Recovery of biological active catechol-O-methyltransferase isoforms from Q-sepharose.

The development of new catechol-O-methyltransferase inhibitors has led to an improvement in the treatment of Parkinson's disease. However, despite the fact that the soluble isoform has been extensively investigated, few studies have been published concerning membrane isoform chromatographic recovery and bioactivity levels. In this work, chromatographic profiles of both catechol-O-methyltransferase isoforms were compared using quaternary amine as a ligand to evaluate its activity levels and recovery rates. Results show that both proteins required different conditions for adsorption; the soluble isoform adsorption was performed at low ionic strength, while the membrane isoform required increasing linear salt gradient. However, the application of 0.5% Triton X-100 promoted membrane isoform adsorption even at low ionic strength. Indeed, chromatographic conditions of both isoforms became similar when detergents were applied. The developed methods also appear to be highly effective in bioactivity recovery, presenting rates of 107% for soluble protein and 67 and 91% for membrane isoform without and with detergents, respectively. The chromatographic strategies with and without detergents resulted in a 4.3- and sevenfold purification, respectively, corresponding to specific activity values of 331 and 496 nmol/h/mg. Thus, the use of Q-sepharose as anion exchanger was effective in the recovery of both enzymes, which is a requirement for further kinetic and pharmacological trials.

Association of a novel high molecular weight, serine-rich protein (SrpA) with fibril-mediated adhesion of the oral biofilm bacterium Streptococcus cristatus.

The surface of the oral plaque bacterium Streptococcus cristatus is decorated with a lateral tuft of fibrils. The fibrillar tuft functions in the adhesion of S. cristatus to heterologous bacterial species in the plaque biofilm. The tuft typically consists of a densely packed fringe of shorter fibrils 238 +/- 19 nm long with longer, less abundant fibrils 403 +/- 66 nm long projecting through the fringe of short fibrils. The two types of fibrils in the tufts of S. cristatus have been refractory to biochemical separation, complicating their characterization. A hexadecane partition assay was used to enrich for subpopulations of S. cristatus CR311 (type strain NCTC 12479) having distinct fibrillar morphotypes. Negative staining in the TEM revealed that cells of a hydrophobic subpopulation of S. cristatus (CR311var1) carried only the long fibrils (395 +/- 32 nm). A hydrophilic subpopulation of S. cristatus (CR311var3) consisted of mixed morphotypes having no fibrils or remnant short fibrils (223 +/- 49 nm). No long fibrils were observed on any cells in the CR311var3 subpopulation. The CR311var3 morphotype, unlike the wild-type strain and CR311var1, was not able to form corncobs with either Corynebacterium matruchotii or Fusobacterium nucleatum. Variant CR311var3 did not express the novel gene srpA, which encodes a high molecular weight (321,882 Da) serine-rich protein, SrpA. The SrpA protein contains two extensive repeat motifs of 17 and 71 amino acids and a gram-positive cell wall anchor consensus sequence (LPNTG). The unusual properties of SrpA most closely resemble those of Fap1, the fimbrial-associated adhesin protein of Streptococcus parasanguis. The association of long fibrils, high surface hydrophobicity, ability to form corncob formations, and expression of the srpA gene suggest that SrpA is a long fibril protein in S. cristatus.

Genome size of human oral Treponema species by pulsed-field gel electrophoresis.

The genome sizes of seven strains of oral treponemes were determined using pulsed-field gel electrophoresis (PFGE). These strains represent members from six of the currently known cultivable oral treponeme groups. The PFGE fragments were digitally recorded and then quantitated using GIMP v 1.2, an image manipulation program. The results show that the six oral treponeme genomes are comparable in size, ranging from approximately 2.2 to 2.5 Mbp. The genome sizes of these strains are 20-25% smaller than Treponema denticola strains, which have genome sizes of approximately 2.8-3.0 Mbp.

Natural transformation of Streptococcus crista.

Over the years Streptococcus gordonii (sanguis) Challis has become the workhorse of genetic manipulations for the sanguis group of oral streptococci. This is because strain Challis was shown in early studies to be highly naturally competent for transformation. However, Challis is not usually the most appropriate strain to use in studies which focus on oral microbial adherence. We report that other members of the newly reorganized sanguis group, particularly within the species S. crista, display reasonable transformation frequencies, with both plasmid and chromosomal DNA, if transformed at the appropriate time during the growth curve. The ability to transform S. crista may be especially important for genetic studies of biological properties that appear to be limited to these specific streptococcal strains.

scbA from Streptococcus crista CC5A: an atypical member of the lraI gene family.

A new member of the lraI family of putative adhesin genes was cloned, from Streptococcus crista CC5A, and sequenced. The gene, scbA appears to be part of an ABC transport operon and encodes a putative peptide of 34.7 kDa. The protein contains a signal sequence with residues 17 to 21 (L-A-A-C-S) matching the consensus sequence for the prolipoprotein cleavage site of signal peptidase II. ScbA is 57 to 93% identical, at the amino acid level, with the five previous sequenced members of the LraI family. Surprisingly, ScbA does not exhibit adhesion properties characteristic of the other LraI proteins. Strain CC5A bound poorly to saliva-coated hydroxyapatite and did not coaggregate with Actinomyces naeslundii PK606. An scbA insertion-duplication mutation that abolished expression (of ScbA was created. There was no difference in fibrin binding between this mutant and wild-type CC5A. Since it is possible that ScbA could play a role in corncob formation between S. crista and Fusobacterium nucleatum, this property was examined. The mutant strain retained the ability to form corncobs. On the basis of the lack of adhesin properties it appears that ScbA is an atypical member of the LraI family.

[Classical pacemaker syndrome. Report of 2 cases with severe clinical manifestations]. / Síndroma do portador de pacemaker clássico. A propósito de dois casos com manifestações clínicas aparatosas.

We report two cases of classic pacemaker syndrome. Both patients developed severe clinical manifestations after implantation of a VVI pacemaker. One patient presented syncopal episodes and the other one manifest cardiac heart failure. Additionally, we review the mechanisms responsible for this clinical entity.

Insertional inactivation of binding determinants of Streptococcus crista CC5A using Tn916.

Intermicrobial binding plays an important role in the ecology of the oral cavity because it represents one mechanism by which specific bacteria colonize dental plaque. The formation of "corncobs", a morphologically distinct microbial unit composed of Streptococcus crista and Fusobacterium nucleatum, is a highly specific binding interaction that depends on the presence of polar tufts of fimbriae on the streptococci. We have used a genetic approach to examine the role of streptococcal cell surface components involved in the binding of S. crista to F. nucleatum. Such binding may be an important component of corncob formation. A method for the genetic transformation of S. crista was used to transfer the broad host range transposon, Tn916, into the bacteria. Cells were grown to early log phase in brain heart infusion broth containing 10% fetal calf serum. The competent cells were mixed with purified DNA from pDL916, a plasmid construct consisting of Tn916 and the streptococcal/Escherichia coli shuttle vector pDL278. Over 300 transformants were screened for a reduction in binding to F. nucleatum. Five of the transformants showed a change in binding ranging from 59% to 29% of the positive control values. Southern blots revealed that the binding-deficient transformants contained the Tn916 element integrated into one of 4 different sites in the chromosome. The transposon, integrated into 4 different sites, appeared to be stable in the absence of selective pressure. Based on these findings, it appears that some strains of S. crista are naturally competent and that insertional inactivation methods can be used to facilitate the study of binding receptors in this group of oral streptococci.

Rescue of a foreign gene by Sendai virus.

A simple protocol for the rescue of a synthetic genome into a paramyxovirus has been developed. First, a synthetic Sendai virus-like RNA, containing the antisense coding region of the chloramphenicol acetyltransferase gene replacing the coding region of the Sendai virus genome, was transcribed from a cDNA. When introduced into cells that are infected with Sendai virus, this RNA construct was transcribed, replicated, and packaged into infectious virions. The addition of infected cell extract to the RNA prior to transfection markedly enhanced levels of chloramphenicol acetyltransferase expression and rescue. However, this enhancement is not due to encapsidation of the RNA into nucleocapsids as the RNA remains nuclease-sensitive. Uninfected cell extract also enhances expression and rescue efficiency, implying involvement of a cellular factor(s) with the synthetic viral-like RNA construct that allows for enhanced polymerase recognition. This system should allow for the dissection of the various cis-acting RNA signals within the paramyxovirus genome.

A family of small repeated elements with some transposon-like properties in the genome of Neisseria gonorrhoeae.

A physical technique known as two-dimensional S1 nuclease heteroduplex mapping has been applied to genomic DNA from the Gram-negative coccus Neisseria gonorrhoeae. This has resulted in the detection of two novel types of repetitive sequences. The first type is a repetitive sequence family of 152 base pairs (bp), whose ends are composed of inverted repeats of 26 bp. There are approximately 20 copies of this sequence, in both N. gonorrhoeae and Neisseria meningitidis (Correia, F., Inouye, S., and Inouye, M. (1986) J. Bacteriol. 167, 1009-1011). The second type of sequence is a 1443-bp duplication in the N. gonorrhoeae genome. The two classes of sequence are linked positionally. Each copy of the long duplicated sequence is adjacent to a member of the 152-bp repetitive sequence. In one instance two copies of the 152-bp repetitive sequence are separated by a 436-bp central region and are in an inverted orientation with respect to one another, resembling a compound transposable element.

A 26-base-pair repetitive sequence specific for Neisseria gonorrhoeae and Neisseria meningitidis genomic DNA.

Two-dimensional heteroduplex mapping of Neisseria gonorrhoeae genomic DNA revealed a number of spots, indicating the existence of repetitive sequences. When one of the spots was extracted and used as a probe for Southern blot analysis, two HindIII bands (11.0 and 3.6 kilobases [kb]) of the genomic digest hybridized with approximately equal intensity. The 3.6-kb fragment was cloned and found to contain two different types of repeated sequence. One type was approximately 1.1 kb in length and was found at least twice in the entire genome. The other consisted of a 26-base-pair family GT(C/A)C(Py)G(Pu)TTTTTGTTAAT(Py)C(Pu)CTATA (Py, pyrimidine; Pu, purine) that was repeated at least 20 times in the entire genome. This repetitive sequence was found also in Neisseria meningitidis but not in various other gram-negative bacteria.