Kaija H. Valkonen

Binding of Phenolic Compounds and Their Derivatives to Bovine and Reindeer β-Lactoglobulin

In plant-based food, phenolic compounds usually do not exist in their native form, but as esters, glycosides, or polymers. The native forms, however, require deglycosylation for their intestinal absorption, and aglycone has been considered to be the potential health-protecting/promoting form. The binding of the aglycones of phenolic compounds to bovine and reindeer beta-lactoglobulins (betaLG) using fluorescence quenching was studied. The effects of pH and storage were also studied. Of the compounds investigated, the majority of flavones, flavonols, flavanones, and isoflavones were bound to betaLG. In the pH studies, no significant effects were found. The fact that the phenolic compounds were not released at pH 2 might indicate that they bind to an external part rather than to the central cavity. Studies implicated that betaLG could act as a binder or carrier for phenolic compounds in acidic, basic, or neutral conditions and that the ligand/betaLG complex can remain stable during storage.

Effect of heat denaturation on beta-lactoglobulin-induced gastrointestinal sensitization in rats: denatured betaLG induces a more intensive local immunologic response than native betaLG.

Beta‐lactoglobulin (βLG) is one of the first foreign antigens encountered by a newborn child, and it is the major allergen causing cows milk allergy. Heat denaturation causes changes to the protein structure, but the significance of heat‐induced changes for immunogenicity or allergenicity is not known. To clarify how heat denaturation affects allergenicity and immunogenicity, we immunized Hooded‐Lister rat pups with intra‐peritoneal injections of native or heat‐denatured βLG at days 43 and 62 after birth. The animals were then fed native and denatured milk products twice weekly from 73 to 101 days of age with a feeding tube, after which they were allowed cheese and milk ad libitum, until they were killed on day 131. Total immunoglobulin (Ig)E and βLG‐specific IgG1 and IgG2a levels were determined from serum samples. Spontaneous interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ) production was measured from duodenal specimens, and specimens of gastrointestinal mucosae were studied for the presence of inflammatory cells. The rats immunized with native βLG had higher levels of total serum IgE than the unimmunized controls or the rats immunized with heat‐denatured βLG, while heat‐denatured βLG induced a significantly more intensive mononuclear inflammatory cell and eosinophil infiltration in the gastroduodenal mucosa. The βLG‐specific IgG antibody and IL‐4 and IFN‐γ responses were similar in the two groups of immunized animals. Hence, denaturation modifies the immunogenic and allergenic properties of βLG. Heat‐denatured βLG induces a more intensive local reaction in the gastrointestinal mucosa, while there is some evidence for enhanced systemic allergic sensitization by native βLG.

Human globin chain separation by isoelectric focusing in ultrathin polyacrylamide gels.

We describe three basic modifications of our previous method for thalassemia screening by isoelectric focusing of heme-free globin chains: the gel thickness is reduced from 2 mm to 240 microns; the level of the detergent Nonidet P-40 in the gels is decreased from 3% to 0.5%; the polyacrylamide slab is covalently fixed to the supporting glass plate by treatment with silane A-174. By the present method the entire focusing process, starting from gel moulding up to gel destaining and drying, is completed within 4 h, a fraction of the time needed in our previous technique. More than a 100 samples can be analyzed per working day. The present technique also affords increased sensitivity: less than 1 microgram protein/band is detected and less than 200 picomol of hemoglobin are needed for each analysis. Band sharpness and resolution in our ultrathin gels is also considerably increased.

Reindeer beta-lactoglobulin crystal structure with pseudo-body-centred noncrystallographic symmetry.

Reindeer beta-lactoglobulin (betaLG) belongs to the lipocalin superfamily. Its DNA and protein sequences have been determined and showed that it had nine residue changes from bovine betaLG. Reindeer betaLG, the structure of which was finally determined at 2.1 A resolution in space group P1, crystallized in a unit cell that is both P2-like and P2(1)-like owing to the presence of an almost perfect (but noncrystallographic) body-centring vector. The non-body-centred data could only be observed using a very bright synchrotron beam and a novel refinement strategy was adopted to enable us to use the weak h + k + l = 2n + 1 reflections.

Isoelectric focusing and isoelectric points of bovine liver histones.

Abstract A technique for isoelectric focusing of total histones in very narrow pH gradients is described. The isoelectric focusing was performed in 5% acrylamide gels at the pH range 9–11 in long quartz tubes (24 cm) in a nitrogen atmosphere. The total bovine liver histones separated into five main fractions which were identified as H1, H3, H2B, H2A, and H4 histones, and their apparent isoelectric points were determined. The main fractions were further divided into several subfractions, the maximal number of bands being 12. The isoelectric point for H1 histone in 6.25 m urea solution in the presence of a nitrogen atmosphere was 8.90, and the corresponding values for H3, H2B, H2A, and H4 histones were 9.80, 9.90, 10.10, and 10.25, respectively. The focusing technique described here has a high resolution, reproducibility, and sensitivity. The technique can be used for preparative and quantitative analysis and for studies on specificity and developmental changes of histones.

Changes in liver nuclear histones during bovine ontogeny.

The function of histones in chromatin is not yet well enough understood. Histones are thought to be involved in the control of genetic activity. Studies have been carried out to find age-related changes in the electrophoretic patterns and in the relative quantities of different histone fractions which could be explained by changes in the template activity of chromatin during development [l-5]. According to model structures chromatin is composed of repeating nucleosome units, each unit consisting of about 200 base pairs of DNA associated with an octamer of pairs of histones H2A, H2B, H3 and H4 [6,7] while the Hl histones are probably bound to DNA on the outside of the nucleosome [8]. Hl histones show species and tissue specificity [9], and the HI histones of mammalian tissues undergo changes during the prenatai and postnatal period [9,10]_ The different location of the Hl histones in chromatin compared with the nucleosome core histones may reflect their specific role in the regulation of the template activity. Therefore the relationship between development and the chemical nature of histones is important. In this work we compared the relative amounts of the main histone types from liver during ontogeny. Our results showed a striking increase in the relative amounts of the HI histones during the whole ontogeny, and the appearance of the HI0 histone during the bovine postnatal period.

Electrophoretic methods for fractionation of native and heat-denatured bovine beta-lactoglobulin.

We have developed three electrophoretic methods for analytical and preparative separation of native and heat-denatured beta-lactoglobulin. The methods can be applied, e.g., to optimize dairy milk processing, especially in laboratories which are not provided with expensive column chromatographic instruments. The methods consist of native PAGE followed by electroelution. Consequently, the eluted proteins are in solution in a biologically active and native form, and can therefore be used immediately for further analyses.

Isoelectric focusing of myelin membrane proteins

Human myelin was isolated from the white matter of autopsy brains. Myelin proteins were characterized by isoelectric focusing in ultrathin slab gels in a pH range from 3.5 to 10 after solubilization with urea and Nonidet P 40. The protein profile in the acidic region (pH below 6.2) revealed at least twelve faint bands which comprised only a few percent of the total myelin proteins. Most of the myelin proteins were focused in the neutral range (pH 6.2–7.8) which showed two sharper and three broader major bands, the total number of bands in this region being about twenty. The basic pH range (pH above 7.8) contained about 30% of the proteins, and revealed a very intense band near the cathode with seven to nine weaker bands below pH 9.0. When the myelin was partially delipidated prior to solubilization, an additional broad “band” was observed at the area pH 8.0–8.5.

Resolution and purification of taurine- and GABA-synthesizing decarboxylases from calf brain

The present work describes a procedure for the co-purification of cysteine sulfinate decarboxylase (CSAD) and glutamate decarboxylase (GAD) from calf brain. A crude enzyme preparation was first made from brain homogenate by acid precipitation and ammonium sulphate fractionation. Subsequent fractionation of the decarboxylase preparation by cation exchange chromatography on CM-Sepharose CL-6B revealed the existence of a specific CSAD enzyme, which has no GAD activity. The GAD activity peak was found to posses CSAD activity. Further fractionation by gel filtration on Sephacryl S-200 separated the specific CSAD activity into two enzyme forms, one of them having a molecular weight of 150,000 and the other of 71,000. GAD activity was eluted from the gel filtration column in a single peak (mol wt 330,000) and showed CSAD activity. The purification of the specific CSAD enzyme was 920-fold and that of GAD activity 850-fold as compared with the starting material, whole calf brain. SDS gel electrophoresis indicated that the purified CSAD and GAD enzymes consisted of two or more subunits. The crude decarboxylase preparation was analysed by isoelectric focusing in ultra-thin polyacrylamide gel in the pH range 3.5–10.0. The most active fraction of CSAD indicated an isoelectric point of 6.5 and that of GAD 6.8. The pH optimum for CSAD activity in the crude preparation was 7.2 and that for GAD activity 7.9.

BCG vaccine modulates intestinal and systemic response to β-lactoglobulin

β‐Lactoglobulin (BLG) is a clinically important antigen in cows milk and one of the major allergens causing cows milk allergy. Bacillus Calmette‐Guérin (BCG) vaccination has been suggested to modify immune response possibly decreasing the risk of allergy to some antigens in both human and experimental animals. In the present study, we have analyzed whether the early BCG vaccination has any effect on the markers of systemic and gastrointestinal (GI) sensitization to BLG. We immunized two groups of Hooded‐Lister rat puppets with intraperitoneal injections of native BLG at 43 and 62 days with pertussis vaccine as adjuvant, one group receiving additionally BCG. The animals were then fed native and denatured milk products twice weekly from 73 to 131 days of age, when they were killed. Control group was not vaccinated and received normal rat forage. Total immunoglobulin E (IgE) levels and BLG‐specific IgG1 and IgG2a concentrations were determined in serum samples. Spontaneous interleukin (IL)‐4 and interferon (IFN)‐γ production from duodenal specimens were measured, and the inflammatory cells were quantitated in specimens from different sections of the GI tract. Administration of BCG simultaneously with BLG resulted in reduced IgE concentration in serum, while the specific IgG1 and IgG2a antibody responses and the spontaneous secretion of IL‐4 and IFN‐γ were not affected. Furthermore, BCG‐induced eosinophilic infiltration and increase of intraepithelial lymphocytes (IEL) in the GI mucosa, and a trend toward increased number of lamina propria mononuclear inflammatory cells in the colon (BCG compared with BLG, p = 0.09; BCG compared with controls, p = 0.02). Controls showed increment of IgG1 response in comparison with the BLG group (p = 0.04) and increase of mucosal eosinophilic infiltration. The BCG modified the response to BLG both at the systemic level as shown by decrease of total IgE and at GI mucosa where increase of eosinophilic infiltration and increased number of IEL were seen. Increment of IgG1 level and eosinophils in the controls might be related with the lack of modulatory effect of pertussis vaccination. A shift of response toward the lower GI tract after BCG immunization as shown by a trend for increase of mononuclear inflammatory cells in colon lamina propria mimics disease development in some cases of clinical food allergy, and emphasizes the need for evaluation of the changes in the whole GI tract in food allergy models.