Hypoxia stimulates osteoclast formation from human peripheral blood

Active pathological bone destruction in humans often occurs in locations where oxygen tension (pO₂) is likely to be low, for example, at the sites of tumours, inflammation, infections and fractures, or the poorly vascularized yellow fatty marrow of the elderly. We examined the effect of pO₂ on formation of osteoclasts, the cells responsible for bone resorption, in 14‐day cultures of normal human peripheral blood mononuclear cells (hPBMCs) on ivory discs. Hypoxia (1–2% O₂) caused threefold increases in the number of osteoclasts formed, compared with 20% O₂. Hypoxia also caused a twofold increase in the number of nuclei per osteoclast, leading to stimulations of resorption pit formation of up to 10‐fold. Exposure to hypoxia led to stabilization of the hypoxia‐inducible factors, HIF1α and HIF2α, and upregulation of vascular endothelial growth factor and interleukin‐6 expression by hPBMCs. These findings help explain why extravasation of mononuclear precursors into relatively O₂‐deficient bone microenvironments could result in osteoclast formation and suggest a new mechanism for the bone loss associated with the pathophysiological conditions where hypoxia commonly occurs. Copyright © 2010 John Wiley & Sons, Ltd.     

Tobacco transgenic for the flax rust resistance gene L expresses allele-specific activation of defense responses

Tobacco was transformed with three different alleles (L2, L6, and L10) of the flax rust resistance gene L, a member of the toll interleukin-1 receptor, nucleotide-binding site, leucine-rich repeat (TIR-NBS-LRR) class of plant disease resistance genes. L6 transgenics had a stunted phenotype, expressed several defense response genes constitutively, and had increased resistance to the fungus Cercospora nicotianae and the oomycete Phytophthora parasitica pv. nicotianae. L2 and L10 transgenics, with one exception for L10, did not express these phenotypes, indicating that the activation of tobacco defense responses is L6 allele-specific. The phenotype of the exceptional L10 transgenic plant was associated with the presence of a truncated L10 gene resulting from an aberrant T-DNA integration. The truncated gene consisted of the promoter, the complete TIR region, and 39 codons of the NBS domain fused inframe to a tobacco retrotransposon-like sequence. A similar truncated L10 gene, constructed in vitro, was transiently expressed in tobacco leaves and gave rise to a strong localized necrotic reaction. Together, these results suggest that defense signaling properties of resistance genes can be expressed in an allele-specific and pathogen-independent manner when transferred between plant genera.     

Oil red-O stains non-adipogenic cells: a precautionary note

Bovine adipofibroblasts, 3T3-L1 cells, L-6 myogenic cells, and sheep satellite cells were allowed to proliferate for 48 h. Oil red-O (ORO) was dissolved in three different solvents isopropanol, propylene glycol and triethyl phosphate. At 48 h, the proliferative cultures were stained with the three stains. ORO stain prepared in both propylene glycol and triethyl phosphate resulted in bright red droplets appearing in all cultures, whereas ORO dissolved in isopropanol was not taken up by any of the cells. These data suggest that certain preparations of ORO may stain cells in non-adipogenic lineages as well as undifferentiated pre-adipocytes. Caution must be exercised when choosing solvents for ORO in differentiation studies using cells of the fat/adipose lineage.     

Design and synthesis of 3,7-diarylimidazopyridines as inhibitors of the VEGF-receptor KDR

3,7-Diarylsubstituted imidazopyridines were designed and developed as a new class of KDR kinase inhibitors. A variety of imidazopyridines were synthesized and potent inhibitors of KDR kinase activity were identified with good aqueous solubility.     

Atypical mechanism of regulation of the Wrch-1 Rho family small GTPase

Rho family GTPases are GDP/GTP-regulated molecular switches that regulate signaling pathways controlling diverse cellular processes. Wrch-1 was identified as a Wnt-1 regulated Cdc42 homolog, upregulated by Wnt1 signaling in Wnt1-transformed mouse mammary cells, and was able to promote formation of filopodia and activate the PAK serine/threonine kinase. Wrch-1 shares significant sequence and functional similarity with the Cdc42 small GTPase. However, Wrch-1 possesses a unique N-terminal 46 amino acid sequence extension that contains putative Src homology 3 (SH3) domain-interacting motifs. We determined the contribution of the N terminus to Wrch-1 regulation and activity. We observed that Wrch-1 possesses properties that distinguish it from Cdc42 and other Rho family GTPases. Unlike Cdc42, Wrch-1 possesses an extremely rapid, intrinsic guanine nucleotide exchange activity. Although the N terminus did not influence GTPase or GDP/GTP cycling activity in vitro, N-terminal truncation of Wrch-1 enhanced its ability to interact with and activate PAK and to cause growth transformation. The N terminus associated with the Grb2 SH3 domain-containing adaptor protein, and this association increased the levels of active Wrch-1 in cells. We propose that Grb2 overcomes N-terminal negative regulation to promote Wrch-1 effector interaction. Thus, Wrch-1 exhibits an atypical model of regulation not seen in other Rho family GTPases.     

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.     

Peptidase activities of the 20/26S proteasome and a novel protease in human brain

Many neurodegenerative diseases are characterized by ubiquitin-positive protein aggregates or inclusion bodies. Ubiquitin-conjugated proteins are degraded by the 20/26S proteasome, and reduced proteasome peptidase activities in brain homogenates have been reported in pathologic lesions of Parkinson's and Alzheimer's diseases. However, it is unknown whether crude extracts of human brain contain other proteases having peptidase activities. We found a novel protease of molecular weight of approximately 105 kDa in normal human brain, which exhibited trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities (corresponding to 52% and 21% of the total activities in crude extracts) but not peptidyl glutamyl peptide hydrolase activity. Both T-L and ChT-L activities of this protease were partially inhibited by proteasome inhibitors (MG132, lactacystin) and, in contrast to those of the proteasome, also by sodium dodecyl sulfate. A simple method to obtain a brain fraction specific to the 20/26S proteasome was developed. Our human brain data suggest that T-L and ChT-L activity levels of the proteasome reported previously may include those of the 105 kDa protease, an enzyme of as yet unknown biological significance, and that it is necessary to separate the proteasome from this protease to evaluate the actual status of the ubiquitin-proteasome system in neurodegenerative disorders.     

Regulation of vascular endothelial growth factor binding and activity by extracellular pH

Angiogenesis, the growth of new blood vessels, is regulated by a number of factors, including hypoxia and vascular endothelial growth factor (VEGF). Although the effects of hypoxia have been studied intensely, less attention has been given to other extracellular parameters such as pH. Thus, the present study investigates the consequences of acidic pH on VEGF binding and activity in endothelial cell cultures. We found that the binding of VEGF165 and VEGF121 to endothelial cells increased as the extracellular pH was decreased from 7.5 to 5.5. Binding of VEGF165 and VEGF121 to endothelial extracellular matrix was also increased at acidic pH. These effects were, in part, a reflection of increased heparin binding, because VEGF165 and VEGF121 showed increased retention on heparin-Sepharose at pH 5.5 compared with pH 7.5. Consistent with these findings, soluble heparin competed for VEGF binding to endothelial cells under acidic conditions. However, at neutral pH (7.5) low concentrations of heparin (0.1-1.0 microg/ml) potentiated VEGF binding. Extracellular pH also regulated VEGF activation of the extracellular signal-regulated kinases 1 and 2 (Erk1/2). VEGF165 and VEGF121 activation of Erk1/2 at pH 7.5 peaked after 5 min, whereas at pH 6.5 the peak was shifted to 10 min. At pH 5.5, neither VEGF isoform was able to activate Erk1/2, suggesting that the increased VEGF bound to the cells at low pH was sequestered in a stored state. Therefore, extracellular pH might play an important role in regulating VEGF interactions with cells and the extracellular matrix, which can modulate VEGF activity.     

Cuticle characteristics and volatile emissions of petals in Antirrhinum majus

Floral volatiles, which are small and generally water-insoluble, must move from their intracellular sites of synthesis through the outermost cuticle membrane before release from the flower surface. To determine whether petal cuticle might influence volatile emissions, we performed the first analysis of petal cuticle development and its association with the emission of flower volatiles using Antirrhinum majus L. (snapdragon) as a model system. Petal cuticular wax amount and composition, cuticle thickness and ultrastructure, and the amounts of internal and emitted methylbenzoate (the major snapdragon floral scent compound) were examined during 12 days, from flower opening to senescence. Normal ( n -) alkanes were found to be the major wax class of snapdragon petals (29.0% to 34.3%) throughout the 12 days examined. Besides n -alkanes, snapdragon petals possessed significant amounts of methyl branched alkanes (23.6–27.8%) and hydroxy esters (12.0–14.0%). Hydroxy esters have not been previously reported in plants. Changes in amount of methylbenzoate inside the petals followed closely with levels of methylbenzoate emission, suggesting that snapdragon petal cuticle may provide little diffusive resistance to volatile emissions. Moreover, clear associations did not exist between methylbenzoate emission and the cuticle properties examined during development. Nevertheless, the unique wax composition of snapdragon petal cuticles shows similarities with those of other highly permeable cuticles, suggesting an adaptation that could permit rapid volatile emission by scented flowers.