Immunomagnetic capture PCR to detect viable Cryptosporidium parvum oocysts from environmental samples

A method to detect viable Cryptosporidium parvum oocysts was developed. Polyclonal immunoglobulin G against C. parvum oocyst and sporozoite surface antigens was purified from rabbit immune serum, biotinylated, and bound to streptoavidin-coated magnetic particles. C. parvum oocysts were captured by a specific antigen-antibody reaction and magnetic separation. The oocysts were then induced to excyst, and DNA was extracted by heating at 95 degrees C for 10 min. A 452-bp fragment of C. parvum DNA was amplified by using a pair of C. parvum-specific primers in PCR. The method detected as few as 10 oocysts in purified preparations and from 30 to 100 oocysts inoculated in fecal samples. The immunomagnetic capture PCR (IC-PCR) product was identified and characterized by a nested PCR that amplified a 210-bp fragment, followed by restriction endonuclease digestion of the IC-PCR and nested-PCR products at the StyI site and a nonradioactive hybridization using an internal oligonucleotide probe labeled with biotin. PCR specificity was also tested, by using DNAs from other organisms as templates. In the control experiments, inactivated oocysts were undetectable, indicating the ability of this method to differentiate between viable and nonviable oocysts. Thus, this system can be used to specifically detect viable C. parvum oocysts in environmental samples with great sensitivity, providing an efficient way to monitor the environment for C. parvum contamination.     

Betaine improves the PCR amplification of GC-rich DNA sequences

Betaine improves the co-amplification of the two alternatively spliced variants of the prostate-specific membrane antigen mRNA as well as the amplification of the coding cDNA region of c-jun. It is suggested that betaine improves the amplification of these genes by reducing the formation of secondary structure caused by GC-rich regions and, therefore, may be generally applicable to ameliorate the amplification of GC-rich DNA sequences.     

Extraction, PCR amplification and sequencing of mitochondrial DNA from human hair shafts

Techniques have been developed for extracting, amplifying and directly sequencing mitochondrial DNA (mtDNA) from human hair shafts. The hair shaft is ground in a glass micro-tissue grinder, and the DNA is extracted with organic solvent and purified by filtration. The filtrate subsequently provides the mtDNA template for the PCR. The two hypervariable segments of the mtDNA control region are amplified in four separate reactions. After a purification step to remove unincorporated PCR primers, amplified products are quantitated by capillary electrophoresis and subjected to cycle sequencing. The products are separated and analyzed on an automated DNA sequencer. The mtDNA sequences from the hair shaft match the mtDNA sequences from blood samples taken from the same donor.     

PCR amplification and comparison of nucleotide sequences from the groESL heat shock operon of Ehrlichia species

Degenerate PCR primers derived from conserved regions of the eubacterial groESL heat shock operon were used to amplify groESL sequences of Ehrlichia equi, Ehrlichia phagocytophila, the agent of human granulocytic ehrlichiosis (HGE), Ehrlichia canis, Bartonella henselae, and Rickettsia rickettsii. The groESL nucleotide sequences were less conserved than the previously determined 16S rRNA gene sequences of these bacteria. A phylogenetic tree derived from deduced GroEL amino acid sequences was similar to trees based on 16S rRNA gene sequences. Nucleotide sequences obtained from clinical samples containing E. equi, E. phagocytophila, or the HGE agent were very similar (99.9 to 99.0% identity), and the deduced amino acid sequences were identical. Some divergence was evident between nucleotide sequences amplified from samples originating from the United States (E. equi and the HGE agent) and sequences from the European species, E. phagocytophila. A single pair of PCR primers derived from these sequences was used to detect E. chaffeensis and HGE agent DNA in blood samples from human patients with ehrlichiosis.     

Atypical herpes simplex virus encephalitis diagnosed by PCR amplification of viral DNA from CSF

The caspases are cysteine proteases that have been implicated in the execution of programmed cell death in organisms ranging from nematodes to humans. Many members of the Bcl-2 family, including Bcl-XL, are potent inhibitors of programmed cell death and inhibit activation of caspases in cells. Here, we report a direct interaction between caspases and Bcl-XL. The loop domain of Bcl-XL is cleaved by caspases in vitro and in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3 withdrawal. Mutation of the caspase cleavage site in Bcl-XL in conjunction with a mutation in the BH1 homology domain impairs the death-inhibitory activity of Bcl-XL, suggesting that interaction of Bcl-XL with caspases may be an important mechanism of inhibiting cell death. However, once Bcl-XL is cleaved, the C-terminal fragment of Bcl-XL potently induces apoptosis. Taken together, these findings indicate that the recognition/cleavage site of Bcl-XL may facilitate protection against cell death by acting at the level of caspase activation and that cleavage of Bcl-XL during the execution phase of cell death converts Bcl-XL from a protective to a lethal protein.     

DNA analysis of cystic fibrosis in Brazil by direct PCR amplification from Guthrie cards

A 3 bp deletion of codon 508 (phenylalanine) of the cystic fibrosis (CF) gene constitutes the mutation of most CF chromosomes. The frequency of this mutation (referred to as delta F508), varies considerably between populations, ranging from 26% of the CF mutations in Turkey to 88% in Denmark. To determine the frequency of the delta F508 mutation in Brazilian Caucasoid CF patients, we used direct polymerase chain reaction (PCR) amplification of DNA obtained from dried blood spots on Guthrie cards, followed by ethidium bromide staining of gels. Although the overall frequency of the delta F508 mutation was 47% of 380 CF chromosomes from Brazilian Caucasoids born in five different states, significant interstate differences were observed, ranging from a delta F508 frequency of 27% to 53%. While our method could be used to screen patients and their relatives for carrier testing and prenatal diagnosis, the efficacy of screening only for the delta F508 mutation would be low, and would vary from state to state. Screening for a panel of local mutations will be needed to increase the mutation detection rate and optimize genetic counseling.     

Improved amplification of microbial DNA from blood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate

Molecular methods are increasingly used to identify microbes in clinical samples. A common technical problem with PCR is failed amplification due to the presence of PCR inhibitors. Initial attempts at amplification of the bacterial 16S rRNA gene from inoculated blood culture media failed for this reason. The inhibitor persisted, despite numerous attempts to purify the DNA, and was identified as sodium polyanetholesulfonate (SPS), a common additive to blood culture media. Like DNA, SPS is a high-molecular-weight polyanion that is soluble in water but insoluble in alcohol. Accordingly, SPS tends to copurify with DNA. An extraction method was designed for purification of DNA from blood culture media and removal of SPS. Blood culture media containing human blood and spiked with Escherichia coli was subjected to an organic extraction procedure with benzyl alcohol, and removal of SPS was documented spectrophotometrically. Successful amplification of the extracted E. coli 16S rRNA gene was achieved by adding 5 microliter of undiluted processed sample DNA to a 50-microliter PCR mixture. When using other purification methods, the inhibitory effect of SPS could be overcome only by dilution of these samples. By our extraction technique, even uninoculated blood culture media were found to contain bacterial DNA when they were subjected to broad-range 16S rRNA gene consensus PCR. We conclude that the blood culture additive SPS is a potent inhibitor of PCR, is resistant to removal by traditional DNA purification methods, but can be removed by a benzyl alcohol extraction protocol that results in improved PCR performance.     

Pretreatment with restriction enzyme or bovine serum albumin for effective PCR amplification of Epstein-Barr virus DNA in DNA extracted from paraffin-embedded gastric carcinoma tissue

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.     

Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions

PCR amplifications of 16S/23S rDNA spacer regions were carried out from conserved 16S and 23S sequences for genomic DNA samples from strains representing 16 bacterial species (12 genera). Multiple products were produced containing conserved homologous sequences at the 3' and 5' ends, separated by highly variable internal spacer sequences. These products cross-hybridized forming heteroduplex DNA structures containing double-stranded ends surrounding an internal single-stranded loop. Single-stranded DNA was also produced in the amplification of rDNA spacer sequences. Fragments comprising the nonhomoduplex DNA components were identified by their susceptibility to removal by digestion with a single-stranded endonuclease. The relative formation of heteroduplex and single-stranded DNA was reduced by reaction conditions favoring primer/template annealing, for example, higher ionic strength, higher primer concentration, and lower annealing temperature, as well as by decreasing the number of amplification cycles. Heteroduplex and single-stranded DNA structures were also generated by denaturing and reannealing spacer amplification products in the absence of polymerase activity. Whereas heteroduplex and single-stranded DNA structures provide additional information that is helpful in distinguishing between species of bacteria that produce similar homoduplex products, the mobility of heteroduplex and single-stranded DNA structures DNA structures is extremely sensitive to electrophoretic conditions.     

Identification of Trichosporon asahii by PCR based on sequences of the internal transcribed spacer regions

Trichosporon asahii is a major causative agent of deep-seated trichosporonosis, which has a high mortality rate. To detect T. asahii, we have developed specific oligonucleotide primers based on the internal transcribed spacer regions of this organism's genome. Amplification products were selectively obtained from only T. asahii DNA; the DNAs of other Trichosporon species, as well as those of medically relevant yeasts such as Candida albicans, Cryptococcus neoformans, and Malassezia furfur, were not amplified. This detection system will be useful as a microbiological tool for the diagnosis of trichosporonosis.     

Comparison of three DNA extraction methods on bone and blood stains up to 43 years old and amplification of three different gene sequences

The objective of this study was to determine the effects of cellobiose and monensin on the in vitro fermentation of organic acids (L-aspartate, fumarate, and DL-malate) by mixed ruminal bacteria. Ruminal fluid was collected from a steer fed 36.7 kg of forage and 4.5 kg of concentrate supplement once per day. Ruminal fluid was centrifuged to sediment feed particles and protozoa, and the resulting supernatant, which contained bacteria, was added (33%, vol/vol) to anaerobic media (500 ml). Incubations (n = 2) were performed in batch culture at 39 degrees C and sampled at 0, 2, 4, 6, 8, 12, and 24 h. Organic acids were added to achieve a final concentration of 7.5 mM. Cellobiose was added to obtain a final concentration of 5 mM, and monensin dissolved in ethanol was included at concentrations of 0 or 5 ppm. Addition of cellobiose to organic acid fermentations increased the rate of organic acid utilization by the mixed bacterial population. Total concentrations of volatile fatty acids were increased by the addition of cellobiose to all fermentations. A lag period (< or = 8 h) occurred in fermentations that were treated with monensin before organic acids were utilized. Total concentrations of volatile fatty acids were increased, and the acetate to propionate ratio was decreased, by monensin treatment. When cellobiose and monensin were added together, propionate production and organic acid utilization were increased. Both cellobiose and monensin affected the in vitro fermentation of organic acids by mixed ruminal bacteria by providing a carbon and energy source and by influencing electron disposal.     

A method to differentiate between thyroglobulin derived from normal thyroid tissue and from thyroid carcinoma based on analysis of reactivity to lectins

BACKGROUND: Positron emission tomography (PET) allows the measurement of benzodiazepine-gamma-aminobutyric acidA (GABA(A)) receptor kinetics. We employed flumazenil radiolabeled with carbon 11, a radioligand that labels the benzodiazepine site on the GABA(A) receptor, and fully quantitative, high-sensitivity PET to test the hypothesis that central benzodiazepine site binding is decreased in medication-free patients with panic disorder. METHODS: We compared 7 patients with panic disorder who had been off medication for at least 6 months and who had never abused alcohol with 8 healthy controls. The resulting parametric voxel-by-voxel maps were analyzed by voxel-based and region of interest-based methods using both parametric and nonparametric statistics. RESULTS: The major finding was that there is a global reduction in benzodiazepine site binding throughout the brain in patients with panic disorder compared with controls. There were sex differences in the 2 samples, but a separate analysis excluding women led to the same conclusions. In addition, the loci with the largest regional decrease in binding (right orbitofrontal cortex and right insula) were areas thought to be essential in the central mediation of anxiety. CONCLUSION: These results must be considered preliminary but are congruous with previous clinical psychopharmacologic evidence of involvement of the benzodiazepine-GABA(A) receptor and demonstrate that decreased flumazenil binding at this site may underlie panic disorder.     

Phylogenetic re-evaluation of Trametes consors based on mitochondrial small subunit ribosomal DNA sequences

Mitochondrial small subunit ribosomal DNAs of Cerrena unicolor and Trametes consors were sequenced and compared with those of known mushroom taxa. Trametes consors is a species recently transferred from Irpex, and Cerrena is a genus closely related to Trametes. The present phylogenetic tree showed that Cerrena unicolor and Trametes consors clustered together and made an independent lineage from the Trametes group. A new combination, Cerrena consors (Berk.) Ko and Jung, comb nov., is proposed here by transferring Trametes consors into Cerrena based on molecular data along with taxonomic evidence.     

Advantages of immunostaining over DNA analysis using PCR amplification to detect p53 abnormality in long-term formalin-fixed tissues of human colorectal carcinomas

The purpose of our studies was to examine differentiation-dependent expression of 15-lipoxygenase (15-LO) and prostaglandin H synthase (PGHS) isoforms in cultured normal human tracheobronchial epithelial cells. In the presence of retinoic acid (RA) the cultures differentiated into a mucociliary epithelium. When cultured in RA-depleted media, the cultures differentiated into a squamous epithelium. In the absence of RA the cultures did not express 15-LO or either of the PGHS isoforms. The PGHS-1 isoform was not expressed in RA-sufficient cultures, but both PGHS-2 messenger RNA (mRNA) and protein were strongly expressed, and prostaglandin E2 (PGE2) was produced during the predifferentiation phase. No PGHS-2 expression or PGE2 could be detected in fully differentiated mucociliary cultures. 15-LO showed the opposite expression pattern: neither mRNA nor protein were detected during the predifferentiation stage, but both were strongly expressed once mucous differentiation had occurred. Cytosolic phospholipase A2 protein was expressed throughout all stages of growth and differentiation. The cultures generated no 15-LO metabolites when incubated with 10 microM to 50 microM arachidonic acid (AA) and stimulated with ionophore. However, lysates prepared from such cultures generated 15-hydroxyeicosatetraenoic acid (15-HETE) and 12-HETE from AA, indicating that the cells contained active enzyme. When cultures expressing 15-LO protein were incubated with 10 microM linoleic acid (LA) instead of AA, and were stimulated with ionophore, they generated 13-hydroxy-9,11-octadecadienoic acid. LA rather than AA appeared to be the preferred substrate for the 15-LO enzyme. Our studies indicated that the expression of 15-LO and PGHS-2 is differentiation dependent in airway epithelial cells.     

DNA-dependent RNA polymerase C from Xenopus laevis ovaries. Ability to transcribe intact double-stranded DNA

When human erythrocyte acetylcholinesterase (AChE) is inhibited by succinyldicholine (SuCh) at low ionic strength, mu = 0.03, two apparent affinity constants for the inhibitor can be distinguished, KI(1) = 5.4 X 10-5 M and KI(2) = 1.3 X 10-5 M. Increasing the ionic strength to mu = 0.58 increases KI(1) to 1.1 X 10-4 M, but abolishes the contribution of KI(2) completely. It is suggested that a similar process may underlie the apparently continuous variation in affinity constant with ionic strength which has been reported for many bifunctional cationic inhibitors. Since KI(2) is in a concentration range commonly attained therapeutically, some of the clinical manifestations of succinyldicholine administration, e.g., muscarinic effects, may be explained by its anticholinesterase action.     

Phylogeny of the Drosophila saltans species group based on combined analysis of nuclear and mitochondrial DNA sequences

Nucleotide sequences from two nuclear loci, alcohol dehydrogenase and internal transcribed spacer-1 of the nuclear ribosomal DNA repeats, and two mitochondrial genes, cytochrome oxidase I and cytochrome oxidase II, were determined from nine species in the Drosophila saltans species group. The partition homogeneity test and partitioned Bremer support were used to measure incongruence between phylogenetic hypotheses generated from individual partitions. Individual loci were generally congruent with each other and consistent with the previously proposed morphological hypothesis, although they differed in level of resolution. Since extreme conflict between partitions did not exist, the data were combined and analyzed simultaneously. The total evidence method gave a more resolved and highly supported phylogeny, as indicated by bootstrap proportions and decay indices, than did any of the individual analyses. The cordata and elliptica subgroups, considered to have diverged early in the history of the D. saltans group, were sister taxa to the remainder of the saltans group. The sturtevanti subgroup, represented by D. milleri and D. sturtevanti, occupies an intermediate position in this phylogeny. The saltans and parasaltans subgroups are sister clades and occupy the most recently derived portion of the phylogeny. As with previous morphological studies, phylogenetic relationships within the saltans subgroup were not satisfactorily resolved by the molecular data.     

Learning of event sequences is based on response-effect learning: Further evidence from a serial reaction task.

Four experiments provide converging evidence that serial learning in a serial reaction task is based on response–effect learning, mediated by the learning of the relations between a response and the stimulus that follows it. In Experiment 1, the authors varied the stimulus sequence and the response–stimulus relations while holding the response sequence constant. Learning effects depended on the complexity of the response–stimulus relations but not on the stimulus–stimulus relations. In Experiment 2, transfer of serial learning from 1 stimulus sequence to another was only found when both sequences had identical response–stimulus relations. In Experiment 3, a variation of the stimulus sequence alone had no effect on serial learning, whereas in Experiment 4 learning effects increased when the response–stimulus relations but not the stimulus–stimulus relations were simplified. These findings suggest that serial learning is based on mechanisms of voluntary action control. (PsycINFO Database Record (c) 2010 APA, all rights reserved)