Butyrate stimulates ApoA-IV-containing lipoprotein secretion in differentiated Caco-2 cells: role in cholesterol efflux

The aim of this study was to determine: (1) whether the Short Chain Fatty Acids (SCFA) Acetate, Propionate, and Butyrate enhance the synthesis and secretion of intestinal apolipoprotein A-IV-containing lipoproteins and (2) if so, whether these particles are able to promote cholesterol efflux in vitro. For this purpose Caco-2 cells were used for their functional properties of differentiated enterocytes. They were incubated with the three SCFA (2, 4, and 8 mM) for 48 h. Only butyrate stimulated apoA-IV gene expression and this was associated with an increase in apoA-IV secretion. A nondenaturing 2D-PAGE (agarose gel was followed by PAGE) was used to identify apoA-IV-containing lipoproteins in various media, and showed that butyrate stimulated the secretion of two small HDL sized particles. The influence of these secreted particles on cholesterol efflux was investigated using incubation of media with (3)H-cholesterol-labeled Fu5AH cells. The data indicate that conditioned media from Caco-2 cells treated with butyrate resulted in an increase of 20-30% in cholesterol efflux. We conclude that butyrate may regulate apoA-IV secretion and, therefore, modulate reverse cholesterol transport.     

A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals

An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees C) in a 30-60% sucrose gradient. Using a combination of SDS-PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS-PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes.     

Purification and characterization of NAD(P)H quinone reductase from the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae)

NAD(P)H quinone reductase [NAD(P)H-QR] present in the latex of Hevea brasiliensis Müll.-Arg. (Euphorbiaceae) was purified to homogeniety from the B-serum fraction obtained by freeze-thawing of the bottom fraction of ultracentrifuged fresh latex. The purification protocol involved acetone fractionation, heat treatment, ion exchange chromatography and affinity chromatography. The M(r) determined by SDS-PAGE for the protein subunit was 21 kDa, and the molecular mass of the native enzyme estimated by gel filtration was 83 kDa, indicating that the native enzyme is a homotetramer. The enzyme showed pH stability over a range of 6 to at least 10 (with an optimum at pH 8) and thermal stability up to 80 degrees C. High NAD(P)H-QR activity (70%) was still retained after 10 h of preincubation at 80 degrees C. A comparable substrate specificity for this enzyme was observed among menadione, p-benzoquinone, juglone, and plumbagin, with only duroquinone generating a lower activity. Positive correlations between latex NAD(P)H-QR activity and rubber yield per tapping [fresh latex (r=0.89, P<0.01), dry rubber (r=0.81, P<0.01)] together with flow time (r=0.85, P<0.01) indicated that enzyme activity could possibly be used as a marker to predict the yield potential of selected clones.     

Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt, 1925: differences in their cell surfaces and in the bacteria-containing vacuoles

Entamoeba histolytica Schaudinn, 1903 and Entamoeba dispar Brumpt. 1925 are two of eight species of Entamoeba that sometimes inhabit the human colon. The former is an invasive organism capable of causing life-threatening intestinal and extra-intestinal disease: the latter appears not to be invasive. Because the two species, when viewed by light microscopy appear morphologically similar, they were long regarded as a single species. However, recent biochemical. immunological, and genetic studies provided convincing evidence that they belong to separate species. Our ultrastructural studies revealed distinct differences in at least two features of the trophozoites. 1) The cell surfaces of the trophozoites of each species differ with regard to structures exposed on the surface, and the distribution and arrangement of intra-membranous proteins. 2) The phagocytosis of bacteria differs in respect to the formation of the phagocytic vacuoles. Loose vacuoles containing several bacteria were seen in E. histolytica whereas tight vacuoles containing a single bacterium were observed in E. dispar. Furthermore, bacteria were found only within vacuoles in E. histolytica; in E. dispar, bacteria were found within vacuoles and some were found free in the cytoplasm.     

In vitro self-assembled HCV core virus-like particles induce a strong antibody immune response in sheep.

The in vitro self-assembly properties of the entire hepatitis C virus core protein (HCcAg) obtained from Pichia pastoris cells and the induction of specific antibody immune response were studied. HCcAg was purified as a low-molecular-weight species by electroelution under denaturing conditions for confirmation of its self-assembly properties. After renaturalization, electron microscopy showed that HCcAg assembled into spherical particles of 30 nm. HCcAg also showed homogeneity and was specifically recognized by serum from a chronic HCV carrier patient. The data indicated that in vitro assembly of HCcAg, into virus-like particles resembling HCV nucleocapsid particles at a mature stage, is an intrinsic quality of this protein. Finally, HCcAg generated a strong antibody immune response in sheep, suggesting its usefulness for stimulating the host immune response against HCV.     

Freeze-fracture and cytochemical studies on the in vitro cyst form of reptilian Blastocystis pythoni

After cultured cysts are osmotically shocked by treating with distilled water, there is an exponential increase in the cyst form of Blastocystis pythoni; this was demonstrated by an immunofluorescence antibody assay against the culture organisms. In 11-day-old cultures of B. pythoni, 68.8% of the organisms (= 2.2 x 10(8) cysts/ml) were in the cyst form. Examination of thin sections of cysts revealed many similarities to the cyst forms of Blastocystis obtained from fecal samples in previous investigations. Freeze-fracture images of the plasma membrane of non-cyst cells also revealed a similar distribution of the intramembrane particles (IMP) when compared to non-cysts of B. hominis, while the plasma membrane of the cyst form showed practically no IMP. The size and morphology of particle-rich small depressions and smooth small protrusions observed on the P face and E face of non-cyst cells, respectively, were similar to endocytic sites reported for B. hominis. In the present study glycogen was cytochemically demonstrated at the ultrastructural level by an alkaline bismuth staining method in both cyst and non-cyst cells.     

Insertion of foreign epitopes in HBcAg: how to make the chimeric particle assemble

Summary. Hepatitis B core antigen is one of the most promising protein carriers of foreign epitopes of various human and animal pathogens. Chimeric HBcAg particles can be used as effective artificial immunogenes. Unfortunately, not all chimeric proteins are able to be particulated. The dependence of correct or incorrect folding of chimeric proteins on physical and chemical properties of inserts was studied with the help of ProAnalyst, SALIX and QSARPro computer programs. We have found that insertion of amino acids with high hydrophobicity, large volume, and high β-strand index prevent self-assembling chimeric proteins. These factors are most important for the C-termini of inserts. Recommendations for obtaining correct folding of chimeric HBcAg particles have been given. Received August 8, 1999, Accepted September 26, 1999     

Localization of the Site of Oxygen Radical Generation inside the Complex I of Heart and Nonsynaptic Brain Mammalian Mitochondria

Mitochondrial production of oxygen radicals seems to be involved in many diseases and aging. Recent studies clearly showed that a substantial part of the free radical generation of rodent mitochondria comes from complex I. It is thus important to further localize the free radical generator site within this respiratory complex. In this study, superoxide production by heart and nonsynaptic brain submitochondrial particles from up to seven mammalian species, showing different longevities, were studied under different conditions. The results, taking together, show that rotenone stimulates NADH-supported superoxide generation, confirming that complex I is a source of oxygen radicals in mammals, in general. The rotenone-stimulated NADH-supported superoxide production of the heart and nonsynaptic brain mammalian submitochondrial particles was inhibited both by p-chloromercuribenzoate and by ethoxyformic anhydride. These results localize the complex I oxygen radical generator between the ferricyanide and the ubiquinone reduction site, making iron—sulfur centers possible candidates, although unstable semiquinones can not be discarded. The results also indicate that the previously described inverse correlation between rates of mitochondrial oxygen radical generation and mammalian longevity operates through mechanisms dependent on the presence of intact functional mitochondria.     

Iron-rich particles in embryos of seeds from the family Pinaceae

Summary Iron-rich particles, previously reported in seeds of members of the genus Pinus, were found in radicle-hypocotyl tissues of dry embryos from eight other genera in the family Pinaceae. Thus, these Fe-rich particles are of common occurrence in seeds of this conifer family. These particles were most difficult to locate inPseudolarix amabilis, which has green embryos. Energy-dispersive X-ray analysis was used to determine the elements present in conifer Fe-rich particles and phytoferritin deposits in pea embryo axes. Ferich particles from all species studied contained mainly Fe and P but also contained considerable K and Mg. Abietoideae group I (genera Cedrus andAbies) had lower Fe P ratios compared to all the other combined subfamilies within the Pinaceae. Pea phytoferritin deposits contained markedly lower amounts of P relative to Fe based on peakto-background ratios and quantitative values calculated by using a ferric phosphate standard. We also found, for the first time, that pea phytoferritin contained considerable K. A strong similarity was found between the energy-dispersive X-ray analysis spectra from Ferich particles and portions of a laboratory-synthesized Fe, K, Mg phytate salt. Phytate is a common mineral-nutrient storage compound in seeds. The possibility of these Fe-rich particles being phytoferritin cannot be ruled out, but if they are phytoferritin, they have lower Fe P ratios than almost all other ferritins reported to date.Abbreviations EDX energy-dispersive X-ray     

Mechanism of selective stabilization of extrinsic polypeptides in PSII particles by glycinebetaine

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