The hydrolysis of primary amide groups in Asn/Gln-containing peptides

Peptides Boc-Ala-Asn/Gln-OH and Boc-Asn/Gln-Ala-OH were saponified with barium hydroxide to corresponding Asp/Glu-containing peptides. Under the conditions of saponification, Boc-Asn-Ala-OH additionally afforded Boc-Asp-OH, isopeptide Boc-Asp(Ala)-OH, and Boc-NHSuc>Ala-OH, with the third being the key intermediate in these transformations. Boc-Asp(OMe)-Ala-OMe underwent similar transformations under treatment with diazomethane or triethylamine. Saponification with barium hydroxide was accompanied by a high epimerization ofN-terminal amino acid residues, whereas the products of the diazomethane treatment of Boc-Asp(OMe)-Ala-OMe had a low degree of epimerization.     

Apoptosis-specific protein (ASP 45 kDa) is distinct from human Apg5, the homologue of the yeast autophagic gene apg5

We have examined whether the apoptosis-specific protein p45ASP and human Apg5 are identical proteins. Like p45ASP, myc-hApg5 cross-reacted with a c-Jun antibody and approximately 50% of myc-hApg5 was bound to a Triton X-100-insoluble fraction in HeLa cells. However, soluble myc-hApg5 was degraded during apoptosis induced by staurosporine or TNFalpha/cycloheximide whilst expression of soluble p45ASP was stabilised. Furthermore, myc-hApg5 degradation was blocked by the caspase inhibitor Boc-Asp(OMe)FMK whilst p45ASP expression was eliminated. Moreover, myc-hApg5 ( approximately 32 kDa) never assumed the size of p45ASP (45 kDa). It is therefore likely that p45ASP and human Apg5 are distinct proteins although they do share some common characteristics.     

p38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x(L)

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor alpha (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x(L) protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine-threonine phosphorylation of Bcl-x(L) in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x(L) and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x(L) in endothelial cells leads to degradation of Bcl-x(L) in proteasomes and subsequent induction of apoptosis.     

Molecular and crystal structures of three monothiated analogues of the terminally blocked ala-aib-ala sequence of peptaibol antibiotics

The molecular and crystal structures of three monothiated analogues of the blocked L -Ala-Aib-L -Ala sequence of peptaibol antibiotics, t-Boc-L -Ala-Aib-ψ(CSNH)-L -Ala-OMe, Ac-L -Ala-Aib-ψ(CSNH)-L -Ala-OMe, and Ac-ψ(CSNH)-L -Ala-Aib-L -Ala-OMe, determined by x-ray diffraction analyses, are reported. In all cases the peptide chain is folded with φ,ψ angles close to or slightly distorted from those expected for a type II β-bend conformation. However, the 4 → 1 H-bond distance falls within the accepted limits only for Ac-L -Ala-Aib-ψ(CSNH)-L -Ala-OMe. The structures are compared with those already published for their two oxygenated analogues.     

Changes in elemental composition of human chromosomes during a G-banding (ASG) and a C-banding (BSG) procedure.

Human chromosomes fixed in methanol-acetic acid have been examined by X-ray microanalysis, before, during and after a G-banding and a C-banding procedure. Phosphorus (representing mainly DNA), sulphur and calcium are the most prominent elements in untreated chromosomes. In the G-banding procedure, the calcium is lost during 2 x SSC treatment. In the C-banding procedure, calcium is lost in the preliminary HCl treatment. During the following barium hydroxide treatment a large amount of barium becomes attached to the chromosomes, but is lost again during the subsequent 2 x SSC treatment. In both banding techniques Giemsa staining produces large peaks for sulphur (thiazine dyes) and bromine (eosin), showing that both types of dyes are involved in the staining. Reduction in the phosphorus peak during these procedures may be partly due to extraction of DNA and other chromosomal components, but could also be due to absorption of phosphorus X-rays by heavy elements (barium and bromine).     

Water unextractable polysaccharides from three milling fractions of rye grain

Water unextractable material from bran, an intermediate milling fraction and sieved flour of rye grain were sequentially extracted at room temperature with saturated barium hydroxide, water, 4 M potassium hydroxide and water followed by extraction with 2 potassium hydroxide in a boiling water bath, giving repeatable recoveries of extracts and polysaccharide residue compositions in collected fractions. Total recoveries of polysaccharide residues in extracts and residue from the different water unextractable materials were 78–88%. Extracts in which 90–93% of the carbohydrates were arabinose and xylose residues were obtained by extraction with saturated barium hydroxide. Subsequent extraction with water yielded a fraction in which 64–68% of the carbohydrates were glucose residues. The extraction with hot alkali resulted in extracts in which 85–89% of the carbohydrates were arabinose and xylose residues. The ara/xyl ratio in the collected fractions ranged from 0.1–1.3, with the lowest ratios in fractions that precipitated after neutralisation of the 4 potassium hydroxide extract and the highest ratios in the unextractable residues. Structural characterisation with ¹H-NMR spectroscopy revealed varying substitution patterns for arabinoxylans in the different extracts and that glucose residues in the extracts essentially originated from mixed-linked β-glucan. The proportion of disubstituted xylose residues was lower in barium hydroxide extracts compared to the other main extracts. A highly branched heteroxylan was extracted with hot alkali. The polysaccharides found in the corresponding extracts for all the starting materials had generally similar structural features, but the yield differed considerably.     

Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases

Mononuclear non-heme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases comprise a large family of enzymes that utilize an Fe(IV)-oxo intermediate to initiate diverse oxidative transformations with important biological roles. Here, four of the major types of Fe(II)/2OG-dependent reactions are detailed: hydroxylation, halogenation, ring formation, and desaturation. In addition, an atypical epimerization reaction is described. Studies identifying several key intermediates in catalysis are concisely summarized, and the proposed mechanisms are explained. In addition, a variety of other transformations catalyzed by selected family members are briefly described to further highlight the chemical versatility of these enzymes.     

Arabinoxylan fractionation on DEAE-cellulose chromatography influenced by protease pre-treatment

An arabinoxylan mixture was extracted with saturated barium hydroxide from a water unextractable residue of rye bran. The mixture was fractionated on an anion exchange column which was eluted with water, 0.0025 M sodium borate, 0.025 M sodium borate and 0.4 M sodium hydroxide. It was possible to fractionate the arabinoxylan mixture into fractions with different structural features. The fractionation was repeated with prior protease treatment of the arabinoxylan mixture, but most of the arabinoxylan did not bind to the column by any mechanism that the protease treatment affected, As the largest fraction was still eluted with 0.4 M sodium hydroxide. Protease treatment affected the proportion of disubstituted xylose residues (dXyl) in the water, 0.0025 M sodium borate and 0.025 M sodium borate fractions, indicating that protein associated with arabinoxylans with a high dXyl content is more liable to the protease treatment or that protein is mainly associated with these structures. This study gives indications that protein is involved in the separation mechanism of rye arabinoxylan on a DEAE–cellulose column.     

Synthesis of 1,4-dideoxy-1,4-imino-D-glucitol, a glucosidase inhibitor

1,2:5,6-Di-O-isopropylidene-D-glucitol was converted via its 1,4-dimethanesulfonate into the 1-azido-4-methanesulfonate which, after deprotection and treatment with barium hydroxide, afforded a 9:1 mixture of the corresponding 3,4- and 4,5-anhydro derivatives. Reduction of this mixture by transfer hydrogenation using ammonium formate in methanol and Pd/C as catalyst afforded 1,4-dideoxy-1,4-imino-D-glucitol (4), the structure of which was proved after acetylation by 1H-n.m.r. spectroscopy. Compound 4 is a potent alpha-D-glucosidase inhibitor (Ki 7 X 10(-4)M) and a less potent beta-D-glucosidase inhibitor (Ki 1.25 X 10(-4)M), and inhibits beta-D-galactosidase non-competitively.     

The temperature dependence and involvement of mitochondria permeability transition and caspase activation in damage to organotypic hippocampal slices following in vitro ischemia

The aggravating effect of hyperglycemia on ischemic brain injury can be mimicked in a model of in vitro ischemia (IVI) using murine hippocampal slice cultures. Using this model, we found that the damage in the CA1 region following IVI in the absence or presence of 40 mm glucose (hyperglycemia) is highly temperature dependent. Decreasing the temperature from 35 to 31 degrees C during IVI prevented cell death, whereas increasing the temperature by 2 degrees C markedly aggravated damage. As blockade of the mitochondrial permeability transition (MPT) is equally effective as hypothermia in preventing ischemic cell death in vivo, we investigated whether inhibition of MPT or of caspases was protective following IVI. In the absence of glucose, the MPT blockers cyclosporin A and MeIle4-CsA but not the immunosuppressive compound FK506 diminished cell death. In contrast, following hyperglycemic IVI, MPT blockade was ineffective. Also, the pan-caspase inhibitor Boc-Asp(OMe)fluoromethyl ketone did not decrease cell death in the CA1 region following IVI or hyperglycemic IVI. We conclude that cell death in the CA1 region of organotypic murine hippocampal slices following IVI is highly temperature dependent and involves MPT. In contrast, cell death following hyperglycemic IVI, although completely prevented by hypothermia, is not mediated by mechanisms that involve MPT or caspase activation.     

Substrate-dependent chemoselective aldose-aldose and aldose-ketose isomerizations of carbohydrates promoted by a combination of calcium ion and monoamines.

Epimerization of aldoses at C-2 has been extensively investigated by using various metal ions in conjunction with diamines, monoamines, and aminoalcohols. Aldoses are epimerized at C-2 by a combination of alkaline-earth or rare-earth metal ions (Ca(2+), Sr(2+), Pr(3+), or Ce(3+)) and such monoamines as triethylamine. In particular, the Ca(2+)-triethylamine system proved effective in promoting aldose-ketose isomerization as well as C-2 epimerization of aldoses. 13C NMR studies using D-(1-(13)C)glucose and D-(1-(13)C)galactose with the CaCl(2) system in CD(3)OD revealed that the C-2 epimerization proceeds via stereospecific rearrangement of the carbon skeleton, or 1,2-carbon shift, and ketose formation proceeds partially through an intramolecular hydrogen migration or 1,2-hydride shift and, in part, via an enediol intermediate. These simultaneous aldose-aldose and aldose-ketose isomerizations showed interesting substrate-dependent chemoselectivity. Whereas the mannose-type aldoses having 2,3-erythro configuration (D-mannose, D-lyxose, and D-ribose) showed considerable resistance to both the C-2 epimerization and the aldose-ketose isomerization, the glucose-type sugars having 2,3-threo and 3,4-threo configurations, D-glucose and D-xylose, are mainly epimerized at C-2 and those having the 2,3-threo and 3,4-erythro configurations, D-galactose and D-arabinose, were mostly isomerized into 2-ketoses. These features are of potential interest in relevance to biomimic sugar transformations by metal ions.     

A study on the mechanism of the epimerization at C-3 of chenodeoxycholic acid by Clostridium perfringens.

The mechanism of 3-hydroxy epimerization of chenodeoxycholic acid by Clostridium perfringens was investigated in 3 alpha, 7 alpha-dihydroxy-[2,2,4,4-2H4]-, 3 alpha, 7 alpha-dihydroxy-[3 beta-2H]- and 3 beta, 7 alpha-dihydroxy-[3 alpha-2H]-5 beta-cholanoic acid transformations. Our findings rule out a dehydration-rehydration pathway and agree with a redox mechanism involving 3-oxochenodeoxycholic acid as intermediate.     

New C-band protocol by heat denaturation in the presence of formamide

C-banding techniques detect the presence of constitutive heterochromatin, which is usually located in centromeric regions of chromosomes in the majority of analysed species. The common method for C-banding used over the last 30 years involves treatment with a mild alkali barium hydroxide 5% Ba(OH)2 at 50 degrees C for 5-15 min and subsequent incubation in salt solution (2 x SSC at 60 degrees C for 1 h). We here present a new, easy and reliable technique for C-banding, which basically involves heat denaturation of chromosomal DNA in the presence of formamide and incubation in 2 x SSC at room temperature.     

Biochemical processing of plant acquired pyrrolizidine alkaloids by the neotropical leaf-beetle Platyphora boucardi

Leaf beetles of the genus Platyphora, feeding on plant species containing pyrrolizidine alkaloids of the lycopsamine type, not only sequester these alkaloids and concentrate them in their exocrine defensive secretions, but also specifically process the plant acquired alkaloids. Using P. boucardi as subject, three mechanisms were studied: (i). utilization of host plant alkaloids that are not sequestered per se; (ii). elucidation of the mechanism of the already documented C-7 epimerization of heliotridine O(9)-monoesters; (iii). the specificity of insect catalyzed necine base esterification. P. boucardi does not sequester the triester parsonsine, the principal alkaloid of its host plant Prestonia portobellensis (Apocynaceae). Beetles fed with a purified mixture of nor-derivatives of parsonsine, obtained from Parsonsia laevigata, did not sequester the triesters but transformed them by partial degradation into monoesters that are accumulated in the defensive secretions. The mechanism of the previously described transformation of rinderine into intermedine by C-7 epimerization was elucidated by feeding C-7 deuterated heliotrine (3'-methylrinderine). The transformation of heliotrine into epiheliotrine (3'-methylintermedine) catalyzed by P. boucardi is accompanied by complete loss of deuterium, indicating the same mechanism of an oxidation-reduction process via a ketone intermediate as recently demonstrated in a pyrrolizidine alkaloid sequestering lepidopteran. P. boucardi is able to form ester alkaloids from five different necine bases fed as radioactively labeled substrates. However, besides C-7 epimerization the beetles are not able to convert simple necine bases into retronecine. The functional importance of the various alkaloid transformations is discussed in comparison to striking parallels of analogous reactions known from pyrrolizidine alkaloid sequestering Lepidoptera.     

N-banding in Triticum aestivum following feulgen hydrolysis

Summary Terminal and/or interstitial N-bands were produced on the seven B-genome chromosomes and chromosomes 4 and 7 of the A-genome of Triticum aestivum cv. Chinese Spring by a modified BSG technique following a standard Feulgen preparation. The banding was accomplished by modifying the barium hydroxide treatment.     

Synthesis of amino acid conjugates of 1,1′-dimethylferrocene: New chiral conjugates

Syntheses of diastereomeric mixtures of 1,1′-dimethylferrocene-2-Ala-OMe (3) and 1,1′-dimethylferrocene-3-Ala-OMe (4) are reported by peptide coupling of L-Ala-OMe to the enantiomeric mixtures of the corresponding acids, 1,1′-dimethylferrocene-2-carboxylic acid (1) and 1,1′-dimethylferrocene-3-carboxylic acid (2).     

Advances in microbial biotechnology of bile acids

The recent advances in microbial biotechnology of production of bile acid metabolites helped to identify a number of neutral and acidic steroidal compounds useful as drugs and drug intermediates on a scale which would not have been possible by classical chemical transformations. Microbial transformations viz., hydroxylation, dehydroxylation, reduction of the carbonyl moieties, epimerization, side-chain metabolism, introduction of carbon-carbon double bonds into the steroid nucleus, deconjugation of bile acid conjugates carried out by various microorganisms for production of useful metabolites with special reference to newer techniques including cell immobilization and transposon mutagenesis for selective transformations are reviewed. The different pathways of microbial degradation of bile acids leading to the formation of various products are discussed. A compilation of the metabolites formed by various microorganisms from the bile acids or their conjugates and reported during the period 1979-1992 is also provided.     

Gas chromatography--mass spectrometry studies of tetramethyl uric acids

Of the 12 possible isomers of tetramethyl uric acid, 6 are obtained by methylation with diazomethane and 4 by on column methylation with trimethylphenyl ammonium hydroxide. Elution patterns on three different columns, quantitative data, and mass spectra are presented. In addition, elution data and the mass spectrum of 8-methoxy-1,3,8-trimethylxanthine are given. The results may be useful for studies of incorporation of [¹³C]glycine and other stable isotope precursors into uric acid.     

Studies on peptides CLVIII. Model experiments for the synthesis of open-chain unsymmetrical cystine-peptides

Treatment of a mixture of Cys(R)(O) and Cys(R) with an acid was found to generate cystine in fairly good yields, when suitable R, R, and an acid were selected. An unsymmetrical cystine peptide was prepared by treatment of a mixture of Z(OMe)-Cys(R) (0)-Ala-NH₂ (R=Acm or MBzl) and Z(OMe)-Cys(MBzl)-Gly-OBzl with TFA or 1 M TFMSA/TFA.³ Oxytocin was obtained in an excellent yield by TFA treatment of the protected peptide containing Cys(Acm)(0) and Cys(MBzl). Thus, formation of the disulfide bond was found feasible at the position of Cys(R) (0).The following abbreviations are used Boc t-butyloxycarbonyl - Z(OMe) p-methoxybenzyloxycarbonyl - MBzl p-methoxybenzyl - Acm acetamidomethyl - Bzl benzyl - Ad l-adamantyl - tBu t-butyl - TFA trifluoroacetic acid - TFMSA trifluoromethanesulfonic acid - TMSOTf trimethylsilyl trifluoromethane sulfonate     

Mechanism of dihydroneopterin aldolase. NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes

Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-D-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-L-monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotonation and reprotonation of C2' of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent. No deuteration of C2' was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation, the DHNA-catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol/aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) were determined by equilibrium binding and transient kinetic studies. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them.