Discrete roles for histone acetylation in human T helper 1 cell-specific gene expression

To better understand the control of T helper (TH) 1-expressed genes, we compared and contrasted acetylation and expression for three key genes, IFNG, TBET, and IL18RAP and found them to be distinctly regulated. The TBET and the IFNG genes, but not the IL18RAP gene, showed preferential acetylation of histones H3 and H4 during TH1 differentiation. Analysis of acetylation of specific histone residues revealed that H3(Lys-9), H4(Lys-8), and H4(Lys-12) were preferentially modified in TH1 cells, suggesting a possible contribution of acetylation of these residues for induction of these genes. On the other hand, the acetylation of IL18RAP gene occurred both in TH1 and TH2 cells the similar kinetics and on the same with residues, demonstrating that selective histone acetylation was not universally the case for all TH1-expressed genes. Histone H3 acetylation of IFNG and TBET genes occurred with different kinetics, however, and was distinctively regulated by cytokines. Interleukin (IL)-12 and IL-18 enhanced the histone acetylation of the IFNG gene. By contrast, histone acetylation of the TBET gene was markedly suppressed by IL-4, whereas IL-12 and IL-18 had only modest effects suggesting that histone acetylation during TH1 differentiation is a process that is regulated by various factors at multiple levels. By treating Th2 cells with a histone deacetylase inhibitor, we restored histone acetylation of the IFNG and TBET genes, but it did not fully restore their expression in TH2 cells, again suggesting that histone acetylation explains one but not all the aspects of TH1-specific gene expression.     

Alteration of the timing of implantation by in vivo gene transfer: delay of implantation by suppression of nuclear factor kappaB activity and partial rescue by leukemia inhibitory factor

Nuclear factor kappaB (NF-kappaB) is activated in the murine endometrium during implantation period [Am. J. Reprod. Immunol. 51 (2004) 16]. Transient transfection of IkappaBalpha mutant (IkappaBalphaM) cDNA into the mouse uterine cavity using hemagglutinating virus of Japan envelope vector suppressed uterine NF-kappaB activity less than half of that observed in control on days 3.5 and 4.5 p.c. IkappaBalphaM cDNA transfection led to significant delay of implantation. After IkappaBalphaM cDNA transfection, LIF mRNA expression in the uterus was significantly suppressed on days 3.5 and 4.5 p.c. Co-transfection of LIF cDNA with IkappaBalphaM cDNA in the uterus partially rescued the delay of implantation induced by suppression of NF-kappaB activity. Taken together, these findings indicate that NF-kappaB activation determines the timing of the implantation, at least in part, via control of LIF expression.     

Noradrenaline stimulates ATP release from DRG neurons by targeting β3 adrenoceptors as a factor of neuropathic pain

Noradrenaline (NA), released in association with sympathetic nerve sprouting into the dorsal root ganglion (DRG) after peripheral nerve injury, may enhance neuropathic pain. ATP serves as a pain mediator; however, NA‐regulated ATP mobilizations in the DRG is far from understanding. In the present study, we analyzed ATP mobilizations in acutely dissociated rat DRG neurons by recording single‐channel currents through P2X receptor channels as an ATP biosensor in an outside‐out patch‐clamp configuration and by monitoring real‐time enzymatic NADPH fluorescent imaging, and examined the role for β₃ adrenoceptors in allodynia using an in vivo rat model. We show here that NA stimulates ATP release from DRG neurons as mediated via β₃ adrenoceptors linked to G_s protein involving PKA activation, to cause allodynia. This represents a fresh regulatory pathway for neuropathic pain relevant to noradrenergic transmission in the DRG. J. Cell. Physiol. 224: 345–351, 2010. © 2010 Wiley‐Liss, Inc.     

Molecular cloning and characterization of novel splicing variants of human decay-accelerating factor

Decay-accelerating factor (DAF) is one of the complement regulatory proteins. Two isoforms of DAF have been identified in humans. In this study, we isolated novel cDNAs encoding five isoforms of DAF from the human lung, which were generated by insertion of new exonic sequences. RT-PCR revealed that all isoforms were expressed in almost all tissues tested, although the expression patterns and levels differed among the tissues. Transfection of isoform vDAF1, 2, and 3 cDNAs into CHO cells showed that these molecules are soluble forms secreted after glycosylation. Isoform vDAF4 and vDAF5 cDNAs included a part of and the entire intron 7 sequence, respectively, and the transfection of vDAF4 cDNA produced a large, glycosylated, membrane-bound form. These results suggest that more than seven isoforms of human DAF are involved in the regulation of complement activation under physiological conditions through their specific structures and localization.     

Defective repair of radiation-induced DNA damage is complemented by a CHORI-230-65K18 BAC clone on rat chromosome 4

The Long Evans cinnamon (LEC) rat is highly susceptible to X-irradiation due to defective DNA repair and is thus a model for hepatocellular carcinogenesis. We constructed a bacterial artificial chromosome (BAC) contig of rat chromosome 4 completely covering the region associated with radiation susceptibility. We used transient and stable transfections to demonstrate that defective DNA repair in LEC cells is fully complemented by a 200-kb BAC, CHORI-230-65K18. Further analysis showed that the region associated with radiation susceptibility is located in a 128,543-bp region of 65K18 that includes the known gene Rpn1. However, neither knockdown nor overexpression of Rpn1 indicated that this gene is associated with radiation susceptibility. We also mapped three ESTs (TC523872, TC533727, and CB607546) in the 128,543-bp region, suggesting that 65K18 contains an unknown gene associated with X-ray susceptibility in the LEC rat.     

Platelet derived growth factor regulates ABCA1 expression in vascular smooth muscle cells

The ATP-binding cassette transporter A1 (ABCA1) regulates lipid efflux from peripheral cells to High-density lipoprotein. The platelet-derived growth factor (PDGF) is a potent mitogen that enables vascular smooth muscle cells to participate in atherosclerosis. In this report, we showed that PDGF suppressed endogenous expression of ABCA1 in cultured vascular smooth muscle cells. Exposure of CRL-208 cells to PDGF elicited a rapid phosphorylation of a kinase downstream from PI3-K, Akt. The constitutively active form of both p110, a subunit of PI3-K, and Akt inhibited activity of the ABCA1 promoter. In conclusion, PI3-K-Akt pathways participate in PDGF-suppression of ABCA1 expression.     

Live imaging of lymphatic development in the zebrafish

The lymphatic system has become the subject of great interest in recent years because of its important role in normal and pathological processes. Progress in understanding the origins and early development of this system, however, has been hampered by difficulties in observing lymphatic cells in vivo and in performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. Here, we show that the optically clear developing zebrafish provides a useful model for imaging and studying lymphatic development, with a lymphatic system that shares many of the morphological, molecular and functional characteristics of the lymphatic vessels found in other vertebrates. Using two-photon time-lapse imaging of transgenic zebrafish, we trace the migration and lineage of individual cells incorporating into the lymphatic endothelium. Our results show lymphatic endothelial cells of the thoracic duct arise from primitive veins through a novel and unexpected pathway.     

WRNIP1 accumulates at laser light irradiated sites rapidly via its ubiquitin-binding zinc finger domain and independently from its ATPase domain

WRNIP1 (Werner helicase-interacting protein 1) was originally identified as a protein that interacts with the Werner syndrome responsible gene product. WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain in the N-terminal region and two leucine zipper motifs in the C-terminal region. In addition, it possesses an ATPase domain in the middle of the molecule and the lysine residues serving as ubiquitin acceptors in the entire of the molecule. Here, we report that WRNIP1 accumulates in laser light irradiated sites very rapidly via its ubiquitin-binding zinc finger domain, which is known to bind polyubiquitin and to be involved in ubiquitination of WRNIP1 itself. The accumulation of WRNIP1 in laser light irradiated sites also required the C-terminal region containing two leucine zippers, which is reportedly involved in the oligomerization of WRNIP1. Mutated WRNIP1 with a deleted ATPase domain or with mutations in lysine residues, which serve as ubiquitin acceptors, accumulated in laser light irradiated sites, suggesting that the ATPase domain of WRNIP1 and ubiquitination of WRNIP1 are dispensable for the accumulation.     

Cell cycle regulation in mouse heart during embryonic and postnatal stages

The regulation of cardiomyocyte proliferation is important for heart development and function. Proliferation levels of mouse cardiomyocytes are high during early embryogenesis and start to decrease at midgestation. Many cardiomyocytes undergo mitosis without cytokinesis, resulting in binucleated cardiomyocytes during early postnatal stages, following which the cell cycle arrests irreversibly. It remains unknown how the proliferation pattern is regulated, and how the irreversible cell cycle arrest occurs. To clarify the mechanisms, fundamental information about cell cycle regulators in cardiomyocytes and cell cycle patterns during embryonic and postnatal stages is necessary. Here, we show that the expression, complex formation, and activity of main cyclins and cyclin‐dependent kinases (CDKs) changed in a synchronous manner during embryonic and postnatal stages. These levels decreased from midgestation to birth, and then showed one wave in which the peak was around postnatal day 5. Detailed analysis of the complexes suggested that CDK activities were inhibited before the protein levels decreased. Analysis of DNA content distribution patterns in mono‐ and binucleated cardiomyocytes after birth revealed changes in cell cycle distribution patterns and the transition from mono‐ to binucleated cells. These analyses indicated that the wave of cell cycle regulator expression or activities during postnatal stages mainly produced binucleated cells from mononucleated cells. The data obtained should provide a basis for the analysis of cell cycle regulation in cardiomyocytes during embryonic and postnatal stages.     

Metabolome analysis of photosynthesis and the related primary metabolites in the leaves of transgenic rice plants with increased or decreased Rubisco content

Because the comprehensive effects on metabolism by genetic manipulation of leaf Rubisco content are unknown, metabolome analysis was carried out on transgenic rice plants with increased or decreased Rubisco content using the capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) technique. In RBCS-sense plants, an increase in Rubisco content did not improve light-saturated photosynthesis. Glyceraldehyde 3-phosphate and sedoheputulose 7-phosphate levels increased, but ribulose bisphosphate (RuBP), ATP and ADP levels were not affected. It is considered from these results that RuBP regeneration independent of ATP supply became a bottleneck for photosynthesis. In RBCS-antisense plants, a decline in Rubisco content decreased photosynthesis with a substantial accumulation of RuBP. ATP and ADP levels also increased and were associated with increases in the diphosphate and triphosphate compounds of other nucleosides. These results imply that a decline in Rubisco content slowed down the Calvin cycle and that the resultant excess energy of ATP was transferred to other nucleoside diphosphates and triphosphates. The levels of amino acids tended to decline in RBCS-sense plants and increase in RBCS-antisense plants, probably reflecting the demand for Rubisco synthesis. Starch and carbohydrate levels decreased only in RBCS-antisense plants. Thus, genetic manipulation of Rubisco contents widely affected C and N metabolism in rice.