Slot-blot Assay Research Articles

Heparin-binding capacity of the HIV-1 NEF-protein allows one-step purification and biochemical characterization.

Recombinant Nef-protein of HIV-1 Bru derived from Escherichia coli revealed heparin-binding activity. This property was used to purify the Nef-protein by a one-step procedure, yielding about 90% homogenous Nef-protein as evaluated by silver staining. The Nef-protein was soluble without denaturing agents. Native folding of Nef was demonstrated with antibodies against conformational epitopes of Nef by a slot blot assay under native conditions. Despite its affinity to heparin and its nuclear localization in persistently HIV-1 infected glioblastoma cells (Kohleisen et al., 1992), Nef did not show DNA-binding properties by slot blot/hybridization assay and South/Western blot. In nucleotide-binding assays a strong autophosphorylation activity with [gamma-32P]ATP was observed. Nef-protein was not a substrate for ADP-ribosylation by bacterial toxins arguing against G-protein-like activities of Nef. Recombinant Nef did not interact with membranes as shown by the lack of increased fluorescence emission of Nef in the presence of liposomes. The recombinant Nef-protein obtained by one-step heparin-based purification shares immunological properties with native Nef and should prove useful for further studies of Nef function and immunogenicity.

Polyadenylated RNA, actin mRNA, and myosin heavy chain mRNA in young and old human skeletal muscle.

The myofibrillar protein synthesis rate in old human skeletal muscle is slower than that in young adult muscle. To examine whether this difference in protein synthesis rate is explained by reduced availability of the mRNAs that encode the most abundant myofibrillar proteins, we determined relative hybridization signals from probes for actin mRNA, myosin heavy chain mRNA, and total polyadenylated RNA in vastus lateralis muscle biopsies taken from young (22- to 31-yr-old) and old (61- to 74-yr-old) human subjects. The mean fractional rate of myofibrillar synthesis was 38% slower in the older muscles, as determined by incorporation of a stable isotope tracer. Total actin and myosin heavy chain mRNAs, and polyadenylated RNA, were determined using slot-blot assays. Isoform-specific determinations of alpha-actin mRNA, type I myosin heavy chain mRNA, and type IIa myosin heavy chain mRNA were done with ribonuclease protection assays. Hybridization signals were expressed relative to tissue DNA content. There was no difference between age groups in total polyadenylated RNA or in any of the specific mRNAs. We conclude that the slower myofibrillar synthesis rate in older muscle is not caused by reduced mRNA availability.

Quantitation ofPhytophthora cinnamomiin Avocado Roots Using a Species-Specific DNA Probe

The proliferation of Phytophthora cinnamomi in avocado roots was quantified with a sensitive, species-specific DNA probe. The probe was isolated from a library of genomic DNA by identifying colonies that strongly hybridized to DNA of P. cinnamomi but not to DNA from other species of Phytophthora. The characterization of five such clones indicated that each detected the same repetitive sequence that was present in genomic DNA at about 12,000 copies per haploid genome. Cross-hybridizing sequences were absent from DNA of other Oomycetes, bacteria, ascomycetes, basidiomycetes, or plants. The probes detected as little as 5 pg of P cinnamomi DNA in dot- and slot-blot assays. The P. cinnamomi probe and a probe for avocado DNA were used to quantitate the growth of the pathogen in roots. The extent of colonization, judged by measuring the relative amounts of pathogen and host DNA, increased over time and with increasing amounts of inoculum.

Detection of Rhizoctonia solani AG-8 in soil using a specific DNA probe

A reliable method of detecting low levels of AG-8 isolates of Rhizoctonia solani in soil samples using the specific DNA probe, pRAG12, has been developed. The procedure involves separation of particulate organic matter from soil by flotation and sieving followed by extraction of DNA from a minimum of 0·1 g of the air dried material. Further purification through a Sepharose CL-6B column is required to remove the humic substances before AG-8 DNA can be measured in a slot blot assay. The procedure gives yields of DNA in excess of 80%. In addition, the DNA is pure enough to be used, without dilution, for RFLP analysis as well as DNA amplification using the PCR. The specificity and high copy number of the AG-8 probe and the rapid DNA purification procedure provide the means for a sensitive diagnostic test for R. solani in the soil and for fundamental studies on the biology of the fungus.

Domain-III Exchanges of Bacillus thuringiensisCryIA Toxins Affect Binding to Different Gypsy Moth Midgut Receptors

Aminopeptidase-N, purified from gypsy moth (Lymantria dispar L.) brush border membrane vesicles, exhibited specific binding to CryIAc toxin but not to CryIAa toxin. CryIAa-CryIAc hybrid toxins were used to localize the aminopeptidase-N binding region on CryIAc. Slot blot assays and ligand blot experiments demonstrated that the hybrid toxins which have the residues 451 to 623, comprising essentially domain III, from CryIAc toxin exhibited strong binding to purified aminopeptidase-N and 120 kDa brush border membrane protein. In contrast, the hybrid toxins which have the residues 451 to 623 from CryIAa toxin failed to bind to aminopeptidase-N, but did bind to another receptor, a 210 kDa protein. This is the first direct evidence that domain III is involved in receptor binding and the first to demonstrate that domain III substitutions direct the binding of these toxins to different gypsy moth midgut receptors.

Topical Tretinoin Increases the Tropoelastin and Fibronectin Content of Photoaged Hairless Mouse Skin

Topical tretinoin treatment of photoaged hairless mice has been shown in previous studies to stimulate formation of a subepidermal zone of new connective tissue characterized by enhanced collagen synthesis. The aims of this study were to localize and/or quantify elastin, fibronectin, and glycosaminoglycans in the same model. Hairless mice (Skh-1) were irradiated thrice weekly for 10 weeks with gradually increasing doses of ultraviolet (up to 4.5 minimal erythema doses per exposure) from Westinghouse FS-40 bulbs. Mice were then treated five times a week with either 0.05% tretinoin, the ethanol:propylene glycol vehicle, or nothing for another 10 weeks. Controls included mice sacrificed after 10 weeks of ultraviolet treatment and age-matched untreated animals. The distribution of elastin and fibronectin was examined by immunofluorescence microscopy, which revealed fine fibrils in the subepidermal zone in tretinoin-treated skin. A quantitative slot-blot immunobinding assay showed that tretinoin induced a threefold higher amount of tropoelastin compared with controls. Insoluble elastin content (desmosine levels) was similar in all groups. Although fibronectin content was increased by ultraviolet radiation, tretinoin treatment induced the largest increase. In contrast, the amount of glycosaminoglycans, although increased by UVB radiation, was reduced by tretinoin treatment.

Differential Polymerase Chain Reaction Assay of Cyclin Dl Gene Amplification in Esophageal Carcinoma

The cyclin D1 gene, located on chromosome 11q13, is frequently rearranged in parathyroid neoplasms and amplified in some carcinomas of other organs. Recent studies have detected amplification of cyclin D1 and other markers on chromosome 11q13 (evaluated by Southern or slot blot assays) in approximately 25-50% of squamous cell carcinomas of the esophagus and noted that amplification was associated with lessened survival time. We applied the technique of differential polymerase chain reaction to the evaluation of cyclin D1 gene amplification in squamous cell carcinomas of the esophagus. Cyclin D1 was found to be amplified in 10 of 45 (22%) primary tumors and three of 12 (25%) lymph node metastases. Lymph node metastases tended to be more common in patients with cyclin D1 amplification (70%) than in those without amplification (37%). In 36 patients with follow-up, cyclin D1 amplification was associated with decreased 1 year survival (28% vs. 59%). Cyclin D1 gene amplification in esophageal carcinomas can be evaluated by differential polymerase chain reaction and may provide useful prognostic information.

Detection of serum hepatitis B virus using a nested polymerase chain reaction assay

We described a nested polymerase chain reaction (PCR) assay to detect serum hepatitis B virus (HBV) DNA in patients. We compared the sensitivity of the PCR assay to that of slot-blot hybridization for detecting HBV DNA in serum and found that analysis by PCR provides a > 10 4 fold increase over the slot-blot assay. Most of the HBe Ag-positive patients were positive in PCR, and most of those being anti-HBe-positive were with this PCR assay.

Identification of a higher molecular weight protein that shows apparent cross-reactivity with anti-p21 ras monoclonal antibodies on Western blots

We have characterized the immune cross-reactivity of several commercially available monoclonal antibodies prepared against the p21 ras proteins and used in Western blotting experiments against human tissue homogenates. Under optimal conditions, only two bands were observed on Western blots. One of these comigrated with control p21 ras protein. A second protein of apparent mobility corresponding to approximately 54 kDa was also observed with all four monoclonal antibodies tested. Protein sequencing by automated Edman degredation indicates that the 54 kDa species corresponds to human immunoglobulin heavy chain. Under suboptimal conditions, another high molecular weight species of apparent mobility 65 kDa was also observed to cross-react with some of the monoclonal antibodies tested. This 65 kDa species was identified by protein sequencing as human serum albumin. Coomassie blue staining of SDS-polyacrylamide gels indicates that serum albumin is a major contaminant of many surgically obtained human tissue samples, while p21 ras and immunoglobulin heavy chain are present at much lower concentrations. These results may be of significance when using monoclonal antibodies to determine p21 ras levels of whole tissue homogenates by dot-blot, slot-blot or microplate assays.

Affinity ranking of influenza neuraminidase mutants with monoclonal antibodies using an optocal biosensor comparison with ELISA and slot blot assays

A recently developed alternative to the more traditional techniques for studying antigen-antibody interactions has been examined. This method involves the use of an optical biosensor employing surface plasmon resonance detection. In this system one of the reactants is immobilized on the sensor surface and other reactants are passed over the sensor surface sequentially at a constant flow rate. Binding phenomena are detected in real time from changes in the angle at which surface plasmon resonance occurs. This is dependent, among other things, on changes in the refractive index (which is directly proportional to the mass) at or near to the sensor surface. Applications of this biosensor technique for comparing the binding of related neuraminidases, purified from escape mutants of influenza virus NWS/G70C/75 (N9), to two antibody Fab fragments, are described. These results were compared with those obtained from ELISA and slot blot assays on the same neuraminidases interacting with the same two monoclonal antibodies. The biosensor method was shown to be highly specific, permitting rapid screening of binding in such antigen-antibody systems.

Comparison of Nucleic Acid Hybridization Assays and Biochemical Characterization Tests for the Confirmation of Listeria monocytogenes

A comparison was made of various biochemical and nucleic acid hybridization assays used for the confirmation of Listeria monocytogenes. Identification and confirmation tests included traditional carbohydrate utilization tests, several types of hemolysis assays, an immunocompromised mouse-pathogenicity test, the API® LISTERIA identification system, the Biolog GP MicroPlate™, a prototype Biolog Listeria MicroPlate™, and two nucleic acid hybridization assays (a slot-blot assay and the commercially available AccuProbe™L. monocytogenes culture confirmation test). A panel of 44 Listeria isolates, including representatives from all six existing Listeria species, was tested. A considerable number of the L. monocytogenes isolates tested gave ambiguous reactions on the CAMP-hemolysis assay. In many instances, the Biolog GP MicroPlate™ was unable to differentiate L. monocytogenes from other Listeria species. Reactions on the prototype Listeria MicroPlate™ mirrored those of traditional biochemical tests, but additional analyses were necessary to differentiate between L. monocytogenes and Listeria innocua. The API® LISTERIA identification system could readily differentiate between the latter two Listeria species without the need for additional tests. Atypical L. monocytogenes isolates (nonhemolytic and nonpathogenic or hemolytic and nonpathogenic) were correctly identified by the API® LISTERIA system. A perfect correlation was observed between results from the slot-blot format hybridization assay and the AccuProbe™ test. The probes used in the two assays hybridized with all L. monocytogenes isolates, regardless of hemolytic reaction or mouse pathogenicity. The commercially available assays examined in this study were all easy to use and capable of providing results within 24 hours.

An Oligonucleotide Probe That Distinguishes Isolates of Low Virulence from the More Pathogenic Strains of Newcastle Disease Virus

A synthesized oligonucleotide, termed cleavage probe (NDV-CL), has been designed to complement the cleavage-activation site of the fusion gene of the Texas GB isolate of Newcastle disease virus (NDV). This oligonucleotide probe, 21 bases in length, bound with RNA from velogenic strains of NDV tested in a slot-blot hybridization assay. The probe also recognized RNA from the mesogenic strains used in this assay, although no signal was observed with RNA isolated from lentogenic NDVs or with that from other common avian viruses used as controls. This probe did not recognize RNA from isolates of other paramyxovirus serotypes (PMV-2 or PMV-3) included in this study. The ability of this probe to distinguish lentogenic NDVs, which cause little or no clinical disease, from those strains that may produce severe morbidity and/or mortality suggests a potential use for the probe in a molecular diagnostic assay.

Chemiluminescent detection of infectious bursal disease virus with a PCR-generated nonradiolabeled probe.

A polymerase chain reaction (PCR)-generated digoxigenin-labeled nonradioactive oligonucleotide probe was developed and utilized in slot-blot hybridization coupled with chemiluminescence for the detection of infectious bursal disease virus (IBDV). The probe was prepared from the RNA of the standard challenge strain (STC) of IBDV serotype 1 by reverse transcription followed by 2 PCR amplifications with 2 separate sets of primers. RNA of STC viruses prepared from bursae infected with STC viruses was subjected to the first PCR with the outer primers V8 and V9 that amplified a 607-base pair (bp) segment. The PCR product from the first PCR was eluted following agarose gel electrophoresis and subjected to the second PCR with the nested primers V6 and V7 that flanked a 351-bp segment. In the second PCR, dTTP was substituted by digoxigenin-11-dUTP in the PCR reaction mixture so that the amplified 351-bp DNA products were labeled with digoxigenin. The specificity of the PCR-generated digoxigenin-labeled probe was tested on different strains of IBDV, several unrelated avian viruses, and bacteria by slot-blot hybridization assay. Hybridization was detected by chemiluminescence. The sensitivity of the probe was assayed using 10-fold serial dilutions of purified RNA from the STC strain of IBDV. The PCR-generated digoxigenin-labeled probe hybridized with genomic RNA of STC and variant strains A, D, E, G, and GLS-5 of IBDV serotype 1 but not OH strain of IBDV serotype 2. The probe did not react with avian reovirus, infectious bronchitis virus, Salmonella enteritidis, Escherichia coli, or Staphylococcus aureus.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related changes in sarcoplasmic reticulum Ca(2+)-ATPase and alpha-smooth muscle actin gene expression in aortas of normotensive and spontaneously hypertensive rats.

The expression of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase gene and the SR Ca2+ pump function were investigated in thoracic aortas of 5- and 17-week-old normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). The relative level of the two isoforms of SR Ca(2+)-ATPase mRNA expressed in the aorta (i.e., SERCA 2a and SERCA 2b) was determined by quantitative S1 nuclease protection analysis and normalized to the level of alpha-smooth muscle (alpha-Sm) actin mRNA. The level of alpha-Sm actin mRNA itself was normalized to the level of 18S ribosomal RNA using slot-blot hybridization assays. Total SR Ca2+ pump activity was estimated by measuring the rate of oxalate-supported Ca2+ uptake in homogenates. At 5 weeks, the amount of SERCA 2a and SERCA 2b mRNA, normalized to 18S ribosomal RNA, and the ratio of alpha-Sm actin mRNA to 18S RNA were identical in SHR and WKY rats. The Ca2+ pump activity was similar in the two strains of rats at 5 weeks. From 5 to 17 weeks, the amount of SERCA 2a mRNA increased in both strains while the level of SERCA 2b mRNA remained constant. The Ca2+ pump activity was unchanged in SHRs and tended to decrease in WKY rats. Accordingly, the change in the ratio of the SR Ca(2+)-ATPase mRNA isoforms does not appear to influence SR function. The level of alpha-Sm actin mRNA and SERCA 2a mRNA increased in parallel from 5 to 17 weeks in both strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Steroid hormone receptor immunohistochemistry and amplification of c-myc protooncogene. Relationship to disease-free survival in breast cancer.

It is important to develop parameters that aid in prognosticating which patients with breast cancer are more likely to have a rapid disease course and therefore might benefit from early aggressive therapies. Specimens from two groups of women, deliberately selected because their clinical courses differed greatly, were studied to detect amplification of the protooncogenes c-myc, int-2, and C-erbB-2/neu by slot-blot assay, the estrogen receptor (ER), and the progesterone receptor (PR) by both biochemical and immunohistochemical procedures (ERICA and PRICA). One group of 50 patients had a prolonged disease-free interval after initial surgery (mean, 6.4 years); the other group of 52 women had had rapid disease recurrence (mean, 1.4 years) or progression (5 patients died of disease within 1 year of diagnosis). The patients were selected from 1700 consecutively accessioned cases if they fit the study criteria and sufficient tissue was available for oncogene hybridization studies. The two groups differed statistically by stage, number of involved axillary lymph nodes, ERICA and PRICA results (P = 0.001), and amplification of c-myc (P = 0.003). The percentage of patients with rapid disease recurrence and progression increased from 0-93% when risk factors changed from best case (ERICA and PRICA results, positive; c-myc, not amplified; and axillary nodes, not involved) to worst case (ERICA and PRICA findings, negative; c-myc, amplified; and axillary nodes, involved). Women with these worst-case parameters were more likely to have a recurrence sooner and rapidly progressive disease. They might benefit from early aggressive therapeutic measures.

Chemical Modification of Alpha Crystallin

Calf lens α-crystallin was isolated and the lysine residues were extensively modified with a variety of chemical agents. The effect of these modifications on elastase inhibitor activity, apparent molecular size, antibody reactivity and solubility were determined. The addition of either a methyl group or a threose residue did not alter the charge on the lysine residues and had little or no effect on either inhibitor activity or apparent molecular size. The introduction of a negative charge by either carboxymethylation or citraconylation caused a marked decrease in size and an almost complete loss of inhibitor activity. The introduction of a hydrophobic residue by reaction with either a trinitrobenzenesulfonic acid or Bolton Hunter reagent caused a slight increase in size, but a 70% increase in elastase inhibitor activity. Reaction with fluorescamine resulted in the dissociation of α-crystallin in a 200-kDa species, yet caused a two to four-fold increase in elastase inhibitor activity, which was similar to the activity of the water-insoluble fraction isolated from aged human lens and cataract.Several of these modified α-crystallins were compared for reactivity with a polyclonal α-crystallin antiserum using a quantitative slot blot assay. Charge neutral modifications resulted in a two to three-fold loss of antibody recognition, whereas the other preparations showed an almost complete loss of antigenic activity. None of the modifications caused the α-crystallin to precipitate at higher salt concentrations (0·3 M) with the exception of threose which caused a 30% decrease in soluble protein. Therefore, none of the modifications caused a significant aggregation of α-crystallin, and glycation, as opposed to hydrophobic modifications, decreased the solubility of α-crystallin at high salt concentrations.

Selective binding of anchorin CII (annexin V) to type II and X collagen and to chondrocalcin (C-propeptide of type II collagen) Implications for anchoring function between matrix vesicles and matrix proteins

Anchorin CII is a collagen binding protein of the annexin family associated with plasma membranes of chondrocytes, osteoblasts, and many other cells. As a major, constituent of cartilage-derived matrix vesicles it has been shown to bind to native type II and X collagen. In accordance with this observation, here we show the localization of anchorin CII in the extracellular matrix of calcifying cartilage in the fetal human growth plate, and that it was restricted to the chondrocyte surface in proliferating and resting cartilage. Furthermore, we present evidence, using a slot blot assay, that anchorin CII not only binds to native type II and X collagen, but also to chondrocalcin, the carboxy-terminal extension of type II procollagen in a calcuim-independent manner, Pepsin digestion of type II collagen results in loss of anchorin CII binding, confirming our previous notion that the telopeptide region of type II collagen carries anchorin CII binding sites.

Dietary Selenium Stabilizes Glutathione Peroxidase mRNA in Rat Liver

The objective of these studies was to determine the step at which dietary selenium (Se) regulates the pre-translational expression of the gene for cytosolic Se-glutathione peroxidase (Se-GPX) in rat liver. Weanling male Sprague-Dawley rats were fed a Torula yeast-based Se-deficient diet, or the same diet supplemented with 0.5 mg/kg as Na2SeO3, for at least 40 d. Livers were excised and divided into three portions for isolation of nuclei, for purification of total RNA and for assay of Se-GPX activity. Nuclei were used in run-on transcription assays and for isolation of total nuclear RNA. Nuclear RNA and total liver RNA were probed with segments from a rat Se-GPX cDNA in Northern blot and slot blot assays. Despite marked differences in Se-GPX activity, there were no significant differences between dietary groups in the transcription rates of the Se-GPX gene. Species hybridizing to Se-GPX were not detected in nuclear RNA. However, steady state levels of Se-GPX mRNA were markedly reduced by Se deficiency. These results suggest that dietary Se exerts its effect on pretranslational Se-GPX gene expression at the level of cytosolic mRNA stabilization.

A Novel Oligonucleotide Probe for the Detection of Newcastle Disease Virus

An approach that possesses high specificity and broad applicability was used to obtain a DNA probe with potential diagnostic value. By utilizing synthesized oligonucleotide DNA, termed NDV probe, all 14 strains of NDV tested under high-stringency conditions were recognized in a slot-blot hybridization assay. The sequence of the NDV probe was generated from a highly conserved region of the NDV genome. No hybridization was observed with RNA isolated from other avian viruses, including avian influenza, infectious bursal disease, and infectious bronchitis. The specificity inherent in using an oligonucleotide probe offers advantages over probes obtained from cloned DNA fragments.

Evaluation of a DNA probe of plasmid origin for the detection of Chlamydia trachomatis in cultures and clinical specimens

This study evaluates five cryptic plasmid-derived DNA probes in a 4-h slot-blot hybridization assay for the detection of Chlamydia trachomatis in cultures and clinical specimens. The probes, consisting of either the entire cloned 7.5 kbp cryptic plasmid pSE8 or one of four Hin dIII/Eco RI fragments measuring 710, 1055, 710, and 500 bp, respectively, were labelled with Photoprobe biotin. The probe was detected using a streptavidin-alkaline phosphatase conjugate followed by addition of BCIP and NBT. The sensitivity of the assay, using 25 ng of probe DNA, ranged from 50 pg (with the entire plasmid as probe) to 5 ng (with the 500 bp fragment as probe). A total of 103 reference strains of Chlamydia trachomatis and other bacteria were tested for reactivity with the probes. All 18 reference strains of C. trachomatis hybridized with the probes. None of the DNA from the heterologous organisms tested was found to hybridize with any of the probes. A total of 174 samples taken from patients visiting the STD clinic at the University Hospital, University of Seville were used in the study. The overall sensitivity of the assay, using the 710 bp biotinylated probe was 94.5% compared to culture while the specificity was 97.5%. Positive and negative predictive values were 96.5% and 97.5%, respectively. It appears that the plasmid-derived probes used in this study could serve as useful tools for the rapid and specific detection of Chlamydia trachomatis in cell cultures and clinical specimens.