Effects of Alkyl Alcohols and Related Chemicals on Rat Liver Structure and Function: III. Physicochemical Properties of Ethanol-, Propanol- and Butanol-treated Rat Liver Mitochondrial Membranes

The physicochemical properties of mitochondria in liver tissue obtained from rats given 32% ethanol, 32% propanol or 6.9% butanol in drinking water for up to 3 months were investigated using differential scanning calo-rimetry and fluorescence polarization measurements. The results obtained were as follows: 1) Phospholipids extracted from mitochondria showed increases in the relative amounts of phosphatidylcholine, phosphatidylinositol and phosphatidylserine, and a decrease in the relative amount of phosphatidylethanolamine. An increase in the unsatu-rated/saturated fatty acid ratio of phospholipids was also observed. 2) Elevation of the thermotropic lipid phase transition temperature with a decrease in the enthalpy value (δ H ) was revealed by differential scanning calo-rimetry. 3) The elevation of the lipid phase transition temperature was detected also by fluorescence polarization measurements using 1,6 diphenyl 1, 3, 5 hexatriene (DPH) as a probe. Elevation of mitochondrial membrane fluidity was found in some of the experimental animals, but most showed no changes in comparison with the control. A possible role of membrane fusion in the mechanism of formation of ethanol-, propanol- and butanol induced hepatic megamitochondria is discussed on the basis of these results. Acta Pathol Jpn 42: 549–557, 1992.     

Potassium induced potential changes in rat diaphragm muscle

Summary The effect of different potassium concentrations on the membrane potential and membrane resistance of rat diaphragm muscle fibres was measured by means of a double sucrose gap method and a microelectrode technique. Concentration measurements showed that the muscle fibres gained sodium and lost potassium in the equilibration period.In the absence of external chloride changing the external potassium concentration from 2.8 mM to potassium-free caused a depolarization of the membrane of about 30 mV and a small increase in membrane resistance.This K-dependent potential change (K-response) was reduced by ouabain, K-strophanthin, 2,4-dinitrophenol and cyanide, indicating that an energy requiring process is involved. The temperature dependence of the K-response found is consistent with this assumption. Variation in potassium permeability in the absence and presence of external potassium could account for only 13% of the K-response.The K-response amplitude appeared to depend on the external potassium and the internal sodium concentration. Hyperpolarization of the membrane could not only be produced after readmission of potassium but also after addition of thallium, the latter being more potent. Raising the external chloride concentration resulted in a decrease of the K-response and membrane resistance. The current, generating the Kresponse was shown to be hardly influenced by conditional polarization of the membrane.It is concluded from these results that the Kresponse is mainly due to the operation of an electrogenic sodium pump.     

In vivo requirements for rDNA chromosome condensation reveal two cell-cycle-regulated pathways for mitotic chromosome folding

Chromosome condensation plays an essential role in the maintenance of genetic integrity. Using genetic, cell biological, and biochemical approaches, we distinguish two cell-cycle-regulated pathways for chromosome condensation in budding yeast. From G(2) to metaphase, we show that the condensation of the approximately 1-Mb rDNA array is a multistep process, and describe condensin-dependent clustering, alignment, and resolution steps in chromosome folding. We functionally define a further postmetaphase chromosome assembly maturation step that is required for the maintenance of chromosome structural integrity during segregation. This late step in condensation requires the conserved mitotic kinase Ipl1/aurora in addition to condensin, but is independent of cohesin. Consistent with this, the late condensation pathway is initiated during the metaphase-to-anaphase transition, supports de novo condensation in cohesin mutants, and correlates with the Ipl1/aurora-dependent phosphorylation of condensin. These data provide insight into the molecular mechanisms of higher-order chromosome folding and suggest that two distinct condensation pathways, one involving cohesins and the other Ipl1/aurora, are required to modulate chromosome structure during mitosis.     

Nano-C60 cytotoxicity is due to lipid peroxidation

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C₆₀, a fullerene aggregate that readily forms when pristine C₆₀ is added to water. Nano-C₆₀ was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses 50 ppb (LC₅₀=2–50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C₆₀ colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C₆₀ inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, l-ascorbic acid, the oxidative damage and resultant toxicity of nano-C₆₀ was completely prevented.     

Genetic Vaccination of Mice with Plasmids Encoding the NS1 Non-structural Protein from Tick-borne Encephalitis Virus and Dengue 2 Virus

Although there is a safe, inexpensive and efficacious vaccine against yellow fever, vaccination against other flavivirus diseases is less successful. There is no licensed vaccine against dengue fever and current vaccines against tick-borne encephalitis (TBE) and Japanese encephalitis are expensive and require several injections. Furthermore novel vaccines containing only virus envelope proteins may raise fears over antibody mediated enhancement (ADE) of disease. Here we report the successful use of genetic vaccination against TBE in an experimental animal model using a plasmid containing the coding sequence of a non-structural protein (NS1). Such vaccines would provide inexpensive protection against disease, without raising concerns over inducing ADE on subsequent exposure to heterotypic infectious virus. Attempts to generate chaemeric plasmids to protect against both TBE and dengue fever were less successful. Although these chaemeric plasmids directed the synthesis and secretion of the virus NS1 protein normally, no protection was observed against either TBE or dengue fever.     

During active viscerocutaneous leishmaniasis the anti-P2 humoral response is specifically triggered by the parasite P proteins.

In this work we show that in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum there are antibodies that react specifically against the parasite acidic ribosomal proteins LiP2a and LiP2b, and that each one of the Leishmania P proteins elicits a specific humoral immune response. Using synthetic peptides, the antigenic epitope of these proteins has been mapped in a single region located adjacent to the C-terminal domain highly conserved among the eukaryotic P proteins. The anti-P antibodies elicited during the Leishmania infection do not recognize the conserved C-terminal domain of the parasite P proteins, in contrast with the findings reported in Chagas' disease or systemic lupus erythematosus. The antigenic epitopes of the LiP2a and LiP2b are almost identical in amino acid sequence. No reactivity against Trypanosoma cruzi and human P proteins was found in sera from L. infantum-infected dogs.     

迁移率改变分析

本文介绍另一种检测特异顺序的DNA结合蛋白的理想方法——迁移率改变法。该方法是在特定的缓冲液中,蛋白质与带有同位素标记的DNA探针进行结合反应,然后在低离子强度的电泳缓冲液中,电泳分离DNA与蛋白质结合的复合物与未结合的游离DNA探针,进而鉴定特异顺序的DNA结合蛋白。     

Disease association of antibodies to human and mycobacterial hsp70 and hsp60 stress proteins.

Structural homology between microbial and human stress proteins has been postulated to be a basis for autoimmunization in chronic inflammatory diseases. Therefore, we estimated by ELISA titration the antibody levels to mycobacterial (M) and human (H) recombinant hsp70 and M-hsp65 heat-shock proteins in sera of patients with Crohn's disease (n = 29), ulcerative colitis (n = 20) and nontuberculous mycobacterial disease of the lungs (n = 20). Antibodies to H-hsp60, separated by two-dimensional gel electrophoresis, were tested in six sera of each group of patients. In Crohn's disease, antibody titres to the M-hsp65 antigen without detectable H-hsp60 binding were significantly elevated in 52% of the patients. In contrast titres to both M-hsp70 and H-hsp70 were demonstrable and correlated, but increased over control values only in four (14%) patients. The antibody pattern in ulcerative colitis was found to be quite different: anti-H-hsp60 binding was demonstrable in most patients, although anti-M-hsp65 titres were not elevated. Furthermore, 25% of patients had significantly elevated titres to M-hsp70, but not to H-hsp70. In non-tuberculous mycobacterial pulmonary disease, about 50% of patients had elevated titres to both hsp65 and hsp71 mycobacterial antigens but not to the corresponding human proteins; patients with Mycobacterium xenopi infection had the highest titres in this group. These results demonstrate the existence of distinct disease-associated patterns in the human antibody response to stress protein antigens. However, these data are not sufficient to imply sensitization with mycobacteria in patients with inflammatory bowel diseases, since certain epitopes of heat-shock proteins are shared by several bacterial genera.     

Autoantibody response to the Ro/La particle may predict outcome in neonatal lupus erythematosus.

This study was undertaken to determine the role of antibodies against both recombinant Ro (r-Ro) and La (r-La) proteins and polypeptides derived from the recombinant La protein in predicting fetal and neonatal outcome in children at risk to develop neonatal lupus erythematosus (NLE). All sera were obtained in the perinatal period and quantitative ELISA assays were used. We collected 41 maternal sera within 2 months of delivery of a child with NLE (21 with congenital heart disease block (CHB) and 20 with dermatologic NLE) and 19 sera from anti-Ro and/or anti-La antibody-positive mothers with systemic lupus erythematosus (SLE) who delivered a child without NLE. All sera were tested for anti-r-La and anti-r-Ro antibodies by ELISA, and most sera were tested for antibodies directed against La polypeptides by immunoblot. We found significantly higher anti-r-La antibody levels in the sera from mothers of children with NLE compared with sera from mothers of unaffected children (0.67 +/- 0.43 versus 0.14 +/- 0.30; P < 0.0001). There was a statistically significant difference in the mean anti-r-La levels between the sera of mothers of children with CHB compared with dermatologic NLE (0.51 +/- 0.45 versus 0.83 +/- 0.37 respectively; P = 0.0091). When we examined antibodies directed against the recombinant 52-kD Ro protein, there was a statistically significant elevation of titres in the sera of mothers of NLE children (0.77 +/- 0.35) compared with non-NLE mothers (0.29 +/- 0.39; P < 0.0001). There was no difference in the r-Ro levels between mothers of children with dermatologic NLE compared with CHB (0.82 +/- 0.37 versus 0.71 +/- 0.74; P = 0.32). When we examined polypeptides derived from the recombinant La protein, the mean number of polypeptides recognized by sera from mothers of children with NLE was significantly higher than the mean number of polypeptides recognized by sera from mothers of unaffected children (5.1 +/- 0.54 versus 2.3 +/- 0.54 respectively; P < 0.001). More importantly, when we examined the individual polypeptides, we found that only sera from mothers of children with NLE and not from mothers of unaffected children recognized a polypeptide designated DD (30% versus 0%, respectively). These studies indicate that the autoantibody response to the Ro/La particle can differentiate sera from mothers of children with NLE and sera from mothers of unaffected children. Furthermore, there was a difference in the anti-La autoantibody response between mothers of children with CHB and dermatologic NLE.(ABSTRACT TRUNCATED AT 400 WORDS)