RNA/DNA co-analysis from blood stains--results of a second collaborative EDNAP exercise.

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 µl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology.

mRNA profiling for the identification of blood--results of a collaborative EDNAP exercise.

A collaborative exercise on mRNA profiling for the identification of blood was organized by the European DNA Profiling Group (EDNAP). Seven blood samples and one blood dilution series were analyzed by the participating laboratories for the reportedly blood-specific markers HBB, SPTB and PBGD, using different kits, chemistries and instrumentation. The results demonstrate that HBB is expressed abundantly in blood, SPTB moderately and PBGD significantly less. All but one of the 16 participating laboratories were able to successfully isolate and detect RNA from the dried bloodstains even though a majority of the laboratories had no prior experience with RNA. Despite some expected variation in sensitivity between laboratories, the method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise support the potential use of mRNA profiling as an alternative to conventional serological tests.

α-(1 → 3)-D-glucans from fruiting bodies of selected macromycetes fungi and the biological activity of their carboxymethylated products.

PURPOSE OF WORK: To show biological activity of carboxymethylated α-(1 → 3)-D-glucans isolated from the selected macromycetes fungi on human tumor and normal cells. Water-insoluble, alkali-soluble polysaccharides (WIP) were isolated from fruiting bodies of four macromycetes fungi: Lentinus edodes, Pleurotus ostreatus, Piptoporus betulinus and Laetiporus sulphureus. The structure of the polysaccharides was determined using composition analysis, methylation analysis, fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The chemical and spectroscopic investigations indicated that the polysaccharides were an α-(1 → 3)-D-glucans. A biological activity analysis of the carboxymethylated (CM) α-(1 → 3)-D-glucans was based on an assessment of their cytotoxic, mitochondrial metabolism-modulating, and free radical scavenging effects. The cytotoxic activity of the CM-glucans was concentration- and cell-type-dependent. The tested CM-glucans, generally, did not have a free radical scavenging effect. The CM-α-(1 → 3)-D-glucans isolated from the selected macromycetes fungi are biologically active and may therefore be used as diet or therapy supplements.

Influence of aromatic compounds on biodegradation of [14C]-labeled xylan and mannan by the white-rot fungus Phlebia radiata.

Radiolabeled [14C]arabinoxylan from wheat meal and [14C]galactoglucomannan from red clover meal were prepared by using 14CO2 as a precursor. Twice as much mannan was mineralized than xylan after 14 days of incubation with Phlebia radiata. Low-molecular-weight phenolic compounds structurally related to lignin increased during mineralization of both hemicellulose fractions. Veratryl alcohol increased degradation of arabinoxylan by approximately 28.5%, whereas veratric acid increased it by only 9.0%. Vanillic acid and ferulic acid also stimulated degradation by 16.6% and 34.7%, respectively. Veratryl alcohol and ferulic acid increased degradation of galactoglucomannan by approximately 75%. Veratraldehyde in both cases repressed the degradation process (23.6% arabinoxylan, 43.8% galactoglucomannan). These results indicate that the degradation of hemicelluloses, e.g., xylan and mannan, by P. radiata is enhanced by addition of aromatic compounds.

Cellular fatty acid composition from Sarcobium lyticum (Legionella lytica comb. nov.)--an intracellular bacterial pathogen of amoebae.

Legionella lytica comb. nov. an intracellular bacterial pathogen of small free-living amoebae was subjected to cellular fatty acid (FA) analysis employing base and acid catalyzed cleavage, gas-liquid chromatography and mass spectrometry. Both unbranched and branched (iso and anteiso) FA of chains ranging from 14 to 30 carbon atoms occurred. The presence of two long-chain FA: 27-oxo-octacosanoic acid and heptacosane-1,27-dioic acid, characteristic for legionellae, was found. Nine amide-linked 3-hydroxy-FA were revealed. The main 3-hydroxy-fatty acids comprise: 3-OH-14:0, 3-OH-16:0, 3-OH-18:0, 3-OH-i18:0, 3-OH-15:OH, 3-OH-i16:0 amd 3-OH-i17:0. The profile of hydroxy FAs permits allocation of L. lytica to group 3 of legionellae which comprise blue-white fluorescent species.

Structure of the O-specific polysaccharide of Mesorhizobium huakuii IFO15243T.

The structure of the O-specific polysaccharide isolated by mild acid hydrolysis of the lipopolysaccharide of Mesorhizobium huakuii IFO15243T was studied using methylation analysis and various one- and two-dimensional 1H and 13C NMR experiments. The O-antigen polysaccharide was found to be linear polymer constituted by a trisaccharide repeating unit of the following structure: --> 2)-alpha-L-6dTalp-(1 --> 3)-alpha-L-6dTalp-(1 --> 2)-alpha-L-Rhap-(1 -->.

Cytokine inducing activities of rhizobial and mesorhizobial lipopolysaccharides of different lethal toxicity.

The lethality and cytokines-inducing activity of lipopolysaccharides (LPS) obtained from nodulating bacteria, Rhizobium leguminosarum and Mesorhizobium loti, were compared to those of Salmonella typhimurium LPS. The activity of R. leguminosarum LPS was almost comparable to Salmonella endotoxin in terms of lethality, Limulus lysate gelating activity and in vivo tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) induction capacity. In contrast to high lethal toxicity of Rhizobium LPS, the lethality of LPS isolated from Mesorhizobium loti was more than 10(3)-fold lower. Weak lethality of LPS from Mesorhizobium correlated with low capacity of this LPS to induce TNF-alpha, IL-1beta, IL-6 and IFN-gamma both in vivo and in vitro in murine splenocytes. The examined overall chemical composition of LPS indicates a considerable distinction in their lipid A regions. Lipid A's obtained from R. leguminosarum and M. loti differed from their enterobacterial counterpart with respect to lipid A sugar backbone, its phosphate content as well as the type and distribution of hydrophobic acyl residues. The relation of lipid A chemotype and bioactivity of LPS from the two Rhizobiaceae genera is discussed.

Occurrence and taxonomic significance of oxo-fatty acids in lipopolysaccharides from members of Mesorhizobium.

Lipopolysaccharides (LPSs) isolated from seven strains of Mesorhizobium were studied for the presence of fatty acids with particular attention for 27-oxooctacosanoic acid and 4-oxo fatty acids. The LPSs from all analysed strains contained various amounts of 27-oxo-28:0 and all of them, with the exception of Mesorhizobium tianshanense, contained also 4-oxo fatty acids (4-oxo-20:0, 4-oxo-i-21:0, 4-oxo-22:0). The group of amide-linked fatty acids consisted of a wide range of 3-hydroxylated and 4-oxo fatty acids whereas all the nonpolar as well as the (omega-1) hydroxylated long-chain acids and the 27-oxo-28:0 fatty acids were ester-linked. The characteristic spectrum of 3-hydroxy fatty acids and presence of 27-OH-28:0 as well as 27-oxo-28:0 acid in LPSs of Mesorhizobium showed that these strains were closely related. Therefore the lipid A fatty acid pattern could be a useful chemotaxonomic marker which helps to isolate the Mesorhizobium group from rhizobium bacteria during the classification process.

Insoluble glucans synthesized by cariogenic streptococci: a structural study.

Of the three cariogenic streptococci grown in four various culture media, the strain Streptococcus mutans 20,381 was found to produce large amounts of extracellular glucosyltransferase and water-insoluble, adhesive exopolysaccharide when grown in batch culture on brain-heart infusion broth. Methylation and nuclear magnetic resonance analyses revealed that the insoluble polymers synthesized by the crude glucosyltransferase preparations were mixed-linkage (1-->3), (1-->6)-alpha-D-glucans (so-called mutans) with a greater proportion of 1,3 to 1,6 linkages and major branch points of 3,6-linked glucose. The percentage content of different types of linkages in glucans varied widely and depended on the strain of cariogenic bacteria used to produce glucosyltransferase, and on the kind of medium utilized to cultivate mutans streptococci. The potential application of insoluble glucan produced by mutans streptococci is discussed.

Occurrence of lipopolysaccharide alterations among Tn5 mutants of Rhizobium leguminosarum bv. trifolii strain 24.1 with altered colony morphology.

Transposon mutants of Rhizobium leguminosarum bv. trifolii 24.1 showing less glossy or smaller colonies were screened for properties usually associated with lipopolysaccharide (LPS) defects in R. leguminosarum, i.e. motility, growth rate, tendency to agglutination in liquid media and symbiotic efficiency. Neither any of the above mutants nor the earlier isolated 24.12 strain, defective in LPS, showed all these properties changed simultaneously. According to PAGE/sodium deoxycholate analysis the mutant 24.12 was the only one producing defective lipopolysaccharide. GC-MS analysis revealed in this mutant qualitative changes in composition of its LPS in comparison with LPS isolated from the parent strain. Other Tn5 mutants produced LPSs similar in composition, however the proportion between LPS I and LPS II differed from that in the parent strain.

Characterization of two lipopolysaccharide types isolated from Rhizobium galegae.

Lipopolysaccharides (LPS) of Rhizobium galegae, a symbiotically nitrogen-fixing species of root-nodule bacteria, were isolated by the phenol-water method from strain HAMBI 1461, the LPS of which resembled enterobacterial smooth type LPS, and from strains HAMBI 1174 and HAMBI 1208, the LPSs of which resembled rough type LPS. The results of PAGE analysis of LPSs, Bio-Gel P2 gel filtration of polysaccharide fractions and the presence of deoxysugars and 4-O-methyl-deoxysugar both in the rough and smooth LPSs suggested that rough LPS contained a short O-antigenic polysaccharide for which we propose the name short O-chain LPS. Accordingly, the smooth LPS is called long O-chain LPS. Despite of the differences in the structure of LPS of R. galegae, all strains were equally effective in nodulating their hosts. The short O-chain LPS of R. galegae showed many features similar to those of phylogenetically related agrobacteria.

Siderophore activity in Rhizobium species isolated from different legumes.

Rhizobium strains isolated from nodules of the different legumes including wild-growing plants were examined for their siderophore activity. Fifteen of the 84 screened rhizobial strains were able to grow under conditions of limited iron supply. Nine of them gave orange halos in the assay with Chrom azurol S. Among these strains were Rhizobium sp. (Ononis) and Rhizobium (Genista), producing hydroxamates and phenolates. These compounds could promote the growth of siderophore-negative bacteria on iron-deficient media. The results imply that the hydroxamates from G1 and O1 strains may belong to the monohydroxamate class of siderophores.

Identification of 3-deoxy-lyxo-2-heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae.

A 3-deoxy-2-heptulosaric acid (DHA), very probably with the lyxo-configuration, was identified in the R-core region of lipopolysaccharides from nodulating strains of Rhizobium leguminosarum, Rhizobium meliloti and from all three biovars of the phytopathogenic Agrobacterium tumefaciens. Its structure could be deduced from the fragmentation pattern of the corresponding alditol acetates obtained after reduction of the 2-keto and the 1.7-carboxy groups by sodium borohydride or sodium borodeuteride. DHA in lipopolysaccharide was not destroyed by periodate and is therefore not in a terminal position. Two DHA-containing oligosaccharides, namely glucosyl (1----4)-3-deoxy-2-heptulosaric acid and rhamnosyl-rhamnosyl-(1----5)-3-deoxy-2-heptulosaric acid could be tentatively identified by mass spectrometric methods amongst the products of mild acidic hydrolysis of lipopolysaccharides of Rhizobium leguminosarum strain 24. The two types of non-nodulating mutants of Rhizobium leguminosarum included in this study did not contain 3-deoxy-2-heptulosaric acid.

Siderophore containing 2,3-dihydroxybenzoic acid and threonine formed by Rhizobium trifolli.

An iron-binding compound was isolated from ethyl acetate extract of culture supernatant fluid of Rhizobium trifolii AR6 and was purified by iron-exchange chromatography. The compound was characterized by UV and IR. It contained 2,3-dihydroxy-benzoic acid and threonine and was accumulated during stationary phase of growth in iron-deficient media. Synthesis of the siderophore was repressed by FeCl3. In iron limited medium the compound promoted growth of R. trifolii strains.