Gradient Sodium Dodecyl Sulfate-polyacrylamide Gel Research Articles

Topical fentanyl stimulates healing of ischemic wounds in diabetic rats.

Topically applied opioids promote angiogenesis and healing of ischemic wounds in rats. We examined if topical fentanyl stimulates wound healing in diabetic rats by stimulating growth-promoting signaling, angiogenesis, lymphangiogenesis and nerve regeneration. We used Zucker diabetic fatty rats that develop obesity and diabetes on a high fat diet due to a mutation in the Leptin receptor. Fentanyl blended with hydrocream was applied topically on ischemic wounds twice daily, and wound closure was analyzed regularly. Wound histology was analyzed by hematoxylin and eosin staining. Angiogenesis, lymphangiogenesis, nerve fibers and phospho-platelet derived growth factor receptor-β (PDGFR-β) were visualized by CD31-, lymphatic vessel endothelium-1, protein gene product 9.5- and anti-phospho PDGFR-β-immunoreactivity, respectively. Nitric oxide synthase (NOS) and PDGFR-β signaling were analyzed using Western immunoblotting. Fentanyl significantly promoted wound closure as compared to phosphate-buffered saline (PBS). Histology scores were significantly higher in fentanyl-treated wounds, indicative of increased granulation tissue formation, reduced edema and inflammation, and increased matrix deposition. Fentanyl treatment resulted in increased wound angiogenesis, lymphatic vasculature, nerve fibers, nitric oxide, NOS and PDGFR-β signaling as compared to PBS. Phospho-PDGFR-β co-localized with CD31 co-staining for vasculature. Topically applied fentanyl promotes closure of ischemic wounds in diabetic rats. Increased angiogenesis, lymphangiogenesis, peripheral nerve regeneration, NO and PDGFR-β signaling are associated with fentanyl-induced tissue remodeling and wound healing.

In vitro identification of nonalcoholic fatty liver disease-related protein hnRNPM.

To study the formation of intracellular glyceraldehyde-derived advanced glycation end products (Glycer-AGEs) in the presence of high concentrations of fructose. Cells of the human hepatocyte cell line Hep3B were incubated with or without fructose for five days, and the corresponding cell lysates were separated by two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Glycer-AGEs were detected with the anti-Glycer-AGEs antibody. Furthermore, the identification of the proteins that are modified by glyceraldehyde in the presence of high concentrations of fructose was conducted using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The protein and mRNA levels were determined by Western blotting and real-time reverse transcription PCR, respectively. The results of the two-dimensional gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a greater amount of Glycer-AGEs in the sample exposed to high concentrations of fructose than in the control. The detected Glycer-AGEs showed isoelectric points in the range of 8.0-9.0 and molecular weights in the range of 60-80 kDa. The heterogeneous nuclear ribonucleoprotein M (hnRNPM), which plays an important role in regulating gene expression by processing heterogeneous nuclear RNAs to form mature mRNAs, was identified as a modified protein using MALDI-TOF-MS. Increasing the concentration of fructose in the medium induced a concentration-dependent increase in the generated Glycer-AGEs. Furthermore, in an experiment using glyceraldehyde, which is a precursor of Glycer-AGEs, hnRNPM was found to be more easily glycated than the other proteins. The results suggest that glyceraldehyde-modified hnRNPM alters gene expression. This change may cause adverse effects in hepatocytes and may serve as a target for therapeutic intervention.

Pin1 Interacts With a Specific Serine-Proline Motif of Hepatitis B Virus X-Protein to Enhance Hepatocarcinogenesis

The peptidyl prolyl isomerase Pin1 frequently is overexpressed in hepatocellular carcinoma (HCC). Hepatitis B virus (HBV) is the most common etiologic agent in HCC, and its encoded X-protein (HBx) is oncogenic and possesses a serine-proline motif that may bind Pin1. The role of Pin1 in hepatocarcinogenesis, particularly in HBV-related HCC, was investigated. Immunohistochemical staining was performed to evaluate the prevalence of Pin1 overexpression in HCCs of different etiologies. Glutathione S-transferase pull-down and co-immunoprecipitation experiments were used to validate the physical interaction between Pin1 and HBx. Reporter assay, cell proliferation assay, and xenotransplantation experiments were used to show the functional consequence and importance of Pin1-HBx interaction in hepatocarcinogenesis. We showed preferential Pin1 overexpression in HBV-related tumors and confirmed the interaction between Pin1 and HBx at the specific serine-proline motif. Pin1 overexpression increased the protein stability of HBx. Furthermore, HBx-mediated transactivation was enhanced by co-expression of Pin1. HepG2 expressing Pin1 and HBx showed a synergistic increase in cellular proliferation, as compared with cells expressing Pin1 or HBx alone. Furthermore, concomitant expression of Pin1 and HBx in the nontumorigenic human hepatocyte cell line MIHA led to a synergistic increase in tumor growth. Finally, in Hep3B cells with suppressed Pin1 expression, HBx-enhanced tumor growth in nude mice was abrogated. Pin1 binds HBx to enhance hepatocarcinogenesis in HBV-infected hepatocytes. The discovery of an interaction between Pin1 and HBx will further our understanding of the molecular pathogenic mechanism of HBV-related HCC in human beings.

Ryanodine Receptor Type 1 (RyR1) Mutations C4958S and C4961S Reveal Excitation-coupled Calcium Entry (ECCE) Is Independent of Sarcoplasmic Reticulum Store Depletion

Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(1s)-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca(2+) channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca(2+)>Sr(2+) approximately Ba(2+), and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys(4958) and Cys(4961) within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.

Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus.

White spot syndrome virus (WSSV) virions were purified from the hemolymph of experimentally infected crayfish Procambarus clarkii, and their proteins were separated by 8 to 18% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to give a protein profile. The visible bands were then excised from the gel, and following trypsin digestion of the reduced and alkylated WSSV proteins in the bands, the peptide sequence of each fragment was determined by liquid chromatography-nano-electrospray ionization tandem mass spectrometry (LC-nanoESI-MS/MS) using a quadrupole/time-of-flight mass spectrometer. Comparison of the resulting peptide sequence data against the nonredundant database at the National Center for Biotechnology Information identified 33 WSSV structural genes, 20 of which are reported here for the first time. Since there were six other known WSSV structural proteins that could not be identified from the SDS-PAGE bands, there must therefore be a total of at least 39 (33 + 6) WSSV structural protein genes. Only 61.5% of the WSSV structural genes have a polyadenylation signal, and preliminary analysis by 3' rapid amplification of cDNA ends suggested that some structural protein genes produced mRNA without a poly(A) tail. Microarray analysis showed that gene expression started at 2, 6, 8, 12, 18, 24, and 36 hpi for 7, 1, 4, 12, 9, 5, and 1 of the genes, respectively. Based on similarities in their time course expression patterns, a clustering algorithm was used to group the WSSV structural genes into four clusters. Genes that putatively had common or similar roles in the viral infection cycle tended to appear in the same cluster.

A rod-linker-contained R-phycoerythrin complex from the intact phycobilisome of the marine red alga Polysiphonia urceolata

A linker-contained R-phycoerythrin (R-PE) complex was obtained by the Sephadex G-150 column chromatography from the Polysiphonia urceolata phycobilisome (PBS) that was disassociated at 37 °C for 6 h in the dilute phosphate buffer (pH 7.0) with 5% (m/v) sodium dodecyl sulfate (SDS). The R-PE complex showed three absorption peaks at 498, 538 and 567 nm, and a fluorescence emission maximum at 578 nm. Polypeptide analysis of the complex by the 8–25% (m/v) gradient SDS–polyacrylamide gel electrophoresis demonstrated that it contained three red subunits, α PE 17.6 , β PE 19.2 and γ PE 31.0 , and a colorless 35.3 kDa rod-linker L R 35.3 . Polypeptide proportion of the complex demonstrated that it was a hexamer in aggregate form γ PE 31.6 α PE 17.6 β PE 19.2 3 L R 35.3 α PE 17.6 β PE 19.2 3 γ PE 31.6 which is proposed to originate from a rod assembly of hexamer–linker–hexamer the substructure α PE 17.6 β PE 19.2 3 of which was decomposed off from the ends of the assembly during the PBS dissociation. The distinctive stability of the prepared hexamer is attributed to a large extent to the electrostatic interaction among its polypeptides, but not to the hydrophobic interaction.

Proteome analysis of human liver tumor tissue by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-mass spectrometry for identification of disease-related proteins.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.

Physicochemical and nitrogen solubility properties of Bacillus proteinase hydrolysates of sodium caseinate incubated with transglutaminase pre- and post-hydrolysis.

Sodium caseinate (NaCN), hydrolyzed with Protamex, a Bacillus proteinase preparation, to 0.5, 1.3, and 17.5% degrees of hydrolysis, was incubated with transglutaminase (TGase) for 3, 42, and 290 min at enzyme/substrate ratios of 1, 1, and 10% (w/w), respectively, pre- and post-hydrolysis. The electrophoretic, reversed-phase high-performance liquid chromatography (RP-HPLC) and nitrogen solubility profiles of the modified products were investigated. Combinations of hydrolysis and incubation with TGase generated products displaying novel physicochemical and nitrogen solubility properties. Significant changes in sodium dodecyl sulfate (SDS) and urea polyacrylamide gel electrophoresis profiles were apparent in the modified caseinate samples. Extensive TGase cross-linking resulted in polymers that were unable to enter the resolving gel during SDS polyacrylamide gradient gel electrophoresis. Extensive combined enzymatic modification resulted in peptides eluting earlier on RP-HPLC than limited combined enzymatic modification or limited hydrolysis. Combination of enzymatic treatments resulted in significantly (P < 0.005) improved solubility around pH 4.6, compared to incubation with TGase or hydrolysis of NaCN alone.

Defective chylomicron synthesis as a cause of delayed particle clearance in diabetes?

Chylomicron metabolism is abnormal in diabetes and the chylomicron particle may play a very important role in atherosclerosis. The aim of this study was to examine the effect of diabetes on the metabolism of chylomicrons in cholesterol-fed alloxan diabetic and nondiabetic rabbits. Five diabetic rabbits and 5 control rabbits were given [14C]linoleic acid and [3H]cholesterol by gavage. Lymph was collected following cannulation of the lymph duct and radiolabelled chylomicrons were isolated by ultracentrifugation. The chylomicrons from each animal were injected into paired control and diabetic recipients. Lymph apolipoprotein (apo) B48, apo B100, and apo E were measured using sodium dodecyl sulfate–polyacrylamide gradient gel electrophoresis. Mean blood sugar of the diabetic donors and diabetic recipients were 19.7 ± 2.3 and 17.2 ± 3.2 mmol/L. Diabetic rabbits had significantly raised plasma triglyceride (10.8 ± 13.9 versus 0.8 ± 0.5 mmol/L, P < 0.02). There was a large increase in apo B48 in lymph chylomicrons in the diabetic donor animals (0.19 ± 0.10 versus 0.04 ± 0.02 mg/h, P < 0.01) and apo B100 (0.22 ± 0.15 versus 0.07 ± 0.07 mg/h, P < 0.05) and a reduction in apo E on the lymph chylomicron particle (0.27 ± 0.01 versus 0.62 ± 0.07 mg/mg apo B, P < 0.001). Diabetic recipients cleared both control and diabetic chylomicron triglyceride significantly more slowly than control recipients (P < 0.05). Clearance of control chylomicron cholesterol was delayed when injected into diabetic recipients compared to when these chylomicrons were injected into control recipients (P < 0.005). Clearance of diabetic chylomicron cholesterol was significantly slower when injected into control animals compared to control chylomicron injected into control animals (P < 0.02). In this animal model of atherosclerosis, we have demonstrated that diabetes leads to the production of an increased number of lipid and apo E–deficient chylomicron particles. Chylomicron particles from the control animals were cleared more slowly by the diabetic recipient (both triglyceride and cholesterol). The chylomicron particles obtained from the diabetic animals were cleared even more slowly when injected into the diabetic recipient. Although there was an initial delay in clearance of chylomicron triglyceride from the diabetic particle when injected into the control animals, the clearance over the first 15 minutes was not significantly different when compared to the control chylomicron injected into the control animal. On the other hand, the cholesterol clearance was significantly delayed. Thus, diabetes resulted in the production of an increased number of lipid- and apo E–deficient chylomicron particles. These alterations account, in part, for the delay in clearance of these particles.

Effect of Incubation Temperature and Salinity on Expression of the Outer Membrane Protein Profile ofEdwardsiella tarda

Outer membrane proteins (OMP) of 10 isolates of Edwardsiella tarda were compared by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The OMP profile of the type strain E. tarda ATCC 15947 cultured at 25°C had five major protein bands of 40, 36.5, 34, 28.5, and 25 kDa and a large number of minor proteins ranging in size from approximately 10 to 120 kDa. Differences between the OMP profiles of the isolates of E. tarda included the inconsistent presence of the 34- or 36.5-kDa proteins in five isolates of E. tarda and two major bands of 47 and 44 kDa that were present in only two isolates of E. tarda. There were no differences in the outer membrane protein profiles of 9 out of 10 isolates of E. tarda incubated at a temperature of 25°C compared with those at 35°C. To evaluate the effect of salinity, 10 isolates of E. tarda were cultured in brain heart infusion broth containing 0.5, 1.5, and 3.0% sodium chloride. Reactions of isolates of E. tarda to the different salinity levels were placed into three groups. The first group expressed more or fewer protein bands at 1.5% sodium chloride. The second group lost major bands at 3% salinity, whereas the third group had no change in the OMP profile with salinity. The OMP profile differences and the different reactions to salinity levels suggest that the isolates are heterogeneous.

Albumin is the major plasma protein target of oxidant stress in uremia

Patients with uremia are exposed to increased oxidative stress. Examination of the oxidation of individual plasma proteins may be useful in establishing specific pathways of oxidative stress in vivo and in determining functional consequences of oxidant stress exposure. We therefore examined oxidative modification of plasma proteins by carbonyl formation using Western blot immunoassay and enzyme-linked immunosorbent assay (ELISA) techniques in patients with chronic renal failure (CRF) and on chronic hemodialysis therapy (HD). Plasma was obtained from 25 HD, 20 CRF, and 20 healthy volunteers, derivatized with 2,4 dinitrophenylhydrazine (DNP) and electrophoresed on duplicate 4 to 12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to nitrocellulose, and stained for DNP for carbonyls and amido black for protein content. Data are recorded as DNP area/protein area and are reported in densitometry units. Total plasma carbonyls were determined by ELISA. Plasma albumin is substantially more oxidized in HD than in healthy volunteers (1.22 +/- 0.14 densitometry units vs. 0.60 +/- 0.08, P = 0.002). There were no significant differences in oxidation of plasma transferrin, immunoglobulin, and fibrinogen in HD versus healthy volunteers. In CRF patients, plasma albumin is more oxidized compared with normal volunteers (1.36 +/- 0.20 densitometry units vs. 0.94 + 0.08, P = 0.09). There were no differences in oxidation of plasma transferrin, fibrinogen, and immunoglobulin in CRF patients versus healthy volunteers. An increased plasma protein carbonyl concentration in CRF patients compared with healthy volunteers was confirmed by ELISA (0.31 +/- 0.07 vs. 0.04 +/- 0.01 nmol/mg protein (P = 0.001). Albumin is the major plasma protein target of oxidant stress in CRF and HD patients.

Cloning and expression of a DNA sequence encoding a 41-kilodalton Cryptosporidium parvum oocyst wall protein.

This study was conducted to produce a recombinant species-specific oocyst wall protein of Cryptosporidium parvum. Antigens unique to C. parvum were identified by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of oocyst proteins from several different Cryptosporidium species. Antiserum was then prepared against a 41-kDa antigen unique to C. parvum and used to identify a recombinant DNA clone, designated rCP41. Expression of CP41 mRNA in C. parvum oocysts was confirmed by reverse transcriptase PCR (RT-PCR). Although the CP41 sequence was shown by PCR to be present in the genome of C. baileyi, CP41 mRNA was not detected in this species by RT-PCR. Immunofluorescence staining with antiserum against recombinant CP41 detected native CP41 antigen on the surface of C. parvum oocysts but failed to detect CP41 on C. baileyi oocysts. Immunoelectron microscopy demonstrated that native CP41 was distributed unevenly on the C. parvum oocyst surface and was associated with amorphous oocyst wall material. In an enzyme-linked immunosorbent assay, purified rCP41 performed as well as native C. parvum oocyst protein in measuring the serological responses of young calves and adult cows to experimental and natural C. parvum infections. These results indicate that recombinant CP41 antigen may have potential in the immunodiagnosis of cryptosporidiosis.

The regulatory particle of the Saccharomyces cerevisiae proteasome.

The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.

Effects of dietary cholesterol on hepatic production of lipids and lipoproteins in isolated hamster liver

The effect of 2-week 2% cholesterol vs. chow feeding on regulation of hepatic lipoprotein, lipids and apoprotein (Apo), and biliary lipids production was evaluated by the isolated perfused hamster liver model. Cholesterol feeding did not change very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) particle size but significantly increased the hepatic production of VLDL-cholesterol fourfold, VLDL-triglyceride two and one-half-fold but not phospholipid in VLDL. It also increased LDL-cholesterol fourfold but not triglyceride or phospholipid in LDL, whereas lipids in HDL remained unchanged. Gradient sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) and Western blot analysis (density of apoprotein/density of albumin/g liver) indicated that cholesterol feeding enhanced Apo B tenfold, Apo A-I fivefold but not Apo E in VLDL. Apo E and Apo B did not change in LDL. Apo E but not Apo A-I increased (threefold) in HDL by cholesterol feeding. Cholesterol feeding decreased bile salt secretion 28% but increased cholesterol secretion 118% in bile, whereas phospholipid and bile volume remained unchanged. Increased Apo A-I in VLDL suggested that Apo A-I is involved in enhanced hepatic export of cholesterol and triglyceride. Different patterns of lipid and Apos in VLDL and LDL after cholesterol feeding also suggested separate VLDL and LDL export mechanisms. Elevated Apo E but not lipids in HDL after cholesterol feeding suggests that hepatic HDL may function as a carrier of newly synthesized hepatic Apo E into the circulation for transfer to other lipoproteins (chylomicron [CM], CMr) to facilitate hepatic cholesterol uptake and clearance. (Hepatology 1996 Aug;24(2):424-34)

Effects of Dietary Cholesterol on Hepatic Production of Lipids and Lipoproteins in Isolated Hamster Liver

The effect of 2-week 2% cholesterol vs. chow feeding on regulation of hepatic lipoprotein, lipids and apoprotein (Apo), and biliary lipids production was evaluated by the isolated perfused hamster liver model. Cholesterol feeding did not change very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) particle size but significantly increased the hepatic production of VLDL-cholesterol fourfold, VLDL-triglyceride two and one-half-fold but not phospholipid in VLDL. It also increased LDL-cholesterol fourfold but not triglyceride or phospholipid in LDL, whereas lipids in HDL remained unchanged. Gradient sodium dodecyl sulfate-polyacrylamide gel electrophesis (SDS-PAGE) and Western blot analysis (density of apoprotein/density of albumin/g liver) indicated that cholesterol feeding enhanced Apo B tenfold, Apo A-I fivefold but not Apo E in VLDL. Apo E and Apo B did not change in LDL. Apo E but not Apo A-I increased (threefold) in HDL by cholesterol feeding. Cholesterol feeding decreased bile salt secretion 28% but increased cholesterol secretion 118% in bile, whereas phospholipid and bile volume remained unchanged. Increased Apo A-I in VLDL suggested that Apo A-I is involved in enhanced hepatic export of cholesterol and triglyceride. Different patterns of lipid and Apos in VLDL and LDL after cholesterol feeding also suggested separate VLDL and LDL export mechanisms. Elevated Apo E but not lipids in HDL after cholesterol feeding suggests that hepatic HDL may function as a carrier of newly synthesized hepatic Apo E into the circulation for transfer to other lipoproteins (chylomicron [CM], CMr) to facilitate hepatic cholesterol uptake and clearance.

Biotinylated hyaluronan: a versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure.

Hyaluronan influences cellular proliferation and migration in developing, regenerating and remodelling tissues and in tissues undergoing malignant tumour-cell invasion. The widespread occurrence of hyaluronan-binding proteins indicates that the recognition of hyaluronan is important to tissue organisation and the control of cellular behaviour. A number of extracellular matrix and cellular proteins, which have been termed the hyaladherins, have specific affinities for hyaluronan. These include cartilage link-protein, hyaluronectin, neurocan, versican and aggrecan, which all bind to HA within the extracellular matrix. Cellular receptors for hyaluronan such as CD44 and RHAMM (receptor for hyaluronate-mediated motility) have also been identified. In the present study biotinylated hyaluronan (bHA) was prepared by reacting adipic dihydrazide with a 170 kDa hyaluronan sample using the bifunctional reagent 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide. The resultant free amine moeity of the hydrazido-hyaluronan was then reacted with biotin succinimidyl ester (sulfo-NHS-biotin) to prepare the bHA. After 4-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotting to nitrocellulose membranes, bHA and avidin alkaline phosphatase conjugate could be used in conjunction with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates to specifically visualise with high sensitivity (> or = 2 ng), bovine nasal cartilage link-protein, aggrecan hyaluronan binding region, and human fibroblast hyaluronan receptors such as CD-44. Conventional Western blotting using specific monoclonal antibodies to these proteins was also used to confirm the identities of these proteins.

The Instability of the Membrane Skeleton in Thalassemic Red Blood Cells

The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came our of our studies on material properties that showed that beta-thalassemia (beta-thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ratio was low in red blood cells (RBCs) of splenectomized beta-thal intermedia patients. The possible explanations for the apparent decrease in spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane changes that lead to spectrin dissociation from the membrane during ghost preparation. To explore the latter alternative, samples from different thalassemic variants were obtained, ie, beta-thal intermedia, HbE/beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in the first lysate of the standard ghost preparation. Normal individuals and patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectrin was detected in identical aliquots of the supernatants of normals and these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibody. Relative amounts of spectrin detected were as follows in decreasing order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-linked immunosorbent assay technique to measure spectrin in the first lysate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably attached in severe thalassemia. From these studies we could postulate that membrane association of abnormal or partially oxidized alpha-globin chains has a more deleterious effect on the membrane skeleton than do beta-globin chains.

A High-Molecular-Weight, Cell-Associated Xylanase Isolated from Exponentially Growing Thermoanaerobacterium sp. Strain JW/SL-YS485

An unusual cell-associated (beta)-1,4-xylanase was purified to gel electrophoretic homogeneity from a cell extract of the bacterium Thermoanaerobacterium sp. strain JW/SL-YS485 harvested at the late exponential growth phase. The molecular mass of the xylanase was 350 kDa as determined by gel filtration and 234 kDa as determined by native gradient gel electrophoresis. The enzyme contained 6% carbohydrates. Heterosubunits of 180 and 24 kDa were observed for the xylanase on sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis gels. The xylanase had a pI of 4.37 and a half-life of 1 h at 70(deg)C. Using a 5-min assay, we observed the highest level of activity at pH 6.2 and 80(deg)C. The K(infm) and k(infcat) values when oat spelt xylan was used were 3 mg/ml and 26,680 U/(mu)mol, respectively. The Arrhenius energy was 41.8 kJ/mol. The purified enzyme differed in size, subunit structure, and location from other xylanases that have been described. The cell-associated enzyme activity appeared in the S-layer fraction.

Left ventricular performance and remodeling in rabbits after myocardial infarction. Effects of a thyroid hormone analogue.

Because the rat postinfarction model differs from human heart failure with respect to the composition of myosin heavy chain (MHC) isoforms and other contractile proteins, alternative animal models are needed for the development of new treatments for human heart failure. The purpose of this study was threefold: (1) to test the feasibility of using the V3(beta,beta) rabbit postinfarction model for the study of heart failure by characterizing the effects of chronic coronary artery occlusion on the left ventricle; (2) to determine whether the thyroid hormone analogue 3,5-diiodothyropropionic acid (DITPA) produces improvements in left ventricular function; and (3) to determine the effects of myocardial infarction and treatment with DITPA on MHC protein isoforms. Male New Zealand White rabbits underwent proximal circumflex coronary artery ligation. After infarction, rabbits were treated with DITPA (3.75 mg/kg body wt) or placebo for 21 days and then underwent conscious and open-chest hemodynamic studies. In separate groups of rabbits, beta- and alpha-MHC isoforms were separated, and relative proportions were measured using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and laser densitometry. Infarction resulted in increased left ventricular end-diastolic pressure and prolonged left ventricular relaxation (tau) (P = .001 for both variables). Postinfarction treatment with DITPA decreased left ventricular end-diastolic pressure and tau (P = .002 and P = .001, respectively) and increased maximum positive and negative dP/dt (P = .002 and P = .016, respectively). Infarcted rabbits treated with DITPA had no significant changes in heart rate or left ventricular systolic pressure compared with untreated rabbits with infarction. There were no significant differences in heart rate, positive dP/dt, peak systolic pressure, or tau between sham-operated rabbits and sham-operated rabbits treated with DITPA. Although infarction resulted in increased left ventricular diameter, there were no effects of DITPA on left ventricular remodeling. Neither myocardial infarction nor treatment with DITPA altered the ratio of MHC isoforms. Rabbits that survive occlusion of the circumflex artery will develop myocardial dysfunction and left ventricular remodeling. Therapy with DITPA, a thyroid hormone analogue, produces improvement in ventricular performance and reduces end-diastolic pressure. The hemodynamic effects of DITPA were not associated with alterations of MHC isoforms. Whether DITPA represents the prototype of a previously undescribed class of agents for the treatment of heart failure will need to be determined by clinical trials.

Seasonal and temperature-induced changes in myosin heavy chain composition of crucian carp hearts.

Myosin heavy chain isoforms of the ventricular myocardium from crucian carp (Carassius carassius L.) hearts were analyzed in different times of the year by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis [K. A. Esser, M. O. Boluyt, and T. P. White, Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H659-H663, 1988]. In winter only one myosin heavy chain type was present, but in summer about one-half of the winter myosin was replaced by more slowly moving summer myosin. The occurrence of summer myosin correlated with seasonal changes in water temperature of the pond, where the fish were caught. Furthermore, the heavy chain composition of the heart was altered by temperature acclimation in the laboratory: cold-acclimated (2 degrees C) fish had only winter myosin, but warm-acclimated (22 degrees C) fish had both summer and winter myosin in about equal amounts. Myosin adenosinetriphosphatase activity of the hearts containing both summer and winter myosin was higher than that of hearts containing only winter myosin. Functionally, changes in myosin heavy chain composition were associated with inverse thermal acclimation in the heart rate. Warm-acclimated fish had higher in vitro heart rate and shorter contraction duration than cold-acclimated animals. Present findings suggest that changes in myosin heavy chain composition together with concomitant changes in Ca2+ activation of contraction make possible large seasonal alterations in the activity of crucian carp hearts. These adjustments are needed to adapt the cardiovascular system to winter hibernation and summer activity, which are dictated by seasonally bound changes in environmental conditions.