and cardiovascular disease. METHODS: We investigated levels of cell-derived microparticles (C-MP) in 23 splenectomized ITP (ITP-S) and 53 unsplenectomized ITP patients (ITP-nS). Assay of C-MP derived from platelets (PMP), leukocytes (LMP), red cells (RMP) and endothelial cells (EMP) were performed by flow cytometry. Coagulation parameters included PT, aPTT and activities of FVIII, IX and XI. Results of all measures were compared between the two groups, ITP-S vs ITP-nS. RESULTS: Levels of all C-MP were higher in ITP-S than ITP-nS but only RMP and LMP reached statistical significance (p = 0.0035 and p < 0.0001, respectively). The aPTT was significantly shorter in ITP-S (p = 0.029). Interestingly, correlation analysis revealed that RMP, but not other C-MP, were associated with shortening of aPTT (p = 0.024) as well as with increased activities of factors VIII (p = 0.023), IX (p = 0.021) and XI (p = 0.0089). CONCLUSIONS: RMP and LMP were significantly elevated in splenectomized compared to non-splenectomized ITP patients. This suggests that the spleen functions to clear procoagulant C-MP, and that elevation of C-MP might contribute to increased risk of thrombosis, progression of atherosclerosis and cardiovascular disease following splenectomy.

Microparticle-mediated thrombin generation assay: increased activity in patients with recurrent thrombosis.

BACKGROUND: Circulating cell-derived microparticles (MP) are important players in thrombogenesis, attributed in part to tissue factor (TF) carried on them. We developed MP-mediated thrombin generation assay (TGA) and measured a series of patients with thrombosis (TBS) and normal controls (NC). METHODS: MP were isolated from plasma of 66 patients with TBS and 34 NC. The MP were resuspended in normal pooled particle-free plasma (PFP) containing corn trypsin inhibitor (to inhibit contact pathway). MP mediated TGA yields three parameters: lag time, peak and rate. This method is not influenced by anticoagulant therapy. Of the TBS patients, 41 had only a single thrombosis (S-TBS) and 25 had recurrences (R-TBS) within a 5-year period. In parallel, MP were quantitated by flow cytometry, and cell origin was determined: endothelial cells (EMP), leukocytes (LMP), red cells (RMP) and platelets (PMP). RESULTS: MP from all TBS patients exhibited higher thrombin generation than NC by all three TGA parameters. R-TBS had significantly greater TGA values than S-TBS, reflected in higher peak and rate, and shorter lag time. MP numbers were also higher in TBS vs. NC, for all MP subtypes, and were significantly higher in R-TBS than S-TBS (except LMP). All MP levels correlated with thrombin generation (P < 0.0001), most closely between PMP and peak (R = 0.47) and rate (R = 0.43). CONCLUSIONS: MP-mediated TGA is a novel way to assess functional procoagulant activity of MP. Enhanced MP-mediated TGA was demonstrated in TBS patients, and significantly higher activity in R-TBS. These findings support a major role of MP in thrombogenesis.

Endothelial microparticles induce formation of platelet aggregates via a von Willebrand factor/ristocetin dependent pathway, rendering them resistant to dissociation.

Endothelial microparticles (EMP) released from activated or apoptotic endothelial cells (EC) are emerging as useful markers for detection of EC dysfunction. Our recent observation that EMP carry von Willebrand factor (vWf) led us to investigate their interaction with platelets. EMP were incubated with normal washed platelets in the presence or absence of ristocetin, then platelet aggregates were measured by flow cytometry. In the absence of ristocetin, negligible EMP conjugated with platelets (< 5%) but in the presence of ristocetin (1 mg mL(-1)), EMP induced up to 95% of platelets to aggregate. EMP-platelet interaction was 80% blocked by anti-CD42b, or by 0.1 microm filtration to remove EMP. Platelet aggregates induced by normal plasma or high molecular weight vWf (Humate-P) dissociated 50% within 15-25 min following 1:20 dilution. In contrast, aggregates formed with EMP persisted two- to threefold longer with the same treatment, indicating greater stability. A similar degree of prolongation of dissociation was observed using plasma from thrombotic thrombocytopenic purpura (TTP) patients compared with normal plasma. Addition of EMP to plasma from severe von Willebrand disease restored his ristocetin-induced platelet aggregation. Multimer analysis of vWf on EMP showed unusually large vWf (ULvWf). In summary, EMP carries ULvWf multimers, promote platelet aggregates, and increase the stability of the aggregates thus formed.

Comparison of unintentional fatal occupational injuries in the Republic of Korea and the United States.

OBJECTIVES: To compare the profile of unintentional fatal occupational injuries in the Republic of Korea and the United States to help establish prevention strategies for Korea and to understand country specific differences in fatality risks in different industries. METHODS: Occupational fatal injury data from 1998-2001 were collected from Korea's Occupational Safety and Health Agency's Survey of Causes of Occupational Injuries (identified by the Korea Labor Welfare Corporation) and from the United States Census of Fatal Occupational Injuries. Employment estimates were obtained in both countries. Industry coding and external cause of death coding were standardized. Descriptive analyses of injury rates and Poisson regression models to examine time trends were conducted. RESULTS: Korea exhibited a significantly higher fatal injury rate, at least two times higher than the United States, after accounting for different employment patterns. The ordering of industries with respect to risk is the same in the two countries, with mining, agriculture/forestry/fishing, and construction being the most dangerous. Fatal injury rates are decreasing in these two countries, although at a faster rate in Korea. CONCLUSIONS: Understanding industrial practices within different countries is critical for fully understanding country specific occupational injury statistics. However, differences in surveillance systems and employment estimation methods serve as caveats to any transnational comparison, and need to be harmonized to the fullest extent possible.

Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines.

In several neuronal systems, nerve growth factor (NGF) and platelet-derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature-sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non-permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A(2) (PLA(2)), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA(2). When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase-inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.

Occupational asthma due to formaldehyde.

Formaldehyde is a low molecular weight chemical and can elicit acute and chronic health related problems. Most of the inhaled formaldehyde is retained in the upper respiratory tract due to its extraordinary solubility. Therefore, cases of formaldehyde-induced occupational asthma are sporadic despite its widespread use in industrial processes. We herein report upon a case of occupational asthma due to formaldehyde, which was confirmed by workplace challenge including working environmental assessments, and by formaldehyde inhalation challenge using a specially designed closed-circuit apparatus. To investigate the possible involvement of an IgE-mediated mechanism, both in vitro and in vivo tests were done. IgE antibody specific for formaldehyde-human serum albumin conjugate (F-HSA) was not detected by ELISA, and no specific cutaneous reactivity to F-HSA was noted by either skin prick or intradermal test. The patient was diagnosed with formaldehyde-induced occupational asthma not associated with an IgE mediated mechanism.

Zn2+-induced ERK activation mediated by reactive oxygen species causes cell death in differentiated PC12 cells.

Recent studies have provided evidence that Zn2+ plays a crucial role in ischemia- and seizure-induced neuronal death. However, the intracellular signaling pathways involved in Zn2+-induced cell death are largely unknown. In the present study, we investigated the roles of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated kinase (ERK), and of reactive oxygen species (ROS) in Zn2+-induced cell death using differentiated PC12 cells. Intracellular accumulation of Zn2+ induced by the combined application of pyrithione (5 microM), a Zn2+ ionophore, and Zn2+ (10 microM) caused cell death and activated JNK and ERK, but not p38 MAPK. Preventing JNK activation by the expression of dominant negative SEK1 (SEKAL) did not attenuate Zn2+-induced cell death, whereas the inhibition of ERK with PD98059 and the expression of dominant negative Ras mutant (RasN17) significantly prevented cell death. Inhibition of protein kinase C (PKC) and phosphatidylinositol-3 kinase had little effect on Zn2+-induced ERK activation. Intracellular Zn2+ accumulation resulted in the generation of ROS, and antioxidants prevented both the ERK activation and the cell death induced by Zn2+. Therefore, we conclude that although Zn2+ activates JNK and ERK, only ERK contributes to Zn2+-induced cell death, and that ERK activation is mediated by ROS via the Ras/Raf/MEK/ERK signaling pathway.

Protein kinase Dyrk1 activates cAMP response element-binding protein during neuronal differentiation in hippocampal progenitor cells.

Dyrk is a dual specific protein kinase thought to be involved in normal embryo neurogenesis and brain development. Defects/imperfections in this kinase have been suggested to play an important role in the mental retardation of patients with Down's syndrome. The transcriptional factor cAMP response element-binding protein (CREB) has been implicated in the formation of many types of synaptic plasticity, such as learning and memory. In the present study we show that Dyrk1 activity is markedly induced during the differentiation of immortalized hippocampal progenitor (H19-7) cells. The addition of a neurogenic factor, basic fibroblast growth factor, to the H19-7 cells results in an increased specific binding of Dyrk1 to active CREB. In addition, Dyrk1 directly phosphorylates CREB, leading to the stimulation of subsequent CRE-mediated gene transcription during the neuronal differentiation in H19-7 cells. Blockade of Dyrk1 activation significantly inhibits the neurite outgrowth as well as CREB phosphorylation induced by basic fibroblast growth factor. These findings suggest that Dyrk1 activation and subsequent CREB phosphorylation is important in the neuronal differentiation of central nervous system hippocampal cells.

IFN-gamma induces cell death in human hepatoma cells through a TRAIL/death receptor-mediated apoptotic pathway.

We demonstrated the induction of cell death in a hepatoma cell line by IFN-gamma and its possible mechanism. Among the 2 hepatitis B virus (HBV)-associated hepatoma cell lines, SNU-354 and SNU-368, IFN-gamma induced cell death and increased caspase-3 activity in SNU-368 but not in SNU-354. IFN-gamma induced several changes in the mRNA expression level of apoptosis-regulating genes, e.g., increased expression of Fas, caspase-1 and TNF-related apoptosis-inducing ligand (TRAIL). In particular, IFN-gamma potently increased the mRNA expression of TRAIL in both cell lines. However, it did not change the mRNA expression level of death-mediating TRAIL receptors, e.g., DR4 and DR5, which were constitutively expressed in both cell lines. In contrast, the decoy receptor DcR1 was expressed in SNU-354 but not in SNU-368, and its expression level in SNU-354 was increased by IFN-gamma. Another decoy receptor, DcR2, was constitutively expressed in both cell lines; however, its expression level in SNU-368 was decreased by IFN-gamma. In addition, exogenous recombinant TRAIL reduced viability in SNU-368, but not in SNU-354, cells. From these findings, we speculated that TRAIL up-regulation and the subsequent TRAIL-mediated apoptosis serve as a mechanism of IFN-gamma-induced cell death in SNU-368. To confirm this hypothesis, we demonstrated that soluble DR4-Fc fusion protein, a TRAIL pathway inhibitor, inhibited IFN-gamma-induced cell death in SNU-368. Our results demonstrated that IFN-gamma acts as an inducer of cell death through TRAIL-mediated apoptosis.

Elevated plasma endothelial microparticles in multiple sclerosis.

OBJECTIVE: To assess endothelial dysfunction in patients with MS and to investigate whether plasma from patients with MS induces endothelial cell dysfunction in vitro. BACKGROUND: Endothelial cell dysfunction may contribute to the pathogenesis of MS. Elevations of soluble adhesion molecules intracellular adhesion molecule, vascular cell adhesion molecule, and platelet-endothelial cell adhesion molecule-1 (CD31) have been reported as markers of blood-brain barrier (BBB) damage in MS, but direct assay of endothelium has been difficult. Endothelial cells release microparticles < approximately 1.5 microm (EMP) during activation or apoptosis. The authors developed a flow cytometric assay of EMP and studied EMP as markers of endothelial damage in MS. METHODS: Platelet-poor plasma (PPP) from 50 patients with MS (30 in exacerbation and 20 in remission) and 48 controls were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD31 and anti-CD51 (vitronectin receptor) antibodies, and two classes of EMP (CD31+ and CD51+) were assayed by flow cytometry. For in vitro studies, patients' plasma was added to the microvascular endothelial cell (MVEC) culture and release of CD31+ and CD51+ EMP were measured in the supernatant. RESULTS: Plasma from patients in exacerbation had 2.85-fold elevation of CD31+ EMP as compared with healthy controls, returning to near control value during remission. The CD31+ EMP concentration showed a positive association with gadolinium enhancement in patients with MS. In contrast, CD51+ EMP remained elevated in both exacerbation and remission. This suggests that CD31+ EMP is a marker of acute injury, whereas CD51+ EMP reflects chronic injury of endothelium. MS plasma induced release of both CD31+ and CD51+ EMP from MVEC culture in vitro. CONCLUSION: Endothelial dysfunction is evident during exacerbation of MS, evidenced by shedding of EMP expressing PECAM-1 (CD31). The in vitro data indicate contribution of one or more plasma factors in endothelial dysfunction of MS.

Induction of neuronal cell death by Rab5A-dependent endocytosis of alpha-synuclein.

The presynaptic alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. Here we examined the pathogenic mechanism of neuronal cell death induced by alpha-synuclein. The exogenous addition of alpha-synuclein caused a marked decrease of cell viability in primary and immortalized neuronal cells. The neuronal cell death appeared to be correlated with the Rab5A-specific endocytosis of alpha-synuclein that subsequently caused the formation of Lewy body-like intracytoplasmic inclusions. This was further supported by the fact that the expression of GTPase-deficient Rab5A resulted in a significant decrease of its cytotoxicity as a result of incomplete endocytosis of alpha-synuclein.

Basic fibroblast growth factor-induced activation of novel CREB kinase during the differentiation of immortalized hippocampal cells.

Growth factors bind to their specific receptors on the responsive cell surface and thereby initiate dramatic changes in the proliferation, differentiation, and survival of their target cells. In the present study we have examined the mechanism by which growth factor-induced signals are propagated to the nucleus, leading to the activation of transcription factor, cis-acting cAMP response element (CRE)-binding protein (CREB), in immortalized hippocampal progenitor cells (H19-7). During the differentiation of H19-7 cells by basic fibroblast growth factor (bFGF) a critical regulatory Ser(133) residue of CREB was phosphorylated followed by an increase of CRE-mediated gene transcription. Expression of S133A CREB mutants blocked the differentiation of H19-7 cells by bFGF. Although the kinetics of CREB phosphorylation by EGF was transient, bFGF induced a prolonged pattern of CREB phosphorylation. Interestingly, bFGF-induced CREB phosphorylation and subsequent CRE-mediated gene transcription is not likely to be mediated by any of previously known signaling pathways that lead to phosphorylation of CREB, such as mitogen-activated protein kinases, protein kinase A, protein kinase C, phosphatidylinositol 3-kinase-p70(S6K), calcium/calmodulin dependent protein kinase, and casein kinase 2. By using in vitro in gel kinase assay the presence of a novel 120-kDa bFGF-inducible CREB kinase was identified. These findings identify a new growth factor-activated signaling pathway that regulates gene expression at the CRE.

Elevated endothelial microparticles in thrombotic thrombocytopenic purpura: findings from brain and renal microvascular cell culture and patients with active disease.

Endothelial injury is believed to be a key initiating event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), leading to platelet activation and formation of platelet-rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and clinical assays to rapidly and reliably monitor endothelial damage are not readily available. Using flow cytometry, we measured endothelial microparticles (EMPs) generated from cultured renal and brain microvascular endothelial cells (MVECs) during activation and apoptosis, and evaluated the effect of TTP plasma on them. EMPs were measured using positivity for monoclonal antibodies (mAbs) CD31 and CD51, and their procoagulant activity was assessed using a Russell viper venom assay. Both cell lines generated procoagulant EMPs when cultured with inducers of activation (tumour necrosis factor alpha; TNF-alpha) or apoptosis (mitomycin C). TTP plasma induced a five- to sixfold increase of EMP generation and a two- to threefold increase of procoagulant activity in cultured brain and renal MVECs. TTP plasma induced a threefold and 13-fold increase of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, respectively, on renal MVECs. Procoagulant activity tended to parallel EMP numbers. The effect of TTP plasma on cell viability was similar to that of TNF-alpha, implying that it induced activation rather than apoptosis. Control plasma and idiopathic thrombocytopenic purpura (ITP) plasma had little effect. In the clinical study, EMP assay of blood from acute TTP patients showed levels markedly elevated compared with normal controls, but values returned to normal in remission. In conclusion, TTP plasma activated and induced injury to MVECs in culture, judged by production of EMP and expression of activation markers. Released procoagulant EMP may play a role in the pathogenesis of TTP. Assay of EMP may be a useful marker of disease activity and endothelial injury in TTP and possibly other thrombotic disorders.

Intracellular calcium mobilization induces immediate early gene pip92 via Src and mitogen-activated protein kinase in immortalized hippocampal cells.

Regulation of intracellular calcium levels plays a central role in cell survival, proliferation, and differentiation. A cell-permeable, tumor-promoting thapsigargin elevates the intracellular calcium levels by inhibiting endoplasmic reticulum Ca(2+)-ATPase. The Src-tyrosine kinase family is involved in a broad range of cellular responses ranging from cell growth and cytoskeletal rearrangement to differentiation. The immediate early gene pip92 is induced in neuronal cell death as well as cell growth and differentiation. To resolve the molecular mechanism of cell growth by intracellular calcium mobilization, we have examined the effect of thapsigargin and subsequent intracellular calcium influx on pip92 expression in immortalized rat hippocampal H19-7 cells. An increase of intracellular calcium ion levels induced by thapsigargin stimulated the expression of pip92 in H19-7 cells. Transient transfection of the cells with kinase-inactive mitogen-activated protein kinase kinase (MEK) and Src kinase or pretreatment with the chemical MEK inhibitor PD98059 significantly inhibited pip92 expression induced by thapsigargin. When constitutively active v-Src or MEK was overexpressed, the transcriptional activity of the pip92 gene was markedly increased. Dominant inhibitory Raf-1 blocked the transcriptional activity of pip92 induced by thapsigargin. The transcription factor Elk1 is activated during thapsigargin-induced pip92 expression. Taken together, these results suggest that an increase of intracellular calcium ion levels by thapsigargin stimulates the pip92 expression via Raf-MEK-extracellular signal-regulated protein kinase- as well as Src kinase-dependent signaling pathways.

Health development experience in North and South Korea.

The purpose of this study is to compare the difference in health status between South Koreans and North Koreans and to identify factors responsible for the remarkable improvements in the health status of South Koreans. In order to examine the causes of the difference in health level, the health indices and their determinants of two Koreas were analyzed in time order. As of the year 2000, the average life expectancy at birth is 71.0 years for men and 78.6 years for women in South Korea, which is longer than that of North Korea by 8.1 for men and 11.2 for women. Infant mortality rate in 1998 was 9.0 per 1,000 live births in South Korea and 54.0 in North Korea. Since being liberated from Japanese ruling in 1945, South Korea has achieved remarkable economic growth under democracy and a market economy system. On the other hand, North Korea has maintained a socialistic system. North Korea has suffered from economic crisis since the 1990s. From this point it could be said that economic status is the major factor for the differences in health level between the two Koreas. Economic status not only directly influences health level but also indirectly affects it through influences on nutrition, hygiene, health resources, and other intervening factors. The South Korean government has concentrated its limited resources on public health activities such as tuberculosis control, family planning (FP), and maternal and child health (MCH) programmes whereas the private sector has taken charge of constructing the health delivery system including health facilities and human resources. In order to solve the problem, which might occur in the private-oriented medical care system, the South Korean government has introduced the national health insurance programme and enforced regulation policies. Many developing countries which are suffering from poverty and disease, can learn from the experience of Korea that had suffered from similar problems up to the early 1970s.