Antioxidant nature of bovine milk beta-lactoglobulin.

Heating is necessary for processing milk in the dairy industry, which evidently produces a conformational change in beta-lactoglobulin (beta-LG). beta-Lactoglobulin, a major protein that accounts for approximately 10 to 15% of total milk proteins, is a globular protein consisting of 162 AA with a relative molecular mass of 18.4 kDa. The purpose of the present study was to determine the antioxidant role of beta-LG in milk and the possible mechanism involved. We showed that beta-LG is a mild antioxidant whose potency is less than that of vitamin E and probucol (the latter being an antioxidant used for clinical therapy). The conversion of the beta-LG monomer to dimer was responsible, in part, for the mode of action in protecting low-density lipoproteins against copper-induced oxidation. Cross-linking the free thiol groups of beta-LG by heating (100 degrees C for 2 min), or chemically modifying the beta-LG by carboxymethylation to block the thiol groups resulted in a substantial loss of antioxidant activity. The data suggest that Cys-121 plays an essential role in the antioxidant nature of beta-LG. By using an anti-LG antibody affinity column to deplete the beta-LG from milk, we observed from the lost antioxidant activity that beta-LG contributes approximately 50% of the total activity. Because beta-LG is extremely sensitive to thermal denaturation, to maintain its antioxidant nature, dairy products consumed daily should not be overheated in order to maintain its antioxidant nature.

A novel conformation-dependent monoclonal antibody specific to the native structure of beta-lactoglobulin and its application.

Molten globules are thought to be general intermediates in protein folding and unfolding. beta-lactoglobulin (beta-LG) is one of the major bovine whey proteins, constituting approximately 10 to 15% of total milk proteins. We have recently identified beta-LG as a superior marker for evaluating thermally processed milk. Strand D of beta-LG participates in irreversible thermal unfolding as probed by a monoclonal antibody (mAb) specific to thermally denatured beta-LG. In the present study, we used native beta-LG as an immunogen to test the hypothesis that a specific mAb against the native beta-LG could be established. As result, a mAb (4H11E8) directed against the native structure of beta-LG was made. The antibody did not recognize the heat-denatured form of beta-LG, such as its dimer and aggregates. Immunoassay using this "native" mAb showed that the stability of beta-LG was at temperatures < or =70 degrees C. beta-Lactoglobulin began to deteriorate between 70 and 80 degrees C over time. The denaturation was correlated with the transition temperature of beta-LG. Further chemical modification of Cys (carboxymethylation) or positively charged residues (acetylation) of beta-LG totally abolished its immunoreactivity, confirming the conformation-dependent nature of this mAb. Using competitive ELISA, the 4H11E8 mAb could determine the native beta-LG content in commercially processed milks. Concentrations of native beta-LG varied significantly among the local brands tested. From a technological standpoint, the mAb prepared in this study is relevant to the design and operation of appropriate processes for thermal sanitation of milk and of other dairy products.

Ion-pair reversed-phase HPLC: assay validation of sodium tanshinone IIA sulfonate in mouse plasma.

Sodium tanshinone IIA sulfonate (STS), a hydrophilic ionic substance, is used as a cardiovascular drug. An ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) method for the determination of STS in mouse plasma was initially developed. The assay involved a rapid and simple extraction process and subsequent detection at 271 nm. The retention time for STS was 7.5 min. Based on extracted STS standard mouse plasma at 1.5,10 and 50 microg/ml, the assay precision were 2.7, 2.1 and 1.7% with a mean accuracy of 96.7, 98.5 and 99.4%, respectively. At plasma concentration of 1.5, 50 and 75 microg/ml, the mean recovery of STS were 93.1, 96.3 and 97.5%. The limit of detection (LOD) and limit of quantification (LOQ) for STS was 0.1 microg/ml and 0.5 microg/ml, respectively. Linear responses were observed over a wide concentration range (0.5-100 microg/ml) for STS in mouse plasma. STS can be detected after intravenous administration. This method was performed for the first time in pharmacokinetic studies of STS in the mouse.

Beta-lactoglobulin is a thermal marker in processed milk as studied by electrophoresis and circular dichroic spectra.

As much of the sterilization process involves heat treatment during the preparation of milk on an industrial scale, the unpredictable measures of the process are an essential issue in determining the quality of the milk. The purpose of the present study was to investigate the major protein change(s) of whey proteins in processed milk and extend the knowledge for future reference in the dairy industry. Using a native polyacrylamide gel electrophoresis, we showed almost a 90% loss and denaturation of beta-lactoglobulin (LG), but not alpha-lactalbumin (LA), in some brands of the processed and dry milks. Immunochemical analysis using Western blotting revealed that part of the loss was attributed to the formation of large multiple forms of LG in the processed product. Such denaturation was presumably associated with the heating procedure used in the process. Essentially, LG was the only major fraction converted to aggregates in milk heated at 95 degrees C for 30 min on 2-dimensional PAGE. The detailed thermal denaturation of purified LG and LA at various temperatures (50 to 95 degrees C) and time (5 to 960 s) were investigated using a circular dichroic analysis. The maximal changes of ellipticity at 205 nm (converting beta-structure to disordered structure) were correlated to heating temperature and time. There were no significant conformational changes of LG at temperatures below 70 degrees C for as long as 480 s. Pronounced and rapid changes occurred between 80 to 95 degrees C in a time-dependent manner. Fifty percent of the maximal changes could be reached within 15 s. In conclusion, the unique chemical and immunochemical loss and conformational changes made LG a superior marker for evaluating the thermal processing of milk. The detailed thermal denaturation curves of LG constructed with its time and temperature in this study provide a valuable reference for the dairy industry. We postulate that heat treatment over 80 degrees C in 15 s may induce a significant denaturation of milk LG.

Distinction between dry and raw milk using monoclonal antibodies prepared against dry milk proteins.

It is well established that the heating process during the preparation of dry milk (DMLK) causes structural changes in some milk proteins. However, because such changes are subtle, whether they can be detected by an immunochemical approach remains questionable. The present study attempted to develop a sensitive mAb that might distinguish the DMLK from freshly prepared raw milk. To test this possibility, we immunized mice with commercially prepared DMLK and produced a panel of mAb. From 900 hybridomas screened using an ELISA, 4 clones were found to be specific to DMLK; the other 68 clones recognized both DMLK and raw milk. In contrast to polyclonal antibodies, only the specific mAb could detect the DMLK spiked into the raw milk at as low as 5% in concentration (vol/vol). Western blot analysis shows that these specific mAb were all directed against beta-lactoglobulin (LG) and LG-milk protein conjugates. These mAb reacted with raw milk heated at 95 degrees for 15 min; the reaction with LG-conjugates, however, was abolished when treated with reducing reagent. Thus, results suggests that a new antigenic epitope was exposed in a heating process, and the thio group of LG cross linked with other protein moiety played a provocative role in mAb recognition. A hypothetical model with respect to the interaction between the mAb and DMLK is proposed and discussed.

Identification of Atractylodes plants in Chinese herbs and formulations by random amplified polymorphic DNA.

AIM: An efficient, precise, and sensitive method for identifying Atractylodes plants has been established and will contribute significantly to quality control and scientific analysis in Chinese traditional medicine. METHODS: Twenty primers were applied for setting up the RAPD (randomly amplified polymorphic DNA) markers of Atractylodes plants, Atractylodes lancea DC (A lancea DC), Atractylodes japonica Koidz (A japonica K), and Atractylodes ovata DC (A ovata DC). The primer OPF03, OPF05, and OPF14 could discriminate them successfully. The results were also able to apply on the Chinese formulations with Atractylodes purchased from local markets. RESULTS: RAPD was used to investigate phylogenetic relationships among and within closely related species. RAPD analysis reflects heritable changes in the nucleotides sequence in both the coding and noncoding regions, because it is conducted directly from the DNA level. This work first conducted RAPD analysis of Atractylodes plants to establish their RAPD makers. CONCLUSION: The RAPD markers could be applied extensively in the Chinese herbal formulations.

Inhibition of neointimal formation in porcine coronary artery by a Ras mutant.

BACKGROUND: Therapeutic approaches to reduce the neointimal formation caused by balloon injury have been focused mainly on experimental models of restenosis in the rat carotid artery. However, restenosis in rat carotid artery may not replicate the coronary arterial responses to injury in larger animals and humans. METHODS: In this study, we used pig coronary arteries as an animal model to evaluate the preventive effects of a virus-mediated dominant negative mutant RasN17 on balloon injury-induced restenosis. The viral particles were delivered to the balloon-injured coronary arteries via a dispatch catheter to keep the virus in a confined arterial segment for 10 min to reach optimal transfection. Six weeks after balloon injury, the pigs were sacrificed and the left anterior descending arteries were isolated for histological analysis. RESULTS: Neointima formation was prominent in the group receiving balloon injury as compared with the uninjured controls. A remodeling process with migration of collagen was also found in the injured coronary arteries. The application of AdRasN17 led to a 56% decrease in neointima formation and a 75% increase in lumen size, as compared with the balloon-injured vessels treated with AdLacZ control. CONCLUSIONS: These results suggest that AdRasN17 is an effective therapeutic gene in preventing balloon injury-induced neointimal formation in pig coronary arteries.

A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs.

Using the binding and translocation domain of Pseudomonas exotoxin A [domain III deleted PE termed PE(DeltaIII)] as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen. PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M. hyopneumoniae 97kDa adhesion were successfully fused to the downstream of PE(DeltaIII) to create a subunit vaccine, i.e. PE(DeltaIII)-RR1. This chimeric protein, over-expressed in inclusion bodies of E. coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97kDa adhesin and was readily purified. The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(DeltaIII)-RR1. Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(DeltaIII)-RR1 was found more marked than that in mice immunized with the M. hyopneumoniae whole cells. Similarly, PE(DeltaIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M. hyopneumoniae vaccine. The PE(DeltaIII)-RR1 would be potentially useful for the future development of a M. hyopneumoniae adhesin vaccine.

A simple technique for the simultaneous determination of molecular weight and activity of superoxide dismutase using SDS-PAGE.

A direct and rapid SDS-PAGE staining method for in situ identification of activity and molecular weight of superoxide dismutase following denaturing treatment has been developed. This technique was based on the removal of SDS after SDS-PAGE and two-step staining procedures of the SDS-polyacrylamide gel to present the achromatic activity-zones of the enzymes. We demonstrated that the detection sensitivity of SDS-PAGE staining method was the same as the traditional xanthine oxidase-NBT solution assay. Through the SDS-PAGE staining method, three classes of superoxide dismutases with distinct molecular sizes were identified in situ. Moreover, activity of copper and zinc containing superoxide dismutase in crude extracts of Escherichia coli and Actinobacillus pleuropneumoniae was significantly enhanced using the two-step staining procedure.

Stimulation of smooth muscle cell proliferation by ox-LDL- and acetyl LDL-induced macrophage-derived foam cells.

To test the hypothesis that LDL lacking of initial oxidation may also anticipate an essential role in the progression for atherosclerotic lesions, we studied the in vitro effect of foam cells induced by low density lipoprotein (LDL), oxidized (ox)-LDL or acetyl-LDL on smooth muscle cell (SMC) proliferation. Intraperitoneal macrophages collected from ICR mice were incubated with buffered saline LDL, ox-LDL or acetyl-LDL to induce foam cell formation. Porcine aortas with atherosclerotic lesions were collected from 5 pigs fed high cholesterol diets. The results indicate that foam cells induced by ox-LDL and acetyl-LDL, but not by LDL, promoted SMC proliferation. SMC proliferation was also increased by ruptured, ox-LDL- and acetyl-LDL- induced foam cells. Immunohistochemically, epitopes of the LDL, ox-LDL, and malondialdelyde (MDA)-LDL were present in atherosclerotic lesions, but the acetyl epitope was not. We suggest that foam cells, whether induced by the oxidized or acetyl or acetyl (unoxidized) form, play an essential role in the pathogenesis of atherosclerosis by stimulating SMC proliferation.

The preventive effects of G115 on balloon injury-induced neointima formation in rats.

After percutaneous transluminal coronary angioplasty (PTCA), 30-50% of the patients may present with restenosis within 6 months. The aim of this study was to search for a preventive remedy against the balloon injury-induced neointima formation. Ginseng, with its wide indications on immune and cardiovascular functions, has prompted us to explore its role in neointima formation. In the present study, we aimed to explore if a standardized Panax Ginseng extract G115 was able to inhibit neointimal formation. With BrdU luminencence assay, maximal proliferation of rat smooth muscle cells was reduced to 24% of control values by G115. Norepinephrine-induced vasocontraction was antagonized in 21% and 44% by 1.44mg/ml and 2.88mg/ml of G115, respectively. Neointima-to-lumen area ratio of balloon-injured rat carotid arteries was reduced 77.3% by G115 as compared to the sham control. These results demonstrate the preventive effects of ginsenosides on angioplasty-mediated neointima formation.

Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin.

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (beta) and light chain (alpha) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing conditions, with a M(r) (molecular mass) of about 42,000 and 14,000 for heavy (beta) and light chains (alpha), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M(r) 42,000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M(r) 42,000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.

Complete nucleotide sequence of pig (Sus scrofa) mitochondrial genome and dating evolutionary divergence within Artiodactyla.

The complete nucleotide sequence of the pig (Sus scrofa) mitochondrial genome, containing 16613bp, is presented in this report. The genome is not a specific length because of the presence of the variable numbers of tandem repeats, 5'-CGTGCGTACA in the displacement loop (D-loop). Genes responsible for 12S and 16S rRNAs, 22 tRNAs, and 13 protein-coding regions are found. The genome carries very few intergenic nucleotides with several instances of overlap between protein-coding or tRNA genes, except in the D-loop region. For evaluating the possible evolutionary relationships between Artiodactyla and Cetacea, the nucleotide substitutions and amino acid sequences of 13 protein-coding genes were aligned by pairwise comparisons of the pig, cow, and fin whale. By comparing these sequences, we suggest that there is a closer relationship between the pig and cow than that between either of these species and fin whale. In addition, the accumulation of transversions and gaps in pig 12S and 16S rRNA genes was compared with that in other eutherian species, including cow, fin whale, human, horse, and harbor seal. The results also reveal a close phylogenetic relationship between pig and cow, as compared to fin whale and others. Thus, according to the sequence differences of mitochondrial rRNA genes in eutherian species, the evolutionary separation of pig and cow occurred about 53-60 million years ago.

Intracellular generation of MDA-LYS epitope in foam cells.

Oxidative stress plays a central role in atherogenesis. Antioxidants, such as probucol, inhibit oxidation of LDL, retard secretion of interleukin-1, growth factors and chemoattractants, and thus inhibit progression of atherosclerosis. Other antioxidants with an ability to inhibit LDL oxidation, however, could not prevent progression of atherosclerosis. The inconsistency between antioxidant potencies indicated oxidative events might have occurred at locations other than LDL. MDA-lysine epitope (MDA-lys) is closely associated with atherogenesis and was recognized as marker for oxidation. We traced formation of MDA-lys during oxidation of LDL and formation of foam cells. The results indicated that thiobarbituric acid reactive substance (TBARS) was primarily present in lipid fraction of ox-LDL not associated with protein fraction after Cu2+ oxidation in vitro. Oxidized LDL did not increase significant immunoreactivity of MDA-lys epitope under our experimental conditions. Foam cells, however, showed the presence of MDA-lys epitope suggesting that intracellular oxidation events occurred to internalized lipids. The uptake of non-oxidatively modified LDL (acetylated LDL) was sufficient to generate MDA-lys epitope in foam cells, consistent with the hypothesis that atherosclerosis is associated with oxidative events in addition to LDL oxidation. We hypothesized that MDA-lys may be generated through intracellular lipid metabolism during the formation of foam cells.

Molecular cloning and characterization of porcine cDNA encoding a 90-kDa heat shock protein and its expression following hyperthermia.

We have isolated and sequenced cDNA clones encoding a 90-kDa heat shock protein (HSP90) from a porcine brain cDNA library. The sequence of the 2202-nucleotide coding region showed 88.6% homology with that of the human homologue. Moreover, the deduced amino acid sequence of the porcine hsp90 cDNA was 99.7% identical to that of the human counterpart, with a difference of only three amino acids in a total of 733 residues. Expression of the gene was greatly increased in cultured cells during recovery from heat shock treatment at 45 degrees C for 60 min. Three major transcripts 2.2, 3.0, and 4.1kb in size were detected by Northern blot hybridization. These transcripts were further identified in a whole-pig hyperthermia experiment. These three hsp90 transcripts were constitutively expressed in porcine tissues including kidney, liver, brain, and heart, and their levels were markedly enhanced during recovery from 30-min hyperthermia treatment at 43 degrees C. Furthermore, we found that HSP90 was preferentially expressed in pituitary gland, brain, adrenal gland, and testis, in comparison to the other tissues.

Characterization and quantitation of extracellular collagen matrix in myocardium of pigs with spontaneously occurring hypertrophic cardiomyopathy.

The extracellular matrix between cardiocytes has been suggested to play an important role in maintaining the structure and function of the heart. The purpose of this study was to elucidate the morphological changes in the collagen of the extracellular matrix (ECM) in the hearts of pigs with hypertrophic cardiomyopathy. Sixty pigs diagnosed with hypertrophic cardiomyopathy from 605 purebred Landrace pigs ages 6 to 9 months were used in this study. Morphologically, these pigs with hypertrophic cardiomyopathy had increased heart weight and heart-to-body weight ratio, thickening of the left ventricular (LV) and right ventricular (RV) free walls and septum, disorientation of cardiocytes, myocardial fibrosis, and intramural coronary arteriosclerosis. Similar observations have been described in our preliminary report (Cardiovasc Pathol 3:261, 1994). In the present study, we have modified the silver impregnation technique to stain paraffin-embedded sections to demonstrate three types of ECM. There were endomysial struts, perimysial weaves, and epimysial coils in the myocardium. The light microscopic findings of the struts, weaves, and coils were also confirmed by scanning electromicroscopic examination. The numbers of these fine structures were increased significantly in the pigs with hypertrophic cardiomyopathy. In addition, the amounts of collagen in the LVs, RVs, and septum (Sep) in pigs with hypertrophic cardiomyopathy (LV = 19.37+/-0.79, RV = 23.72+/-0.72, Sep = 20.38+/-0.94 microg/mg, n = 60) were significantly higher (p < 0.01) than that in similar areas of normal pigs (LV = 14.56+/-1.11, RV = 18.90+/-1.02, Sep = 14.99+/-1.33 microg/mg, n = 30, respectively). Our findings of an overall increase of collagen content suggested that the accumulation of collagen matrix might be another factor responsible for the diastolic dysfunction of hypertrophic cardiomyopathy. These results might also infer that the increased collagen matrix could contribute to the stiffness of the cardiac chambers, thereby markedly affecting systolic and diastolic function of the heart. These observations provide further support that the pig may be an animal model for human cardiovascular disease.

Effect of antioxidant in endothelial cells exposed to oxidized low-density lipoproteins.

Antioxidants such as probucol and alpha-tocopherol have been shown to attenuate the oxidation of low-density lipoproteins (LDL) and atherosclerotic lesions in animal models of atherosclerosis. The purpose of this study is to determine the protection effect of antioxidants on endothelial cells when exposed to oxidized and native LDL. In a cell-free system, we found that probucol, alpha-tocopherol, and ascorbic acid inhibited copper-induced LDL oxidation by a dose-dependent fashion (from 1 microM to 10 mM). In porcine aortic endothelial cells, antioxidants alone did not change basal endothelin-1 (ET-1) secretion. When porcine aortic endothelial cells were exposed to LDL and oxidized-LDL, both of them stimulated ET-1 secretion dose-dependently, whereas oxidized-LDL elicited higher ET-1 secretion. However, probucol, alpha-tocopherol, and ascorbic acid did not prevent LDL or oxidized-LDL induced ET-1 secretion. Furthermore, nimodipine inhibited both of native and oxidized LDL induced ET-1 secretion. Since Ca2+ channel blocker reduced the elevation of induced ET-1 secretion, the [Ca2+]i is possibly involved for the regulation of ET-1 secretion. Our results suggest that antioxidants can only prevent the oxidation of LDL rather than oxidized and native LDL-induced ET-1 secretion in vascular endothelial cells. The increase in the [Ca2+]i of endothelial cells through the opening of voltage-dependent Ca2+ channels may be involved in the LDL-induced ET-1 release.

Intramural coronary artery disease in swine with naturally occurring hypertrophic cardiomyopathy.

Intramural coronary artery disease (ICAD) has been reported in myocardium affected with hypertrophic cardiomyopathy (HCM), but has never been studied in detail with respect to the cell type or lipid infiltration involved in the wall-thickening. The lack of heart samples may be one of the rationales to hamper the progress in investigating this disease. Recently, the discovery of naturally occurring HCM in swine has provided an excellent opportunity for the study of ICAD because of the high prevalence of ICAD in this animal. The present study provides a detailed structure feature in the thickened arterial wall of ICAD by both histologic and electron microscopic means. Morphologically, the feature of ICAD is due primarily to the neointimal thickening. Smooth muscle cells (SMC) and extracellular matrix (collagen and elastic fibers) are the major components responsible for the thickened neointima. Fragmentation of the internal elastic membrane is associated with the migration and proliferation of SMC from the media to the intima. Therefore, pigs with HCM may be a potential animal model not only for the study of the mechanism by which SMC migrate and proliferate into intima, but also for the future investigation of interventions in coronary artery occlusion.

One-step HPLC purification procedure for porcine brain 90-kDa heat shock protein.

The 90-kDa heat shock protein (HSP90) was purified from porcine brain by a novel single-step purification procedure using diethylaminoethyl high-performance liquid chromatography (HPLC). About 4.8 mg of HSP90 was isolated from 25 g wet wt porcine brain tissue. The purified protein possessed a single moiety on one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. Western blotting using monoclonal antibody prepared against human HSP90 confirmed its identity as HSP90. These results indicate that small-scale HPLC purification of HSP90 from porcine brain tissue can be readily accomplished, with high yield, using a convenient one-step purification method. The procedure described in this paper represents a significant improvement in current purification methods for the isolation of HSP90 from porcine brain.