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Cyfluthrin is a pyrethroid insecticide and common household pesticide. The effect of cyfluthrin on Ca2+-related physiology in human osteosarcoma is unclear. This study investigated the effect of cyfluthrin on cytosolic-free Ca2+concentrations ([Ca2+]i) and viability in MG63 human osteosarcoma cells. Cyfluthrin concentration-dependently induced [Ca2+]irises. Cyfluthrin-induced Ca2+entry was confirmed by the Mn2+-induced quench of fura-2 fluorescence. Cyfluthrin at concentrations of 10–100 μM induced [Ca2+]irises. Ca2+removal reduced the signal by approximately 50%. Cyfluthrin (100 μM) induced Mn2+influx suggesting Ca2+entry. Cyfluthrin-induced Ca2+entry was inhibited 50% by protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) and inhibitor (GF109203X) and also by three inhibitors of store-operated Ca2+channels: nifedipine, econazole, and SKF96365. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+pump inhibitor thapsigargin (TG) completely inhibited cyfluthrin-evoked [Ca2+]irises. Conversely, treatment with cyfluthrin abolished TG-evoked [Ca2+]irises. Inhibition of phospholipase C (PLC) with 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dion abolished cyfluthrin-induced [Ca2+]irises. Cyfluthrin at 25–65 μM decreased cell viability, which was not reversed by pretreatment with the Ca2+chelator 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid–acetoxymethyl ester. Together, in MG63 cells, cyfluthrin induced [Ca2+]irises by evoking PLC-dependent Ca2+release from the endoplasmic reticulum and Ca2+entry via PKC-sensitive store-operated Ca2+entry. Cyfluthrin also caused Ca2+-independent cell death.

Bifenthrin, a commonly used pyrethroid pesticide, evokes various toxicological effects in different models. However, the effect of bifenthrin on cytosolic-free Ca2+ level ([Ca2+] i) and cytotoxicity in human prostate cancer cells is unclear. This study examined whether bifenthrin altered Ca2+ homeostasis and cell viability in PC3 human prostate cancer cells. [Ca2+] i in suspended cells were measured using the fluorescent Ca2+-sensitive dye fura-2. Cell viability was examined by 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 assay. Bifenthrin (100–400 μM) concentration-dependently induced [Ca2+] i rises. Ca2+ removal reduced the signal by approximately 30%. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished bifenthrin-evoked [Ca2+] i rises. Conversely, treatment with bifenthrin abolished BHQ-evoked [Ca2+] i rises. Inhibition of phospholipase C (PLC) with U73122 significantly inhibited bifenthrin-induced [Ca2+] i rises. Mn2+ has been shown to enter cells through similar mechanisms as Ca2+ but quenches fura-2 fluorescence at all excitation wavelengths. Bifenthrin (400 μM)-induced Mn2+ influx implicates that Ca2+ entry occurred. Bifenthrin-induced Ca2+ entry was inhibited by 30% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate) and inhibitor (GF109203X) and three inhibitors of store-operated Ca2+ channels: nifedipine, econazole, and SKF96365. Bifenthrin at 175–275 μM decreased cell viability, which was not reversed by pretreatment with the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane- N, N, N′, N′-tetra acetic acid-acetoxymethyl ester. Together, in PC3 cells, bifenthrin-induced [Ca2+] i rises by evoking PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive store-operated Ca2+ entry. Bifenthrin also caused Ca2+-independent cell death.

Edmund Cheung So,1,2 Yu-Xuan Lin,3 Chi Hao Tseng,1 Bo-Syong Pan,3 Ka-Shun Cheng,2 Kar-Lok Wong,2 Lyh-Jyh Hao,4 Yang-Kao Wang,5 Bu-Miin Huang2 1Department of Anesthesia, Tainan Municipal An Nan Hospital, China Medical University, Tainan, Taiwan; 2Department of Anesthesia, China Medical University, Taichung, Taiwan; 3Department of Cell Biology and Anatomy, National Cheng Kung University, Tainan, Taiwan; 4Department of Internal Medicine, Division of Endocrinology and Metabolism, Kaohsiung Veteran General Hospital Tainan Branch Tainan, Taiwan; 5Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei, Taiwan Purpose: The present study aims to investigate how midazolam, a sedative drug for clinical use with cytotoxicity on neuronal and peripheral tissues, induced apoptosis in MA-10 mouse Leydig tumor cells. Methods: The apoptotic effect and underlying mechanism of midazolam to MA-10 cells were investigated by flow cytometry assay and Western blotting methods. Results: Data showed that midazolam induced the accumulation of the MA-10 cell population in the sub-G1 phase and a reduction in the G2/M phase in a time- and dose-dependent manner, suggesting an apoptotic phenomenon. Midazolam could also induce the activation of caspase-8, -9, and -3 and poly (ADP-ribose) polymerase proteins. There were no changes in the levels of Bax and cytochrome-c, whereas Bid was significantly decreased after midazolam treatment. Moreover, midazolam decreased both pAkt and Akt expression. In addition, midazolam stimulated the phosphorylation of p38 and c-Jun NH2-terminal kinase but not extracellular signal-regulated kinase. Conclusion: Midazolam could induce MA-10 cell apoptosis through the activation of caspase cascade, the inhibition of pAkt pathway, and the induction of p38 and c-Jun NH2-terminal kinase pathways. Keywords: midazolam, apoptosis, MA-10 cell, caspase, Akt, MAPKs

A staphylococcus aureus protein A co-operated ELISA (SPA-ELISA) for the detection of anti-HCV-IgM has been established using HCV antigenic polypeptide, SPA-bearing germs and horseradish peroxidase labelled anti-human IgM. The specificity of SPA-ELISA has been confirmed by some substitution tests, blocking tests and destroying test with 2-mercaptoethanol. The results showed that the rate of anti-HCV-IgG in a group of patients with acute hepatitis and there were significant difference in anti-HCV-IgM was higher than that of anti-HCV-IgM detected rates between patients with acute hepatitis and those with chronic hepatitis (32.26%, P < 0.01). On the other hand, the positive rates of anti-HCV-IgM were 53.66% and 63.41% in transfusion associated hepatitis, 38.10% and 42.86% in sporadic hepatitis, 6.11% and 16.33% in people who have had active social activities, 40.00% and 10.00% in a group of blood donors respectively. Furthermore, taking into account the characteristics of HCV polypeptide used, its easiness of manipulation, and elimination of the interference of anti-HCV-IgG in sera, the new SPA-ELISA is believed to be of practical value in clinical and epidemiological studies of hepatitis C.

2346 liver samples from 17 areas in China were investigated for intrahepatic hepatitis D antigen (HDAg) by direct enzyme-labelled technique. HDAg was detected in 167 out of 1764 samples of HBsAg positive individuals with the detection rate of 9.47%. Hepatitis D virus (HDV) infection existed in all the examined areas without any significant difference regarding HDAg detection rate. A relationship of intrahepatic HDAg to the pathologic type of the liver disease was observed. The HDAg detection rate in chronic liver diseases and severe hepatitis was higher than in other liver diseases. It suggested that HDV infection was associated with the progression and chronicity of the liver disease. Studies on the relationship between HDV infection and HBV replication showed that HBV replication might be suppressed by HDV infection. Both HDV and HBV, however, could replicate in the same hepatocyte simultaneously.

28 cases of hemophilia were examined for HCV infection status by using the Kehua anti-HCV ELISA kit of second generation. It was found that the infection rate was 78.5% and the infection rate was even higher with patients who had received transfusions or preparations of coagulatory factors. 10 families of 15 patients were also investigated. It was found that of 15 hemophilia patients, 12 showed positive anti-HCV, while none of their 53 family members exhibited any positive anti-HCV. In 8 children of 9 couples no positive anti-HCV was found. Our results revealed that the hemophilia patient may get infected with HCV by receiving multiple transfusions or preparation of coagulatory factors. The risk of getting infected with HCV via daily-life contact including sexual contact is extremely low.

Intrahepatic HBVDNA of patients with chronic hepatitis B was detected by in situ cytohybridization assay in combination with demonstration of HBsAg and HBcAg expression, and correlated with the foci of hepatic necrosis. It was found that HBsAg and HBcAg expressing sites appeared in the same areas where HBVDNA replicated; when HBsAg and HBcAg simultaneously expressed in the liver, one antigen usually dominated, the other declined. This unbalanced expression may be caused by the gene or transcripts variance which encodes the Surface or core antigen, or by the activity of each replication. The other observation is that HBVDNA-positive hepatocytes outnumbered liver cells containing HBsAg and HBcAg in a few cases. It is supposed that a part of cytoplasmic HBVDNA was on replicative phase with gene production expression, on the contrary, another part of cytoplasmic HBVDNA may be out of replication with no gene product expression during chronic hepatitis B. Finally HBsAg and HBcAg expressing sites corresponding to HBVDNA-positive hepatocytes were closely attached to the foci of hepatic necrosis. There was no significant difference in the frequence of HBsAg and HBcAg expression correlating to hepatic necrosis.

To explore etiology of acute exacerbation in severe chronic active hepatitis, in situ HBVDNA hybridization was carried out combined with detection of HBV markers in the serum and the liver as well as intrahepatic HDAg in 15 cases. Four subgroups were identified based on the etiological evidence: 1) 9 cases were still undergoing HBV active replication or reactivation with cytoplasmic and membraneous HBcAg expression, often associated with the hepatic necrosis foci; 2) 3 cases showed HBsAg or/and HBVDNA positivity despite absence of HBcAg expression, the membranous and homogeneous HBsAg expression being closely related with hepatic necrosis; 3) 2 cases were HDAg positive; 4) the remaining case exhibited no HBV infection evidence. All findings suggested that HBV active replication or reactivation was the major cause of the exacerbation in severe chronic active hepatitis. In addition, HBV superinfection accounted for over 10% of cases with acute exacerbation. Hepatitis A or C may contribute to some episodes of exacerbation.

A biotin-labeled DNA probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue of 129 cases with liver disease. It was found that HBV DNA was predominantly visualized in cytoplasm of hepatocytes in three patterns: cytoplasmic compact, discrete and inclusion pattern. Its distribution in parenchyma in the sections of specimens may be defined as lobular, focal and spotty. The detection of intrahepatic HBV DNA depended on two factors, at least in the present study: 1) liver disease activity; chronic active hepatitis (CAH) group had a significant higher prevalence (81%) as compared to cirrhosis, chronic lobular hepatitis (CLH), acute hepatitis and hepatocellular carcinoma (HCC) groups; 2) HBV infection state: HBV DNA was more easily detected in HBeAg positive or intrahepatic HBcAg positive patients than in single HBsAg positive or anti-HBc cases. The findings that hepatocytes expressing HBV DNA, particularly in focal distribution, were closely related to hepatic necrosis sites suggested that HBV replication might occur in conjunction with hepatic necrosis.

Bei 163 Patienten mit Virushepatitis verschiedener Typen bzw. mit schweren Lebernekrosen wurden mittels der modifizierten indirekten Immunfluoreszenztechnik nach Meyer zum Buschenfelde die Leberzellmemtran-Antikorper (LMA) untersucht. 42 gesunde Erwachsene dienten als Kontrolle. Die Nachweisrate der LMA war bei akuter Hepatitis 24,24% (8 von 33 Patienten), bei chronisch-persistierender Hepatitis 25% (15 von 60 Patienten) und bei chronisch-aktiver Hepatitis 71,11% (32 von 45 Patienten). Unter 22 HBsAg-Tragern erwies sich ein Fall als LMA-positiv, dagegen keiner von 3 Patienten mit Lebernekrose infolge von Intoxikation. Bei den 42 Kontrollpersonen erhielten wir mit Ausnahme von 1 Fall negative Resultate. Die Nachweisrate der LMA war bei Patienten mit CAH bei der HBsAg-negativen Gruppe signifikant hoher als bei der HBsAg-positiven Gruppe (8/8 bzw. 24/37), wahrend bei der anderen Gruppe mit anderen Hepatitiden keine deutliche Differenz beobachtet wurde. Die immunologische Bedeutung der LMA in der Pathogenese der CAH wird diskutiert.

SDS-polyacrylamide gel electrophoresis and Western blotting analysis by using 9 clones of monoclonal antibodies specific for hemorrhagic fever with renal syndrome (HFRS) viruses were carried out on the virion proteins of 17 strains of HFRS viruses isolated from different areas in Asia and different hosts such as Apodemus agrarius, Rattus norvegicus, domestic cat and HFRS patients. Polypeptide with apparent molecular weight about 50 kitodalton (Kd) could be detected in all 17 strains of HFRS viruses. On this polypeptide there are two different antigenic determinants and one of them might be genus specific.

Das Lipoprotein der Leberzellmembran, das LSP, wurde durch Chromographie nach der Methode, die von McFarlane beschrieben wurde, aus normalem Lebergewebe isoliert. Zur Charakterisierung des isolierten LSP-Praparates wurde Polyakrylamid-Gel-Elektrophorese durchgefuhrt. Enzym-gekoppeltes Staphylokokken-Protein A Immunosorbens Assay (SPA-ELISA) mit dem gereinigten LSP-Praparat als Antigen wurde zur Bestimmung von Anti-LSP-Antikorper in Seren von Patienten verwendet. Insgesamt wurden 277 Serumproben untersucht. Die Resultate der Untersuchungen in der Hepatitis-Gruppe zeigten, da\ die Nachweisrate von Anti-LSP bei Patienten mit schwerer Hepatitis am hochsten (8/9) und bei Patienten mit CAH die nachsthochste (75%) war. Die Nachweisrate von Anti-LSP in Seren von Patienten mit AH, CPH und von HBsAg-Tragern betrug 47,6%, 38,4% bzw. 30,4%. In den Seren von 46 Normalpersonen zeigte sich aber nur ein Fall Anti-LSP-positiv. In der Gruppe mit anderen Erkrankungen war Anti-LSP positiv in 8 von 13 Fallen mlt Leberzirrhose, 5 von 7 Fallen mit SLE und 2 von 7 Fallen mit chronischer Nephritis. Die Resultate der Untersuchungen zeigten, da\ SPA-ELISA eine empfindliche Methode ist, die in der Klinik leicht durchgefuhrt werden kann. Es bestand eine Korrelation zwischen den Nachweisraten bzw. den Titern von Anti-LSP und Leberschadigung bei Hepatitis. Die Bestimmung von Anti-LSP durfte deshalb eine brauchbare Methode zur Beurteilung der Leberschadigung bei Patienten mit Hepatitis sein.

To explore the role of HBV antigen expression and monocyte infiltrate in situ in chronic hepatitis, HBV markers and compositions and number of monocytes in the liver of chronic hepatitis B were immunohistochemically located and identified, and correlated with hepatocyte necrosis. It was found that hepatic necrosis frequently took place in the centre or boundary of membranous HBsAg and/or HBcAg expression and the majority of monocytes accumulated in the necrotic areas showed CD8+ cell token in HBeAg-positive stage of chronic active hepatitis. On the other hand, with HBeAg positivity converted into HBeAg-negativity or anti-HBe-positivity, HBV antigen expression significantly declined, CD4+ cells remarkably increased as compared to HBeAg-positive status. These findings suggest that there may be distinct pathogenesis of hepatic necrosis in different stages of chronic HBV infection.