Western blotting analysis of heat shock proteins of Rickettsiales and other eubacteria

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits alpha(v), beta3, beta1, were examined. Results were as follows: 1) PMA induced OPN mRNA and OPN secretion into media; 2) untreated cells expressed beta1 and CD44 mRNA, and PMA induced alpha(v), and beta3 mRNA and increased beta1 and CD44 mRNA expression; 3) PMA increased levels of alpha(v), beta3, beta1 and CD44 protein on the cell surface; and 4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, alpha(v), beta3, beta1, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells.     

The efficacy of local anaesthesia for percutaneous epididymal sperm aspiration and testicular sperm aspiration

The actin filament-disrupting agent cytochalasin D strikingly increased tyrosine phosphorylation of a 75 kDa protein (p75) in rabbit aortic vascular smooth muscle cells. The microtubule-disrupting agent, colchicine had no effect on p75 tyrosine phosphorylation. Cytochalasin D also stimulated p75-directed kinase activity as determined by kinase assays of anti-Tyr(P) immunoprecipitates. Cytochalasin D stimulated tyrosine phosphorylation of the F-actin-binding protein, p80/85 cortactin, but p75 was not immunologically related either to cortactin, the phosphatidylinositol 3'-kinase p85 alpha subunit, or the 80 kDa isoform of caldesmon. These results suggest that p75 may represent a cytochalasin D-inducible kinase or kinase-associated component and provide evidence for the existence of a potentially novel kinase pathway regulated by disruption of the actin cytoskeleton.     

Thiopental alters contraction, intracellular Ca2+, and pH in rat ventricular myocytes

We have shown that osteogenic protein-1 (OP-1) (bone morphogenetic protein-7) is responsible for the induction of nephrogenic mesenchyme during embryonic kidney development. Gene knock-out studies showed that OP-1 null mutant mice die of renal failure within the first day of postnatal life. In the present study, we evaluated the effect of recombinant human OP-1 for the treatment of acute renal failure after 60 min bilateral renal artery occlusion in rats. Bioavailability studies in normal rats indicate that approximately 1.4 microg OP-1/ml is available in the circulation 1 min after intravenous administration of 250 microg/kg, which then declines steadily with a half life of 30 min. About 0.5% of the administered OP-1 dose/g tissue is targeted for OP-1 receptors in the kidney. We show that OP-1 preserves kidney function, as determined by reduced blood urea nitrogen and serum creatinine, and increased survival rate when administered 10 min before or 1 or 16 h after ischemia, and then at 24-h intervals up to 72 h after reperfusion. Histochemical and molecular analyses demonstrate that OP-1: (a) minimizes infarction and cell necrosis, and decreases the number of plugged tubules; (b) suppresses inflammation by downregulating the expression of intercellular adhesive molecule, and prevents the accumulation and activity of neutrophils; (c) maintains the expression of the vascular smooth muscle cell phenotype in pericellular capillaries; and (d) reduces programmed cell death during the recovery. Collectively, these data suggest that OP-1 prevents the loss of kidney function associated with ischemic injury and may provide a basis for the treatment of acute renal failure.     

Vascular integrin alpha(v)beta3: a new prognostic indicator in breast cancer

Blood vessel density is a prognostic indicator of multiple tumor types. Recently, it has been established that tumor-associated blood vessels express elevated levels of integrin alpha(v)beta3. In fact, there is evidence that integrin alpha(v)beta3 identifies the most proliferative endothelial cells within human breast carcinomas. Therefore, we evaluated breast cancer tissue in terms of both blood vessel density and alpha(v)beta3 expression. We found that the antibody LM609 to integrin alpha(v)beta3 preferentially stains the blood vessels of small caliber. Furthermore, comparative studies between LM609 and anti-CD31 antibodies on normal breast indicate that very low and weak expression of integrin alpha(v)beta3 was found on vessels within normal tissue, whereas CD31 antigen was expressed in almost all vasculature. Indeed, expression of integrin alpha(v)beta3 was significantly higher in tumors of patients with metastasis than in those without metastasis. In a series of 197 consecutive patients with invasive breast cancer and long follow-up, vascular expression of integrin alpha(v)beta3 in tumor vascular "hot spots" was found to be the most significant prognostic factor predictive of relapse-free survival in both node-negative and node-positive patients. These findings support the contention that angiogenesis plays a critical role in breast cancer progression and suggest that integrin alpha(v)beta3 is an endothelial cell marker with significant prognostic value and potential usefulness as a target for specific antiangiogenic therapy.     

Variation in HLA-DM expression influences conversion of MHC class II alpha beta:class II-associated invariant chain peptide complexes to mature peptide-bound class II alpha beta dimers in a normal B cell line

The maturation of invariant chain (Ii):MHC class II complexes into peptide-loaded alpha beta dimers occurs by proteolytic removal of Ii chain and binding of antigenic peptides derived from exogenous and endogenous Ags. A fragment of the Ii chain (class II-associated invariant chain peptide (CLIP) remains associated with class II alpha beta and is an intermediate in this process. Conversion of alpha beta:CLIP complexes into alpha beta:peptide complexes is facilitated by HLA-DM. Two unique mAbs, specific for I-Ab bound to human CLIP and I-Ab bound to DR alpha peptide, were used to assess the formation of these peptide:class II complexes in a human B lymphoblastoid cell line (B-LCL) (Swei) transfected with I-A(b). In multiple independent Swei:I-Ab transfectants, the amount of human CLIP (hCLIP):I-Ab expressed was inversely proportional to the amount of DR alpha 52-68:I-Ab; quantitative differences in HLA-DM expression accounted for this phenotype. In the low DM transfectant, a substantial proportion of I-Ab, but not DR molecules, was altered structurally and unable to present native protein Ags. Addition of DM transgenes to the DM-low cells resulted in an increase in DR alpha 52-68:I-Ab coupled with a decrease in hCLIP:I-Ab complexes and restoration of exogenous protein Ag presentation. The DR5 molecules in Swei cells, which have a lower affinity for hCLIP than I-Ab, were not affected by low DM expression, suggesting that the amount of DM required for conversion of CLIP:class II to peptide:class II may depend on the affinity of the class II molecules for CLIP or DM.     

Rickettsia conorii infection enhances vascular cell adhesion molecule-1- and intercellular adhesion molecule-1-dependent mononuclear cell adherence to endothelial cells

Leukocyte adherence to the endothelium is an essential component of the inflammatory response during rickettsial infection. In vitro, Rickettsia conorii infection of endothelial cells enhances the expression of adhesive molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in a time- and dose-dependent manner. Rickettsial lipopolysaccharide does not seem to be involved, because polymyxin B does not reduce their expression. The intracellular presence of the organism and de novo host protein synthesis are required for expression of cell adhesive molecules, since rickettsial inactivation by formol and pretreatment of cells with cycloheximide inhibits an increase in expression. The contribution of interleukin-1alpha (IL-1alpha) to this endothelial adhesive phenotype was shown by inhibitory experiments 8 and 24 h after infection with IL-1 receptor antagonist and IL-1alpha blocking antibodies. Enhanced adherence of mononuclear cells to infected endothelial cells involved VCAM-1- and ICAM-1-dependent mechanisms at the late phase of the inflammatory response. This endothelial adhesive phenotype may constitute a key pathophysiologic mechanism in R. conorii-induced vascular injury.     

Ultrastructural osteopontin localization in papillary stones induced in rats

OBJECTIVES: To detect in situ the precise osteopontin (OPN) localization in papillary stones. METHODS: Immunocytochemical labelling procedures are applied to detect OPN localizations in crystalline material of renal papillary stones. The tissue-processing procedure for electron microscopy, which includes OsO4 postfixation, preserves both immunocytochemical OPN reactivity and cellular membrane contrast up to the ultrathin section. Reflection-contrast light microscopical images are correlated with high resolution transmission-electron microscopical observations from consecutive ultrathin epon sections. RESULTS: Preserved crystalline material in interstitial and peripheral papillary stones is recognized as calcium oxalate monohydrate. After section incubation with markers conjugated to an antibody against OPN (alpha OPN) the crystals are converted into ghosts. In the ghosts, alpha OPN markers are present around microcrystals. The size of these microcrystals ranges from several nanometers to micrometers. It is observed (due to the OsO4-preserved membranes) that interstitial cells are separated from the stone surfaces by unidentified extracellular material, also present in the center as a stone matrix. CONCLUSION: The microcrystal-growth inhibitor OPN is detected in situ in interstitial stones induced in the rat's papilla and at the surface of the papilla.     

Intracrine and autocrine effects of basic fibroblast growth factor in vascular smooth muscle cells

In order to elucidate the effects of the different basic fibroblast growth factor (bFGF) isoforms on vascular smooth muscle, we examined aorta-derived vascular smooth muscle cells from transgenic mice expressing the human isoforms of bFGF. Four cell lines were examined from mice in which transgene expression was driven by the ubiquitous phosphoglycerate kinase promoter. Overexpression and cellular localization was confirmed by Western blot analysis in vascular smooth muscle cells from mice expressing: all four human bFGF isoforms (24, 22, 21, and 18 kDa); all three nuclear targeted isoforms (24, 22, and 21 kDa); only the 24 kDa isoform; and the only secreted/non-nuclear targeted isoform, 18 kDa. All lines showed approximate four-fold increases in bFGF expression, nuclear localization of all nuclear targeted bFGF isoforms, and cytosolic localization of only the 18 kDa bFGF. Measurement of [3H]thymidine incorporation into quiescent cells stimulated with increasing concentrations of serum, showed increased DNA synthesis in cell lines expressing any bFGF isoform when compared to non-transgenic control cells, and a further increase in DNA synthesis in cells expressing the nuclear targeted isoforms (24, 22, and 21 kDa) over the 18 kDa bFGF expressing cell line at any concentration of serum. All cells showed equal label incorporation when stimulated with 10 ng/ml of platelet-derived growth factor confirming an equal potential for DNA synthesis. Neutralizing the bFGF antibody markedly decreased serum-stimulated DNA synthesis, but only in the cell lines overexpressing the secreted/non-nuclear targeted 18 kDa isoform. These results suggest amplification of DNA synthesis through synergistic intracrine and autocrine effects of the nuclear targeted and non-nuclear targeted bFGF isoforms in vascular smooth muscle cells.     

Specific cross-links between fragments of proteolyzed Na,K-ATPase induced by o-phthalaldehyde

We have used o-phthalaldehyde (OPA) to cross-link adjacent fragments of "19 kDa membranes", a tryptic preparation of Na,K-ATPase lacking the ATP site but retaining cation occlusion sites. Treatment with OPA of "19 kDa membranes" or detergent-solubilized membranes containing occluded Rb ions [Or, E., Goldshleger, R., Tal, D. M., and Karlish, S. J. D. (1996) Biochemistry 35, 6853-6864] yielded cross-linked products of 25 and 31 kDa. Both species contained the 19 kDa fragment of the alpha subunit (transmembrane segments M7-M10). In addition, the 25 kDa product contained the fragment including M5-M6, while the 31 kDa product contained a 16 kDa fragment of the beta subunit. Cross-linking was unaffected by the absence or presence of ligands (Na, Rb, or Mg and ouabain). Cross-linking was largely abolished in thermally inactivated "19 kDa membranes". When proteolytic digestion of the 25 and 31 kDa products was combined with antibody binding, PKA-dependent phosphorylation, and sequencing of fragments, approximate positions of the cross-links were established. In the 25 kDa product, the cross-link was located within the short cytoplasmic segment Asn831-Arg841 of the 19 kDa fragment preceding M7 and within Ala749-Ala770 preceding M5. Thus, M7 and M5 are likely to be in close proximity. In the 31 kDa product, the cross-link was located in the extracellular loop of the alpha subunit between M7 and M8, close to residues which are known to interact with the beta subunit. Functional implications of the interactions between the fragments of the alpha (M5-M6 and M7-M10) and beta subunits are discussed.     

Concentrations of cytokines in plasma of patients with large burns: their relation to time after injury, burn size, inflammatory variables, infection, and outcome

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) are structurally related growth factors that exert their biological actions by binding to the same cell-surface receptor, EGF receptor. However, in chicken cells, human EGF binds with approximately 100-fold lower affinity than human TGF-alpha. In a previous study, we localized EGF/TGF-alpha receptor immunohistochemically in the granulosa and theca of the developing follicles of laying hens. We have also shown that TGF-alpha binds to cell-surface receptors of the granulosa cells. The present study characterizes the nature of the EGF/TGF-alpha receptor. Immunoprecipitation of receptor proteins from cultured granulosa cells with an anti-EGF receptor antibody (12E) shows the expression of a 170-kDa receptor protein. The expression of the receptor protein decreases with follicular enlargement between the F3 and F1. Incubation of the cells with [125I]TGF-alpha followed by cross-linking with bis(sulphosuccinimidyl)suberate showed that TGF-alpha binds a similar (170 kDa) receptor protein immunoprecipitated with the 12E anti-EGF receptor antibody. The binding of TGF-alpha to granulosa cells caused receptor protein oligomerization, yielding the monomeric (170 kDa) and dimeric (340 kDa) protein forms. Oligomerization seemed to favour the formation of the dimeric rather than the monomeric form. Culturing granulosa cells with luteinizing hormone or follicle-stimulating hormone increased the expression of both monomer and dimer forms of the receptor proteins compared with the control. Western blotting analysis with anti-phosphotyrosine antibody revealed that the lysates of TGF-alpha-stimulated cells express phosphotyrosine-containing receptor proteins of 170 kDa and 340 kDa. The results show that chicken granulosa cells express the 170-kDa EGF/TGF-alpha receptor protein, which dimerizes on binding to TGF-alpha, suggesting that the receptor protein may be involved in the signal transduction of TGF-alpha actions in the chicken granulosa cells.     

Protein kinases of cultured chicken osteoblasts that phosphorylate extracellular bone proteins

Cytosolic and microsomal protein kinase preparations from cultured chicken osteoblasts were found to phosphorylate up to six major proteins with Mrs 66, 58, 50, 36, 32, and 22 kDa in chicken bone extract. Use of heparin led to the conclusion that these proteins were predominantly phosphorylated by factor-independent protein kinase (FIPK) present both in microsomal and cytosolic preparations. It was confirmed that microsomal preparation contained predominantly FIPK, whereas cytosolic preparation contained additional kinases, that can phosphorylate the bone proteins. Use of purified chicken bone osteopontin (OPN) (58 kDa) and recombinant OPN led to the same conclusions. The identify of the protein kinases was clearly established by using a series of synthetic peptide substrates. Quantitative analysis utilizing pure protein kinases and purified chicken bone OPN, recombinant mouse OPN, and bovine bone OPN and BSP led to introduction of approximately 9 moles of phosphate/mole of OPN and 6.6 moles phosphate/mole bovine bone sialoprotein (BSP) by casein kinase II. cGMP-dependent protein kinase and protein kinase C both introduced 0.5-1.2 moles phosphate/mole of OPN and BSP, whereas cAMP-dependent protein kinase led to no significant phosphorylation of OPN or BSP. Consistent with the above results, sites of phosphorylation identified for OPN (metabolically labeled) and BSP (labeled by casein kinase II) revealed that predominant phosphorylated sites have recognition sequences for FIPK.     

Protection of B lymphocyte hybridoma against starvation-induced apoptosis: survival-signal role of some amino acids

Immunofluorescence microscopic observations indicated that a monoclonal antibody, Vmp 18, raised against the peptide 199-208 of murine interleukin 1 alpha, cross-reacted with an antigenic determinant of Drosophila thorax muscles. Immunoelectron microscopic analysis showed that the gold particles were mainly localized in the Z-line which is the attachment site of thin filaments from adjacent sarcomeres. On the contrary, the antibody failed to mark the Z-line in vertebrate skeletal muscle. A Western blot of total protein extract from Drosophila thorax muscles bound a protein of 43 kDa. Our observations suggest that the Vmp 18 antibody could contribute to clarify the composition of the Z-line in insect's flight muscles.     

FISH-based analysis of stable translocations in a Techa River population

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.     

The CD8+ cell phenotype mediating antiviral activity in feline immunodeficiency virus-infected cats is characterized by reduced surface expression of the CD8 beta chain

The acute stage of feline immunodeficiency virus (FIV) infection is characterized by a CD8+ anti-FIV response that parallels the appearance of a CD8+ subpopulation with reduced expression of the beta chain (CD8 alpha + beta lo). The relationship between the CD8 alpha + beta lo phenotype and CD8+ anti-FIV activity was examined. Flow cytometric analysis of peripheral blood mononuclear cells with anti-CD8 beta chain monoclonal antibody 117 revealed that the CD8 alpha + beta lo phenotype expanded throughout the asymptomatic infection, constituting 80%-90% of the CD8 beta + cells in long-term-infected cats. Purified CD8 alpha + beta hi and CD8 alpha + beta lo subpopulations were analyzed for anti-FIV activity in an acute infection assay. Anti-FIV activity resided principally in the CD8 alpha + beta lo population and was demonstrated in acute FIV infections, as well as in long-term asymptomatic infections. These data suggest that a unique CD8 alpha + beta lo anti-FIV phenotype arises early in infection and may play a major role in eliminating virus and maintaining the asymptomatic infection.     

Modulation of in vivo migratory function of alpha 2 beta 1 integrin in mouse liver

We report herein that expression of alpha 2 beta 1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that alpha 2 beta 1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of alpha 2 beta 1 on KX2C2 and RDX2C2 cells using a alpha 2 beta 1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated alpha 2 beta 1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn2+, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that alpha 2 beta 1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that alpha 2 beta 1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.