Analysis of the SDS−Lysozyme binding isotherm

A scheme to describe SDS−lysozyme complex formation has been proposed on the basis of isothermal titration calorimetry (ITC) and FTIR spectroscopy data. ITC isotherms are convoluted and reveal a marked effect of both SDS and lysozyme concentration on the stoichiometry of the SDS−lysozyme complex. The binding isotherms have been described with the aid of FTIR spectroscopy in terms of changes in the lysozyme structure and the nature of the SDS binding. At low SDS concentrations, ITC isotherms feature an exothermic region that corresponds to specific electrostatic binding of SDS to positively charged amino acid residues on the lysozyme surface. This leads to charge neutralization of the complex and precipitation. The number of SDS molecules that bind specifically to lysozyme is approximately 8, as determined from our ITC isotherms, and is independent of lysozyme solution concentration. At high SDS concentrations, hydrophobic cooperative association dominates the binding process. Saturated binding stoichiometries as a molar ratio of SDS per molecule of lysozyme range from 220:1 to 80:1, depending on the lysozyme solution concentration. A limiting value of 78:1 has been calculated for lysozyme solution concentrations above 0.25 mM.

Analysis of the SDS-lysozyme binding isotherm

A scheme to describe SDS-lysozyme complex formation has been proposed on the basis of isothermal titration calorimetry (ITC) and FTIR spectroscopy data. ITC isotherms are convoluted and reveal a marked effect of both SDS and lysozyme concentration on the stoichiometry of the SDS-lysozyme complex. The binding isotherms have been described with the aid of FTIR spectroscopy in terms of changes in the lysozyme structure and the nature of the SDS binding. At low SDS concentrations, ITC isotherms feature an exothermic region that corresponds to specific electrostatic binding of SDS to positively charged amino acid residues on the lysozyme surface. This leads to charge neutralization of the complex and precipitation. The number of SDS molecules that bind specifically to lysozyme is approximately 8, as determined from our ITC isotherms, and is independent of lysozyme solution concentration. At high SDS concentrations, hydrophobic cooperative association dominates the binding process. Saturated binding stoichiometries as a molar ratio of SDS per molecule of lysozyme range from 220: 1 to 80: 1, depending on the lysozyme solution concentration. A limiting value of 78: 1 has been calculated for lysozyme solution concentrations above 0.25 mM.

Adjuvant-free immunization with hemagglutinin-Fc fusion proteins as an approach to influenza vaccines

The hemagglutinins (HAs) of human H1 and H3 influenza viruses and avian H5 influenza virus were produced as recombinant fusion proteins with the human immunoglobulin Fc domain. Recombinant HA-human immunoglobulin Fc domain (HA-HuFc) proteins were secreted from baculovirus-infected insect cells as glycosylated oligomer HAs of the anticipated molecular mass, agglutinated red blood cells, were purified on protein A, and were used to immunize mice in the absence of adjuvant. Immunogenicity was demonstrated for all subtypes, with the serum samples demonstrating subtype-specific hemagglutination inhibition, epitope specificity similar to that seen with virus infection, and neutralization. HuFc-tagged HAs are potential candidates for gene-to-vaccine approaches to influenza vaccination.

In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria. In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism. Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied. The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.

Development of antimicrobial synbiotics using potentially-probiotic faecal isolates of Lactobacillus fermentum and Bifidobacterium longum

The aims of the present study were to investigate in vitro the antimicrobial activity of Lactobacillus fermentum and Bifidobacterium longum, isolated from faeces of healthy elderly individuals, against enterohaemorrhagic Escherichia coli (E. coli O157:H7) and enteropathogenic E. coli (E. coli O86), to determine the capability of the selected strains to tolerate acid and bile in vitro, to select suitable carbohydrates in order to enhance the growth and maximise antimicrobial activity of the putative probiotic organisms and examine the adhesion properties of the synbiotics. Antimicrobial activity of the putative probiotics and synbiotics was investigated by a microtitre method using cell-free culture supernatants (CFCS). Results of the antimicrobial assay showed that both putative probiotic strains produced compounds at pH 5 that lead to higher lag phases of both E. coli O157:H7 and E. coli O86. When half the quantity of cell-free culture supernatants of both probiotic strains was used at pH 5, B. longum maintained the same antimicrobial effect against both strains of E. coli, whereas L. fermentum lead to a higher lag phase of E. coli O86 only. Neutralization of the culture supernatants with alkali reduced the antimicrobial effect with only cell-free supernatant of L. fermentum causing lower maximum growth rates of E. coli O157:H7 and E. coli O86. L. fermentum appeared to be acid tolerant whereas B. longum was more susceptible to acid and both isolates were bile tolerant. A short chain fructooligosaccharide (scFOS) and an isomalto-oligosaccharide (IMO) proved to be the most effective substrates, enhancing antimicrobial activity for L. fermentum and B. longum respectively. The adhesion of the synbiotic combinations showed that L. fermentum, exhibited higher percentage of adhesion when grown on glucose and as a synbiotic combination with scFOS whereas B. longum exhibited lowest percentage of adhesion when grown on both glucose and IMO.

Nonlinear saturation of baroclinic instability: part II: continuously stratified fluid

Rigorous upper bounds are derived that limit the finite-amplitude growth of arbitrary nonzonal disturbances to an unstable baroclinic zonal flow in a continuously stratified, quasi-geostrophic, semi-infinite fluid. Bounds are obtained bath on the depth-integrated eddy potential enstrophy and on the eddy available potential energy (APE) at the ground. The method used to derive the bounds is essentially analogous to that used in Part I of this study for the two-layer model: it relies on the existence of a nonlinear Liapunov (normed) stability theorem, which is a finite-amplitude generalization of the Charney-Stern theorem. As in Part I, the bounds are valid both for conservative (unforced, inviscid) flow, as well as for forced-dissipative flow when the dissipation is proportional to the potential vorticity in the interior, and to the potential temperature at the ground. The character of the results depends on the dimensionless external parameter γ = f02ξ/β0N2H, where ξ is the maximum vertical shear of the zonal wind, H is the density scale height, and the other symbols have their usual meaning. When γ ≫ 1, corresponding to “deep” unstable modes (vertical scale ≈H), the bound on the eddy potential enstrophy is just the total potential enstrophy in the system; but when γ≪1, corresponding to ‘shallow’ unstable modes (vertical scale ≈γH), the eddy potential enstrophy can be bounded well below the total amount available in the system. In neither case can the bound on the eddy APE prevent a complete neutralization of the surface temperature gradient which is in accord with numerical experience. For the special case of the Charney model of baroclinic instability, and in the limit of infinitesimal initial eddy disturbance amplitude, the bound states that the dimensionless eddy potential enstrophy cannot exceed (γ + 1)2/24&gamma2h when γ ≥ 1, or 1/6;&gammah when γ ≤ 1; here h = HN/f0L is the dimensionless scale height and L is the width of the channel. These bounds are very similar to (though of course generally larger than) ad hoc estimates based on baroclinic-adjustment arguments. The possibility of using these kinds of bounds for eddy-amplitude closure in a transient-eddy parameterization scheme is also discussed.

Neural stem cells adopt tumorigenic properties by constitutively activated NF-kappaB and subsequent VEGF up-regulation

One of the challenges in stem cell research is to avoid transformation during cultivation. We studied high passage subventricular zone derived neural stem cells (NSCs) cultures of adult rats in the absence of growth factors epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). We termed this culture exogenous growth factor independent neural stem cells (GiNSCs). GiNSCs expressed stemness markers, displayed a high constitutive NF-kappaB activity and an increased, aberrant, polyploid DNA content. GiNSCs showed a tumorigenic phenotype and formed colonies in a soft agar assay. Microarray analysis showed the up-regulation of the NF-kappaB target gene vascular endothelial growth factor (VEGF). In contrast, proneuronal genes were down-regulated. Under neuronal differentiation conditions GiNSCs adopted a glioma-like phenotype, with nuclear p53, preserving high amounts of Nestin positive cells and prolonged proliferation. Neutralization of VEGF strongly inhibited proliferation and induced differentiation. In a gain of function approach, the transfection of NSCs with constitutively active upstream kinase IKK-2 led to constitutively activated NF-kappaB, proliferation in absence of growth factors and augmented VEGF secretion. In a rescue experiment a reduction of NF-kappaB activity by overexpression of IkappaB-AA1 was able to shift the morphology toward an elongated cell form, increased cell death, and decreased proliferation. Thus GiNSCs may provide a potent tool in cancer research, as their exogenous cytokine independent proliferation and their constitutively high NF-kappaB expression presumes cancerous properties observed in gliomas. In addition, this study might add a novel mechanism for detecting oncogenic transformation in therapeutic stem cell cultures.

Comparison of bioactivities, binding properties and intra-follicular levels of bovine follistatins

Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, 41kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin- and heparan sulphate proteoglycan (HSP)-binding properties and bio-potencies in neutralization of activin-A action in vitro revealed that all five isoforms bound activin-A, but with different affinities. Only the 31kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency with 35 and 41kDa isoforms being least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analyzed bFF from dominant (DF) and subordinate (SF) follicles collected at strategic times during a synchronized estrous cycle. Total FST, activin-A and activin-AB were measured by immunoassay while individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with estrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin-A (r=-0.34), activin-AB (r=-0.80) and ‘total’ FST (r=-0.70) levels. Follicle diameter was positively correlated with abundance of the 41 kDa isoform (r=0.59) but negatively correlated with abundance of 33 and 31 kDa isoforms (r=-0.56, -0.41). Both follicle status (DF vs SF) and cycle stage affected total FST, activin-A, activin-B levels while follicle status, but not cycle stage, affected abundance of 41, 37, 33 and 31kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms that differ in their ability to bind and neutralise activins and associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may have an important negative role, either directly or via inhibition of the positive effects of activins, on follicle growth and function during follicular waves.