Search PubMed⌕ Search

Biomedical subjects

D Ralph

Publications and source records attributed to D Ralph.

At least 37 records · Page 2Linked to original sources
PubMed1995

Genetic and phenotypic analysis of Borrelia miyamotoi sp. nov., isolated from the ixodid tick Ixodes persulcatus, the vector for Lyme disease in Japan.

The ixodid tick Ixodes persulcatus is the most important vector of Lyme disease in Japan. Most spirochete isolates obtained from I. persulcatus ticks have been classified as Borrelia burgdorferi sensu lato because of their genetic, biological, and immunological characteristics. However, we found that a small number of isolates obtained from I. persulcatus contained a smaller 38-kDa endoflagellar protein and single 23S-5S rRNA gene unit. Representative isolate HT31T (T = type strain) had the same 23S rRNA gene physical map as Borrelia turicatae. The DNA base composition of strain HT31T was 28.6 mol% G+C. DNA-DNA hybridization experiments revealed that strain HT31T exhibited moderate levels of DNA relatedness (24 to 51%) with Borrelia hermsii, B. turicatae, Borrelia parkeri, and Borrelia coriaceae. However, the levels of DNA reassociation with the previously described Lyme disease borreliae (B. burgdorferi, Borrelia garinii, and Borrelia afzelii) were only 8 to 13%. None of the previously described species examined exhibited a high level of DNA relatedness with strain HT31T. In addition, the 16S rRNA gene sequence (length, 1,368 nucleotides) of strain HT31T was determined and aligned with the 16S rRNA sequences of other Borrelia species. Distance matrix analyses were performed, and a phylogenetic tree was constructed. The results showed that isolate HT31T is only distantly related to both previously described Lyme disease borreliae and relapsing fever borreliae. Thus, the spirochetes isolated from I. persulcatus and closely related isolates should be classified as members of a new Borrelia species. We propose the name Borrelia miyamotoi sp. nov. for this spirochete; strain HT31 is the type strain.

Animals↗
PubMed1994

Interactions among regulators of RNA abundance characterized using RNA fingerprinting by arbitrarily primed PCR.

Using RNA fingerprinting by arbitrarily primed PCR it is possible to infer convergent transcript regulatory pathways from the coordinate behavior of subsets of anonymous transcripts without cloning any genes. The number of transcripts in each response category can be estimated. The same may be true for differential display. We demonstrate these claims by treating a cell line with two known modulators of RNA abundance, transforming growth factor-beta (TGF beta) and cycloheximide (CX), used together and alone. The responses of over 1700 anonymous transcripts were monitored under these three conditions and in an untreated control. Eight of the twenty-seven [3(3)] possible transcript response categories were observed among 86 differentially expressed transcripts. For example, CX stabilizes or induces as many as 2.7% of transcripts of which about one third do not accumulate when TGF beta is also present. This intersection may reflect CX stabilization or induction of an important class of RNAs that otherwise usually have short half-lives. We predict that RNAs in this class constitute the majority of transcripts targeted for rapid down regulation in response to TGF beta and perhaps most other natural transcriptional modulators.

Animals↗
PubMed1994

Rapid isolation of tissue-specific and developmentally regulated brain cDNAs using RNA arbitrarily primed PCR (RAP-PCR).

RNA arbitrarily primed PCR (RAP-PCR) was used to isolate cDNAs that represent developmentally regulated brain-specific genes. Five clones with a restricted pattern of expression were identified and sequenced. Four cDNAs had no obvious homology to the sequences in GenBank. One clone had over 95% homology to a Ca2+/calmodulin-insensitive adenylyl cyclase, a recently cloned gene that was isolated from rat brain and was shown to be expressed only in adult brain and lung. Two novel cDNAs were investigated further by Northern blot analysis and were found to be expressed differentially during development; their expression was confined to the forebrain in the adult mouse. Further characterization by in situ hybridization showed that the mRNA corresponding to one clone was localized to a limited number of differentiating functional structures in the developing nervous system. In the adult brain, this message is confined to the forebrain with the highest level of expression in the cortex. These data suggest that the product of this gene is involved in the establishment of neuronal networks during brain development and in synaptic plasticity in the mature cortex. This work demonstrates that RAP-PCR is a powerful method for the simultaneous detection of differences between multiple RNA populations and, as such, can be used to study differential gene expression in the brain.

Animals↗
PubMed1994

Phylogenetic evidence for horizontal transfer of an intervening sequence between species in a spirochete genus.

The 23S rRNA genes (rrl genes) of some strains of certain species of the spirochete genus Leptospira carry an intervening sequence (IVS) of 485 to 759 bases flanked by terminal inverted repeat and encoding an open reading frame for a putative protein of over 120 amino acids. The structure and the sporadic distribution of the IVS suggest that it might be a mobile element that can be horizontally transferred within or between species. Phylogenetic hypotheses based on the sequences for six IVS open reading frames from various species were compared with hypotheses constructed by using DNA sequences from the 16S rRNA gene (rrs), which is not closely linked to rrl in this genus. The predicted phylogenies for the IVS and rrs differed in a major respect: one strain that claded with L. weillii in the tree based on the rrs data claded with L. noguchi in the tree based on the IVS data. Neither set of data supported a tree in which this strain was constrained to be in the same clade as was supported by the other set of data. This result indicates a probable horizontal transfer of the IVS from a recent ancestor of L. noguchi to a recent ancestor of one of the L. weillii strains. This observation is the first indication of horizontal transfer of elements encoded on the chromosomes of spirochetes.

Amino Acid Sequence↗
PubMed1993

RNA fingerprinting using arbitrarily primed PCR identifies differentially regulated RNAs in mink lung (Mv1Lu) cells growth arrested by transforming growth factor beta 1.

RNA fingerprinting using arbitrarily primed PCR (RAP) samples an RNA population and allows the detection of differentially expressed genes between two or more populations. This method was applied to mink lung epithelial cells, which respond to treatment with transforming growth factor beta (TGF-beta) by undergoing cell cycle arrest at or near the G1/S-phase boundary. The steady-state abundances of approximately 200 RNAs were surveyed, a few of which displayed differential regulation in response to TGF-beta 1. Three products were isolated, cloned, and sequenced. One differentially regulated RNA corresponded to cyclin A, a gene known to be required for the progression of mammalian fibroblasts through S phase. Northern blot analysis confirmed that the cyclin A mRNA steady-state abundance decreased dramatically in response to a 24-hr TGF-beta 1 treatment and also in response to cell cycle arrest caused by contact inhibition. A second RAP product corresponded to a previously unknown 7.5-kb mRNA, the level of which decreased dramatically in response to TGF-beta 1 treatment. Unlike the cyclin A mRNA, the abundance of this transcript did not decrease in response to growth arrest induced by contact inhibition. A third RAP product corresponded to the mRNA for osteonectin, an extracellular matrix protein. The abundance of this mRNA increased at least 2-fold during TGF-beta 1 treatment. This observation is consistent with other reports of increases in extracellular matrix proteins during TGF-beta treatment. RAP should be able to identify many of the genes that change in steady-state expression during the cell cycle.

Animals↗
PubMed1993

Species of Borrelia distinguished by restriction site polymorphisms in 16S rRNA genes.

Three phyletic groups of Borrelia associated with Lyme disease, B. burgdorferi, B. garinii and group VS461 can be distinguished from each other and other species of Borrelia by BfaI restriction site polymorphisms in PCR amplified 16S rRNA genes. One strain isolated from an Ixodes pacificus tick in California that was previously unclassifiable was distinguishable from B. burgdorferi by an Mn/I restriction site polymorphism.

Base Sequence↗
PubMed1993

Intervening sequence with conserved open reading frame in eubacterial 23S rRNA genes.

An intervening sequence (IVS) occurred in the 23S rRNA genes (rrl) of some, but not all, strains of four species of the spirochete genus Leptospira and was absent from strains in three other species. The IVS varied in size from 485 to 759 base pairs and replaced bases 1224-1245 in both copies of rrl. The two ends of each IVS shared 22-35 bases of complementarity that could form a stable double helix. The presence of an IVS correlated with a cleaved mature 23S rRNA that probably results from removal of the IVS without religation. The 3' site of cleavage was mapped within the inverted repeat of the IVS. An open reading frame of 121-133 amino acids was conserved in the IVS in all four species, oriented so that the sense strand was in the rRNA transcript. When the open reading frames were compared between species, they predicted polypeptides that showed between 51% and 78% amino acid conservation and similar DNA sequence conservation, indicating selection for protein function.

Amino Acid Sequence↗
PubMed1993

Arbitrary primed PCR fingerprinting of RNA applied to mapping differentially expressed genes.

Differential gene expression between various tissues and developmental stages or between cells in vitro under different growth conditions can be rapidly and efficiently compared using the RNA arbitrarily primed polymerase chain reaction (RAP) fingerprinting method (Welsh et al., 1992b; Liang and Pardee, 1992). In RAP, a primer of arbitrary sequence primes both first and second strand cDNA synthesis. The mixture of products is then PCR amplified and resolved electrophoretically, yielding highly reproducible fingerprints that are tissue-specific or growth condition-specific. Differences between fingerprints arise from differentially expressed genes, as verified by Northern blot analysis. RAP can be performed on the RNA samples using various DNA primers. Each two day experiment yields a sample of approximately twenty cDNA products per lane making the identification of differentially or developmentally regulated genes no longer rate limiting. Those PCR products representing genes that are regulated can be cloned from the gel and sequenced. Sequences can be compared to the DNA and protein sequence databases to identify homologs, motifs and members of gene families. The clones can be placed on the genetic map as Expression Tagged Sites (ETS, Adams et al., 1991a).

Animals↗
PubMed1993

Leptospira genomes are modified at 5'-GTAC.

Genomic DNAs of 14 strains from seven species of the spirochete Leptospira were resistant to cleavage by the restriction endonuclease RsaI (5'-GTAC). A modified base comigrating with m4C was detected by chromatography. Genomic DNAs from other spirochetes, Borrelia group VS461, and Serpulina strains were not resistant to RsaI digestion. Modification at 5'-GTAm4C may occur in most or all strains of all species of Leptospira but not in all genera of spirochetes. Genus-wide DNA modification has rarely been observed in bacteria.

5-Methylcytosine↗
PubMed1993

Leptospira species categorized by arbitrarily primed polymerase chain reaction (PCR) and by mapped restriction polymorphisms in PCR-amplified rRNA genes.

Reference strains from 48 selected serovars representing eight species of Leptospira were examined by two polymerase chain reaction (PCR)-based strategies. First, mapped restriction site polymorphisms (MRSP) were examined in PCR products from portions of rrs (16S rRNA gene) and rrl (23S rRNA gene). Twenty MRSP and 2 length polymorphisms were used to group reference strains into 16 MRSP profiles. Species assignments were consistent with those obtained by a second method, genomic fingerprinting with arbitrarily primed PCR, in which strains within a species were characterized by many shared arbitrarily primed PCR products. The results of both of these methods were in general agreement with those of previous studies that used DNA-DNA relatedness and confirmed the high level of divergence among the recognized species of Leptospira. However, Leptospira meyeri serovar ranarum and evansi strains were indistinguishable from some strains of Leptospira interrogans sensu stricto. Intervening sequences of about 485 to 740 bp were located near base 1230 in rrl of some strains.

Base Sequence↗
PubMed1992

Arbitrarily primed PCR fingerprinting of RNA.

Fingerprinting of RNA populations was achieved using an arbitrarily selected primer at low stringency for first and second strand cDNA synthesis. PCR amplification was then used to amplify the products. The method required only a few nanograms of total RNA and was unaffected by low levels of genomic double stranded DNA contamination. A reproducible pattern of ten to twenty clearly visible PCR products was obtained from any one tissue. Differences in PCR fingerprints were detected for RNAs from the same tissue isolated from different mouse strains and for RNAs from different tissues from the same mouse. The strain-specific differences revealed are probably due to sequence polymorphisms and should be useful for genetic mapping of genes. The tissue-specific differences revealed may be useful for studying differential gene expression. Examples of tissue-specific differences were cloned. Differential expression was confirmed for these products by Northern analysis and DNA sequencing uncovered two new tissue-specific messages. The method should be applicable to the detection of differences between RNA populations in a wide variety of situations.

Animals↗
PubMed1991

A consumer's guide to commercially available urodynamic equipment.

Eight commercially available urodynamic machines from 6 major manufacturers (Aspen Medical, Dantec, Electro-Medical Supplies (EMS), Uro-gyn, Ormed and Wiest) were assessed for accuracy, construction and user-friendliness. There was less than 10% inaccuracy with regard to fill volume, void rate and pressure measurements. The Wiest 6000 plus system was between 11 and 14% inaccurate during voided volume measurement and other systems were less than 10% inaccurate. User-friendliness was assessed during routine cystometry and is mainly dependent on the software supplied with the equipment. The merits and faults of each urodynamic system with regard to performing a urodynamic investigation, running a urodynamic service and data management are compared with an "ideal" urodynamic system.

Diagnostic Errors↗
PubMed1990

The distribution and dynamics of Rickettsia in the tick population of Ohio.

A five-year survey of ticks and associated rickettsia in Ohio shows a partitioning of spotted fever group rickettsia (namely, Rickettsia montana and R. rickettsii) into three areas of the state where the majority of human spotted fever cases occur. The percentage of ticks infected, even in areas with a high incidence of disease, is remarkably low, less than 1%. The statewide infection rate for R. bellii approaches 3%, and this organism appears to be more evenly distributed throughout the state. Dermacentor variabilis, which accounts for 97% of Ohio ticks received for testing, is the only species which has been found to harbor rickettsia. Ohio's tick testing program is not an effective surveillance tool and is of no use in diagnosing tick-borne disease. However, the program does function as an excellent means of increasing public awareness of tick-borne disease.

Animals↗
PubMed1990

Genetic relationships among the members of the family rickettsiaceae as shown by DNA restriction fragment polymorphism analysis.

The genetic diversity of members of the genus Rickettsia was examined using restriction site polymorphisms found within a series of DNA fragments scattered throughout the genome. Rickettsia belli, R. akari, and R. australis were the most divergent species when compared to the other species examined. These three species were also not closely related to each other. The other examined species were more tightly clustered. This survey also examined the genetic diversity within several species. The unexpected finding of this survey is that several species of rickettsia are as closely related to the surveyed strains of R. rickettsii as these strains are to each other. These results indicate that R. sibirica, R. parkeri, R. rickettsii, and an unnamed isolate from Africa are likely to be strains of a single rickettsial species of worldwide distribution. R. conorii was very closely related to this R. rickettsii-containing group but is likely to remain in a genetically distinct category as the data base expands.

DNA, Bacterial↗
PubMed1988

Physical identification of branched intron side-products of splicing in Trypanosoma brucei.

Every mRNA in trypanosomes consists of two exons, a common 5' capped mini-exon or spliced leader and a coding-exon. All evidence suggests that the exons are joined by trans-splicing of two individual precursor RNAs, the mini-exon donor RNA or spliced leader precursor RNA (medRNA) and the pre-mRNA. We studied intermediates of the splicing reaction using denaturing two-dimensional PAGE and structurally identified a group of small (approximately 180-300 nt) non-polyadenylated, Y-shaped branched RNAs. The branched Y-shaped RNAs contain the 105 nt medRNA derived intron, joined in a 2'-5' phosphodiester bond to small heterogeneously sized RNAs. These non-polyadenylated branched Y-shaped RNA molecules are analogous to the lariat shaped introns of higher eukaryotes and presumably represent the released intron-like by-products of a trans-splicing reaction which joins the mini-exon and the major coding-exon.

Animals↗
PubMed1987

Enzyme-linked immunosorbent assay for Potomac horse fever disease.

An enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) and IgM in natural and experimental infections of equids with Ehrlichia risticii was developed. Ehrlichial organisms purified from an infected mouse macrophage cell line were used as the antigen. IgM was separated from serum IgG by the expedient of spun-column chromatography, allowing the use of an indirect ELISA for quantitation of both IgG and IgM in the test sera. Among 16 paired sera from horses exhibiting clinical signs of Potomac horse fever, 8 were positive by the indirect fluorescent-antibody test (IFA), 11 were positive by the IgG ELISA, and 8 were positive by the IgM ELISA. All IFA-positive specimens were positive by the IgG ELISA, which appeared to be more sensitive than the IFA. In all cases, the IgG ELISA alone would have sufficed for diagnosis when acute- and convalescent-phase sera were available. When 26 single acute- or convalescent-phase serum samples were tested, the IFA detected 8, the IgG ELISA detected 10, and the IgM ELISA detected 6 positive serum specimens. The kinetics of IgG and IgM responses as determined by ELISA in two experimentally infected ponies which survived infection and challenges revealed that specific IgM was short-lived, falling to undetectable levels by day 60 postinoculation, whereas specific IgG persisted for more than 1 year. IgM and IgG were detected as early as days 1 and 10, respectively, postinoculation. The results suggest that the ELISA is more sensitive than the IFA and that the IgM ELISA may provide a means for early diagnosis of Potomac horse fever at or before the onset of clinical signs.

Animals↗
PubMed1985

Rapid separation of IgM from whole serum using spun column chromatography.

Spun columns were used to separate IgM from serum samples by exploiting molecular weight and size difference between IgM and other serum antibodies. Miniature gel chromatography columns were prepared in 3 ml syringe barrels. A polyacrylamide liquid chromatography gel, Bio-Gel P-200, was chosen to exclude proteins with molecular weights greater than 200,000. IgM, 900,000 MW, was excluded while IgG, 150,000 MW, was retained in the gel column. Centrifugation of the serum-loaded columns in test tubes accomplished the separation in one step by eluting a void volume equal to the sample volume from each column into a holding tube. IgM recovery in the eluent exceeded 96% of the total serum IgM in the pre-column sample. No IgG was detected in the eluent. IgM separated from La Crosse encephalitis immune human serum retained immunological activity in a viral neutralization test. Spun column chromatography is eminently suitable for diagnostic laboratories as more than 100 sera may be fractionated in one day using inexpensive materials and a low-speed centrifuge equipped with a swinging-bucket rotor.

Antibodies, Viral↗