SDS-polyacrylamide Gel Electrophoresis Analysis Research Articles

Combined treatment of porcine plasma with microbial transglutaminase and cysteine: Effects on the heat-induced gel properties

The effects of the treatment of porcine plasma with microbial transglutaminase (MTGase) and cysteine on its heat-induced gelation at pH 5.5 were studied. Four different conditions, besides the control samples, were considered in this work: cysteine addition; MTGase alone treatment; MTGase and cysteine simultaneously treatment; and MTGase treatment first, adding then cysteine just before the thermal gelation. Texture (hardness, springiness and cohesiveness) and water-holding capacity (WHC) were the measured gel properties. Scanning electron microscopy images were also taken in order to evaluate the effects on gel microstructure, and differential scanning calorimetry (DSC) and SDS-polyacrylamide gel electrophoresis analysis (SDS-PAGE) analyses were carried out to get information about the implied proteins in the cross-linking reactions. The results suggest that gel properties were differently affected due to of the applied treatment. All of them improved the texture of plasma gels with respect to the control samples; however, in the case of the combined treatments the order of addition of cysteine did not affect the obtained results and their effects seemed to be additive more than synergic. The MTGase treatment was the only one capable of improving WHC. Cysteine alone did not improve this parameter but, on the contrary, the improvements achieved with MTGase were lost when treating also with cysteine. From DSC and SDS-PAGE we postulate that at least fibrinogen and globulins would be proteins participating in the MTGase reaction.

Evaluation of the Residual Antigenicity of Dairy Whey Hydrolysates Obtained by Combination of Enzymatic Hydrolysis and High-Pressure Treatment

Dairy whey was hydrolyzed for 15 min with five food-grade enzymes (Alcalase, Neutrase, Corolase 7089, Corolase PNL, and Papain) at atmospheric pressure (0.1 MPa) and in combination with high pressure (HP) at 100, 200, and 300 MPa, applied prior to or during enzymatic digestion. The peptide profile of the hydrolysates obtained was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and their residual antigenicity was assessed by immuno-blotting with anti–β-lactoglobulin monoclonal antibodies and the sera from pediatric patients allergic to cow's milk proteins. Moreover, to evaluate the presence of residual trace amounts of casein in bovine whey hydrolysates, immunoblotting with anti–cow's milk protein polyclonal antibodies was performed. SDS-PAGE analysis showed that HP treatment increased hydrolysis by the proteases assayed, especially when it was applied during the enzymatic digestion. Positive reactions at the band corresponding to β-lactoglobulin were detected for Corolase PN-L and Corolase 7089 hydrolysates, except for those obtained under 300 MPa by the last protease. However, the immunochemical reaction was lower in the hydrolysis products obtained under HP than in those obtained at atmospheric pressure and after the HP treatment. On the contrary, no residual immunochemical reactivity associated with β-lactoglobulin was observed in the hydrolysates obtained by Alcalase and Neutrase under HP, and none was observed in any of the hydrolysis products obtained by Papain. The presence of traces of casein was not significant. These results suggest that HP combined with selected food-grade proteases is a treatment that can remove the antigenicity of whey protein hydrolysates for their use as ingredients of hypoallergenic infant formulae.

The Schizosaccharomyces pombe Replication Inhibitor Spd1 Regulates Ribonucleotide Reductase Activity and dNTPs by Binding to the Large Cdc22 Subunit

Ribonucleotide reductase (RNR) is an essential enzyme that provides the cell with a balanced supply of deoxyribonucleoside triphosphates for DNA replication and repair. Mutations that affect the regulation of RNR in yeast and mammalian cells can lead to genetic abnormalities and cell death. We have expressed and purified the components of the RNR system in fission yeast, the large subunit Cdc22p, the small subunit Suc22p, and the replication inhibitor Spd1p. It was proposed (Liu, C., Powell, K. A., Mundt, K., Wu, L., Carr, A. M., and Caspari, T. (2003) Genes Dev. 17, 1130-1140) that Spd1 is an RNR inhibitor, acting by anchoring the Suc22p inside the nucleus during G1 phase. Using in vitro assays with highly purified proteins we have demonstrated that Spd1 indeed is a very efficient inhibitor of fission yeast RNR, but acting on Cdc22p. Furthermore, biosensor technique showed that Spd1p binds to the Cdc22p with a KD of 2.4 microM, whereas the affinity to Suc22p is negligible. Therefore, Spd1p inhibits fission yeast RNR activity by interacting with the Cdc22p. Similar to the situation in budding yeast, logarithmically growing fission yeast increases the dNTP pools 2-fold after 3 h of incubation in the UV mimetic 4-nitroquinoline-N-oxide. This increase is smaller than the increase observed in budding yeast but of the same order as the dNTP pool increase when synchronous Schizosaccharomyces pombe cdc10 cells are going from G1 to S-phase.

Genetic Variation of Glutelin Acidic Subunit Polypeptides in Bangladesh Rice Genetic Resources

Bangladesh rice genetic resources collected from six distinct regions of the country were examined to obtain the diversity in glutelin acidic subunit polypeptides. Seed glutelins from 576 Bangladesh rice cultivars representing seven ecotypes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF) electrophoresis and two-dimensional electrophoresis (2-DE: SDS-PAGE/IEF) analyses. Glutelin acidic subunit was separated into four bands, α-1, α-2, α-3 and α-4, and the variation in each of bands was detected by SDS-PAGE analysis. A higher molecular size component of α-1(α-1′) was identified in the cultivars tested. In case of α-2, α-2H with high molecular mass, α-2L with low molecular mass and α-2H/L were detected. α-3 band variation showed α-3H, α-3L, and α-3H/L, while for α-4, α-4H, α-4L bands were identified. In IEF analysis, a total of 16 bands with independent pI ranging from pI 6.30 to 7.52 were identified for the glutelin acidic subunit among the cultivars. The maximum and minimum numbers of IEF bands found were 13 and 9, respectively. The α-2L less cultivars were also lacking in pI 6.80 polypeptide in IEF. Result of 2-DE showed that pI 6.80 polypeptide was the main component of α-2L band. Transplanted Aman ecotype was the most diverse with respect to glutelin variation. Geographical distribution of glutelin variation in the Transplanted Aman ecotype differed according to the regions. The above results indicate that Bangladesh rice cultivars possess great genetic diversity in glutelin acidic subunit polypeptides. This study of indigenous rice cultivars from Bangladesh provides useful information regarding their breeding potential.

Characterization of STAT5B phosphorylation correlating with expression of cytokine-inducible SH2-containing protein (CIS)

Cytokine-inducible SH2-containing protein (CIS) is the first identified member of genes encoding for the suppressor of cytokine signaling (SOCS). CIS is also a well-known target gene of signal transducer and activator of transcription 5 (STAT5) pathways, providing normal negative feedback control of signaling by cytokines and growth factors. Three other SOCS genes, SOCS1, SOCS2, and SOCS3, can be silenced by DNA hypermethylation in human cancers, suggesting a potential mechanism for constitutive STAT activation. However, it is not known whether CIS expression is similarly perturbed in tumor cells. We report here the absence of CIS expression in T lymphoma LSTRA that overexpresses the Lck protein tyrosine kinase and exhibits elevated STAT5 activity. Pervanadate-induced CIS expression and STAT5 binding to the CIS promoter in vivo over a short time course implies that mechanisms other than DNA hypermethylation may contribute to defective CIS expression in LSTRA cells. Comparison with cytokine-dependent BaF3 cells stimulated with interleukin-3 (IL-3) further reveals that CIS induction correlates with specific STAT5b post-translational modifications. It exhibits as the slowest migrating form through SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. This distinctly modified STAT5b is the predominant form that binds to the consensus STAT5 sites in the CIS promoter and accumulates in the nucleus. In vitro phosphatase assays and phosphoamino acid analysis suggest the involvement of phosphorylation on residues other than the highly conserved tyrosine and serine sites in this distinct STAT5b mobility shift. All together, our results provide a novel link between incomplete STAT5b phosphorylation and defective SOCS gene expression in cancer cells.

Thioredoxin Reductase System Mediates Iron Binding in IscA and Iron Delivery for the Iron-Sulfur Cluster Assembly in IscU*

IscA is a key member of the iron-sulfur cluster assembly machinery found in bacteria and eukaryotes. Previously, IscA was characterized as an alternative iron-sulfur cluster assembly scaffold, as purified IscA can host transient iron-sulfur clusters. However, recent studies indicated that IscA is an iron-binding protein that can provide iron for the iron-sulfur cluster assembly in a proposed scaffold IscU (Ding H., Clark, R. J., and Ding, B. (2004) J. Biol. Chem. 279, 37499-37504). To further elucidate the roles of IscA in the biogenesis of iron-sulfur clusters, we reevaluate the iron binding activity of IscA under physiologically relevant conditions. The results indicate that in the presence of the thioredoxin reductase system, Escherichia coli IscA binds iron with an iron association constant of 2.0 x 10(19) M(-1) in vitro. Whereas all three components (thioredoxin 1, thioredoxin reductase and NADPH) in the thioredoxin reductase system are essential for mediating the iron binding in IscA, only catalytic amounts of thioredoxin 1 and thioredoxin reductase are required. In contrast, IscU fails to bind iron in the presence of the thioredoxin reductase system, suggesting that the iron binding in IscA is specific. Nevertheless, the thioredoxin reductase system can promote the iron-sulfur cluster assembly in IscU in the presence of the iron-loaded IscA, cysteine desulfurase (IscS), and L-cysteine, demonstrating a physiologically relevant system for the biogenesis of iron-sulfur clusters. The results provide additional evidence for the hypothesis that IscA is capable of recruiting intracellular "free" iron and delivering the iron for the iron-sulfur cluster assembly in IscU.

A small ciliary surface glycoprotein of the monogenean parasiteNeobenedenia girellaeacts as an agglutination/immobilization antigen and induces an immune response in the Japanese flounderParalichthys olivaceus

The capsalid monogenean Neobenedenia girellae, a parasite of seawater fishes, was found to express an antigen that elicits antibodies in rabbits, and these antibodies had agglutination/immobilization activity against N. girellae larvae (oncomiracidia) in vitro. Indirect immunofluorescence staining of N. girellae oncomiracidia showed that this agglutination/immobilization antigen was expressed on the surface of cilia. An intraperitoneal injection of ciliary proteins (either sonicated or intact) with adjuvant also elicited agglutinizing/immobilizing antibodies in sera from Japanese flounder, Paralichthys olivaceus. These antisera showed a clear correlation between anti-ciliary antibody levels (measured by enzyme-linked immunosorbent assays) and their agglutination/immobilization activity. Anti-ciliary antibody levels in Japanese flounder reached a plateau at 39 days after booster immunization and were significantly higher in the two immunized groups (injection of sonicated or intact cilia) as compared with control fish (injection of bovine serum albumin; ANOVA, Tukey's test, P < 0.01). Anti-ciliary antibodies were also found in fish mucus; however, there was no correlation between fish serum anti-ciliary antibody levels and mucus antibody levels. A Triton X-114-soluble 8 kDa glycoprotein of the ciliary integral membrane fraction is a plausible candidate for the agglutination/immobilization antigen based on SDS-polyacrylamide gel electrophoresis and immunoblot analyses with rabbit and fish antisera.

Formation and Growth of Bryopsis hypnoides Lamouroux Regenerated from Its Protoplasts

Tissue culture, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and spectra analysis were used for studying the aggregation mechanism of protoplasts from Bryopsis hypnoides Lamouroux and the discrepancy between the protoplast-regenerated plants and the wild type. The aggregation of protoplasts from B. hypnoides was observed in natural seawater and artificial seawater with different pH values, and the location and mechanism of the materials causing the aggregation were also studied. Results showed that the protoplasts could aggregate into some viable spheres in natural seawater and subsequently grow into mature individuals. Aggregation of the protoplasts depended exclusively upon the pH value (6-11), and the protoplasts aggregated best at pH 8-9. Some of the extruded protoplasts were separated into two parts by centrifugation: the pellet (PO) and the supernatant (PL). The PO could aggregate in artificial seawater (pH 8.3) but not in PL. No aggregation was found in PO cultured in natural seawater containing nigericin, which can dissipate the proton gradients across the membrane. These experiments suggest that the aggregation of protoplasts is proton-gradient dependent and the materials causing the aggregation were not in the vacuolar sap, but located on the surface or inside the organelles. Furthermore, the transfer of the materials across the membrane was similar to Delta pH-based translocation (Delta pH/TAT) pathway that occurs in the chloroplasts of higher plants and bacteria. Obvious discrepancies in both the total soluble proteins and the ratio of chlorophyll a to chlorophyll b between the regenerated B. hypnoides and the wild type were found, which may be related to the exchange of genetic material during aggregation of the organelles. In the process of development, diatom Amphora coffeaeformis Agardh attached to the protoplast aggregations, retarding their further development, and once they were removed, the aggregations immediately germinated, which showed that diatoms can affect the development of other algae.

Poly(ADP-Ribose) Polymerase-1 Is a Component of the Oncogenic T-Cell Factor-4/β-Catenin Complex

T-cell factor (TCF)-4 regulates a certain set of genes related to growth and differentiation of intestinal epithelial cells. Aberrant transactivation of these TCF-4-regulated genes by beta-catenin protein plays a crucial role in early intestinal carcinogenesis, and the transcriptional machinery of the TCF-4/beta-catenin complex is likely to contain targets for molecular therapy. We explored the molecular composition of the TCF-4/beta-catenin transcriptional complex by means of proteomics. A protein of approximately 112 kilodaltons was consistently coimmunoprecipitated with FLAG-tagged TCF-4 transiently expressed in HEK293 cells, and the protein was identified by mass spectrometry as poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 physically interacted with TCF-4 and augmented the transcriptional activity of the beta-catenin/TCF-4 complex. Knockdown of PARP-1 by RNA interference significantly suppressed both transcriptional activity and proliferation by colorectal cancer cells. Auto-polyADP-ribosylation of the PARP-1 protein induced by DNA damage inhibited the functional interaction of PARP-1 with TCF-4. PARP-1 was overexpressed in the intestinal adenomas of patients with familial adenomatous polyposis and multiple intestinal polyposis mice. The expression of PARP-1 was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. In this study, we identified PARP-1 as a novel coactivator of the beta-catenin/TCF-4 complex. Although PARP-1 has been believed to play a protective role against carcinogenesis, these expression patterns and functional properties of PARP-1 were highly suggestive of its participation in early colorectal carcinogenesis.

Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly( d, l-lactic-co-glycolic acid) versus chitosan microspheres

Stable polymeric microspheres capable of controlled release of tetanus toxoid (TT) for periods ranging from days to over months were developed. TT was stabilized, encapsulated in microspheres prepared from poly( d, l)-lactide-co-glycolide (PLGA) and chitosan by using protein stabilizer (trehalose) and its immune response was compared. The influence of co-encapsulated protein stabilizer on tetanus toxoid's stability and release from the microspheres was studied. The protein stabilizer (trehalose) prevented structural losses and aggregation of microencapsulated TT. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polymer, we also co-incorporated into the polymer an antacid, (Mg(OH) 2), which neutralized the acidity during degradation of the polymer and also prevented TT structural losses and aggregation. The in vitro release experiments with PLGA and chitosan microspheres were performed and the release of TT was increased up to 80–90%. The antigen integrity was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by coomassie brilliant blue staining. The SDS-PAGE analysis confirmed that antigen integrity was not affected by the encapsulation procedure. In addition, the immunogenicity of PLGA and chitosan microspheres based single dose vaccine was evaluated in guinea pigs and compared with multiple doses of alum adsorbed TT. Results indicated that a single injection of PLGA and chitosan microspheres containing TT could maintain the antibody response at a level comparable to the booster injections of conventional alum adsorbed vaccines. The both PLGA and chitosan based stable vaccine formulations produced an equal immune response. Hence chitosan can be used to replace the expensive polymer PLGA. This approach should have potential application in the field of vaccine delivery.

Determination of Prolamins in Beers by ELISA and SDS-PAGE

Coeliac disease is triggered by exposure to the prolamin protein fraction of wheat, barley, or rye. The prolamin content of five lager beers and one wheat beer were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting and seven lager beers and three wheat beers were analyzed by enzyme-linked immunosorbent assay (ELISA). Most of the lager beers were made from barley and some had varying amounts of rice or corn as adjuncts. One of the beers was “gluten-free”, having been produced from corn and buckwheat without barley. The lager beer samples were gel-filtered before ELISA or SDS-PAGE analysis. Prolamin proteins were found in all but one beer which was made of corn, rice and barley and which was not the “gluten-free” beer. ELISA analysis was done using a commercially available gluten assay kit. For lager beers, a barley prolamin standard for ELISA was propanol-extracted from barley malt instead of using the prolamin standard of the gluten assay kit. As expected, the wheat beers contained much higher amounts of prolamins than the lager beers. The samples were studied by SDS-PAGE to identify different prolamin fractions. Proteins having a relative molecular mass in the range of 8000–17,000 and 38,000 and above were detected in immunoblotting by the prolamin sensitive antibody in the lager beers.

Proteoglycans Released from Cultured Bovine Aortic Endothelial Cell Layers by Sodium Spirulan Are Both Perlecan and Biglycan

Sodium spirulan (Na-SP) is a sulfated polysaccharide isolated from the blue-green alga Spirulina platensis. Na-SP has anticoagulant and fibrinolytic properties in vitro, including activation of heparin cofactor II, enhancement of vascular endothelial cell fibrinolytic activity and stimulation of endothelial proteoglycan (PG) release. In the present study, we investigated the types of endothelial PGs whose release is stimulated by Na-SP. Na-SP stimulated the PG release in a dose- and time-dependent manner. However, heparin, dextran sulfate and hyaluronan stimulated the release of heparan sulfate PGs rather than chondroitin/dermatan sulfate PGs, whereas the release of both types of PGs was strongly stimulated by Na-SP. Sepharose CL-6B chromatography of [35S]sulfate-labeled PGs showed that PGs were partially released after partial degradation of the core proteins without a change in chain length of the glycosaminoglycan chains after Na-SP treatment. On the other hand, SDS-polyacrylamide gel electrophoresis and Western blot analysis of the PG core proteins indicated that the Na-SP-releasable PGs are both a large heparan sulfate PG, perlecan, and a small chondroitin/dermatan sulfate PG, biglycan, without change in the size of the core proteins. Taken together, these results suggest that Na-SP stimulates the endothelial release of both perlecan and biglycan with the intact structures, possibly by mechanisms different from those of heparin, dextran sulfate and hyaluronan; a part of the PG core proteins may be degraded after Na-SP treatment. Since perlecan and biglycan have antithrombin activities, the present data support the hypothesis that Na-SP may enhance local anticoagulant activity in the liquid phase on the endothelium via stimulation of endothelial PG release.

The Grapefruit Juice Effect Is Not Limited to Cytochrome P450 (P450) 3A4: Evidence for Bergamottin-Dependent Inactivation, Heme Destruction, and Covalent Binding to Protein in P450s 2B6 and 3A5

Bergamottin (BG), a component of grapefruit juice, is a mechanism-based inactivator of cytochromes P450 (P450) 2B6 and 3A5 in the reconstituted system. The inactivation of both P450s was NADPH-dependent and irreversible. The kinetic constants for the inactivation of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of P450 2B6 were: K(I), 5 microM; k(inact) 0.09 min(-1); and t1/2, 8 min. The kinetic constants obtained for the inactivation of the testosterone 6beta-hydroxylation activity of P450 3A5 were: K(I), 20 microM; k(inact) 0.045 min(-1); and t1/2, 15 min. Incubations of P450s 2B6 and 3A5 with 20 microM BG at 37 degrees C for 20 min resulted in an approximately 60% loss in the catalytic activity that was accompanied by a significant loss in intact heme and a similar decrease in the reduced CO difference spectrum. The extrapolated partition ratios for BG with P450s 2B6 and 3A5 were approximately 2 and approximately 20, respectively. Liquid chromatography-mass spectroscopy analysis of the BG-inactivated samples showed that the mass of the inactivated apoprotein had increased by approximately 388 Da for both P450 2B6 and P450 3A5. SDS-polyacrylamide gel electrophoresis analysis demonstrated that [14C]BG was irreversibly bound to the apoprotein in the BG-inactivated samples. The stoichiometry of binding was approximately 0.5 mol BG metabolite/mol of each P450 inactivated. High-pressure liquid chromatography analysis of the metabolites of BG showed that P450 2B6 generated two major metabolites, whereas P450 3A5 generated three additional metabolites. Two of metabolites were identified as 6',7'-dihydroxybergamottin and bergaptol.

식염 및 지방을 함유한 분쇄돈육의 이화학적 성상 및 최종가열온도 측정을 위한 근장단백질의 이용

본 연구는 식염과 지방 그리고 여러 가열조건에 의한 pH 및 일반성분, 가열감량 및 단백질 용해성의 변화에 따른 단백질 분획의 변화에 대해 분석하였다 처리구에 따른 가열감량 (CL, %)은 지방의 첨가에 상관없이 식염을 2%첨가한 경우 식염을 첨가하지 않은 경우에 비하여 약 1/2배 정도 낮은 가열감량을 보였다. 한편, 식염 무첨가구의 경우 지방을 첨가한 경우 가열감량은 유의적으로 낮았으나, 식염 2%첨가구는 지방 첨가에 따른 가열감량의 유의적 차이가 나타나지 않았다. 그리고 가열온도에 따른 가열감량은 <TEX>$66^{\circ}C$</TEX>까지는 점진적으로 증가하다가 <TEX>$68^{\circ}C$</TEX> 보다 높은 가열온도에서 유의적으로 증가하는 경향을 보였다. 식염 무첨가구와 식염 2% 첨가구 사이에서는 단백질 용해성의 유의적인 차이가 없었으나 지방이 첨가된 경우 상대적인 단백질 함량의 감소로 단백질 용해성이 유의적으로 감소하였다. 가열 온도가 높아짐에 따라 용해성의 감소가 현저히 나타났으나 <TEX>$68^{\circ}C$</TEX> 이상의 가열온도에서는 단백질 용해성이 유의적 차이 없이 일정한 수준을 유지하였다. 추출한 근장 단백질이 각 처리구별 가열온도에 따른 단백질 분획을 분석하기 위해 전기영동을 실시한 결과 식염, 지방 그리고 가열에 따라 특이적으로 반응한 것은 36 과 66 kDa의 분자량을 가진 근장 단백질 분획이었다. 이러한 단백질 분획은 최종가열온도가 66 <TEX>${\sim} 70^{\circ}C$</TEX>에서 급격히 thtlfehladmfhTJ 돈육의 최종 가열온도 측정을 위한 지시제로 사용될 수 있을 것으로 판단된다. Processed meals, such as a ground meat and hamburger patty, are required to ensure that no pathogens remain in the final products. However, there was no rapid method available to verify that the recommended end-point cooking temperature(EPT) was reached. Thus, the objective of this study was to rapidly determine EPT of ground pork hams using sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SOS-PAGE), based on the disappearance of sarcoplasmic proteins after cooking. Fresh pork hams were added two levels of salt(0, 2%) and fat(15, 25%) combinations, and stored in refrigerator overnight, and cooked to internal cooking temperatures of <TEX>$64^{\circ}C$</TEX> to <TEX>$74^{\circ}C$</TEX> with <TEX>$2^{\circ}C$</TEX> increments. Cooked pork hams were measured cooking loss(CL, %), protein solubility(PS) and SOS-PAGE. CL(%) was reduced with the addition of 2% salt, as compared to the control, regardless of fat contents. It was also increased with increasing eooking temperature. Protein solubility was affected by the cooking temperature, resulting in reduced PS up to <TEX>$64^{\circ}C$</TEX>(P < 0.05), but remained constant higher than <TEX>$68^{\circ}C$</TEX>. In SOS-PAGE analysis, protein bands with the molecular weights of 36 and 66 kDa were affected by the addition of salt and fat combinations. regardless of treatments. These protein fractions were decreased gradually with increased cooking temperatures up to <TEX>$68^{\circ}C$</TEX> <TEX>${\sim}$</TEX> <TEX>$70^{\circ}C$</TEX> and might be good indicators for the determination of EPT in ground pork hams.

The role of cathepsin B and cystatin C in the mechanisms of invasion by ovarian cancer

Objective. The aim of this study was to investigate the contribution of cathepsin B and cystatin C to the mechanisms of invasion by ovarian cancer. Materials and methods. Using surgical materials from patients with ovarian cancer, immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis were performed using antibodies against cathepsin B or cystatin C. Serum levels of cathepsin B and cystatin C in patients with benign and malignant ovarian lesions were determined by enzyme-linked immunosorbent assay (ELISA). An invasion assay using an ovarian cancer cell line was performed by addition of cystatin C or specific inhibitors of cathepsin B. Results. While immunohistochemical staining of cathepsin B and cystatin C was evident in cancer cells and associated stromal tissue, this was not the case in benign tumors. The malignancies were also found to be positive for cathepsin B and cystatin C by SDS-PAGE and Western blotting analysis. No significant difference in serum cathepsin B levels was observed between patients with benign and malignant disease. However, the concentration of cystatin C in cases with ovarian cancer was significantly higher in benign cases ( P < 0.0001) and in healthy controls ( P < 0.0001). Invasion by cancer cells was dose-dependently suppressed by cystatin C and cathepsin B inhibitors. Conclusion. The results provided convincing evidence that cathepsin B and cystatin C may contribute to the mechanisms of invasion of ovarian cancer.

Analysis of zein by matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was used to analyze the protein composition of corn prolamine (zein). Mass spectra were obtained from commercial zein and zein extracted with aqueous 2-propanol and aqueous ethanol from consumer corn meal. For the commercial zein, three major zein fractions with m/z 26.8k, 24.1k, and 23.4k were clearly seen with two minor fractions (m/z 14.5k and 20.4k) also present. As compared with the results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these three fractions were identified as alpha-zeins (24.1k and 23.4k combined as Z19; 26.8k as Z22). When extracted with 55% aqueous 2-propanol, three alpha-zein fractions with m/z 26.8k, 24.1k, and 23.4k were predominant. When extracted with ethanol, extraction temperature had an effect on the final products. When extracted with 75% aqueous ethanol at room temperature, alpha-zein and some 17-18k species were observed, whereas at 60 degrees C, a small amount of delta-zein was also present. Comparison of the MALDI/MS results with SDS-PAGE and gene sequence analysis shows that the MALDI/MS method is superior to SDS-PAGE in having higher resolution and mass accuracy.

Expression and function of the endothelial protein C receptor in human neutrophils

AbstractActivation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase–polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti–endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation–blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil’s respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C–based treatment of severe sepsis.

A New Fungal Lectin Recognizing α(1–6)-linked Fucose in the N-Glycan

In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies.

Capturing ion exchanger-bound infectious pancreatic necrosis virus—design and application for large volume water samples

A method was designed for rapid and reliable demonstration of the presence of infectious pancreatic necrosis virus retrieved from 5 l water samples. Viruses together with an added carrier protein were adsorbed to a resin of an added anion exchanger. Then the resin was collected rapidly and quantitatively through a specific device which we designed. The resin was transferred to a column from which the viruses were eluted, and subsequently further concentrated by acid precipitation, followed by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Serological results were obtained within 24 h after the water sample was introduced into the laboratory. Proteins were recovered at an efficiency between 70 and 80% and the total concentration factor ranged between 150 000 and 250 000 times, depending on the requirements and the methods of choice.

Relationship between cardiac protein tyrosine phosphorylation and myofibrillogenesis during axolotl heart development

The axolotl, Ambystoma mexicanum, is a useful system for studying embryogenesis and cardiogenesis. To understand the role of protein tyrosine phosphorylation during heart development in normal and cardiac mutant axolotl embryonic hearts, we have investigated the state of protein tyrosine residues (phosphotyrosine, P-Tyr) and the relationship between P-Tyr and the development of organized sarcomeric myofibrils by using confocal microscopy, two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting analyses. Western blot analyses of normal embryonic hearts indicate that several proteins were significantly tyrosine phosphorylated after the initial heartbeat stage (stage 35). Mutant hearts at stages 40–41 showed less tyrosine phosphorylated staining as compared to the normal group. Two-dimensional gel electrophoresis revealed that most of the proteins from mutant hearts had a lower content of phosphorylated amino acids. Confocal microscopy of stage 35 normal hearts using phosphotyrosine monoclonal antibodies demonstrated that P-Tyr staining gradually increased being localized primarily at cell–cell boundaries and cell–extracellular matrix boundaries. In contrast, mutant embryonic hearts showed a marked decrease in the level of P-Tyr staining, especially at sites of cell–cell and cell–matrix junctions. We also delivered an anti-phosphotyrosine antibody (PY 20) into normal hearts by using a liposome-mediated delivery method, which resulted in a disruption of the existing cardiac myofibrils and reduced heartbeat rates. Our results suggest that protein tyrosine phosphorylation is critical during myofibrillogenesis and embryonic heart development in axolotls.