Visualization of multivalent histone modification in a single cell reveals highly concerted epigenetic changes on differentiation of embryonic stem cells

Combinations of histone modifications have significant biological roles, such as maintenance of pluripotency and cancer development, but cannot be analyzed at the single cell level. Here, we visualized a combination of histone modifications by applying the in situ proximity ligation assay, which detects two proteins in close vicinity (∼30 nm). The specificity of the method [designated as imaging of a combination of histone modifications (iChmo)] was confirmed by positive signals from H3K4me3/acetylated H3K9, H3K4me3/RNA polymerase II and H3K9me3/H4K20me3, and negative signals from H3K4me3/H3K9me3. Bivalent modification was clearly visualized by iChmo in wild-type embryonic stem cells (ESCs) known to have it, whereas rarely in Suz12 knockout ESCs and mouse embryonic fibroblasts known to have little of it. iChmo was applied to analysis of epigenetic and phenotypic changes of heterogeneous cell population, namely, ESCs at an early stage of differentiation, and this revealed that the bivalent modification disappeared in a highly concerted manner, whereas phenotypic differentiation proceeded with large variations among cells. Also, using this method, we were able to visualize a combination of repressive histone marks in tissue samples. The application of iChmo to samples with heterogeneous cell population and tissue samples is expected to clarify unknown biological and pathological significance of various combinations of epigenetic modifications.     

Homeobox Genes in the Rodent Pineal Gland: Roles in Development and Phenotype Maintenance

The pineal gland is a neuroendocrine gland responsible for nocturnal synthesis of melatonin. During early development of the rodent pineal gland from the roof of the diencephalon, homeobox genes of the orthodenticle homeobox (Otx)- and paired box (Pax)-families are expressed and are essential for normal pineal development consistent with the well-established role that homeobox genes play in developmental processes. However, the pineal gland appears to be unusual because strong homeobox gene expression persists in the pineal gland of the adult brain. Accordingly, in addition to developmental functions, homeobox genes appear to be key regulators in postnatal phenotype maintenance in this tissue. In this paper, we review ontogenetic and phylogenetic aspects of pineal development and recent progress in understanding the involvement of homebox genes in rodent pineal development and adult function. A working model is proposed for understanding the sequential action of homeobox genes in controlling development and mature circadian function of the mammalian pinealocyte based on knowledge from detailed developmental and daily gene expression analyses in rats, the pineal phenotypes of homebox gene-deficient mice and studies on development of the retinal photoreceptor; the pinealocyte and retinal photoreceptor share features not seen in other tissues and are likely to have evolved from the same ancestral photodetector cell.     

A role for LFA‐1 in delaying T‐lymphocyte egress from lymph nodes

Lymphocytes use the integrin leukocyte function‐associated antigen‐1 (LFA‐1) to cross the vasculature into lymph nodes (LNs), but it has been uncertain whether their migration within LN is also LFA‐1 dependent. We show that LFA‐1 mediates prolonged LN residence as LFA‐1^?/? CD4 T cells have significantly decreased dwell times compared with LFA‐1^+/+ T cells, a distinction lost in hosts lacking the major LFA‐1 ligand ICAM‐1. Intra‐vital two‐photon microscopy revealed that LFA‐1^+/+ and LFA‐1^?/? T cells reacted differently when probing the ICAM‐1‐expressing lymphatic network. While LFA‐1^+/+ T cells returned to the LN parenchyma with greater frequency, LFA‐1^?/? T cells egressed promptly. This difference in exit behaviour was a feature of egress through all assessed lymphatic exit sites. We show that use of LFA‐1 as an adhesion receptor amplifies the number of T cells returning to the LN parenchyma that can lead to increased effectiveness of T‐cell response to antigen. Thus, we identify a novel function for LFA‐1 in guiding T cells at the critical point of LN egress when they either exit or return into the LN for further interactions.     

A study of wall shear stress in 12 aneurysms with respect to different viscosity models and flow conditions

Recent computational fluid dynamics (CFD) studies relate abnormal blood flow to rupture of cerebral aneurysms. However, it is still debated how to model blood flow with sufficient accuracy. Common assumptions made include Newtonian behaviour of blood, traction free outlet boundary conditions and inlet boundary conditions based on available literature. These assumptions are often required since the available patient specific data is usually restricted to the geometry of the aneurysm and the surrounding vasculature. However, the consequences of these assumptions have so far been inadequately addressed.     

Effects of small interfering RNA-mediated hepatic glucagon receptor inhibition on lipid metabolism in db/db mice

Hepatic glucose overproduction is a major characteristic of type 2 diabetes. Because glucagon is a key regulator for glucose homeostasis, antagonizing the glucagon receptor (GCGR) is a possible therapeutic strategy for the treatment of diabetes mellitus. To study the effect of hepatic GCGR inhibition on the regulation of lipid metabolism, we generated siRNA-mediated GCGR knockdown (si-GCGR) in the db/db mouse. The hepatic knockdown of GCGR markedly reduced plasma glucose levels; however, total plasma cholesterol was increased. The detailed lipid analysis showed an increase in the LDL fraction, and no change in VLDL HDL fractions. Further studies showed that the increase in LDL was the result of over-expression of hepatic lipogenic genes and elevated de novo lipid synthesis. Inhibition of hepatic glucagon signaling via siRNA-mediated GCGR knockdown had an effect on both glucose and lipid metabolism in db/db mice.     

Substituted imidazopyridazines are potent and selective inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1)

A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria.     

Energetic Pathway Sampling in a Protein Interaction Domain

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The development and characterization of a competitive ELISA for measuring active ADAMTS-4 in a bovine cartilage ex vivo model

ADAMTS-4 (aggrecanase1) is believed to play an important role in the degradation of aggrecan during the progression of joint diseases. ADAMTS-4 is synthesized as a latent pro-enzyme that requires the removal of the pro-domain, exposing the N-terminal neoepitope, to achieve activity. We developed a monoclonal antibody against this neoepitope of active ADAMTS-4. Furthermore, we established and characterized a competitive ELISA for measuring active ADAMTS-4 form applying the specific antibody. We used this assay to profile the presence of active ADAMTS-4 and its aggrecan degradation product (NITEGE³⁷³) in a bovine cartilage ex vivo model. We found that after stimulation with catabolic factors, the cartilage initially released high levels of aggrecanase-derived aggrecan fragments into supernatant but subsequently decreased to background levels. The level of active ADAMTS-4 released into the supernatant and retained in the cartilage matrix increased continuously throughout the 21 days of the study. The activity of ADAMTS-4 on the last day of catabolic stimulation was verified in vitro by adding deglycosylated or native aggrecan to the conditioned medium. Samples of human cartilage affected by varying degrees of osteoarthritis stained strongly for active ADAMTS-4 where surface fibrillation and clustered chondrocytes were observed. This assay could be an effective tool for studying ADAMTS-4 activity and for screening drugs regulating ADAMTS-4 activation. Moreover, it could be a potential biomarker for degenerative joint disease.