Comprehensive identification of PIP3-regulated PH domains from C. elegans to H. sapiens by model prediction and live imaging

Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3) control cell growth, migration, and other processes by recruiting proteins with pleckstrin homology (PH) domains and possibly other domains to the plasma membrane (PM). However, previous experimental and structural work with PH domains left conflicting evidence about which ones are PIP3 regulated. Here we used live-cell confocal imaging of 130 YFP-conjugated mouse PH domains and found that 20% translocated to the PM in response to receptor-generated PIP3 production. We developed a recursive-learning algorithm to predict PIP3 regulation of 1200 PH domains from different eukaryotes and validated that it accurately predicts PIP3 regulation. Strikingly, this algorithm showed that PIP3 regulation is specified by amino acids across the PH domain, not just the PIP3-binding pocket, and must have evolved several times independently from PIP3-insensitive ancestral PH domains. Finally, our algorithm and live-cell experiments provide a functional survey of PH domains in different species, showing that PI3K regulation increased from approximately two C. elegans and four Drosophila to 40 vertebrate proteins.     

FACT-mediated exchange of histone variant H2AX regulated by phosphorylation of H2AX and ADP-ribosylation of Spt16

The phosphorylation of histone variant H2AX at DNA double-strand breaks is believed to be critical for recognition and repair of DNA damage. However, little is known about the molecular mechanism regulating the exchange of variant H2AX with conventional H2A in the context of the nucleosome. Here, we isolate the H2AX-associated factors, which include FACT (Spt16/SSRP1), DNA-PK, and PARP1 from a human cell line. Our analyses demonstrate that the H2AX-associated factors efficiently promote both integration and dissociation of H2AX and this exchange reaction is mainly catalyzed by FACT among the purified factors. The phosphorylation of H2AX by DNA-PK facilitates the exchange of nucleosomal H2AX by inducing conformational changes of the nucleosome. In contrast, poly-ADP-ribosylation of Spt16 by PARP1 significantly inhibits FACT activities for H2AX exchange. Thus, these data establish FACT as the major regulator involved in H2AX exchange process that is modulated by H2AX phosphorylation and Spt16 ADP-ribosylation.     

Epinephrine increases DNA synthesis via ERK1/2s through cAMP, Ca(2+)/PKC, and PI3K/Akt signaling pathways in mouse embryonic stem cells

Epinephrine is a catecholamine that plays important roles in regulating a wide variety of physiological systems by acting through the adrenergic receptors (ARs). The cellular responses to AR stimulation are mediated through various signaling pathways. Therefore, this study examined the effects of epinephrine on DNA synthesis and related signaling molecules in mouse embryonic stem cells (ESCs). Epinephrine increased DNA synthesis in a dose- and time-dependent manner, as determined by the level of [(3)H]-thymidine incorporation. AR subtypes (alpha1(A), alpha2(A), beta1, beta2, and beta3) were expressed in mouse ESCs and their expression levels were increased by epinephrine. In this experiment, epinephrine increased cAMP levels, intracellular Ca(2+) concentration ([Ca(2+)](i)), and translocation of protein kinase C (PKC) from the cytosol to the membrane compartment. In addition, we observed Akt phosphorylation in response to epinephrine; this was stimulated by phosphorylation of the epidermal growth factor receptor (EGFR). Epinephrine also induced phosphorylation of ERK1/2 (p44/42 MAPKs), while inhibition of PKC or Akt blocked this phosphorylation. Epinephrine increased the mRNA levels of proto-oncogenes (c-fos, c-jun, c-myc), while inhibition of ERK1/2 decreased these mRNA levels. In experiments aimed at examining the involvement of cell cycle regulatory proteins, epinephrine increased the levels of cyclin E/cyclin-dependent kinase 2 (CDK2) and cyclin D1/cyclin-dependent kinase 4 (CDK4). In conclusion, epinephrine stimulates DNA synthesis via ERK1/2 through cAMP, Ca(2+)/PKC, and PI3K/Akt signaling pathways in mouse ESCs.     

An exploration of LEAFY expression in independent evolutionary origins of rosette flowering in Brassicaceae

Whereas most Brassicaceae produce flowers on an elongated inflorescence, a few lineages produce flowers directly from the vegetative rosette on elongated pedicels. Knowing the extent to which independent origins of rosette flowering involve the same developmental and genetic mechanisms could clarify the constraints acting on plant architectural evolution. Prior work in Idahoa, Ionopsidium, and Leavenworthia suggested that changes in the activity or expression of the flower meristem identity gene, LEAFY (LFY), played a role in all three origins of rosette flowering. Here we studied the developmental morphology of L. crassa and immunolocalization of LFY protein in Leavenworthia and Ionopsidium to further compare independent origins of rosette flowering. Leavenworthia crassa differs from Ionopsidium and Idahoa in producing ebracteate flowers. Flowers are, however, associated with "squamules," here interpreted as stipules of a cryptic bract. LFY was detected in L. crassa flower primordia but not in inflorescence meristems. In contrast, the rosette flowering Io. acaule accumulated LFY protein in the inflorescence meristem, whereas its inflorescence-flowering close relative, Io. prolongoi, did not. Thus, although different cases of rosette flowering likely entailed modifications of the same meristem identity program, distinct developmental genetic mechanisms appear to be involved in each case.     

Effects of dietary energy and lysine intake during late gestation and lactation on blood metabolites, hormones, milk composition and reproductive performance in multiparous sows

This study was conducted to investigate the effects of different energy and lysine intake on reproductive performance of multiparous sows during late gestation and lactation. Thirty-six sows were allocated to six dietary treatments in a 3 x 2 factorial arrangement and each treatment had six replicates. Three energy levels and two lysine levels were used during late gestation and lactation. The results demonstrated that higher lysine intake improved (p < 0.05) the body condition of sows during both periods and their reproductive performance during lactation. Both, dietary energy and lysine level had a significant effect (p < 0.05) on the composition of colostrum and milk. In general, dietary treatments had no effect on blood metabolites and hormones. These results suggest that energy levels recommended by the National Research Council (1998) for sows are enough; however, higher lysine levels than those recommended could improve reproductive performance during late gestation and lactation in multiparous sows.     

T-type Ca2+ channel blockers suppress the growth of human cancer cells

In order to further clarify the role of T-type Ca²⁺ channels in cell proliferation, we have measured the growth inhibition of human cancer cells by using our potent T-type Ca²⁺ channel blockers. As a result, KYS05090, a most potent T-type Ca²⁺ channel blocker, was found to be as potent as doxorubicin against some human cancer cells without acute toxicity. Therefore, this letter provides the biological results that T-type calcium channel is important in regulating the important cellular phenotype transition leading to cell proliferation, and thus novel T-type Ca²⁺ channel blocker presents new prospects for cancer treatment.     

Changes in Triphasic Mechanical Properties of Proteoglycan-Depleted Articular Cartilage Extracted from Osmotic Swelling Behavior Monitored Using High-Frequency Ultrasound

This study aims to obtain osmosis-induced swelling strains of normal and proteoglycan (PG) depleted articular cartilage using an ultrasound system and to investigate the changes in its mechanical properties due to the PG depletion using a layered triphasic model. The swelling strains of 20 cylindrical cartilage-bone samples collected from different bovine patellae were induced by decreasing the concentration of bath saline and monitored by the ultrasound system. The samples were subsequently digested by a trypsin solution for approximately 20 min to deplete proteoglycans, and the swelling behaviors of the digested samples were measured again. The bi-layered triphasic model proposed in our previous study (Wang et al., J Biomech Eng-Trans ASME 2007; 129: 413-422) was used to predict the layered aggregate modulus Hafrom the data of depth-dependent swelling strain, fixed charge density and water content. It was found that the region near the bone, for the normal specimens, had a significantly higher aggregate modulus (Ha₁= 20.6±18.2 MPa) in comparison with the middle zone and the surface layer (Ha₂= 7.8±14.5 MPa and Ha₃= 3.6±3.2 MPa, respectively) (p < 0.001). The normalized thickness of the deep layer h₁ was 0.68±0.20. After the trypsin digestion, the parametric values decreased to Ha₁ = 13.6±9.6 MPa, Ha₂ = 6.7±11.5 MPa, Ha₃ = 2.7±3.2 MPa, and h₁ = 0.57±0.28. Other models were also used to analyze data and the results were compared. This study showed that high-frequency ultrasound measurement combined with the triphasic modeling was capable of nondestructively quantifying the alterations in the layered mechanical properties of the proteoglycan-depleted articular cartilage.     

Free radical scavenging and hepatoprotective actions of Quercus aliena acorn extract against CCl4-induced liver

In the present study, we investigated the protective effect of Quercus aliena acorn extracts against CCl₄-induced hepatotoxicity in rats, and the mechanism underlying the protective effects. Aqueous extracts of Quercus aliena acorn had higher superoxide radical scavenging activity than other types of extracts. The Quercus aliena acorn extracts displayed dose-dependent superoxide radical scavenging activity (IC₅₀ = 4.92 μg/ml), as assayed by the electron spin resonance (ESR) spin-trapping technique. Pretreatment with Quercus aliena acorn extracts reduced the increase in serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. The hepatoprotective action was confirmed by histological observation. The aqueous extracts reversed CCl₄-induced liver injury and had an antioxidant action in assays of FeCl₂- ascorbic acid induced lipid peroxidation in rats. Expression of cytochrome P450 2E1 (CYP2E1) mRNA, as measured by RT-PCR, was significantly decreased in the livers of Quercus aliena acorn-pretreated rats compared with the livers of the control group. These results suggest that the hepatoprotective effects of Quercus aliena acorn extract are related to its antioxidative activity and effect on the expression of CYP2E1.