Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity.

Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit protein kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors (Ki approx. 12 and 59 microM) of PKC and derivatives containing a tertiary amino head group were approx. 4-20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N,N-dimethylethylenediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.     

Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [N]H(4) Assimilation in Lemna minor L

Rapid, sensitive, and selective methods for the determination of the ¹⁵N abundance of amino acids in isotopic tracer experiments with plant tissues are described and discussed. Methodology has been directly tested in an analysis of the kinetics of [¹⁵N]H₄⁺ assimilation in Lemna minor L. The techniques utilize gas chromatography-mass spectrometry selected ion monitoring of major fragments containing the N moiety of N-heptafluorobutyryl isobutyl esters of amino acids. The ratio of selected ion pairs at the characteristic retention time of each amino acid derivative can be used to calculate ¹⁵N abundance with an accuracy of ±1 atom% excess ¹⁵N using samples containing as little as 30 picomoles of individual amino acids. Up to 11 individual amino acid derivatives can be selectively monitored in a single chromatogram of 30 minutes. It is suggested that these techniques will be useful in situations where the small quantities of N available for analysis have hitherto hindered the use of ¹⁵N-labeled precursors.     

New derivatives of eremomycin containing 15N or F atoms for NMR study

New semisynthetic derivatives of eremomycin containing ¹⁵N or F atoms were obtained for studying the antibiotic-target interaction in intact cells of Gram-positive bacteria by REDOR NMR method. Interaction of the terminal carboxyl group of amino acid 7 (AA7) of eremomycin with amines in the presence of PyBOP and TBTU reagents resulted in the corresponding [¹⁵N]-amide, p-fluorobenzylamide, p-fluorophenylpiperazide, and 6-N-(p-fluorobenzyl)aminohexylamide. A selective method of [¹⁵N]-amidation of carboxyl group of amino acid 3 (AA3) of carboxyeremomycin was developed, and the amide of eremomycin containing [¹⁵N] in AA3 amide group near the antibiotic binding pocket was obtained. Carboxyeremomycin bisamides substituted at AA3 and AA7 and containing two atoms of [¹⁵N] or F were obtained from carboxyeremomycin and [¹⁵N]NH₄Cl or the corresponding p-fluorobenzylamine hydrochloride in the presence of PyBOP at pH ～8. The Edman degradation of eremomycin p-fluorobenzylamide gave de-(D-MeLeu)-eremomycin p-fluorobenzylamide, a hexapeptide derivative incapable of the antibiotic binding with-D-Ala-D-Ala fragment of growing cell wall peptidoglycan. Among the compounds studied, carboxyeremomycin bis-p-fluorobenzylamide showed the best activity against both the glycopeptides-sensitive and glycopeptides-resistant strains of staphylococci and enterococci.     

A series of 2-O-trifluoromethylsulfonyl-d-mannopyranosides as precursors for concomitant F-labeling and glycosylation by click chemistry

A series of ‘clickable’ mannopyranosides bearing a triflate leaving group at C-2 position were synthesized and tested for their potential as ¹⁸F-labeling precursors. 3,4,6-Tri-O-acetyl-2-O-trifluoromethanesulfonyl-β-d-mannopyranosyl azide (2β) was the most convenient precursor for a site-specific and reliable click chemistry-based three-step, two-pot concomitant ¹⁸F-labeling and glycosylation of an alkyne-functionalized amino acid derivative.     

A practical post-modification synthesis of oligodeoxynucleotides containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside

We describe herein the practical post-modification synthesis of oligodeoxynucleotides (ODNs) containing 4,7-diaminoimidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine nucleoside (ImN^N). Since the ImN^N nucleoside unit possessing tribenzoyl groups on its exocyclic amino groups as the protecting group was quite unstable under acidic conditions, cleavage of its glycosidic linkage in ODN has been suggested throughout the conditions of solid-phase synthesis. As an alternative approach, we investigated a post-modification synthesis of the desired ODNs containing the ImN^N unit. Starting with protected 4-amino-7-chloro-1-(2-deoxy-β-d-ribofuranosyl)imidazo[5′,4′:4,5]pyrido[2,3-d]pyrimidine derivative 1, conversion into the corresponding phosphoramidite unit was examined. The p-bromobenzoyl group (p-BrBz) was the best protecting group of 4-amino group of 1 to give the phosphoramidite unit 9 for the post-modification synthesis. After carrying out the ODN synthesis linked to the controlled pore glass (CPG) support, the support was treated with ammonium hydroxide at 55 °C to remove the protecting groups, detach the ODN form the CPG support, and convert the 7-chloro group into a desired amino group. As a result, the desired ODNs containing ImN^N were obtained in good yield.     

1,6-diamino-2,5-anhydro-1,6-dideoxy-l-iditol and some derivatives thereof

1,6-Diamino-2,5-anhydro-1,6-dideoxy-l-iditol (31) and its derivatives were synthesized, starting from 2,4-O-benzylidene-1,6-di-O-tosyl-d-glucitol. The 1,6-bis-(acetamido)-l-talo epoxide was readily hydrolyzed to the corresponding l-iditol derivative under anchimeric assistance of the 1-acetamido group. On treatment with formaldehyde-formic acid, diamine 31 gave a tricyclic, 1,4:3,6-bis(N,O-methylene) derivative which was stable under acidic conditions but, according to ¹³C-n.m.r. spectroscopy, was readily hydrolyzed to an equilibrium mixture in neutral, aqueous solution. The corresponding 1,6-bis(dimethylamino) derivative could be obtained by reducing this equilibrium mixture with borohydride. The different, quaternary salts obtained on methylation of the corresponding 1,6-bis(dimethylamino) derivatives with methyl iodide (aiming at the structure of epi-allo-muscarine) showed no muscarine-like, biological activity.     

Methionine Changes in Bacteriorhodopsin Detected by FTIR and Cell-Free Selenomethionine Substitution

Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR→M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR→M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1284 and 900 cm⁻¹ due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.     

QTL mapping for brown rot (Monilinia fructigena) resistance in an intraspecific peach (Prunus persica L. Batsch) F1 progeny

Brown rot (BR) caused by Monilinia spp. leads to significant post-harvest losses in stone fruit production, especially peach. Previous genetic analyses in peach progenies suggested that BR resistance segregates as a quantitative trait. In order to uncover genomic regions associated with this trait and identify molecular markers for assisted selection (MAS) in peach, an F1 progeny from the cross “Contender” (C, resistant)?×?“Elegant Lady” (EL, susceptible) was chosen for quantitative trait loci (QTL) analysis. Over two phenotyping seasons, skin (SK) and flesh (FL) artificial infections were performed on fruits using a Monilinia fructigena isolate. For each treatment, infection frequency (if) and average rot diameter (rd) were scored. Significant seasonal and intertrait correlations were found. Maturity date (MD) was significantly correlated with disease impact. Sixty-three simple sequence repeats (SSRs) plus 26 single-nucleotide polymorphism (SNP) markers were used to genotype the C?×?EL population and to construct a linkage map. C?×?EL map included the eight Prunus linkage groups (LG), spanning 572.92 cM, with an average interval distance of 6.9 cM, covering 78.73 % of the peach genome (V1.0). Multiple QTL mapping analysis including MD trait as covariate uncovered three genomic regions associated with BR resistance in the two phenotyping seasons: one containing QTLs for SK resistance traits near M1a (LG C?×?EL-2, R ²?=?13.1–31.5 %) and EPPISF032 (LG C?×?EL-4, R ²?=?11–14 %) and the others containing QTLs for FL resistance, near markers SNP_IGA_320761 and SNP_IGA_321601 (LG3, R ²?=?3.0–11.0 %). These results suggest that in the C?×?EL F1 progeny, skin resistance to fungal penetration and flesh resistance to rot spread are distinguishable mechanisms constituting BR resistance trait, associated with different genomic regions. Discovered QTLs and their associated markers could assist selection of new cultivars with enhanced resistance to Monilinia spp. in fruit.     

Resistance Training Improves Hemodynamic Function,Collagen Deposition and Inflammatory Profiles: Experimental Model of Heart Failure

The role of resistance training on collagen deposition, the inflammatory profile and muscle weakness in heart failure remains unclear. Therefore, this study evaluated the influence of a resistance training program on hemodynamic function, maximum strength gain, collagen deposition and inflammatory profile in chronic heart failure rats. Thirty-two male Wistar rats submitted to myocardial infarction by coronary artery ligation or sham surgery were assigned into four groups: sedentary sham (S-Sham, n = 8); trained sham (T-Sham, n = 8); sedentary chronic heart failure (S-CHF, n = 8) and trained chronic heart failure (T-CHF, n = 8). The maximum strength capacity was evaluated by the one maximum repetition test. Trained groups were submitted to an 8-week resistance training program (4 days/week, 4 sets of 10–12 repetitions/session, at 65% to 75% of one maximum repetition). After 8 weeks of the resistance training program, the T-CHF group showed lower left ventricular end diastolic pressure (P<0.001), higher left ventricular systolic pressure (P<0.05), higher systolic blood pressure (P<0.05), an improvement in the maximal positive derivative of ventricular pressure (P<0.05) and maximal negative derivative of ventricular pressure (P<0.05) when compared to the S-CHF group; no differences were observed when compared to Sham groups. In addition, resistance training was able to reduce myocardial hypertrophy (P<0.05), left ventricular total collagen volume fraction (P<0.01), IL-6 (P<0.05), and TNF-α/IL-10 ratio (P<0.05), as well as increasing IL-10 (P<0.05) in chronic heart failure rats when compared to the S-CHF group. Eight weeks of resistance training promotes an improvement of cardiac function, strength gain, collagen deposition and inflammatory profile in chronic heart failure rats.     

Electrogenic transport properties of bacteriorhodopsin containing chemically modified retinal analogues

The electrical activity of bacteriorhodopsin (BR) containing the 13-substituted retinal analogues 13-demethyl and 13-methoxy as well as the naturally occurring retinal carrying a methyl group at C13 is compared. White membrane patches reconstituted with the different retinals are attached to a black lipid membrane, and the dependency of the photocurrent on light intensity is measured. This allows a comparison of the overall photocycle time and the number of protons transported per cycle for the various preparations. From previous work (Gärtner, W., Towner, P., Hopf, H. and Oesterhelt, D. (1983) Biochem. 22, 2637–2644, see also Gärtner, W. and Oesterhelt, D., unpublished data) the equilibrium isomeric distribution (all-trans and 13-cis) of the different retinals in the binding site is known. Taking into account that only all-trans retinal BR contributes to the pumping activity (Fahr, A. and Bamberg, E. (1982) FEBS Lett. 140, 251–253), it is shown, that the cycle time for the modified BRs is moderately changed, whereas the number of protons transported per cycle and transporting all-trans BR molecule is not affected by the substituent. It is concluded, that substituting the methyl group at position 13 of the retinal molecule by a hydrogen atom or a methoxy group only slightly affects the pumping activity of the trans-photocycle, but rather controls the biological function of BR via the equilibrium isomeric distribution of the retinal molecule in the binding site.     

Influence of amino acid supplements on the metabolism of Clostridium acetobutylicum

The effects of organic nitrogen on the metabolism of Clostridium acetobutylicum were investigated in batch fermentations. For this study, amino acids were added to a chemically defined medium in groups from the same biosynthetic pathways. In all cases the addition of amino acids shifted the solvent ratio to higher butanol production at the expense of that of acetone (except for the glutamic acid group) and ethanol (except for histidine). Highest biomass production was obtained from media containing aromatic amino acids and histidine (4.57 g · l⁻¹ and 5.4 g · l⁻¹, respectively). However, the solvent production (ca. 20 g · l⁻¹) and the solvent yield (ca. 33%) in both cases, were similar to those obtained from the synthetic medium. Lower values were obtained from fermentations carried out with other families of amino acids. The strongest inhibition of cell growth (1.13 g · l⁻¹) which related to the lowest solvent production (3.15 g · l⁻¹) was observed on a medium complemented with amino acids of the pyruvic acid group. During the second phase of fermentation, amino acids-complemented media caused a less efficient remetabolization of acetic and butyric acids. Highest production of acids was obtained with the aspartic acid group (7.4 g · l⁻¹). These observations suggest that amino acids can be used as a competitive nitrogen source and also modify the level of enzyme activities involved in acid and solvent production.     

Transformation of 3-(3-carboxyphenyl)alanine in iris species An example of diversity in catabolism of secondary plant products

3-(3-Carboxyphenyl)-DL-[2-¹⁴C]alanine has been incorporated into four species of iris. In all species extensive metabolization takes place. In Iris × hollandica, in which both the alanine derivative and 3′-carboxyphenylglycine occur, the products identified are the glycine derivative, 3′-carboxyphenylacetate acid, 3′-carboxymandelic acid, and 3′-carboxyphenylglyoxylic acid. In I. sanguinea, in which the alanine and glycine derivatives also occur, the products identified are the glycine and acetic acid derivatives but the major product is 3-(3-hydroxymethylphenyl)alanine, a naturally occurring amino acid in this species. In I. tectorum, in which only the carboxy-substituted alanine derivative occurs, the products identified are the acetic acid and glyoxylic acid derivatives. In I. pallida, not containing any of the meta-substituted amino acids, the products identified are again the acetic acid and glyoxylic acid derivatives. The results have been further substantiated by incorporation of labelled 3′-carboxyphenylacetic acid and 3′-carboxymandelic acid into I. × hollandica and I. sanguinea.The results demonstrate three different metabolic patterns for the alanine derivative and confirm previous results on the pathway from the alanine to the glycine derivative. Furthermore, the results may be of significance for the elucidation of the catabolism of phenylalanine.     

Amino acid sequence of a peptide containing an essential cysteine residue of pig heart aconitase

Pig heart aconitase reacts with one mole of phenacyl bromide per molecule to give complete inactivation due to the alkylation of a cysteine residue at the active site. A tryptic peptide containing this essential residue has been isolated and its amino acid sequence determined as Ile-Gln-Leu-Leu-Cys ¹-Pro-Leu-Leu-Asn-Gln-Phe-Asp-Lys by manual methods and by the use of an automated solid phase sequencer. There is a limited similarity in amino acid sequence between this peptide and other peptides containing the cysteine residues involved in the binding of the iron-sulfur clusters of high-potential iron-sulfur protein of Rhodopseudomonas gelatinosa and rubredoxins from various bacteria.     

Synthesis of P 1-(11-phenoxyundecyl)-P 2-(��-D-galactopyranosyl) diphosphate and P 1-(11-phenoxyundecyl)-P 2-(��-D-glucopyranosyl) diphosphate and investigation on their acceptor properties in the reaction of mannosyl residue transfer catalyzed by mannosyltransferase from Salmonella newport

P ¹-(11-phenoxyundecyl)-P ²-(??-D-galactopyranosyl) diphosphate and P ¹-(11-phenoxyundecyl)-P ²-(??-D-glucopyranosyl) diphosphate have been synthesized for the first time, and their ability to serve as a mannosyl residue substrate-acceptors in the enzymatic reaction, catalyzed by mannosyltransferase membrane preparation from Salmonella newport cells, was investigated. It was demonstrated that the derivative containing galactopyranose residue is able to accept the mannosyl residue from GDP-Man, while the derivative containing glucopyranose residue does not have such an ability.     

Homogeneous synthesis and characterization of quaternized chitin in NaOH/urea aqueous solution

Water-soluble and white quaternized chitin (QC) was homogeneously synthesized by stirring transparent chitin solution (2%) in 8 wt%NaOH/4 wt% urea aqueous solution containing 2,3-Epoxypropyltrimethylammonium Chloride (EPTMAC) at 10 °C for 24 h. The structure and properties of quaternized chitin were characterized by FT-IR, XRD, ¹H NMR, GPC, element analysis and ζ-potential. The results indicate that quaternary groups were successfully incorporated onto chitin backbones and the degree of substitution (DS) of quaternary groups can be easily adjusted by changing the molar ratio of chitin unit to EPTMAC. Additionally, quaternized chitin shows better antibacterial activity against Escherichia coli and Staphylococcus aureus as compared with chitosan. Thus, this work provides a simply and “green” method to functionalize chitin and the resulting quaternized chitin may have potential applications in environmental, food and biomedical fields.     

Specificity of aspartate aminotransferases from leguminous plants for 4-substituted glutamic acids

Aspartate aminotransferase (glutamate-oxalacetate transaminase) was partially purified from extracts of germinating seeds of peanut (Arachis hypogaea), honey locust (Gleditsia triacanthos), soybean (Glycine max), and Sophora japonica. The ability of these enzyme preparations, as well as aspartate aminotransferase purified from pig heart cytosol, to use 4-substituted glutamic acids as amino group donors and their corresponding 2-oxo acids as amino group acceptors in the aminotransferase reaction was measured. All 4-substituted glutamic acid analogs tested were poorer substrates than was glutamate or 2-oxoglutarate. 2-Oxo-4-methyleneglutarate was least effective (lowest relative V_m/K_m) as a substrate for the enzyme from peanuts and honey locust, which are the two species studied that accumulate 4-methyleneglutamic acid and 4-methyleneglutamine. Of the different aminotransferases tested, the enzyme from honey locust was the least active with 2-oxo-4-hydroxy-4-methylglutarate, the corresponding amino acid of which also accumulates in that species. These results suggest that transamination of 2-oxo-4-substituted glutaric acids is not involved in the biosynthesis of the corresponding 4-substituted glutamic acids in these species. Rather, accumulation of certain 4-substituted glutamic acids in these instances may be, in part, the result of the inefficacy of their transamination by aspartate aminotransferase.     

Pseudomonas aeruginosa Chemotaxis Associated with Blooms of N2-Fixing Blue-Green Algae (Cyanobacteria)

Pseudomonas aeruginosa (Schroeter) Migula, a numerically significant bacterium found during N₂-fixing blooms of the blue-green algae (cyanobacteria) Anabaena sp. in the Chowan River, North Carolina, was chemotactically attracted to amino acids when tested in a radioassay. The bacterium was labeled with ³²P_i, and the disintegrations per minute determined by liquid scintillation counting were proportional to the number of cells accumulating in microcapillaries containing amino acids. Positive chemotaxis was observed toward all of the amino acids tested, although the degrees of response varied. Since many nitrogen-fixing blue-green algae secrete nitrogenous compounds, this attraction may be instrumental in establishing a symbiotic relationship between this bacterium and blue-green algae in freshwater.     

Genetic mapping and QTL analysis for yield and agronomic traits with an F2:3 population derived from a waxy corn × sweet corn cross

We constructed a framework map using SSR markers in the F₂ population derived from a cross between a waxy corn inbred line and a sweet corn inbred line. We constructed a genetic linkage map of the F_2:3 population employing 295 SSR markers on 158 F₂ individuals produced from the cross. The map comprised a total genomic length of 2,626.5 cM in 10 linkage groups and an average distance between markers of 8.9 cM. The number of loci per linkage group ranged from 27 (chr. 5) to 34 (chr. 7). The genetic distance per linkage group ranged from 213.6 cM (chr. 10) to 360.6 cM (chr. 2). Χ ² tests revealed that 254 markers (86.1 %) distributed over all 10 chromosomes exhibited a Mendelian segregation ratio of 1:2:1. A total of 14 quantitative trait loci (QTLs) for days to silking (DTS), plant height (PH), ear height (EH), ear height ratio (ER), ear length (L-ear), and setted ear length (L-sear) were found in the 158 F₂ progeny. They were mapped to chromosomes 1, 2, 3, 7, 8, and 10. Among them, one QTL was associated with DTS, three with PH, six with EH, one with ER, two with L-ear, and one QTL was related to L-sear. In our study, we found that four QTLs: qDTS1, qEH1a, qEH1b, and qPH1, were clustered between umc2390 and umc1603 on chromosome 1. These new QTLs identified by the present study could serve as useful molecular markers in selecting for yield and agronomic traits in maize. The results of this study may improve the identification and characterization of genes responsible for yield and agronomic traits in waxy corn and sweet corn.