Analysis of Socio Demographic Risk Factors of Cervical Intraepithelial Neoplasia at Colposcopic Clinic in Mymensingh Medical College Hospital.

Cervical intraepithelial neoplasia (CIN) is a pre-malignant lesion of the cervix of uterus. Several risk factors increased the risk of developing CIN. Purpose of this study was to evaluate the socio-demographic risk factors related to CIN at our setting. This Cross sectional observational study was performed at Colposcopic clinic of Mymensingh Medical College Hospital (MMCH), Mymensingh, Bangladesh from 9th November 2017 to 8th May 2018. Overall demographic features of 50 patients of precancerous cervical lesion show that, most of the patients belonged to the age group 30-39 years (46.0%), mean age was 32.7±10.3 years. Maximum numbers of respondents came from rural area (58.0%), followed by urban area (42.0%). Among them house wife- 46.0%, daily worker- 30.0% and illiterate 36.0%, primary level of education 32.6%. Among the patients the poor class 46.0% and 58.0% of the respondents were married at age ≤19 year. Among the respondents, (26.0%) were conceived their first child 1 month after their marriage and 54.0% of the women within 12 months of marriage. In this study multipara were (62.0%). Oral contraceptive pill was taken by (42.0%) of patients. The association of risk factors revealed that betel or tobacco chewing present in 28.0% cases; history of menstrual regulation (MR), dilatation, evacuation and curettage (DE&C), miscarriage were in 26.0% cases, family history of cancer were in 16.0% cases and multiple sexual exposure was in 10.0% cases. Women develop pre-malignant cervical lesions require early treatment. It is recommended that provision of proper health care support, early detection of CIN and proper management, can reduce the fatal outcome of the disease.

Relaparotomy after Cesarean Section: Experience in a Tertiary Referral Hospital.

This study was aimed at exploring the causes of relaparotomy following caesarean section. The surgical procedures performed during relaparotomy were also discussed. This was a prospective study conducted in the Department of Obstetrics and Gynaecology, Mymensingh Medical College Hospital (MMCH), Mymensingh, Bangladesh from November 2020 to May 2021. MMCH is the largest referral Hospital in Mymensingh. During this study period 48 puerpera needed relaparotomy after caesarean section within 6 weeks of caesarean section. The frequency of relaparotomy was 2.6%. Of the 48 cases, 28(58.33%) cases needed relaparotomy due to post partum haemorrhage (PPH). Among them 9(18.75%) had primary PPH, 19(39.58%) patients had secondary PPH. Here 7(14.58%) patients suffered from sub rectus hematoma, 5(10.42%) patients had puerperal sepsis, 3(6.23%) had internal haemorrhage and 4(8.33%) women had wound dehiscence. Foreign body was removed in 1 case (2.08%). Main procedure performed was subtotal (45.83%) and total hysterectomy (25%). Coagulation failure and septicaemia were causes of maternal death. Case fatality rate was 4.17%. Obstetric patients who need relaparotomy face potential death. This study will help us to identify the causes for relaparotomy. Due precautions should be taken as far as possible to avoid this complications following caesarean section and thereby reduce maternal mortality and morbidity.

Pattern of Fetal Congenital Anomalies and Its Relation with Maternal Factors.

Congenital anomalies are one of the four leading causes of neonatal mortality in Bangladesh. The risk factors which are predictive of congenital anomaly in babies vary from country to country. In a developing country like Bangladesh many possible factors are present which should be identified & frequency needs to be assessed to understand the burden. The aim of this study was to determine the patterns and related maternal factors of fetal congenital anomaly. This cross-sectional type of comparative study was conducted at Department of Obstetrics & Gynecology in Mymensingh Medical College Hospital, Mymensingh, Bangladesh from September 2019 to August 2020. All the births occurring in the labor room were recorded. All newborn babies born with congenital anomalies were identified & included in this study. The rate of congenital anomalies was estimated and common types of congenital anomalies were noted. This study was conducted involving all women who had babies with congenital anomalies and the same number whose babies had no congenital anomalies. A structured questionnaire was used during data collection. Data was analyzed by Chi square test, bivariate analysis & multivariate logistic regression using statistical package for social sciences (SPSS) version 26.0. During the study period, 11479 deliveries were conducted. Among them 87 cases with congenital anomalies were identified. Frequency of congenital anomaly was 0.8%. Central nervous system was the predominant system involved (49.4%). Regarding risk assessment, Maternal age >30 years (OR 2.96, 95% CI 1.10-7.93, p value 0.032), consanguinity (OR 7.73, 95% CI 1.79-33.39, p value 0.006), first degree relative with history of congenital anomaly (OR 35.52, 95% CI 4.31-292.86, p value 0.001) and no intake of folic acid (OR 15.99, 95% CI 5.28-48.52, p value <0.001), passive smoking (OR 6.45, 95% CI 1.66-25.09, p value 0.007) were independent risk factors for congenital anomalies.

Effect of Amniotomy on Outcome of Spontaneous Labour.

Intentional artificial rupture of the amniotic membranes during labour, called amniotomy or 'breaking of the water's, is one of the most commonly performed procedures in modern obstetric and midwifery practice. The primary aim of amniotomy is to speed up uterine contractions and therefore shorten the length of labour. However there are concerns regarding unintended adverse effects on the woman and baby. A prospective observational study was conducted to determine the effectiveness and safety of routine procedure of amniotomy to shorten the duration of labour (prolonged or not) in Mymensingh Medical College & Hospital, Mymensingh, Bangladesh from July 2011 to December 2011. One hundred low-risk women with spontaneous onset of labour at term with singleton fetus in cephalic presentation and intact amniotic membranes and a cervical dilatation between 4 and 5cm were conventionally assigned to have amniotomy during the course of labour. Maternal demographics, duration of labour (prolonged or not), maternal and perinatal outcome were considered as major outcome. Majority (49.0%) of the patients belonged to 21-25 years age group and primigravida was predominant and most of them had middle socio-economic conditions. More the three-fourth (89.0%) of the patients had head engaged. Rh-positive and negative were found 96.0% and 4.0% respectively. The primigravidae required 10.07±2.17 hours in 1st stage of labour and had 1.51±0.5 hours duration of 2nd stage of labour. In case of multi-gravidae it was 6.07±2.06 hours in 1st stage of and 1±0.5 hours in 2nd stage of labour. There was a marked reduction of amniotomy-delivery interval time in this study, which was 3 hours 40 minutes and whereas mean cervical dilatation was 4cm during amniotomy. Almost three fourth (72.0%) cases delivered vaginally among which, with episiotomy in 49.0% and without episiotomy in 23.0%. Instrumental delivery was in 9.0% of which 4.0% by forceps, 5.0% by vaccum extraction and 14.0% underwent LUCS. Still birth was found 2.0%, asphyxiated 3.0% and prenatal death 1.0%. In terms of referral to neonatal care unit it was found that 7.0% were asphyxiated. Asphyxia and low APGAR score was 4.0%, low birth weight 9.0%, instrumental delivery was 5.0%, Rh incompatibility was 2.0%. Only 1.0% babies needed admission to neonatal care unit and were intubated. So, Amniotomy significantly reduced the duration of the first stage of labour without affecting the oxytocin requirement, the rate of caesarean section and newborn outcome.

Gastrointestinal Manifestations among COVID-19 Patients in Bangladesh: A Cross Sectional Study.

This study was done to find out the frequency of gastrointestinal manifestations among adult COVID-19 patients in Bangladesh. This descriptive cross sectional retrospective study was conducted from 27 May till 20 June 2020. Data were collected retrospectively from three different hospitals of Dhaka, Bangladesh. Patients were interviewed over telephone and verbal consents were taken. Their demographic and clinical profiles were put in the questionnaires by the investigators themselves. Statistical analysis was done thereafter. Out of 226 patients diagnosed with COVID-19, 145(61.4%) patients had gastrointestinal symptoms. Five of them had none other than gastrointestinal symptoms. Twenty six patients were asymptomatic. Mean age of the patients with were 41.6±14.8 years. Males were affected almost equally as the females (51.72% vs. 48.27%) (Ratio 1.1:1). Anorexia (44.7%) followed by diarrhoea (35%) and nausea (22.6%) were the predominant symptoms. Patients with co-morbidities (74.7%) were more prone to develop GI symptoms. Family members (71.6%) of COVID-19 patients with GI symptomatic patients were more affected than the others. Hospitalizations (77.6%) were more among GI symptomatic patients than the others. Thirty Five percent (35%) patients had diarrhoea. Mean duration of diarrhoea was 2.7±1.7 days with a frequency of were 4±1.8 days per day. Gastrointestinal manifestations are common among patients with COVID-19. Clinicians need to be aware of the GI features amongst COVID-19 patients so that they can be addressed and treated effectively and immediately. Further large scale study is needed to predict the disease outcome among COVID 19 patients with GI symptoms.

Influence of the Interval between Antenatal Corticosteroid Therapy and Delivery on the Incidence of Respiratory Distress Syndrome in Neonate.

In spite of the recommendation for rescue antenatal corticosteroids (ACS), the optimal time interval between primary and rescue courses has not been clearly demonstrated. The aim of this retrospective study was to determine the effects of the interval between a single ACS (Dexamethasone) course and delivery on the incidence of respiratory distress syndrome (RDS) in neonates at Mymensingh Medical College Hospital Center from 1st January 2017 to 30th June 2017. Injection Dexamethasone 2 doses (12.5mg IM 12 hourly for 2 doses) or 4 doses (6mg IM every 12 hours for 4 doses) use to arrest preterm labor as well as to prevent RDS delivered beyond 48 hours after ACS administration between 24 and 34 weeks gestation. The risk of RDS was compared between patients who delivered within seven days (Group I) and 7-14 days (Group II) after ACS administration. We included 140 and 60 patients in Group I and Group II respectively. After adjusting for confounders, the ACS delivery interval was significantly associated with RDS in Group II (adjusted odds ratio 12.8, 95% confidence interval 1.31-164.7). A longer ACS delivery interval is associated with a higher risk of RDS. Thus, the use of a rescue course could be expected to reduce the incidence of RDS in patients beyond seven days after ACS administration who remain at risk for preterm delivery within seven days, especially in cases of placenta previa and/or women bearing a male fetus.

Clinical Study on Rupture Uterus in Mymensingh Medical College Hospital.

Uterine rupture is a devastating situation, has claimed innumerable lives of both the mother and the fetus. Even today, it is one of the common obstetric complications and a significant cause of maternal and fetal death. Several factors are responsible for this including-inadequate antenatal and intra partum care, poor communications and inadequate logistic support, above all, illiteracy and lack of knowledge of the people. To evaluate the patients with rupture uterus A cross sectional descriptive study was carried out in the department of Obstetrics & Gynaecology, Mymensingh Medical College Hospital, Mymensingh, Bangladesh from July 2012 to June 2013. Total 100 cases of rupture uterus were included in this study within this period. Data were processed and analyzed by Computer software SPSS-16 version (Statistical Package for Social Science) & cases were selected purposively. Incidence of rupture uterus was 1.43%, common age group was 20-30 years, majority (67%) came from rural areas, multi-gravid patients were mainly affected (98%). Most of the patients (68%) had no antenatal check-up and 46% were handled by untrained Dai at home during labour pain and 56% exposed to oxytocic drugs. Unscarred uterus was more common (61%) & common clinical presentation was hypo-volumic shock (64%). Subtotal hysterectomy (51%) had done as surgical procedure in 51% patients and average duration of hospital stay was 11±4 days. The common post operative complications were sepsis (20%), wound infection (13%), shock (10%) and urinary fistula (8%). Incidence of peri-natal mortality was 89% and maternal mortality 14%.This study suggests maternal and perinatal morbidity and mortality is high due to rupture uterus. So prevention and proper management is necessary to overcome this problem. This study will hopefully give us some guide to take the strategies in improving the care of rupture uterus.

Cadmium(II) complexes of (arylazo)imidazoles: synthesis, structure, photochromism, and density functional theory calculation.

Reaction between CdX2 and 1-alkyl-2-(phenylazo)imidazole (RaaiR') has isolated complexes of composition Cd(RaaiR')2X2 in MeOH or MeCN. Crystallization of Cd(RaaiR')2I2 from N,N-dimethylformamide (DMF) has separated [Cd(RaaiR')I2.DMF], while Cd(RaaiR')2X2 (X = Cl and Br) remains unchanged in its composition upon crystallization under identical conditions. The structure has been established by spectral (UV-vis and 1H NMR) data and confirmation in the latter case by a single-crystal X-ray diffraction study of [Cd(TaiMe)I2.DMF] [where TaiMe = 1-methyl-2-(p-tolylazo)imidazole]. UV-light irradiation in a MeCN solution of Cd(RaaiR')2I2 and [Cd(RaaiR')I2.DMF] shows trans-to-cis isomerization of coordinated azoimidazole. The reverse transformation, cis-to-trans, is very slow with visible light irradiation. Quantum yields (phit-->c) of trans-to-cis isomerization are calculated, and the free ligand shows higher phi values than their cadmium(II) iodo complexes. The cis-to-trans isomerization is a thermally induced process. The activation energy (Ea) of cis-to-trans isomerization is calculated by a controlled-temperature experiment. The effects of the anions (Cl-, Br-, I-, and ClO4-) and the number of coordinated azoimidazoles (RaaiR') [Cd(RaaiR') or Cd(RaaiR')2] on the rate and quantum yields of photochromism are established in this work. A slow rate of photoisomerization of [Cd(RaaiR')4](ClO4)2 compared to Cd(RaaiR')I2 or Cd(RaaiR')2X2 may be associated with the increased mass and rotor volume of the complexes. The rate of isomerization is also dependent on the nature of X and follows the sequence Cd(RaaiR')2Cl2 < Cd(RaaiR')2Br2 < Cd(RaaiR')2I2. It may be related to the size and electronegativity of halide, which reduces the effective molar association in the order of I < Br < Cl and hence the rate. Gaussian 03 calculations of representative complexes and free ligands are used to explain the difference in the rates and quantum yields of photoisomerization.

Structural studies and photochromism of mercury(II)-iodo complexes of (arylazo)imidazoles.

Neat reaction between HgI2 and 1-methyl-2-(phenylazo)imidazole (Pai-Me) under microwave irradiation has isolated a novel compound whose structure shows intercalated HgI2 in the layers of Pai-Me. They exist independently in interpenetrated arrays. In a solution phase study, the same reaction has synthesized an iodo-bridged azoimidazole-Hg(II) complex, [Hg(RaaiR')(mu-I)(I)]2 (RaaiR' = 1-alkyl-2-(arylazo)imidazole). The structures have been characterized by X-ray diffraction studies. Chloro-bridged Hg(II) complexes of azoimidazoles, [Hg(RaaiR')(mu-Cl)(Cl)]2, are also known. These complexes upon irradiation with UV light show trans-to-cis isomerization. The reverse transformation, cis-to-trans isomerization, is very slow with visible light irradiation. Quantum yields (phi t-->c) of trans-to-cis isomerization are calculated, and the free ligand shows higher phi than their Hg(II) complexes. The cis-to-trans isomerization is a thermally induced process. The activation energy (Ea) of cis-to-trans isomerization is calculated by controlled temperature reaction. The Ea's of free ligands are much higher than that of halo-bridged Hg(II)-azoimidazole complexes. Chloro-bridged Hg(II) complexes show lower Ea's than those of iodo-bridged complexes. DFT calculation has been adopted to rationalize the experimental results.

Highly accumulated platelet vascular endothelial growth factor in coagulant thrombotic region.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific potent mitogen that induces angiogenesis and microvascular hyperpermeability. Recently, it has been reported that megakaryocytes and platelets contain VEGF in their cytoplasm. OBJECTIVES: To elucidate and confirm the bioactivity and role of VEGF in platelets (platelet VEGF), which may be closely related to vascular thrombosis and atherosclerosis. METHODS: The VEGF localization in megakaryocytes on bone marrow smears was analyzed by immunofluorescence and confocal laser scanning microscopic analysis. The intracellular VEGF expressed in platelets was determined by flow cytometric analysis. Platelet-rich plasma and washed platelets were used to analyze the secretion of VEGF during platelet aggregation by thrombin or gelatinase A (matrix metalloproteinase-2) stimulation. Immunohistochemical studies for VEGF in the thrombotic region were performed. RESULTS AND CONCLUSIONS: Megakaryocytes and platelets are a very rich source of circulating VEGF. Gelatinase A, which is closely associated with vascular remodeling, enhances the VEGF levels released from platelets. VEGF was clearly detected in the fibrin nets of a thrombus. Taken together, platelet VEGF is bioactive as a direct angiogenic growth factor, and may play a very important role in wound healing and atherosclerosis in conjunction with other platelet cytokines such as platelet-derived growth factor, platelet-derived endothelial cell growth factor, transforming growth factor (TGF)-alpha, and TGF-beta.

The agonist of the protease-activated receptor-1 (PAR) but not PAR3 mimics thrombin-induced vascular endothelial growth factor release in human smooth muscle cells.

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, induces vascular endothelial growth factor-(VEGF) release. However, the molecular mechanism of thrombin-induced VEGF release is largely unknown. Anagonist of protease-activated receptor-i (PARI), SFLL-RNPNDKYEPF, mimicked thrombin-induced VEGF release in human vascular smooth muscle (HVSM) cells, as determined by enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and Northern blotting. In contrast, the agonist of PAR3, TFR- GAP, did not affect VEGF release or expression. SFLL-RNPNDKYEPF, but not TFRGAP, up-regulated [Ca2-]i.Moreover, the calcium ionophone A23187 was found to trigger VEGF release in HVSM cells. Thrombin-inducedVEGF release was blocked by anti-thrombin, heparin, a synthetic thrombin receptor inhibitor E5510, the calcium chelator BAPTA, the protein kinase C inhibitor calphostin C, and the MEK1/2 inhibitor U0126. Thus, our data show that thrombin caused VEGF release via PARI activation in a manner dependent on [Ca2+]i and p44/42 downstream from the receptor activation.

Anandamide induces cell death independently of cannabinoid receptors or vanilloid receptor 1: possible involvement of lipid rafts.

Anandamide triggers various cellular activities by binding to cannabinoid (CB1/CB2) receptors or vanilloid receptor 1 (VR1). However, the role of these receptors in anandamide-induced apoptosis remains largely unknown. Here, we show that SR141716A, a specific inhibitor of cannabinoid receptor (CB1-R), did not block anandamide-induced cell death in endogenously CB1-R expressing cells. In addition, CB1-R-lacking Chinese hamster ovary (CHO) cells underwent cell death after anandamide treatment. SR144528, a specific inhibitor of CB2-R also failed to block anandamide-induced cell death in HL-60 cells. Capsazepine, a specific antagonist of VR1 could not prevent anandamide-induced cell death in constitutively and endogenously VR1 expressing PC12 cells. Moreover, anandamide noticeably triggered cell death in VR1-lacking human embryonic kidney (HEK) cells. In contrast, methyl-beta cyclodextrin (MCD), a membrane cholesterol depletor, completely blocked anandamide-induced cell death in a variety of cells, including PC12, C6, Neuro-2a, CHO, HEK, SMC, Jurkat and HL-60 cells. MCD also blocked anandamide-induced superoxide generation, phosphatidyl serine exposure and p38 MAPK/JNK activation. Thus, our data imply a novel role for of membrane lipid rafts in anandamide-induced cell death.

Bioactivity of the vascular endothelial growth factor trapped in fibrin clots: production of IL-6 and IL-8 in monocytes by fibrin clots.

The blood coagulation cascade is activated following vascular-wall injury. The serine protease thrombin is the final protease in this cascade that causes the formation of fibrin from fibrinogen. Thrombin also causes the activation of platelets, which are trapped in a fibrin net followed by hemostasis. Platelets gathered into fibrin clots release several growth factors such as platelet-derived growth factor and transforming growth factor beta. In the present study, we demonstrated that the vascular endothelial growth factor (VEGF) could be bound to fibrin clots in the plasma, and that incubation of the endothelial cells with these VEGF-bound fibrin clots induced proliferation of endothelial cells. Thus, it suggests that clot-bound VEGF may play a role in wound healing through the proliferation of endothelial cells and vascular smooth-muscle cells. On the other hand, a noticeable migration of monocytes was observed when they were cultured on dishes in the presence of VEGF-bound fibrin clots. Moreover, peripheral blood monocytes incubated in the presence of VEGF-bound fibrin clots strikingly increased the production of IL-6 and IL-8, demonstrating that VEGF trapped in fibrin clots not only induces proliferation of human umbilical vein endothelial cells and migration of monocytes but also enhances secretion of IL-6 and IL-8. Thus, our data suggest that fibrin clots that contain several growth factors act as a bioactive reservoir and may play an important role in hemostasis as well as wound healing.

Inhibitory effect of epigallocatechin-gallate on brain tumor cell lines in vitro.

We investigated the effect of epigallocatechin-gallate (EGCG), the main constituent of green tea polyphenols, on human glioblastoma cell lines U-373 MG and U-87 MG, rat glioma cell line C6, and rat nonfunctioning pituitary adenoma cell line MtT/E. Cell viability was determined by assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and the extent of apoptosis was studied by flow cytometric analysis. Apoptosis was also characterized by morphology using fluorescent microscopy. The role of insulin-like growth factor-I (IGF-I) was studied by assay with MTT, immunohistochemistry, and immunoradiometric assay. After 72-h exposure, a statistically significant loss of viability (P = < 0.0001) was observed at concentrations of 12.5, 25, 50, and 100 microg/ml in U-373 MG cells and U-87 MG cells. EGCG at concentrations of 50 microg/ml and higher significantly reduced the viability of C6 cells. EGCG inhibited viability of MtT/E cells only at a concentration of 100 microg/ml. Quantitative study by flow cytometry demonstrated that lower doses of EGCG (12.5, 25, 50 microg/ml) induced apoptosis in U-373 MG, U-87 MG, and C6 cells; however, only the highest dose (100 microg/ml) induced apoptosis in MtT/E cells. Compared with other cell lines, MtT/E cells showed stronger IGF-I immunoreactivity. Neutralization of IGF-I with an antihuman IGF-I antibody reduced viability of the cell lines. It can be concluded that EGCG has an inhibitory effect on malignant brain tumors, and IGF-I may be involved in the effects of EGCG.

Anandamide induces apoptosis of PC-12 cells: involvement of superoxide and caspase-3.

Anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand has been suggested to have physiological role in mammalian nervous system. However, little is known about the role of anandamide on neuronal cells. Here, we demonstrate that anandamide causes death of PC-12 cells, showing marked DNA condensation and fragmentation, appearance of cells at sub-G(0)/G(1) and redistribution of phosphatidyl serine, the hallmark features of apoptosis. Anandamide raised intracellular superoxide level and CPP32-like protease activity in PC-12 cells markedly. Furthermore, antioxidant N-acetyl cysteine prevented anandamide-induced superoxide anion formation and cell death, implying that intracellular superoxide is a novel mediator of anandamide-induced apoptosis of PC-12 cells.

Polymyxin B binds to anandamide and inhibits its cytotoxic effect.

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)-immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

Inhibition of caspase-3 activation by SB 203580, p38 mitogen-activated protein kinase inhibitor in nitric oxide-induced apoptosis of PC-12 cells.

Mitogen-activated protein kinase (MAPK) p38 plays pivotal role in cell proliferation, differentiation, and apoptosis when cysteine protease caspase induces apoptosis in different cell systems. SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole) is widely used as a specific inhibitor of p38 MAPK, and prevents apoptosis induced by various agents. The effect of SB 203580 on nitric oxide(NO)- or peroxynitrite-induced cell death is not known. Western blotting results indicate that p38 MAPK was activated significantly in NO- or peroxynitrite-induced cell death in a time-dependent manner, and subsequently this cell death was markedly inhibited by SB 203580, as determined by fluorescence-activated cell sorting (FACS)-can analyzer. Furthermore, NO/peroxynitrite-induced caspase-3 activation was notably inhibited by SB 203580, however, phosphorylation of either p38 MAPK or p44/42 was not influenced by SB 203580. Thus, it is likely that SB 203580 prevents NO/peroxynitrite-induced cell death by inhibiting caspase-3 activation in PC-12 cells.

Inhibition of thrombin-induced neuronal cell death by recombinant thrombomodulin and E5510, a synthetic thrombin receptor signaling inhibitor.

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a pivotal role in thrombosis and haemostasis. Thrombin acts as a mitogen and apoptosis inducer in a dose-dependent fashion. We have previously shown that thrombin caused proliferation of vascular smooth muscle cells (VSMCs). Here, we show that a low concentration of thrombin caused proliferation of mouse neuroblastoma (Neuro-2a) and human neuroblastoma (NB-1) cells, while higher concentrations affected cell viability in a time-dependent manner. Similar effects were observed when thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) was applied. The dying cells showed nuclear condensation and fragmentation, suggesting that cell death occurred by apoptosis. The extent to which thrombin induced cell death was significantly attenuated by recombinant thrombomodulin (rTM), or by a minimum functional domain of TM, termed E456. Furthermore, a synthetic compound that inhibits signaling from the thrombin receptor, 4-cyano-5,5-bis (4-methoxyphenyl)-4-pentanoic acid (E5510), and the antioxidant N-acetyl L-cysteine (NAC), efficiently prevented thrombin-induced Neuro-2a cell death. Thus, thrombin inhibitors and antioxidant appear to neutralize thrombin toxicity.

Recombinant thrombomodulin inhibits thrombin-induced vascular endothelial growth factor production in neuronal cells.

Thrombin is a serine protease which is generated from its precursor prothrombin by the activation of the blood coagulation cascade. Thrombin converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a central role in thrombosis and hemostasis by regulating platelet aggregation and blood coagulation. Here, we show that thrombn enhanced vascular endothelial growth factor (VEGF) production in a dose- and time-dependent manner in the supernatant of cultured PC-12 cells, as determined by enzyme-linked immunosorbent assay (ELISA). Thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) exerted an effect similar to thrombin on VEGF production. Thrombin-induced VEGF production was significantly attenuated by recombinant human thrombomodulin (rTM) and its minimal functional domain E456. Furthermore, the antioxidant N-acetyl-L-cysteine (NAC) markedly inhibited thrombin-induced VEGF production. Thus, rTM and NAC apparently inhibited the effect of thrombin on VEGF production in neuronal cells.