A Simple Method for Enzymatic Synthesis of Unlabeled and Radiolabeled Hydroxycinnamate-CoA

Hydroxycinnamate coenzyme A (CoA) thioesters are substrates for biosynthesis of lignin and hydroxycinnamate esters of polysaccharides and other polymers. Hence, a supply of these substrates is essential for investigation of cell wall biosynthesis. In this study, three recombinant enzymes, caffeic acid 3-O-methyltransferase, 4-coumarate-CoA ligase 1, and 4-coumarate-CoA ligase 5, were cloned from wheat, tobacco, and Arabidopsis, respectively, and were used to synthesize ¹⁴C-feruloyl-CoA, caffeoyl-CoA, p-coumaroyl-CoA, feruloyl-CoA, and sinapoyl-CoA. The corresponding hydroxycinnamoyl-CoA thioesters were high-performance liquid chromatography purified, the only extraction/purification step necessary, with total yields between 88–95%. Radiolabeled ¹⁴C-feruloyl-CoA was generated from caffeic acid and S-adenosyl-¹⁴C-methionine under the combined action of caffeic acid 3-O-methyltransferase and 4-coumarate-CoA ligase 1. About 70% of ¹⁴C-methyl groups from S-adenosyl methionine were incorporated into the final product. The methods presented are simple, fast, and efficient for the preparation of the hydroxycinnamate thioesters.     

Laccase-catalyzed polymerization of tyrosine-containing peptides

Laccase-catalyzed polymerization of tyrosine and tyrosine-containing peptides was studied in the presence and absence of ferulic acid (FA). Advanced spectroscopic methods such as MALDI-TOF MS, EPR, FTIR microscopy and HPLC-fluorescence, as well as more conventional analytical tools: oxygen consumption measurements and SDS/PAGE were used in the reaction mechanism studies. Laccase was found to oxidize tyrosine and tyrosine-containing peptides, with consequent polymerization of the compounds. The covalent linkage connecting the compounds was found to be an ether bond. Only small amounts of dityrosine bonds were detected in the polymers. When FA was added to the reaction mixtures, it was found to be incorporated into the polymer structure. Thus, in addition to homopolymers, different heteropolymers containing two or four FA residues were formed in the reactions.     

Immunological investments reflect parasite abundance in island populations of Darwin's finches

The evolution of parasite resistance can be influenced by the abundance of parasites in the environment. However, it is yet unresolved whether vertebrates change their investment in immune function in response to variation in parasite abundance. Here, we compare parasite abundance in four populations of small ground finches (Geospiza fuliginosa) in the Galapagos archipelago. We predicted that populations exposed to high parasite loads should invest more in immune defence, or alternatively use a different immunological defence strategy. We found that parasite prevalence and/or infection intensity increased with island size. As predicted, birds on large islands had increased concentrations of natural antibodies and mounted a strong specific antibody response faster than birds on smaller islands. By contrast, the magnitude of cell-mediated immune responses decreased with increasing parasite pressure, i.e. on larger islands. The data support the hypothesis that investments into the immune defence are influenced by parasite-mediated selection. Our results are consistent with the hypothesis that different immunological defence strategies are optimal in parasite-rich and parasite-poor environments.     

Erythrocyte membrane modifying agents and the inhibition of Plasmodium falciparum growth: structure-activity relationships for betulinic acid analogues

The natural triterpene betulinic acid and its analogues (betulinic aldehyde, lupeol, betulin, methyl betulinate and betulinic acid amide) caused concentration-dependent alterations of erythrocyte membrane shape towards stomatocytes or echinocytes according to their hydrogen bonding properties. Thus, the analogues with a functional group having a capacity of donating a hydrogen bond (COOH, CH(2)OH, CONH(2)) caused formation of echinocytes, whereas those lacking this ability (CH(3), CHO, COOCH(3)) induced formation of stomatocytes. Both kinds of erythrocyte alterations were prohibitive with respect to Plasmodium falciparum invasion and growth; all compounds were inhibitory with IC(50) values in the range 7-28 microM, and the growth inhibition correlated well with the extent of membrane curvature changes assessed by transmission electron microscopy. Erythrocytes pre-loaded with betulinic acid or its analogues and extensively washed in order to remove excess of the chemicals could not serve as hosts for P. falciparum parasites. Betulinic acid and congeners can be responsible for in vitro antiplasmodial activity of plant extracts, as shown for Zataria multiflora Boiss. (Labiatae) and Zizyphus vulgaris Lam. (Rhamnaceae). The activity is evidently due to the incorporation of the compounds into the lipid bilayer of erythrocytes, and may be caused by modifications of cholesterol-rich membrane rafts, recently shown to play an important role in parasite vacuolization. The established link between erythrocyte membrane modifications and antiplasmodial activity may provide a novel target for potential antimalarial drugs.     

Physical training may enhance beta-cell function in type 2 diabetes

In healthy young subjects, training increases insulin sensitivity but decreases the capacity to secrete insulin. We studied whether training changes beta-cell function in type 2 diabetic patients. Patients, stratified into "moderate" and "low" secretors according to individual C-peptide responses to an intravenous glucagon test, were randomly assigned to a training program [ergometer cycling 30-40 min/day, including at least 20 min at 75% maximum oxygen consumption (Vo(2 max)), 5 days/wk for 3 mo] or a sedentary schedule. Before and after the intervention (16 h after last training bout), a sequential hyperglycemic (90 min at 11, 18, and 25 mM) clamp was performed. An intravenous bolus of 5 g of arginine was given at the end. Training increased Vo(2 max) 17 +/- 13% and decreased heart rate during submaximal exercise (P < 0.05). During the 3 mo of sedentary lifestyle, insulin and C-peptide responses to the clamp procedures were unchanged in both moderate and low secretors. Likewise, no change in beta-cell response was seen after training in the low secretors (n = 5). In contrast, moderate secretors (n = 9) showed significant increases in beta-cell responses to 18 and 25 mM hyperglycemia and to arginine stimulation. Glucagon responses to arginine as well as measures of insulin sensitivity and Hb A(1c) levels were not altered by training. In conclusion, in type 2 diabetic patients, training may enhance beta-cell function if the remaining secretory capacity is moderate but not if it is low. The improved beta-cell function does not require changes in insulin sensitivity and Hb A(1c) concentration.     

ALK7, a receptor for nodal, is dispensable for embryogenesis and left-right patterning in the mouse

Mesendoderm formation and left-right patterning during vertebrate development depend upon selected members of the transforming growth factor beta superfamily, particularly Nodal and Nodal-related ligands. Two type I serine/threonine kinase receptors have been identified for Nodal, ALK4 and ALK7. Mouse embryos lacking ALK4 fail to produce mesendoderm and die shortly after gastrulation, resembling the phenotype of Nodal knockout mice. Whether ALK4 contributes to left-right patterning is still unknown. Here we report the generation and initial characterization of mice lacking ALK7. Homozygous mutant mice were born at the expected frequency and remained viable and fertile. Viability at weaning was not different from that of the wild type in ALK7(-/-); Nodal(+/-) and ALK7(-/-); ALK4(+/-) compound mutants. ALK7 and ALK4 were highly expressed in interdigital regions of the developing limb bud. However, ALK7 mutant mice displayed no skeletal abnormalities or limb malformations. None of the left-right patterning abnormalities and organogenesis defects identified in mice carrying mutations in Nodal or in genes encoding ActRIIA and ActRIIB coreceptors, including heart malformations, pulmonary isomerism, right-sided gut, and spleen hypoplasia, were observed in mice lacking ALK7. Finally, the histological organization of the cerebellum, cortex, and hippocampus, all sites of significant ALK7 expression in the rodent brain, appeared normal in ALK7 mutant mice. We conclude that ALK7 is not an essential mediator of Nodal signaling during mesendoderm formation and left-right patterning in the mouse but may instead mediate other activities of Nodal and related ligands in the development or function of particular tissues and organs.     

Desulfosporomusa polytropa gen. nov., sp. nov., a novel sulfate-reducing bacterium from sediments of an oligotrophic lake

Five strains of sulfate-reducing bacteria were isolated from the highest positive dilutions of a most probable number (MPN) series supplemented with lactate and inoculated with sediments from the oligotrophic Lake Stechlin. The isolates were endospore-forming and were motile by means of laterally inserted flagella. They stained Gram-negative and contained b-type cytochromes. CO difference spectra indicated the presence of P582 as a sulfite reductase. Phylogenetic analyses of the 16S rDNA sequences revealed that the isolates were very closely affiliated with the genus Sporomusa. However, sulfate and amorphous Fe(OH)₃, but not sulfite, elemental sulfur, MnO₂, or nitrate were used as terminal electron acceptors. Homoacetogenic growth was found with H₂/CO₂ gas mixture, formate, methanol, ethanol, and methoxylated aromatic compounds. The strains grew autotrophically with H₂ plus CO₂ in the presence or absence of sulfate. Formate, butyrate, several alcohols, organic acids, carbohydrates, some amino acids, choline, and betaine were also utilized as substrates. The growth yield with lactate and sulfate as substrate was 7.0 g dry mass/mol lactate and thus two times higher than in sulfate-free fermenting cultures. All isolates were able to grow in a temperature range of 4–37°C. Physiologically and by the presence of a Gram-negative cell wall, the new isolates resemble known Desulfosporosinus species. However, phylogenetically they are affiliated with the Gram-negative genus Sporomusa belonging to the Selenomonas subgroup of the Firmicutes. Therefore, the new isolates reveal a new phylogenetic lineage of sulfate-reducing bacteria. A new genus and species, Desulfosporomusa polytropa gen. nov., sp. nov. is proposed.Dedicated to Prof. H. G. Schlegel on the occasion of his 80th birthday.     

Variants of CYP46A1 may interact with age and APOE to influence CSF Aβ42 levels in Alzheimer’s disease

Recent studies have suggested that variants of CYP46A1, encoding cholesterol 24-hydroxylase (CYP46), confer risk for Alzheimers disease (AD), a prospect substantiated by evidence of genetic association from several quantitative traits related to AD pathology, including cerebrospinal fluid (CSF) levels of the 42 amino-acid cleavage product of -amyloid (A42) and the tau protein. In the present study, these claims have been explored by the genotyping of previously associated markers in CYP46A1 in three independent northern European case-control series encompassing 1323 individuals and including approximately 400 patients with measurements of CSF A42 and phospho-tau protein levels. Tests of association in case-control models revealed limited evidence that CYP46A1 variants contributed to AD risk across these samples. However, models testing for potential effects upon CSF measures suggested a possible interaction of an intronic marker (rs754203) with age and APOE genotype. In stratified analyses, significant effects were evident that were restricted to elderly APOE 4 carriers for both CSF A42 (P=0.0009) and phospho-tau (P=0.046). Computational analyses indicate that the rs754203 marker probably does not impact the binding of regulatory factors, suggesting that other polymorphic sites underlie the observed associations. Our results provide an important independent replication of previous findings, supporting the existence of CYP46A1 sequence variants that contribute to variability in -amyloid metabolism.     

Allergy vaccine engineering: epitope modulation of recombinant Bet v 1 reduces IgE binding but retains protein folding pattern for induction of protective blocking-antibody responses

Human type 1 immediate allergic response symptoms are caused by mediator release from basophils and mast cells. This event is triggered by allergens aggregating preformed IgE Abs bound to the high-affinity receptor (FcepsilonRI) on these cells. Thus, the allergen/IgE interaction is crucial for the cascade leading to the allergic and anaphylactic response. Two genetically engineered forms of the white birch pollen major allergen Bet v 1 with point mutations directed at molecular surfaces have been characterized. Four and nine point mutations led to a significant reduction of the binding to human serum IgE, suggesting a mutation-induced distortion of IgE-binding B cell epitopes. In addition, the mutated allergens showed a decrease in anaphylactic potential, because histamine release from human basophils was significantly reduced. Retained alpha-carbon backbone folding pattern of the mutated allergens was indicated by x-ray diffraction analysis and circular dichroism spectroscopy. The rBet v 1 mutants were able to induce proliferation of T cell lines derived from birch pollen allergic patients. The stimulation indices were similar to the indices of nonmutated rBet v 1 and natural Bet v 1 purified from birch pollen. The ability of anti-rBet v 1 mutant specific mouse IgG serum to block binding of human serum IgE to rBet v 1 demonstrates that the engineered rBet v 1 mutants are able to induce Abs reactive with nonmodified Bet v 1. rBet v 1 mutants may constitute vaccine candidates with improved efficacy/safety profiles for safer allergy vaccination.     

A previously found thylakoid membrane protein of 14kDa (TMP14) is a novel subunit of plant photosystem I and is designated PSI-P

We show that the thylakoid membrane phosphoprotein TMP14 is a novel subunit of plant photosystem I (PSI). Blue native/SDS-PAGE and sucrose gradient fractionation demonstrated the association of the protein exclusively with PSI. We designate the protein PSI-P. The presence of PSI-P subunit in Arabidopsis mutants lacking other PSI subunits was analyzed and suggested a location in the proximity of PSI-L, -H and -O subunits. The PSI-P protein was not differentially phosphorylated in state 1 and state 2.