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.     

p51/p63, a novel p53 homologue, potentiates p53 activity and is a human cancer gene therapy candidate

BACKGROUND: p51 (p73L/p63/p40/KET), a recently isolated novel p53 homologue, binds to p53-responsive elements to upregulate some p53 target genes and has been suggested to share partially overlapping functions with p53. p51 may be a promising candidate target molecule for anti-cancer therapy. METHODS: In this study, we adenovirally transduced p51A cDNA into human lung, gastric and pancreatic cancer cells and analyzed the intracellular function of p51 in anti-oncogenesis in vitro and in vivo. RESULTS: Overexpression of p51A revealed an anti-proliferative effect in vitro in all the cancer cells examined in this study. The anchorage-dependent and -independent cell growth of EBC1 cells carrying mutations in both p51 and p53 was suppressed and significant apoptosis following adenoviral transduction with p51 and/or p53 was seen. This growth suppression was cooperatively enhanced by the combined infection with adenoviral vectors encoding both p51 and p53. Furthermore, p51 activated several, but not all, p53-inducible genes, indicating that the mechanisms controlling p51- and p53-mediated tumor suppression differed. CONCLUSIONS: Our observations indicate that, although p51 exhibited reduced anti-oncogenetic effects compared with p53, it cooperatively enhanced the anti-tumor effects of p53. Our results suggest that p51 functions as a tumor suppressor in human cancer cells in vitro and in vivo and may be useful as a potential tool for cancer gene therapy.     

Selective regulation of CD8 effector T cell migration by the p110 gamma isoform of phosphatidylinositol 3-kinase

Chemokine-mediated T cell migration is essential to an optimal immune response. The p110gamma isoform of PI3K is activated by G protein-coupled receptors and regulates neutrophil and macrophage chemotaxis. We used p110gamma-deficient mice to examine the role of p110gamma in CD8 T cell migration and activation in response to viral challenge. Naive CD8 T cell migration in response to CCL21 in vitro and trafficking into secondary lymphoid organs in vivo was unaffected by the loss of p110gamma. Furthermore, loss of p110gamma did not affect CD8 T cell proliferation and effector cell differentiation in vitro in response to anti-CD3 stimulation or in vivo in response to vaccinia virus (VV) challenge. However, there was reduced migration of p110gamma knockout (p110gamma(-/-)) CD8 effector T cells into the peritoneum following i.p. challenge with VV. The role of p110gamma in CD8 effector T cell migration was intrinsic to T cells, as p110gamma(-/-) CD8 effector T cells exhibited impaired migration into the inflamed peritoneum following secondary transfer into wild-type recipients. In addition, p110gamma(-/-) CD8 effector T cells exhibited impaired migration in vitro in response to inflammatory chemoattractants. Although wild-type mice efficiently cleared VV at high viral doses, infection of p110gamma knockout mice resulted in visible illness and death less than a week after infection. Thus, p110gamma is dispensable for constitutive migration of naive CD8 T cells and subsequent activation and differentiation into effector CD8 T cells, but plays a central role in the migration of effector CD8 T cells into inflammatory sites.     

The WAVE2 complex regulates T cell receptor signaling to integrins via Abl- and CrkL-C3G-mediated activation of Rap1

WAVE2 regulates T cell receptor (TCR)–stimulated actin cytoskeletal dynamics leading to both integrin clustering and affinity maturation. Although WAVE2 mediates integrin affinity maturation by recruiting vinculin and talin to the immunological synapse in an Arp2/3-dependent manner, the mechanism by which it regulates integrin clustering is unclear. We show that the Abl tyrosine kinase associates with the WAVE2 complex and TCR ligation induces WAVE2-dependent membrane recruitment of Abl. Furthermore, we show that WAVE2 regulates TCR-mediated activation of the integrin regulatory guanosine triphosphatase Rap1 via the recruitment and activation of the CrkL–C3G exchange complex. Moreover, we demonstrate that although Abl does not regulate the recruitment of CrkL–C3G into the membrane, it does affect the tyrosine phosphorylation of C3G, which is required for its guanine nucleotide exchange factor activity toward Rap1. This signaling node regulates not only TCR-stimulated integrin clustering but also affinity maturation. These findings identify a previously unknown mechanism by which the WAVE2 complex regulates TCR signaling to Rap1 and integrin activation.     

Recurrent pneumonia with mild hypogammaglobulinemia diagnosed as X-linked agammaglobulinemia in adults

Background  

X-linked agammaglobulinemia (XLA) is a humoral immunodeficiency caused by disruption of the Bruton's tyrosine kinase (BTK) gene. Typical XLA patients suffer recurrent and severe bacterial infections in childhood.     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

P2Y6 receptor‐Gα12/13 signalling in cardiomyocytes triggers pressure overload‐induced cardiac fibrosis

Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)‐β. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (Gα_12/13) in cardiomyocytes suppressed pressure overload‐induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF‐β, connective tissue growth factor, and periostin) and Ang‐converting enzyme (ACE) was suppressed by the functional inhibition of Gα_12/13. The expression of these fibrogenic genes through Gα_12/13 by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G‐protein‐coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of Gα_12/13 in cardiomyocytes by the extracellular nucleotides‐stimulated P2Y₆ receptor triggers fibrosis in pressure overload‐induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF‐β.     

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.     

Production of functional spermatids from mouse germline stem cells in ectopically reconstituted seminiferous tubules

Testicular germ cell transplantation into the seminiferous tubules is at present the only way to induce spermatogenesis from a given source of spermatogonial stem cells. Here we show an alternative method that harnesses the self-organizing ability of testicular somatic cells. The testicular cells of embryonic or neonatal mice or rats and of newborn pigs were dissociated into single cells. Each of them reorganized into a tubular structure following implantation into the subcutis of immunodeficient mice. When mouse germline stem (GS) cells derived from spermatogonial stem cells and expanded in culture were intermingled with testicular cells of rodents, they were integrated in the reconstituted tubules and differentiated beyond meiosis into spermatids. Normal offspring were produced by the microinjection of those spermatids into oocytes. This method could be applicable to various mammalian species and useful for producing functional gametes from GS cells in a xenoectopic environment.