Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1

Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.     

The role of tumor necrosis factor-α for interleukin-10 production by murine dendritic cells

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α^−/−) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α^−/− DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α^−/− DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α^−/− DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α^−/− DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs.     

Investigation of the microbial community in a microbiological additive used in a manure composting process

The objectives of this study were to investigate the fate of microorganisms by using cultivation methods as well as DNA analyses in a commercial microbiological additive (MA) in the course of the composting. Almost all the predominant species in the microbial succession during composting process determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were in disagreement with those determined by the clone library method. None of the microbial species in the composting stages corresponded to the microorganisms identified in the MA either by the cultivation method or DNA analysis. The results in regard to predominant microorganisms of the MA detected from the liquid medium by the PCR-DGGE did not correspond with those detected from the MA itself and composting processes. Although no evidence was found that predominant species in the MA itself dominate in the composting process, predominant species diversity in the MA itself was markedly changed after culturing at different thermophilic temperatures. These results suggested that cultivable microorganisms in the MA did not become predominant in the composting process: however, some microorganisms that are detected from the MA itself by the DNA analysis may act effectively in the composting process.     

Involvement of aldolase A in X-ray resistance of human HeLa and UV(r)-1 cells

To find novel proteins involved in radio-resistance of human cells, we searched for nuclear proteins, whose expression levels alter after X-ray irradiation in HeLa cells, using agarose fluorescent two-dimensional differential gel electrophoresis following mass spectrometry. We identified 6 proteins, whose levels were increased in nuclei 24 h after irradiation at 5 Gy, including aldolase A. Nuclear aldolase A levels increased twofold after the irradiation, however, total aldolase A levels did not change. When the expression of aldolase A was suppressed by its specific siRNA, sensitization of the suppressed cells to X-ray-induced cell death was observed. In addition, UV^r-1 cells with higher aldolase A expression exhibited lower sensitivity to X-ray-induced cell death than the parental RSa cells with lower aldolase A expression. These results suggest that aldolase A may play a role in the radio-response of human cells, probably in nuclei, in addition to its glycolytic role in the cytosol.     

The cleavage site preference of the porcine pepsin on the N-terminal α1 chain of bovine type I collagen: a focal analysis with mass spectrometry

Bovine type I collagen consists of two α1 and one α2 chains, containing the internal triple helical regions and the N- and C-terminal telopeptides. In industries, it is frequently digested with porcine pepsin to produce a triple helical collagen without the telopeptides. However, the digestion mechanism is not precisely understood. Here, we performed a mass spectrometric analysis of the pepsin digest of the N-terminal telopeptide pQLSYGYDEKSTGISVP (1–16) in the α1 chain. When purified collagen was digested, pQLSYGY (1–6) and pQLSYGYDEKSTG (1–12) were identified, while DEKSTG (7–12) was not. When the N-terminal telopeptide mimetic synthetic peptide pQLSK(MOCAc)GYDEKSTGISK(Dnp)P-NH₂ was digested, pQLSK(MOCAc)GYDEKSTG (1–12) and ISK(Dnp)P-NH₂ (13?16) were readily identified, pQLSK(MOCAc)GY (1?6) and DEKSTGISK(Dnp)P-NH₂ (7?16) were weakly detected, and DEKSTG (7–12) was hardly identified. These results suggest that pepsin preferentially cleaves Tyr6–Asp7 and less preferentially Gly12–Ile13. They also suggest that the former cleavage requires native collagen structure, while the latter cleavage does not.     

Peroxisome proliferator-activated receptor-γ ligands attenuate brain natriuretic peptide production and affect remodeling in cardiac fibroblasts in reoxygenation after hypoxia

Cardiac fibroblasts (CFs) participate in cardiac remodeling after hypoxic cardiac damage, and remodeling is thought to be mediated by CF synthesis of brain natriuretic peptide (BNP). It is unknown whether the peroxisome proliferator-activated receptors (PPARs), which mediate cellular signaling for growth and migration, affect BNP synthesis and whether PPARs participate in regulation of extracellular matrix protein (ECM) expression for remodeling. We examined the production of BNP in cultured neonatal ventricular CFs and its signaling system on collagen synthesis and on activation of matrix metalloproteinases (MMPs) in reoxygenation after hypoxia. BNP mRNA was detected in CFs, and a specific BNP protein, BNP1-32, was secreted into the media. Abundance of collagen I and III was increased in the media at reoxygenation. mRNA and protein levels for MMP-2 and the tissue inhibitor of metalloproteinase (TIMP)-1 were enhanced in CFs at reoxygenation. These observations also were noted in CFs after incubation with angiotensin II (10 μM) for 24 h. Pretreatment with pioglitaozone (0.1–10 μM) attenuated BNP mRNA and protein abundance of collagen III, MMP-2, and TIMP-1 in CFs at reoxygenation. The secreted BNP was also decreased by pioglitaozone in the media. Furthermore, PPAR activators inhibited reoxygenation-induced activation of nuclear factor (NF)-kB. These results demonstrate that PPAR activators inhibit BNP synthesis in CFs and imply that PPAR activators may regulate ECM remodeling partially through the NF-kB-mediated pathway.     

Comparison of cell lines for stable production of fucose-negative antibodies with enhanced ADCC

Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.     

Effect of indomethacin, tiaprofenic acid and dicrofenac on rat gastric mucosal damage and content of prostacyclin and prostaglandin E2

Gastric ulcerogenicity and depletion of endogenous prostaglandins (PGs) content induced by tiaprofenic acid, dicrofenac and indomethacin were examined using the same antiinflammatory effective doses. Male Wistar rats were given each of these drugs intragastrically 24, 18, and 3 hrs before sacrifice in the following doses (mg/kg): indomethacin (0.8, 4 and 20); tiaprofenic acid (1.2, 6 and 30); dicrofenac (0.8, 4 and 20). Endogenous prostacyclin (PGI2) and PGE2 in fundic mucosa were determined by radioimmunoassay. The three compounds produced fundic mucosal lesions in a dose-dependent manner. However, tiaprofenic acid and dicrofenac were both less potent than indomethacin in producing gastric mucosal lesions at similar antiinflammatory doses. Mucosal PGE2 content was abolished by the three compounds in the following doses (mg/kg): indomethacin (4 and 20); tiaprofenic acid (6 and 30); dicrofenac (20). Mucosal PGI2 was maintained around 50% of the control value in rats given tiaprofenic acid in a dose of 6 mg/kg or dicrofenac in a dose of 4 mg/kg, while indomethacin in a dose of 4 mg/kg markedly reduced mucosal PGI2 to 17% of the control value. In larger doses, tiaprofenic acid and dicrofenac were also significantly less potent in reducing mucosal PGI2 than indomethacin. These results suggest that the difference in ulcerogenicity between indomethacin and the other two compounds was closely related to their potency in decreasing PGI2 in the gastric (fundic) mucosa.     

Comparison of urine cytology between the ileal conduit and Indiana pouch

OBJECTIVE: To compare the cytomorphologic features of urine obtained from two different kinds of urinary diversions constructed after total bladder resection. STUDY DESIGN: The smears of urine from 11 ileal conduits and 6 Indiana pouches were evaluated. All patients underwent total bladder resection due to transitional cell carcinoma (TCC) or other kinds of cancer before urine diversion. RESULTS: The cytologic features of Indiana pouch urine include degenerated, small, round cells without columnar cells derived from intestinal epithelium. In ileal conduit urine, well-preserved columnar cells and degenerated, small, round cells were frequently observed. The columnar cells in ileal conduit urine exhibited cytologic features that should be distinguished from TCC cells. CONCLUSION: The method of reconstructing the urinary tract is important in urine cytology from urine diversions because the cytomorphologic features of urine are different between the two kinds of urinary diversions. Since columnar cells in ileal conduit urine might lead to misdiagnosis as TCC, special consideration is required to examine ileal conduit urine.