Glycan structures and intrageneric variations of venom acidic phospholipases A(2) from Tropidolaemus pitvipers

Most of the phospholipases A(2) (PLA(2) ; EC3.1.1.4) variants isolated so far from snake venoms are nonglycosylated enzymes. In the present study, we purified an active glycosylated PLA(2) and an inactive nonglycosylated Lys49-like PLA(2) from two geographical venom samples of Tropidolaemus. The PLA(2) variants from the two samples have rather different N-terminal sequences, implying that the samples were probably derived from two species (Tropidolaemus?subannulatus and Tropidolaemus?wagleri). The active PLA(2) s from Sulawesi and Sumatra venoms were designated as Tsu-E6 and Twa-E6, respectively, as a result of the presence of their conserved Glu6 residue. Tsu-E6 inhibited ADP-induced aggregation of mouse and human platelets. Twa-E6 stimulated the aggregation of mouse platelets but inhibited the aggregation of human platelets. Both PLA(2) s were found to be glycosylated at Asn14. Using MALDI-TOF analysis, the released glycans were shown to comprise complex type oligosaccharides without sialylation. This is the first glycan structure of the snake venom PLA(2) to be solved. Furthermore, the enzymatic removal of glycans from both PLA(2) s did not significantly alter their effects on lipid hydrolysis and platelet aggregation. The thermostability of glycosylated Twa-E6 was also found to be as good as that of other homologous PLA(2) s. The presence of these oligosaccharides in PLA(2) s warrants further analyses, which may provide useful insights into the functional regulation of these biomolecules.     

Effect of (R)‐salbutamol on the switch of phenotype and metabolic pattern in LPS‐induced macrophage cells

Evidence demonstrates that M1 macrophage polarization promotes inflammatory disease. Here, we discovered that (R)‐salbutamol, a β₂ receptor agonist, inhibits and reprograms the cellular metabolism of RAW264.7 macrophages. (R)‐salbutamol significantly inhibited LPS‐induced M1 macrophage polarization and downregulated expressions of typical M1 macrophage cytokines, including monocyte chemotactic protein‐1 (MCP‐1), interleukin‐1β (IL‐1β) and tumour necrosis factor α (TNF‐α). Also, (R)‐salbutamol significantly decreased the production of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and reactive oxygen species (ROS), while increasing the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. In contrast, (S)‐salbutamol increased the production of NO and ROS. Bioenergetic profiles showed that (R)‐salbutamol significantly reduced aerobic glycolysis and enhanced mitochondrial respiration. Untargeted metabolomics analysis demonstrated that (R)‐salbutamol modulated metabolic pathways, of which three metabolic pathways, namely, (a) phenylalanine metabolism, (b) the pentose phosphate pathway and (c) glycerophospholipid metabolism were the most noticeably impacted pathways. The effects of (R)‐salbutamol on M1 polarization were inhibited by a specific β₂ receptor antagonist, ICI‐118551. These findings demonstrated that (R)‐salbutamol inhibits the M1 phenotype by downregulating aerobic glycolysis and glycerophospholipid metabolism, which may propose (R)‐salbutamol as the major pharmacologically active component of racemic salbutamol for the treatment of inflammatory diseases and highlight the medicinal value of (R)‐salbutamol.     

Roles of db-cAMP,IBMX and RA in Aspects of Neural Differentiation of Cord Blood Derived Mesenchymal-Like Stem Cells

Mesenchymal stem cells (MSCs) have multilineage differentiation potential which includes cell lineages of the central nervous system; hence MSCs might be useful in the treatment of neurodegenerative diseases such as Parkinson''s disease. Although mesenchymal stem cells have been shown to differentiate into the neural lineage, there is still little knowledge about the underlying mechanisms of differentiation particularly towards specialized neurons such as dopaminergic neurons. Here, we show that MSCs derived from human umbilical cord blood (MSC^hUCBs) are capable of expressing tyrosine hydroxylase (TH) and Nurr1, markers typically associated with DA neurons. We also found differential phosphorylation of TH isoforms indicating the presence of post-translational mechanisms possibly activating and modifying TH in MSC^hUCB. Furthermore, functional dissection of components in the differentiation medium revealed that dibutyryl-cAMP (db-cAMP), 3-isobutyl-1-methylxanthine (IBMX) and retinoic acid (RA) are involved in the regulation of Nurr1 and Neurofilament-L expression as well as in the differential phosphorylation of TH. We also demonstrate a possible inhibitory role of the protein kinase A signaling pathway in the phosphorylation of specific TH isoforms.     

Syndecan-2 regulation of morphology in breast carcinoma cells is dependent on RhoGTPases

Background

While syndecan-2 is usually considered a mesenchymal transmembrane proteoglycan, it can be upregulated in some tumour cells, such as the malignant breast carcinoma cell line, MDA-MB231. Depletion of this syndecan by siRNA, but not other syndecans, has a marked effect on cell morphology, increasing spreading, microfilament bundle and focal adhesion formation, with reduced cell migration.

Methods

A combination of siRNA transfection, immunofluorescence microscopy, phosphoprotein analysis and migration assays was used to determine how syndecan-2 may influence the cytoskeleton.

Results

The altered adhesion upon syndecan-2 depletion was dependent on the RhoGTPases. p190ARhoGAP relocated to the margins of spreading cells, where it codistributed with syndecan-4 and active β₁-integrin. This was accompanied by increased RhoGAP tyrosine phosphorylation, indicative of activity and RhoGTPase suppression. Consistent with this, GTP-RhoA was strongly present at the edges of control cells, but lost after syndecan-2 reduction by siRNA treatments. Further, RhoA, but not RhoC was shown to be essential for the anchored phenotype of these breast carcinoma cells that accompanied siRNA-mediated loss of syndecan-2.

Conclusions

Syndecan-2 has a key role in promoting the invasive activity of these cells, in part by regulating the RhoGTPases.

General significance

Syndecan-2, as a cell surface receptor is accessible for targeting to determine whether breast tumour progression is altered. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Functions of the segment polarity genes midline and H15 in Drosophila melanogaster neurogenesis

The Drosophila melanogaster ventral nerve cord derives from neural progenitor cells called neuroblasts. Individual neuroblasts have unique gene expression profiles and give rise to distinct clones of neurons and glia. The specification of neuroblast identity provides a cell intrinsic mechanism which ultimately results in the generation of progeny which are different from each other. Segment polarity genes have a dual function in early neurogenesis: within distinct regions of the neuroectoderm, they are required both for neuroblast formation and for the specification of neuroblast identity. Previous studies of segment polarity gene function largely focused on neuroblasts that arise within the posterior part of the segment. Here we show that the segment polarity gene midline is required for neuroblast formation in the anterior-most part of the segment. Moreover, midline contributes to the specification of anterior neuroblast identity by negatively regulating the expression of Wingless and positively regulating the expression of Mirror. In the posterior-most part of the segment, midline and its paralog, H15, have partially redundant functions in the regulation of the NB marker Eagle. Hence, the segment polarity genes midline and H15 play an important role in the development of the ventral nerve cord in the anterior- and posterior-most part of the segment.     

Adsorption and Distribution of Fluorescent Solutes near the Articular Surface of Mechanically Injured Cartilage

The development of cartilage-specific imaging agents supports the improvement of tissue assessment by minimally invasive means. Techniques for highlighting cartilage surface damage in clinical images could provide for sensitive indications of posttraumatic injury and early stage osteoarthritis. Previous studies in our laboratory have demonstrated that fluorescent solutes interact with cartilage surfaces strongly enough to affect measurement of their partition coefficients within the tissue bulk. In this study, these findings were extended by examining solute adsorption and distribution near the articular surface of mechanically injured cartilage. Using viable cartilage explants injured by an established protocol, solute distributions near the articular surface of three commonly used fluorophores (fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and carboxytetramethylrhodamine (TAMRA)) were observed after absorption and subsequent desorption to assess solute-specific matrix interactions and reversibility. Both absorption and desorption processes demonstrated a trend of significantly less solute adsorption at surfaces of fissures compared to adjacent intact surfaces of damaged explants or surfaces of uninjured explants. After adsorption, normalized mean surface intensities of fissured surfaces of injured explants were 6%, 40%, and 32% for FITC, TRITC, and TAMRA, respectively, compared to uninjured surfaces. Similar values were found for sliced explants and after a desorption process. After desorption, a trend of increased solute adsorption at the site of intact damaged surfaces was noted (316% and 238% for injured and sliced explants exposed to FITC). Surface adsorption of solute was strongest for FITC and weakest for TAMRA; no solutes negatively affected cell viability. Results support the development of imaging agents that highlight distinct differences between fissured and intact cartilage surfaces.     

<Emphasis Type="Italic">Halymenia johorensis</Emphasis> sp. nov. (Halymeniaceae,Rhodophyta), a new foliose red algal species from Malaysia

A new Halymenia species, Halymenia johorensis sp. nov., from southern Peninsular Malaysia is proposed based on plastid-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) gene analyses and detailed morphological observations. The new species is characterized by having (1) elliptical, oblong, or irregularly shaped blades, incised with some perforations, arising from a narrow-cuneate stipe attached to a discoid holdfast; (2) blades with a cartilaginous and gelatinous texture, a smooth to rugose surface, and irregularly dentate and cleft margins; and (3) isodiametric outer cortical cells and rounded to stellate inner cortical cells. RbcL sequence analyses have shown H. johorensis to be genetically distinct from other Halymenia species. Although H. johorensis is sister to Halymenia plana, these two species can be distinguished both molecularly and morphologically. Further studies are necessary to investigate the phylogenetic relationships and species diversity in this genus.     

A nitrocellulose membrane based IgM capture enzyme immunoassay for etiological diagnosis of dengue virus infections

A nitrocellulose membrane based immunoassay for the detection of dengue virus specific IgM suitable for use in field situations or in peripheral laboratories would be useful for disease surveillance and control. This paper describes such an assay in an IgM capture format (MAC DOT) similar to the microplate based MAC ELISAs currently in use in several research and reference laboratories around the world. The MAC DOT was tested on several sample sets including a retrospective study of 119 patients from Children's Hospital, Bangkok, Thailand, with confirmed dengue infection. The sensitivity of the test was shown to be 94% taking only admission sera into consideration but rising to 99% when both an admission and a discharge specimen were considered. Other sample sets confirmed the high sensitivity and a study of 494 unselected febrile children showed that the specificity of the MAC DOT was 98%.