TGF-β1 as possible link between loss of bone mineral density and chronic inflammation

Background

The TGF family plays a key role in bone homeostasis. Systemic or topic application of proteins of this family apparently positively affects bone healing in vivo. However, patients with chronic inflammation, having increased TGF-β₁ serum-levels, often show reduced bone mineral content and disturbed bone healing. Therefore, we wanted to identify intracellular mechanisms induced by chronic presence of TGF-β₁ and their possible role in bone homeostasis in primary human osteoblasts.

Methodology/Principal Findings

Osteoblasts were isolated from femur heads of patients undergoing total hip replacement. Adenoviral reporter assays showed that in primary human osteoblasts TGF-β₁ mediates its signal via Smad2/3 and not Smad1/5/8. It induces proliferation as an intermediate response but decreases AP-activity and inorganic matrix production as a late response. In addition, expression levels of osteoblastic markers were strongly regulated (AP↓; Osteocalcin↓; Osteopontin↑; MGP↓; BMP 2↓; BSP2↓; OSF2↓; Osteoprotegerin↓; RANKL↑) towards an osteoclast recruiting phenotype. All effects were blocked by inhibition of Smad2/3 signaling with the Alk5-Inhibitor (SB431542). Interestingly, a rescue experiment showed that reduced AP-activities did not recover to base line levels, even 8 days after stopping the TGF-β₁ application.

Conclusions/Significance

In spite of the initial positive effects on cell proliferation, it is questionable if continuous Smad2/3 phosphorylation is beneficial for bone healing, because decreased AP-activity and BMP2 levels indicate a loss of function of the osteoblasts. Thus, inhibition of Smad2/3 phosphorylation might positively influence functional activity of osteoblasts in patients with chronically elevated TGF-β₁ levels and thus, could lead to an improved bone healing in vivo.     

Phenocopy--a strategy to qualify chemical compounds during hit-to-lead and/or lead optimization

A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype-determined phenotype of another individual. The phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of biological systems with new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor β Receptor I (TGF-βR1) were qualified by high-throughput RNA expression profiling. This chemical genomics approach resulted in a precise time-dependent insight to the TGF-β biology and allowed furthermore a comprehensive analysis of each NCE's off-target effects. The evaluation of off-target effects by the phenocopy approach allows a more accurate and integrated view on optimized compounds, supplementing classical biological evaluation parameters such as potency and selectivity. It has therefore the potential to become a novel method for ranking compounds during various drug discovery phases.     

A large plant beta-tubulin family with minimal C-terminal variation but differences in expression

Tubulins, as the major structural component of microtubules (MT), are highly conserved throughout the entire eukaryotic kingdom. They consist of alpha/beta heterodimers. Both monomers, at least in multicellular organisms, are encoded by gene families. In higher plants up to eight beta-tubulin isotypes, mostly differing in their very C-termini, have been described. These variable beta-tubulin C-termini have been discussed in the context of functional microtubule diversity. However, in plants, in contrast to vertebrates, functional isotype specificity remains yet to be demonstrated. Unlike higher plants, unicellular green algae in general do not exhibit isotypic variations. The moss Physcomitrella patens is a phylogenetic intermediate between higher plants and green algae. We isolated six beta-tubulin genes from Physcomitrella, named PpTub1 to 6. We show that the exon/intron structure, with the exception of one additional intron in PpTub6, is identical with that of higher plants, and that some members of the family are differentially expressed. Moreover, we find that all Physcomitrella isotypes are highly conserved and, most strikingly, are almost identical within their C-terminal amino acids (aa). This evolutionary ancient and large beta-tubulin gene family without significant isotypic sequence variation points to a role of differential regulation in the evolution of plant tubulin isotypes.     

Random mating with respect to mating status in the simultaneously hermaphroditic land snail Arianta arbustorum

In promiscuous species with sperm storage, males are expected to show a preference for mating with virgin and young females to reduce the risk of sperm competition. In the simultaneous hermaphrodite land snail Arianta arbustorum, sperm production precedes egg production by 2–4 weeks, resulting in a short period of protandric hermaphroditism before shell growth is completed. In natural populations, copulating pairs involving individuals which have not yet completed shell growth (virgins) have been observed. We ran a series of mate-choice experiments to examine whether virgin and nonvirgin (experienced) individuals of A. arbustorum discriminate between virgin and nonvirgin mating partners. We also assessed the number of sperm delivered to partners with different mating status. Neither virgin nor nonvirgin snails showed any preference for mating with a virgin partner. In all test situations mating was random and the number of sperm delivered was not adjusted to the mating status of the partner. Mating success was mainly determined by the activity of the individual. The random mating pattern does not imply random fertilization of eggs because the presence of a sperm-digesting organ and the morphology of the sperm storage organ allow a selective storage and use of sperm in A. arbustorum.     

Moderate concentrations of 4-O-methylhonokiol potentiate GABAA receptor currents stronger than honokiol

Background

Magnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABA_A receptors. 4-O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABA_A receptors and its GABA_A receptor subtype-specificity was established.

Methods

Different recombinant GABA_A receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4-O-methylhonokiol.

Results

3 μM 4-O-methylhonokiol is shown here to potentiate responses of the α₁β₂γ₂ GABA_A receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4-O-methylhonokiol is also detailed for 12 GABA_A receptor subtypes to assess GABA_A receptor subunits that are responsible for the potentiating effect.

Conclusion

The much higher potentiation of GABA_A receptors at identical concentrations of 4-O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4-O-methylhonokiol over honokiol.

General significance

The results point to the use of 4-O-methylhonokiol as a lead for GABA_A receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine.     

Rates of cuticular penetration of 1-naphthylacetic acid (NAA) as affected by adjuvants, temperature, humidity and water quality

When NAA is used for fruit thinning, results can be unpredictable. Problemswith foliar penetration of NAA can contribute to this variability. Usingisolated pear leaf cuticles we have investigated effects of humidity,temperature, hard water and selected adjvants on rates of cuticularpenetration of NAA. If NAA was dissolved in deionised water about 40% ofthe applied dose penetrated in 8 h, while water having a pH > 7 practicallyeliminated cuticular penetration of NAA, even at high humidity (100%) andmoderate temperatures (20°C). Rates of penetration were much lowerat 10°C and 55% humidity. Adding urea, ammonium sulphate or Tween20 increased rates of penetration but not to a great extent. Highest rates ofpenetration were obtained when solutions were buffered at pH 4 with DL-lactic acid and the accelerator adjuvant Genapol C-100 was added. Withthis combination, about 70% of the applied NAA dose penetrated within 8 hat 20°C and 40% at 10°C, respectively, even at low humidity(55 to 60%) and when hard water was used as carrier. It is argued thatpoor performance of NAA as chemical thinner must be expected when hardwater is used for spraying and when the pH of the spray liquid is notadjusted to values around 4, such that a significant proportion of the NAAis non-ionised. Only non-ionised NAA can penetrate cuticles. Rates of foliarpenetration of NAA can be greatly increased, especially at low temperatures,by adding 0.2 to 2.0 g l^–1 Genapol C-100. Since NAA is destroyed byUV light, spraying should be done in the evening. High humidity during thenight is favourable for foliar penetration, but low temperatures aredetrimental and addition of an accelerator adjuvant (i.e. Genapol C-100) isnecessary. If NAA must be sprayed during cold weather (5–10°C),higher NAA concentrations may be used because rates of penetrationduring the night are proportional to dose and excess NAA left on the foliagewill be destroyed by UV light after sunrise.     

Oviposition preference and larval development of the invasive moth Cydalima perspectalis on five European box‐tree varieties

The box‐tree pyralid Cydalima perspectalis (Walker 1859) (Lepidoptera: Pyralidae), native to Eastern Asia, is a newly introduced species causing severe damage to box‐trees (Buxus sp.) in private and public gardens as well as in semi‐natural box‐tree forests in Central Europe. It is so far not known whether different box‐tree subspecies (or varieties) are similarly affected by this invasive moth. In a choice experiment offering branches of five different box‐tree varieties as oviposition sites, we found a preference of female moths for laying their egg clusters on the variety ‘Rotundifolia’, while other varieties were less frequently considered. The preference for ‘Rotundifolia’, the variety with the largest leaves in the tests, remained when intervariety differences in foliar area (mean leaf size × number of leaves) were taken into account. Feeding larvae on leaves of either of the five box‐tree varieties revealed a significant effect of the seasonal generation of C. perspectalis on the growth rate of individuals but no influence of the box‐tree variety. Larvae from the spring generation show the highest growth rate, those from the summer generation a moderate and those from the autumn generation the lowest growth rate. The moths used in the experiments may belong to the 10th to 12th generation present in Europe. The time elapsed since their introduction may be too short for an optimal adaptation to the partly novel diet encountered by the invasive moth.