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PubMedDate not supplied

Rheumatoid arthritis: new findings on the failure to isolate or detect mycoplasmas by multiple cultivation or serologic procedures and a review of the literature.

Using 12 different and elaborate broth, agar, and cell culture procedures, we failed to isolate mycoplasmas, ureaplasmas, spiroplasmas, or chlamydiae from the synovial fluid of 10 patients with rheumatoid arthritis (RA) and from six patients with non-rheumatoid arthritis (NRA). In addition, sera from 35 patients with RA and 12 patients with NRA also were examined. Although some of the sera had moderately high titers of metabolism-inhibiting antibody to some of the 10 human Mycoplasma species, especially to the common respiratory pathogen Mycoplasma pneumoniae, and to some of the eight Ureaplasma urealyticum serovars, especially serovars V and VII, there were no significant differences between titers of these antibodies in the two groups of patients. Among RA patients serum antibody titers to M. pneumoniae were 1:32 in five and 1:16 in eight; two patients had higher synovial fluid titers (1:16) than serum titers (1:4). The geometric mean titer (GMT) of antibody to serovar V in synovial fluid was higher in RA patients than in NRA patients, but the difference did not reach significance (P = .056). Reports on the possible role of infectious agents in the pathogenesis of rheumatoid arthritis are reviewed.

Adult↗
PubMed1993

Probing DNA-protein interactions in vitro with the CpG DNA methyltransferase.

A sensitive method was devised to monitor the in vitro binding of nuclear proteins from HeLa cells presumably to the major groove of DNA. Upon the incubation of DNA with nuclear extracts, the complexed DNA was incubated with the CpG DNA methyltransferase from Spiroplasma species. Subsequently, the DNA was repurified, and the location of the methylated cytidine residues was determined by the hydrazine reaction of the DNA sequencing method. By using as DNA substrate the VAI (virus associated) region of human adenovirus type 2 (Ad2) DNA or specific Alu sequences associated with a number of human genes, it was documented that those segments of DNA that were protected by bound proteins against the reaction with DNasel also escaped in vitro methylation by the CpG DNA methyltransferase. This new footprinting method provides a sensitive indicator for in vitro DNA--protein interactions which are specific for the major groove of DNA.

Adenoviridae↗
PubMed1994

The topology of the promoter of RNA polymerase II- and III-transcribed genes is modified by the methylation of 5'-CG-3' dinucleotides.

In eukaryotic cells, RNA polymerase II- and III-transcribed promoters can be inactivated by sequence-specific methylation. For some promoter motifs, the introduction of 5-methyldeoxycytidine (5-mC) residues has been shown to alter specific promoter motif-protein interactions. To what extent does the presence of 5-mC in promoter or regulatory DNA sequences affect the structure of DNA itself. We have investigated changes in DNA bending in three naturally occurring DNA elements, the late E2A promoter of adenovirus type 2 (Ad2) DNA, one of our main model systems, the VAI (virus-associated) RNA gene of Ad2 DNA, and an Alu element associated with the human angiogenin gene. Alterations in electrophoretic mobility of differently permuted promoter segments in non-denaturing polyacrylamide gels have been used as assay system. In the late E2A promoter of Ad2 DNA, a major and possibly some minor DNA bending motifs exist which cause deviations in electrophoretic mobility in comparison to coelectrophoresed marker DNA fragments devoid of DNA bending motifs. DNA elements have been specifically in vitro methylated by the HpaII (5'-CCGG-3'), the FnuDII (5'-CGCG-3'), or the CpG DNA methyltransferase from Spiroplasma species (M-SssI; 5'-CG-3'). Methylation by one of these DNA methyltransferases influences the electrophoretic mobility of the three tested promoter elements very strikingly, though to different extents. It cannot be predicted whether sequence-specific promoter methylation increases or decreases electrophoretic mobility; these changes have to be experimentally determined. Methylation of the E. coli dcm (5'-CCA/TGG-3') sites in some of the DNA constructs does not make a contribution to mobility changes. It is concluded that sequence-specific methylations in promoter or regulatory DNA elements can alter the bending of DNA very markedly. This parameter may contribute significantly to the silencing of promoters, probably via altering spatial relationships among DNA-bound transcription factors.

Adenovirus E2 Proteins↗
PubMed2003

Host specificity of mollicutes oriC plasmids: functional analysis of replication origin.

Recently, artificial oriC plasmids containing the chromosomal dnaA gene and surrounding DnaA box sequences were obtained for the mollicutes Spiroplasma citri and Mycoplasma pulmonis. In order to study the specificity of these plasmids among mollicutes, a set of similar oriC plasmids was developed for three mycoplasmas belonging to the mycoides cluster, Mycoplasma mycoides subsp. mycoides LC (MmmLC), M.mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capricolum. Mycoplasmas from the mycoides cluster, S.citri and M.pulmonis were used as recipients for transformation experiments by homologous and heterologous oriC plasmids. All five mollicutes were successfully transformed by homologous plasmids, suggesting that the dnaA gene region represents the functional replication origin of the mollicute chromosomes. However, the ability of mollicutes to replicate heterologous oriC plasmids was found to vary noticeably with the species. For example, the oriC plasmid from M.capricolum did not replicate in the closely related species MmmSC and MmmLC. In contrast, plasmids harbouring the oriC from MmmSC, MmmLC and the more distant species S.citri were all found to replicate in M.capricolum. Our results suggest that the cis-elements present in oriC sequences are not the only determinants of this host specificity.

Amino Acid Sequence↗
PubMed1998

Sequences of the 16S rRNA genes and phylogeny of the goat mycoplasmas Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii.

The nucleotide sequences of the 16S rRNA genes from the type strains of four goat mycoplasmas, Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii, were determined by direct solid-phase DNA sequencing. Polymorphisms were found in two of the 16S rRNA gene sequences, showing the existence of two different rRNA operons. Three polymorphisms were found in M. adleri, and one was found in M. yeatsii. The sequence information was used for the construction of phylogenetic trees. M. adleri was included in the Mycoplasma lipophilum cluster within the hominis group. M. auris was comprised in the Mycoplasma hominis cluster of the hominis group. M. cottewii and M. yeatsii were found to be very closely related with only four nucleotide differences, and they grouped with Mycoplasma putrefaciens in the Mycoplasma mycoides cluster within the spiroplasma group. Sequencing of two field isolates of M. cottewii and M. yeatsii, geographically distant from the type strains, showed that the 16S rRNA gene from the field isolate of M. cottewii was identical to the one from the type strain. The field isolate of M. yeatsii had only two nucleotide differences to the type strain and these were present in only one of the two rRNA operons. Sequencing of the 16S rRNA genes from two unidentified mycoplasma isolates from Nepal indicated that they should both be regarded as M. auris strains.

Animals↗
PubMed1995

Expression of foreign genes and selection of promoter sequences in Acholeplasma laidlawii.

The stable maintenance and expression of foreign genes in mollicutes (mycoplasmas) have been difficult to achieve due to the lack of suitable vectors. In this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic resistance genes in Acholeplasma laidlawii. Plasmids derived from the lactococcal vector pNZ18 could introduce and maintain four different genes for many generations in A. laidlawii. One of these, encoding the dominant membrane lipoprotein spiralin from the mollicute Spiroplasma citri, was expressed; however, expression was weak, the signal peptide of spiralin was not cleaved and the protein was not covalently modified by fatty acids. This resulted in a hydrophilic character of spiralin and its cytoplasmic localization in A. laidlawii. To increase the expression of foreign genes, random A. laidlawii DNA fragments were cloned into a pNZ18-related plasmid and expression signals were selected using the Bacillus licheniformis alpha-amylase gene as a probe. Selection was done in Escherichia coli as well as directly in A. laidlawii. Active recombinants from E. coli were also able to express alpha-amylase activities and an enzyme of native size in A. laidlawii. The highest activity was obtained from a recombinant selected directly in A. laidlawii. This is the first example of a promoter sequence selected in a mollicute. Analysis of the putative promoters in seven clones revealed similar -10 and -35 regions, and similar spacer distances in A. laidlawii, Acholeplasma oculi, Lactococcus and E. coli. Vectors related to pNZ18 should be useful for the genetic analysis of specific A. laidlawii proteins and functions.

Acholeplasma laidlawii↗
PubMed2001

The Stockholm populations of Adalia bipunctata (L) (Coleoptera: Coccinellidae)--a case of extreme female-biased population sex ratio.

The genetic composition and sex ratio in the Stockholm populations of Adalia bipunctata have been studied. The overall frequency of melanics is 3.2%, which is significantly lower than in the populations of St. Petersburg and other large cities along the Baltic Sea. The secondary sex ratio in the Stockholm populations is female-biased 82:18. More than half of A. bipunctata females are infected with the male-killing Spiroplasma bacterium. Beetles of the co-existing species Adalia decempunctata are infected with a different bacterium belonging to the genus Rickettsia.

Animals↗
PubMed2005

A functional dosage compensation complex required for male killing in Drosophila.

Bacteria that selectively kill males ("male-killers") were first characterized more than 50 years ago in Drosophila and have proved to be common in insects. However, the mechanism by which sex specificity of virulence is achieved has remained unknown. We tested the ability of Spiroplasma poulsonii to kill Drosophila melanogaster males carrying mutations in genes that encode the dosage compensation complex. The bacterium failed to kill males lacking any of the five protein components of the complex.

Acetyltransferases↗
PubMed1977

Mycoplasma-dependent activation of normal lymphocytes: mitogenic potential of mycoplasmas for mouse lymphocytes.

Nonviable preparations of a wide variety of glucose-utilizing mycoplasma species, including Acholeplasma laidlawii and Spiroplasma citri, were found to be mitogenic for mouse lymphocytes. Particularly strong reactions were obtained with Mycoplasma synoviae, M. gallisepticum, M. pneumoniae, S. citri, and a strain of M. fermentans that was previously isolated from a leukemic patient. Nonviable preparations of arginine-utilizing mycoplasmas inhibited the uptake of [3H]thymidine by lymphocytes, but this effect could be reversed by heat treatment or arginine supplementation, and a stimulatory effect was then observed. Viable M. arthritidis was also found to have a mitogenic effect, as detected by an increased uptake of [3H]thymidine by normal lymphocytes and by autoradiographic techniques in which an increase in the numbers of transformed cells was seen. These observations provide the potential for enhanced immunological responsiveness or lymphokine-mediated inflammation in mycoplasma-infected hosts.

Acholeplasma laidlawii↗
PubMed1976

Distribution and composition of lipopolysaccharides from mycoplasmas.

Polymeric carbohydrates containing glycerol and fatty acids were isolated from whole cells and membranes of mycoplasmas by hot aqueous phenol extraction and gel filtration. Lipopolysaccharides were found to occur in four species of Acholeplasma, two of Anaeroplasma, and in Mycoplasma neurolyticum. None were detected in Spiroplasma citri or in five species of Mycoplasma. All lipopolysaccharides contained both neutral and N-acylated amino sugars in ratios varying from 1:1 to 3:1. The neutral sugars found in varying distribution were glucose, galactose, and mannose. The amino sugars included fucosamine, an unidentified deoxyhexosamine, galactosamine, and glucosamine. Fucosamine and glucose were the only sugars common to all lipopolysaccharides. The fatty acids were similar to those found in the lipids of each organism.

Acholeplasma↗
PubMed1984

Mycoplasmas (Mollicutes) have a low number of rRNA genes.

DNA from Mycoplasma, Ureaplasma, Acholeplasma, and Spiroplasma species digested by restriction endonucleases was hybridized with probes consisting of portions of the rrnB rRNA operon of Escherichia coli and the rRNA operon of Mycoplasma capricolum. The results indicate the presence of only one or two sets of rRNA genes in the genome of Mollicutes linked in the procaryotic fashion, 16S-23S-5S.

Acholeplasma↗
PubMed1989

Topology and acylation of spiralin.

Of the 51 polypeptides detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the plasma membrane of the helical mollicute Spiroplasma melliferum, 21 are acylated, predominantly with myristic (14:0) and palmitic (16:0) chains. This is notably the case for spiralin, the major membrane protein of this bacterium, which contains an average of 0.7 acyl chains per polypeptide, attached very probably by ester bonds to alcohol amino acids. The amphiphilicity of spiralin was demonstrated by the behavior of the protein in charge-shift electrophoresis, its incorporation into liposomes, and its ability to form in the absence of lipids and detergents, globular protein micelles (diameter, approximately 15 nm). The presence of epitopes on the two faces of the cell membrane, as probed by antibody adsorption and crossed immunoelectrophoresis, and the strong interaction between spiralin and the intracytoplasmic fibrils show that spiralin is a transmembrane protein. The mean hydropathy of the amino acid composition of spiralin (-0.30) is on the hydrophilic side of the scale. Surprisingly, the water-insoluble core of spiralin micelles, which is the putative membrane anchor, has a still more hydrophilic amino acid composition (mean hydropathy, -0.70) and is enriched in glycine and serine residues. Taking into account all these properties, we propose a topological model for spiralin featuring a transbilayer localization with hydrophilic domains protruding on the two faces of the membrane and connected by a small domain embedded within the apolar region of the lipid bilayer. In this model, the membrane anchoring of the protein is strengthened by a covalently bound acyl chain.

Acylation↗
PubMed1991

Adenovirus type 2 VAI RNA transcription by polymerase III is blocked by sequence-specific methylation.

Sequence-specific methylation of the promoter and adjacent regions in mammalian genes transcribed by RNA polymerase II leads to the inhibition of these genes. So far, RNA polymerase III-transcribed genes have not been investigated in depth. We therefore studied methylation effects on the RNA polymerase III-transcribed VAI gene of adenovirus type 2 DNA. The VAI gene contains 20 5'-CG-3' dinucleotides, of which 4 (20%) can be methylated by HpaII (5'-CCGG-3') and HhaI (5'-GCGC-3'). Three of these 5'-CG-3' sequences are located close to the internal regulatory region of the VAI segment. An unmethylated, a 5'-CCGG-3'- and 5'-GCGC-3'-methylated, and a 5'-CG-3'-methylated pUC18 construct containing the VAI and VAII regions were transfected into mammalian cells. In many experiments, an inactivating effect of 5'-CCGG-3' and 5'-GCGC-3' DNA methylation on the VAI region was not observed. In contrast, methylation of all 20 5'-CG-3' sequences in the VAI region by a CpG-specific DNA methyltransferase from Spiroplasma species did interfere with VAI transcription. Transcription of the VAI- and VAII- and of the VAI-containing constructs was also shown to be inhibited in an in vitro cell-free transcription system after the constructs had been methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences or at all 5'-CG-3' sequences. When an oligodeoxyribonucleotide which carried the internal control block A of the VAI region was methylated at three 5'-CG-3' sequences, the formation of a complex with HeLa nuclear proteins was abrogated. The results presented support the notion that the VAI gene transcribed by the DNA-dependent RNA polymerase III is also inactivated by methylation of the decisive 5'-CG-3' sequences.

Adenoviruses, Human↗
PubMed1988

Mycoplasma viruses.

Unlike bacterial viruses that infect cells bounded by a cell wall, mycoplasma viruses have evolved to enter and propagate in mycoplasma cells bounded only by a single lipid-protein cell membrane. In addition, mycoplasmas have the smallest amount of genetic information of any known cells, so their complexity is constrained by a limited genetic coding capacity. As a consequence of these host cell differences, mycoplasma viruses have been found to have a variety of structures and replication strategies which are different from those of the bacterial viruses. This article is a critical review of mycoplasma viruses infecting the genera Acholeplasma, Spiroplasma, and Mycoplasma; included are data on classification, morphology and structure, biological and physical properties, chemical composition, and productive and lysogenic replication cycles.

Acholeplasma↗
PubMed1976

In vitro anti-mycoplasmal activity of amphotericin B methyl ester.

The polyene macrolide antibiotic amphotericin B (AB) and its chemically modified derivative amphotericin B methyl ester (AME) were tested for in vitro activity against Acholeplasma laidlawii, Spiroplasma citri and Mycoplasma gallisepticum. Both polyene macrolide preparations demonstrated anti-mycoplasmal activity. However, AME was mycoplasmacidal toward all three strains of mycoplasma at levels which previous studies have indicated would be permissible for most cell culture systems, whereas the levels of AB required for similar activity would be physiologically intolerable for tissue culture cells. In addition, AME was 100 fold more active than AB toward A. laidlawii, 10 fold more active than AB toward S. citri and demonstrated equivalent activity as AB toward M gallisepticum. The in vitro anti-mycoplasmal activity of AME and AB was directly correlated with polyene macrolide antibiotic levels and the number of treated mycoplasma.

Acholeplasma laidlawii↗
PubMed1999

[Sex ratio and male killing in Siberian populations of Harmonia axyridis (Pass.)].

In populations of Harmonia axyridis Pall. from Novosibirsk and Kyzyl, females (three out of 34 studied) that produce exclusively female progeny were found. In one of the families studied, the inheritance of the male-killing trait was monitored over five generations. The male-killing trait was maternally inherited. The beetles of this family were infected with the bacteria that, according to the sequence analysis of the gene fragment for 16S rRNA, belong to the genus Spiroplasma (VI group).

Animals↗
PubMedDate not supplied

[The characteristics of embryogenesis and oogenesis in Adalia bipunctata L. and Harmonia axyridis Pall. beetles in all-female families].

We describe abnormal embryonic development leading to death of 50% of embryos in all-female lines of Adalia and Harmonia infected with an androcide Spiroplasma. The arrest of embryonic development takes place at different stages throughout embryogenesis. Oogenesis of the infected beetles proceeds without any significant morphological changes.

Animals↗
PubMed1987

Localization of spiralin in Escherichia coli cells transformed with the recombinant plasmid pESI.

The expression of spiralin in the transformant strain HB101 Tsp of Escherichia coli has been investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunodetection after electrotransfer, and crossed immunoelectrophoresis. The protein has been sought in whole cells, cytoplasm, and plasma and outer membranes of the bacterium. Untransformed E. coli cells and Spiroplasma citri cells were used as negative and positive references, respectively. Contrary to earlier claims, spiralin was detected not only in the cytoplasm of E. coli, but also in the inner and outer membranes. In addition, our results show that the protein was not secreted by the bacterium. Three forms of spiralin could be distinguished with respect to differences in apparent molecular mass: 27.5, 29.5 and 30 kilodalton (kDa). The presence of the high molecular mass polypeptide in the two membranes of E. coli invalidates the hypothesis according to which the 27.5-kDa (or 28-kDa) species is a mature form derived from the 30-kDa (or 30.5-kDa) species by the amputation of a signal sequence. Since spiralin is an acyl protein, the possibility of variation in the extent of acylation of the protein in E. coli, with subsequent variation of the apparent molecular mass, should be taken into account.

Acylation↗