Liquid chromatography/tandem mass spectrometry to monitor acrylamide adducts with bovine beta-lactoglobulin B

Complexation of acrylamide with bovine beta-lactoglobulin B and some of its tryptic fragments have been examined by liquid chromatography coupled to tandem mass spectrometry. Such complexation was investigated both in the presence and in the absence of dithiothreitol as a reducing agent. Under the latter conditions, the intact protein exhibited a single cysteine-acrylamide complex which both the present work and previous studies attribute to Cys160. The involvement of this particular residue is tentatively attributed to an intramolecular disulphide exchange which results in its disengagement from the S-S bridge to offer a free SH group for reaction with the acrylamide monomer. In the absence of dithiothreitol, both free and complexed cysteine-containing tryptic fragments were present, while in its presence, one of the tryptic fragments, which contains three cysteine residues was fully absent, instead a part of this fragment containing two cysteines complexed with two acrylamide monomers was observed. The absence of any analytical information in the literature regarding the latter complexes underlines the potential of liquid chromatography coupled to mass spectrometry in the characterization of this commonly occurring modification.     

Structural basis of the Tanford transition of bovine beta-lactoglobulin

The structures of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 6.2, 7.1, and 8.2 have been determined by X-ray diffraction methods at a resolution of 2.56, 2. 24, and 2.49 A, respectively. The corresponding values for R (Rfree) are 0.192 (0.240), 0.234 (0.279), and 0.232 (0.277). The C and N termini as well as two disulfide bonds are clearly defined in these models. The glutamate side chain of residue 89 is buried at pH 6.2 and becomes exposed at pH 7.1 and 8.2. This conformational change, involving the loop 85-90, provides a structural basis for a variety of pH-dependent chemical, physical, and spectroscopic phenomena, collectively known as the Tanford transition.     

High-level expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its physical properties

Bovine beta-lactoglobulin (BLG) variant A has been expressed in the methylotropic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the alpha-mating factor prepro-leader peptide from Saccharomyces cerevisiae. P. pastoris Mut+ transformants were obtained by single cross-over integration of the BLG-containing vector into the AOX1 locus. In a fed-batch fermenter, a cell density of approximately 300 mg/ml was achieved by controlled glycerol feeding for a total of 24 h. After 72 h of methanol induction, the secreted BLG reached levels of > 1 g/l. The secreted protein could be purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the secreted BLG revealed that the Glu-Ala spacer repeats inserted between the mature protein and the alpha-factor prepro-leader were still present. The purified protein was characterized by a number of methods, including CD spectroscopy, guanidine-HCl unfolding, crystallization and two-dimensional 1H-NMR spectroscopy. By all of these measures, the physical characteristics of recombinant BLG were indistinguishable from those of the native purified bovine BLG, making it useful as a model for protein folding and other biophysical studies.     

Identification of a conserved hydrophobic cluster in partially folded bovine beta-lactoglobulin at pH 2

BACKGROUND: NMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions formed during folding. Although the complete structural characterization of partially folded proteins is a very difficult task, the identification of structured subsets, such as hydrophobic clusters, is of value in understanding the structural organization of such states. Here, we report the NMR characterization, in acidic conditions (pH 2), of a well-defined hydrophobic cluster localized in the core of bovine beta-lactoglobulin. RESULTS: The existence of a small hydrophobic cluster present in the lipocalin protein family has been assessed on the basis of structural alignment and NRM data obtained for the partially folded bovine beta-lactoglobulin. The presence of the cluster had been predicted identifying those residues that are highly conserved in most members of the family. An NMR study conducted at pH 2, where the protein exhibits a very stable beta-core together with disordered regions, reveals the presence of NOEs among sidechains of 11 hydrophobic residues centered around Trp19 and pointing towards the interior of the protein. This buried cluster is found to be unusually stable at pH 2, not only at room temperature but also at 323K. Furthermore, conserved hydrophobic residues pointing towards the surface of the protein define a hydrophobic surface patch located in a groove between the strands and the helix. CONCLUSIONS: The detected buried cluster most likely plays an important role in bovine beta-lactoglobulin stability. The analysis of five structurally related proteins reveals that the same extended cluster is present in these structures. We propose that the buried cluster may represent the internal binding site as well and that the hydrophobic surface patch is involved in a second external binding site.     

Spectroscopic and thermodynamic evidence for a complex denaturation mechanism of bovine beta-lactoglobulin A

PURPOSE/OBJECTIVES: To investigate the patterns of functioning and psychosocial adjustment of midlife and older women following surgery for breast cancer. Differences between those who received follow-up adjuvant therapy and those who did not also were compared. DESIGN: 2 x 3 mixed design with one between-groups factor (type of treatment) and one within-subjects factor (time). SETTING: Four midwestern hospitals. SAMPLE: 46 patients with breast cancer who are age 55 or older. METHODS: Baseline data about presurgical functional status and other variables were obtained during the first week after surgery. Follow-up data were obtained at six weeks, three months, and six months postsurgery. Data were collected via telephone interviews and mailed questionnaires. MAIN RESEARCH VARIABLES: Functional status, patient symptomatology, quality of life (QOL), demands of illness, and type of treatment (surgery only versus surgery plus adjuvant therapy). FINDINGS: No differences existed between the two treatment groups at baseline, with the exception of lower functional status reported by the surgery-only group. In the surgery-only group, functional status improved significantly from six weeks to three months postsurgery. The most frequently reported symptoms of both groups included fatigue and pain. CONCLUSIONS: These results suggest that both groups did equally well, regardless of whether they received adjuvant therapy (radiation or chemotherapy). Neither QOL nor demands of illness differed between the two groups, nor did these scores change significantly over time following surgery. IMPLICATIONS FOR NURSING PRACTICE: These findings suggest that women undergoing surgery for breast cancer, whether they receive adjuvant therapy or not, may have functional and psychosocial needs that could be effectively addressed by nursing interventions pre- and postsurgery.     

Expression of bovine beta-lactoglobulin/human erythropoietin fusion protein in the milk of transgenic mice and rabbits

We have generated several transgenic mouse lines and rabbits expressing efficiently (up to 0.3 mg/ml in mice and up to 0.5 mg/ml in rabbits) human erythropoietin in their milk as bovine beta-lactoglobulin fusion protein. Human erythropoietin cDNA was inserted in frame into exon 5 of the bovine beta-lactoglobulin gene with a linker oligonucleotide encoding the cleavage site for bacterial IgA protease. RNA analysis performed on one lactating transgenic mouse and one transgenic rabbit revealed that the fusion gene was expressed almost exlusively in the mammary gland, although low amounts of transgene-derived RNA were detectable in salivary glands and uterus or in the kidney. The fusion protein was specifically cleaved with IgA protease. The erythropoietin part obtained upon digestion had a lower molecular mass than recombinant erythropoietin produced in Chinese hamster ovary cells. By deglycosylation analysis it was shown that the difference in size was due to a different type of glycosylation. Biological activity of the fusion protein, as determined by growth stimulation of TF-1 erythroleukemia cells, was less than 15% of that of human recombinant erythropoietin. Upon digestion of the fusion protein with IgA protease, biological activity comparable to that of the recombinant erythropoietin was recovered. Transgenic males and virgin females did not show signs of enhanced erythropoiesis, but lactating females expressing the transgene displayed transient increases in their hematocrit values.     

Nonenzymatic lactosylation of bovine beta-lactoglobulin under mild heat treatment leads to structural heterogeneity of the glycoforms

Lactose reacts nonenzymatically with beta-lactoglobulin (beta-LG), the major whey protein, under mild heat treatment and the formation of the complex may be monitored by mass spectrometry. Using Reverse Phase HPLC coupled with Electrospray Ionization MS (ESI-MS) we have measured the global extent of glycosylation and examined the distribution of lactose among the beta-LG glycoforms. Identification of lactosylated sites by trypsinolysis and Tandem MS indicate that, although the glycosylation reaction was non-specific and potentially involved all the reactive sites (alpha- and epsilon-amino groups), beta-LG appeared to have at least two populations of lysine with the distinct ability to react with lactose. These results underline the structural heterogeneity of beta-LG glycoforms, with respect to the number of lactose linked per molecule and to the binding sites involved, which could affect the biological function of beta-LG.     

Structural changes of beta-lactoglobulin B induced by urea. Evidence of residual structure

Several spectroscopic methods have been used to study the structure of beta-lactoglobulin B at pH 2.1 in the presence of 8M urea. Fluorescence and polarization of fluorescence spectroscopy measurements indicate that the two tryptophanyl residues of the protein are exposed to the solvent in the denatured state. CD in the far-UV indicates that the amount of secondary structure in the denatured state is comparable to that found in the native state, whereas the CD spectrum in the near-UV shows that the tertiary structure is not completely disordered. The results of one-dimensional 1H NMR spectroscopy show that some local non-random structure is maintained in the denatured state, but most of the polypeptide chain has an extended non-globular conformation under the conditions of the present experiments. This conclusion is reinforced by the results of two-dimensional 1H NMR conducted on denatured samples of beta-lactoglobulin B. The study of states with intermediate levels of order will aid the understanding of how the native structure of beta-lactoglobulin B is organised during the refolding pathways.     

Role of B.A. Petrov in foundation and development of free flap skin transplantation problem

The article reviews the history of development of free-flap skin transplantation and basic role of acad. B.A. Petrov who was the first to show the role of dermatom skin transplantation in vast granulating wounds as the only method saving the life of patients with severe burns. He also suggested to solve an actual problem--deficiency of resources of donor skin--by repeated excision of skin flaps from previously used donor sites and use of cadaver skin. Very important is ales the role of B.A. Petrov in development of such particular questions as the role of patient's condition, wound bed am microbial flora of the wound in survival of skin transplants. In aspect of historical relationships the perspective of new approach to treatment of deep burns by transplantation of ceratinocytes, cultivated outside the patient's organism is shown.     

Role of phosphatidylethanolamine lipids in the stabilization of protein-lipid contacts

We have investigated the effect of lipids with phosphatidylethanolamine (PE) head groups on the stabilization of contacts between the tryptophan side chains of gramicidin and the lipid head groups. We initially developed two fluorescence methods that can be correlated to the spontaneous curvature of DOPC/DOPE and DOPC/DOPEme. One is based on bilayer structure and measures the rotational motion of a probe located close to the membrane surface relative to a more deeply-buried probe. The second is based on surface hydration/polarity and measures the emission energy of a polarity-sensitive probe located on the membrane surface. We used these methods to estimate the pseudo-curvature (i.e., curvature obtained by fluorescence measurements) of lipids with dimyristyl chains, and their pressure and temperature dependence. We then investigated the stability of gramicidin tryptophan-lipid contacts in DMPC/DMPE as a function of temperature and pressure. Stability was assessed by tryptophan rotational motion as determined by fluorescence anisotropy, since rotational motion is limited when the indoles are hydrogen bonded to the lipid head groups. The results suggest that the presence of PE lipids destabilizes these contacts due to either their smaller size relative to PC head groups, or their tendency to self-interact. Fluorescence quenching studies support these results.     

Expression, purification and immunochemical characterization of recombinant bovine beta-lactoglobulin, a major cow milk allergen

The immunological characteristics of a recombinant beta-lactoglobulin were studied using monoclonal antibodies, polyclonal antiserum and sera from allergic patients. Recombinant beta-lactoglobulin (rBLG) was expressed in Escherichia coli strain DH5alpha and purified as described previously [Cho et al. (1994) J. Biol. Chem. 269, 11 102-11 107]. The method has been modified by adding an immunoaffinity purification step. A quantity of 5-10mg of purified rBLG per liter of medium culture can be produced. rBLG shared the same molecular weight as the natural BLG (nBLG) and also possessed at least one intrachain disulfide bridge. In HPLC, rBLG appeared as a single peak, and the purity was estimated to be greater than 95%. All the monoclonal antibodies (mAbs) used in this study recognized different epitopes of the BLG and presented compatible binding. No differences could be detected between rBLG and nBLG when tested in a Western blot with rabbit polyclonal antiserum or with three mAbs that bound preferentially the reduced and S-carboxymethylated form of BLG. In a competitive enzyme immunoassay (EIA) using either a rabbit polyclonal antiserum or four mAbs that recognized conformational epitopes, we could not distinguish between rBLG or nBLG. In direct ELISA using nBLG or rBLG as the immobilized allergen, we measured a similar concentration of specific anti-BLG IgE in five sera from allergic patients. The results of this study indicate that we have obtained a rBLG with biochemical and immunological properties very similar to nBLG.     

In vitro renaturation of bovine beta-lactoglobulin A leads to a biologically active but incompletely refolded state

When bovine beta-lactoglobulin (beta-LG) was refolded after extensive denaturation in 4.8 M guanidine hydrochloride (GuHCl), the functional activity of the protein, retinol binding, as measured by the enhancement of this ligand's fluorescence, was completely recovered. In contrast, the room-temperature tryptophan phosphorescence lifetime of the refolded protein, a local measure of the residue environment, was approximately 10 ms, significantly shorter than the phosphorescence lifetime of the untreated native protein (approximately 20 ms). The lability of the freshly refolded protein, as monitored by following the time course of its unfolding when incubated in 2.5 M GuHCl through the change in fluorescence intensity at 385 nm, was also determined and found to be increased significantly relative to untreated native protein. In contrast to the long term postactivation conformational changes detected previously in Escherichia coli alkaline phosphatase (Subramaniam V, Bergenhem NCH, Gafni A, Steel DG, 1995, Biochemistry 34:1133-1136), we found no changes in either the lability or phosphorescence decays of beta-LG during a period of 24 h. Our results are in agreement with the report by Hattori et al. (1993, J Biol Chem 268:22414-22419), using conformation-specific monoclonal antibodies to recognize native-like structure, that long-term changes occur in the protein conformation, compared with the native structure, on refolding.     

The whey proteins of the milk of red deer (Cervus elaphus L.). A homologue of bovine beta-lactoglobulin

Increasing plasma free fatty acids decreased the degree of glycogen depletion, and increased the citrate concentration, in slow-red (soleus) and fast-red (deep portion of vastus lateralis) muscle during exercise (approx. 50% depletion of glycogen, as against 75% in control animals). There was no effect in fast-white muscle (superficial portion of vastus lateralis). Glycogen concentration in the liver decreased by 83% in controls, but only by 23% in animals with increased free fatty acids during exercise. The decreased glycogen depletion may be partly explained by the findings that (a) plasma-insulin concentration was two- to three-fold higher in animals with increased plasma free fatty acids and (b) the exercise-induced increase in plasma glucagon was lessened by increased free fatty acids. Blood glucose was higher in the animals with increased free fatty acids after the exercise. The rats with increased plasma free fatty acids utilized approx. 50% as much carbohydrate as did the controls during the exercise.     

Comparative production of beta-lactoglobulin and orotic acid with lactose in bovine mammary cell cultures: effects of cell density and constituent inhibition

Abilities to accumulate beta-lactoglobulin and ortic acid were compared to lactose in dispersed cell cultures of lactating bovine mammary tissue. The inverse of the amount accumulated of each milk constituent at a given time in the culture medium was a linear function of the inverse of the cell density. The amount of lactose had no effect on its own subsequent accumulation, but added orotic acid and beta-lactoglobulin inhibited their own production. The accumulation of certain milk constituents in the culture medium is a factor in the expression and loss of normal function in the in vitro cultures which may be related to the observed effects of milk accumulation in vivo on the rate of milk synthesis and mammary involution.     

Role of B cells in antigen presentation of the hepatitis B core

The hepatitis B virus (HBV) nucleocapsid or core antigen (HBcAg) is extremely immunogenic during infection and after immunization. For example, during many chronic infections, HBcAg is the only antigen capable of eliciting an immune response, and nanogram amounts of HBcAg elicit antibody production in mice. Recent structural analysis has revealed a number of characteristics that may help explain this potent immunogenicity. Our analysis of how the HBcAg is presented to the immune system revealed that the HBcAg binds to specific membrane Ig (mIg) antigen receptors on a high frequency of resting, murine B cells sufficiently to induce B7.1 and B7.2 costimulatory molecules. This enables HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive Th cells in vivo and to T cell hybridomas in vitro approximately 10(5) times more efficiently than classical macrophage or dendritic antigen-presenting cells (APC). These results reveal a structure-function relation for the HBcAg, confirm that B cells can function as primary APC, explain the enhanced immunogenicity of HBcAg, and may have relevance for the induction and/or maintenance of chronic HBV infection.     

Role of antioxidants and intracellular free radicals in retinamide-induced cell death

The cancer chemopreventive synthetic retinoid N-(4-hydroxyphenyl)retinamide (HPR) possesses antiproliferative and apoptotic activity at pharmacological doses. In this study we show that addition of antioxidants to HL-60 cells cultured in the presence of 3 microM HPR, markedly suppresses the apoptopic effect of the retinoid and significantly prolongs cell survival (48-96 h). We also show, by the use of the oxidation-sensitive probe 2',7'-dichlorofluorescin diacetate (DCF-DA) and in combination with flow cytometric and spectrofluorimetric analysis, that treatment of cells with 3 microM HPR results in an immediate and sustained production of intracellular free radicals, most likely hydroperoxides. Interestingly, the formation of these HPR-induced free radicals is effectively blocked by the water soluble antioxidants L-ascorbic acid and N-acetyl-L-cysteine. Neither 3-15 microM N-(4-methoxyphenyl) retinamide (MPR), the structurally similar but biologically inert analog of HPR, nor 3 microM doses of the retinoids all-trans retinoic acid, 9-cis-retinoic acid, TTNPB and SR11237 induce intracellular free radicals, thus indicating that the specificity of this phenomenon is restricted to HPR. Altogether, we provide the first direct evidence that HPR stimulates the generation of intracellular free radicals, which appear to have a causative role in the induction of apoptosis in vitro. Our findings raise the possibility that the therapeutic efficacy of HPR may, at least in part, depend on these apoptosis-inducing oxidative phenomena.     

Role of increased cytosolic free calcium concentration in myocardial ischemic injury

Increases in cytosolic free calcium concentration ([Ca2+]I) may play an important role in myocardial ischemic injury. An early effect of the rise in [Ca2+]I may be impaired postischemic contractile function if the ischemic myocardium is reperfused during the reversible phase of ischemic injury; furthermore, if the rise in [Ca2+]I is prolonged, a cascade of events may be initiated which ultimately results in lethal injury. With the development of methods for measuring [Ca2+]I, it has become possible to evaluate directly the role of increased [Ca2+]I in myocardial ischemic injury. Although it has been possible to show that inhibition of the transport processes which contribute to the early rise in [Ca2+]I attenuates stunning and the rise in [Ca2+]I concurrently, if increased [Ca2+]I plays an important role in ischemic injury, then it should be possible to show that interventions which alter the timecourse of ischemic injury also alter the timecourse of the rise in [Ca2+]I in a parallel manner. Recently, considerable effort has been expended to investigate the mechanisms underlying the preconditioning phenomenon, whereby repetitive brief periods of ischemia prior to a sustained period of ischemia protects the myocardium from injury during the sustained period of ischemia, and this has stimulated additional work to understand the possible involvement of adenosine as a mediator of preconditioning as well as to understand the protective effects of adenosine. Measurements of [Ca2+]I using 19F NMR of 5FBAPTA-loaded hearts have shown that preconditioning attenuates the rise in [Ca2+]I during 30 min of ischemia and reduces stunning during reflow. Adenosine pretreatment mimics the effects of preconditioning on the rise in [Ca2+]I and on stunning, but adenosine receptor antagonists do not eliminate the protective effects of preconditioning, although some adenosine antagonists also block hexose transport and under these conditions, the ability of preconditioning to attenuate the rise in [Ca2+]I is abolished and there is a corresponding loss of the protective effect of preconditioning on stunning. Although it has been suggested that the beneficial effect of preconditioning on infarct size can be eliminated by pretreatment with glibenclamide, in the isolated rat heart glibenclamide does not affect the attenuation of the rise in [Ca2+]I induced by preconditioning and does not affect stunning. All of these studies show a consistent relationship between the magnitude of the rise in [Ca2+]I during ischemia and the degree of stunning during reperfusion. The data suggest that increased [Ca2+]I plays a very important role in myocardial ischemic injury.