A case of methanol intoxication caused by methomyl pesticide ingestion.

When clinicians treat patients with pesticide poisoning, they often pay attention only to the chief toxic agent and ignore the toxicity of the pesticide's additives or solvents. Occasionally, however, a solvent (e.g. methanol) may itself be the cause of poisoning. We report a case of acute methanol intoxication that occurred after ingestion of a methomyl pesticide that contained methanol as an additive. A 49-year-old man was brought to the emergency department in an unconscious state after ingestion of 20 ml of a carbamate pesticide (chief ingredient: methomyl; active ingredient: methanol). Upon arrival, he was semicomatose and did not breathe spontaneously; however, his cholinesterase level was within normal limits and cholinergic symptoms were not observed. High anion gap metabolic acidosis was present. His blood ethanol level was 74.8 mg/dL. The urine methanol level was 55.60 mg/dL, and urine ethanol level was 22.0 mg/dL. He was treated with hemodialysis; subsequently, his metabolic acidosis resolved and he returned to normal mental status. We guessed that methanol, as the solvent of the methomyl, had produced the symptoms. When treating pesticide-poisoned patients, clinicians should identify the solvent used in the pesticide, because solvents such as methanol may exacerbate the symptoms of poisoned patients.

Aquatic birnavirus infection activates the transcription factor NF-kappaB via tyrosine kinase signalling leading to cell death.

Our previous studies found that infectious pancreatic necrosis virus (IPNV) induces host apoptotic cell death, possibly through a newly synthesized protein trigger. Here, we examine whether IPNV infection can induce NF-kappaB activation through tyrosine kinase signalling of CHSE-214 cell death (host cell death). Using the electrophoretic mobility shift assay (EMSA) to detect transcription factor activation, we found that NF-kappaB is apparently activated 6-8 h post-IPNV infection. Using genistein (100 microg mL(-1); a tyrosine kinase inhibitor) to determine whether NF-kappaB activation requires tyrosine kinase activation, we found genistein blocks NF-kappaB activation at 8 h post-infection (p.i), and either enhances cell viability up to 50% at 12 h p.i. or blocks DNA fragmentation at 24 h p.i. Furthermore, the proteasome inhibitors PSI-I and PSI-II (both at 40 microm) also effectively blocked the NF-kappaB activation as well as stimulating a 30% increase in cell viability (30% decrease in apoptosis) at 8 and 12 h p.i. Taken together our data suggest that IPNV may induce NF-kappaB activation through tyrosine kinase signalling, which may be associated with induction of apoptosis.

Enhanced viability of a nervous necrosis virus-infected stable cell line over-expressing a fusion product of the zfBcl-xL and green fluorescent protein genes.

Nervous necrosis virus (NNV) infection induces host cell apoptosis by an ill-understood process. We utilized a fusion between enhanced green fluorescent protein (EGFP) and the zfBcl-x(L) gene in GL-av cells to select for zfBcl-x(L) stable cell lines and to assess the effectiveness of the anti-apoptotic protein Bcl-x(L) in circumventing NNV-induced cell death. Stable EGFP and EGFP-Bcl-x(L)-expressing clones were obtained at high purity within 2.5-3 months. In the latter, the EGFP-Bcl-x(L) fusion protein (approximately 58.2 kDa, as ascertained by Western blot) was predominantly targeted to mitochondria. We assayed for apoptosis in red-spotted grouper NNV Tainan no. 1 (RGNNV TN1)-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different virus doses. NNV infection of NNV Bcl-x(L) GL-av cell line revealed a protective effect, with a decrease in TUNEL-positive cells of 7%, 8% and 31.8% at 24, 48 and 72 h, respectively. In addition, RGNNV infection of the Bcl-x(L) GL-av cell line revealed a protective effect, with an enhanced viability of 3%, 40% and 73% at 24, 48, and 72 h, respectively. We conclude that NNV-induced apoptotic cell death can be lessened in transgenic grouper fish cells.

Aquatic birnavirus induces apoptosis through activated caspase-8 and -3 in a zebrafish cell line.

In this study, the possible influence of temperature on infectious pancreatic necrosis virus (IPNV)-induced apoptosis in a zebrafish liver epithelium (ZLE) cell line was investigated. At a lower temperature (18 degrees C), there was expression of viral proteins VP2 and VP3 at 4 h post-infection (p.i.). At this time no expression was found in the high temperature group at 28 degrees C. The cell survival ratio was 52 and 18% at 24 and 48 h p.i., respectively, during IPNV infection at 18 degrees C. In addition, we assayed for apoptosis in IPNV-infected cells with terminal deoxynucleotidyl transferase (TdT)-mediated end labelling (TUNEL) of DNA at different dosages of virus. We found a ratio of apoptotic cells of 8 and 25% at 12 and 18 h p.i., respectively, in the multiplicity of infection (MOI) 1 group. The MOI 10 group had 20 and 45% apoptotic cells at 12 and 18 h, respectively. Furthermore, at 18 degrees C IPNV activated the caspase-8 and 3 from 1.5 to 2 times at 12 and 18 h p.i., respectively. Taken together, these findings suggest that successful virus replication occurs at the low temperature (18 degrees C) compared with the non-permissive temperature of 28 degrees C. Thus, IPNV replication is capable of activating caspase-8 and -3 and inducing host apoptosis.

Induction of apoptotic death in cells via Bad gene expression by infectious pancreatic necrosis virus infection.

A Bcl-2 related family member, Bad, promotes cell death, and its function is regulated by phosphorylation. In this study, we show how the IPNV elicits the induction of Bad gene expression and promotes host apoptotic death. Anti-IPNV-E1S polyclonal and anti-VP3 monoclonal antibodies are used to neutralize the virus that blocks the prime death signal via the virus receptor. In the viability assay, each antibody could also enhance cell viability during IPNV infection. We tested tyrosine kinase inhibitors on IPNV-infected cells in order to assess their effect on blocking the death signal. With 100 microg/ml genistein treatment, Bad-like gene expression was blocked, either by rescuing the IPNV-infected CHSE-214 cells or by blocking internucleosomal DNA cleavage; but the tyrphostin group did not block Bad expression. For CHSE-214 cells, treatment with the protein synthesis-inhibitor, cycloheximide (1microg/ml), blocked new protein synthesis via activated tyrosine kinase during IPNV infection. We found that Bad protein expression could be blocked, and apoptotic death prevented. Together, these results demonstrate that the IPNV exerts up-regulation of a pro-apoptotic death gene (Bad), the expression of which serves to trigger apoptotic cell death. Our data also suggests that the IPNV induces apoptotic death via a viral receptor which triggers death effector Bad gene expression, possibly through a tyrosine kinase-dependent pathway.

Cloning and characterization of zfBLP1, a Bcl-XL homologue from the zebrafish, Danio rerio.

The importance of the Bcl-2 family proteins in normal vertebrate embryogenesis is being recognized; however, their regulatory mechanism is poorly understood. We report here the cloning and characterization of a novel zebrafish Bcl-2 family protein, zfBLP1. The zfBLP1 cDNA is 1942 nucleotides long, encoding a polypeptide of 238 amino acids. The primary sequence of zfBLP1 shares 50% identity to human Bcl-XL, and contains all four conserved BH domains of the Bcl-2 family proteins. Primary sequence analysis identified a consensus ER retention signal at the C-terminal end of zfBLP1. Northern blot analysis indicated that there were two major and two minor zfBLP1 mRNA species expressed during embryonic development. Among the two major mRNA species, the short one, approx. 3 kb in size, was expressed throughout embryonic development, while the long one, approx. 7 kb long, was not detectable until the gastrula stage. These results suggest that zfBLP1 is a novel Bcl-2 family protein under complicated regulations, and is likely to play an important role in zebrafish oogenesis and embryogenesis.

Cloning and characterization of a novel nuclear Bcl-2 family protein, zfMcl-1a, in zebrafish embryo.

The importance of the Bcl-2 family proteins in normal vertebrate embryogenesis is being recognized; however, their regulatory mechanism is poorly understood. To elucidate the embryonic roles of Bcl-2 family proteins, we cloned and characterized the first zebrafish Bcl-2 family protein, zfMcl-1a. Zebrafish Mcl-1a shows the highest homology to rat Mcl-1 and contains several conserved BH domains of the Bcl-2 family proteins. It also contains a nuclear localization signal (NLS). Using EGFP reporter analysis, we verified the nuclear localization of zfMcl-1a. Deletion of the NLS resulted in distribution of the fusion protein in the cytoplasm. Northern blot analysis indicated that zfMcl-1a mRNA is 1.5 kb and was expressed in oocytes and throughout embryonic development. Notably, the expression of zfMcl-1a transcript was significantly downregulated during gastrulation. These results suggest that zfMcl-1a is a novel nuclear Bcl-2 family protein and is likely to play an important role in zebrafish oogenesis and embryogenesis.

Infectious pancreatic necrosis virus induces apoptosis due to down-regulation of survival factor MCL-1 protein expression in a fish cell line.

Infectious pancreatic necrosis virus (IPNV), a member of the virus family Birnaviridae, causes an acute, contagious disease in a number of economically important fish species. CHSE-214, a Chinook salmon embryonic cell line, when infected by IPNV showed morphological and biochemical features of apoptosis, including an intense DNA laddering pattern and blebbing of the plasma membrane, followed by formation of apoptotic bodies. The Mcl-1 gene product proved to be a member of the Bcl-2 gene family, and like Bcl-2 had the capacity to promote cell viability. Here, we investigated the pattern of expression of Mcl-1 in CHSE-214 cells infected by IPNV. We found that the Mcl-1 level decreased markedly in cells undergoing apoptosis after IPNV infection. This decrease was rapid during the first 8 h postinfection and preceded cell death. Furthermore, we found that drugs including cycloheximide, genistein and EDTA either prevented the decline in Mcl-1 levels or blocked the intense DNA laddering pattern. Other drugs like serine proteinase inhibitor, 400 microg/ml aprotinin, 400 microg/ml leupeptin and 100 microg/ml tryphostin did not. The virus gene expression pattern was examined by Western blot using antivirion polyclonal antibody and was blocked during treatment with cycloheximide, genistein and EDTA but not by serine proteinase, aprotinin, leupeptin or tryphostin. Together the data showed a striking correlation between virus replication and Mcl-1 expression in CHSE-214 cells, suggesting that the virus gene expression has a possible involvement with Mcl-1 in the regulation of apoptosis in these cells.

Dynamics of nontypical apoptotic morphological changes visualized by green fluorescent protein in living cells with infectious pancreatic necrosis virus infection.

Morphologically, apoptotic cells are characterized by highly condensed membrane blebbing and formation of apoptotic bodies. Recently, we reported that apoptosis precedes necrosis in a fish cell line infected with infectious pancreatic necrosis virus (IPNV). In the present study, we tested the possibility that nontypical apoptosis is a component of IPNV-induced fish cell death. A variant type of green fluorescent protein (EGFP) was expressed in a fish cell line such that EGFP served as a protein marker for visualizing dynamic apoptotic cell morphological changes and for tracing membrane integrity changes during IPNV infection. Direct morphological changes were visualized by fluorescence microscopy by EGFP in living cells infected with IPNV. The nontypical apoptotic morphological change stage occurred during the pre-late stage (6 to 7 h postinfection). Nontypical apoptotic features, including highly condensed membrane blebbing, occurred during the middle apoptotic stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and were finally pinched off from the cell membrane. At the same time, at this pre-late apoptotic stage, apoptotic cells formed unique small holes in their membranes that ranged from 0.39 to 0.78 micrometer according to examination by scanning electron microscopy and immunoelectron microscopy. Quantitation of the intra- and extracellular release of EGFP by CHSE-214-EGFP cells after IPNV infection was done by Western blotting and fluorometry. Membrane integrity was quickly lost during the late apoptotic stage (after 8 h postinfection), and morphological change and membrane integrity loss could be prevented and blocked by treatment with apoptosis inhibitors such as cycloheximide, genistein, and EDTA before IPNV infection. Together, these findings show the apoptotic features at the onset of pathology in host cells (early and middle apoptotic stages), followed secondarily by nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (late apoptotic stage), of a fish cell line. Our results demonstrate that nontypical apoptosis is a component of IPNV-induced fish cell death.

Apoptosis precedes necrosis of fish cell line with infectious pancreatic necrosis virus infection.

The current view of infectious pancreatic necrosis virus (IPNV) infection includes a necrotic process that relies primarily on the histological appearance of tissue after the degenerative process. We tested this view by examining the possibility that apoptosis is a component of double-stranded RNA virus (IPNV) that induces fish embryonic cell death. Four kinds of assays for apoptosis were used in analyzing IPNV-infected CHSE-214 cells: (1) assay with terminal deoxynucleotidyl transferase (TdT)-mediated end-labeling of DNA in nuclei of intact cells during virus infection, (2) assay for procoagulant activity, (3) assay for DNA ladders, and (4) electron microscopic assays for the ultrastructural changes in characteristic apoptotic cells. In all p.i. samples, both low and high m.o.i. groups contained apoptotic nuclei, according to TdT-mediated dUTP labeling of intact cells, but in control CHSE-214 cells, apoptotic nuclei were rare at all levels of incubation sampled by TdT-mediated dUTP labeling. Prenecrotic or postnecrotic cells were found to express phosphatidylserine on the surface by annexin V-FITC labeling, but normal cells did not. DNAs from both 4 h p.i. of high m.o.i. and 8 h p.i. of low m.o.i. were found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. The IPNV-infected CHSE-214 cells were analyzed with an electron microscope and showed a pattern of ultrastructural change, indicating that apoptosis appears before pathological changes of necrosis, including condensed chromatin, fragmented nuclei, nuclei with chromatin marginations, and secondary necrosis from prenecrotic cells in IPNV-infected CHSE-214 cells. Together, these findings show that apoptosis precedes any detectable necrotic change in CHSE-214 cells that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in host cells and is followed by necrotic processes.

In vivo and in vitro studies on the antitumor activities of MCP (Malva crispa L. Powder).

Four short-term in vivo and in vitro tests were used to further confirm the antitumor activities of MCP, a vegetable powder, prepared from Malva crispa L. (i) In the H22 hepatoma-transplanting test, MCP had antitumor action, but MCP residue did not show such action; 5-FU appeared to have more potent antitumor activities and more harmful effects than MCP. (ii) In the micronucleus (MN) test, MCP significantly decreased MN frequency. (iii) In the cancer cell culture systems, the MCP fat-soluble extract revealed inhibitory effects on the growth and proliferation of the human hepatoma and the gastric cancer cells in a dose-response manner. (iv) In the colony formation test, MCP also altered the morphology of human gastric cancer cells. It was suggested that MCP could be consumed not only by healthy subjects for cancer prevention but also by patients with cancer as supplementary treatment in combination with anticarcinogenic drug such as 5-FU, cyclophosphamide (CP).

Involvement of serine proteinase in infectious pancreatic necrosis virus capsid protein maturation and NS proteinase cleavage in CHSE-214 cells.

An investigation of virus-specific protein maturation in infectious pancreatic necrosis virus (IPNV) infected Chinook salmon embryo cells (CHSE-214) was undertaken. The precursor protein (pVP2-1) of the major mature capsid protein (VP2) was processed sequentially from pVP2-1 to pVP2-2 and VP2. Experiments using serine proteinase inhibitors showed that the maturation of the VP2 was blocked in the pVP2-1 post-translational cleavage steps. A protinin, a potent proteinase inhibitor, at 800 µg ml(-1) blocked pVP2-2 to VP2 and the cleavage of VP4 (28 kDa) to VP4-1 (25 kDa). Therefore, our data showed that the maturation of the capsid protein (VP2) and cleavage of VP4 (NS proteinase) can be blocked by serine proteinase inhibitors.

3-Year-old phenotypic female with campomelic dysplasia and bilateral gonadoblastoma.

The authors report the case of bilateral gonadoblastomas in a phenotypic female, with a 46,XY karyotype, with campomelic dysplasia. Although campomelic dysplasia with gonadal dysgenesis should be expected to contribute to an increased risk of gonadoblastoma, this is the first documented case report of campomelic dysplasia and gonadoblastoma. Phenotypic females with campomelic dysplasia should be karyotyped once the skeletal dysplasia is recognized. phenotypic females with campomelic dysplasia should undergo gonadectomy if their karyotype includes a Y chromosome or fragment.

Weaning practices and growth in rural Sichuan infants: a positive deviance study.

To understand some of the factors involved in weaning and growth faltering in rural China, a cross-sectional positive deviance study was undertaken among 389 rural 4-12-month-old infants from two townships of a county in Sichuan. The infants' mothers were interviewed about their child-feeding practices and other sociodemographic information, and anthropometric measurements were made on their infants. Positive deviant infants (those growing adequately in environments in which the majority of the children suffer from growth retardation and malnutrition) were identified from the Chinese WAZ-scores calculated from the anthropometric measurements. Feeding practices found to be associated with the better growth of the positive deviant infants included breastfeeding through age 12 months, feeding soybean milk, liver and pork blood products on a more than weekly basis during the ages of 7-9 months, not feeding rice flour (mifen) before age 7 months, and not giving supplements or tonics. Mothers' nutrition knowledge was also associated with positive deviance status. The relevance of the findings is discussed with respect to designing nutrition education interventions for rural Sichuan.

Effect of Konjac food on blood glucose level in patients with diabetes.

Seventy-two type II diabetic subjects were given Konjac food for 65 days. The data analyzed by multiple F test indicate that the fasting blood glucose (FBG) and the 2-h postprandial blood glucose (PBG) on the 30th and the 65th days after the food was ingested were significantly reduced (P = 0.001, P less than 0.001, respectively), as was the glycosylated hemoglobin level at the end of the trial (P less than 0.05). The final FBG and PBG of the subjects with initial FBG-O greater than 200 mg% decreased on the average by 51.8 and 84.6 mg%, respectively; those with FBG-O 150-200 mg% decreased by 24.1 and 68.7 mg%; and those with FBG-O less than 150 mg% decreased by 4.8 and 21.4 mg%. No significant changes in blood lipid indexes were observed, except that the triglyceride values of subjects with hypertriglyceridemia (greater than 200 mg%) significantly decreased by 118.7 mg%. It was concluded that Konjac food is very useful in the prevention and treatment of hyperglycemia.

The effect of foods containing refined Konjac meal on human lipid metabolism.

A total of 110 elderly people with hyperlipidemia were randomly assigned to one of two groups. The experimental group consumed an ordinary diet plus foods containing refined Konjac meal, and the control group consumed only the ordinary diet. The experiment was carried out for 45 days. The results indicate that for the experimental group blood levels of triglycerides (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) were significantly lowered (P less than 0.01) at the end of the trial, whereas high-density lipoprotein cholesterol (HDL-C) and apoprotein (AI) levels were significantly elevated (P less than 0.01). In contrast, for the control group, the changes in the above parameters were insignificant. The differences in TC, TG, LDL-C, and HDL-C levels between the two groups were statistically significant. The effects of refined Konjac meal on lipid levels in the blood were somewhat different between patients with hyperlipidemia and subjects with risk critical values only. For the former, TG and TC were decreased by 83.8 +/- 133.5 mg/dl, and 42.4 +/- 23.4 mg/dl, respectively; but for the latter, they are decreased only by -1.1 +/- 23.1 mg/dl and 8.3 +/- 18.2 mg/dl, respectively; the difference mentioned above is statistically significant (P less than 0.01).