Dynamic expression of the cell adhesion molecule cell-CAM 105 in fetal and regenerating rat liver

Cell-CAM 105 is an integral cell surface glycoprotein that is involved in cell-cell adhesion of adult rat hepatocytes in vitro. In the present report we used a radio-immunoassay, a quantitative immunoblotting technique and immunofluorescence microscopy to investigate the expression of cell-CAM 105 in fetal and regenerating rat liver. In the fetal liver cell-CAM 105 did not appear until day 16 of the gestation, when it increased rapidly to reach the level found in adult liver, 3 weeks after birth. In liver regenerating after partial hepatectomy a transient decrease in the amount of cell-CAM 105 was observed in the plasma membranes of the hepatocytes. A significant decrease was observed as early as 12 h after partial hepatectomy, reaching a minimum by 3 days after the operation, corresponding to approx. 35% of the amount of cell-CAM 105 in normal liver. The amount then increased slowly and was back to the normal level by about 15 days after partial hepatectomy. The results indicate that cell-CAM 105 exerts its major function in terminally differentiated cells. An excellent correlation was seen between the kinetics of the expression of cell-CAM 105 and of reported changes of both enzymatic and organizational patterns of hepatocytes in regenerating and fetal liver. This suggests that cell-CAM 105 could be important for the development and maintenance of the cell-cell binding and organizational pattern characteristic of terminally differentiated hepatocytes.     

Nitric oxide binding properties of neuroglobin. A characterization by EPR and flash photolysis

Neuroglobin is a recently discovered member of the globin superfamily. Combined electron paramagnetic resonance and optical measurements show that, in Escherichia coli cell cultures with low O(2) concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe(2+)-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO. Analogous studies for mutated neuroglobin (mutation of E7-His to Leu, Val, or Gln) reveal the predominant presence of the nitrosyl ferrous form. After sonication of the cells wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric form, whereas NO ligation initially protects the mutants from oxidation. Flash photolysis studies of wild-type neuroglobin and its E7 mutants show high recombination rates (k(on)) and low dissociation rates (k(off)) for NO, indicating a high intrinsic affinity for this ligand similar to that of other hemoglobins. Since the rate-limiting step in ligand combination with the deoxy-hexacoordinated wild-type form involves the dissociation of the protein ligand, NO binding is slower than for the related mutants. Structural and kinetic characteristics of neuroglobin and its mutants are analyzed. NO production in rapidly growing E. coli cell cultures is discussed.     

Multifrequency EPR study of carbonate- and sulfate-derived radicals produced by radiation in shells and corallite

Shells of two sea mollusks (Venus sp.), pearl oyster (Meleagrina vulgaris) and corallite (white coral) were exposed to ionizing radiation (gamma and X rays) and then examined by EPR spectroscopy in X, Q and W band. The resulting spectra were analyzed and the g values of the EPR lines in the multicomponent spectra were determined. The increased resolution in Q- and W-band spectra allowed us to assign the observed lines to CO(2)(-) ion radicals (isotropic and orthorhombic), SO(2)(-) isotropic, SO(3)(-) (isotropic and axial), and Mn(2+) species. The assignments were confirmed by simulations of the spectra. Practical implications for the use of Q and/or W band in low-dose quantitative EPR measurements for dating and for accidental dose estimation are discussed.     

Mycotoxin fumonisin B1 increases intestinal colonization by pathogenic Escherichia coli in pigs

Fumonisin B(1) (FB(1)) is a mycotoxin that commonly occurs in maize. FB(1) causes a variety of toxic effects in different animal species and has been implicated as a contributing factor of esophageal cancers in humans. In the present study, we examined the effect of dietary exposure to FB(1) on intestinal colonization by pathogenic Escherichia coli associated with extraintestinal infection. Three-week-old weaned pigs were given FB(1) by gavage as a crude extract or as a purified toxin at a dose of 0.5 mg/kg of body weight daily for 6 days. On the last day of the toxin treatment, the pigs were orally inoculated with an extraintestinal pathogenic E. coli strain. All animals were euthanized 24 h later, necropsies were performed, and tissues were taken for bacterial counts and light microscopic examination. Ingestion of FB(1) had only a minimal effect on animal weight gain, did not cause any macroscopic or microscopic lesions, and did not change the plasma biochemical profile. However, colonization of the small and large intestines by an extraintestinal pathogenic E. coli strain was significantly increased. Our results show that FB(1) is a predisposing factor to infectious disease and that the pig can be used as a model for the study of the consequences of ingesting mycotoxin-contaminated food.     

Auxotrophic Mutations Reduce Tolerance of Saccharomyces cerevisiae to Very High Levels of Ethanol Stress

Very high ethanol tolerance is a distinctive trait of the yeast Saccharomyces cerevisiae with notable ecological and industrial importance. Although many genes have been shown to be required for moderate ethanol tolerance (i.e., 6 to 12%) in laboratory strains, little is known of the much higher ethanol tolerance (i.e., 16 to 20%) in natural and industrial strains. We have analyzed the genetic basis of very high ethanol tolerance in a Brazilian bioethanol production strain by genetic mapping with laboratory strains containing artificially inserted oligonucleotide markers. The first locus contained the ura3Δ0 mutation of the laboratory strain as the causative mutation. Analysis of other auxotrophies also revealed significant linkage for LYS2, LEU2, HIS3, and MET15. Tolerance to only very high ethanol concentrations was reduced by auxotrophies, while the effect was reversed at lower concentrations. Evaluation of other stress conditions showed that the link with auxotrophy is dependent on the type of stress and the type of auxotrophy. When the concentration of the auxotrophic nutrient is close to that limiting growth, more stress factors can inhibit growth of an auxotrophic strain. We show that very high ethanol concentrations inhibit the uptake of leucine more than that of uracil, but the 500-fold-lower uracil uptake activity may explain the strong linkage between uracil auxotrophy and ethanol sensitivity compared to leucine auxotrophy. Since very high concentrations of ethanol inhibit the uptake of auxotrophic nutrients, the active uptake of scarce nutrients may be a major limiting factor for growth under conditions of ethanol stress.     

Catalase and estradiol inhibit mitochondrial protein S-glutathionylation

Regulation and downstream effects of mitochondrial protein S-glutathionylation in response to oxidative stress are poorly understood. The study aim was to determine whether anti-oxidants such as catalase and estradiol alter mitochondrial protein S-glutathionylation and in turn affect apoptosis following ultraviolet B (UV-B) light irradiation. HeLa cells were transduced with increasing amounts of adenovirus encoding catalase (Ad-Cat) and β-galactosidase (Ad-Lac Z) or pre-incubated with estradiol before induction of apoptosis by UV-B light exposure. Inhibition of mitochondrial protein S-glutathionylation was assessed using autoantibodies specific for the non-S-glutathionylated form of PDC-E2. The percentage of apoptotic cells following UV-B irradiation were not significantly different between mock cells (cells with no virus infection) and Ad-Cat and Ad-Lac Z infected cells at all viral doses (all p > 0.050). Autoantibody staining of non-S-glutathionylated PDC-E2 in apoptotic cells was three times greater in only Ad-Cat infected cells compared to only Ad-Lac Z infected cells (81.3 ± 16.7 vs 26 ± 7.2 %, respectively, p = 0.030). Similarly estradiol treatment (33 and 100 nM) also significantly increased PDC-E2 staining in apoptotic cells compared to non-treated cells (both p < 0.010). The percentage of apoptotic cells was not significantly different with any of the estradiol concentrations (all p > 0.100). The observed procaspase 12 cleavage following UV-B irradiation suggests that a mitochondrial-independent apoptotic pathway was activated. In conclusion, following an apoptotic stimulus, estradiol may inhibit mitochondrial protein S-glutathionylation without inhibiting apoptosis. This effect may play a role in ninefold greater prevalence of autoantibodies against PDC-E2 in women with primary biliary cirrhosis.     

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.