Intragastric ethanol infusion model for cellular and molecular studies of alcoholic liver disease

The intragastric alcohol infusion rat model (IAIRM) of alcoholic liver disease (ALD) has been utilized in various laboratories to study various aspects of ALD pathogenesis including oxidative stress, cytokine upregulation, hypoxic damage, apoptosis, ubiquitin-proteasome pathway and CYP2E1 induction. The basic value of the model is that it produces pathologic changes which resemble ALD including microvesicular and macrovesicular fat, megamitochondria, apoptosis, central lobular and pericellular fibrosis, portal fibrosis, bridging fibrosis, central necrosis, and mixed inflammatory infiltrate including PMNs and lymphocytes. The model is valuable because the diet and ethanol intake are totally under the control of the investigator. A steady state can be maintained with high or low blood alcohol levels for long periods. The cycling of the blood alcohol levels, when a constant infusion rate of alcohol is maintained, simulates binge drinking. Using this model the importance of dietary fat, especially the degree of saturation of the fatty acids on the induction of liver pathology, has been documented. The role of endotoxin, the Kupffer cell, TNFalpha, and NADPH oxidase have been demonstrated. The importance of 2E1 in oxidative stress induction has been shown using inhibitors of the isozyme. The importance of dietary iron in the pathogenesis of cirrhosis has been documented. Acetaldehyde has been shown to play a role in preventing liver pathology by preventing NFkappaB activation. Using the model, to maintain high blood alcohol levels is found to be necessary to demonstrate proteasomal peptidase inhibition. Ubiquitin synthesis is also inhibited at high blood alcohol levels in the IAIRM model. Oxidized proteins accumulate in the liver at high blood alcohol levels. Neoantigens derived from protein adducts formed with products of oxidation induce autoimmune mechanisms of liver injury. Thus, in many ways the model has revolutionized our understanding of the pathogenesis of ALD.     

Immunolocalization of fibroblast growth factor receptors 1 and 2 in mouse palate development

Recent evidence has implicated mutations of fibroblast growth factor receptors (FGF-R) in the pathogenesis of craniosynostotic syndromes. Cleft palate can be a component of such syndromes. The expression of FGF-R1 and FGF-R2 has been delineated in normally developing cranium, where they seem to regulate cellular differentiation and proliferation, respectively. The specific role of fibroblast growth factor signaling in mammalian palate development is unclear. The authors investigated the patterns of expression of FGF-R1 and FGF-R2 throughout mouse palatal development in the embryo. Time-dated CD-1 mouse heads (n = 135) were harvested at embryonic ages 12.5, 13.5, 14.5, 15.5, and 16.5 days (term gestation = 19.5 days), fixed in paraformaldehyde, embedded in paraffin, and sectioned. In addition, paired palatal shelves (n = 30) were isolated by means of microdissection from embryonic day--13.5 embryos, grown on Millipore filters in serum-free medium in vitro for 24, 48, 72, or 96 hours and processed for histological analysis. Immunohistochemical analysis for FGF-R1 and FGF-R2 was performed on the in vivo and in vitro specimens. FGF-R1 and FGF-R2 were found to be specifically expressed in the epithelium of the developing palatal shelves from the time of their outgrowth from the maxillary processes through completion of fusion in vivo and in vitro. Expression of both receptors was particularly strong during the phases of medial epithelial-medial epithelial contact between the individual shelves, through the formation of the medial epithelial seam, to the ultimate dissolution of the seam. Such a pattern of expression seems to implicate fibroblast growth factor signaling in the regulation of the critical phase of fusion of the bilateral shelves. The expression of both FGF-R1 and FGF-R2 in the lateral palatal mesenchyme, where such secondary structures as tooth primordia and bone begin to appear, also suggests a role for fibroblast growth factor signaling in the induction of ongoing differentiation and maturation of the palate after fusion. These data suggest that fibroblast growth factor signaling may play a role in the epithelial-mesenchymal interactions that dictate fusion and maturation of the developing palate. Furthermore, the data are consistent with the correlation of cleft palate formation with aberrant fibroblast growth factor signaling.     

Intraneuronal amyloid precursor protein (APP) and appearance of extracellular beta-amyloid peptide (abeta) in the brain of aging kokanee salmon

Antibodies to human amyloid precursor protein (APP(695)) and beta-amyloid peptide (A beta(1-42)) were used to determine timing of amyloidosis in the brain of kokanee salmon (Oncorhynchus nerka kennerlyi) in one of four reproductive stages: immature (IM), maturing (MA), sexually mature (SM), and spawning (SP), representing a range of aging from somatically mature but sexually immature to spawning and somatic senescence. In IM fish, immunoreactive (ir) intracellular APP occurred in 18 of 23 brain regions. During sexual maturation and aging, the number of neurons expressing APP increased in 11 of these APP-ir regions. A beta-ir was absent in IM fish, present in seven regions in MA fish, moderately abundant in 15 regions in SM fish, and was most abundant in all brain regions of SP fish exhibiting A beta-ir. Intracellular APP-ir was observed in brain regions involved in sensory integration, olfaction, vision, stress responses, reproduction, and coordination. Intra- and extracellular A beta(1-42) immunoreactivity (A beta-ir) was present in all APP-ir regions except the nucleus lateralis tuberis (hypothalamus) and Purkinje cells (cerebellum). APP-ir and A beta deposition increase during aging. APP-ir is present in IM fish; A beta-ir usually appears first in MA or SM fish and increases in SM fish as does APP-ir. Extracellular A beta deposition dramatically increases between SM and SP stages (1-2 weeks) in all fish, indicating an extremely rapid and synchronized process. Rapid senescence observed in pacific salmon could make them a useful model to investigate timing of amyloidosis and neurodegeneration during brain aging.     

Ectopic expression of MHC class II genes (RT1.B(I) beta/alpha) in rat hepatocytes in vivo and in culture can be elicited by treatment with the pregnane X receptor agonists pregnenolone 16 alpha-carbonitrile and dexamethasone

The synthetic steroid, pregnenolone-16-alpha-carbonitrile (PCN), has served for decades as a probe for a postulated series of hepatic defenses activated under situations of environmental "stress". PCN, an antiglucocorticoid, and also such glucocorticoids as dexamethasone (Dex) appear to stimulate hepatic metabolism and elimination of xenobiotics by binding to the nuclear pregnane X receptor (PXR) which then interacts with a distinct DNA response element associated with induction of cytochrome P450 3A genes. To explore the full domain of genes controlled by PCN/PXR, we used differential display to detect rat liver mRNA species selectively induced by PCN or by Dex. Sequence analysis identified one of many PCN induced cDNA fragments as RT1.B(I)beta, a member of the major histocompatability class II (MHC) gene family usually found only in antigen presenting cells. Northern blot analysis of RNA from rat liver or from cultured hepatocytes confirmed that amounts of RT1.B(I)beta mRNA and also of its companion gene, RT1.B(I)alpha mRNA, became readily detectable within 3-6 hours following treatment with PCN or Dex, whereas no induction was observed in spleen RNA. Induction by PCN of RT1.B(I)beta immunoreactive protein was localized to the hepatocytes as judged by immunofluorescence. We conclude that ectopic expression of MHC II genes, an unprecedented effect of steroids or drugs, is rapidly evoked by PCN acting on the liver, directly. The concept of a set of genes coordinately controlled to maintain homeostasis in parenchymal tissues during toxic stress must now be extended to include the immune system.     

Regulation of the production of polygalacturonase by Aspergillus niger

Synthesis of ethylene in static cultures as well as the effect of endogenous and exogenous ethylene on the synthesis of polygalacturonase byAspergillus niger were determined. This strain produced maximum ethylene amounts when cultured at 30 °C for 3 d. The effect of adding ethylene precursors (citrate-cycle intermediates) on ethylene production was investigated. Best intracellular and extracellular polygalacturonase production was obtained with 2-oxoglutaric, pyruvic and fumaric acids, and with glutamic acid too. Addition of ethylene to the culture medium also increased the synthesis of polygalacturonase, although to a lower degree than when glutamic acid was added.     

Pharmaco-toxicological study of Kageneckia oblonga, Rosaceae

The probable antipyretic, antiinflammatory, analgesic and antioxidant properties of Kageneckia oblonga, Rosaceae, were investigated and the major compounds of its active extracts were isolated. The study comprised the acute toxicity of the extracts of global methanol, hexane, dichloromethane and methanol. The cytotoxicity of global methanol extract was studied in three tumoral cell lines. All the extracts exhibited the pharmacological activities under study. Methanol and dichloromethane were the most toxic extracts. From the global methanol extract, isolations were performed of prunasin, 23,24- dihydro-cucurbitacin F, and a new cucurbitacin, 3beta-(beta-D-glucosyloxy)-16alpha,23alpha-epoxycucurbita-5,24-diene-11-one. The cytotoxicity of both cucurbitacins on human neutrophils at the assayed concentrations was not statistically significant. In-vitro assays showed that both cucurbitacins can be partly responsible for the analgesic, antipyretic, and anti-inflammatory activities. Evaluation was done of the cytotoxicity of global methanol extract, 23, 24-dihydrocucurbitacin F, aqueous extracts and prunasin against P-388 murine leukaemia, A-549 human lung carcinoma and HT-29 colon carcinoma. Since global methanol extract presented a strong cytotoxicity against P-388 murine leukaemia, A-549 human lung carcinoma, and HT-29 cell lines, it is highly probable that this extract contain one or more cytotoxic compounds that could be investigated for their potential use as an agent against cancer.     

Oxygen and nitrate-dependent regulation of <Emphasis Type="Italic">dmsABC</Emphasis> operon expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>: sites for Fnr and NarL protein interactions

Background  

Escherichia coli can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation. Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present.     

A cytoplasmically inherited mutant controlling early chloroplast development in barley seedlings

Cytoplasmic line 2 (CL2) has been previously reported as a cytoplasmically inherited chlorophyll-deficient mutant selected from a chloroplast-mutator genotype of barley. It was characterized by a localized effect on the upper part of the first-leaf blade. At emergence the CL2 seedlings-phenotype varied from a grainy light green to an albino color. They gradually greened during the following days, starting from the base of the blade and extending to cover most of its surface when it was fully grown. The present results, from both light microscopy and transmission electron microscopy (TEM), confirmed the previously described positional and time-dependent expression of the CL2 syndrome along the first-leaf blade. During the first days after emergence, light microscopy showed a normally developed chloroplast at the middle part of the CL2 first-leaf blade, meanwhile at the tip only small plastids were observed. TEM showed that the shapes and the internal structure of the small plastids were abnormal, presenting features of proplastids, amyloplasts and/or senescent gerontoplasts. Besides, they lack plastid ribosomes, contrasting with what was observed inside chloroplasts from normal tips, which presented abundant ribosomes. Phenotypic observations and spectrophotometric analysis of seedlings produced by mother plants that had been grown under different temperatures indicated that higher temperatures during seed formation were negatively associated with pigment content in CL2 seedlings. In contrast, higher temperatures during the growth of CL2 seedlings have been associated with increased pigment content. Aqueous solution with kanamycin and streptomycin, which are antibiotics known to interfere with plastid gene translation, were used for imbibition of wild-type and CL2 seeds. Antibiotic treatments differentially reduced the chlorophyll content in the upper part of the first-leaf blade in CL2, but not in wild-type seedlings. These results suggest that in the wild-type, plastid-gene proteins which are necessary for chloroplast development and chlorophyll synthesis in the upper part of the first-leaf blade are usually synthesized during embryogenesis. However, under certain circumstances, in CL2 seedlings, they would be synthesized after germination. In addition, a shortening of the sheath has been observed in association with pigment decrease suggesting the existence of plastid factors affecting the expression of some nuclear genes. We consider the CL2 mutant a unique experimental material useful to study biological phenomena and external factors regulating plastid, and nuclear gene expression during embryogenesis and early seedling development.Communicated by R. Hagemann