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M Ranki

Publications and source records attributed to M Ranki.

At least 37 records · Page 2Linked to original sources
PubMed1986

Time-resolved fluorometry: a sensitive method to quantify DNA-hybrids.

Europium and other lanthanides can be excitated with UV-radiation, whereafter the energy is released as fluorescence, delayed in time up to 1 ms after the excitation. Eu can be used as a sensitive label in biological assays. Here we report on the application of time-resolved fluorometry to detect nucleic acid hybrids. The probe DNA was tagged with a hapten, either a fluorene or a sulfone group. After hybridization the probe DNA was detected by a two-step immunological assay with the second antibody labelled with Eu. The method is quantitative with a detection limit of 0.3 pg of actual target regions of immobilized adenovirus genomic DNA. The label was also used in sandwich hybridization, which allowed analyzing nasopharyngeal mucus for the presence of adenovirus.

2-Acetylaminofluorene↗
PubMed1986

Detection of human papillomavirus DNA by the nucleic acid sandwich hybridization method from cervical scraping.

Cervical scrapes collected from 100 consecutive patients participating in a prospective follow-up study for cervical human papillomavirus (HPV) infections were tested for the presence of HPV 11 DNA by the nucleic acid sandwich hybridization method, which allows testing the specimens in a crude form. Part of each specimen was processed through phenol extraction and DNA purification to a dot blot hybridization assay. The dot blots were serially hybridized with HPV 6, 11, 16, and 18 probes as well as with an Alu-repeat probe to estimate the number of cells in the specimen. In PAP smears, HPV-infection was suspected in 63 patients whereas in 37 patients the smear was negative. In the first group, the dot blot assay revealed three cases of HPV 11, two of HPV 16, and one of HPV 18 infection. In the second group with normal PAP smear, one additional HPV 18 infection was found. The sandwich hybridization assay detected 5 HPV 11 infections, including the three mentioned above. All HPV DNA-positive samples contained at least 1.6 X 10(6) cells. Since we considered this a prerequisite for successful diagnosis, only 25 specimens in the first group and 15 in the second were adequate specimens. Thus the HPV-DNA detection rate was 32% (8/25) in the first group and 1/15 in the second. This study demonstrates that sandwich hybridization, detecting 1-3 X 10(5) HPV 11 molecules is a reliable diagnostic method. Cervical scrape is a valuable alternative to punch biopsy, but the number of cells collected is critical for the outcome of the assay.

Cervix Uteri↗
PubMed1986

Serological evidence for the role of bacterial infections in the pathogenesis of thyroid diseases.

Subacute thyroiditis is generally believed to be of viral origin, and infection is also suspected of playing a role as a triggering factor in the pathogenesis of autoimmune thyroid diseases. We have measured a broad spectrum of bacterial and viral antibodies in paired sera of 32 patients with thyroid disease of recent onset. The data indicate a preceding infection in 14 (44%) of the patients, enterobacterial in 5, streptococcal in 4 and staphylococcal in 2. A viral infection was suggested in 6 patients, in each case caused by different agents; 3 of them also showed evidence of a bacterial infection. Patients with positive microbial serology were found in all diagnostic groups, including subacute thyroiditis, Graves' disease and Hashimoto's disease. These results suggest an association between a preceding bacterial infection and the development of thyroid disease in some patients.

Adult↗
PubMed1986

Rapid diagnosis of respiratory adenovirus infections in young adult men.

Rapid viral diagnosis was attempted in 106 military conscripts with pneumonia and in 101 military conscripts with other types of respiratory infections. Nasopharyngeal suction specimens (NPS) were assayed for viral antigens by immunofluorescence and enzyme immunoassay (EIA). Sputum specimens from 97 pneumonia patients were assayed for viral antigens by EIA. Also, 71 NPS and 13 sputum specimens were examined for the presence of adenovirus DNA by a sandwich hybridization (HYB) method. The reference test was adenovirus isolation in cell culture from the NPS. Adenoviruses were isolated from 6 pneumonia patients and from 20 patients with other respiratory infections. Of these 26 NPS, rapid diagnosis was successful in 13, 16, and 14 cases by EIA, immunofluorescence, and HYB, respectively. Four antigen-positive specimens were found among the 181 specimens which were negative by virus isolation. Sputum was found to contain adenovirus antigen by EIA in 5 of 97 tested specimens. Of these 97 specimens, 13 were selectively tested in HYB, and a positive signal was observed in 4 cases. Serological testing of paired sera revealed 23 adenovirus infections in the pneumonia group and 42 in the group with other respiratory infections. Other viral infections were found only sporadically. All rapid virus detection methods showed excellent specificity but had a lower sensitivity (60%) than virus isolation. Our results show that rapid methods for diagnosing respiratory adenovirus infections can be successfully used in selected groups of adults.

Adenoviridae Infections↗
PubMed1985

Triggering infections in reactive arthritis.

Certain microbes like yersinia, salmonella, shigella, campylobacter, chlamydia, and possibly gonococcus can trigger reactive arthritis especially in patients of the HLA-B27 type. In the present study we have used serological and culture methods to identify the probable triggering infection in 50 consecutive HLA-B27 positive patients diagnosed as having reactive arthritis. The two most common triggering agents thus identified were Yersinia enterocolitica (12 patients) and Chlamydia trachomatis (11 patients). In addition six patients had high antistreptolysin O titres and two high teichoic acid antibody titres suggesting group A streptococci and Staphylococcus aureus as triggering agents. In 13 patients no preceding infection could be identified. The identity of the infective agent seems to have very little effect on the clinical picture of the reactive arthritis - the only difference between the various aetiological groups in the present material was absence of fever in the patients with a preceding C. trachomatis infection, of whom only one out of 11 had a temperature greater than or equal to 38 degrees C, whereas 13 of 16 patients with a preceding enterobacterial, and five of the eight patients with a streptococcal or staphylococcal infection had raised temperatures.

Arthritis, Infectious↗
PubMed1985

Microbial diagnosis by nucleic acid sandwich hybridization.

Sandwich hybridization is a three-component nucleic acid hybridization method suitable for the identification of microbes. In this method, one specific DNA fragment on solid support acts as catching reagent, and the second reagent is a labeled probe. The labeling of the support is mediated by a specimen nucleic acid homologous to both reagents. Because the specimen is kept in solution, relatively crude specimens not requiring elaborate pretreatments can be tested without background problems. The utility of the method in microbial diagnosis (adenovirus, cytomegalovirus, and Chlamydia trachomatis) has been demonstrated. Increased sensitivity and nonradioactive detection methods will no doubt further extend the applicability of the sandwich hybridization method.

Adenoviridae↗
PubMed1984

Cytomegalovirus in urine: detection of viral DNA by sandwich hybridization.

A cytomegalovirus (CMV)-specific sandwich hybridization test was constructed by using two adjacent BamHI DNA fragments of CMV DNA as reagents. The fragments were cloned into two different vectors. One of the recombinants was attached to the filter, and the other was the labeled probe. When present in the sample, CMV DNA mediated labeling of the filter by hybridizing to both the filter-bound DNA and the probe. The sandwich hybridization test was applied for the detection of CMV DNA from urine. DNA was released from virus by 2% Sarkosyl, concentrated by 2-butanol extraction and isopropanol precipitation, denatured, and finally subjected to the sandwich hybridization test. As a result, 70 to 90% of the original viral DNA could be recovered and demonstrated by the quantitative hybridization reaction. Urine could be stored at room temperature in Sarkosyl for at least 2 days without affecting the detectability of CMV. The clinical applicability of the test was evaluated by studying urine samples from four infants excreting CMV. Sandwich hybridization demonstrated the presence of CMV DNA in all of the specimens. These contained originally 10(5) to 10(8) CMV DNA molecules per ml.

Cytomegalovirus↗
PubMed1984

Inhibition of cell functions by RNA-virus infections.

The studies with poliovirus have revealed that cells have a full capacity to translate uncapped mRNA, although their own mRNAs are capped and methylated. These observations have been confirmed by translation studies in fractionated systems in vitro. In poliovirus-infected cells, and in cell extracts prepared from them, a well-documented loss of capacity to translate capped mRNAs has been shown. This is due to the dissociation of cap-binding protein(s) from the initiation factor eIF-3. It has not been possible to show which virus-specific protein, if any, is responsible for this dissociation. It may be that once not used, the cap-binding protein is excluded passively from its normal association cycle with eIF-3, rendering the cells incapable of translating capped mRNAs. Late in reovirus infection a similar phenomenon is observed. The secondary transcription products of reovirus subviral particles are uncapped mRNAs, which are synthesized in abundance. Their capping and methylation is prevented in the SVPs due to a masking of the respective enzymes. The inability to translate capped mRNAs in lysates prepared from reovirus-infected cells, late in infection, may be due to a similar mechanism, as in poliovirus-infected cells. Simply, a large excess of uncapped mRNA competes with initiation factors without specific need for the cap-binding protein(s), which becomes dissociated from the eIF-3 and loses its activity. Inhibition of host protein synthesis, whether due to simple competition or specified by a viral gene product, leads apparently to a shortage of initiation factors and ribosomes, causing the cessation of translation of both capped and uncapped mRNA, and finally to cell death. The studies with UV-inactivated VSV, its temperature-sensitive mutants and defective-interfering LT-particles clearly suggest that inhibition of host-cell macromolecular synthesis can be induced by mechanisms that do not operate during normal VSV-infection. It may be that similar phenomena occur in guanidine-treated poliovirus-infected cells. In these cells the host protein synthesis is inhibited as a result of the translation of a few hundred incoming viral RNAs. It would be interesting to know whether the cap-binding protein is dissociated from initiation factors under these conditions. If not, another mechanism has to be revealed, which again may not be manifest during normal poliovirus infection. The inhibition of host RNA and DNA synthesized by various viruses has been less intensively studied than inhibition of protein synthesis. Apparently, good in vitro systems are needed to solve these difficult problems.

Alphavirus↗
PubMed1983

Novel test for rapid viral diagnosis: detection of adenovirus in nasopharyngeal mucus aspirates by means of nucleic-acid sandwich hybridisation.

Nucleic-acid sandwich hybridisation detected adenovirus in nasopharyngeal aspirates from children with acute respiratory infections within 20 h. The differentiation between subgroups of adenovirus (B and C) and the lack of false positives indicated the specificity of the method. The reference test was detection of an adenovirus protein (hexon) by means of radioimmunoassay. In nucleic-acid sandwich hybridisation the sample DNA mediates the binding of a labelled "probe" DNA fragment to a second DNA fragment bound on filter. The sample DNA itself is not fixed to a solid support, and the only pretreatment required is boiling in a detergent solution. The results indicate that the sandwich hybridisation is specific, quantitative, easy to do, and only marginally affected by the crude nature of the sample. It may therefore be a valuable addition to the range of rapid methods in microbial diagnosis.

Acute Disease↗
PubMed1980

Semliki Forest virus replication complex capable of synthesizing 42S and 26S nascent RNA chains.

A complex synthesizing Semliki Forest virus (SFV)-specific RNAs was purified from infected HeLa cells. During purification, the RNA-synthesizing complex was monitored by the presence of RNA chains synthesized during a 1 min pulse in vivo and the ability to synthesize 42S and 26S RNAs in vitro. Finally, the protein composition of the replication complex was analysed. Thirty to 40% of the pulse-labelled RNAs and 10 to 25% of the polymerase activity present in the postnuclear supernatant were recovered in smooth membranes. At this stage of purification single stranded 42S and 26S RNA were synthesized and released from the replication complex in vitro. After treatment of the smooth membrane fraction with Triton X-100 the replication complex was solubilized. When analysed by sucrose gradient centrifugation, the solubilized replication complex distributed heterogeneously. It had reduced RNA polymerase activity, but was still able to synthesize both 42S and 26S nascent RNA chains which were not released from RIs and RFs. The non-structural protein ns70 was the major virus-specified component associated with the replication complex.

DNA-Directed RNA Polymerases↗
PubMed1976

Initiation of translation directed by 42S and 26S RNAs from Semliki Forest virus in vitro.

The proteins synthesized in vitro in response to 42S and 26S RNAs from Semliki Forest virus were labeled with formyl-[35S]methionine from initiator tRNA. One protein which comigrated with viral capsid protein was labeled under the direction of 26S RNA, and only one labeled peptide was detected after digestion with trypsin. Further digestion with pronase gave rise to the dipeptide fMet-AsN. Several labeled polypeptides were found in the 42S RNA directed product and these had molecular weights of up to 150,000. However, tryptic digestion of the product yielded only one formylmethionyl-labeled peptide, which had a different mobility from that directed by the 26S RNA. Further digestion with pronase gave a single dipeptide, fMet-Ala. This indicates that nonstructural proteins as large as 150,000 daltons are probably synthesized from one initiation site on the 42S RNA. Translation starting from the internal initiation site on the 42S RNA, which is equivalent to that on the 26S RNA, could not be detected under the conditions used. Internal initiation sites which are similarly inactive have also been detected in other viral RNAs (e.g., brome mosaic virus, tobacco mosaic virus, and polyoma 19S RNA) and this suggests that, although eukaryotic mRNAs can contain more than one initiation site for protein synthesis, only the site nearer the 5' terminus is active in vitro.

Cell-Free System↗