Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.     

C-Terminal peptidyl-L-proline hydrolase activity of Aspergillus acid carboxypeptidase.

Aspergillus saitoi acid carboxypeptidase hydrolyzed C-terminal peptidyl-L-proline bonds and released the C-terminal proline from Z-Gly-Pro-Leu-Gly-Pro and Z-Gly-Pro at pH 3.3. Proline liberated by the enzymic reaction was measured by a sensitive colorimetric ninhydrin method in glacial acetic acid at 513 nm. A Km value of 1.0 mM and a kcat value of 0.09 s-1 for Z-Gly-Pro-Leu-Gly-Pro hydrolysis, and a Km value of 5.0 mM and a kcat value of 0.0045 s-1 for Z-Gly-Pro hydrolysis were calculated from Lineweaver-Burk plots.     

CD44 stimulation by fragmented hyaluronic acid induces upregulation and tyrosine phosphorylation of c-Met receptor protein in human chondrosarcoma cells

Hepatocyte growth factor/scatter factor (HGF/SF) can induce proliferation and motility and promote invasion of tumor cells. Since HGF/SF receptor, c-Met, is expressed by tumor cells, and since stimulation of CD44, a transmembrane glycoprotein known to bind hyaluronic acid (HA) in its extracellular domain, is involved in activation of c-Met, we have studied the effects of CD44 stimulation by ligation with HA upon the expression and tyrosine phosphorylation of c-Met on human chondrosarcoma cell line HCS-2/8. The current study indicates that (a) CD44 stimulation by fragmented HA upregulates expression of c-Met proteins; (b) fragmented HA also induces tyrosine phosphorylation of c-Met protein within 30 min, an early event in this pathway as shown by the early time course of stimulation; (c) the effects of HA fragments are critically HA size-dependent. High molecular weight HA is inactive, but lower molecular weight fragments (M(r) 3.5 kDa) are active with maximal effect in the microg/ml range; (d) the standard form of CD44 (CD44s) is critical for the response because the effect on c-Met, both in terms of upregulation and phosphorylation, is inhibited by preincubation with an anti-CD44 monoclonal antibody; and (e) phosphorylation of c-Met induced by CD44 stimulation is inhibited by protein tyrosine kinase inhibitor, tyrphostin. Therefore, our study represents the first report that CD44 stimulation induced by fragmented HA enhances c-Met expression and tyrosine phosphorylation in human chondrosarcoma cells. Taken together, these studies establish a signal transduction cascade or cross-talk emanating from CD44 to c-Met.     

TG1 integrase-based system for site-specific gene integration into bacterial genomes

Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ～2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ～10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ～10-kbp DNA into the genome with an efficiency of ～10⁴ transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli “centromere” analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC–migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.     

Microwounding is a pivotal factor for the induction of actin-dependent penetration resistance against fungal attack

Induced penetration resistance is triggered by failed penetration attempts of nonpathogenic fungi. The resistance mechanism is an important nonhost reaction in plants that can block the invasion of filamentous pathogens such as fungi and oomycetes. However, it remains unclear whether the mechanical stimuli accompanying fungal penetration play a role in induced penetration resistance, whereas the perforation of the cell wall may provide significant stimuli to plant cells. Here, we used microneedles or biolistic bombardment to mimic fungal penetration pegs and a micromanipulation transfer technique of the bio-probe, a germling of Blumeria graminis hordei, to the wounded cells to demonstrate that microwounds derived from fungal penetration attempts may trigger induced penetration resistance in plant cells. When preinoculated with the nonpathogenic fungi Erysiphe pisi and Colletotrichum orbiculare, which were unable to penetrate a barley cell, the penetration of a bio-probe that was transferred by micromanipulation onto the same cell was completely blocked. Fungal penetration was essential to the triggering of induced penetration resistance because a penetration-peg-defective mutant of C. orbiculare completely lacked the ability to trigger resistance. The artificial microwounds significantly, but not completely, blocked the penetration of the bio-probe. Treatment with the actin polymerization inhibitor cytochalasin A or expression of the actin depolymerizing protein HvPro1 caused complete ablation of the induced penetration resistance triggered by either failed fungal penetration or artificial microwounds. These results strongly suggest that microwounding may trigger actin-dependent induced penetration resistance. Manipulation of induced penetration resistance may be a promising target to improve basic disease resistance in plants.     

Rapid Retting of Kôzo (Paper Mulberry) Bast with Erwinia carotovora

The degradation of nitrite and the inhibition towards formation of carcinogenic nitrosamines by melanoidins, produced from the glucose-glycine system were investigated at various conditions. The degradation of nitrite was highest at pH 1.2 (29%), when the ratio of melanoidins to nitrite was 1: 3. The inhibition towards formation of nitrosamines by melanoidins had the same tendency as the degradation of nitrite, the inhibition also being highest at pH 1.2 (99%). In addition, melanoidins after nitrite treatment exhibited a little higher mutagenicity and much stronger desmutagenicity than those of the original melanoidins. The change of the structure of melanoidins after treating with nitrite was also investigated by HPLC and CP-MAS NMR.     

Effects of gestational exposure to 1.95‐GHz W‐CDMA signals for IMT‐2000 cellular phones: Lack of embryotoxicity and teratogenicity in rats

The present study was designed to evaluate whether gestational exposure to an EMF targeting the head region, similar to that from cellular phones, might affect embryogenesis in rats. A 1.95‐GHz wide‐band code division multiple access (W‐CDMA) signal, which is one applied for the International Mobile Telecommunication 2000 (IMT‐2000) system and used for the freedom of mobile multimedia access (FOMA), was employed for exposure to the heads of four groups of pregnant CD(SD) IGS rats (20 per group) for gestational days 7–17. The exposure was performed for 90 min/day in the morning. The spatial average specific absorption rate (SAR) for individual brains was designed to be 0.67 and 2.0 W/kg with peak brain SARs of 3.1 and 7.0 W/kg for low (group 3) and high (group 4) exposures, respectively, and a whole‐body average SAR less than 0.4 W/kg so as not to cause thermal effects due to temperature elevation. Control and sham exposure groups were also included. At gestational day 20, all dams were killed and fetuses were taken out by cesarean section. There were no differences in maternal body weight gain. No adverse effects of EMF exposure were observed on any reproductive and embryotoxic parameters such as number of live (243–271 fetuses), dead or resorbed embryos, placental weights, sex ratios, weights or external, visceral or skeletal abnormalities of live fetuses. Bioelectromagnetics 30:205–212, 2009. © 2008 Wiley‐Liss, Inc.