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A M Alves

Publications and source records attributed to A M Alves.

At least 19 recordsLinked to original sources
PubMed2001

Different physiological roles of ATP- and PP(i)-dependent phosphofructokinase isoenzymes in the methylotrophic actinomycete Amycolatopsis methanolica.

Cells of the actinomycete Amycolatopsis methanolica grown on glucose possess only a single, exclusively PP(i)-dependent phosphofructokinase (PP(i)-PFK) (A. M. C. R. Alves, G. J. W. Euverink, H. J. Hektor, J. van der Vlag, W. Vrijbloed, D.H.A. Hondmann, J. Visser, and L. Dijkhuizen, J. Bacteriol. 176:6827-6835, 1994). When this methylotrophic bacterium is grown on one-carbon (C(1)) compounds (e.g., methanol), an ATP-dependent phosphofructokinase (ATP-PFK) activity is specifically induced, completely replacing the PP(i)-PFK. The two A. methanolica PFK isoenzymes have very distinct functions, namely, in the metabolism of C(6) and C(1) carbon substrates. This is the first report providing biochemical evidence for the presence and physiological roles of PP(i)-PFK and ATP-PFK isoenzymes in a bacterium. The novel ATP-PFK enzyme was purified to homogeneity and characterized in detail at the biochemical and molecular levels. The A. methanolica ATP-PFK and PP(i)-PFK proteins possess a low level of amino acid sequence similarity (24%), clearly showing that the two proteins are not the result of a gene duplication event. PP(i)-PFK is closely related to other (putative) actinomycete PFK enzymes. Surprisingly, the A. methanolica ATP-PFK is most similar to ATP-PFK from the protozoon Trypanosoma brucei and PP(i)-PFK proteins from the bacteria Borrelia burgdorferi and Treponema pallidum, both spirochetes, very distinct from actinomycetes. The data thus suggest that A. methanolica obtained the ATP-PFK-encoding gene via a lateral gene transfer event.

Actinomycetales↗
PubMed2000

DNA immunisation against the CFA/I fimbriae of enterotoxigenic Escherichia coli (ETEC).

The CFA/I fimbria promotes the attachment of enterotoxigenic Escherichia coli (ETEC) to the surface of human enterocytes. The generation of a protective immune response requires the induction of antibodies able to block the CFA/I-mediated binding of ETEC to receptors located on the small intestine epithelium or on the surface of human red blood cells, in hemagglutination tests. An eukaryotic expression plasmid, pBLCFA, encoding the CFA/I gene under the control of the human cytomegalovirus major immediate-early promoter was constructed as a prototype DNA vaccine against ETEC. pBLCFA-tranfected BHK-21 cells secreted a peptide cross-reacting with a monoclonal antibody raised against CFA/I subunits. BALB/c mice immunized intramuscularly with one or two doses of purified pBLCFA developed CFA/I-specific serum antibodies for at least 52 weeks, composed predominantly of the IgG1 subclass. pBLCFA-induced antibodies bind mainly to epitopes exposed on the surface of intact CFA/I fimbriae and do not react with immune recessive epitopes found in other ETEC fimbra sharing amino acid homologies with CFA/I. Furthermore, pBLCFA-induced antibodies were able to block the adhesive properties of the CFA/I fimbriae, as evaluated by the ability to inhibit the hemagglutination promoted by CFA/I-expressing ETEC cells. These results suggest that secretion of CFA/I encoded by pBLCFA preserves important conformational epitopes required for the generation of protective antibodies against the adhesive properties of the CFA/I fimbriae and open new perspectives for the development of DNA vaccines against enteric bacterial pathogens.

Animals↗
PubMed1999

Antibody response in mice immunized with a plasmid DNA encoding the colonization factor antigen I of enterotoxigenic Escherichia coli.

The colonization factor antigen I (CFA/I) is one of the most epidemiologically relevant enterotoxigenic Escherichia coli (ETEC) fimbrial adhesins, which mediates the binding to human small intestine epithelium. A recombinant eukaryotic expression plasmid, pRECFA, encoding the CFA/I protein fused to the glycoprotein D of herpes simplex type 1 virus, was used to generate an antibody response in a murine model following intramuscular inoculation of purified DNA. Eukaryotic cells (BHK-21) transfected with pRECFA expressed the CFA/I protein in vitro, as revealed by Western blot and immunofluorescence microscopy. Administration of a single pRECFA 100-microg dose induced a long-term CFA/I-specific antibody response in BALB/c mice composed mainly of IgG and, to a lesser extent, IgA isotypes. The major CFA/I-specific IgG subclass was IgG2a, suggesting a Th-1-type immune response. A second dose with the same amount of purified DNA, given 2 weeks later, caused a booster effect on the immunoglobulin levels, but did not qualitatively alter the isotypes and subclasses of the induced antibody response. Immunization with different amounts of purified DNA and/or number of doses showed that maximal transient CFA/I-specific antibody levels could be obtained after two 100-microg doses of pRECFA given 2 weeks apart, but long-term antibody levels were similar.

Animals↗
PubMed1999

New vaccine strategies against enterotoxigenic Escherichia coli. I: DNA vaccines against the CFA/I fimbrial adhesin.

Stimulation of the mammalian immune system by administration of plasmid DNA has been shown to be an important approach for vaccine development against several pathogens. In the present study we investigated the use of DNA vaccines to induce immune responses against an enteric bacterial pathogen, enterotoxigenic Escherichia coli (ETEC). Three plasmid vectors encoding colonization factor antigen 1 (CFA/I), an ETEC fimbrial adhesin, were constructed. Eukaryotic cells transfected with each of these plasmids expressed the heterologous antigen in different compartments: bound to the cytoplasmic membrane (pRECFA), accumulated in the cytoplasm (pPolyCFA) or secreted to the outside medium (pBLCFA). BALB/c mice were intramuscularly (i.m.) inoculated with purified plasmid DNA and the systemic, cellular and secreted CFA/I-specific immune responses were analyzed. The results showed that all three DNA vaccine formulations could elicit CFA/I-specific immune responses. Moreover, cellular location of the plasmid-encoded CFA/I seems to have an important role in the induced immune response. Taken together, these results indicate that DNA vaccines also represent a promising approach against enteric bacterial pathogens.

Adhesins, Escherichia coli↗
PubMed1999

New vaccine strategies against enterotoxigenic Escherichia coli. II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine.

The induction of systemic (IgG) and mucosal (IgA) antibody responses against the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC) was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen. The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine. The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone. These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

Animals↗
PubMed1998

Immunoglobulin G subclass responses in mice immunized with plasmid DNA encoding the CFA/I fimbria of enterotoxigenic Escherichia coli.

Balb/c mice immunized either with a plasmid vector (pRECFA), encoding the CFA/I fimbrial adhesin of enterotoxigenic Escherichia coli (ETEC), or purified CFA/I fimbriae induced similar antibody responses in regard to the kinetics of serum total immunoglobulins and IgG. Nonetheless, the IgG subclass responses in mice vaccinated with DNA were composed predominantly by IgG2a (ranging from 39 to 74% of the total anti-CFA/I IgG) and, to a lesser extent, by IgG (ranging from 15 to 24% of the total anti-CFA/I IgG) during a 12 week observation period. On the other hand, mice immunized with purified CFA/I presented mainly an IgG1 antibody response (ranging from 39 to 67% of the total anti-CFA/I IgG). These results indicated that, irrespective of the immunogenic properties and-or origin of the encoded antigen, immunization with pRECFA elicited an specific IgG subclass response which may affect the ability of DNA vaccines to induce a protective immune response against CFA/I mediated colonization of ETEC bacterial cells.

Animals↗
PubMed1998

Epitope specificity of antibodies raised against enterotoxigenic Escherichia coli CFA/I fimbriae in mice immunized with naked DNA.

The cfaB gene, coding for the CFA/I fimbrial adhesin of enterotoxigenic Escherichia coli (ETEC), was cloned and expressed as a fusion peptide with the glycoprotein D (gD) from herpes simplex virus type 1 (HSV) in the pRE4 eukaryotic expression vector, resulting in the recombinant plasmid pRECFA. All BALB/c mice injected intramuscularly (i.m.) with a single dose (100 micrograms) of the purified plasmid developed antibodies against epitopes found on dissociated CFA/I subunits as well as other homologous ETEC fimbriae. Surface-exposed epitopes found on intact CFA/I fimbriae were also recognized by antibodies derived from DNA immunization, but they did not overlap with those generated with purified CFA/I fimbriae. None of the sera raised in mice immunizated with pRECFA were able to agglutinate bacterial cells or inhibit haemagglutination promoted by CFA/I bearing ETEC cells. These results show that pRECFA can elicit CFA/I-specific antibodies, which may have different epitope specificities and functional properties compared with those generated with purified bacterial protein.

Animals↗
PubMed1997

Identification of ATP-dependent phosphofructokinase as a regulatory step in the glycolytic pathway of the actinomycete Streptomyces coelicolor A3(2).

The ATP-dependent phosphofructokinase (ATP-PFK) of Streptomyces coelicolor A3(2) was purified to homogeneity (1,600-fold) and characterized (110 kDa, with a single type of subunit of 40 kDa); it is allosterically inhibited by phosphoenolpyruvate. Cloning of the pfk gene of S. coelicolor A3(2) and analysis of the deduced amino acid sequence (343 amino acids; 36,667 Da) revealed high similarities to the PPi-PFK enzyme from Amycolatopsis methanolica (tetramer, nonallosteric; 70%) and to the allosteric ATP-PFK enzymes from other bacteria, e.g., Escherichia coli (tetramer; 37%) and Bacillus stearothermophilus (tetramer, 41%). Further structural and functional analysis of the two actinomycete PFK enzymes should elucidate the features of these proteins that determine substrate specificity (ATP versus PPi) and allosteric (in)sensitivity.

Adenosine Triphosphate↗
PubMed1996

Genomic variation in Trypanosoma cruzi clonal cultures.

Spontaneous changes in restriction DNA profiles and pulsed-field gel electrophoresis (PFGE) patterns, along with a concomitant loss of infectivity, were observed in infective clones of Trypanosoma cruzi strain Y either following a number of passages during the exponential growth phase of after subcloning in liver infusion tryptone (LIT) medium using as the probe a genomic fragment of the parasite (pMYP16), indicating naturally occurring rearrangements of DNA sequences. No variation could be detected when the genomic DNA was probed with conserved T. cruzi tubulin and actin genes. There was no correlation between such rearrangements and the life-cycle forms of the parasites, since trypomastigote forms showed the same karyotype and hybridization patterns as did epimastigote forms. The variations observed could be reverted and infectivity, recovered after inoculation of the parasites in newborn mice.

Actins↗
PubMed1996

Characterization and phylogeny of the pfp gene of Amycolatopsis methanolica encoding PPi-dependent phosphofructokinase.

The actinomycete Amycolatopsis methanolica employs a PPi-dependent phosphofructokinase (PPi-PFK) (EC 2.7.1.90) with biochemical characteristics similar to those of both ATP- and PPi-dependent enzymes during growth on glucose. A 2.3-kb PvuII fragment hybridizing to two oligonucleotides based on the amino-terminal amino acid sequence of PPi-PFK was isolated from a genomic library of A. methanolica. Nucleotide sequence analysis of this fragment revealed the presence of an open reading frame encoding a protein of 340 amino acids with a high degree of similarity to PFK proteins. Heterologous expression of this open reading frame in Escherichia coli gave rise to a unique 45-kDa protein displaying a high level of PPi-PFK activity. The open reading frame was therefore designated pfp, encoding the PPi-PFK of A. methanolica. Upstream and transcribed divergently from pfp, a partial open reading frame (aroA) similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes was identified. The partial open reading frame (chiA) downstream from pfp was similar to chitinase genes from Streptomyces species. A phylogenetic analysis of the ATP- and PPi-dependent proteins showed that PPi-PFK enzymes are monophyletic, suggesting that the two types of PFK evolved from a common ancestor.

Actinobacteria↗
PubMed1994

Enzymes of glucose and methanol metabolism in the actinomycete Amycolatopsis methanolica.

The actinomycete Amycolatopsis methanolica was found to employ the normal bacterial set of glycolytic and pentose phosphate pathway enzymes, except for the presence of a PPi-dependent phosphofructokinase (PPi-PFK) and a 3-phosphoglycerate mutase that is stimulated by 2,3-bisphosphoglycerate. Screening of a number of actinomycetes revealed PPi-PFK activity only in members of the family Pseudonocardiaceae. The A. methanolica PPi-PFK and 3-phosphoglycerate mutase enzymes were purified to homogeneity. PPi-PFK appeared to be insensitive to the typical effectors of ATP-dependent PFK enzymes. Nevertheless, strong N-terminal amino acid sequence homology was found with ATP-PFK enzymes from other bacteria. The A. methanolica pyruvate kinase was purified over 250-fold and characterized as an allosteric enzyme, sensitive to inhibition by P(i) and ATP but stimulated by AMP. By using mutants, evidence was obtained for the presence of transketolase isoenzymes functioning in the pentose phosphate pathway and ribulose monophosphate cycle during growth on glucose and methanol, respectively.

Actinobacteria↗
PubMed1993

Reversible changes in the isoenzyme electrophoretic mobility pattern and infectivity in clones of Trypanosoma cruzi.

The stability of zymodemes in clonal cultures of Trypanosoma cruzi derived from strain Y was followed. Reversible changes in the isoenzyme electrophoretic mobility pattern from type A to types B and C were observed after subculturing of cloned cultures in medium of different composition or after passage in newborn mice. Type A zymodeme was observed in clones grown in blood-containing media, while types B and C were found in clones and subclones grown in media progressively less rich in nutrients (10 and 5% fetal calf serum, respectively) and containing no blood. The change in zymodeme from type A to type B or C was associated with loss of infectivity, which could be recovered by passages in newborn mice. Parasites infective for mice always showed zymodeme A. Simultaneously with zymodeme change from type A to types B and C there is a decrease in the specific activity of G6PD and 6PGD.

Animals↗
PubMed1986

Measurement of lipogenesis in isolated preputial gland cells of the rat and the effect of oestrogen.

Lipogenesis was measured in isolated preputial gland cells of female rats after ovariectomy and after the administration of oestradiol benzoate. Ovariectomy decreased preputial gland cell lipogenesis and also altered the pattern of lipid synthesis, producing a relative decrease in the proportion of polar lipids and an increase in the proportion of 'triglycerides'. Although daily administration of 2 or 10 micrograms oestradiol benzoate for 7 days produced slight increases in preputial gland cell lipogenesis in ovariectomized rats, the effects were not significant. A single injection of 10 micrograms oestradiol benzoate, however, produced significant increases in preputial gland cell lipogenesis of ovariectomized rats at both 2 and 24 h and, moreover, at 24 h the pattern of polar lipid and triglyceride labelling was restored to normal. Prior administration of actinomycin D reduced the lipogenic effect of oestradiol benzoate. Oestradiol benzoate had little or no effect on preputial gland cell lipogenesis in male rats. These results confirm that oestrogen is able to stimulate preputial lipogenesis in female rats. Whether this action of oestrogen is related to its pheromone-producing effect on the preputial glands is not yet known.

Animals↗
PubMed1983

Noninvasive versus invasive evaluation of the extracranial circulation in patients with suspected cerebral vascular disease.

This is the second in an ongoing series of evaluations in an attempt to find a combined system of noninvasive cerebral vascular evaluation of the extracranial circulation reliable enough so that decisions can be made, utilizing this system regarding further diagnostic studies and management. The current study evaluates a system of combined doppler arterial scanning of the extracranial circulation with Real Time B-Mode imaging of the territory of the carotid bifurcations and show that such a system, in skilled hands, is capable of very high levels of accuracy both quantitatively and qualitatively regarding the status of the carotid bifurcations as well as giving much indirect information about the cerebral vascular and subclavian circulations. The paper ends with a strong statement supporting the routine utilization of noninvasive screening of patients with suspected cerebral vascular disease. This conclusion is reached after comparing 59 patients representing 118 carotid bifurcations studied; first by physical examination and noninvasive evaluation and subsequently by angiography.

Adult↗