Production and purification of Escherichia coli fimbrial antigen F165

Abstract The production of fimbrial antigen F165 by Escherichia coli strains was found to be dependent on the composition of the culture medium and was repressed in the presence of alanine or high levels of glucose, in anaerobic conditions or at growth temperatures of lower than 37°C. Optimal F165 production was found on a minimal medium containing 1% (w/v) casamino acids (MD-1). F165 antigen was isolated from bacteria by mechanical shearing, precipitated with ammonium sulfate, and purified by deoxycholate treatment and gel filtration on Superose 12. The purified fimbriae retained their native morphology as observed by electron microscopy and consisted of two separate protein subunits with apparent molecular weights of 17 500 and 19 000 on sodium dodecyl sulfate-polyacrylamide gels.     

Replication banding in the chromosomes of the European eel (Anguilla anguilla)

The chromosomes of the European eel Anguilla anguilla have been analyzed with a replication banding technique from lymphocyte cultures treated with 5-BrdU. This technique allows us to identify with high resolution the individual chromosome pairs and to differentiate classes of chromatin by the order of replication. The replication banding obtained on the chromosomes of European eel can be related with the structural bands described in this species.     

Purification and characterization of 4-hydroxybenzoate 3-hydroxylase from aKlebsiella pneumoniae mutant strain

Unlike the parent wild-type strain, theKlebsiella pneumoniae mutant strain MAO4 has a 4-HBA⁺ phenotype. The capacity of this mutant to take up and metabolize 4-hydroxybenzoate (4-HBA) relies on the expression of a permease and an NADPH-linked monooxygenase (4-HBA-3-hydroxylase). Both enzymes are normally expressed at basal levels, and only the presence of 4-HBA in the media enhances their activities. Strikingly, when theAcinetobacter calcoaceticus pobA gene encoding 4-hydroxybenzoate-3-hydroxylase was expressed in hydroxybenzoateK. pneumoniae wild-type, the bacteria were unable to grow on 4-HBA, suggesting that the main difference between the wild-type and the mutant strain is the capability of the latter to take up 4-HBA. 4-HBA-3-hydroxylase was purified to homogeneity by affinity, gel-filtration, and anion-exchange chromatography. The native enzyme, which appeared to be a dimer of identical subunits, had an apparent molecular mass of 80 kDa and a pI of 4.6. Steady-state kinetics were analyzed; the initial velocity patterns were consistent with a concerted substitution mechanism. The purified enzyme had 362 amino acid residues, and a tyrosine seemed to be involved in substrate activation.     

Some peptide-like colocalizations in endocrine cells of the pyloric caeca and the intestine of Oncorhynchus mykiss (Teleostei)

Summary The coexistence of immunoreactivities to cholecystokinin, glucagon, glucagon-like peptide 1, salmon pancreatic polypeptide, neuropeptide tyrosine, and peptide tyrosine tyrosine was studied immunocytochemicaly, revealing for the first time in fish intestine the existence in the same cell of immunoreactivities to cholecystokinin-glucagon/glucagon-like peptide 1, cholecystokinin-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-salmon pancreatic polypeptide, glucagon/glucagon-like peptide 1-neuropeptide tyrosine, salmon pancreatic polypeptide tyrosine tyrosine, and glucagon/glucagon-like peptide 1-peptide tyrosine tyrosine. Colocalization of cholecystokinin-salmon pancreatic polypeptide was observed only in the pyloric caeca of the rainbow trout Oncorhynchus mykiss, while the other colocalizations also occurred in proximal and middle intestinal segments. In all cases, endocrine cells immunoreactive to only one of the paired antisera were detected except for anti-glucagon and anti-glucagon-like peptide 1, which always immunostained the same cells.     

Antibody response to Candida albicans cell wall antigens

The cell wall of Candida albicans is not only the structure where many essential biological functions reside but is also a significant source of candidal antigens. The major cell wall components that elicit a response from the host immune system are proteins and glycoproteins, the latter being predominantly mannoproteins. Both carbohydrate and protein moieties are able to trigger immune responses. Proteins and glycoproteins exposed at the most external layers of the wall structure are involved in several types of interactions of fungal cells with the exocellular environment. Thus, coating of fungal cells with host antibodies has the potential to profoundly influence the host-parasite interaction by affecting antibody-mediated functions such as opsonin-enhanced phagocytosis and blocking the binding activity of fungal adhesins to host ligands. In this review we examine various members of the protein and glycoprotein fraction of the C. albicans cell wall that elicit an antibody response in vivo. Some of the studies demonstrate that certain cell wall antigens and anti-cell wall antibodies may be the basis for developing specific and sensitive serologic tests for the diagnosis of candidiasis, particularly the disseminated form. In addition, recent studies have focused on the potential of antibodies against the cell wall protein determinants in protecting the host against infection. Hence, a better understanding of the humoral response triggered by the cell wall antigens of C. albicans may provide the basis for the development of (i) effective procedures for the serodiagnosis of disseminated candidiasis, and (ii) novel prophylactic (vaccination) and therapeutic strategies to control this type of infections.     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

Factors that affect the re-attachment of Chondracanthus chamissoi (Rhodophyta, Gigartinales) thalli

Vegetative reproduction of Chondracanthus chamissoi by means of fragmentation and re-attachment of thalli is considered an effective strategy for maintaining natural populations of this species. Here, we evaluate the effects of (1) time of drifting thallus, (2) type of substratum, and (3) photon flux density, on the re-attachment capacity of thallus fragments of C. chamissoi. The results show that re-attachment decreases with the time after detachment, and was higher at the lower photon flux densities tested (10 and 40 μmol photons m⁻²s⁻¹), and on calcareous substratum. Secondary attachment discs are formed along the entire surface of the fragment.     

Stability of <Emphasis Type="Italic">Potato virus X</Emphasis> expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert–deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.     

Microsatellite and mtDNA analysis of the population structure of grey seals (Halichoerus grypus) from three breeding areas in the Baltic Sea

The growing number of grey seals in the Baltic Sea has led to a dramatic increase in interactions between seals and fisheries. The conflict has become such a problem that hunting was introduced in Finland in 1998 and the Swedish Environment Protection Agency recommended a cull of grey seals starting in 2001. Culling has been implemented despite the lack of data on population structure. Low levels of migration between regions would mean that intensive culling in specific geographic areas would have disproportionate effects on local population structure and genetic diversity. We used eight microsatellite loci and a 489 bp section of the mtDNA control region to examine the genetic variability and differentiation between three breeding sites in the Baltic Sea and two in the UK. We found high levels of genetic variability in all sampled Baltic groups for both the microsatellites and the control region. There were highly significant differences in microsatellite allele frequencies between all three Baltic breeding sites and between the Baltic sites and the UK sites. However, there were no significant differences in mtDNA control region haplotypes between the Baltic sites. This genetic substructure of the Baltic grey seal populations should be taken into consideration when managing the seal population to prevent the hunting regime from having an adverse effect on genetic diversity by setting hunting quotas separately for the different subpopulations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.