Survival Signals of Hepatic Stellate Cells in Liver Regeneration Are Regulated by Glycosylation Changes in Rat Vitronectin,Especially Decreased Sialylation

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO- or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn¹⁶⁷. Highly sialylated O-glycans were found to be present in the Thr¹¹⁰–Thr¹²⁴ region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.     

Quantification and cluster analysis of actin cytoskeletal structures in plant cells: role of actin bundling in stomatal movement during diurnal cycles in Arabidopsis guard cells

Manual evaluation of cellular structures is a popular approach in cell biological studies. However, such approaches are laborious and are prone to error, especially when large quantities of image data need to be analyzed. Here, we introduce an image analysis framework that overcomes these limitations by semi-automatic quantification and clustering of cytoskeletal structures. In our framework, cytoskeletal orientation, bundling and density are quantified by measurement of newly-developed, robust metric parameters from microscopic images. Thereafter, the microscopic images are classified without supervision by clustering based on the metric patterns. Clustering allows us to collectively investigate the large number of cytoskeletal structure images without laborious inspection. Application of this framework to images of GFP-actin binding domain 2 (GFP-ABD2)-labeled actin cytoskeletons in Arabidopsis guard cells determined that microfilaments (MFs) are radially oriented and transiently bundled in the process of diurnal stomatal opening. The framework also revealed that the expression of mouse talin GFP-ABD (GFP-mTn) continuously induced MF bundling and suppressed the diurnal patterns of stomatal opening, suggesting that changes in the level of MF bundling are crucial for promoting stomatal opening. These results clearly demonstrate the utility of our image analysis framework.     

Distinct localization of histone H3 methylation in the vegetative nucleus of lily pollen

We analysed the distribution of histone H3 modifications in the nucleus of the vegetative cell (the vegetative nucleus) during pollen development in lily (Lilium longiflorum). Among the modifications specifically and/or abundantly present in the vegetative nucleus, dimethylation of histone H3 at lysine 9 (H3K9me2) and lysine 27 (H3K27me2) were found in heterochromatin, whereas trimethylation of histone H3 at lysine 27 (H3K27me3) was localized in euchromatin in the vegetative nucleus. Such unique localization of the histone H3 methylation marks, particularly of H3K27me3, within a nucleus was not observed in lily nuclei other than the vegetative nucleus. The level of H3K27me3 increased in the euchromatic region of the vegetative nucleus during pollen maturation. The results suggest that H3K27me3 controls the gene expression of the vegetative cell during pollen maturation.     

Inheritance of acquired traits in plants: Reinstatement of Lamarck

Since Lamarck proposed the idea of inheritance of acquired traits 200 years ago, much has been said for and against it, but the theory was finally declined after the 1930s. Despite of the negative opinions of the majority of geneticists, botanists and plant breeders have long recognized that altered properties during the growth were occasionally transmitted to the offspring. This was also the case with artificially altered properties such as dwarfism, flowering timing and plant stature, which were induced by a non-mutagenic chemical, 5-azacytidine and its derivatives. As these drugs are powerful inhibitors of DNA methylation in vivo, a close correlation between methylation and phenotypic expression was suggested. Subsequent studies showed that rice plants acquired disease resistance upon demethylation of the corresponding resistant gene, and that both resistant trait and hypomethylated status were inherited by the progeny up to nine generations. Whether or not the methylation pattern changes under natural condition was then questioned, and recent studies have indicated that it indeed naturally changes in response to environmental stresses. Whether or not the altered methylation pattern during the vegetative growth is heritable was also questioned, and studies on toadflax and rice affirmed the question, showing stable maintenance of hypermethylation in the former and hypomethylation in the latter for 250 and 10 years, respectively. The observation strongly suggested that acquired traits can be heritable as far as the acquired methylation pattern is stably transmitted. This concept is consistent with the Lamarck''s theory of the inheritance of acquired traits, which therefore should be carefully reevaluated to reestablish his impaired reputation.Key words: acquired traits, cytosine methylation, disease resistance, environmental stress, epigenetics, Lamarckian inheritanceIn 1809, the French naturalist, Jean Baptiste de Lamarck (1744–1829) proposed two laws of evolution—the law of use/disuse and the law of inheritance of acquired traits. The theory was declined almost completely after the 1930s. In plants, however, phenomena showing apparent inheritance of acquired traits have long been observed. This article briefly summarizes the current view of the “Lamarckian inheritance” in higher plants. Many excellent review articles related to this topic have been published, and readers are strongly suggested to refer to them for further information on molecular aspects.^1–3     

<Emphasis Type="Italic">Actinocephalus verae</Emphasis> (Eriocaulaceae), a new species from the Brazilian Campos Rupestres

We describe and illustrate the new species Actinocephalus verae (Eriocaulaceae: Paepalanthoideae). This species is endemic to the rocky outcrops of the Espinhaço range in Minas Gerais, Brazil. We make comparisons with Actinocephalus ithyphyllus and Actinocephalus ochrocephalus, the morphologically most similar species. The new species’ morphological variation, habitat, geographic distribution, and conservation status are discussed.     

CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the receptor complex of Toll-like receptors 2 and 1 without binding to the complex

It has demonstrated that the recognition of triacylated lipopeptides by Toll-like receptor (TLR) 2 requires TLR1 as a coreceptor. In the NF-kappaB reporter assay system in which human embryonic kidney 293 cells were transfected with TLR2 and TLR1 together with an NF-kappaB luciferase reporter gene, S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-Lys-Lys-Lys-Lys (Pam(3)CSK(4)) and Pam(3)CSSNA were recognized by TLR2/TLR1, but the recognition level was unexpectedly very low. However, cotransfection of CD14 drastically enhanced the recognition of triacylated lipopeptides by TLR2/TLR1. The CD14-induced enhancement did not occur without cotransfection of TLR1. Both CD14(dS39-A48), a mutant with deletion of the part of possible N-terminal ligand-binding pocket, and anti-CD14 monoclonal antibody reduced the CD14-induced enhancement. Transfection of a TIR domain-deficient mutant of TLR2 (TLR2(dE772-S784)) or TLR1 (TLR1(dQ636-K779)) completely abrogated the CD14-induced enhancement. Soluble recombinant CD14 added extracellularly enhanced the recognition of Pam(3)CSSNA by TLR2/TLR1. Immunoprecipitation analysis demonstrated that CD14 was not associated with TLR2 but that TLR1 was associated with TLR2. In addition, surface plasmon resonance-based assay demonstrated that CD14 binds to Pam(3)CSK(4) at a dissociation constant of 5.7 microM. This study suggests that CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the TLR2/TLR1 complex without binding to the receptor complex.     

Sphingosine 1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner

Sphingosine 1-phosphate (S1P) is accumulated in platelets and released on stimulation by thrombin or Ca(2+). Thrombin-stimulated S1P release was inhibited by staurosporin, whereas Ca(2+)-stimulated release was not. When the platelet plasma membrane was permeabilized with streptolysin O (SLO), S1P leaked out with cytosol markers, whereas granular markers remained in the platelets. The SLO-induced S1P leakage required BSA, probably for solubilization of S1P in the medium. These results indicate that S1P is localized in the inner leaflet of the plasma membrane and that its release is a carrier-mediated process. We also used alpha-toxin (ATX), which makes smaller pores in the plasma membrane than SLO and depletes cytosolic ATP without BSA-dependent S1P leakage. The addition of ATP drove S1P release from ATX platelets. The ATP-driven S1P release from ATX platelets was greatly enhanced by thrombin. An ATP binding cassette transporter inhibitor, glyburide, prevents ATP- and thrombin-induced S1P release from platelets. Ca(2+) also stimulated S1P release from ATX platelets without ATP, whereas the Ca(2+)-induced release was not inhibited by glyburide. Our results indicate that two independent S1P release systems might exist in the platelet plasma membrane, an ATP-dependent system stimulated by thrombin and an ATP-independent system stimulated by Ca(2+).     

Chondroitin acts in the guidance of gonadal distal tip cells in C. elegans

In Caenorhabditis elegans hermaphrodites, the U-shaped gonad arms are formed by directed migration of the gonadal distal tip cells (DTCs). The stereotyped pattern of DTC migration is carefully controlled by extracellular and cell surface molecules during larval development. Here we report that two proteins, SQV-5 (chondroitin synthase) and its cofactor MIG-22 (chondroitin polymerizing factor), are required for chondroitin biosynthesis and are essential for the dorsally guided migration of DTCs. We found that MIG-22 is expressed in migrating DTCs, hypodermal seam cells, developing vulva and oocytes. The expression of SQV-5 or MIG-22 in both DTCs and hypodermis rescued the DTC migration defects of the relevant mutants more efficiently than when they were expressed in either single tissue. Furthermore, the expression of SQV-5 by the mig-22 promoter significantly rescued sqv-5 mutants, implying that these two proteins act in the same tissues and that chondroitin proteoglycans produced in both of these tissues are required for DTC migration. The DTC migration defects caused by sqv-5 or mig-22 mutations were partially suppressed in the anterior and enhanced in the posterior DTCs in unc-6, unc-5 or unc-40 mutant backgrounds, suggesting that chondroitin proteoglycans play roles in the UNC-6/netrin-dependent guidance of DTCs.