The importance of critically short telomere in myelodysplastic syndrome.

A few critically short telomeres trigger genomic instability regardless of average telomere length (TL). Recently, the telomere shortest length assay (TeSLA) was developed to detect critically short telomeres and measure absolute telomeres. Using TeSLA with the internally labeled biotin probe, we measured the TL of bone marrow (BM) aspirates from 52 patients with myelodysplastic syndrome (MDS). A percentage of shortest telomeres (< 1.0 kb (ShTL1.0)) were calculated. ShTL1.0 was correlated to IPSS-R risk (spearman's rho = 0.35 and p = 0.0196), and ShTL1.0 and BM blast (2.61% in < 5% blast, 4.15% in 5-10% blast, and 6.80% in 10-20% blast, respectively, p = 0.0332). Interestingly, MDS patients with a shortest TL ≥ 0.787 kb at the time of diagnosis showed better overall survival (OS) and progression-free survival (PFS) than patients with a shortest TL < 0.787 kb in the multivariate analyses (HR = 0.13 and 0.30, p = 0.011 and 0.048 for OS and PFS, respectively). Our results clearly show the presence and abundance of critically short telomeres in MDS patients. These pathologic telomeres are associated with IPSS-R which is a validated prognostic scoring system in MDS. Furthermore, they are independent prognostic factors for OS in MDS patients. Future prospective studies are needed to validate our results.

Increased Apoptotic Activity in Low-Risk Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous hematopoietic disorder associated with cellular proliferative and apoptotic activity. We retrospectively investigated these activities in bone marrow samples from 76 MDS patients using immunohistochemical staining for Ki-67 and cleaved caspase-3. We divided cleaved caspase-3 into two groups based on median value and compared the differences according to MDS risk scoring systems. We compared MDS patient indices with idiopathic cytopenia of undetermined significance (ICUS) and healthy control (HC) indices using our previously published data. Cleaved caspase-3 immunohistochemistry was highest in MDS patients, followed by ICUS patients and HCs. Similarly, the mean Ki-67 grade was also highest in MDS patients, followed by ICUS patients and HCs. Higher cleaved caspase-3 grade was significantly associated with lower IPSS-R score (p = 0.020), whereas Ki-67 was not associated with MDS. Interestingly, TET2 mutation was associated with decreased cleaved caspase-3 levels (p = 0.03). However, there was no significant association between proliferative/apoptotic activity and survival. Our results suggest that apoptotic activity gradually increases from healthy controls and ICUS patients to MDS patients. Furthermore, higher apoptotic activity was associated with better MDS patient prognostic scores. Further studies are needed to reveal the differences in apoptotic activity between lower- and higher-risk MDS.

Mutation of ten-eleven translocation-2 is associated with increased risk of autoimmune disease in patients with myelodysplastic syndrome.

BACKGROUND/AIMS: Myelodysplastic syndrome (MDS) is caused by genetic and epigenetic alteration of hematopoietic precursors and immune dysregulation. Approximately 20% of patients with MDS develop an autoimmune disease (AID). Here, we investigated whether particular genetic mutations are associated with AID in patients with MDS. METHODS: Eighty-eight genetic mutations associated with myeloid malignancy were sequenced in 73 MDS patients. The association between these mutations and AID was then analyzed. RESULTS: The median age of the 73 MDS patients was 70 years (interquartile range, 56 to 75), and 49 (67.1%) were male. AID was observed in 16 of 73 patients (21.9%). Mutations were detected in 57 (78.1%) patients. The percentage (68.8% vs. 80.7%, p = 0.32) and the mean number of mutations (1.8 ± 1.6 vs. 2.2 ± 1.8, p = 0.34) in MDS patients with or without AID were similar. However, the ten-eleven translocation- 2 (TET2) mutation rate was significantly higher in patients with AID than in those without (31.3% vs. 5.3%, respectively; p = 0.001). All TET2 mutations were variants of strong clinical significance. CONCLUSION: Mutation of TET2 in patients with MDS may be associated with increased risk of developing AID.

Replicative senescence of hematopoietic cells in patients with idiopathic cytopenia of undetermined significance.

We hypothesized that a subset of idiopathic cytopenia of undetermined significance (ICUS) is associated with an increased autonomous proliferation with exhaustion of hematopoiesis. The aim of this study was to investigate the cell turnover rate and replicative history of the bone marrow cells of ICUS patients. To this end, we examined telomere length (TL), proliferation, and apoptosis of the bone marrow cells of ICUS patients and healthy controls (HCs) using telomere quantitative fluorescence in situ hybridization and immunohistochemical staining for Ki-67 and cleaved caspase-3. We also performed targeted sequencing of 88 myeloid-associated genes. A total of 37 patients with ICUS were enrolled in this study, with a median age of 66 years (range: 31-83). TLs were significantly shorter in patients with ICUS than in the HCs (8.8, interquartile range [IQR] 6.8-12.1 vs 18.4, IQR 14.4-22.0, p < 0.0001). Proliferation (Ki-67-positive) and apoptosis (cleaved caspase-3-positive) were significantly increased in patients with ICUS compared to HCs (median = 20.0% vs 5.0%, p = 0.0003; 45.0% vs 22.5%, p = 0.0005, respectively). The shortening of TL and the increased proliferation and apoptotic activity was also prominent in patients with ICUS without mutation and dysplasia than in HCs (p < 0.0001, p < 0.0001, and p = 0.0093, respectively). TL was not associated with mutational profile and clinical characteristics as well in patients with ICUS. To our knowledge, this is the first study to show that ICUS is associated with premature replicative senescence with increased proliferation and apoptosis of bone marrow cells. Further study is needed to address the cause of replicative exhaustion in ICUS patients.

ASXL1 is a molecular predictor in idiopathic cytopenia of undetermined significance.

We analyzed the mutational profile of idiopathic cytopenia of undetermined significance (ICUS) compared with that of myelodysplastic syndrome (MDS). Targeted sequencing of 88 genes associated with myeloid malignancies was performed using samples of bone marrow mononuclear cells from ICUS and MDS patients. Forty patients with ICUS and 128 patients with MDS were included in this study. The median mutational burden was 0.7 mutation/person in the ICUS group and 2.2 mutation/person in the MDS group. ASXL1 (seven patients) was the most frequently mutated gene. ASXL1 was an independent significant prognostic factor for event-free survival (EFS) and overall survival (OS) (hazard ratio (HR) = 10.07 and 30.63, p = .004 and .003, respectively). The ASXL1 mutation which is frequently detected in elderly patients is a molecular predictor for pancytopenia and survival in patients with ICUS. A larger prospective study is needed to validate the role of this genetic mutation in an ICUS prognosis.

Manufacture of Clinical-Grade Human Clonal Mesenchymal Stem Cell Products from Single Colony Forming Unit-Derived Colonies Based on the Subfractionation Culturing Method.

Stem cell products derived from mesenchymal stem cells (MSCs) have been widely used in clinical trials, and a few products have been already commercialized. However, the therapeutic effects of clinical-grade MSCs are still controversial owing to mixed results from recent clinical trials. A potential solution to overcome this hurdle may be to use clonal stem cells as the starting cell material to increase the homogeneity of the final stem cell products. We have previously developed an alternative isolation and culture protocol for establishing a population of clonal MSCs (cMSCs) from single colony forming unit (CFU)-derived colonies. In this study, we established a good manufacturing practice (GMP)-compatible procedure for the clinical-grade production of human bone marrow-derived cMSCs based on the subfractionation culturing method. We optimized the culture procedures to expand and obtain a clonal population of final MSC products from single CFU-derived colonies in a GMP facility. The characterization results of the final cMSC products met our preset criteria. Animal toxicity tests were performed in a good laboratory practice facility, and showed no toxicity or tumor formation in vivo. These tests include single injection toxicity, multiple injection toxicity, biodistribution analysis, and tumorigenicity tests in vivo. No chromosomal abnormalities were detected by in situ karyotyping using oligo-fluorescence in situ hydridization (oligo-FISH), providing evidence of genetic stability of the clinical-grade cMSC products. The manufacture and quality control results indicated that our GMP methodology could produce sufficient clonal population of MSC products from a small amount of bone marrow aspirate to treat a number of patients.

Stochastic acquisition of a stem cell-like state and drug tolerance in leukemia cells stressed by radiation.

A rare population of leukemia cells have the properties of leukemia stem cells (LSCs) and cause resistance to therapy, but their development is not clearly understood. In the current study, we show that a higher resistance to cytotoxic drug (Ara-C) can be developed in the subpopulation of promyelocytic leukemia cells that survived radiation treatment. These drug-tolerant leukemia cells (DTLs) are not observed immediately after radiation despite extensive genetic instability in the cells, but appear in 3 weeks of recovery culture. Moreover, when the single cell-derived clones were examined by clonal trafficking, no correlation between radio-resistant and chemo-resistant leukemic clones was detected, indicating that the resistance is developed by active acquisition of the resistance without clonal predisposition. Interestingly, the DTLs mimicked the characteristics of LSCs exhibiting leukemia-initiating activities and lower levels of reactive oxygen species or a higher level expression of bmi-1 as well as higher resistance to retinoic acid-induced differentiation compared to parental leukemic cells. These studies show that an active reacquisition of stem cell-like properties can occur in the leukemia cells to develop resistance to treatments and that such reacquisition process of leukemic cells occurs in a stochastic manner triggered by radiation stress on leukemic cells.

The relationship between the presence of chromosomal instability and prognosis of squamous cell carcinoma of the lung: fluorescence in situ hybridization analysis of paraffin-embedded tissue from 47 Korean patients.

To evaluate the prognostic importance of chromosomal instability (CIN) in squamous cell carcinoma (SCC) of the lung, the relationship between CIN detected by fluorescence in situ hybridization (FISH) and survival in SCC patients was examined. Forty-seven surgical specimens of lung SCC were analyzed. To identify tumors with CIN, p16 and multi-target DNA FISH assays for c-myc, chromosome 6, EGFR, and chromosome 5 (LAVysion, Vysis) were performed on nuclei extracted from paraffin-embedded tumor tissues. Survival rates were compared in terms of age, T factor, N factor, CIN, and smoking status. A sample was defined as CIN-positive if at least four of the five chromosomes were positive. Among the 47 specimens, 9 (19%) were CIN-positive. The overall survival rate was 66%. Overall survival rates were estimated as 33.3% for CIN-positive patients and 76.7% for CIN-negative patients (Hazard ratio 3.47; 95% Confidence interval, 1.25-9.67; P=0.017). In multivariate analysis, the presence of CIN was a predictive factor for survival. CIN-positive based on FISH can be prognostic factor of lung SCC.

Sexual activity and hepatitis B and C virus infection among young adults after introduction of a vaccination program in an area of high endemicity.

BACKGROUND: In areas where hepatitis is endemic, little is known about the sexual transmission of HBV after introduction of an HBV vaccination program. METHODS: We used a self-administered questionnaire and serological tests for HBsAg, anti-HBs, anti-HBc, and anti-HCV to examine the role of sexual activity, as well as sociodemographic status, lifestyle habits, and a history of vaccinations, transfusions, and surgery, in the transmission of HBV and HCV in Korea. The subjects were 865 female and 541 male university students (median age, 19 years; age range, 16-25). RESULTS: Overall seropositivity was 8.1% for HBsAg, 69.3% for anti-HBs, 21.3% for anti-HBc, and 0.4% for anti-HCV. Regarding HBV, 8% of the subjects were chronic carriers or had recently been infected, 22.8% were never exposed and nonvaccinated, 16.6% were exposed noncarriers, and 52.7% had most likely been vaccinated. We found a significant association between HBsAg seropositivity and history of sexual intercourse (Odds Ratio, 1.8; 95% CI, 1.1-2.8). Students without serologic evidence of immunization against HBV were more likely to have become HBsAg-positive after becoming sexually active. CONCLUSIONS: Our findings suggest that sexual transmission does occur among adolescents and young adults who have not been vaccinated, whereas vaccination protects individuals from becoming an HBV carrier after becoming sexually active.

Clinical predictors of recurrent venous thromboembolism: a single institute experience in Korea.

INTRODUCTION: Racial disparities in incidence rate as well as risk factors for venous thromboembolism (VTE) exist between Asian and Western populations. Moreover, predictors for recurrent VTE were not identified in Asians. Thus, this study was undertaken to investigate risk factors for recurrent VTE events in Korean people. MATERIALS AND METHODS: Three hundred-three patients newly diagnosed as VTE were enrolled from Seoul National University Hospital. Recurrence rate based on risk factors for VTE were investigated. Cumulative incidence of recurrent VTE was calculated by the Kaplan and Meier method. Independent predictors for VTE were determined using Cox proportional hazards model. RESULTS: After a median follow-up of 44 months, 24 (8%) of 303 patients relapsed for a total observation time of 1,217 patient-year. Cumulative incidences of recurrent VTE were 3% at 1 year, 10% at 5 years, and 18% at 8 years. Independent predictors for recurrent VTE were presence of residual thrombosis (hazard ratio [HR]=3.1, 95% confidence interval [CI] 1.0-9.3; p=0.044), antiphospholipid syndrome (APS) (HR=4.3, 95% CI 1.0-19.0; p=0.052), and age 50 years or younger (HR=2.5, 95% CI 1.0-6.6; p=0.053) by multivariate analysis. Residual thrombosis and APS remained predictive of recurrence by the anticoagulation-period stratified analysis. CONCLUSIONS: In contrast to Western populations, Korean patients with VTE had the lower recurrent rate. Extended anticoagulation is necessary for Korean patients with residual thrombosis or APS.

Chromosomal instability is a risk factor for poor prognosis of adenocarcinoma of the lung: Fluorescence in situ hybridization analysis of paraffin-embedded tissue from Korean patients.

BACKGROUND: In this study, we sought to evaluate the prognostic importance of chromosomal instability (CIN) in adenocarcinoma (AC) of the lung. The relationship between CIN detected by fluorescence in situ hybridization (FISH) and survival in AC patients was examined. METHODS: Sixty-three surgical specimens of lung AC were analyzed. To identify tumors with CIN, p16 and multi-target DNA FISH assays for c-myc, chromosome 6, EGFR, and chromosome 5 (LAVysion, Vysis) were performed on nuclei extracted from paraffin-embedded tumor tissues. Survival rates were compared in terms of sex, age, histology, T factor, N factor, CIN, and smoking status. A sample was classified as CIN-positive if at least three of the five chromosomes were positive. RESULTS: Out of the 63 specimens, 32 (39.7%) were CIN-positive. The 5-year overall disease-free survival rate was 58.7% as a whole, 46.9% for CIN-positive patients and 71.0% for the CIN-negative patients [hazard ratio (HR), 2.34; 95% confidence interval (CI), 1.04-5.26; p = 0.04]. The 5-year overall survival rate was 81.0%, 68.7% for CIN-positive patients and 93.5% for the CIN-negative patients (HR, 5.64; 95% CI, 1.23-25.70; p = 0.026). In multivariate analysis after adjusting for pathologic nodal staging, tumor staging, sex, age, and smoking history, compared with the CIN-negative patients, the CIN-positive status remained significantly associated with decreased overall survival (HR, 8.48; 95% CI, 1.66-43.42; p = 0.010). CONCLUSIONS: CIN can be effectively detected in primary AC of lung using FISH analysis. CIN is associated with poor prognosis for AC, and may thus be utilized as an independent prognostic factor for the disease.

Trastuzumab inhibits the growth of human gastric cancer cell lines with HER2 amplification synergistically with cisplatin.

HER2 has been found to be amplified in 10-20% of gastric cancers, and is correlated with poor outcome. The aims of this study were to recognize HER2 amplification in gastric cancer cell lines via fluorescence in situ hybridization and to evaluate the growth inhibitory effect of trastuzumab in HER2-amplified cell lines. To elucidate the mechanism of the growth inhibition, we performed cell cycle analysis and immunoblotting of downstream molecules. We also conducted drug interaction studies of trastuzumab with other chemotherapeutic agents. HER2 amplification was newly identified only in SNU-216 cells, and trastuzumab moderately inhibited the growth of SNU-216 cells and positive controls. Trastuzumab-mediated G1 arrest occurred with increased expression of p27(KIP1) and decreased cyclins. Phosphorylation of HER2 and downstream molecules, STAT3, AKT, and ERK, was also inhibited by trastuzumab. Treatment of SNU-216 cells with trastuzumab plus cisplatin resulted in a synergistic inhibitory effect, whereas treatment of SNU-216 cells with trastuzumab plus 5-FU, or trastuzumab plus oxaliplatin produced an additive effect. These results suggest that trastuzumab combined with chemotherapeutic agents can be active against gastric cancer with HER2 amplification.

Combination of SK-7041, one of novel histone deacetylase inhibitors, and STI571-induced synergistic apoptosis in chronic myeloid leukemia.

Although STI571 still plays a key role in the treatment of chronic myeloid leukemia, emergence of resistance to STI571 is a major obstacle to successful outcome. Therefore, new agents that increase the sensitivity of chronic myeloid leukemia cells to STI571 are urgently required. SK-7041 is a novel hybrid synthetic histone deacetylase inhibitor derived from the hydroxamic acid of trichostatin A and pyridyl ring of MS-275. Its cytotoxic effects were examined both as a single agent and in combination with STI571 in acute and chronic myeloid leukemia. SK-7041 exhibited growth inhibition of leukemia cells by downregulation of CDK4, cyclin E and cyclin B1 expression, and by upregulation of p21 expression with subsequent activation of the mitochondria-mediated caspase pathway. SK-7041 showed synergism on growth inhibition, cell cycle arrest and induction of apoptosis in chronic myeloid leukemia (K562) when combined with STI571. The synergistic effect was mediated through the same mechanism as in SK-7041 alone, involving reduction of cyclin D1 and induction of p21. Taken together, our findings suggest that SK-7041 is active against leukemia and offers new prospects for overcoming STI571 resistance in chronic myeloid leukemia.

Increased risk of atherothrombotic events associated with cytochrome P450 3A5 polymorphism in patients taking clopidogrel.

BACKGROUND: Clopidogrel is a prodrug requiring metabolism by cytochrome P450 3A (CYP3A) isoenzymes, including CYP3A5, in order to be active. It is controversial whether clopidogrel interacts with CYP3A inhibitors. We investigated the influence of CYP3A5 polymorphism on the drug interaction of clopidogrel. METHODS: In phase 1 of the study, we administered clopidogrel to 16 healthy volunteers who had the CYP3A5 non-expressor genotype (*3 allele) and 16 who had the CYP3A5 expressor genotype (*1 allele) with and without pretreatment with itraconazole, a potent CYP3A inhibitor. A platelet aggregation test was performed at baseline, 4 hours, 24 hours and 6 days after clopidogrel administration. In phase 2, we compared clinical outcomes of 348 patients treated with clopidogrel after successful coronary angioplasty with bare-metal stent implantation according to their CYP3A5 genotype; the primary end point was a composite of atherothrombotic events (cardiovascular death, myocardial infarction and non-hemorrhagic stroke) within 1 and 6 months after stent implantation. RESULTS: In phase 1, the change in platelet aggregation after clopidogrel administration and pretreatment with itraconazole was greater among the subjects with the CYP3A5 expressor genotype than among those with the non-expressor genotype: 24.9% (standard deviation [SD] 13.9%) v. 6.2% (SD 13.5%) at 4 hours (p < 0.001); 27.7% (SD 16.5%) v. 2.5% (SD 8.3%) at 24 hours (p < 0.001); and 33.5% (SD 18.6%) v. 17.8% (SD 13.8%) at day 7 (p < 0.01). In phase 2, atherothrombotic events occurred more frequently within 6 months after stent implantation among the patients with the non-expressor genotype than among those with the expressor genotype (14/193 v. 3/155; p = 0.023). Multivariable analysis showed that the CYP3A5 polymorphism was a predictor of atherothrombotic events in clopidogrel users. INTERPRETATION: People with the CYP3A5 non-expressor genotype are vulnerable to drug interactions between clopidogrel and CYP3A inhibitors. This phenomenon may be associated with worse outcomes in patients with the non-expressor genotype who are given clopidogrel after coronary angioplasty and implantation of bare-metal stents.

Green tea extract inhibits paraquat-induced pulmonary fibrosis by suppression of oxidative stress and endothelin-l expression.

Paraquat-induced pulmonary fibrosis involves two factors, direct injury by oxygen free radicals and indirect injury by inflammatory cells and fibroblasts. Endothelin-1 (ET-1) has been shown to act as a mediator of pulmonary fibrosis, and its formation increases during oxidative stress. We investigated whether green tea extract (GTE), which has antioxidant properties, inhibits paraquat-induced pulmonary fibrosis and whether ET-1 is involved in this process. Paraquat (0.3 mg/kg) was instilled into the right lungs of rats, following which the rats were either not further treated (Group P, n = 7), or they were administered 1% GTE mixed with feed (Group PG; n = 7) or the ET(A) receptor antagonist ZD2574 (10 mg/kg through gavage; Group PZ; n = 7) for two weeks. As control, we used rats instilled with saline (Group N; n = 6). Two weeks after paraquat instillation, we assayed the degree of pulmonary fibrosis by light microscopic morphometry and hydroxyproline content; lipid peroxidation as a marker of oxidative stresses by measurement of malondialdehyde (MDA); ET-1 by immunohistochemistry; and prepro-ET-1 mRNA expression by reverse transcription-polymerase chain reaction. Compared with Group N, significant pulmonary fibrosis was observed in Group P, accompanied by increases in MDA, ET-1, and prepro-ET-1 mRNA expression. Compared with Group P, Group PG showed significant decreases in pulmonary fibrosis, along with decreases in MDA, ET-1, and prepro-ET-1 mRNA expression. We also observed significant decreases in pulmonary fibrosis in Group PZ compared with Group P. These findings suggest that GTE inhibits paraquat-induced pulmonary fibrosis by suppression of oxidative stress and ET-1 expression.

Identification of differentially expressed proteins in imatinib mesylate-resistant chronic myelogenous cells.

Resistance to imatinib mesylate (also known as Gleevec, Glivec, and STI571) often becomes a barrier to the treatment of chronic myelogenous leukemia (CML). In order to identify markers of the action of imatinib mesylate, we used a mass spectrometry approach to compare protein expression profiles in human leukemia cells (K562) and in imatinib mesylate-resistant human leukemia cells (K562-R) in the presence and absence of imatinib mesylate. We identified 118 differentially regulated proteins in these two leukemia cell-lines, with and without a 1 microM imatinib mesylate challenge. Nine proteins of unknown function were discovered. This is the first comprehensive report regarding differential protein expression in imatinib mesylate-treated CML cells.

Differential tyrosine phosphorylation of leukemic cells during apoptosis as a result of treatment with imatinib mesylate.

Bcr-Abl fusion tyrosine kinase contributes to leukemic transformation. Imatinib mesylate inhibits Bcr-Abl tyrosine kinase, resulting in a blockage of tyrosine phosphorylation in its downstream pathways. We analyzed the alteration of tyrosine phosphorylation, on BCR/ABL+ chronic myelogenous leukemia cells, after treatment with imatinib mesylate. Data were collected using a two-dimensional gel electrophoresis followed by Western blot and mass spectrometry. The inhibition of Bcr-Abl tyrosine kinase by 2.5 microM imatinib mesylate caused both cell cycle arrest in the G0/G1 phase and increased the portion of apoptotic cells. As a result, the population of leukemic cells decreased by 30% and 70% compared to controls at 24 and 72 h, respectively. Furthermore, treatment with imatinib mesylate altered tyrosine phosphorylation of 24 protein spots as the incubation time proceeded from 0 to 24 and 72 h. Ten of the 24 protein spots are visible at all three times. Four are detectable at both the 0 and 24 h points in time. Eight were detectable only at time 0.

Simvastatin inhibits cigarette smoking-induced emphysema and pulmonary hypertension in rat lungs.

RATIONALE: In cigarette smoking-induced chronic obstructive pulmonary disease, structural and functional derangements are characterized by parenchymal destruction and pulmonary hypertension. Statins are 3-hydroxy-3-methyl-glutaryl-coenzyme-A reductase inhibitors that have been used as lipid-lowering agents. These drugs also have additional pharmacologic properties, including antiinflammation, scavenging reactive oxygen species, restoring endothelial function, and antithrombogenesis, all of which can counteract the harmful effects of cigarette smoking. OBJECTIVE: We performed assays to determine whether simvastatin could attenuate lung damage induced by chronic cigarette smoking in rats. METHODS: In Sprague-Dawley rats exposed to cigarette smoke for 16 weeks, morphologic changes in the lungs and pulmonary arterial pressure were examined. MAIN RESULTS: Simvastatin inhibited lung parenchymal destruction and development of pulmonary hypertension, and also inhibited peribronchial and perivascular infiltration of inflammatory cells and induction of matrix metalloproteinase-9 activity in lung tissue. Simvastatin additionally prevented pulmonary vascular remodeling and the changes in endothelial nitric oxide synthase expression induced by smoking. In human lung microvascular endothelial cells, simvastatin increased expression of endothelial nitric oxide synthase mRNA. CONCLUSIONS: Simvastatin ameliorated the structural and functional derangements of the lungs caused by cigarette smoking, partly by suppressing inflammation and matrix metalloproteinase-9 induction and preventing pulmonary vascular abnormality. These findings indicate that statins may play a role in the treatment of cigarette smoking-induced chronic obstructive pulmonary disease.

A bound water molecule is crucial in initiating autocatalytic precursor activation in an N-terminal hydrolase.

Cephalosporin acylase is a member of the N-terminal hydrolase family, which is activated from an inactive precursor by autoproteolytic processing to generate a new N-terminal nucleophile Ser or Thr. The gene structure of the precursor cephalosporin acylases generally consists of a signal peptide that is followed by an alpha-subunit, a spacer sequence, and a beta-subunit. The cephalosporin acylase precursor is post-translationally modified into an active heterodimeric enzyme with alpha- and beta-subunits, first by intramolecular cleavage and, second, by intermolecular cleavage. Intramolecular autocatalytic proteolysis is initiated by nucleophilic attack of the residue Ser-1beta onto the adjacent scissile carbonyl carbon. This study determined the precursor structure after disabling the intramolecular cleavage. This study also provides experimental evidence showing that a conserved water molecule plays an important role in assisting the polarization of the OG atom of Ser-1beta to generate a strong nucleophile and to direct the OG atom of the Ser-1beta to a target carbonyl carbon. Intramolecular proteolysis is disabled as a result of a mutation of the residues causing conformational distortion to the active site. This is because distortion affects the existence of the catalytically crucial water at the proper position. This study provides the first evidence showing that a bound water molecule plays a critical role in initiating intramolecular cleavage in the post-translational modification of the precursor enzyme.