Evidence that C4b-binding protein (proline-rich protein) is synthesized by hepatocytes

C4b-binding protein (C4bp), a glycoprotein involved in regulating the classical pathway of the complement system, binds the activated form of C4b and accelerates the decay rate of the C4b, C2a complex. Recently, sequence analysis of the cDNA for proline-rich protein (PRP) demonstrated that PRP is identical with C4bp. We measured the concentration of C4bp in serum by single radial immunodiffusion in patients with various liver diseases. Concentration of C4bp was significantly lower in hepatic cirrhosis (P = 0.001) and higher in fatty liver (P = 0.0002) than the control values, after adjusting for age, sex, and concentration of total cholesterol, triglyceride, and C-reactive protein. Significant positive correlations were observed between the concentration of C4bp in serum and total protein, albumin, cholinesterase level, and lecithin-cholesterol acyltransferase activity. Immunohistochemical analysis of human liver with specific antiserum to human C4bp demonstrated reaction endproducts in the hepatocytes around the central veins. These observations provide evidence that C4bp is synthesized by hepatocytes.     

Specific interaction of angiostatin with integrin alpha(v)beta(3) in endothelial cells

Angiostatin, the N-terminal four kringles (K1-4) of plasminogen, blocks tumor-mediated angiogenesis and has great therapeutic potential. However, angiostatin's mechanism of anti-angiogenic action is unclear. We found that bovine arterial endothelial (BAE) cells adhere to angiostatin in an integrin-dependent manner and that integrins alpha(v)beta(3), alpha(9)beta(1), and to a lesser extent alpha(4)beta(1), specifically bind to angiostatin. alpha(v)beta(3) is a predominant receptor for angiostatin on BAE cells, since a function-blocking antibody to alpha(v)beta(3) effectively blocks adhesion of BAE cells to angiostatin, but an antibody to alpha(9)beta(1) does not. epsilon-Aminocaproic acid, a Lys analogue, effectively blocks angiostatin binding to BAE cells, indicating that an unoccupied Lys-binding site of the kringles may be required for integrin binding. It is known that other plasminogen fragments containing three or five kringles (K1-3 or K1-5) have an anti-angiogenic effect, but plasminogen itself does not. We found that K1-3 and K1-5 bind to alpha(v)beta(3), but plasminogen does not. These results suggest that the anti-angiogenic action of angiostatin may be mediated via interaction with alpha(v)beta(3). Angiostatin binding to alpha(v)beta(3) does not strongly induce stress-fiber formation, suggesting that angiostatin may prevent angiogenesis by perturbing the alpha(v)beta(3)-mediated signal transduction that may be necessary for angiogenesis.     

Amino acid residues in the alpha IIb subunit that are critical for ligand binding to integrin alpha IIbbeta 3 are clustered in the beta-propeller model

Several distinct regions of the integrin alpha(IIb) subunit have been implicated in ligand binding. To localize the ligand binding sites in alpha(IIb), we swapped all 27 predicted loops with the corresponding sequences of alpha(4) or alpha(5). 19 of the 27 swapping mutations had no effect on binding to both fibrinogen and ligand-mimetic antibodies (e.g. LJ-CP3), suggesting that these regions do not contain major ligand binding sites. In contrast, swapping the remaining 8 predicted loops completely blocked ligand binding. Ala scanning mutagenesis of these critical predicted loops identified more than 30 discontinuous residues in repeats 2-4 and at the boundary between repeats 4 and 5 as critical for ligand binding. Interestingly, these residues are clustered in the predicted beta-propeller model, consistent with this model. Most of the critical residues are located at the edge of the upper face of the propeller, and several critical residues are located on the side of the propeller domain. None of the predicted loops in repeats 1, 6, and 7, and none of the four putative Ca(2+)-binding predicted loops on the lower surface of the beta-propeller were important for ligand binding. The results map an important ligand binding interface at the edge of the top and on the side of the beta-propeller toroid, centering on repeat 3.     

Fine needle aspiration of toxoplasmic lymphadenitis in an intramammary lymph node. A case report

BACKGROUND: Cytologic findings of toxoplasmic lymphadenitis (TL) have been only sporadically reported. Intramammary lymph node is an extremely rare site for TL. CASE: A 47-year-old, healthy, female presented with a breast tumor, which was aspirated. The cytomorphologic features were interpreted as suggestive of TL. Histopathology of the excisional biopsy specimen and subsequent serologic examination confirmed the diagnosis. CONCLUSION: We obtained several characteristic findings in aspiration of TL. Of these, epithelioid cell clusters and monocytoid cells were the most diagnostic.     

Evidence for the association of ultraviolet-C and H(2)O(2)-induced apoptosis with acid sphingomyelinase activation

We measured the temperature dependence of oxygen evolution in thylakoids from tobacco using mass spectrometry and high resolution polarography. We determined the initial S-state distribution and the efficiency of the transition between these states including the probability of the O(2) yield through a fast mode. We observed discontinuous changes of the parameters at the temperatures 11 degrees C, 15 degrees C and 21 degrees C. Due to the mass spectroscopy data we think that the irregularity observed at 11 degrees C is due to conformational changes within the water catalytic site. We show that the different contributions of the slow and fast modes of oxygen evolution and of the water molecule exchange are correlated and that their behavior can be explained in terms of the H(2)O accessibility to the water splitting enzyme.     

Replacement of the Epstein-Barr virus plasmid with the EBER plasmid in Burkitt's lymphoma cells

Transfection of an Epstein-Barr virus (EBV)-encoded plasmid containing EBER caused a substantial decrease in the level of plasmid containing EBV in Akata and Mutu Burkitt's lymphoma (BL) lines, but failed to do so in other BL lines. The results suggest that EBER could replace the role of EBV, but other EBV products also play a role in the growth of BL.     

Role of bcl-2 in Epstein-Barr virus-induced malignant conversion of Burkitt's lymphoma cell line Akata

We have demonstrated that Epstein-Barr virus (EBV) confers enhanced growth capability in soft agarose, tumorigenesis in the SCID mouse, and resistance to apoptosis in the Burkitt's lymphoma cell line Akata. Subsequently, we have shown that EBV-encoded small RNAs (EBERs) are responsible for these phenotypes. We constantly observed the upregulation of bcl-2 oncoprotein expression upon EBV infection and expression of EBERs. To test whether these phenotypes were due to the upregulation of bcl-2 expression, we introduced bcl-2 into EBV-negative Akata cells at various levels encompassing the range at which EBV-positive cells expressed it. As cells expressed bcl-2 at higher levels, they became more capable of growing in soft agarose and became resistant to apoptosis. However, clones expressing bcl-2 at a higher level than EBV-positive Akata cells were negative in the tumorigenesis assay in the SCID mouse. On the other hand, introduction of bax into EBV-positive Akata cells reduced the resistance to apoptosis; however, it failed to reduce the growth capability in soft agarose. These data indicate that EBV targets not only bcl-2, but also an unknown pathway(s) to enhance the oncogenic potential of Akata cells.     

Bezafibrate attenuates the overexpression of plasminogen activator inhibitor-1 messenger RNA by a combination of mono-unsaturated fatty acid and insulin in hepG2 cells

The effects of bezafibrate (PPAR alpha activator) and troglitazone (PPAR gamma activator) on the expression of plasminogen activator inhibitor type-1 (PAI-1) in HepG2 cells were investigated. Exposure of the cells for 24 hours to either oleic acid or insulin showed no obvious effects on PAI-1 synthesis, whereas the combination of the two agents induced a 2.3-fold increase in PAI-1 synthesis, which was accompanied by a 3-fold increase in both the 2.2 kb and 3.2 kb forms of PAI-1 mRNA. This up-regulation of PAI-1 synthesis was attenuated by bezafibrate in a dose-dependent manner (1-100 microM) with 30% reversal at 100 microM. In contrast, troglitazone further stimulated PAI-1 synthesis to 140% of the level obtained in the presence of both oleic acid and insulin. This attenuation by bezafibrate and enhancement by troglitazone required the presence of both oleic acid and insulin. It is interesting that PAI-1 expression was affected so differently by these two PPAR activators.     

Mutational analysis of cell cycle inhibition by integrin beta1C

Integrin beta1C is an alternatively spliced cytoplasmic variant of the beta1 subunit that potently inhibits cell cycle progression. In this study, we analyzed the requirements for growth suppression by beta1C. A chimera containing the extracellular/transmembrane domain of the Tac subunit of the human interleukin 2 receptor (gp55) fused to the cytoplasmic domain of beta1C (residues 732-805) strongly inhibited growth in mouse 10T1/2 cells even at low expression levels, whereas chimeras containing the beta1A, beta1B, beta1D, beta3, and beta5 cytoplasmic domains had weak and variable effects. The beta1C cytoplasmic domain is composed of a membrane proximal region (732-757) common to all beta1 variants and a COOH-terminal 48-amino acid domain (758-805) unique to beta1C. The beta1C-specific domain (758-805) was sufficient to block cell growth even when expressed as a soluble cytoplasmic green fluorescent protein fusion protein. These results indicate that growth inhibition by beta1C does not require the intact receptor and can function in the absence of membrane targeting. Analysis of deletions within the beta1C-specific domain showed that the 18-amino acid sequence 775-792 is both necessary and sufficient for maximal growth inhibition, although the 13 COOH-terminal residues (793-805) also had weak activity. Finally, beta1C is known to be induced in endothelial cells in response to tumor necrosis factor and is down-regulated in prostate epithelial cells after transformation. The green fluorescent protein/beta1C (758-805) chimera blocked growth in the human endothelial cell line EV304 and in the transformed prostate epithelial cell line DU145, consistent with a role for beta1C as a growth inhibitor in vivo.