Obtaining and characterization of “Holospora curviuscula” and Holospora obtusa, bacterial symbionts of the macronuclei of Paramecium bursaria and Paramecium caudatum

The symbionts of the macronuclei of Paramecium bursaria and P. caudatum, “Holospora curviuscula” 02AZ16 and H. obtusa 88Ti, respectively, were obtained and investigated. The 16S rDNA nucleotide sequences of “Holospora curviuscula” were obtained for the first time. The differences in 16S rDNA (3.4%) suggest their classification within the genus Holospora. Molecular phylogenetic analysis of the symbionts revealed that these intranuclear symbionts of the ciliates belonged to the order Rickettsiales, forming within a compact cluster of related species.     

Comparison of mutagenicity and chemical properties of N-methyl-N′-alkyl-N-nitrosoureas

Thed mutagenic activities of 11 N-methyl-N′-alkyl-N-nitrosoureas were tested on Samonellatyphimurium TA1535 and compared with chemical properties (alkylating activity and decompostion rate). In their relative mutagenicities the N-nitrosoureas that had a cyclic N′-alkyl group showed far more mutagenic activity than those having a chain N′-alkyl group. M(1-A)NU and M(2-A)NU, which had the most bulky N′-alkyl group in this series, exhibited lethal effects at high concentrations. The mutagenicity showed a small positive correlation with decomposition rates but not with alkylating activities on 4-(p-nitrobenzyl_prridine. The highest mutagenicity in this series was observed in N-methyl-N′-cyclobutyl-N-nitrosourea.These results suggest that, in this series of N-methyl-M′-alkyl-N-nitrosoureas, structural differences in the N′-alkyl groups had great significance in mutagenicity.     

Isolation and synthesis of trans- and cis-(−)-clovamides and their deoxy analogues from the bark of Dalbergia melanoxylon

N-(3′,4′-Dihydroxy-trans-cinnamoyl)-3-(3,4-dihydroxyphenyl)-L-alanine [(?)-clovamide], the major phenolic metabolite (0.1%) in the bark of Dalbergia melanoxylon, is associated with minor proportions of its cis-isomer, and similar pairs of geometrical isomers of their deoxy analogues N-(4′-hydroxycinnamoyl)-3-(3,4-dihydroxyphenyl)-L-alanine and N-(4′-hydroxycinnamoyl)-3-(4-hydroxyphenyl)-L-alanine. (?)-Trans-clovamide is synthesized by direct condensation of the acid chloride of caffeic acid with L-DOPA. Diagnostic CD spectra of these compounds and ¹³C spectra of (?)-trans- and (?)-cis-clovamides are recorded.     

Preparation and conformational analysis of C-glycosyl β- and β/β-peptides

Ten C-glycosyl β²- and β/β²-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble β²-amino acids in which C-glycosylic substituents are attached to the C-2 position of β-amino acids. Conformational analysis of the C-glycosyl β²-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl β/β²-peptides are governed by the configuration at the α-carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl β²-amino acids.     

Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a δ-aminolevulinic acid-dependent mutant strain

Summary The Rhodobacter capsulatus hemA gene, coding for the enzyme -aminolevulinic acid synthase (ALAS), was isolated from a genome bank by hybridization with a hemT probe from Rhodobacter sphaeroides. Subcloning of the initial 3.9 kb HindIII fragment allowed the isolation of a 2.5 kb HindIII-BglII fragment which was able to complement the -aminolevulinic acid-requiring (ALA-requiring) Escherichia coli mutant SHSP19. DNA sequencing revealed an open reading frame coding for a protein with 401 amino acids which displayed similarity to the amino acid sequences of other known ALASs. However, no resemblance was seen to the HemA protein of E. coli K12. Based on the sequence data, an ALA-requiring mutant strain of R. capsulatus was constructed by site-directed insertion mutagenesis. Introduction of a plasmid, containing the hemA gene of R. capsulatus on the 3.9 kb HindIII fragment, restored ALA-independent growth of the mutant indicating that there is only one gene for ALA biosynthesis in R. capsulatus. Transfer of the R factor pRPS404 and hybridization analysis revealed that the ALAS gene is not located within the major photosynthetic gene cluster.Part of this research was presented at the Symposium on Molecular Biology of Membrane-Bound Complexes in Phototrophic Bacteria, Freiburg, FRG, 2–5 August 1989     

α-Onocerin and sterol content of twelve species of Ononis

The sterols and triterpenoids of 12 species of the genus Ononis were analysed by GLC. α-Onocerin was found in all but one of these species, although in some others its concentration was low. In all species examined, sitosterol was the major sterol; stigmasterol, campesterol, cholesterol and the triterpenoids cycloartenol and 24-methylene cycloartanol also occurred. The patterns of α-onocerin and sterols found seem to be consistent with the accepted classification of species within the genus.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

Synthesis of (±)-7,3′- and 7,4′-di-O-methyleriodictyol and of velutin and pilloin

Synthetic 2′-hydroxy-3,4′,6′-trimethoxy-4-benzyloxychalcone (I) affords (±)-7,3′-di-O-methyleriodictyol (II) and 7,3′-di-O-methylluteolin (or velutin, VII) identical with natural samples. Similarly synthetic 2′-hydroxy-4,4′,6′-trimethoxy-3-benzyloxychalcone (X) gives natural (±)-7,4′-di-O-methyleriodictyol (XI) and 7,4′-di-O-methylluteolin (or pilloin, IX). However, attempts to partially etherify II with one mole of prenyl bromide to obtain the natural prenyl ether failed; only the corresponding diprenyloxychalcone (IV) was obtained.     

The role of O-linked and N-linked oligosaccharides on the structure–function of glycoprotein hormones: Development of agonists and antagonists

Thyrotropin (TSH) and the gonadotropins; follitropin (FSH), lutropin (LH) and human chorionic gonadotropin (hCG) are a family of heterodimeric glycoprotein hormones. These hormones composed of two noncovalently linked subunits; a common α and a hormone specific β subunits. Assembly of the subunits is vital to the function of these hormones. However, genetic fusion of the α and β subunits of hFSH, hCG and hTSH resulted in active polypeptides. The glycoprotein hormone subunits contain one (TSH and LH) or two (α, FSHβ and hCGβ) asparagine-linked (N-linked) oligosaccharides. CGβ subunit is distinguished among the β subunits because of the presence of a carboxyl-terminal peptide (CTP) bearing four O-linked oligosaccharide chains. To examine the role of the oligosaccharide chains on the structure–function of glycoprotein hormones, chemical, enzymatic and site-directed mutagenesis were used. The results indicated that O-linked oligosaccharides play a minor role in receptor binding and signal transduction of the glycoprotein hormones. In contrast, the O-linked oligosaccharides are critical for in vivo half-life and bioactivity. Ligation of the CTP bearing four O-linked oligosaccharide sites to different proteins, resulted in enhancing the in vivo bioactivity and half-life of the proteins. The N-linked oligosaccharide chains have a minor role in receptor binding of glycoprotein hormones, but they are critical for bioactivity. Moreover, glycoprotein hormones lacking N-linked oligosaccharides behave as antagonists. In conclusion, the O-linked oligosaccharides are not important for in vitro bioactivity or receptor binding, but they play an important role in the in vivo bioactivity and half-life of the glycoprotein hormones. Addition of the O-linked oligosaccharide chains to the backbone of glycoprotein hormones could be an interesting strategy for designing long acting agonists of glycoprotein hormones. On the other hand, the N-linked oligosaccharides are not important for receptor binding, but they are critical for bioactivity of glycoprotein hormones. Deletion of the N-linked oligosaccharides resulted in the development of glycoprotein hormone antagonists. In the case of hTSH, development of an antagonist may offer a novel therapeutic strategy in the treatment of thyrotoxicosis caused by Graves' disease and TSH secreting pituitary adenoma.     

13C NMR and CD of some 3,8″-biflavanoids from Garcinia species and of related flavanones

The ¹³C NMR signals for most of the carbon atoms of some 3,8″-biflavanoids could be assigned with the help of the spectra of the corresponding monomers. The CD spectra of such compounds containing one flavanone and one chalcone chromophore can be used to determine the absolute configuration.     

Why do O-alkylguanine and O-alkylthymine miscode? The relationship between the structure of DNA containing O-alkylguanine and O-alkylthymine and the mutagenic properties of these bases

The carcinogenic and mutagenic N-nitroso compounds produce GC to AT and TA to GC transition mutations because they alkylate O⁶ of guanine and O⁴ of thymine. It has been generally assumed that these mutations occur because O⁶-alkylguanine forms a stable mispair with thymine and O⁴-alkylthymine forms a mispair with guanine. Recent studies have shown that this view is mistaken and that the alkylG·T and alkylT·G mispairs are not more stable than their alkylG·C or alkylT·A counterparts. Two possible explanations based on recent structural studies are put forward to account for the miscoding. The first possibility is that the DNA polymerase might mistake O⁶-alkylguanine for adenine, and O⁴-alkylthymine for cytosine, because of the physical similarity of these bases. O⁶-Methylguanine and adenine are similarly lipophilic and X-ray crystallography of the nucleosides has shown a close similarity in bond angles and lengths between O⁶-methylguanine and adenine, and between O⁴-methylthymine and cytosine. The second possible explanation is that the important factor in the miscoding is that the alkylG·T and alkylT·G mispairs retain the Watson-Crick alignment with N1 of the purine juxtaposed to N3 of the pyrimidine while the alkylG·C and alkylT·A pairs adopt a wobble conformation. ³¹P NMR of DNA duplexes show that the phosphodiester links both 3′ and 5′ to the C have to be distorted to accomodate the O⁶-ethylguanine:C pair, whereas there is less distortion of the phosphodiesters 3′ and 5′ to the T in an ethylG·T pair. Recent kinetic measurements show that the essential aspect of base selection in DNA synthesis is the ease of formation of the phosphodiester links on both the 3′ and 5′ side of the incoming base. The Watson-Crick alignment of the alkylG·T and alkylT·G mispairs may facilitate formation of these phosphodiester links, and this alignment rather than the strength of the base pairs and the extent of hydrogen bonding between them may be the crucial factor in the miscoding. If either hypothesis is correct it suggests that previously too much emphasis has been placed on the stability of the normal pairs in the replication of DNA.     

Resolution and reconstitution of succinate-cytochrome c reductase. Preparations and properties of high purity succinate dehydrogenase and ubiquinol—cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c₁ III complex).An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinate dehydrogenase fraction and the cytochrome b-c₁ complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c₁ complex was separated into cytochrome b-c₁ III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate.The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 and 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low K_m ferricyanide reductase activity of 85 μmol succinate oxidized per min per mg protein at 38°C.Chemical composition analysis of cytochrome b-c₁ III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation.When these three components were mixed in a proper ratio, a thenoyl-trifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.     

Synthesis and properties of 3′-C-methylnucleosides and their phosphoric esters

3′-C-Methyluridine and 3′-C-methylcytidine were synthesized in 11 steps starting from d-glucose. The conformation of 3′-C-methylnucleosides was studied in solution and in the crystal by using the techniques of c.d., ¹H-n.m.r. spectroscopy, and X-ray diffraction analysis. 3′-C-Methyluridine 2′,3′-cyclophosphate was prepared, and its hydrolysis with nucleases was studied. 3′-C-Methyluridine 5′- mono- and 5′-tri-phosphate were also synthesized.     

Regio- and stereospecific synthesis of rac-carbasugar-based cyclohexane pentols; Investigations of their α- and β-glucosidase inhibitions

In the present study, (3aR,7aS)-1,3,3a,4,7,7a-hexahydroisobenzofuran was submitted to photooxygenation and two isomeric hydroperoxides were successfully obtained. Without any further purification, reduction of the hydroperoxides with titanium tetraisopropoxide catalyzed by dimethyl sulfide gave two alcohol isomers in high yields. After acetylation of alcohol with Ac₂O in pyridine, epoxidation reaction of formed monoacetates with m-CPBA, then chromatographed and followed by hydrolysis of the acetate groups with NH₃ in CH₃OH resulted in the formation of epoxy alcohol isomers respectively. These epoxy alcohol isomers were subjected to trans-dihydroxylation reaction with acid (H₂SO₄) in the presence of water to afford triols. Acetylation of the free hydroxyl groups produced benzofuran triacetates in high yields. Ring-opening reaction of furan triacetates with sulfamic acid catalyzed in the presence of acetic acid/acetic anhydrate and subsequently hydrolysis of the acetate groups with ammonia gave the targeted cyclohexane carbasugar-based pentols. All products were separated and purified by chromatographic and crystallographic methods. Structural analyses of all compounds were conducted by spectral techniques including NMR and X-ray analyses. The biological inhibition activity of the target compounds was tested against glycosidase enzymes, α- and β-glucosidase.     

Biosynthetic origin of the saw-toothed profile in δC and δΗ of n-alkanes and systematic isotopic differences between n-, iso- and anteiso-alkanes in leaf waxes of land plants

The n-fatty acids containing an even number of carbons (ECN-n-FAs) in higher plants are biosynthesised by repetitive addition of a two carbon unit from malonyl-ACP. The n-alkanes containing an odd number of carbon atoms (OCN-n-alkanes) are generally formed by the decarboxylation of ECN-n-FAs, but it is unknown how the less abundant even-carbon-numbered alkanes (ECN-n-alkanes) are biosynthesised in higher plants.There is a distinctive compositional pattern of incorporation of stable carbon (¹³C) and hydrogen (²H) isotopes in co-existing ECN- and OCN-n-alkanes in leaves of higher plants, such that the OCN n-alkanes are relatively enriched in ¹³C but relatively depleted in ²H against the ECN-n-alkanes. This is consistent with the OCN-n-fatty acids having a propionate precursor which is derived from reduction of pyruvate. A tentative pathway is presented with propionate produced by enzymatic reduction of pyruvate which is then thio-esterified with CoSH (coenzyme A thiol) in the chloroplast to form the terminal precursor molecule propionyl-CoA. This is then repetitively extended/elongated with the 2-carbon unit from malonyl-ACP to form the long chain OCN-n-fatty acids.The anteiso- and iso-alkanes in Nicotiana tabacum leaf waxes have previously been found to be systematically enriched in ¹³C compared with the n-alkanes by Grice et al. (2008). This is consistent with the isotopic composition of their putative respective precursors (pyruvate as precursor for n-alkanes, valine for iso-alkanes and isoleucine for anteiso-alkanes). The current study complements that of Grice et al. (2008) and looks at the distribution of hydrogen isotopes. The n-alkanes were found to be more enriched in deuterium (²H) than the iso-alkanes which in turn were more enriched than the anteiso-alkanes. We propose therefore that the depletion of ²H in the iso-alkanes, relative to the n-alkanes is the consequence of accepting highly ²H-depleted hydrogen atoms from NADPH during their biosynthesis. The anteiso-alkanes are further depleted again because there are three NADPH-derived hydrogen atoms in their precursor isoleucine, as compared with only one NADPH-derived hydrogen in valine, the precursor of the iso-alkanes.     

Cloning and comparison of Aα mating-type alleles of the basidiomycete Schizophyllum commune

Summary An A mating-type allele (A4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the A1 allele isolated from the walk was used as a probe to recover the A1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an A allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that A encodes a diffusible product. Restriction mapping shows that A1 and A4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of A1 or A4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other A alleles. A1 and A4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.     

ζ-Potential and surface charge of Thermoplasma acidophila

The surface charge density and the ζ-potential of Thermoplasma acidophila was estimated from microscopic electrophoresis experiments. The cells moved towards the positive electrode. The mobility remained constant from pH 2 to 5, and increased for pH values higher than 6. The mobility at pH 6 decreased dramatically with increased external Ca²⁺ concentration. At pH 2 and an ionic strength similar to that of the growth medium, the ζ-potential was about 8 mV, negative relative to the bulk medium; the surface charge density was 1360esu/cm^-2 which corresponds to one elementary charge per 3500 A².