Biomedical subjects

Airi Palva

Publications and source records attributed to Airi Palva.

27 records · Page 2Linked to original sources

PubMed2003

Cowpox with severe generalized eruption, Finland.

Cowpox with a severe, generalized eruption was diagnosed in an atopic 4-year-old girl by electron microscopy, virus isolation, polymerase chain reaction, and immunoglobulin (Ig) M and low-avidity IgG antibodies. The hemagglutinin gene of the isolate clustered with a Russian cowpox virus strain, and more distantly, with other cowpox and vaccinia virus strains. The patient's dog had orthopoxvirus-specific antibodies, indicating a possible transmission route. In Finnish wild rodents, orthopoxvirus seroprevalences were 0%-92%, in humans the seroprevalence was 100% in the age group >50, decreasing towards younger age groups.

Animals↗

PubMed2002

Genetic characterization of an oligopeptide transport system from Lactobacillus delbrueckii subsp. bulgaricus.

The operon of the putative lactobacillar oligopeptide transport system (Opp) from Lactobacillus delbrueckii subsp. bulgaricus B14 was cloned and characterized. The opp operon was found to consist of five genes, oppD, oppF, oppB, oppC and oppA (1). In addition, an oppA (1) homolog, oppA (2), was found downstream of the operon. Sequence comparisons of the L. delbrueckii subsp. bulgaricus Opp system with other bacterial transport systems revealed the highest similarity to the oligopeptide transport system of Lactococcus lactis. Northern analyses of oppmRNAs revealed 6.1-kb and 2.1-kb transcripts, confirming that, in addition to the operon structure oppDFBCA (1), the oppA (1) gene was also expressed as a monocistronic transcript. The oppA (2) gene was expressed as a separate 2.1-kb monocistronic transcript with a low expression level. Primer-extension mapping of the 5'end of oppDFBCA (1) mRNA revealed two adjacent transcriptional start sites, and primer extension analyses of oppA (1) and oppA (2) mRNAs confirmed the location of the predicted promoters of these genes. For complementation analysis, oppA (1) alone and the operon constructs oppDFBCA (1) and oppDFBCA (2) were fused with the nisA promoter and expressed in Lactococcus lactisNZ9000Delta oppA strain. Only the L. delbrueckii subsp. bulgaricus oppDFBCA (1)genes were able to complement the L. lactis oppA mutation.

Amino Acid Sequence↗

PubMed2002

PCR-ELISA II: Analysis of Bifidobacterium populations in human faecal samples from a consumption trial with Bifidobacterium lactis Bb-12 and a galacto-oligosaccharide preparation.

A PCR-ELISA method was extended for detection of most common Bifidobacterium species in humans and applied to a feeding trial including administration of Bifidobacterium lactis Bb-12 and galacto-oligosaccharide (GOS)-containing syrup as probiotic and prebiotic preparations, respectively. For PCR-ELISA, oligonucleotide probes based on 16S rDNA sequences were designed and tested for specificity and sensitivity with nine different bifidobacterial species followed by analysis of faecal samples. Bifidobacteria were monitored for their fluctuations during and after the feeding trial. Bifidobacterium longum was the most common species found in the faecal samples, followed by B. adolescentis and B. bifidum. During ingestion of the probiotic B. lactis Bb-12, the strain appeared in the faeces but was absent again one week after finishing of the trial. The species that were observed in the faecal samples taken prior to the feeding experiments persisted also in samples derived from the pre-feeding and feeding periods. The most consistent change observed was the decrease in the relative amount of B. longum in the test group ingesting either B. lactis Bb-12 alone or in combination with GOS-syrup. Since the amounts of B. longum increased again in the post-feeding sample with these subjects, it may suggest that to some extent B. lactis Bb-12 is able to transiently replace B. longum.

Animals↗

PubMed2002

PCR-ELISA I: Application to simultaneous analysis of mixed bacterial samples composed of intestinal species.

Sixteen oligonucleotide identification probes, designed in this study or adapted from literature, were tested for a PCR-ELISA application to simultaneously detect under standardised conditions selected intestinal bacteria, lactobacilli and bifidobacteria. The level of specificity obtained with most of the probes fulfilled the set criteria. The lack of efficiency of PCR performed with the primers, proposed to be specific for the entire eubacteria domain, and compromises made in hybridisation conditions due to simultaneous usage of multiple probes reduced the sensitivity of the PCR-ELISA test. The method was, however, found to be suitable for detecting predominant members of the intestinal flora. Applicability of the PCR-ELISA test could be further widened using primers with a more restricted specificity in the PCR step, as was demonstrated for the detection of Bifidobacterium with genus-specific primers. Advantages of the PCR-ELISA method include convenient performance and the possibility to test rapidly large amounts of samples with a multitude of probes.

Bacteria↗

PubMed2002

Surface display of foreign epitopes on the Lactobacillus brevis S-layer.

So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.

Bacterial Proteins↗

PubMed2002

Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis.

Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliC(H7) gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.

Bacterial Adhesion↗

PubMed2002

Isolation of three new surface layer protein genes (slp) from Lactobacillus brevis ATCC 14869 and characterization of the change in their expression under aerated and anaerobic conditions.

Two new surface layer (S-layer) proteins (SlpB and SlpD) were characterized, and three slp genes (slpB, slpC, and slpD) were isolated, sequenced, and studied for their expression in Lactobacillus brevis neotype strain ATCC 14869. Under different growth conditions, L. brevis strain 14869 was found to form two colony types, smooth (S) and rough (R), and to express the S-layer proteins differently. Under aerobic conditions R-colony type cells produced SlpB and SlpD proteins, whereas under anaerobic conditions S-colony type cells synthesized essentially only SlpB. Anaerobic and aerated cultivations of ATCC 14869 cells in rich medium also resulted in S-layer protein patterns similar to those of the S- and R-colony type cells, respectively. Electron microscopy suggested the presence of only a single S-layer with an oblique structure on the cells of both colony forms. The slpB and slpC genes were located adjacent to each other, whereas the slpD gene was not closely linked to the slpB-slpC gene region. Northern analyses confirmed that both slpB and slpD formed a monocistronic transcription unit and were effectively expressed, but slpD expression was induced under aerated conditions. slpC was a silent gene under the growth conditions tested. The amino acid contents of all the L. brevis ATCC 14869 S-layer proteins were typical of S-layer proteins, whereas their sequence similarities with other S-layer proteins were negligible. The interspecies identity of the L. brevis S-layer proteins was mainly restricted to the N-terminal regions of those proteins. Furthermore, Northern analyses, expression of a PepI reporter protein under the control of the slpD promoter, and quantitative real-time PCR analysis of slpD expression under aerated and anaerobic conditions suggested that, in L. brevis ATCC 14869, the variation of S-layer protein content involves activation of transcription by a soluble factor rather than DNA rearrangements that are typical for most of the S-layer phase variation mechanisms known.

Aerobiosis↗

PubMed1997

Lactobacilli carry cryptic genes encoding peptidase-related proteins: characterization of a prolidase gene (pepQ) and a related cryptic gene (orfZ) from Lactobacillus delbrueckii subsp. bulgaricus.

Two genes, pepQ and orfZ, encoding a prolidase and a prolidase-like protein, respectively, were cloned and characterized from Lactobacillus delbrueckii subsp. bulgaricus. The identity of the pepQ and orfZ genes with the Lactobacillus delbrueckii subsp. lactis prolidase gene (pepQ) was shown to be 98% and 60%, respectively. Both pepQ and orfZ were preceded by a putative promoter region. Northern analysis of pepQ mRNA revealed a 1.1 kb transcript indicating that pepQ forms a monocistronic transcriptional unit. Under the growth conditions used, no evidence was obtained that orfZ was expressed, either by mRNA size determination in Northern analysis or by primer extension analysis. With reverse transcription-PCR, however, the presence of monocistronic orfZ transcripts was established. The orfZ gene could also be overexpressed in E. coli using the vector pKK223-3. The size of the protein synthesized, 41 kDa, confirmed the molecular mass of OrfZ calculated according to DNA sequence analysis. In contrast to PepQ, which showed a substrate specificity characteristic of prolidase enzymes, no enzymic activity for the orfZ-encoded protein was found with the peptide substrates tested. These results indicate that orfZ is a cryptic gene, which is expressed at a very low level under the growth conditions used. It is noteworthy that homologues of the Lb. delbrueckii subsp. bulgaricus orfZ and pepQ genes appeared to be present in both Lb. delbrueckii subsp. lactis and Lactobacillus helveticus.

Amino Acid Sequence↗

PubMed1996

Characterization of a prolinase gene and its product and an adjacent ABC transporter gene from Lactobacillus helveticus.

A prolinase (pepR) gene was cloned from an industrial Lactobacillus helveticus strain (53/7). Three clones, hybridizing with a gene probe specific for a peptidase shown to have activity against di- and tripeptides, were detected from a L. helveticus genomic library constructed in Escherichia coli. None of the three clones, however, showed enzyme activity against the di- or tripeptide substrates tested. One of the clones, carrying a vector with a 5.5 kb insert, was further characterized by DNA sequencing. The sequence analysis revealed the presence of two ORFs, ORF1 and ORF2 of 912 and 1602 bp, respectively. ORF2, located upstream of and in the opposite orientation to ORF1, had a promoter region overlapping that of ORF1. ORF1 had the capacity to encode a 35083 Da protein. When amplified by PCR, ORF1 with its control regions specified a 35 kDa protein in E. coli that was able to hydrolyse dipeptides, with highest activity against Pro-Leu, whereas from the tripeptides tested, only Leu-Leu-Leu was slowly degraded. By the substrate-specificity profile and protein homologies, the 35 kDa protein was identified as a prolinase. The activity of the cloned prolinase was inhibited by p-hydroxymercuribenzoate. Northern and primer-extension analyses of ORF1 revealed a 1.25 kb transcript and two adjacent transcription start sites, respectively, thus confirming the DNA sequence data. ORF2 had encoding capacity for a 59.5 kDa protein that showed significant homology to several members of the family of ABC transporters. Determination of the mRNA levels at different growth phases revealed that the pepR gene and ORF2 are transcribed in L. helveticus at the exponential and stationary phases of growth, respectively. Furthermore, two ORF2 deletion constructs, carrying the intact pepR gene, showed that this upstream operon adversely affected PepR activity in E. coli, which explains the enzymic inactivity of the original clones.

ATP-Binding Cassette Transporters↗