Correlations of pre- and post-operative symptoms with cytokines in different phenotypes and endotypes of chronic rhinosinusitis.

BACKGROUND: Recognising inflammatory endotypes in chronic rhinosinusitis (CRS) has become more important, especially with the advent of biological treatments. In this study, we investigated the correlations of pre- and post-operative symptoms with cytokine positivity in different endotypes and phenotypes of CRS. METHODOLOGY: In total, 102 patients undergoing routine functional endoscopic sinus surgery were enrolled. The endotype classification (type 1, 2, or 3 CRS) was defined based on positivity for interferon-γ, interleukin (IL)-5, or IL-17 respectively, in sinonasal tissue samples. Clinical symptom scores were evaluated pre- and post-operatively using the 22-item Sinonasal Outcome Test and its four symptom subdomains: sleep, nasal, otologic/facial symptoms, and emotional function. Symptoms were compared between endotypes and phenotypes, and exploratory factor analysis (EFA) based on principal component analysis (PCA) was performed. The correlations of cytokine levels with baseline symptoms and changes in symptoms after 1 year were analysed. RESULTS: Symptoms in the otologic/facial pain category were associated with non-type 2 endotypes in PCA and confirmatory analysis. Non-type 2 CRS patients exhibited significantly more improvement in facial symptoms 1 year after surgery. Neutrophil-associated cytokines, such as IL-17, matrix metalloproteinase 9, and myeloperoxidase, were significantly correlated with baseline otologic/facial pain symptoms and changes in those symptoms after surgery. CONCLUSIONS: Otologic/facial pain symptoms may be indicative of non-type 2 endotypes. Neutrophil-associated cytokines, such as IL-17, MMP-9, and MPO, were significantly correlated with these symptoms. The establishment of links between specific symptoms and certain cytokines may help use and develop biological therapies for CRS.

Airway Epithelial Cells Drive Airway Smooth Muscle Cell Phenotype Switching to the Proliferative and Pro-inflammatory Phenotype.

The increased mass of airway smooth muscle (ASM) in the airways of asthmatic patients may contribute to the pathology of this disease by increasing the capacity for airway narrowing. Evidence for the airway epithelium as a participant in ASM remodeling is accruing. To investigate mechanisms by which airway epithelial cells induce ASM cell (ASMC) proliferation, we have employed a co-culture model to explore markers of ASMC proliferative phenotype. Co-culture with epithelial cells led to incorporation of bromodeoxyuridine into ASMCs, indicating augmented proliferation and an associated increase in mRNA of the pro-proliferative co-transcription factor Elk1. Although the mitogen heparin-binding epidermal growth factor (HB-EGF) was augmented in the co-culture supernatant, the ASMC epidermal growth factor receptor (EGFR), an effector of HB-EGF induced proliferation, did not mediate epithelial-induced proliferation. The co-culture increased the expression of ASMC mRNA for the pro-inflammatory cytokines IL-6 and IL-8 as well as the pro-proliferative microRNA miR-210. The transcriptional repressor Max-binding protein (Mnt), a putative target of miR-210, was transcriptionally repressed in co-cultured ASMCs. Together, these data indicate that the airway epithelium-induced proliferative phenotype of ASMCs is not driven by EGFR signaling, but rather may be dependent on miR210 targeting of tumor suppressor Mnt.

Simultaneous determination of arbutin and its decomposed product hydroquinone in whitening creams using high-performance liquid chromatography with photodiode array detection: Effect of temperature and pH on decomposition.

OBJECTIVE: Arbutin is an effective agent for the treatment of melanin disorders. Arbutin may be converted to hydroquinone under conditions of high temperature, ultraviolet (UV) radiation and dilute acid. The aim of the current study was to develop an analytical method to determine the levels of arbutin and hydroquinone in whitening cosmetic products using high-performance liquid chromatography with photodiode array detection (HPLC-DAD). In addition, we investigated the effects of high temperature and pH on the decomposition of arbutin. METHODS: Samples extracted using two-step sonications were separated on a C18 column using a gradient mobile phase consisting of water and methanol. A 60-mm (40 µL) DAD cell was used to enhance the sensitivity of hydroquinone determination. Thermal decomposition of arbutin was evaluated at temperatures ranging from 60 to 120°C for 1-36 h. RESULTS: The method showed good linearity (R(2) ≥ 0.9997), precision (relative standard deviation, RSD < 5%) and acceptable extraction recovery (90-102.6%). The limits of quantitation for arbutin and hydroquinone were 0.0085 and 0.0119 µg mL(-1) , respectively. One sample of 21 cosmetic products tested contained arbutin at a concentration 1.61 g 100 g(-1) cream and 0.12 g 100 g(-1) cream of hydroquinone. Arbutin (327.18 ppm) decomposed after 6 h at 120°C and produced 10.73 ppm of hydroquinone. CONCLUSION: The developed method is simple to detect both arbutin and hydroquinone simultaneously in cosmetic products, at an adequate level of sensitivity. Notably, temperature and pH did not influence the decomposition of arbutin to hydroquinone in a 2% arbutin cream.

Successful rescue of early graft failure in pediatric patients using T-cell-depleted haploidentical hematopoietic SCT.

Graft failure (GF) is a significant complication after allogeneic hematopoietic stem cell transplantation (HCT) and is associated with a high mortality rate. We performed re-transplantation using haploidentical-related donors to rescue children with early GF. Between 2008 and 2013, 10 patients received re-transplantation from haploidentical family donors. The median age at HCT was 13.5 years and the median time between transplantations was 52.5 days. Conditioning regimen with fludarabine and CY was used in seven patients, and TBI was added in three patients. All 10 patients received T-cell-depleted grafts using CD3 or CD3/CD19 MoAb. The median numbers of CD34(+) and CD3(+) cells were 5.52 × 10(6)/kg and 1.08 × 10(6)/kg, respectively. For GVHD prophylaxis, mycophenolate mofetil (MMF) and tacrolimus or MMF and CYA were used. All 10 patients achieved a sustained neutrophil engraftment and maintained a complete donor chimerism at the time of analysis (median 23 months, range 6-62 months). Nine of 10 patients were alive, and one patient with moyamoya disease with AML died of encephalopathy 7 months post transplant. This study suggests that fludarabine- and CY-based conditioning with T-cell-depleted haploidentical HCT is a feasible option to rescue pediatric patients with primary GF.

Anti-obesity effect of kimchi fermented with Weissella koreensis OK1-6 as starter in high-fat diet-induced obese C57BL/6J mice.

AIMS: In this study, we investigated the anti-obesity effects of kimchi (Korean traditional fermented vegetable) fermented either without starter culture or with a specific starter culture, Weissella koreensis OK1-6. METHODS AND RESULTS: C57BL/6J mice were divided into four groups (n = 7); normal diet, HF (high-fat diet), HF-KC (high-fat diet containing 3% kimchi manufactured without starter) and HF-KCO (high-fat diet containing 3% kimchi manufactured with the starter culture W. koreensis OK1-6). After 12 weeks of dietary intervention, the mice were killed, and serum and tissue samples were examined. Serum and hepatic lipid profile, insulin, leptin concentration and expression level of lipid anabolic genes like peroxisome proliferator-activated receptor γ, stearoyl-CoA desaturase-1, liver X receptor α and SREBP2 were significantly decreased (<0.05) along with body and epididymal fat pad weight in the HF-KCO group compared with the HF-KC and HF group. CONCLUSIONS: These results suggested that kimchi fermented with the starter W. koreensis OK1-6 has anti-obesity effects in HF-induced obese mice. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may contribute to nutraceutical and food industries in developing functional food and probiotics based therapies for the treatment and prevention of obesity.

Adenosine A2A receptor polymorphisms in Korean patients with systemic sclerosis.

Adenosine A2A receptor (ADORA2A) regulates inflammation, promotes tissue repair and collagen production by human dermal fibroblasts. We investigated the genetic polymorphisms of ADORA2A in susceptibility to systemic sclerosis (SSc). We genotyped 142 Korean SSc patients and 150 controls for polymorphisms of -1751A/C (rs5996696) and 1976C/T (rs5751876), to cover the promoter and all exon sequences of ADORA2A in Koreans, using TaqMan fluorogenic 5' nuclease assay and single base primer extension assay. Neither -1751A/C nor 1976C/T polymorphism showed difference in the distribution of alleles or genotypes between patients and controls with allele frequency of 89.9% v 91.0% for -1751A (p=0.64) and 56.5% v 54.0% for 1976C (p=0.55). Our findings suggest that the role of ADORA2A in SSc may not be genetically related.

The safety of simultaneous bilateral versus unilateral total knee arthroplasty: the experience in a Korean hospital.

INTRODUCTION: The safety of simultaneous bilateral total knee arthroplasty (SBTKA) remains controversial. This study aimed to compare the postoperative outcomes of SBTKA versus unilateral total knee arthroplasty (UTKA) performed by a single surgeon at a tertiary teaching hospital in Korea. METHODS: 629 female patients with total knee arthroplasty (308 patients for SBTKA and 321 for UTKA) performed under combined spinal epidural anaesthesia (CSE) were selected, and their medical records during admission and follow-up visits for a duration of six months after discharge were reviewed. RESULTS: Although significantly higher incidences of postoperative confusion and hypoxia during hospitalisation and a longer hospital stay were demonstrated in the SBTKA group, the rates of serious postoperative complications, such as myocardial infarction and deep venous thrombosis, were not different between the groups during the hospital stay and six months afterwards. No death associated with the surgery was encountered in both groups. CONCLUSION: It was concluded that SBTKA under CSE may be considered to be relatively safe in Korean female patients.

Hepatic veno-occlusive disease in children after hematopoietic stem cell transplantation: incidence, risk factors, and outcome.

Four hundred and sixty-seven hematopoietic stem cell transplantations (HSCTs) (217 autologous and 250 allogeneic HSCT) were performed in 374 children at four pediatric HSCT centers in Korea from January 2005 to December 2007. Among 467 transplants, veno-occlusive disease (VOD) developed in 72 transplants (15.4%) at a median of 10 days after HSCT. Multivariate analysis showed that BU or TBI-containing regimen (P=0.002), VOD prophylaxis without lipo-prostaglandin E1 (PGE1) (P=0.012), number of previous HSCT (P=0.014), and pretransplant serum ferritin (P=0.018) were independent risk factors for developing VOD. Mean serum ferritin levels were significantly higher in HSCT with VOD (2109.6+/-2842.5 ng/ml) than in HSCT without VOD (1315.9+/-1094.4 ng/ml) (P<0.001). The relative risk of death within 100 days of HSCT in transplants with VOD compared with transplants without VOD was 3.39 (confidence interval: 1.78-6.45). Our results suggest that lipo-PGE1 might have a protective effect against the development of VOD, and pretransplant serum ferritin could act as a risk factor for VOD. A larger prospective study is needed to confirm a possible role of lipo-PGE1 and iron chelation therapy in reducing the incidence of VOD.

Serum levels of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) and pentraxin 3 (PTX3) as markers of infection in febrile patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the role of sTREM-1 and PTX3 as markers of infection in febrile patients with SLE. METHODS: In febrile (body temperature > or =38 degrees C) patients with SLE, blood samples of day 0, 1, 2, and 14 after presentation were drawn and relevant clinical data were collected. The patients were allocated to an infection group (n=19) or disease flare group (n=14). Serum levels of sTREM-1 and PTX3 were measured by ELISA using the serum samples of SLE patients and age- and sex-matched healthy controls (n=31). RESULTS: A total of 33 febrile episodes occurred in 32 SLE patients (19 infections, 14 flares) were studied. sTREM-1 levels on day 0 were significantly higher in the infection group than in the flare group (109.9 pg/ml (median) vs. 48.0 pg/ml, p=0.002), but PTX3 levels were similar in these two groups. The difference of sTREM-1 levels between infection group and flare group was persistent on day 1 and 2 (day 1, p=0.007; day 2, p=0.034). The highest diagnostic value (sensitivity=1.0, specificity=0.664) of sTREM-1 was obtained at the threshold value of 53.2 pg/mL. CONCLUSION: Serum sTREM-1 levels were significantly higher in the infection group than in the flare group of febrile SLE patients. Our findings suggest that serum sTREM-1 levels could be used to determine whether SLE patients have contracted an infection.

Serotonin transporter gene polymorphism and irritable bowel syndrome.

Polymorphisms in the promoter region of the serotonin reuptake transporter (SERT) gene may underlie the disturbance in gut function in patients with irritable bowel syndrome (IBS). Association studies of SERT polymorphisms and IBS have shown diverse results among different countries, which might be due to racial and subject composition differences. The aim of this study was to assess the potential association between SERT polymorphisms and IBS in Koreans. A total of 190 IBS patients, who met the Rome II criteria, and 437 healthy controls were subjected to genotyping. SERT polymorphisms differed in the IBS and control groups (P = 0.014). The SERT deletion/deletion genotype occurred with greater frequency in the diarrhoea-predominant IBS group than in the controls. A strong genotypic association was observed between the SERT deletion/deletion genotype and diarrhoea-predominant IBS (P = 0.012). None of the clinical symptoms analysed was significantly associated with the SERT genotypes. The frequency of the SERT insertion/insertion genotype was much lower than that of the other two genotypes. A significant association was observed between the SERT polymorphism and IBS, especially diarrhoea-predominant IBS, suggesting that the SERT gene is a potential candidate gene involved in IBS in Korea.

Regional differences of superoxide dismutase activity enhance the superoxide-induced electrical heterogeneity in rabbit hearts.

During myocardial ischemia and the subsequent reperfusion, free radicals are important intermediates of the cellular damage and rhythm disturbances. We examined the effects of superoxide radicals or hydrogen peroxide (H(2)O(2)) on the action potentials in isolated rabbit Purkinje fibers, atrial muscle and ventricular muscle. Reactive oxygen species (ROS) donors such as adriamycin, xanthine/xanthine oxidase and menadione induced prolongation of APD(90) in Purkinje fibers. Menadione (30 microM), the most specific superoxide radical donor, prolonged the action potential duration at 90% repolarization (APD(90)) by 17% in Purkinje fibers, whereas it shortened the APD by 57% in ventricular muscle, and it did not affect the atrial APD. All these menadione-induced effects were completely blocked by 2,2,6,6-tetramethyl- 1-peperadinyloxy, a superoxide radical scavenger. Superoxide dismutase (SOD) activity was lowest in Purkinje fibers, it was moderate in atrial muscle and highest in ventricular muscle. H(2)O(2) shortened the APDs of all three cardiac tissues in a concentration-dependent manner. These results suggest that the different electrical responses to O(2) ([Symbol: see text]-) in different cardiac regions may result from the regional differences in the SOD activity, thereby enhancing the regional electrical heterogeneity.

Generation of the dominant-negative mutant of hArpNbeta: a component of human SWI/SNF chromatin remodeling complex.

hArpNbeta, an actin-related protein located within the nucleus, is a subunit of the human SWI/SNF chromatin remodeling complex. hArpNbeta has been proposed to regulate the assembly and activity of the hSWI/SNF complex. Sequence comparisons of the potential ArpN homologs with beta-actin showed that the ArpNs have the divergent subdomains Ib and IIb in addition to the unique N-terminal short insert, MS(G/A)-(V/L)YGG. Since the proposed function of hArpNbeta requires more than two distinct but concurrently operating surfaces, we examined whether the disruption of one operating surface of hArpNbeta results in dominant-negative phenotype. When overexpressed in HeLa or 293T cells, the subdomain Ib or IIb hybrids, in which the subdomain Ib or IIb of hArpNbeta was replaced with that of beta-actin, respectively, showed no effect on cell survival. On the other hand, the overexpression of the N-terminal deletion mutant of hArpNbeta resulted in cell death probably through apoptotic process. These results indicate that the proper function of hArpNbeta is essential for cell survival in human cells. Furthermore, they suggests the possibility that the N-terminal short sequence is indispensable for the chromatin remodeling activity or the assembly of the hSWI/SNF complex after the binding of hArpNbeta with functionally essential partner proteins.

Effects of occupational metallic mercury vapour exposure on suppressor-inducer (CD4+CD45RA+) T lymphocytes and CD57+CD16+ natural killer cells.

OBJECTIVES: To examine the effects of metallic mercury vapour on the cellular and humoral immune system. METHODS: We measured T lymphocyte and natural killer (NK) cell subpopulations, B lymphocytes, and serum immunoglobulins (i.e. IgG, IgA and IgM) together with total T (CD3 +) lymphocytes and total lymphocytes in blood samples from 20 male, fluorescent-lamp makers (mercury workers) and the same number of gender-, age- and smoking-matched controls. Urinary concentrations of inorganic mercury (UHg) in the 20 workers ranged from 1.8 to 163.5 (mean 44.8) microg/l. They had been exposed to mercury vapour for 4 to 62 (mean 31) months. RESULTS: Numbers of CD4+CD45RA+ (suppressor-inducer) T lymphocytes and total CD4+ T lymphocytes in the mercury workers were significantly smaller than those in the controls (paired-sample t-test, P < 0.01). The number of CD57+CD16+ NK cells was inversely correlated with UHg. CONCLUSION: It is suggested that numbers of CD4+CD45RA+ T lymphocytes and CD57+CD16+ NK cells are inversely affected by exposure to metallic mercury vapour in workers, with an average urinary inorganic mercury concentration of 45 microg/l being found.

Factors associated with false-positive staging of lung cancer by positron emission tomography.

BACKGROUND: Positron emission tomography imaging is gaining popularity as a noninvasive staging tool in non-small cell lung cancer. Nonmalignant processes can also affect radio-tracer uptake. This study seeks to identify factors associated with false-positive staging of mediastinal metastases. METHODS: A retrospective review was performed of 100 patients with early stage non-small cell lung cancer referred for positron emission tomography scan evaluation. All had pathologic confirmation of their disease. Positron emission tomography scans, radiology records, operative reports, and pathology results were reviewed. Patients with positron emission tomography scans interpreted as positive for mediastinal involvement and negative pathology at operation were selected. RESULTS: Seven patients were found to have a false-positive positron emission tomography evaluation for mediastinal metastases. All but 1 patient had a concurrent inflammatory process or an anatomic factor associated with the false positive. The sensitivity and specificity in detecting involved mediastinal nodes was 87.5% and 90.7%, respectively. The negative predictive value was 95.8%. CONCLUSIONS: Although positron emission tomography has been established as an accurate modality to stage non-small cell lung cancer, false-positive evaluation of mediastinal metastases can occur in the setting of concurrent inflammatory lung diseases or for centrally located tumors. Pathologic evaluation of mediastinal disease should be pursued whenever suggested by a positive positron emission tomography scan especially in the face of those factors described.

Structure and stress-related expression of two cDNAs encoding proteinase inhibitor II of Nicotiana glutinosa L.

Two cDNAs, pNGPI-1 and pNGPI-2, encoding Nicotiana glutinosa proteinase inhibitor II (PI-II) have been cloned, sequenced and identified. The deduced amino acid sequences are 54-82% identical to those of other plant PI-II. The NGPI-1 protein is composed of eight repeated domains, while NGPI-2 contains six repeated regions, each with a putative reactive site. The expression of NGPI-1 is highly regulated in a developmental- and tissue-specific manner, with the transcript being detected in young leaves and floral organs of N. glutinosa plants. In mature leaves, the NGPI-1 gene is rapidly activated by distinct temporal induction patterns in response to pathogen-related (biotic) and wound-related (abiotic) stresses.

TCR v(beta) usage and clonality of T cells isolated from progressing and rejected tumor sites before and after in vitro culture.

A gelatin sponge model of concomitant tumor immunity was employed in order to examine the clonality of T cells associated with progressing and rejected tumor sites. Here we show that freshly isolated T cells bearing TCR V(beta)1, CDR3 RPGTGN, J(beta)1.1 and TCR V(beta)8, CDR3 GD, J(beta)1.6 predominated progressing and rejected tumor sites. Despite the similarity in T cell populations, the T cells from rejected tumor sites were capable of killing the autologous tumor cells, whereas T cells from progressing tumor sites were not able to do so. The differing cytolytic ability could not be attributed to a difference in TCR zeta chain protein expression levels between both T cell populations. After a 5 day mixed lymphocyte tumor culture the T cells from the progressing tumor site were capable of killing autologous tumor cells, which suggested changes took place within the cell population during in vitro culture. Further TCR analysis revealed T cells bearing TCR V(beta)1, CDR3 RPGTGN, J(beta)1.1 and TCR V(beta)8, CDR3 GD, J(beta)1.6 were not expanded following the in vitro culture. These data suggest that the lack of cytotoxicity of freshly isolated tumor-infiltrating lymphocytes (TIL) was not due to abnormal TCR zeta chain expression or major differences in the TCR V(beta) usage. Additionally, the gain of TIL effector function did not correlate with an expansion of the TCR bearing T cells found to predominate the in vivo response. These data suggest that the predominant TCR V(beta) used by lymphocytes infiltrating regressing or rejected tumors may not represent the tumor reactive T cells that grow in culture or respond to the autologous tumor in vitro.

Co-induction of p75NTR and p75NTR-associated death executor in neurons after zinc exposure in cortical culture or transient ischemia in the rat.

Recently, a 22 kDa protein termed p75(NTR)-associated death executor (NADE) was discovered to be a necessary factor for p75(NTR)-mediated apoptosis in certain cells. However, the possible role for p75(NTR)/NADE in pathological neuronal death has yet been undetermined. In the present study, we have examined this possibility in vivo and in vitro. Exposure of cortical cultures to zinc induced both p75(NTR) and NADE in neurons, whereas exposure to NMDA, ionomycin, iron, or H(2)O(2) induced neither. In addition, zinc exposure increased neuronal NGF expression and its release into the medium. A function-blocking antibody of p75(NTR) (REX) inhibited association between p75(NTR) and NADE as well as neuronal death induced by zinc. Conversely, NGF augmented zinc-induced neuronal death. Caspase inhibitors reduced zinc-induced neuronal death, indicating that caspases were involved. Because reduction of NADE expression with cycloheximide or NADE antisense oligonucleotides attenuated zinc-induced neuronal death, NADE appears to contribute to p75(NTR)-induced cortical neuronal death as shown in other cells. Because zinc neurotoxicity may be a key mechanism of neuronal death after transient forebrain ischemia, we next examined this model. After ischemia, p75(NTR) and NADE were induced in degenerating rat hippocampal CA1 neurons. There was a close correlation between zinc accumulation and p75(NTR)/NADE induction. Suggesting the role of zinc here, injection of a metal chelator, CaEDTA, into the lateral ventricle completely blocked the induction of p75(NTR) and NADE. Our results suggest that co-induction of p75(NTR) and NADE plays a role in zinc-triggered neuronal death in vitro and in vivo.

Induction of an immediate early gene egr-1 by zinc through extracellular signal-regulated kinase activation in cortical culture: its role in zinc-induced neuronal death.

Egr-1 is one of the immediate early transcription factors that are induced after brain insults. However, the mechanism and the role of Egr-1 induction are not yet determined. In the present study, using mouse cortical cultures, we examined the ionic mechanism of Egr-1 induction and its role in neuronal death. Although zinc, NMDA, or ionomycin induced comparable neuronal death in cortical culture, only zinc increased Egr-1 expression, which was attenuated by blocking zinc influx. It is intriguing that brief exposure to zinc induced sustained extracellular signal-regulated kinase (Erk) activation. PD098059, an inhibitor of the Erk 1/2 upstream kinase mitogen-activated protein kinase kinase 1 (MEK1), blocked Erk 1/2 activation, Egr-1 induction, and neuronal death by zinc. The present study has demonstrated that zinc, rather than calcium, induces lasting Egr-1 expression in cortical culture by activating Erk 1/2, which is part of a cascade that may play an active role in zinc neurotoxicity. We propose that translocation of endogenous zinc may be the key mechanism of Egr-1 induction and neuronal death in brain ischemia.

Caspase 3 specifically cleaves p21WAF1/CIP1 in the earlier stage of apoptosis in SK-HEP-1 human hepatoma cells.

We report here that p21WAF1/CIP1, an inhibitor of cyclin kinases, underwent proteolytic processing into a smaller fragment, p14, in the early stage of apoptosis in SK-HEP-1 cells. Apoptosis was induced by either staurosporine or ginsenoside Rh2, a ginseng saponin with a dammarane skeleton. Proteolytic processing was the result of caspase-3 activity, which accompanied the early changes in cell morphology and DNA fragmentation. p21WAF1/CIP1 translated in vitro was cleaved into a p14 fragment when incubated with cell extracts obtained from either ginsenoside Rh2-treated or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-CHO, a specific caspase-3 inhibitor, but not by Ac-YVAD-CHO, a specific caspase-1 inhibitor. Similarly, p21WAF1/CIP1 was efficiently cleaved by recombinant caspase-3, overexpressed in Escherichia coli. Moreover, the endogenous p21WAF1/CIP1 of untreated cell extracts was also cleaved by recombinant caspase 3, as measured by immunoblotting. Mutation analysis allowed identification of two caspase-3 cleavage sites, DHVD112/L and SMTD149/F, which are located within or near the interaction domains for cyclins, Cdks, and proliferating cell nuclear antigen (PCNA). Taken together, these results show that ginsenoside Rh2 and staurosporine increase caspase-3 activity, which in turn directly cleaves p21WAF1/CIP1 during the early stages of apoptosis. We propose that proteolytic cleavage of p21WAF1/CIP1 is a functionally relevant event that allows release of the cyclin/Cdk complex from the p21WAF1/CIP1 inhibitor, resulting in the elevated levels of cyclin/Cdk kinase activity seen in the earlier stage of apoptosis.