Modulation of bone morphogenetic protein antagonists to stimulate clinical osteogenesis

Bone morphogenetic proteins (BMPs) are important for the development and functioning of a wide variety of tissues and organ systems. Their ability to induce bone formation has been harnessed for clinical application. Specifically, local application of BMPs into fractures and fusions has shown some efficacy in inducing bone formation. However, clinical success has not been as robust as might be expected from the results obtained using animal models. This difference may be due to a number of mechanisms regulating BMP activity in vivo. One class of major regulators is the extracellular antagonist (e.g. Noggin, Gremlin, DAN), the dysfunction of which has been shown to result in ectopic bone formation in animal models and human disease. We hypothesize that local application of BMPs at high concentrations induces increased production of BMP antagonists, thereby limiting BMP activity and clinical efficacy. Therapies blocking the function of BMP antagonists should therefore result in enhanced BMP activity and increased bone formation. Furthermore, titrated systemic regulation of BMP antagonist may potentially reverse osteoporosis. Our collective experience with the clinical use of BMP illustrates the importance of understanding mechanisms of endogenous antagonism and regulation in the exogenous application of a protein as a therapeutic.     

Metabolic engineering of monoterpene biosynthesis: two-step production of (+)-trans-isopiperitenol by tobacco

Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.     

Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway,through o-phthalic acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Characterization of the effects of vasoactive substances on the bulbus arteriosus of the eel,Anguilla rostrata

The fish bulbus arteriosus (BA) smooths cardiac output by expanding during cardiac systole and rebounding during diastole, thereby providing constant perfusion of the gills downstream. Published data have demonstrated innervation of the teleost BA and shown that the tension and compliance of the BA responded to vasoactive agonists, such as epinephrine and acetylcholine, suggesting that the BA was more than a mere "windkessel." To examine vasoactivity in the BA more directly, we measured the responses of isolated tissue rings from the BA of the eel, Anguilla rostrata to a suite of putative vasoactive agonists, which had been shown to affect vascular smooth muscle in a variety of teleosts. The BA of the eel was insensitive to acetylcholine but constricted when endothelin (ET-1) was applied. Nitric oxide, sodium nitroprusside (SNP; NO donor), natriuretic peptides (NP), and prostaglandin E1 (but not the prostacyclin agonist carbaprostacyclin) produced significant dilation in the BA. Since both ET-1 and sarafotoxin S6c produced concentration-dependent constriction, it appears that endothelin receptor B-type (ETB) receptors (and possibly ETA receptors) are present. The dilation produced by SNP was also concentration dependent, as were the dilations produced by porcine C-type natriuretic peptide, eel atrial natriuretic peptide (NP receptor agonists), Sulprostone and Butaprost (PGE receptor agonists). Our data demonstrate that the BA of eel is responsive to a variety of vasoactive agonists, suggesting that the BA is under neurohumoral control. The role of agonist-induced changes in BA tension in fish cardiovascular physiology remains to be determined, as do the specific receptor types involved.     

Design and synthesis of a novel series of cyanoindole derivatives as potent γ-secretase modulators

The discovery, design and synthesis of a new series of GSMs is described. The classical imidazole heterocycle has been replaced by a cyano group attached to an indole nucleus. The exploration of this series has led to compound 26-S which combined high in vitro and in vivo potency with an acceptable drug-like profile.     

Identification of acid-sensing ion channels in bone

Bone balances serum pH variations and both osteoclasts and osteoblasts are regulated by subtle changes in pH. The aim of the current study was to identify molecules in bone that can sense pH. Interesting candidates are the acid-sensing ion channels (ASICs). In bone, ASIC2 and ASIC3 were most abundant, while in chondrocytes it was ASIC1. Isolated human monocytes expressed ASIC1, -2, and -3, which persisted after induction to osteoclast differentiation, albeit to a lower level. In human osteoblasts ASIC1, ASIC2, and ASIC3 mRNAs were shown. Western blot and immunostaining confirmed this at protein level. ASIC4 expression was always very low abundant. For the first time, we demonstrated ASICs in human skeleton, providing a means to sense and respond to differences in extracellular pH.     

Negative regulation of the platelet Na+/H+ exchanger by trimeric G-proteins.

Human platelets contain a Na+/H+ exchanger (NHE) that regulates the cytosolic pH. The role of trimeric G-proteins in NHE control was investigated in plasma membrane vesicles by measuring exchange of intravesicular protons for extravesicular Na+. Exchange was saturable, independent of membrane potential and inhibited by ethylisopropyl amiloride (Ki 0.05 micromol.L-1), demonstrating the involvement of NHE-1. The G-protein activators AlF4- and GMP-P(NH)P reduced exchange by increasing the Km for Na+ from 11.3 +/- 2.1 mM to 21.6 +/- 1.4 mM (AlF4-) and 19.8 +/- 1.1 mM (GMP-P(NH)P), leaving Vmax and the Hill coefficient unchanged. This effect was abolished by inhibitors of Gi-proteins (N-ethylmaleimide, holoenzyme- and A-protomer of pertussis toxin) and by an anti-Galpha Ig and GDP(beta)S. Activation of Gi-proteins by mastoparan and its synthetic analogue Mas7 also strongly reduced NHE activity. These data show that in platelets NHE-1 is under negative control of the Gi-family of trimeric G-proteins.     

Introduction of foreign genes into potato cultivars Bintje and Désirée using an Agrobacterium tumefaciens binary vector

Tuber discs of Solanum tuberosum cv Bintje and Désirée were cocultivated with an Agrobacterium tumefaciens binary vector, carrying both the neomycine phosphotransferase and the E. coli -glucuronidase gene fused to resp. the nopaline synthase and Cauliflower mosaic virus 35S promotor.Inoculated tuber discs produce transgenic shoots in selective media containing kanamycin. The transgenic plants are phenotypically normal and contain the euploid number of chromosomes. Both the neomycin phosphotransferase as well as the -glucuronidase gene are expressed conferring resp. kanamycin resistance and -glucuronidase activity to the plants.Abbreviations GUS -glucuronidase - NPT neomycin phosphotransferase - CaMV Cauliflower Mosaic Virus - BAP 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthalineacetic acid - LB Luria Broth - MU methylumbelliferone