Characterisation of Leucaena condensed tannins by size and protein precipitation capacity

The ability of differently sized condensed tannins from the genus Leucaena, a fodder tree-legume, to bind protein at different pH values was evaluated to characterise their potential biological effects. Two factors affecting the ability of condensed tannin to bind protein, its major biological activity, have been purported to be the condensed tannin size and the pH of the reaction environment. To test these hypotheses, the protein-precipitating capacities of condensed tannin extracted from four Leucaena genotypes, L leucocephala (UHK636), L pallida (CQ3439), L trichandra (CP146568) and L collinsii (OFI52/88), were assessed. Condensed tannin from L leucocephala had approximately 50% of the ability to precipitate protein on a gg(-1) basis than L pallida or L trichandra, while L collinsii gave no measurable ability to precipitate protein (reaction environment pH 5.0). Increasing or decreasing the pH of the reaction solution away from pH 5.0 (approximately the isoelectric point of the protein) reduced the ability of condensed tannin from all the species to precipitate protein, the decrease being higher at pH 2.5 than at pH 7.5. Condensed tannins from each Leucaena species were also separated by size exclusion chromatography, and the fractions examined for protein-precipitating capacity. In general, it was found that the larger-sized condensed tannin of the accessions L pallida and L trichandra could precipitate more protein than the smaller-sized condensed tannin. This pattern was not found for L leucocephala. (C) 2001 Society of Chemical Industry.

Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as,well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.

Effect of supplementation with a by-product of molasses fermentation or a non-protein nitrogen/mineral mix on feed intake and microbial protein supply in sheep consuming chopped oat (Avena sativa) hay

Crossbred ewes, weighing 30-40 kg, were assigned to three groups of six animals. One group of sheep was fed chopped oat hay (control), the second group was fed the control diet plus 30 g per head per day spray dried residue from the fermentation of molasses and the third group was fed the control diet plus 30 g per head per day of a non-protein nitrogen/mineral mix. Voluntary feed intake, digestibility of DM, OM and nitrogen, nitrogen balance and microbial nitrogen flow to the intestines were significantly increased by supplementation but efficiency of microbial protein production was not affected. (C) 2001 Elsevier Science BN. All rights reserved,

Relationships between crude protein and mineral concentrations in alfalfa and value of alfalfa silage as a mineral source for periparturient cows

One hundred and eight samples from three cultivars of alfalfa were obtained from three cuttings in 1996-1998 to evaluate the relationship between crude protein (CP) and mineral concentrations of alfalfa with cutting and maturation. The CP content drastically decreased from 27.9 to 11.4% on DM with maturity. Highly positive correlations were observed between CP and K in the first and the second cutting of alfalfa. The Ca content remained almost constant throughout the growth period. Four multiparous Holstein cows were assigned an alfalfa silage diet or an orchardgrass silage diet from 3 weeks prepartum to 1 week postpartum to examine the effect on the mineral balance. In the prepartum and postpartum diet, the roughage to concentrate ratio was 70:30 and 50:50, with alfalfa being 50 and 100% of the roughage, respectively. The alfalfa contained 1.93% of K. No metabolic disorders occurred, but the body weight decreased drastically from 1 to 6 days postpartum with each diet because of the high milk production immediately after the parturition. Positive retention of N, Ca, P, Mg, and K was observed prepartum, whereas the cows had negative N and mineral retention from 2 to 4 days postpartum. The Ca and P absorption, and Mg retention of cows with the alfalfa diet were higher than with the grass diet. The plasma Ca and inorganic P were not affected by diet, but the plasma PTH at parturition and plasma hydroxyproline from 1 week prepartum to 1 week postpartum were higher with the alfalfa diet. These results suggest that the low K alfalfa is suitable not only to prevent the incidence of milk fever but also to increase Ca, P and Mg utilization of periparturient cows, but the mineral supplementation is needed for the postpartum cows immediately after the parturition. (C) 2001 Elsevier Science B.V. All rights reserved.

Thiopalmitoylation of myelin proteolipid protein epitopes enhances immunogenicity and encephalitogenicity

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP104-117 and PLP139-151) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8(+) cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4(+) T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.

Exploiting the versatility of human cytochrome P450 enzymes: The promise of blue roses from biotechnology

The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY 1002/3A4. which express respective human P450 enzymes and NADPH-cytochrome P350 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA 1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double me promoter and the other, pOA 102, carrying O-AT and umuClacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 135 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 1-Amino-1,4-dimethyl-5H-pyrido[4.3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B-1 exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta -Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrom P450 enzyme involved in bioactivation of HCAs. (C) 2001 Elsevier Science B.V. All rights reserved.

Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with G beta

There is increasing evidence that heterotrimeric G-proteins (G-proteins) are involved in many plant processes including phytohormone response, pathogen defence and stomatal control. In animal systems, each of the three G-protein subunits belong to large multigene families; however, few subunits have been isolated from plants. Here we report the cloning of a second plant G-protein γ-subunit (AGG2) from Arabidopsis thaliana. The predicted AGG2 protein sequence shows 48% identity to the first identified Arabidopsis Gγ-subunit, AGG1. Furthermore, AGG2 contains all of the conserved characteristics of γ-subunits including a small size (100 amino acids, 11.1 kDa), C-terminal CAAX box and a N-terminal α-helix region capable of forming a coiled-coil interaction with the β-subunit. A strong interaction between AGG2 and both the tobacco (TGB1) and Arabidopsis (AGB1) β-subunits was observed in vivo using the yeast two-hybrid system. The strong association between AGG2 and AGB1 was confirmed in vitro. Southern and Northern analyses showed that AGG2 is a single copy gene in Arabidopsis producing two transcripts that are present in all tissues tested. The isolation of a second γ-subunit from A. thaliana indicates that plant G-proteins, like their mammalian counterparts, may form different heterotrimer combinations that presumably regulate multiple signal transduction pathways.

Antibody levels to the class I and II epitopes of the M protein and myosin are related to group A streptococcal exposure in endemic populations

Rheumatic fever (RF)/rheumatic heart disease (RHD) and post-streptococcal glomerulonephritis are thought to be autoimmune diseases, and follow group A streptococcal (GAS) infection. Different GAS M types have been associated with rheumatogenicity or nephritogenicity and categorized into either of two distinct classes (I or II) based on amino acid sequences present within the repeat region ('C' repeats) of the M protein. Sera from ARF patients have previously been shown to contain elevated levels of antibodies to the class I-specific epitope and myosin with the class I-specific antibodies also being cross-reactive to myosin, suggesting a disease association. This study shows that immunoreactivity of the class I-specific peptide and myosin does not differ between controls and acute RF (ARF)/RHD in populations that are highly endemic for GAS, raising the possibility that the association is related to GAS exposure, not the presence of ARF/RHD. Peptide inhibition studies suggest that the class I epitope may be conformational and residue 10 of the peptide is critical for antibody binding. We demonstrate that correlation of antibody levels between the class I and II epitope is due to class II-specific antibodies recognizing a common epitope with class I which is contained within the sequence RDL-ASRE. Our results suggest that antibody prevalence to class I and II epitopes and myosin is associated with GAS exposure, and that antibodies to these epitopes are not an indicator of disease nor a pathogenic factor in endemic populations.

Specificity of 17 beta-oestradiol and benzo[a] pyrene oxidation by polymorphic human cytochrome P4501B1 variants substituted at residues 48, 119 and 432

1. Eight human cytochrome P4501B1 (CYP1B1) allelic variants, namely Arg(48)Ala(119)Leu(432), Arg(48)Ala(119)Val(432), Gly(48)Ala(119)Leu(432), Gly(48)Ala(119)Val(432), Arg(48)Ser(119)Leu(432), Arg(48)Ser(119)Val(432), Gly(48)Ser(119)Leu(432) and Gly(48)Ser(119)Val(432) (all with Asn(453)), were expressed in Escherichia coli together with human NADPH-P450 reductase and their catalytic specificities towards oxidation of 17 beta -oestradiol and benzo[a]pyrene were determined. 2. All of the CYP1B1 variants expressed in bacterial membranes showed Fe2+. CO versus Fe2+ difference spectra with wavelength maxima at 446 nm and they reacted with antibodies raised against recombinant human CYP1B1 in immunoblots. The ratio of expression of the reductase to CYP1B1 in these eight preparations ranged from 0.2 to 0.5. 3. CYP1B1 Arg(48) variants tended to have higher activities for 17 beta -oestradiol 4-hydroxylation than Gly(48) variants, although there were no significant variations in 17 beta -oestradiol 2-hydroxylation activity in these eight CYP1B1 variants. Interestingly, ratios of formation of 17 beta -oestradiol 4-hydroxylation to 2-hydroxylation by these CYP1B1 variants were higher in all of the Val(432) forms than the corresponding Leu(432) forms. 4. In contrast, Leu(432) forms of CYP1B1 showed higher rates of oxidation of benzo[a]pyrene (to the 7, 8-dihydoxy-7,8-dihydrodiol in the presence of epoxide hydrolase) than did the Val(432) forms. 5. These results suggest that polymorphic human CYP1B1 variants may cause some altered catalytic specificity with 17 beta -oestradiol and benzo[a]pyrene and may influence susceptibilities of individuals towards endogenous and exogenous carcinogens.

Apoptotic deletion of Th cells specific for the 19-kDa carboxyl-terminal fragment of merozoite surface protein 1 during malaria infection

Immunity induced by the 19-kDa fragment of merozoite surface protein 1 is dependent on CD4(+) Th cells. However, we found that adoptively transferred CFSE-labeled Th cells specific for an epitope on Plasmodium yoelii 19-kDa fragment of merozoite surface protein 1 (peptide (p)24), but not OVA-specific T cells, were deleted as a result of P. yoelii infection. As a result of infection, spleen cells recovered from infected p24-specific T cell-transfused mice demonstrated reduced response to specific Ag. A higher percentage of CFSE-labeled p24-specific T cells stained positive with annexin and anti-active caspase-3 in infected compared with uninfected mice, suggesting that apoptosis contributed to deletion of p24-specific T cells during infection. Apoptosis correlated with increased percentages of p24-specific T cells that stained positive for Fas from infected mice, suggesting that P. yoelii-induced apoptosis is, at least in part, mediated by Fas. However, bystander cells of other specificities also showed increased Fas expression during infection, suggesting that Fas expression alone is not sufficient for apoptosis. These data have implications for the development of immunity in the face of endemic parasite exposure.

Protein targeting to the plasma membrane of adult skeletal muscle fiber: An organized mosaic of functional domains

The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta -dystroglycan, dystrophin, and caveolin-3, These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta -dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta -dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expressed domain-specific markers, We found that each marker protein was targeted to distinct microdomains, The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division. (C) 2001 Academic Press.