Monoribosomal attachment to messenger ribonucleic acid in sodium fluoride-treated rabbit reticulocytes (Short Communication)

Mild proteolysis with Pronase selectively dissociates ribosomes not attached to mRNA into subunits; ribosomes attached to mRNA remain intact. A portion of monoribosomes from reticulocytes incubated with NaF resisted proteolytic dissociation. Recovery of mRNA from monoribosomes of NaF-treated reticulocytes therefore may be explained by persistent attachment of some monoribosomes to mRNA.     

Carbohydrate Recognition Specificity of Trans-sialidase Lectin Domain from Trypanosoma congolense

Fourteen different active Trypanosoma congolense trans-sialidases (TconTS), 11 variants of TconTS1 besides TconTS2, TconTS3 and TconTS4, have been described. Notably, the specific transfer and sialidase activities of these TconTS differ by orders of magnitude. Surprisingly, phylogenetic analysis of the catalytic domains (CD) grouped each of the highly active TconTS together with the less active enzymes. In contrast, when aligning lectin-like domains (LD), the highly active TconTS grouped together, leading to the hypothesis that the LD of TconTS modulates its enzymatic activity. So far, little is known about the function and ligand specificity of these LDs. To explore their carbohydrate-binding potential, glycan array analysis was performed on the LD of TconTS1, TconTS2, TconTS3 and TconTS4. In addition, Saturation Transfer Difference (STD) NMR experiments were done on TconTS2-LD for a more detailed analysis of its lectin activity. Several mannose-containing oligosaccharides, such as mannobiose, mannotriose and higher mannosylated glycans, as well as Gal, GalNAc and LacNAc containing oligosaccharides were confirmed as binding partners of TconTS1-LD and TconTS2-LD. Interestingly, terminal mannose residues are not acceptor substrates for TconTS activity. This indicates a different, yet unknown biological function for TconTS-LD, including specific interactions with oligomannose-containing glycans on glycoproteins and GPI anchors found on the surface of the parasite, including the TconTS itself. Experimental evidence for such a scenario is presented.     

Integrin α4 Enhances Metastasis and May Be Associated with Poor Prognosis in MYCNlow Neuroblastoma

High-risk neuroblastoma is associated with an overall survival rate of 30–50%. Neuroblastoma-expressed cell adhesion receptors of the integrin family impact cell adhesion, migration, proliferation and survival. Integrin α4 is essential for neural crest cell motility during development, is highly expressed on leukocytes, and is critical for transendothelial migration. Thus, cancer cells that express this receptor may exhibit increased metastatic potential. We show that α4 expression in human and murine neuroblastoma cell lines selectively enhances in vitro interaction with the alternatively spliced connecting segment 1 of fibronectin, as well as vascular cell adhesion molecule-1 and increases migration. Integrin α4 expression enhanced experimental metastasis in a syngeneic tumor model, reconstituting a pattern of organ involvement similar to that seen in patients. Accordingly, antagonism of integrin α4 blocked metastasis, suggesting adhesive function of the integrin is required. However, adhesive function was not sufficient, as mutants of integrin α4 that conserved the matrix-adhesive and promigratory function in vitro were compromised in their metastatic capacity in vivo. Clinically, integrin α4 is more frequently expressed in non-MYNC amplified tumors, and is selectively associated with poor prognosis in this subset of disease. These results reveal an unexpected role for integrin α4 in neuroblastoma dissemination and identify α4 as a potential prognostic indicator and therapeutic target.     

Identification of a multigene family for small heat shock proteins in soybean and physical characterization of one individual gene coding region

When soybean seedlings are tranferred from 28 to 40 ° C, a heat shock (hs) response is elicited. This is characterized by the synthesis of a new set of proteins (hs-proteins) and by cessation of normal protein synthesis (8). At the level of poly(A)mRNA, a new class of highly abundant RNAs appears which encodes a group of hs-proteins in the low molecular weight range of 15–18 kD (11). The classification of these proteins/genes into several sub-classes is based on a complex sequence relationship for class I protein/genes.This was confirmed by both the complexity and the similarity of southern blot hybridization patterns of genomic DNA digests with class I cDNA-probes. Genomic DNA clones (obtained from -libraries by screening with cDNA-probes) for the class I gene 1968 showed cross hybridization with all other class I cDNA-probes. Higher specificity of gene/protein correlation was obtained by variation of hybridization criteria. The specificity of cDNA clone 1968 for the genomic DNA clone hs68-7 was demonstrated by thermal stability of hybridization at 55 ° C and 65 ° C in 50% formamide compared to other cross-reacting probes. The correlation of clone 1968 with a specific hs-protein was obtained by temperature dependent release of hybrid selected hs-mRNAs at 50, 60, 70 and 85 ° C followed byin vitro translation and two-dimensional gel analysis. The coding regions of hs-genes on genomic DNA clones were mapped by R-loop formation. The position of R-loops was mapped relative to certain restriction sites on subclones of hs68-7 DNA. The polarity of hs-genes was determined by attaching X174RF-DNA labels to the 3 poly(A)-tails of the mRNAs of R-loops.     

Mutual and directional allogeneic cytotoxic reaction of hemocytes in the solitary ascidian Halocynthia roretzi revealed by one-step quantitative fluorimetric assay

A novel one-step microplate cytotoxicity assay using the cytoplasmic fluorescent viability dye calcein AM was established for simple, rapid, sensitive, and quantitative measurements of the allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the ascidian Halocynthia roretzi. The mutual and directional ACR was distinguishable by the assay using the hemocytes from pairs of animals with different alloreactivities. The ACR assay may allow more precise genetic analysis of the gene that controls alloreactivity of hemocytes, since the mutual and directional ACR may be related to levels of expression or numbers of the gene product or products on the target cells. The directional ACR will be useful in elucidating the cellular and molecular mechanisms of self-recognition in H. roretzi, since it allowed us to equate hemocytes from one animal with "effector cells" and those from the other animal of the pair with "target cells". In addition, the quantitative ACR assay in a large number of samples is possible and it will allow production of monoclonal antibodies that may recognize receptors or ligands functioning in self-recognition processes by the H. roretzi hemocytes.     

Formin leaky cap allows elongation in the presence of tight capping proteins

Formins, characterized by formin homology domains FH1 and FH2, are required to assemble certain F-actin structures including actin cables, stress fibers, and the contractile ring. FH1FH2 in a recombinant fragment from a yeast formin (Bni1p) nucleates actin filaments in vitro. It also binds to the filament barbed end where it appears to act as a "leaky" capper, slowing both polymerization and depolymerization by approximately 50%. We now find that FH1FH2 competes with tight capping proteins (including gelsolin and heterodimeric capping protein) for the barbed end. We also find that FH1FH2 forms a tetramer. The observation that this formin protects an end from capping but still allows elongation confirms that it is a leaky capper. This is significant because a nucleator that protects a new barbed end from tight cappers will increase the duration of elongation and thus the total amount of F-actin. The ability of FH1FH2 to dimerize probably allows the formin to walk processively with the barbed end as the filament elongates.     

Identification of a variant form of PZR lacking immunoreceptor tyrosine-based inhibitory motifs

PZR is an immunoglobulin superfamily protein that specifically binds tyrosine phosphatase SHP-2 through its intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Here we report a novel isoform of the protein designated PZR1b. PZR1b shares the same extracellular region with PZR, but it lacks intracellular ITIMs and thus the ability to recruit SHP-2. Genomic sequence analysis revealed that PZR1b is resulted from alternative gene splicing of the PZR gene localized at chromosome 1q24. Like PZR, PZR1b is widely expressed. However, the relative ratio of two forms varies in different human tissues and cells. More importantly, overexpression of PZR1b in human HT-1080 cells had a dominant negative effect by blocking concanavalin A-induced tyrosine phosphorylation of full-length PZR and recruitment of tyrosine phosphatase SHP-2. Therefore, PZR1b may have an important role in cell signaling by counteracting with PZR.     

ClC chloride channels

Chloride-conducting ion channels of the ClC family are emerging as critical contributors to a host of biological processes. These polytopic membrane proteins form aqueous pathways through which anions are selectively allowed to pass down their concentration gradients. The ClCs are found in nearly all organisms, with members in every mammalian tissue, yet relatively little is known about their mechanism or regulation. It is clear, however, that they are fundamentally different in molecular construction and mechanism from the well-known potassium-, sodium-, and calcium-selective channels. The medical importance of ClC channels - four inherited diseases have been blamed on familial ClC dysfunction to date - highlights their diverse physiological functions and provides strong motivation for further study.     

Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells

Regulation of endothelial cell (EC) permeability by bioactive molecules is associated with specific patterns of cytoskeletal and cell contact remodeling. A role for mechanical factors such as shear stress (SS) and cyclic stretch (CS) in cytoskeletal rearrangements and regulation of EC permeability becomes increasingly recognized. This paper examined redistribution of focal adhesion (FA) proteins, site-specific focal adhesion kinase (FAK) phosphorylation, small GTPase activation and barrier regulation in human pulmonary EC exposed to laminar shear stress (15 dyn/cm2) or cyclic stretch (18% elongation) in vitro. SS caused peripheral accumulation of FAs, whereas CS induced randomly distributed FAs attached to the ends of newly formed stress fibers. SS activated small GTPase Rac without effects on Rho, whereas 18% CS activated without effect on Rac. SS increased transendothelial electrical resistance (TER) in EC monolayers, which was further elevated by barrier-protective phospholipid sphingosine 1-phosphate. Finally, SS induced FAK phosphorylation at Y576, whereas CS induced FAK phosphorylation at Y397 and Y576. These results demonstrate for the first time differential effects of SS and CS on Rho and Rac activation, FA redistribution, site-specific FAK phosphorylation, and link them with SS-mediated barrier enhancement. Thus, our results suggest common signaling and cytoskeletal mechanisms shared by mechanical and chemical factors involved in EC barrier regulation.