Slot-blot Assay Research Articles

Increased airway mucins after cardiopulmonary bypass associated with postoperative respiratory complications in children

ObjectiveAirway mucins may play an important role in the mechanism of respiratory complications after cardiopulmonary bypass in infants and children. Our aim was to measure airway mucin levels before and after cardiopulmonary bypass and to determine whether changes in mucin levels were associated with the development of respiratory complications. MethodsAirway glycoprotein and mucins (MUC5AC, MUC5B, and MUC2) in serial small-volume airway lavage samples from 39 young children who underwent cardiac operations with cardiopulmonary bypass were measured by slot-blot assay with specific antimucin peptide antibodies. The relationship between mucin changes and post–cardiopulmonary bypass respiratory complications was investigated. Airway lavage samples were also collected from 11 children before and after operation without cardiopulmonary bypass, and changes in mucin levels were compared with those in subjects who underwent cardiopulmonary bypass. Airway lavage sample DNA was also measured to investigate the relationship between mucin changes and lung injury. ResultsGlycoprotein, MUC5AC, and MUC5B levels were significantly increased after cardiopulmonary bypass (P < .001) whereas MUC2 level was not. Children with respiratory complications showed significantly higher glycoprotein and MUC5AC levels than did children without respiratory complications before and after cardiopulmonary bypass (P < .05). Increase of total mucin (MUC5AC, MUC5B, and MUC2) during cardiopulmonary bypass showed positive correlation with DNA increase during cardiopulmonary bypass (r = 0.73), Paco2 (r = 0.62) and alveolar-arterial oxygen difference (r = 0.55) immediately after cardiopulmonary bypass. Increase of total mucin was associated with postoperative respiratory complications and their severity. There were no significant changes detected in airway mucin during operations without cardiopulmonary bypass. ConclusionsAirway mucins were increased during cardiopulmonary bypass, and this increase was associated with markers of lung injury after cardiopulmonary bypass and with the development of postoperative respiratory complications.

Reactivity with Tris(hydroxymethyl)aminomethane confounds immunodetection of acrolein-adducted proteins.

The toxic alpha,beta-unsaturated aldehyde acrolein readily attacks proteins, generating adducts at cysteine, histidine, and lysine residues. In this study, rabbit antiserum was raised against acrolein-modified keyhole limpet hemocyanin in the expectation that it would allow immunodetection of adducted proteins in biological samples. Using slot-blot and enzyme-linked immunosorbent assays, the antiserum detected acrolein-modified protein with high sensitivity and specificity. Adduct immunodetection was strongly inhibited by acrolein-modified polylysine but not polyhistidine. Efforts to develop a Western blotting method for detecting adducted proteins in cell lysates were hampered by irreproducible outcomes, evidently due to adduct instability during SDS-PAGE. Indeed, adducts generated via brief exposure of a model protein to acrolein displayed pH- and concentration-dependent instability to tris(hydroxymethyl)aminomethane (Tris), a nucleophilic buffer used in protein electrophoresis. The effect was most striking when Tris solutions were buffered to pH 8.0 and higher. In contrast, adducts formed during extended exposure to acrolein (> or =60 min) were completely stable to Tris. The time dependence of susceptibility raised the possibility that Tris interfered with specific steps in lysine modification, which involves stepwise Michael addition of two molecules of acrolein to the same residue, followed by condensation and dehydration to form a heterocyclic adduct, N(epsilon)-(3-formyl-3,4-dehydropiperidino)lysine. We hypothesize that carbonyl-retaining Michael adducts may react with Tris by forming imines with the primary amine of the buffer. Consistent with this idea, triethanolamine, a tertiary amine buffer unable to form imines, had no effect on acrolein-adducted protein. These effects of Tris may explain difficulties in the detection of acrolein-adducted proteins during conventional Western blotting procedures.

Development of an Alu-Based, QSY 7-Labeled Primer PCR Method for Quantitation of Human DNA in Forensic Samples

Determining the amount of human DNA extracted from a crime scene sample is an important step in DNA profiling. The forensic community relies almost entirely upon a technique (slot blot) to quantitate human DNA that is imprecise, time consuming, and labor intensive. This paper describes the development of a new technique based on PCR amplification of a repetitive Alu sequence. Specific primers were used to amplify a 124-bp fragment of Alu sequence; amplification was detected by SYBR Green I staining in a fluorescent plate reader. To reduce background in the plate reader assay, QSY-7 labeled primers were utilized. The assay was tested on animal DNAs, human blood spots, mock crime samples, and degraded DNA in comparison with the slot blot technique. The QSY Alu assay has a dynamic range of 10 ng to 10 pg, and is sensitive, specific, fast, quantitative, and comparable in cost to the slot blot assay.

Chemiluminescent measurement of telomere DNA content in biopsies.

Telomeres are nucleoprotein complexes that protect the ends of chromosomes from fusion and degradation. They are typically shorter in tumor cells than in paired normal cells, and shorter telomeres are associated with poor outcome in cancer. We previously described a slot blot-based methodfor measuring telomere DNA content, a proxy for telomere length. Although this method represented an improvement over existing methods, its 30-ng limit of sensitivity was insufficient for use with biopsy or other scant tissues. Here we describe a chemiluminescent slot blot assay for telomere DNA content that has the sensitivity required for use with biopsy materials. The results obtained with DNA derived from human placental, HeLa, human peripheral blood lymphocytes, sham-needle core prostate biopsies, and archival prostatectomy tissues demonstrated that telomere DNA content can be reliably and reproducibly measured in 5 ng, and sometimes as little as 2 ng, genomic DNA. Sham-needle core prostate biopsy and prostatectomy specimens processed in parallel produced comparable results. The contribution of truncated telomeres in admixtures containing as much as 75% normal placental DNA could be established. We also demonstrated that the treatment of tissue with formalin before DNA purification does not decrease the efficacy of the assay.

Vitellogenin mRNA regulation and plasma clearance in male sheepshead minnows, ( Cyprinodon variegatus) after cessation of exposure to 17β-estradiol and p-nonylphenol

Research was conducted to determine the kinetics of hepatic vitellogenin (VTG) mRNA regulation and plasma VTG accumulation and clearance in male sheepshead minnows ( Cyprinodon variegatus) during and after cessation of exposure to either 17β-estradiol (E2) or para-nonylphenol (NP). Adult fish were continuously exposed to aqueous measured concentrations of 0.089 and 0.71 μg E2 per l, and 5.6 and 59.6 μg NP per l for 16 days using an intermittent flow-through dosing apparatus. Fish were sampled on days 8 and 16 of exposure followed by sampling at discrete intervals for up to 96 days post-exposure. At each interval five fish were randomly sampled from each concentration and hepatic VTG mRNA and serum VTG levels for individual fish determined by slot blot and direct enzyme-linked immunosorbent assay (ELISA), respectively. Exposure to E2 and NP resulted in a dose dependent increase in hepatic VTG mRNA and plasma VTG over the course of the 16-day exposure period. Mean plasma VTG levels at day 16 were >100 mg/ml for both high doses of E2 and NP, and >20 mg/ml for the low exposure treatments. Within 8 days post-exposure, hepatic VTG mRNA levels returned to baseline in both high and low E2 treatments but remained elevated 2–4 fold in the NP treatments. Due to a shortened sampling period, a clearance rate for plasma VTG in the 5.6 μg NP per l treatment could not determined. In the 0.089, 0.71 μg E2 per l, and 59.6 μg NP per l treatments, VTG levels began decreasing within 4 days after exposure cessation and exhibited an exponential rate of elimination from plasma. Clearance rates for 0.71 μg E2 per l and 59.6 μg NP per l were not significantly different ( P=0.47), however, both demonstrated significantly higher rates of clearance ( P<0.02) than observed in the 0.089 μg E2 per l treatment. Our results indicate that hepatic VTG mRNA rapidly diminishes after cessation of estrogenic exposure in sheepshead minnows, but plasma VTG clearance is concentration and time dependent and may be detected at measurable levels for months after initial exposure to an estrogenic compound.

Combined PCR and slot blot assay for detection of Salmonella and Listeria monocytogenes

Detection of Salmonella typhimurium and Listeria monocytogenes by the polymerase chain reaction (PCR) assay coupled with slot blot detection was investigated in this study. After being extracted from diluted bacterial culture with the extraction buffer, bacterial DNA was subjected to PCR. The slot blot assay was optimized and used to detect PCR products. The lowest detection level of this method was 10 3 cfu/ml in the original culture media for both pathogens, or 5 bacterial cells in the PCR reaction. Combined with immunomagnetic separation (IMS) to separate and concentrate bacteria from samples, the detection limit could be 40 cfu/ml of bacteria from milk samples. The whole detection procedure was completed within 7 h. After multiplex PCR (amplification of DNA from two different bacteria in the same PCR tube) and slot blot, a detection level of 10 3 cfu/ml was achieved in the simultaneous detection for both pathogens, which was similar to that of individual detection for each pathogen. The combination of PCR and slot-blot seems to be highly sensitive and time-efficient, and is therefore promising for routine use in the detection of Salmonella and L. monocytogenes in food samples such as milk.

ANTIOXIDANT AND INFLAMMATORY RESPONSE AFTER ACUTE NITROGEN DIOXIDE AND OZONE EXPOSURES IN C57Bl/6 MICE

Ozone (O3) and nitrogen dioxide (NO2) are highly reactive and toxic oxidant pollutants. The objective of this study is to compare chemokine, cytokine, and antioxidant changes elicited by acute exposures of O3 and NO2 in a genetically sensitive mouse. Eight-weekold C57Bl/6J mice were exposed to 1 or 2.5 ppm ozone or 15 or 30 ppm NO2 for 4 or 24 h. Changes in mRNA abundance in lung were assayed by slot blot and ribonuclease protection assay (RPA). Messages encoding metallothionein (Mt), heme oxygenase I (HO-I), and inducible nitric oxide synthase (iNOS) demonstrated increased message abundance after 4 and 24 h of exposure to either O3 or NO2. Furthermore, increases in message abundance were of a similar magnitude for O3 and NO2. Messages encoding eotaxin, macrophage inflammatory protein (MIP)-1 a, and MIP-2 were elevated after 4 and 24 h of exposure to 1 ppm ozone. Interleukin-6 was elevated after 4 h of exposure to ozone. After 4 h of 2.5 ppm ozone exposure, increased mRNAs of eotaxin, MIP-1 a, MIP-2, Mt, HO-I, and iNOS were elevated to a higher magnitude than were detected after 1 ppm ozone. Monocyte chemoattractant protein (MCP-1) was elevated following 15 ppm NO2 exposure. After 4 h of 30 ppm NO2 exposure, messages encoding eotaxin, MIP-1 a, MIP-2, and MCP-1 were elevated to levels similar to those detected after ozone exposure. Our results demonstrate a similar antioxidant and chemokine response during both O3 and NO2 exposure. Induction of these messages is associated with the duration and concentration of exposure. These studies suggest that these gases exert toxic action through a similar mechanism.

Genome plasticity during the acquisition of embryogenic competence

Hypocotyl explants from carrot and other species experience concomitant segregation events and differentiation of homeotic structures during the first 20 days of culture on 2,4-dichlorophenoxyacetic acid (2,4-D). In addition to these cyto-morphological changes, significant amounts of nuclear DNA are lost, the molecular details of which we investigate in this paper. We have developed a slot-blot analysis assay to study the DNA content of a series of carrot samples; besides the leaves, this survey ranged over different culture timepoints: hypocotyls, cell lines, and somatic embryo stages. We carried on to study the relationship between this DNA loss and sequence complexity modulation. Results from probing sequences that correspond to different degrees of complexity, such as medium repetitive and unique sequences as well as sequences belonging to both classes (ribosomal cistrons, ubiquitin, actin, and chalcone synthase), consistently manifested a reduction in DNA levels during the acquisition of embryogenic competence. In some cases, the cultured cells would contain only 10% of the gene copies observed in the reference tissues. Modulation trends also showed that DNA levels of most sequences recover at the torpedo-plantlet stage, which again correlates DNA modulation and the acquisition of embryogenic competence. These results suggest that similar DNA variations may occur in plants in vivo during meiosis, possibly so that meiotic division may be properly completed.Key words: Daucus carota L., DNA reduction, somatic embryogenesis, totipotency, commitment.

Marked Intratumoral Heterogeneity of the Proto-Oncogene Her-2/neu Determined by Three Different Detection Systems.

It is customary to submit only one portion of a breast cancer to determine if there is amplification or overexpression of the proto-oncogene HER-2/neu. In routine studies of the expression of neu in breast cancer, however, we noted discrepancies in intratumoral positivity. To investigate this phenomenon further, multiple tumor specimens (129 samples) from 41 women with breast cancer were examined. Forty cases were analyzed for neu amplification by slot blot assay and 18 with fluorescent in situ hybridization. Neu overexpression was determined using four different specific antibodies. In more than 50% of cases there were discrepancies in results between the tissue blocks examined. This was evident in both inter- and intra-assay comparisons. It is concluded that intratumoral heterogeneity of neu amplification/overexpression in breast cancer exists to a far greater degree than previously recognized and could be a responsible factor for conflicting published data regarding neu's prognostic significance. Examination of only one tumor sample may not give a true indication of either amplification or overexpression of this oncogene.

Cancer initiation by polycyclic aromatic hydrocarbons results from formation of stable DNA adducts rather than apurinic sites.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with high carcinogenic potencies that have been linked to the etiology of human cancers through their presence in cigarette smoke and environmental mixtures. They are metabolically activated in cells by cytochrome P450 enzymes and/or peroxidases to reactive intermediates that damage DNA. One pathway of activation forms dihydrodiol epoxides that covalently bind to exocyclic amino groups of purines in DNA to form stable adducts. Another pathway involves formation of radical cations that bind to the N7 or C8 of purines to form unstable adducts that depurinate to leave apurinic (AP) sites in DNA. In the present study the proportions of stable DNA adducts and AP sites formed by the carcinogenic PAHs dibenzo[a,l]-pyrene (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P) have been investigated in a target tissue for carcinogenesis, mouse epidermis. After topical application of the PAHs on the skin of female SENCAR mice epidermal DNA was isolated and the formation of stable DNA adducts was measured by (33)P-postlabeling and HPLC analysis. AP sites in DNA were measured with an aldehyde reactive probe in a slot-blot assay. At both 4 and 24 h after exposure, DB[a,l]P formed significantly higher amounts of stable DNA adducts than DMBA, and B[a]P exhibited the lowest level of binding. In contrast, the number of AP sites present in mice treated with these PAHs was in the order: DMBA > B[a]P >> DB[a,l]P. The level of AP sites was significantly lower than the level of stable adducts for each PAH. The most potent carcinogen, DB[a,l]P, induced the highest level of stable adducts and the lowest level of AP sites in epidermal DNA. These results indicate that stable DNA adducts rather than AP sites are responsible for tumor initiation by carcinogenic PAHs.

Effects of vanadate on expression of liver arginase in experimental diabetic rats.

This work was carried out to study the effects of vanadate on the expression of liver-type arginase in experimentally induced diabetes in the rat. The results showed that the activity and mRNA levels of arginase were increased significantly in the diabetic condition. Vanadate treatment reversed the increased activity and restored mRNA levels of arginase almost to the control values. The reversal effects of vanadate were found to be similar to those of insulin, and this further confirms the insulin-like effects of vanadate. ELISA and slot-blot assay observations were consistent with the biochemical measurements of enzyme activity. The increase in arginase activity and mRNA levels in diabetes and the decrease in insulin- and vanadate-treated animals may be due to the transcriptional regulation of arginase.

Dissociation of surfactant protein B from canine surfactant large aggregates during formation of small surfactant aggregates by in vitro surface area cycling

Pulmonary surfactant isolated by lavage can be separated into large aggregates (LA) and small aggregates (SA). Pulse labeling experiments have shown that the LA subtype is the precursor of the SA subtype. Conversion of LA to SA can be demonstrated in vitro using the technique of surface area cycling. The precise mechanisms of surfactant subtype conversion remain unknown. We have previously reported a decline in surfactant-associated protein B (SP-B) during in vitro subtype conversion of canine surfactant. This led to the hypothesis that SP-B may be degraded by a serine protease ‘convertase’ during cycling. The current studies used a quantitative slot-blot assay to investigate the fates of SP-A and SP-B during in vitro cycling. These studies confirmed some SP-A is present in SA, but SP-B is confirmed to LA. Conversion leads to an apparent loss of SP-B during cycling. However, SP-B can be recovered from the walls of polypropylene and Teflon tubes by washing with chloroform:methanol. Recovered SP-B migrated on non-reducing tricine gels as a single band with an apparent molecular weight of 17 kDa, corresponding to intact SP-B dimer. Reconstitution studies demonstrated that the recovered SP-B retained its surface active properties as determined on a pulsating bubble surfactometer. We conclude in vitro surface area cycling of canine LA results in the dissociation of SP-B from surfactant lipids resulting in an apparent decline in SP-B levels.

Genome plasticity during the acquisition of embryogenic competence

Hypocotyl explants from carrot and other species experience concomitant segregation events and differentiation of homeotic structures during the first 20 days of culture on 2,4-dichlorophenoxyacetic acid (2,4-D). In addition to these cyto-morphological changes, significant amounts of nuclear DNA are lost, the molecular details of which we investigate in this paper. We have developed a slot-blot analysis assay to study the DNA content of a series of carrot samples; besides the leaves, this survey ranged over different culture timepoints: hypocotyls, cell lines, and somatic embryo stages. We carried on to study the relationship between this DNA loss and sequence complexity modulation. Results from probing sequences that correspond to different degrees of complexity, such as medium repetitive and unique sequences as well as sequences belonging to both classes (ribosomal cistrons, ubiquitin, actin, and chalcone synthase), consistently manifested a reduction in DNA levels during the acquisition of embryogenic competence. In some cases, the cultured cells would contain only 10% of the gene copies observed in the reference tissues. Modulation trends also showed that DNA levels of most sequences recover at the torpedo-plantlet stage, which again correlates DNA modulation and the acquisition of embryogenic competence. These results suggest that similar DNA variations may occur in plants in vivo during meiosis, possibly so that meiotic division may be properly completed.

Specific detection of native multi-subunit proteins by slot-blot assay.

Specific detection of native multi-subunit proteins by slot-blot assay.

Prolonged depletion of AH receptor without alteration of receptor mRNA levels after treatment of cells in culture with 2,3,7,8-tetrachlorodibenzo-p-dioxin

Previous experiments have shown that the total cellular content of the AH receptor (AHR) drops rapidly after exposure of mouse hepatoma cells (Hepa-1) to the potent AHR ligand 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD); within 6 hr after treatment, less than 20% of the original cell content of AHR can be detected by radioligand binding or by immunoblotting. The goals of our current study were to determine the duration of receptor depletion following treatment with ligand and to determine if depletion is due to decreased expression of the Ahr gene that encodes the AHR. We found that depletion of AHR persisted for at least 72 hr after exposure to TCDD. Treatment with 3-methylcholanthrene caused a transient drop in total cell AHR, but the AHR levels returned to near pretreatment levels within 72 hr after the first exposure. TCDD treatment did not alter the levels of AHR mRNA as assessed by reverse transcription-polymerase chain reaction or slot blot assays. Thus, the decrease in AHR protein cannot be attributed to depression of transcription of the Ahr gene by TCDD. TCDD treatment did not alter the levels of the dimerization partner of the AHR, the AH receptor nuclear translocator protein (ARNT), or ARNT mRNA. In the presence of TCDD, both the AHR and the ARNT protein can be maintained at high levels in the nucleus if transcription is inhibited with actinomycin-D. In the absence of actinomycin-D, the AHR protein was lost rapidly, but the ARNT protein level in the cell was maintained. Together, these results suggest that the AHR protein is degraded through a selective mechanism that spares the ARNT protein and that the degradation pathway involves a protein that itself has a short half-life.

The use of DIG-labelled cRNA probes for the detection of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) in orchids

DIG-labelled sense and antisense cRNA probes were synthesized from cDNA clones of CymMV and ORSV for virus detection in infected plants. A slot-blot hybridization assay was developed using either crude leaf extracts or total RNA from infected leaves. The assay could detect 50 and 250 pg of purified CymMV and ORSV RNA, respectively. As little as 30 mg of Nicotiana benthamiana infected leaves was sufficient to provide positive detection. CymMV and ORSV were detected at 3125 and 625 times dilution of leaf extracts, respectively. The DIG-labelled cRNA probes are stable for more than a year. This method is sensitive, reliable and suitable for large-scale routine testing of plant viruses. By using the two DIG-labelled cRNA probes in situ, CymMV and ORSV were localized in systemically infected leaves and stems of N. benthamiana and orchids.

Identification of a heparin-releasable hepatic lipase binding protein from rat liver.

Hepatic lipase (HL) plays a key role in the metabolism of several lipoproteins. Metabolically active HL is bound in liver parenchymal cells to specific binding sites. We studied the nature of the HL binding in rat liver. Rat livers were perfused with heparin, which lead to a loss of 80% of the HL binding capacity of the liver. The heparin-containing perfusates possessed HL binding capacity, determined by slot-blot assay. The perfusates were loaded on to a heparin-Sepharose column and eluted with a linear salt gradient (0.2-1 M). HL binding activity, assessed by a slot-blot binding assay, eluted both at 0.3 M and at 0.8 M NaCl. A 0.5 M NaCl eluate was used to further characterize the HL binding activity. In this fraction the major protein had a molecular mass of 70 kDa. The fraction showed saturable HL binding in a solid-phase binding assay. Cross-linking of the 0.5 M NaCl fraction to 125I-labelled HL yielded a complex of 130 kDa, suggesting the cross-linking of the 57 kDa 125I-labelled HL to a protein of about 73 kDa. We concluded that heparin releases a protein of about 73 kDa from rat liver, which associates with HL. This protein may represent the HL binding site in liver.

Correlation of take-all disease severity and inoculum level of Gaeumannomyces graminis var. tritici using a slot-blot hybridization assay

The amount of Gaeumannomyces graminis var. tritici inoculum was quantified by slot-blot hybridization assay, using a specific DNA probe, pG158. Estimates of the amount of G. graminis var. tritici DNA in naturally infested and inoculated soils were made and correlated with take-all disease severity as determined by a conventional soil bioassay (expressed as a percentage of seminal roots with G. graminis var. tritici lesions). Disease severity in the glasshouse assay and DNA levels were highly correlated. The level of take-all disease severity in naturally infested soil can be predicted using the equation y i = 53.078 log ( x i ) + 88.866, where y i = the predicted value of the disease severity (%) and x i = amount of G. graminis var. tritici DNA (ng) in 100 g soil. The DNA-based assay for quantifying the amount of G. graminis var. tritici has been further developed and improved in terms of the sampling techniques and efficiency. Soils of different types were collected to a depth of 10cm and organic matter between 0.5—1.4 mm in size was separated from this soil. Using a slot-blot hybridization assay, it was found that G. graminis var. tritici was mostly present in the organic matter fraction greater than a size of 0.5 mm and in soil at a depth of 5 cm. This rapid and reliable DNA-based assay is being used in conjunction with a model of take-all disease development to predict disease levels in the field due to G. graminis var. tritici .

Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a primary regulator of the initiation of blood coagulation. TFPI is internalized and degraded by HepG2 cells through the low-density-lipoprotein receptor-related protein (LRP) but also binds another molecule present on the cell surface at approx. 10-fold the abundance of LRP [Warshawsky, Broze and Schwartz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668]. When HepG2 cells are washed with heparin or dextran sulphate, a substance that binds TFPI is removed from the cell surface and can be detected in a slot-blot assay. Preincubation with trypsin destroys the reactivity of the TFPI-binding component in the slot-blot assay, suggesting that it is a protein. In addition, when the sulphation of glycosaminoglycans (GAGs) is prevented by growing the HepG2 cells in the presence of 30 mM sodium chlorate, TFPI binding is unaffected, whereas the binding of bovine lipoprotein lipase, a protein known to associate with cell-surface GAGs, falls to 50% of control levels. Dextran sulphate washes of HepG2 cells grown in sodium chlorate have an equal reactivity in slot-blot experiments to that of non-treated cells, suggesting that GAGs are not totally responsible for the binding activity observed. By using the slot blot to follow binding activity and conventional protein purification techniques, a protein species that migrates at 40 kDa after reduction was identified in the HepG2 cell wash. The binding of this protein to TFPI was confirmed with immobilized TFPI. Amino acid sequence analysis identified this protein species as a proteolytic fragment of glypican-3 (also called OCI-5), a member of the glypican family of glycosylphosphatidylinositol-anchored proteoglycans.

Quantification of Gaeumannomyces graminis var. tritici in infected roots and soil using slot-blot hybridization

A slot-blot hybridization assay for quantifying Gaeumannomyces graminis var. tritici, in diseased roots and in soil, using a specific and sensitive DNA probe (pG158), has been developed. The pG158 probe hybridized strongly to pathogenic isolates of G. graminis var. tritici, moderately to G. graminis var. avenae and did not hybridize to non-pathogenic G. graminis var. graminis and to a wide range of other soil fungi. The pG158 contains a dispersed highly repeated sequence which provides the high level of sensitivity needed for a quantitative diagnostic test. The amount of Gaeumannomyces graminis var. tritici in soil can be estimated directly from the test soil without growing the susceptible plants or culturing the fungus. pG158 detects approximately 1·0 ng and 0·3 ng of G. graminis var. tritici DNA in 1 μg total DNA extracted from diseased roots and from highly infested soil organic matter, respectively. Both the high copy clone, pG158 and a single copy clone, pG217, clearly distinguished the three varieties of G. graminis and can be used for accurate intra-specific classifications of G. graminis isolates. The pG158 probe provides a tool for field researchers to determine levels of pathogenic isolates of G. graminis in soils and to study the ecology of this fungus.