Antioxidants prevent UV-induced apoptosis by inhibiting mitochondrial cytochrome c release and caspase activation in Spodoptera frugiperda (Sf9) cells

Oxidative stress has been shown to be associated with apoptosis (programmed cell death) in a number of cell systems. We earlier reported in vitro cultured Spodoptera frugiperda (Sf9) cells as a model system to study oxidative stress induced apoptosis (J Biosciences 24 (1999) 13) and the inhibition of UV-induced apoptosis by the baculovirus antiapoptotic p35 protein that acts as a sink to sequester reactive oxygen species (Proc Natl Acad Sci USA 96 (1999) 4838). We now show that UV-induced apoptosis in Sf9 cells, is preceded by the release of mitochondrial cytochrome c into the cytosol and consequent activation of Sf-caspase-1. The inhibitory effect of different antioxidants including scavengers of oxygen radicals such as butylated hydroxyanisole (BHA), alpha tocopherol acetate, benzoate and reduced-glutathione (GSH) on ultra violet B (UVB)-induced apoptosis in cultured Sf9 cells was assessed. Both, cytochrome c release as well as Sf-caspase-1 activation was inhibited by pre-treatment with antioxidants such as BHA and alpha tocopherol acetate, suggesting that these antioxidants inhibit apoptosis by acting quite upstream in the apoptosis cascade at the mitochondrial level, as well as downstream at the caspase level.     

The 14-3-3 proteins Rad24 and Rad25 negatively regulate Byr2 by affecting its localization in Schizosaccharomyces pombe

In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis. In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase [MAPK] kinase kinase). Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells. In contrast, the mating and sporulation efficiency of rad24- or rad25-null cells was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1(S214DT218D). Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined. The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation. Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants. In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein. Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation. Deletion of rad24 or rad25 fastened the timing of Byr2 translocation. Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.     

Differential protein expression at the stage of neural tube closure in the mouse embryo

Analysis of the protein complement of a biological system through proteomics provides the opportunity to directly monitor the functional readout of gene expression. In this study, proteomics was applied to the mouse embryo to investigate the molecular events underlying the processes occurring at the stage of neural tube closure. Protein profiles of embryos between embryonic days 8.5 and 10.5 exhibited a number of stage-specific changes. Identification of developmentally regulated proteins by mass spectrometry revealed several groups of functionally related proteins including circulatory, cytoskeletal, and stress proteins. Additional proteins of unknown function were identified, such as Copine 1 and PICOT, whose developmental regulation was previously unsuspected.     

Cardiovascular risk factors in subjects with Helicobacter pylori infection

Background. It has been proposed that Helicobacter pylori infection is related to cardiovascular disease, although this has not been fully investigated. The aim of this study was to investigate whether H. pylori in‐fection is associated with cardiovascular risk factors. Subjects and Methods. One thousand six hundred and fifty people undergoing annual medical checks at Shimane Institute of Health Science between September 1998 and August 1999 were enrolled. Gender, age, body mass index, habitual smoking and drinking, systolic and diastolic blood pressure, serum level of total cholesterol, triglyceride, high‐density lipoprotein cholesterol (HDLC), blood glucose, leukocyte count and hemoglobin were compared between H. pylori seropositive and seronegative cases. Results. In H. pylori seropositive individuals, HDLC was significantly lower than that in seronegative individuals. After adjustment for possible confounding factors (gender, age, BMI, smoking and drinking habits), mean HDLC in H. pylori‐seropositive and seronegative individuals were 56.1 and 58.2 mg/dl, respectively (p < .005). The percentage of the elderly (over 50 years old) individuals with HDLC < 35 mg/dl in H. pylori seropositive and seronegative groups were 7.4% and 4.7%, respectively (p < .001). In addition, the lower HDLC level was accompanied by an increased leukocyte count. Conclusion. Long‐term infection with H. pylori may have an important role in decreasing the serum HDLC concentration.     

Biochemical and genetic characterization of mundticin KS,an antilisterial peptide produced by Enterococcus mundtii NFRI 7393

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.     

Programmed cell death and its clinical implications

Cell death is a highly regulated process that is ubiquitous in all eukaryotes. Programmed cell death (PCD) is an integral part of both animal and plant development. Studies on apoptosis, the well characterized form of programmed cell death led to the identification of a central tripartite death switch i.e. apoptosome consisting of Apaf-1, Apaf-2 and Apaf-3. The caspases, a family of cysteine-dependent aspartate directed-proteases, constitute the central executioners of apoptosis. Much of the attention on programmed cell death is focused on caspases, however, cell death can still occur even when the caspase cascade is blocked, revealing the existence of nonapoptotic alternative pathway(s) of cell death. The mitochondrial release of cytochrome C following a PCD inducing stimulus in both plants and animals suggests the evolutionary conservation of death pathways. Dysregulation of apoptosis may be related to the development of several disease states as well as ageing. Excessive apoptosis is associated with neurodegenerative disorders, AIDS etc., whereas deficient apoptosis is associated with cancer, auto-immunity, viral infections etc. Understanding the regulation of programmed cell death would throw light in designing drugs and gene therapies that can target specific molecules in the apoptotic pathway opening the vistas for new therapeutic endeavors in many areas of medicine.     

Determination of soil-to-plant transfer factors of 137Cs and 90Sr in the tropical environment of Bangladesh

Soil-to-plant transfer factors (TF) of ¹³⁷Cs and ⁹⁰Sr have been determined for different plants/crops, such as rice, beans, peanuts, pineapple, cabbage, tomato, spinach and grass. They were obtained from radioisotope experiments on plants grown in pots under outdoor ambient tropical conditions for three growing seasons (1994–1996). In the case of ¹³⁷Cs and concerning the above mentioned plants/crops, the average TFs were found to be 0.28, 0.25, 0.77, 0.19, 0.23, 0.28, 0.59 and 0.18, respectively. In the case of ⁹⁰Sr, the average TFs were found to be 0.82, 0.51, 0.20, 0.82, 0.69, 0.59, 0.91 and 0.84, respectively. A minor seasonal variation was observed. This study provides a database of TFs for tropical environments to be used, e.g., for radiological safety assessment models. Received: 14 October 1997 / Accepted in revised form: 9 February 1998     

Vascular smooth muscle cell growth and insulin regulation of mitogen-activated protein kinase in hypertension

Hyperinsulinemia (HI) and insulin resistance (IR) are frequentlyassociated with hypertension and atherosclerosis. However, the exactroles of HI and IR in the development of hypertension are unclear.Mitogen-activated protein kinases (MAPK) are well-characterized intracellular mediators of cell proliferation. In this study, weexamined the contribution of MAPK pathway in insulin-stimulated mitogenesis using primary vascular smooth muscle cells (VSMCs) isolatedfrom aortas of normotensive Wistar-Kyoto rats (WKY) and spontaneoushypertensive rats (SHR). VSMCs were grown to confluence in culture,serum starved, and examined for DNA synthesis {using [³H]thymidine (TDR),immunoprecipitated MAPK activity, and MAPK phosphatase (MKP-1)induction}. Basal rate of TDR incorporation into DNA was twofoldhigher in SHR compared with WKY (P < 0.005). Insulin caused a dose-dependent increase in TDR incorporation (150% over basal levels with 100 nM in 12 h). Stimulation was sustained for 24 h with a decline toward basal in 36 h. Pretreatment with insulin-like growth factor I (IGF-I) receptor antibody did notabolish mitogenesis mediated by 10-100 nM insulin, suggesting thatinsulin effect is mediated via its own receptors. Insulin had a smallmitogenic effect in WKY (33% over basal). Insulin-stimulated mitogenesis was accompanied by a dose-dependent increase in MAPK activity in SHR, with a peak activation (>2-fold over basal) between 5 and 10 min with 100 nM insulin. Insulin had very small effects onMAPK activity in WKY. In contrast, serum-stimulated MAPK activation wascomparable in WKY and SHR. Pretreatment with MEK inhibitor, PD-98059,completely blocked insulin's effect on MAPK activation andmitogenesis. Inhibition of phosphatidylinositol 3-kinase with wortmannin also prevented insulin's effects on MAPK activation andmitogenesis. In WKY, insulin and IGF-I treatment resulted in a rapidinduction of MKP-1, the dual-specificity MAPK phosphatase. Incontrast, VSMCs from SHR were resistant to insulin with respect toMPK-1 expression. We conclude that insulin is mitogenic in SHR, and theeffect appears to be mediated by sustained MAPK activation due toimpaired insulin-mediated MKP-1 mRNA expression, which may act asan inhibitory feedback loop in attenuating MAPK signaling.

     

Ordered Cloned DNA Map of the Genome of Vibrio cholerae 569B and Localization of Genetic Markers

By using a low-resolution macrorestriction map as the foundation (R. Majumder et al., J. Bacteriol. 176:1105–1112, 1996), an ordered cloned DNA map of the 3.2-Mb chromosome of the hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. A cosmid library the size of about 4,000 clones containing more than 120 Mb of V. cholerae genomic DNA (40-genome equivalent) was generated. By combining landmark analysis and chromosome walking, the cosmid clones were assembled into 13 contigs covering about 90% of the V. cholerae genome. A total of 92 cosmid clones were assigned to the genome and to regions defined by NotI, SfiI, and CeuI macrorestriction maps. Twenty-seven cloned genes, 9 rrn operons, and 10 copies of a repetitive DNA sequence (IS1004) have been positioned on the ordered cloned DNA map.     

Further constituents from the bark of Holarrhena pubescens.

Three compounds, pubadysone [11 alpha-hydroxy-18,20-oxido-3-oxo-pregna-1,4,17(20)-triene] (1), puboestrene [3-acetoxy-17-oxo-1,3,5(10)-estratriene] (2) and pubamide [3,18-dioxo-11 alpha-hydroxycona-1,4-diene] (3), have been isolated from the bark of Holarrhena pubescens. Their structures have been established through spectroscopic studies.