Analysis of complex protein-polypeptide systems for proteomic studies

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), followed by protein extraction and characterization with chemical sequencing or mass spectrometry (MS), is the most commonly used method to analyze complex protein systems such as cells and organelles. However, it is claimed that 2-D PAGE is a slow and labor-intensive technique and also needs subsequent efforts for one-by-one identification of proteins. Recently, the combined methods of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, with preceding separation techniques such as capillary isoelectric focusing (CIEF) or liquid chromatography, have been demonstrated as high-throughput techniques suitable for proteomic analysis of protein systems. The studies which employ FTICR MS, aimed at the analysis of complex protein systems, have been reviewed, comparing their performance with that of 2-D PAGE. Also, the possibilities of combining 2-D PAGE and the FTICR MS method to analyze and reconstruct the structures and functions of complex systems are discussed.     

Detection of protein-protein interactions and a group of immunoglobulin G-associated minor proteins in human plasma by nondenaturing and denaturing two-dimensional gel electrophoresis

The dissociation of noncovalently associated protein-protein complexes in human plasma was examined by comparing two-dimensional gel electrophoresis (2-DE) patterns obtained in two different electrophoretic conditions. A type I 2-DE pattern was obtained running nondenaturing isoelectric focusing (IEF) followed by nondenaturing gel electrophoresis and a type II 2-DE pattern was nondenaturing IEF followed by sodium dodecyl sulfate gel electrophoresis. Micro-sized gels (internal diameter(id) 1.3 x 35 mm polyacrylamide IEF gels and 38 x 38 x 1 mm polyacryamide slab gels) were used to follow the dissociation processes of major plasma proteins. Larger gel sizes (id 3.4 x 160 mm agarose IEF gels and 160 x 120 x 2.8 mm polyacrylamide slab gels) were used to detect minor plasma proteins dissociated from major proteins. About 110 spots, which have not been detected on type I (nondenaturing) 2-D gels, newly appeared on type II large-sized 2-D gels at molecular masses smaller than 67 kDa. Some of these spots had been analyzed and identified, but about 70 minor spots (isoelectric point 5.5-7.5 and relative molecular mass 8-45 kDa) were detected for the first time by applying large volumes of human plasma samples to the large type II 2-D gels. These minor spots could be concentrated on type II 2-D gels by enriching the immunoglobulin G (IgG) fraction under nondenaturing conditions, and they disappeared when IgG was removed from the fraction. These results strongly suggest that many of the minor spots newly detected were bound to IgG in physiological conditions.     

Balance of tumor necrosis factor alpha and interleukin-10 in a buccal infection in a streptozotocin-induced diabetic rat model

This study evaluates the local levels of proinflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), and anti-inflammatory cytokine, interleukin-10 (IL-10), in an experimental buccal abscess of a diabetic rat model. We prepared a buccal cavity induced by injection of carrageenin in a diabetic rat (blood glucose, 460.6 +/- 54.7 mg/dl, mean +/- SE) induced by streptozotocin (STZ). The buccal abscess was formed by the direct inoculation of Streptococcus pyogenes S-8 (2 x 10(7) cfu) into the buccal cavity at day 5 after carrageenin injection. Cytokine levels in the exudate of the buccal abscess were measured by enzyme-linked immunosorbent assay for 48 h after infection. Bacterial counts, weighing of exudate, and histological analysis were also performed. The mean TNF-alpha levels in the buccal abscess exudate of the diabetic group, which were generally higher than those of the control group, tended to increase over time until 48 h after infection, while the TNF-alpha levels in the control group peaked at 24 h after infection and then decreased. The IL-10 levels in the diabetic group remained almost unchanged until 48 h after infection, while the IL-10 levels in the control group were significantly higher than in the diabetic group at 12-24 h after infection. The mean ratio of TNF-alpha to IL-10 levels was 1.17-1.67 in the diabetic group, which was higher than the 0.26-0.69 of the control group. The bacterial counts in the buccal abscess and the weight of exudate were significantly higher in the diabetic group compared to the control group at 36-48 h. Histological findings showed that inflammatory cell infiltration was remarkable in the diabetic group compared to that of the control group. These results suggest that the elevated proinflammatory TNF-alpha levels and decreased anti-inflammatory IL-10 levels, which are produced at local infection sites, may at least in part be related to the severity of inflammation in this rat model with diabetes induced by STZ.     

Suppression of anoikis by v-Src but not by activated c-H-ras in human gallbladder epithelial cells

Detachment of anchorage-dependent normal epithelial cells from their substratum causes the type of apoptosis known as anoikis, whereas malignant cells can proliferate independently of anchorage. Because src and ras oncogenes are activated in many human cancers, we investigated their role and downstream signaling pathways in anoikis resistance, using HAG-1 human epithelial cells transfected with v-src or activated H-ras. Consequently, anchorage-dependent mock- or ras-transfected cells underwent anoikis. In contrast, anchorage-independent v-Src-transformed cells did not exhibit such apoptotic features. Focal adhesion kinase (FAK), a transducer of integrin, was only activated in v-Src-transformed cells. Herbimycin A, an Src kinase inhibitor, reduced tyrosyl phosphorylation of FAK and reversed resistance to anoikis. However, both protein kinase C (PKC) and phophatidylinositol-3 (PI-3) kinase inhibitors failed to induce anoikis. These data suggest that the ability of activated Src to prevent anoikis may be mediated by Src to a downstream signaling pathway involving FAK, but not Ras, PI-3 kinase, or PKC.     

Phenotypic characterization of a human synovial sarcoma cell line, SW982, and its response to dexamethasone

SW982 cells are characterized by expression of inflammatory cytokine and matrix metalloproteinase (MMP) genes and by their response to dexamethasone at different cell densities. They express genes encoding interleukin (IL)-1 beta; IL-6; transforming growth factor-beta; intercellular adhesion molecule-1; cycloxygenase (COX)-2; and MMPs, including MMP-1, MMP-2, MMP-13, and MT1-MMP; tissue inhibitor of metalloproteinase-2; and a disintegrin and metalloproteinase with thrombospondin motifs-4. Expression of all the genes examined was induced with 2 ng/ml IL-1 beta at low cell density. The cells, however, failed to express tumor necrosis factor-alpha, COX-1, and MMP-9, regardless of the presence of IL-1 beta. Dexamethasone significantly reduced IL-1 beta, IL-6, COX-2, and MMP-1 expression at high cell density. The results suggest that SW982 cells are a useful tool for studying the expression of inflammatory cytokine or MMP genes.     

A linkage map of the basidiomycete Coprinus cinereus based on random amplified polymorphic DNAs and restriction fragment length polymorphisms

A genetic linkage map of the basidiomycete Coprinus cinereus was constructed on the basis of the segregation of 219 RAPD markers, 28 RFLP markers and the A and B mating-type loci among 40 random basidiospore progeny from a single cross between a wild-type homokaryon, KF(3)#2, and an AmutBmut strain, #326. Thirteen linkage groups covering a total of 1346cM were identified and correlated to the 13 chromosomes of this fungus by hybridization of RFLP and RAPD marker probes to CHEF blots. These probes also revealed chromosome length polymorphisms (CLP), which could be associated with haplotype plots of the progeny. The average kb/cM ratio in this cross was approximately 27.9kb/cM. The AmutBmut strain undergoes sexual development without mating, because of mutations in both A and B mating-type loci, and has been used to identify mutations affecting developmental processes such as dikaryosis, fruit body morphogenesis, and meiosis. The markers in the map, especially the RAPD ones, would facilitate mapping of genes responsible for such mutations induced in the AmutBmut strain.     

ATP-sensitive K+ channel-mediated glucose uptake is independent of IRS-1/phosphatidylinositol 3-kinase signaling

We previously found that disruption of Kir6.2-containing ATP-sensitive K+ (KATP) channels increases glucose uptake in skeletal muscle, but the mechanism is not clear. In the present study, we generated knockout mice lacking both Kir6.2 and insulin receptor substrate-1 (IRS-1). Because IRS-1 is the major substrate of insulin receptor kinase, we expected disruption of the IRS-1 gene to reduce glucose uptake in Kir6.2 knockout mice. However, the double-knockout mice do not develop insulin resistance or glucose intolerance. An insulin tolerance test reveals the glucose-lowering effect of exogenous insulin in double-knockout mice and in Kir6.2 knockout mice to be similarly enhanced compared with wild-type mice. The basal 2-deoxyglucose uptake rate in skeletal muscle of double-knockout mice is increased similarly to the rate in Kir6.2 knockout mice. Accordingly, disruption of the IRS-1 gene affects neither systemic insulin sensitivity nor glucose uptake in skeletal muscles of Kir6.2-deficient mice. In addition, no significant changes were observed in phosphatidylinositol 3-kinase (PI3K) activity and its downstream signal in skeletal muscle due to lack of the Kir6.2 gene. Disruption of Kir6.2-containing Katp channels clearly protects against IRS-1-associated insulin resistance by increasing glucose uptake in skeletal muscles by a mechanism separate from the IRS-1/PI3K pathway.     

Skin temperature changes induced by strong static magnetic field exposure

High intensity static magnetic fields, when applied to the whole body of the anesthetized rat, have previously been reported to decrease skin temperature. The hypothesis of the present study was that in diamagnetic water, molecules in the air play significant roles in the mechanism of skin temperature decrease. We used a horizontal cylindrical superconducting magnet. The magnet produced 8 T at its center. A thermistor probe was inserted in a subcutaneous pocket of the anesthetized rats to measure skin temperature. Animals (n=10) were placed in an open plastic holder in which the ambient air was free to move in any direction (group I). Animals (n=10) were placed in a closed holder in which the air circulation toward the direction of weak magnetic field was restricted (group II). Each holder was connected to a hydrometer to measure humidity around the animal in the holder. The data acquisition phase consisted of a 5 min baseline interval, followed by inserting the animal together with the holder into the center of the magnet bore for a 5 min exposure and a 5 min postexposure period outside the bore. In group I, skin temperature and humidity around the animal significantly decreased during exposure, followed by recovery after exposure. In group II, skin temperature and humidity did not decrease during the measurement. The skin temperature decrease was closely related to the decrease in humidity around the body of the animal in the holder, and the changes were completely blocked by restricting the air circulation in the direction of the bore entrance. Possible mechanisms responsible for the decrease in skin temperature may be associated with magnetically induced movement of water vapor at the skin surface, leading to skin temperature decrease.     

Amphibian in vitro heart induction: a simple and reliable model for the study of vertebrate cardiac development

Amphibian embryos are an excellent model system for analyzing the mechanisms of vertebrate cardiogenesis. Studies of heart development in Xenopus have, for example, revealed that the inductive interaction of the heart primordia with the adjacent underlying endoderm and dorsal lip starts at the early stages of gastrulation. However, the molecular basis of those early inductive events and the genes expressed during the early phases of heart differentiation remain largely unknown. Amphibian blastula embryos contain pluripotent cells in their ectodermal region, called the "animal cap," which fortunately can be exploited for understanding a variety of organogenesis processes. Despite an enormous potential for analysis, the use of this system in cardiogenesis research has languished due to a lack of information concerning appropriate culture methods. Herein we report conditions for generating an in vitro heart induction system and present evidence from two types of in vivo transplantations, that the cultured heart rudiment can develop and function in the adult organism. It is expected that the fundamental principles established in this model system will provide a versatile research platform for a variety of organ engineering projects, including modifying in vitro organ growth with exogenous components (e.g. various growth factors) and developing methods for preparing tissue for transplantation.