Label-free electrochemical genosensor based on mesoporous silica thin film.

A novel label-free electrochemical strategy for nucleic acid detection was developed by using gold electrodes coated with mesoporous silica thin films as sensing interface. The biosensing approach relies on the covalent attachment of a capture DNA probe on the surface of the silica nanopores and further hybridization with its complementary target oligonucleotide sequence, causing a diffusion hindering of an Fe(CN)6 (3-/4-) electrochemical probe through the nanochannels of the mesoporous film. This DNA-mesoporous silica thin film-modified electrodes allowed sensitive (91.7 A/M) and rapid (45 min) detection of low nanomolar levels of synthetic target DNA (25 fmol) and were successfully employed to quantify the endogenous content of Escherichia coli 16S ribosomal RNA (rRNA) directly in raw bacterial lysate samples without isolation or purification steps. Moreover, the 1-month stability demonstrated by these biosensing devices enables their advanced preparation and storage, as desired for practical real-life applications. Graphical abstract Mesoporous silica thin films as scaffolds for the development of novel label-free electrochemical genosensors to perform selective, sensitive and rapid detection of target oligonucleotide sequences. Application towards E. coli determination.

Antileishmanial activity of compounds produced by endophytic fungi derived from medicinal plant Vernonia polyanthes and their potential as source of bioactive substances.

The purpose of this work was to evaluate the antileishmanial activity of endophytic fungi isolated from leaves of Vernonia polyanthes plant and their prospective use in the discovery of bioactive compounds. Sixteen endophytes were isolated by using potato dextrose agar medium and submitted to cultivation in rice medium. The fungal cultures were extracted with ethanol and used as crude extracts for testing their antileishmanial activity. The most active ethanol extract was obtained from P2-F3 strain, which was identified as Cochliobolus sativus by ITS rRNA gene sequence data. Followed by a bioassay-guided fractionation, the cochlioquinone A, isocochlioquinone A and anhydrocochlioquinone A compounds were isolated from the crude extracts and demonstrated to inhibit the parasites. From the present work, it is possible to conclude that endophytic fungi derived from medicinal plant V. polyanthes may be considered promising source for the discovery of bioactive compounds.

Activated charcoal-mediated RNA extraction method for Azadirachta indica and plants highly rich in polyphenolics, polysaccharides and other complex secondary compounds.

BACKGROUND: High quality RNA is a primary requisite for numerous molecular biological applications but is difficult to isolate from several plants rich in polysaccharides, polyphenolics and other secondary metabolites. These compounds either bind with nucleic acids or often co-precipitate at the final step and many times cannot be removed by conventional methods and kits. Addition of vinyl-pyrollidone polymers in extraction buffer efficiently removes polyphenolics to some extent, but, it failed in case of Azadirachta indica and several other medicinal and aromatic plants. FINDINGS: Here we report the use of adsorption property of activated charcoal (0.03%-0.1%) in RNA isolation procedures to remove complex secondary metabolites and polyphenolics to yield good quality RNA from Azadirachta indica. We tested and validated our modified RNA isolation method across 21 different plants including Andrographis paniculata, Aloe vera, Rosa damascena, Pelargonium graveolens, Phyllanthus amarus etc. from 13 other different families, many of which are considered as tough system for isolating RNA. The A260/280 ratio of the extracted RNA ranged between 1.8-2.0 and distinct 28S and 18S ribosomal RNA bands were observed in denaturing agarose gel electrophoresis. Analysis using Agilent 2100 Bioanalyzer revealed intact total RNA yield with very good RNA Integrity Number. CONCLUSIONS: The RNA isolated by our modified method was found to be of high quality and amenable for sensitive downstream molecular applications like subtractive library construction and RT-PCR. This modified RNA isolation procedure would aid and accelerate the biotechnological studies in complex medicinal and aromatic plants which are extremely rich in secondary metabolic compounds.

A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome-arm identification, integration of genetic linkage groups and analysis of major repeat family distribution.

We developed a reference karyotype for B. vulgaris which is applicable to all beet cultivars and provides a consistent numbering of chromosomes and genetic linkage groups. Linkage groups of sugar beet were assigned to physical chromosome arms by FISH (fluorescent in situ hybridization) using a set of 18 genetically anchored BAC (bacterial artificial chromosome) markers. Genetic maps of sugar beet were correlated to chromosome arms, and North-South orientation of linkage groups was established. The FISH karyotype provides a technical platform for genome studies and can be applied for numbering and identification of chromosomes in related wild beet species. The discrimination of all nine chromosomes by BAC probes enabled the study of chromosome-specific distribution of the major repetitive components of sugar beet genome comprising pericentromeric, intercalary and subtelomeric satellites and 18S-5.8S-25S and 5S rRNA gene arrays. We developed a multicolor FISH procedure allowing the identification of all nine sugar beet chromosome pairs in a single hybridization using a pool of satellite DNA probes. Fiber-FISH was applied to analyse five chromosome arms in which the furthermost genetic marker of the linkage group was mapped adjacently to terminal repetitive sequences on pachytene chromosomes. Only on two arms telomere arrays and the markers are physically linked, hence these linkage groups can be considered as terminally closed making the further identification of distal informative markers difficult. The results support genetic mapping by marker localization, the anchoring of contigs and scaffolds for the annotation of the sugar beet genome sequence and the analysis of the chromosomal distribution patterns of major families of repetitive DNA.

Assessment of arbuscular mycorrhizal fungal diversity in roots of Solidago gigantea growing in a polluted soil in Northern Italy.

The arbuscular mycorrhizal (AM) status of Solidago gigantea was investigated in a contaminated site of Northern Italy, where the chemical industry ACNA (Associated National Chemical Companies) was active till 1999. To counteract the devastating effects of chemicals and to allow re-vegetation, soil from an uncontaminated area was used to cover the highly polluted hills of the industrial site about 25 years ago. On the basis of the current floristic features, the hill was divided into four areas. Heavy metal content in soil and in plant shoots and roots was determined by chemical analysis. The AM fungal community colonizing S. gigantea was investigated from a morphological and a molecular point of view. All plants were modestly colonized, but the fungal structures within the roots were normal. By PCR-RFLP and sequencing of 18S rDNA, 14 AM fungal types were identified: three of them were present in all the considered areas and nine appeared to be specific to certain areas. Glomus was the predominant AM genus. Our analysis demonstrates the presence and the relatively high level of AM species variety and shows how a remediation programme based on cover-soil has been efficient to restore a community of AM fungi, tolerant enough to proliferate in a still contaminated soil.

Rhizosphere bacteria affected by transgenic potatoes with antibacterial activities compared with the effects of soil, wild-type potatoes, vegetation stage and pathogen exposure.

A greenhouse experiment was performed to analyze a potential effect of genetically modified potatoes expressing antibacterial compounds (attacin/cecropin, T4 lysozyme) and their nearly isogenic, nontransformed parental wild types on rhizosphere bacterial communities. To compare plant transformation-related variations with commonly accepted impacts caused by altered environmental conditions, potatoes were cultivated under different environmental conditions, for example using contrasting soil types. Further, plants were challenged with the blackleg pathogen Erwinia carotovora ssp. atroseptica. Rhizosphere soil samples were obtained at the stem elongation and early flowering stages. The activities of various extracellular rhizosphere enzymes involved in the C-, P- and N-nutrient cycles were determined as the rates of fluorescence of enzymatically hydrolyzed substrates containing the highly fluorescent compounds 4-methylumbelliferone or 7-amino-4-methyl coumarin. The structural diversity of the bacterial communities was assessed by 16S rRNA-based terminal restriction fragment length polymorphism analysis, and 16S rRNA gene clone libraries were established for the flowering conventional and T4 lysozyme-expressing Desirée lines grown on the chernozem soil, each line treated with and without E. carotovora ssp. atroseptica. Both genetic transformation events induced a differentiation in the activity rates and structures of associated bacterial communities. In general, T4 lysozyme had a stronger effect than attacin/cecropin. In comparison with the other factors, the impact of the genetic modification was only transient and minor, or comparable to the dominant variations caused by soil type, plant genotype, vegetation stage and pathogen exposure.

Comparison between direct microscopy and flow cytometry for rRNA-based quantification of Candidatus Accumulibacter phosphatis in activated sludge.

A comparison of the quantification of a specific microbial group in activated sludge by fluorescent in-situ hybridization, coupled with either direct microscopic counting or flow cytometry, was performed using an enhanced-biological-phosphorus-removal, sequencing-batch reactor. The population dynamics of Candidatus Accumulibacter phosphatis (Cand. A. phosphatis) was evaluated during two separate runs of the reactor. With the operational conditions used, Cand. A. phosphatis was enriched until a failure in the pH controller eliminated its ecological advantage. As a result, the comparison of quantification techniques included Cand. A. phosphatis concentrations as low as 11% and as high as 96% of the total cells in the samples. The analysis demonstrated that, regardless of the particular limitations of each technique, both provided similar results when the activated-sludge flocs were easily dispersed. However, when the activated-sludge samples contained flocs that were difficult to disperse, flow cytometry failed to provide quantitative results.

Investigation of infectious agents associated with arthritis by reverse transcription PCR of bacterial rRNA.

In reactive and postinfectious arthritis the joints are generally sterile but the presence of bacterial antigens and nucleic acids has been reported. To investigate whether organisms traffic to affected joints in these conditions, we performed reverse transcription PCR using universal primers to amplify any bacterial 16S rRNA sequences present in synovial fluid. Bacterial sequences were detected in most cases, even after treatment of the synovial fluid with DNase, implying the presence of bacterial RNA and therefore of transcriptionally active bacteria. Analysis of a large number of sequences revealed that, as reported in rheumatoid arthritis, most were derived from gut and skin commensals. Organisms known to have triggered arthritis in each case were not found by sequencing the products obtained using universal primers, but could in some cases be shown to be present by amplifying with species specific primers. This was the case for Yersinia pseudotuberculosis and Chlamydia trachomatis. However, in arthritis thought to be related to Campylobacter infection the sequences obtained were not from Campylobacter jejuni or C. coli, but from other Campylobacter spp. that are not known to be associated with reactive arthritis and are probably present as commensals in the gut. We conclude that although rRNA from reactive arthritis associated organisms can be detected in affected joints, bacterial RNA from many other bacteria is also present, as was previously noted in studies of other forms of inflammatory arthropathy.

Antitumor effects of curcin from seeds of Jatropha curcas.

AIM: To study the antitumor effects of curcin from Jatropha curcas. METHODS: Antitumor activity of curcin was tested by MTT assay. The N-glycosidase activity of curcin was determined by characterization of R-fragment in gel. A cell-free system, rabbit reticulocyte lysate, was introduced to quantify the inhibitory activity of curcin on protein biosynthesis. RESULTS: The curcin had a powerful inhibitory action upon protein synthesis in reticulocyte lysate with an IC50 (95 % confidence limits) value of 0.19 (0.11-0.27) nmol/L. The IC50 (95 % confidence limits) of curcin on SGC-7901, Sp2/0, and human hepatoma was 0.23 (0.15-0.32) mg/L, 0.66 (0.35-0.97) mg/L, 3.16 (2.74-3.58) mg/L, respectively. Curcin was found to have no toxic to Hela cells and normal cells (MRC). After the rRNA of ribosome was treated with curcin and aniline at acidic condition, a cleaved R-fragment of approximately 450 nt appeared, but this fragment did not occur after treatment with curcin only. A comparison of the amino acid sequences of curcin, ricin A-chain and trichosanthin revealed that there were relatively high similarities among them. The percentages of homology between curcin and ricin A chain, between curcin and trichosanthin were found to be 54 % and 57 % respectively. Especially, the conserved residues forming the active sites of the A chain of ricin and trichosanthin occurred in curcin. CONCLUSION: Curcin has an obvious antitumor effect and its mechanisms are related to the N-glycosidase activity.

Authentication of Chinese crude drug, Gecko, by allele-specific diagnostic PCR.

Based on the sequences of the mitochondrial 12S rRNA gene fragment of 17 samples from Gekkonidae, Salamandridae, Agamidae and Hynobiidae, respectively, a pair of allele-specific primers was designed for differentiating the Chinese medicinal material Gecko from its adulterants by PCR. The results of amplification with the primers indicate that amplicons from the templates of Gekko gecko were clearly revealed by agarose gel electrophoresis, whereas no evident amplicons were found from other species. The primers were employed to identify crude drug samples from different sources. Among a total of 9 samples, 3 were diagnosed as genuine Gecko. This result is consistent with morphological identification and DNA sequence analyses.

Effect of short-chain carbohydrates on human intestinal bifidobacteria and Escherichia coli in vitro.

Plate counts and small subunit (SSU) rRNA abundance were used to study the effects of fructo-oligosaccharides (FOS), fructose, or galacto-oligosaccharides (GOS) on bifidobacterial populations in human faecal microbiotas. The bacteria were grown in pH-controlled anaerobic fermentation vessels. Untreated cultures and fructose-amended fermenters were used as controls. Bifidobacterium longum, B. adolescentis and B. angulatum comprised the dominant bifidobacterial populations throughout the experiment. No major differences were found in the four treatments, in terms of viable counts of the organisms or of total populations of bifidobacteria at any time point. However, large differences were observed with respect to the abundance of bifidobacterial SSU rRNA between the treatments. Greatest bifidobacterial SSU rRNA abundance was seen in FOS cultures, with the lowest in the untreated control fermentation. GOS and fructose also increased bifidobacterial SSU rRNA. Cultures supplemented with FOS and GOS were also associated with lower colony counts and SSU rRNA abundance for Escherichia coli, compared with fructose-supplemented and control fermenters. At the 24-h time point, the untreated control contained 19.8 microg of enterobacterial SSU rRNA/ml of culture fluid, compared with 11.4 microg/ml for the fructose fermentation, and 2.6 and 0.5 microg/ml for the FOS and GOS culture vessels, respectively.

Effect of the tropical forage calliandra on microbial protein synthesis and ecology in the rumen.

AIMS: To determine the effect of condensed tannins in Calliandra calothyrsus (calliandra) on rumen microbial function. METHODS AND RESULTS: Microbial populations, ruminal protein synthesis and fermentation end-products were measured in sheep fed roughage hay supplemented with calliandra (30%), with and without inclusions of polyethylene glycol (PEG) to counteract the effect of tannin. Molecular and conventional enumeration techniques were used to quantify rumen bacteria, fungi and protozoa, and protein synthesis was predicted from estimates of urinary purine excretion. The total number of cellulolytic bacteria, including populations of Fibrobacter succinogenes and Ruminococcus spp., was significantly lower in sheep supplemented with calliandra and these populations increased when animals were treated with PEG. By contrast, protozoa and fungi and the microbial group containing Bacteroides-Porphyromonas-Prevotella bacteria appeared to be less affected. The efficiency of microbial protein synthesis in the rumen was not altered significantly. CONCLUSION: Calliandra caused significant shifts in rumen microbial populations without changing the efficiency of protein synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of calliandra tannins on rumen digestion may result more from complexing with nutrients than direct inhibition of micro-organisms.

Cryptic peripheral ribosomal domains distributed intermittently along mammalian myelinated axons.

A growing body of metabolic and molecular evidence of an endogenous protein-synthesizing machinery in the mature axon is a challenge to the prevailing dogma that the latter is dependent exclusively on slow axoplasmic transport to maintain protein mass in a steady state. However, evidence for a systematic occurrence of ribosomes in mature vertebrate axons has been lacking until recently, when restricted ribosomal domains, called "periaxoplasmic plaques," were described in goldfish CNS myelinated axons. Comparable restricted RNA/ribosomal "plaque" domains now have been identified in myelinated axons of lumbar spinal nerve roots in rabbit and rat on the basis of RNase sensitivity of YOYO-1-binding fluorescence, immunofluorescence of ribosome-specific antibodies, and ribosome phosphorus mapping by electron spectroscopic imaging (ESI). The findings were derived from examination of the axoplasm isolated from myelinated fibers as axoplasmic whole mounts and delipidated spinal nerve roots. Ribosomal periaxoplasmic plaque domains in rabbit axons were typically narrow ( approximately 2 microm), elongated ( approximately 10 microm) sites that frequently were marked by a protruding structure. The domain complexity included an apparent ribosome-binding matrix. The small size, random distribution, and variable intermittent axial spacing of plaques around the periphery of axoplasm near the axon-myelin border are likely reasons why their systematic occurrence has remained undetected in ensheathed axons. The periodic but regular incidence of ribosomal domains provides a structural basis for previous metabolic evidence of protein synthesis in myelinated axons.

Chlamydia pneumoniae present in the human synovium are viable and metabolically active.

We demonstrated that chromosomal DNA from Chlamydia pneumoniae is present in synovial tissue in at least some patients with reactive arthritis/Reiter's syndrome and other arthritides. Here, we provide initial molecular evidence that the bacterium is viable and metabolically active when present in the synovium. We used reverse transcription-polymerase chain reaction (RT-PCR) assays targeting primary transcripts from the chlamydial rRNA operons, and mRNA from several C. pneumoniae genes (hsp60, ompA, KDO transferase, Mr=76000 protein), to analyse RNA preparations from synovial tissue of 10 patients with various forms of arthritis; each patient was known to be PCR-positive for C. pneumoniae DNA in synovium prior to RT-PCR assays. Two PCR-negative patients served as controls for RT-PCR assays. In the 10 patients PCR-positive for C. pneumoniae DNA, RT-PCR assays targeting primary transcripts from the rRNA operons of the organism showed that these molecules were present in each sample, as were transcripts from the bacterial hsp60 gene. Assays targeting mRNAs from the Mr=76000 protein and the KDO transferase genes of C. pneumoniae gave positive results for 6/10 preparations. We were unable to identify mRNA from the chlamydial major outer membrane protein gene (ompA) in any preparation. RNA preparations from the two control patients were negative in all RT-PCR assays targeting C. pneumoniae transcripts. These results indicate that in patients infected with the organism, synovial C. pneumoniae are viable and metabolically active, as are C. trachomatis cells in the same context. Such viability is consistent with a role in long-term contribution to pathogenesis in joint disease.

Protective effects of dimethyl-4,4'-dimethoxy-5,6,5',6'-dimethylene dioxybiphenyl-2,2'-dicarboxylate on damages of isolated rat hepatocytes induced by carbon tetrachloride and D-galactosamine.

The protective effect of biphenyl dimethyl dicarboxylate (DDB) on chemically induced damages was studied in isolated suspended rat hepatocytes. The experimental results showed that DDB (200 micrograms/10(6) cells) efficiently protected the hepatocytes against carbon tetrachloride (CCl4 10 mmol.L-1) and D-galactosamine (1 mmol.L-1) induced damages. Membranal lipid peroxidation (malondialdehyde, MDA formation) and glutamic pyruvic transaminase (GPT) release from the hepatocytes were markedly decreased. The damage of the cell surfaces of the hepatocytes were also reduced as seen under a scanning electron microscope (SEM). Pretreatment with DDB (300 mg.kg-1) orally ameliorated the reduction of liver glycogen and blood glucose caused by ip injection of D-galactosamine (800 mg.kg-1) in mice. When normal rats were given DDB 300 mg.kg-1 once daily for 10 d, the free ribosomal protein and RNA in the liver increased significantly. These results indicate that DDB is of beneficial effects on both damaged and normal hepatocytes.

A novel diagnostic method of Pneumocystis carinii. In situ hybridization of ribosomal ribonucleic acid with biotinylated oligonucleotide probes.

The reactivity and specificity of the in situ hybridization of ribosomal RNA in the diagnosis of Pneumocystis carinii were investigated. Three complementary oligonucleotide probes, 22 and 25 nucleotides long, corresponding to the species specific regions of 5S and 18S ribosomal RNA of Pneumocystis carinii were synthesized and labeled with biotinylated dUPT at the 3' termini. In situ hybridization was performed on formalin-fixed paraffin-embedded human lung tissues using the mixture of these probes and detected with the avidin-biotin peroxidase complex method. The reactions were positive in all 12 cases of Pneumocystis carinii pneumonia, but in none of the infections with other pathogenic agents, including virus (6 cases), mycobacteria (4 cases), protozoa (4 cases) and fungi (8 cases). The reactivity and specificity of this method was comparable with that of immunohistochemistry using a monoclonal anti-human Pneumocystis carinii antibody. Because the specificity of in situ hybridization is based on nucleotide sequences of ribosomal RNAs, that are constant among species, contrary to morphology of protista or expression of antigens, it should complement conventional staining and immunohistochemical methods, and provide a useful tool for the diagnosis of Pneumocystis carinii.

Transfer RNAs of potato (Solanum tuberosum) mitochondria have different genetic origins.

Total transfer RNAs were extracted from highly purified potato mitochondria. From quantitative measurements, the in vivo tRNA concentration in mitochondria was estimated to be in the range of 60 microM. Total potato mitochondrial tRNAs were fractionated by two-dimensional polyacrylamide gel electrophoresis. Thirty one individual tRNAs, which could read all sense codons, were identified by aminoacylation, sequencing or hybridization to specific oligonucleotides. The tRNA population that we have characterized comprises 15 typically mitochondrial, 5 'chloroplast-like' and 11 nuclear-encoded species. One tRNA(Ala), 2 tRNAs(Arg), 1 tRNA(Ile), 5 tRNAs(Leu) and 2 tRNAs(Thr) were shown to be coded for by nuclear DNA. A second, mitochondrial-encoded, tRNA(Ile) was also found. Five 'chloroplast-like' tRNAs, tRNA(Trp), tRNA(Asn), tRNA(His), tRNA(Ser)(GGA) and tRNA(Met)m, presumably transcribed from promiscuous chloroplast DNA sequences inserted in the mitochondrial genome, were identified, but, in contrast to wheat (1), potato mitochondria do not seem to contain 'chloroplast-like' tRNA(Cys) and tRNA(Phe). The two identified tRNAs(Val), as well as the tRNA(Gly), were found to be coded for by the mitochondrial genome, which again contrasts with the situation in wheat, where the mitochondrial genome apparently contains no tRNA(Val) or tRNA(Gly) gene (2).

Phosphorus imaging of the 7-S ribonucleoprotein particle.

In this study an unprecedented demonstration of the detection sensitivity of electron spectroscopic imaging (ESI) is reported. This microanalytical technique is capable of forming elemental maps of a specimen with high sensitivity and resolution by forming images with electrons that have lost particular amounts of energy due to interactions with the atoms of the specimen. The 7-S ribonucleoprotein particle, composed of one molecule of 5-S RNA and one molecule of the 40K MW protein, TFIIIa, was used for this demonstration. As few as 120 phosphorus atoms of the 5-S RNA have been detected and their localization in the particle determined. The shape of the 7-S particle is ellipsoidal with a long axis of 15.0 nm and a shorter axis between 8 and 9 nm. Similarly, the 5-S RNA is also an elongated structure located asymmetrically on one side of the particle. The average signal-to-noise ratio over the particle in the net phosphorus images is 14 whereas the ratio measured for the nucleosome containing 2.4-fold more phosphorus is 30.

Immunological capture of nucleic acid hybrids and application to nonradioactive DNA probe assay.

Antibodies specific for DNA:RNA hybrids were coated onto polystyrene test tubes and applied to hybridization assays involving DNA and RNA. Synthetic DNA probes complementary to 16S rRNA of Campylobacter were labeled with biotin and hybridized to ribosomal RNA directly in lysates of bacterial cells. After hybridization, DNA:RNA hybrids were captured with immobilized anti-DNA:RNA antibody, and the biotinylated probe was detected with streptavidin-horseradish peroxidase (EC 1.11.1.7) conjugate. The assay was optimized to detect as few as 70,000 Campylobacter cells in a sample. We compared the utility of this hybridization assay with that of conventional microbiology methods by examination of 1448 stool samples from hospital clinical laboratories. The DNA hybridization assay had a sensitivity of 98.7% (75/76) and a specificity of 98.2% (1347/1372) and overall agreed with 98.2% of the conventional results for a test population that had a 5.2% incidence (76/1448) of Campylobacter infection. The assay is simple to perform and yields results within 2.5 h.