Bio-silica and bio-polyphosphate: applications in biomedicine (bone formation)

Bio-silica represents the main mineral component of the sponge skeletal elements (siliceous spicules), while bio-polyphosphate (bio-polyP), a multifunctional polymer existing in microorganisms and animals acts, among others, as reinforcement for pores in cell membranes. These natural inorganic bio-polymers, which can be readily prepared, either by recombinant enzymes (bio-silica and bio-polyP) or chemically (polyP), are promising materials/substances for the amelioration and/or treatment of human bone diseases and dysfunctions. It has been demonstrated that bio-silica causes in vitro a differential effect on the expression of the genes OPG and RANKL, encoding two mediators that control the tuned interaction of the anabolic (osteoblasts) and catabolic (osteoclasts) pathways in human bone cells. Since bio-silica and bio-polyP also induce the expression of the key mediator BMP2 which directs the differentiation of bone-forming progenitor cells to mature osteoblasts and in parallel inhibits the function of osteoclasts, they are promising candidates for treatment of osteoporosis.     

Genome Sequence of an Unusual Human G10P[8] Rotavirus Detected in Vietnam

A rare human G10P[8] rotavirus with a reassortment between bovine and human viruses was detected from a patient with acute gastroenteritis in Vietnam. Genetic analysis using complete coding sequences of all segments showed a genomic constellation of this virus of G10-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Its VP7 region was genetically related to that of a bovine rotavirus derived from Australia (strain VICG10.01), whereas all other genes were identical to those of a human rotavirus derived from Australia (strain Victoria/CK00047). These results indicate a possibility that the reassortment of the rotavirus was caused by immune escape in Australia and the rotavirus was carried to Vietnam. Additionally, this finding will help further understanding the evolution of rotaviruses circulating in Vietnam.     

Adenylate cyclase system of bovine adrenal plasma membranes.

The adenylate cyclase system present in a preparation enriched in plasma membranes derived from bovine adrenal cortex was investigated in considerable detail. This system is stimulated by adrenocorticotropic hormone (ACTH), by biologically active analogs of this hormone, and by fluoride ion. The preparation contains sodium-potassium- and magnesium-dependent ATPases that are markedly inhibited by 50 mM sodium fluoride. Incorporation of a pyruvate phosphokinase ATP generating system into the adenylate cyclase assay medium provided constant substrate levels. In the presence of the ATP generating system, the rate of cyclic AMP formation (basal, fluoride, and ACTH-activated) was proportional to enzyme concentration and was linear with time. Proportionality with respect to enzyme concentration as concerned the hormone-activated adenylate cyclase was achieved only when the ratio of hormone to enzyme protein was kept constant. The temperature optimum of the adenylate cyclase, basal or activated, was approximately 30 degrees. Michaelis-Menten kinetics were observed when the ratio of Mg2+ to ATP was approximately 6:1. Both calcium and ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid completely inhibited the adenylate cyclase system at concentrations of 5 and 0.5 mM, respectively. GTP was inhibitory at concentrations of 10-2 M but had little effect at lower concentrations. Freezing in liquid nitrogen and storage at -60 degrees exerted little effect on the fluoride-stimulated enzyme but lowered hormone stimulated activity. Preincubation in the presence of ACTH afforded a high degree of stabilization of the enzyme system while preincubation with a biologically inactive analog afforded no protection.     

Association of AUUUA-binding protein with A+U-rich mRNA during nucleo-cytoplasmic transport.

Resealed nuclear envelope (NE) vesicles from rat liver containing entrapped exogenous RNA were used to study the effect of adenosine+uridine binding factor (AUBF), present in cytosolic cell extracts, on ATP-dependent transport of A+U-rich RNA (AU+RNA) and A+U-free RNA (AU-RNA) across the NE. This factor specifically binds to A+U-rich sequences present in the 3' untranslated regions of lymphokine and cytokine mRNAs, containing overlapping AUUUA boxes (granulocyte-macrophage colony stimulating factor, interleukin-3). Addition of AUBF to the extravesicular compartment markedly increased the efflux of the in vitro transcribed, capped and polyadenylated AU+ RNAs. Export of entrapped AU- control RNA, such as beta-globin RNA, was not affected by AUBF, in contrast to chimeric AU+ beta-globin RNA containing the A+U-rich sequence of human interferon-alpha mRNA (6 reiterated AUUUA motifs). Competition experiments revealed that AUBF enhances the affinity of poly(A)-containing AU+ RNAs to the NE poly(A)-binding component (poly(A)-recognizing mRNA carrier p106), and thereby accelerates nuclear export of these RNAs. We could demonstrate that AUBF added to the extravesicular space forms stable complexes with polyadenylated AU+ RNA with relative molecular masses of about 45,000, 62,000 and 70,000 inside the vesicles or during ATP-dependent export. In addition we determined that AUBF may affect mRNA stability by protecting A+U-rich RNA against degradation by trans-acting, nuclear matrix-associated and A+U-specific endoribonuclease V.     

Expression and function of TETRAN, a new type of membrane transporter

In contrast to transport across basolateral membranes, the mechanism governing transport of organic anions across the luminal membranes of proximal tubules has remained unclear. We recently found Tetracycline transporter-like protein (TETRAN), a human ortholog of yeast Tpo1p that can transport anionic Non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we examine the expression and function of TETRAN. TETRAN mRNA is expressed in various human tissues, including kidney. When overexpressed in cultured cells, TETRAN was predominantly localized on cytoplasmic membranes. Immunohistochemical analysis of human and mouse kidney tissue showed that TETRAN was expressed at the luminal membranes of proximal tubules. Overexpression of TETRAN in cultured cells facilitated the uptake of organic anions such as indomethacin (a NSAID) and fluorescein. The results suggest that TETRAN is a novel human organic anion transporter, and that it serves as a transporter for some NSAIDs and various other organic anions at the final excretion step.     

Bioorganic/inorganic hybrid composition of sponge spicules: matrix of the giant spicules and of the comitalia of the deep sea hexactinellid Monorhaphis

The giant basal spicules of the siliceous sponges Monorhaphis chuni and Monorhaphis intermedia (Hexactinellida) represent the largest biosilica structures on earth (up to 3m long). Here we describe the construction (lamellar organization) of these spicules and of the comitalia and highlight their organic matrix in order to understand their mechanical properties. The spicules display three distinct regions built of biosilica: (i) the outer lamellar zone (radius: >300 microm), (ii) the bulky axial cylinder (radius: <75 microm), and (iii) the central axial canal (diameter: <2 microm) with its organic axial filament. The spicules are loosely covered with a collagen net which is regularly perforated by 7-10 microm large holes; the net can be silicified. The silica layers forming the lamellar zone are approximately 5 microm thick; the central axial cylinder appears to be composed of almost solid silica which becomes porous after etching with hydrofluoric acid (HF). Dissolution of a complete spicule discloses its complex structure with distinct lamellae in the outer zone (lamellar coating) and a more resistant central part (axial barrel). Rapidly after the release of the organic coating from the lamellar zone the protein layers disintegrate to form irregular clumps/aggregates. In contrast, the proteinaceous axial barrel, hidden in the siliceous axial cylinder, is set up by rope-like filaments. Biochemical analysis revealed that the (dominant) molecule of the lamellar coating is a 27-kDa protein which displays catalytic, proteolytic activity. High resolution electron microscopic analysis showed that this protein is arranged within the lamellae and stabilizes these surfaces by palisade-like pillars. The mechanical behavior of the spicules was analyzed by a 3-point bending assay, coupled with scanning electron microscopy. The load-extension curve of the spicule shows a biphasic breakage/cracking pattern. The outer lamellar zone cracks in several distinct steps showing high resistance in concert with comparably low elasticity, while the axial cylinder breaks with high elasticity and lower stiffness. The complex bioorganic/inorganic hybrid composition and structure of the Monorhaphis spicules might provide the blueprint for the synthesis of bio-inspired material, with unusual mechanical properties (strength, stiffness) without losing the exceptional properties of optical transmission.     

Expression of Eph receptor tyrosine kinases and their ligands in chick embryonic motor neurons and hindlimb muscles

Evidence is accumulating that Eph receptor tyrosine kinases and their ligands regulate cell migration and axonal guidance during development. It was previously found that one of the Eph receptors, EphA4, is transiently expressed in subsets of chick embryonic motor neurons. Here, the expression of EphA and ephrin-A subfamily members was further examined, and the dynamic patterns of expression in chick embryonic motor neurons found. EphA3, EphA4, ephrin-A2, and ephrin-A5 were also expressed in the connective tissues of limb muscles and EphA3 and EphA4 expressing motor neurons innervated EphA3 and EphA4 expressing limb muscles, respectively. These spatiotemporal expression patterns suggest that EphA and ephrin-A proteins play important roles in muscle patterning and motor axonal guidance.