Infiltration of H-2d-specific cytotoxic macrophage with unique morphology into rejection site of allografted meth A (H-2d) tumor cells in C57BL/6 (H-2b) mice

It is assumed that CD8(+) cytotoxic T lymphocytes (CTLs) mediate direct lysis of allografts and that their growth, differentiation, and activation are dependent upon cytokine production by CD4(+) helper T lymphocytes. In the present study, the effector cells responsible for the rejection of i.p. allografted, CTL-resistant Meth A tumor cells from C57BL/6 mice were characterized. The cytotoxic activity was associated exclusively with peritoneal exudate cells and not with the cells in lymphoid organs or blood. On day 8, when the cytotoxic activity reached a peak, 3 types of cells (i.e., lymphocytes, granulocytes, and macrophages) infiltrated into the rejection site; and allograft-induced macrophages (AIM) were cytotoxic against the allograft. Bacterially-elicited macrophages also exhibited cytotoxic activity (approximately 1/2 of that of AIM) against Meth A cells, whereas the cytotoxic activity of AIM against these cells but not that of bacterially-elicited macrophages was completely inhibited by the addition of donor (H-2(d))-type lymphoblasts, suggesting H-2(d)-specific cytotoxicity of AIM against Meth A cells. In contrast, resident macrophages were inactive toward Meth A cells. Morphologically, the three-dimensional appearance of AIM showed them to be unique large elongated cells having radiating peripheral filopodia and long cord-like extensions arising from their cytoplasmic surfaces. The ultrastructural examination of AIM revealed free ribosomes in their cytoplasm, which was often deformed by numerous large digestive vacuoles. These results indicate that AIM are the H-2(d)-specific effector cells for allografted Meth A cells and are a more fully activated macrophage with unique morphological features.     

Microbial communities associated with acetate-rich gas-petroleum reservoir surface facilities

We evaluated the microbial communities in acetate-rich production waters from separators of a high-temperature gas-petroleum reservoir in Higashi-Niigata, Japan. Bacterial and archaeal 16S rRNA gene libraries constructed from these waters were dominated by Acetobacterium-, Methanofollis-, and Methanosarcina-related sequences. The libraries constructed from enrichment cultures of the production waters were dominated by sequences related to the Acetobacterium- and Methanofollis-related sequences.     

A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation

The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 μM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.     

β-Actin specifically controls cell growth, migration, and the G-actin pool

Ubiquitously expressed β-actin and γ-actin isoforms play critical roles in most cellular processes; however, their unique contributions are not well understood. We generated whole-body β-actin-knockout (Actb(-/-)) mice and demonstrated that β-actin is required for early embryonic development. Lethality of Actb(-/-) embryos correlated with severe growth impairment and migration defects in β-actin-knockout primary mouse embryonic fibroblasts (MEFs) that were not observed in γ-actin-null MEFs. Migration defects were associated with reduced membrane protrusion dynamics and increased focal adhesions. We also identified migration defects upon conditional ablation of β-actin in highly motile T cells. Of great interest, ablation of β-actin altered the ratio of globular actin (G-actin) to filamentous actin in MEFs, with corresponding changes in expression of genes that regulate the cell cycle and motility. These data support an essential role for β-actin in regulating cell migration and gene expression through control of the cellular G-actin pool.     

A Src family inhibitor (PP1) potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells

Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity. We have recently reported that Cx32 acts as a tumor suppressor gene in renal cancer cells due to the inhibition of Src-dependent signaling. In line with the previous study, here we examined if a Src family inhibitor (PP1) could potentiate tumor-suppressive effect of Cx32 in Caki-2 cell from human renal cell carcinoma. In order to clarify the potentialization of PP1, using Cx32-transfected Caki-2 cells and mock-transfected Caki-2 cells, we estimated difference in cytotoxic effect of PP1 on the two cell clones in vitro as well as in vivo. PP1 showed more cytotoxic effect on Caki-2 cells having Cx32 positive expression than that of Cx32 negative expression at lower doses. This potentialization was also observed in xenograft model of nude mice. The potentialization of the effect mainly depended on the induction of apoptosis but not the control of cell growth. In conjugation with this event, the reduction of anti-apoptotic molecules (Bcl-2 and Bcl-xL) was caused by the combination of Cx32 expression and PP1 treatment in Caki-2 cells. These results suggest that PP1 potentiates tumor-suppressive effect of connexin 32 gene in renal cancer cells through the reduction of anti-apoptotic molecules.     

Engineering issues of microbial ecology in space agriculture]

Closure of the materials recycle loop for water-foods-oxygen is the primary purpose of space agriculture on Mars and Moon. A microbial ecological system takes a part of agriculture to process our metabolic excreta and inedible biomass and convert them to nutrients and soil substrate for cultivating plants. If we extend the purpose of space agriculture to the creation and control of a healthy and pleasant living environment, we should realize that our human body should not be sterilized but exposed to the appropriate microbial environment. We are proposing a use of hyper-thermophilic aerobic composting microbial ecology in space agriculture. Japan has a broad historical and cultural background on this subject. There had been agriculture that drove a closed loop of materials between consuming cities and farming villages in vicinity. Recent environmental problems regarding garbage collection and processing in towns have motivated home electronics companies to innovate "garbage composting" machines with bacterial technology. Based on those matured technology, together with new insights on microbiology and microbial ecology, we have been developing a conceptual design of space agriculture on Moon and Mars. There are several issues to be answered in order to prove effectiveness of the use of microbial systems in space. 1) Can the recycled nutrients, processed by the hyper-thermal aerobic composting microbial ecology, be formed in the physical and chemical state or configuration, with which plants can uptake those nutrients? A possibility of removing any major components of fertilizer from its recycle loop is another item to be evaluated. 2) What are the merits of forming soil microbial ecology around the root system of plants? This might be the most crucial question. Recent researches exhibit various mutually beneficial relationships among soil microbiota and plants, and symbiotic ecology in composting bacteria. It is essential to understand those features, and define how to conduct preventive maintenance for keeping cultivating soil healthy and productive. 3) Does microbial ecology contribute to building sustainable and expandable human habitation by utilizing the on site extraterrestrial resources? We are assessing technical feasibility of converting regolith to farming soil and structural materials for space agriculture. In the case of Mars habitation, carbon dioxide and a trace amount of nitrogen in atmosphere, and potassium and phosphor in minerals are the sources we consider. Excess oxygen can be accumulated by woods cultivation and their use for lumber. 4) Is the operation of space agriculture robust and safe, if it adopts hyper-thermophilic aerobic microbial ecology? Any ecological system is complex and non-linear, and shows latency and memory effects in its response. It is highly important to understand those features to design and operate space agriculture without falling into the fatal failure. Assessment should be made on the microbial safety and preparation of the preventive measures to eliminate negative elements that would either retard agricultural production or harm the healthy environment. It is worth to mention that such space agriculture would be an effective engineering testbed to solve the global problem on energy and environment. Mars and Moon exploration itself is a good advocate of healthy curiosity expressed by the sustainable civilization of our humankind. We propose to work together towards Mars and Moon with microbial ecology to assure pleasant habitation there.     

Maternal Effect by Stem Females in <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>: Effect of Starvation on Mixis Induction in Offspring

We examined whether starvation during the initial period of life in stem females affected reproductive characteristics of the offspring. Starvation treatment had different effects on rotifers hatched from resting eggs and those hatched from amictic eggs. When stem females experienced starvation after hatching, this induced a higher percentage of mixis in their offspring. When the same starvation treatment was applied to rotifers hatched from amictic eggs, there was no effect on the induction of mixis. It is probable that stem females hatched from resting eggs have specific features that are vulnerable to unfavorable environmental conditions, and that these features can be inherited by their offspring through the maternal cytoplasm.     

Validation of an enzyme-linked immunosorbent assay kit using herpesvirus papio 2 (HVP2) antigen for detection of herpesvirus simiae (B virus) infection in rhesus monkeys

Serologic testing for antibody to monkey B virus (BV) in macaque sera is problematic due to the biohazardous nature of BV antigens. Herpesvirus papio 2 (HVP2), a herpesvirus of baboons, is nonpathogenic to humans and is genetically and antigenically more closely related to BV than is human herpes simplex virus 1. This paper describes the results of our in-house laboratory that compared a BV antigen-based enzyme-linked immunosorbent assay (ELISA) by commercial testing laboratory and an HVP2-based ELISA in our laboratory by using 447 sera from 290 rhesus monkeys. The HVP2-based ELISA identified as positive 99.11% of the sera identified as BV-positive by the BV ELISA. The BV antigen-based ELISA identified as positive 98.21% of the sera identified as BV-positive by the HVP2-based ELISA. The HVP2 ELISA also identified two BV-negative and six BV-equivocal sera as positive. Both ELISAs identified the same 85 negative and three equivocal samples as negative and equivocal, respectively. The high degree of correlation (weighted kappa coefficient, 0.94) between the two tests indicates that the HVP2 ELISA is a sensitive and reliable assay for in-house testing of the BV status of rhesus monkeys.     

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.