Development of multiple embryos in polyembryonic insertional mutant OsPE of rice

A T-DNA insertional mutant OsPE of rice gives twin and triplet seedlings in up to 20?% of the seeds. Detailed cytological and histological analysis of OsPE indicated normal male and female gametogenesis in the OsPE mutant. Confocal laser scanning microscopic (CLSM) analysis of the developing seeds of OsPE showed multiple embryo development in up to 60?% of the ovules. The multiple embryos, mostly twins and triplets, and rarely quadruplets, developed through sequential cleavage from a single zygotic embryo in each ovule. The reduced number of multiple seedlings compared with multiple embryos observed in CLSM study may be attributed to their inability to develop further due to competition in a single embryo sac. Key message Multiple seedlings in the OsPE mutant are due to sequential proliferation and cleavage of the zygotic embryos. The nucellar tissue was not involved in multiple embryo development.     

PATTERNS OF OSSIFICATION IN SOUTHERN VERSUS NORTHERN PLACENTAL MAMMALS

Consensus on placental mammal phylogeny is fairly recent compared to that for vertebrates as a whole. A stable phylogenetic hypothesis enables investigation into the possibility that placental clades differ from one another in terms of their development. Here, we focus on the sequence of skeletal ossification as a possible source of developmental distinctiveness in “northern” (Laurasiatheria and Euarchontoglires) versus “southern” (Afrotheria and Xenarthra) placental clades. We contribute data on cranial and postcranial ossification events during growth in Afrotheria, including elephants, hyraxes, golden moles, tenrecs, sengis, and aardvarks. We use three different techniques to quantify sequence heterochrony: continuous method, sequence‐ANOVA (analysis of variance) and event‐paring/Parsimov. We show that afrotherians significantly differ from other placentals by an early ossification of the orbitosphenoid and caudal vertebrae. Our analysis also suggests that both southern placental groups show a greater degree of developmental variability; however, they rarely seem to vary in the same direction, especially regarding the shifts that differ statistically. The latter observation is inconsistent with the Atlantogenata hypothesis in which afrotherians are considered as the sister clade of xenarthrans. Interestingly, ancestral nodes for Laurasiatheria and Euarchontoglires show very similar trends and our results suggest that developmental homogeneity in some ossification sequences may be restricted to northern placental mammals (Boreoeutheria).     

Fatty acid regulation of liver X receptors (LXR) and peroxisome proliferator-activated receptor alpha (PPARalpha ) in HEK293 cells

Fatty acids bind to and regulate the activity of peroxisome proliferator-activated (PPAR) and liver X receptors (LXR). However, the role lipid metabolism plays in the control of intracellular fatty acid ligands is poorly understood. We have identified two strains of HEK293 cells that display differences in fatty acid regulation of nuclear receptors. Using full-length and Gal4-LBD chimeric receptors in functional assays, 20:4,n6 induced PPARalpha activity approximately 2.2-fold and suppressed LXRalpha activity by 80% (ED50 approximately 25-50 microm) in HEK293-E (early passage) cells but had no effect on PPARalpha or LXRalpha receptor activity in HEK293-L (late passage) cells. LXRbeta was insensitive to fatty acid regulation in both HEK293 strains. Metabolic labeling studies using [14C]20:4,n6 (at 100 microm) indicated that the uptake of 20:4,n6 and its assimilation into triacylglycerol, diacylglycerol, and polar lipids revealed no difference between the two strains. Such treatment increased total cellular 20:4,n6 ( approximately 11-fold) and its elongation product, 22:4,n6 ( approximately 3.6-fold), within 6 h. Non-esterified 20:4,n6 and 22:4,n6 represented 相似文献   

Mutational analysis reveals an essential role for the LXXLL motif in the transformation function of the human herpesvirus-8 oncoprotein, kaposin

Human herpesvirus-8 (HHV-8) is causally linked to Kaposi's sarcoma (KS). Sequence analysis of the genome and subsequent studies revealed several genes including kaposin, with transformation properties in cell culture. In this study, we have analyzed the requirement of Kaposin A for cellular transformation in an effort to understand its contribution towards KS pathogenesis. Comparative analysis of Kaposin with other proteins identified the LXXLL motif spanning from residues 31-35 (LVCLL). The observation that the LXXLL motif is present in nuclear receptor coactivators that mediate the interaction of coactivators with nuclear receptors has prompted us to investigate the relevance of this motif for Kaposin's function(s). Kaposin A coding sequences were cloned into a eukaryotic expression plasmid with the Flag (FL) epitope fused in-frame at the C-terminus (Kap-FL). To evaluate the role of leucine residues in the motif, site-directed mutagenesis was utilized, whereby alanine was substituted for the leucine residues (Kap-AXXAA-FL). Both Kap-FL and Kap- AXXAA-FL exhibited similar levels of expression in cells. Interestingly, the Kap-AXXAA-FL mutant failed to show transforming activity by two independent assays: anchorage-independent growth, and focus formation. Immunofluorescence (IFA) and FACS analysis indicated that Kap-FL was localized around the nucleus and at the cell surface, respectively. However, Kap-AXXAA-FL exhibited diffuse cytoplasmic staining as measured by IFA yet was still detectable on the cell surface by FACS. Ironically, both Kap-FL and Kap-AXXAAFL were able to activate the AP-1 promoter. These results support an important role for the LXXLL motif in the ability of Kaposin to induce transformation.     

Functional replacement of a retroviral late domain by ubiquitin fusion

Retroviral Gag polyprotein precursors are both necessary and sufficient for the assembly and release of virus-like particles (VLPs) from infected cells. It is well established that small Gag-encoded motifs, known as late domains, promote particle release by interacting with components of the cellular endosomal sorting and ubiquitination machinery. The Gag proteins of a number of different retroviruses are ubiquitinated; however, the role of Gag ubiquitination in particle egress remains undefined. In this study, we investigated this question by using a panel of equine infectious anemia virus (EIAV) Gag derivatives bearing the wild-type EIAV late domain, heterologous retroviral late domains or no late domain. Ubiquitin was fused in cis to the C-termini of these Gag polyproteins, and the effects on VLP budding were measured. Remarkably, fusion of ubiquitin to EIAV Gag lacking a late domain (EIAV/DeltaYPDL-Ub) largely rescued VLP release. We also determined the effects of ubiquitin fusion on the sensitivity of particle release to budding inhibitors and to depletion of key endosomal sorting factors. Ubiquitin fusion rendered EIAV/DeltaYPDL-Ub sensitive to depletion of cellular endosomal sorting factors Tsg101 and Alix and to overexpression of dominant-negative fragments of Tsg101 and Alix. These findings demonstrate that ubiquitin can functionally compensate for the absence of a retroviral late domain and provide insights into the host-cell machinery engaged by ubiquitin during particle egress.     

Human brain glycogen content and metabolism: implications on its role in brain energy metabolism

The adult brain relies on glucose for its energy needs and stores it in the form of glycogen, primarily in astrocytes. Animal and culture studies indicate that brain glycogen may support neuronal function when the glucose supply from the blood is inadequate and/or during neuronal activation. However, the concentration of glycogen and rates of its metabolism in the human brain are unknown. We used in vivo localized 13C-NMR spectroscopy to measure glycogen content and turnover in the human brain. Nine healthy volunteers received intravenous infusions of [1-(13)C]glucose for durations ranging from 6 to 50 h, and brain glycogen labeling and washout were measured in the occipital lobe for up to 84 h. The labeling kinetics suggest that turnover is the main mechanism of label incorporation into brain glycogen. Upon fitting a model of glycogen metabolism to the time courses of newly synthesized glycogen, human brain glycogen content was estimated at approximately 3.5 micromol/g, i.e., three- to fourfold higher than free glucose at euglycemia. Turnover of bulk brain glycogen occurred at a rate of 0.16 micromol.g-1.h-1, implying that complete turnover requires 3-5 days. Twenty minutes of visual stimulation (n=5) did not result in detectable glycogen utilization in the visual cortex, as judged from similar [13C]glycogen levels before and after stimulation. We conclude that the brain stores a substantial amount of glycogen relative to free glucose and metabolizes this store very slowly under normal physiology.     

Point mutations in the HIV-1 matrix protein turn off the myristyl switch

During the late phase of human immunodeficiency virus type-1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P(2)) and Gag's N-terminally myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P(2) binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P(2) is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P(2) or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr(s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5)P(2) binding. This mutant binds PI(4,5)P(2) with twofold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding.     

Improving Phenotypic Prediction by Combining Genetic and Epigenetic Associations

We tested whether DNA-methylation profiles account for inter-individual variation in body mass index (BMI) and height and whether they predict these phenotypes over and above genetic factors. Genetic predictors were derived from published summary results from the largest genome-wide association studies on BMI (n ∼ 350,000) and height (n ∼ 250,000) to date. We derived methylation predictors by estimating probe-trait effects in discovery samples and tested them in external samples. Methylation profiles associated with BMI in older individuals from the Lothian Birth Cohorts (LBCs, n = 1,366) explained 4.9% of the variation in BMI in Dutch adults from the LifeLines DEEP study (n = 750) but did not account for any BMI variation in adolescents from the Brisbane Systems Genetic Study (BSGS, n = 403). Methylation profiles based on the Dutch sample explained 4.9% and 3.6% of the variation in BMI in the LBCs and BSGS, respectively. Methylation profiles predicted BMI independently of genetic profiles in an additive manner: 7%, 8%, and 14% of variance of BMI in the LBCs were explained by the methylation predictor, the genetic predictor, and a model containing both, respectively. The corresponding percentages for LifeLines DEEP were 5%, 9%, and 13%, respectively, suggesting that the methylation profiles represent environmental effects. The differential effects of the BMI methylation profiles by age support previous observations of age modulation of genetic contributions. In contrast, methylation profiles accounted for almost no variation in height, consistent with a mainly genetic contribution to inter-individual variation. The BMI results suggest that combining genetic and epigenetic information might have greater utility for complex-trait prediction.     

Exploration of the correlation between solvation dynamics and internal dynamics of a protein

Protein function is intimately related to the dynamics of the protein as well as to the dynamics of the solvent shell around the protein. Although it has been argued extensively that protein dynamics is slaved to solvent dynamics, experimental support for this hypothesis is scanty. In this study, measurements of fluorescence anisotropy decay kinetics have been used to determine the motional dynamics of the fluorophore acrylodan linked to several locations in a small protein barstar in its various structural forms, including the native and unfolded states as well as the acid and protofibril forms. Fluorescence upconversion and streak camera measurements have been used to determine the solvation dynamics around the fluorophore. Both the motional dynamics and solvent dynamics were found to be dependent upon the location of the probe as well as on the structural form of the protein. While the (internal) motional dynamics of the fluorophore occur in the 0.1-3 ns time domain, the observed mean solvent relaxation times are in the range of 20-300 ps. A strong positive correlation between these two dynamical modes was found in spite of the significant difference in their time scales. This observed correlation is a strong indicator of the coupling between solvent dynamics and the dynamics in the protein.