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AbstractBackground: A new type of glomerulonephritis following a methicillin-resistant Staphylococcus aureus (MRSA) infection has been reported. The purpose of this study is to elucidate the clinicopathological features and the responsiveness to treatment of the disease. Methods: We studied the treatment of 8 patients with glomerulonephritis related to MRSA infection. We observed the eight cases and analyzed clinical features, laboratory findings and histopathological data. Results: On admission, all patients had no renal abnormalities. One to four months after suffering from MRSA infection, severe proteinuria and hematuria developed. Renal biopsy specimens revealed moderate to severe mesangial proliferative glomerulonephritis with various degrees of crescent formation. Immunofluorescence studies showed IgA and C3. Antibiotic therapy was performed in six cases, resulting in successfully reducing the proteinuria in parallel with the decreased activity of MRSA infection in five cases. The other 2 cases received corticosteroid treatment after complete cessation of MRSA infection, but they had a relapse of MRSA infection and later died from sepsis. Conclusions: These results suggested that MRSA-associated glomerulonephritis might respond to antibiotic treatment in most cases. This also indicated that special care must be taken in the application of steroid therapy for the glomerulonephritis with crescents, even though the MRSA infection has gone into an inactive state.Copyright © 2002 S. Karger AG, Basel

Human platelet myosin forms 10S and 6S conformations, and its Ca(2+)- and Mg(2+)-ATPase activities are parallel with the transition between 10S and 6S conformation, as judged by the gel filtration, intrinsic fluorescence, and viscosity methods. The 20,000-dalton myosin light chain (LC20) is phosphorylated by both myosin light chain kinase (MLC kinase) and Ca2+, phospholipid-dependent protein kinase (protein kinase C [PKC]). The phosphorylation (1 mol of phosphate/mol of LC20) by MLC kinase shifts the equilibrium toward the 6S conformation, but that by PKC does not. The prephosphorylation of myosin by PKC prevents the effect of phosphorylation by MLC kinase on actin-activated Mg(2+)- ATPase activity, but not the effect on conformational change. Inhibition of actin-activated ATPase activity by PKC is due to a decreased affinity of myosin for actin, and no change in Vmax is observed. These results suggest that sequential phosphorylation of myosin by both kinases plays an important role in the ATPase activities of human platelet myosin.

We identified and cloned cDNAs for two novel CD30 mRNAs of 2.3 kb that are induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in the human myeloid leukemia cell line HL-60. These transcripts were transcribed from the intronic region just upstream of the exon coding for the transmembrane domain of the CD30 protein. The shorter cDNA had a deletion of 54 nucleotides corresponding to the 3′ region of the transmembrane domain of the CD30 and which was probably caused by alternative splicing. Translation of these transcripts appeared to start from the internal methionine codon at nucleotide position 289 that corresponds to that of 1612 in the CD30 cDNA, and encode a protein of 132 amino acid residues which corresponds exactly to the C-terminal cytoplasmic domain of CD30 protein. The calculated molecular mass of this variant CD30 (CD30v) protein was 14,087. Thus, the predicted CD30v protein retains most of the cytoplasmic region, but lacks the extracellular and transmembrane domains. Northern blots detected the expression of CD30v transcripts only in the lung and the TPA-stimulated HL-60 cell line. Translation of this mRNA in vitro produced a protein of 25 kD. Immunoblotting analysis with HCD30C1, a rabbit polyclonal antibody raised against the cytoplasmic domain of CD30 protein, detected proteins with an apparent Mr 25 kD expressed in TPA-stimulated HL-60 and COS-7 cells that were transfected with both types of CD30v cDNAs. Constitutive phosphorylation of the CD30v protein was demonstrated by in vitro labeling with [32P]. Immunohistochemical studies demonstrated CD30v protein was in alveolar macrophages. Cotransfection experiments using a kappa B-site-dependent reporter construct showed that CD30v can transactivate gene expression through activation of NF kappa B, as was noted on the authentic CD30 protein. Overexpression of the CD30v induced differentiation of HL-60 cells as evidenced by an increased NBT reduction activity. These observations provided new insights into the molecular heterogeneity and biological function of CD30 in myeloid cells.

A monoclonal anti-human platelet antibody, TP82, is described, which caused irreversible aggregation of platelets in association with the release of adenosine triphosphate or [14C] serotonin, and which inhibited ristocetin-induced agglutination. Immunofluorescence assay showed that the antibody binds to platelets, megakaryocytes, and common acute lymphoblastic leukemia cells. The antibody (IgG1) immunoprecipitated a polypeptide of 23,000 daltons with an isoelectric point of about 7.0. The aggregation induced by the purified antibody and/or F(ab')2 fragments occurred in platelet-rich plasma and with washed platelets, but not with formalin-fixed washed platelets. TP82- induced aggregation was completely inhibited by disodium ethylendiaminotetraacetate, diltiazem, W-7, PGE1, and several metabolic inhibitors. At a concentration of apyrase or CP/CPK, which inhibited adenosine 5-diphosphate-induced aggregation. TP82-induced aggregation was only partially affected. Thrombin was not required for the antibody- mediated effects, since two thrombin inhibitors failed to block the reaction. The antibody, at least at a high concentration, induced platelet aggregation by a mechanism almost independent of thromboxane A2 formation, since cyclooxygenase inhibitors had little inhibitory effect on aggregation. TP82 monoclonal antibody is a new platelet- aggregating substance that interacts with a low-molecular-weight binding site on the platelet membrane.

We produced two hybridomas by fusion of mouse myeloma cells with splenocytes from a mouse immunized with the THP-1 human monocytoid leukemia cell line. Two cloned hybridoma cell lines, designated as TM2 and TM3, were obtained. They secreted antibodies against a unique cell surface antigen expressed on all normal peripheral blood monocytes, neutrophilic granulocytes, platelets, and mitogen-induced lymphoblasts, some cells from patients with immature-type lymphoid leukemias. However, the antibodies reacted neither with large numbers of peripheral blood lymphocytes nor with red cells. Cross-blocking studies showed that these monoclonal antibodies recognized the same or a nearly positioned antigen epitope. Immunoprecipitation of THP-1 cell extract with TM2 or TM3 under reducing and nonreducing conditions yielded a specific band of mol wt equal to 120,000 daltons. This determinant appeared to be involved in granulocyte chemotaxis, since neutrophilic granulocytes exposed to TM2 or TM3 showed a significant decrease in chemotaxis toward endotoxin-activated serum. These two monoclonal antibodies did not affect O2- release or luminol-dependent chemiluminescence of neutrophils. Moreover, they did not alter platelet aggregation induced by thrombin. TM2 and TM3 will provide a new reagent in defining the linkage between lymphoid and myeloid differentiation and intermyeloid development.

Summary We purified three populations of virus particles, F7, F9 and F17, with buoyant densities of 1.34, 1.33 and 1.29 g/ml, respectively, in CsCl equilibrium density gradients from cultures of chick embryo liver cells infected with the H-162 strain of the virus of egg drop syndrome 1976. F9 particles were infectious complete virions and most F17 particles were empty particles. F7 particles were less infectious, and had little capacity of hemagglutination (HA). HA titers were the same at 4° and 37° C and maximal between pH 6.4 and 8.4 and ionic strength from 0.14 to 0.54m of NaCl. HA titer was inversely proportional to erythrocyte concentration. Potassium periodate destroyed markedly the infectivity of the virus and partially its HA activity at 37° C. HA activity was stable at 56° C or lower temperatures and destroyed at 80° C. Trypsin, a-chymotrypsin, papain, ficin and neuraminidase had no effect on HA activity. Alpha-chymotrypsin destroyed the receptor for the virus on chicken erythrocytes, whereas trypsin and neuraminidase did not affect the receptor.

About 100 amino acid residues in the C-terminal region are conserved among human, rat and murine CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, and can be separated into three subdomains with relatively higher conservation (D1, D2 and D3). Activation of NFκB by CD30 was shown to be mediated through interaction of TNFR-associated factor (TRAF) 1, 2 and 5 with the D2 and D3 subdomains. However, the function of the other conserved subdomain, D1, remained to be determined. Deletion of the D2 and D3 subdomains abolished interactions with TRAF2 and 5 but it did not affect NFκB activation. Reporter gene assays using deletion and mutant constructs of CD30 revealed that the D1 subdomain is sufficient for NFκB activation, without interaction with TRAF2 or 5, and that each subdomain alone can activate NFκB. Electrophoretic mobility shift assays revealed constitutive and CD30-induced NFκB activation in stable transformants of 293 cells expressing CD30 or a deletion mutant lacking D2 and D3 subdomains. Deletion of C-terminal 19 amino acid residues of the D1 subdomain abolished activation of NFκB. Substitution of alanine for one of the two threonine residues (amino acid position 524 and 529), one of which is a potential phosphorylation site in the D1 subdomain, also abolished the NFκB activation. Overexpression of the TRAF domain of TRAF2 or 5 had a dominant negative effect on the NFκB activation mediated by the D1 subdomain, thereby suggesting involvement of TRAF proteins in the signaling. Thus, the C-terminal 100 amino acid region of CD30 is composed of three independent functional subdomains, two of which contain binding sites for TRAF proteins. A novel domain in the cytoplasmic tail mediates NFκB activation, without direct interaction of TRAF2 or 5. Our observations suggest involvement of an unknown TRAF protein(s) in the signal transduction pathway of CD30.
Keywords:CD30, NFκB, tumor necrosis factor receptor-associated factor (TRAF), tumor necrosis factor receptor superfamily

About 100 amino acid residues in the C-terminal region are conserved among human, rat and murine CD30, a member of the tumor necrosis factor receptor (TNFR) superfamily, and can be separated into three subdomains with relatively higher conservation (D1, D2 and D3). Activation of NFkappaB by CD30 was shown to be mediated through interaction of TNFR-associated factor (TRAF) 1, 2 and 5 with the D2 and D3 subdomains. However, the function of the other conserved subdomain, D1, remained to be determined. Deletion of the D2 and D3 subdomains abolished interactions with TRAF2 and 5 but it did not affect NFkappaB activation. Reporter gene assays using deletion and mutant constructs of CD30 revealed that the D1 subdomain is sufficient for NFkappaB activation, without interaction with TRAF2 or 5, and that each subdomain alone can activate NFkappaB. Electrophoretic mobility shift assays revealed constitutive and CD30-induced NFkappaB activation in stable transformants of 293 cells expressing CD30 or a deletion mutant lacking D2 and D3 subdomains. Deletion of C-terminal 19 amino acid residues of the D1 subdomain abolished activation of NFkappaB. Substitution of alanine for one of the two threonine residues (amino acid position 524 and 529), one of which is a potential phosphorylation site in the D1 subdomain, also abolished the NFkappaB activation. Overexpression of the TRAF domain of TRAF2 or 5 had a dominant negative effect on the NFkappaB activation mediated by the D1 subdomain, thereby suggesting involvement of TRAF proteins in the signaling. Thus, the C-terminal 100 amino acid region of CD30 is composed of three independent functional subdomains, two of which contain binding sites for TRAF proteins. A novel domain in the cytoplasmic tail mediates NFkappaB activation, without direct interaction of TRAF2 or 5. Our observations suggest involvement of an unknown TRAF protein(s) in the signal transduction pathway of CD30.

MEG-01s, an established human megakaryoblastic leukemia cell line, exhibited specific high-affinity binding sites for [3H]iloprost, a stable prostaglandin (PG) I2 analogue, for [3H]SQ-29548, a stable thromboxane (TX) A2 antagonist and, for [3H]PGE2/PGE1, but not for [3H]PGD2. In the MEG-01s cells, iloprost/PGI2, or PGE1 stimulated cAMP production with ED50 values practically identical to the IC50 values for the [3H] iloprost binding. STA2 and U46619, TXA2/PGH2 agonists, PGE2/PGE1, iloprost/PGI2, and thrombin elevated the intracellular concentrations of Ca2+ ([Ca2+]i), as determined by Fura-2 fluorescence signals. Elevation of [Ca2+]i by PGE2/PGE1 and iloprost, but not that by TX-agonists or thrombin, was totally dependent on the presence of extracellular Ca2+. This effect by PGE2/PGE1 was partially inhibited by prior treatment of the cells with islet-activating protein (IAP), while that by TX-agonists or by PGI2/iloprost was not affected. We tentatively conclude from these results that: (1) MEG-01s cells express (a) PGI2/PGE1 receptor(s) coupled to adenylate cyclase and Ca2+ influx, a TXA2/PGH2 receptor coupled to the phosphatidylinositol-turnover-Ca2+ system, and the PGE2/PGE1 receptor coupled to Ca2+ influx; (2) the receptors for TXA2/PGH2 and iloprost and those for PGE2/PGE1 and thrombin are coupled to IAP-insensitive and IAP-sensitive GTP-binding proteins, respectively, and function in a different manner to elevate [Ca2+]i. Thus, the MEG-01s cell line is a pertinent model for studying eicosanoid receptor-mediated signal transduction in platelet/megakaryocyte systems.