Estudo comparativo do espermograma em portadores e näo portadores de Ureaplasma urealyticum no líquido seminal / Seminal parameters in patients with and without Ureaplasma urealyticum

Foram analisadas 47 amostras de líquido seminal com o objetivo de determinar se alteraçöes nos valores de referência do espermograma poderiam estar associadas à presença de Ureaplasma urealyticum na amostra. Observamos positividade em 24(51,1%) dos materiais examinados. Os valores de referência para vitalidade foram considerados satisfatórios em ambos os grupos. Portadores de ureaplasmas apresentaram contagem menor do número de espermatozóides em relaçäo ao grupo näo portador. A presença de títulos elevados de U. urealyticum (> ou = 10**3 CFU/ml)näo altera significativamente os valores de referência dos testes do espermograma

Preliminary characterization of the urease and a 96 kDa surface-expressed polypeptide of Ureaplasma urealyticum.

Analysis by SDS-PAGE of Ureaplasma urealyticum (predominantly serotype 8), propagated in a growth medium containing 10% (v/v) foetal calf serum, revealed a complex series of polypeptides apparently free of medium contaminants. Serological analysis using an immobilized antibody reagent, and immunoblotting using a polyclonal serum, showed the presence of two major and several minor antigens. One major antigen, a putative surface component of apparent molecular mass 96 kDa was shown, with a monoclonal antibody, to be serotype-specific. Growth of the organism was partially suppressed in the presence of the antibody. The second major antigen had an apparent molecular mass of 76 kDa and was presumed to be an internal component since it failed to label with the Bolton and Hunter reagent, in contrast to the 96 kDa antigen. Another monoclonal antibody was characterized which detected the canonical urease enzyme of the organism serotype 8 and of the two other human serotypes tested. Purification of this urease antigen by affinity chromatography and electrophoretic analysis of polypeptides after denaturation revealed a single polypeptide of molecular mass 76 kDa, putatively related to the above major antigen. Enzymic activity could be recovered after purification and demonstrated by in situ techniques only when electrophoretic analysis was done under non-denaturing conditions suggesting that the functional enzyme is a multimeric complex.

DNA probes for the detection of mycoplasmas in genital specimens.

The utility of whole-genomic DNA probes for the detection of infections by genital mycoplasmas was investigated in 220 men attending a sexually transmitted diseases clinic. In 144 patients, probe results were compared with quantitative culture results. The prevalence of Mycoplasma hominis was 11% by culture, whereas the prevalence of ureaplasmas was 38%. The M. hominis DNA probe detected 9 of 16 M. hominis culture-positive specimens and 2 of 128 culture-negative specimens. The Ureaplasma urealyticum DNA probe detected 36 of 57 U. urealyticum culture-positive specimens and 18 of 87 culture-negative specimens. Most of the probe-negative culture-positive specimens had colony counts of less than 10(3) organisms/ml of specimen. The DNA probe does not require viable organisms, and the probe-positive, culture-negative specimens suggest that false-negative cultures occurred, perhaps due to specimen handling or insensitivity of culture methods for some strains of mycoplasmas.

Mass cultivation of ureaplasmas and some applications.

A procedure for the mass cultivation of Ureaplasma urealyticum is described. Yields of about 100 mg, dry weight, were obtained form 100-liter cultures of three serovars, 3, 4 and 8. Although not totally free from precipitated medium components, the use of radiolabeling permitted chemical analyses of organism-derived lipids and lipoglycans. All three serovars contained lipoglycans composed of hexoses, glycoerol, fatty acid esters and phosphorus.

Detection of lipoglycans in ureaplasmas.

Serotypes 3, 4, and 8 of Ureaplasma urealyticum were found to contain lipoglycans. Although the ratios of their components differed, all contained neutral sugars, fatty acids, glycerol, and phosphorus. All three became labeled when the organisms were cultivated in the presence of [14C]glucose, [14C]palmitic or [14C]oleic acids, and inorganic 32P. Only neutral sugars were found, and these consisted of mannose, glucose, and galactose. Hot phenol extracts of uninoculated and supernatant culture media contained polymeric carbohydrate, but this differed in composition from ureaplasmal lipoglycans and did not become radiolabeled. Since lipoglycans contained phosphorus but no amino sugars, they could be separated from contaminating polysaccharides by anion exchange chromatography.

Cytochemical evidence of extramembranous carbohydrates on Ureaplasma urealyticum (T-strain Mycoplasma).

Cells of Ureaplasma urealyticum that were prepared by a ruthenium red technique demonstrated an extramembranous layer of polyanions of about 15 to 30 nm in width. Application of the concanavalin A-iron dextran strain indicated that the outer surface of this layer contained glucosyl-like residues.

Isoelectric focusing of mycoplasma proteins.

Polyacrylamide gel isoelectric focusing (PAGIF) in thin layer was used to resolve proteins of Mycoplasma spp., Acholeplasma spp., and eight strains of Ureaplasma urealyticum (T-strain). A mixture of urea, Triton X-100, and dithioerythritol was used to solubilize sonically disrupted cells. PAGIF was performed in the range of pH 3 to 10. Protein patterns were carefully compared, demonstrating resolved and distinguishable species-specific protein bands. The eight serotypes of U. urealyticum (T-strain) gave identical protein patterns in the pH 3 to 10 range. The characteristic "fingerprints" of a species appeared to correlate with the biochemical nature and not the habitat in each case. Arginine-hydrolyzing species seemed to show more diverse focusing than those that ferment glucose, or prefer an acid environment. Characterization and identification of highly resolved species-specific proteins, ease of performance, and reproducibility of this method suggest that PAGIF might be employed as a taxonomic aid.

A study of the amino acids and proteins of some human T-mycoplasma membranes.

Purified membranes were prepared from seven human T-mycoplasmas. Their amino acid composition was determined and was similar to that of other biological membranes. Their proteins were examined by isoelectric focusing and 7 to 10 protein bands were detected mostly in the pH 4 to 7 range of the gel. T-mycoplasma T-McA also had one distinctive band at pH 3 while T-mycoplasma T-213 had a prominent basic band at pH 9. The proteins were denatured by storage at -25 degrees. Five to seven Periodic Schiff-positive bands also were observed but were not identified.

The preparation of membranes of some human T-mycoplasmas and the analysis of their carbohydrate content.

Purified membranes were prepared from seven human T-mycoplasmas of which four are laboratory strains and three isolates from patients with nonspecific urethritis. The T-mycoplasmas were resistant to osmotic shock and sonication. Membranes were obtained only after lysing most of the T-mycoplasmas by four passes through a cell fractionator at 40,000 psi and separating the membranes from the unlysed cells by differential centrifugation. The membranes contained between 1 and 7% carbohydrates by dry weight. Mannose, galactose, and glucose were identified with glucose in the largest concentration.