Sesquiterpene lactones from the fruits of smyrnium rotundifolium

In addition to known compounds an investigation of the fruits of Smyrnium rotundifolium afforded four new germacrane derivatives, all closely related to glechomanolide, one being a secogermacrane anhydride. The new compounds were 8,9-epoxy-8,9-secoglechomanolide, 1β,10α-epoxy-4-methoxy-8-hydroxyglechomanolide and 8-oxo-8,9-secoglechomanolide. The structures were assigned by spectral methods as well as by some chemical reactions.     

Eudesmanolides from Pluchea dioscoridis

Reinvestigation of the aerial parts of Pluchea dioscoridis afforded, in addition to known compounds, five new eudesmanolides and a new thiophene derivative. The stereochemistry of one of the eudesmanolides isolated previously has been corrected. The structures were elucidated by spectroscopic methods.     

Androgen receptor down regulation by small interference RNA induces cell growth inhibition in androgen sensitive as well as in androgen independent prostate cancer cells

We investigated the effects of androgen receptor (AR) down regulation with a small interference RNA molecule (siRNA_AR(start)) on androgen sensitive LNCaP and androgen independent LNCaPabl prostate cancer cells, the latter representing an in vitro model for the development of therapy resistance in prostate cancer. Although LNCaPabl cells express increased levels of AR in comparison with androgen sensitive LNCaP cells, the protein was significantly down regulated in response to siRNA_AR(start) treatment. This AR down regulation resulted in a marked cell growth inhibition in both cell lines. By contrast, DU-145 prostate cancer cells, which lack AR expression, were not inhibited by the siRNA_AR(start). In consequence to AR down regulation, both cell lines, LNCaP and LNCaPabl, shared a highly similar gene expression profile in terms of major changes in cell cycle regulatory genes. The cell cycle inhibitor p21(Waf1/Cip1) as well as cyclin D1 were significantly up regulated by siRNA_AR(start) treatment, considering a switch in cyclin expression towards cell cycle retardation. Control molecules had moderate effects on cell proliferation and gene expression, respectively. In summary, we found that AR inhibition with siRNA induces cell growth retardation in androgen sensitive as well as in androgen independent prostate cancer cells and thus may represent an interesting approach to combat hormone-refractory prostate cancer.     

Intra-articular hyaluronic acid for the treatment of osteoarthritis of the knee: systematic review and meta-analysis

Background

Osteoarthritis of the knee affects up to 10% of the elderly population. The condition is frequently treated by intra-articular injection of hyaluronic acid. We performed a systematic review and meta-analysis of randomized controlled trials to assess the effectiveness of this treatment.

Methods

We searched MEDLINE, EMBASE, CINAHL, BIOSIS and the Cochrane Controlled Trial Register from inception until April 2004 using a combination of search terms for knee osteoarthritis and hyaluronic acid and a filter for randomized controlled trials. We extracted data on pain at rest, pain during or immediately after movement, joint function and adverse events.

Results

Twenty-two trials that reported usable quantitative information on any of the predefined end points were identified and included in the systematic review. Even though pain at rest may be improved by hyaluronic acid, the data available from these studies did not allow an appropriate assessment of this end point. Patients who received the intervention experienced a reduction in pain during movement: the mean difference on a 100-mm visual analogue scale was –3.8 mm (95% confidence interval [CI] –9.1 to 1.4 mm) after 2–6 weeks, –4.3 mm (95% CI –7.6 to –0.9 mm) after 10–14 weeks and –7.1 mm (95% CI –11.8 to –2.4 mm) after 22–30 weeks. However, this effect was not compatible with a clinically meaningful difference (expected to be about 15 mm on the visual analogue scale). Furthermore, the effect was exaggerated by trials not reporting an intention-to-treat analysis. No improvement in knee function was observed at any time point. Even so, the effect of hyaluronic acid on knee function was more favourable when allocation was not concealed. Adverse events occurred slightly more often among patients who received the intervention (relative risk 1.08, 95% CI 1.01 to 1.15). Only 4 trials explicitly reported allocation concealment, had blinded outcome assessment and presented intention-to-treat data.

Interpretation

According to the currently available evidence, intra-articular hyaluronic acid has not been proven clinically effective and may be associated with a greater risk of adverse events. Large trials with clinically relevant and uniform end points are necessary to clarify the benefit–risk ratio.Osteoarthritis affects about 10% of the population over 55 years of age. Of those, one-quarter are severely disabled.¹ The condition is characterized by degeneration of the articular cartilage and subsequent subchondral bone changes. The underlying mechanisms remain unknown, but the glycosaminoglycan–proteoglycan matrix may play a major role.²Hyaluronic acid, a glycosaminoglycan, is widely used for the treatment of osteoarthritis of the knee. A survey of 2 general practices in the United Kingdom showed that about 15% of patients with osteoarthritis received intra-articular treatment with glucosamine sulfates.³ The costs of such treatment are significant. At present, 1 syringe of hyaluronic acid costs at least Can$130 (US$110). The treatment of knee osteoarthritis is covered by the US Medicare program but not by provincial formularies in Canada. In Austria (which has 8 million inhabitants) more than 10 million euros (approximately US$12 million or Can$15 million) is spent by social insurance programs annually for hyaluronic acid preparations (excluding the cost of application).Hyaluronic acid has beneficial effects in vitro.⁴ Because of its viscoelastic quality, it may replace synovial fluid. Furthermore, it may reduce the perception of pain. Beneficial molecular and cellular effects have also been reported.^2,4 Hyaluronic acid is frequently applied by intra-articular injection, but the evidence concerning its clinical relevance is conflicting. The European League against Rheumatism (EULAR) recommends the intra-articular application of hyaluronic acid as “category 2” evidence (at least 1 controlled study without randomization).⁵ The American College of Rheumatology recommends intra-articular hyaluron therapy for patients with no response to nonpharmacologic therapy and simple analgesics.⁶ In contrast, other specialists have concluded that “hyaluronate sodium is not efficacious” in the treatment of osteoarthritis.⁷ The first state-of-the-art systematic review and meta-analysis was published recently,⁸ and its authors concluded “that intra-articular hyaluronic acid, at best, has a small effect.” We performed a systematic review and meta-analysis of the effect of intra-articular hyaluronic acid for the treatment of osteoarthritis of the knee. In contrast to 2 previous meta-analyses on this subject,^8,9 we used a different approach to data synthesis and interpretation: instead of analyzing a composite effect size over time, we allocated trial data, when possible, to 3 outcome groups that we assumed would be relevant for patients with osteoarthritis. We specifically looked at pain at rest, pain during exercise and joint function as distinct outcomes, measured repeatedly over time. In addition, we assessed adverse events and the impact of both trial quality and molecular mass of the product. This analysis allows us to provide important additional insight into the effects of intra-articular administration of hyaluronic acid for the treatment of osteoarthritis of the knee.     

Morphology and transfection study of human microvascular endothelial cell angiogenesis: an in vitro three-dimensional model

Microvascular endothelial cells from human neonatal foreskin were grown in vitro until a three-dimensional network of capillary-like structures was formed. All stages of the angiogenic cascade could be observed in this in vitro model, including the formation of an internal lumen. The microscopy focused on morphology, formation of an internal lumen, role of the extracellular matrix, polarity of the cells, and the time-course of the angiogenic cascade. Bright-field microscopy revealed cells arranged circularly side by side and the internal lumen of capillary-like structures was verified by electron microscopy. Immunolabeling revealed a peritubular localization of collagen IV. Reporter gene expression after the formation of capillary-like structures was marginally higher than control expression, but clearly lower than the expression of cells at the stage of proliferation. Highest transfection efficiencies were obtained using vectors with the CMV promoter and the long fragment of the Ets-1 promoter. This is a first study of transfection efficiencies mapped for stages of in vitro angiogenesis. We describe here the morphological features of a long-term in vitro model of angiogenesis of human microvascular endothelial cells that could be used for transfection studies, without the provision of an extracellular matrix substrate. The cells self-create their own extracellular matrix to proliferate and form a three-dimensional network of capillary-like structures with an internal lumen.     

Caveolin-1 deficiency stimulates neointima formation during vascular injury

Neointima formation is a process characterized by smooth muscle cell (SMC) proliferation and extracellular matrix deposition in the vascular intimal layer. Here, we critically evaluate the role of caveolin-1 (Cav-1) in the pathogenesis of neointima formation. Cav-1 and caveolae organelles are particularly abundant in SMCs, where they are thought to function in membrane trafficking and signal transduction events. To directly evaluate the role of Cav-1 in the pathogenesis of neointimal lesions, we used Cav-1-deficient (Cav-1 -/-) mice as a model system. The right common carotid artery of wild-type and Cav-1 -/- mice was ligated just proximal to its bifurcation. Specimens were then harvested 4-weeks postligation and processed for morphometric and immunohistochemical analyses. The carotids of Cav-1 -/- mice showed significantly more intimal hyperplasia with subtotal luminal obstruction, as compared to wild-type mice. These neointimal lesions consisted mainly of SMCs. Mechanistically, neointimal lesions derived from Cav-1 -/- mice exhibited higher levels of phospho-p42/44 MAP kinase and cyclin D1 immunostaining, consistent with the idea that Cav-1 functions as a negative regulator of signal transduction. A significant increase in phospho-Rb (Ser780) immunostaining was also observed, in line with the upregulation of cyclin D1. In conclusion, using a carotid artery blood-flow cessation model, we show that genetic ablation of Cav-1 in mice stimulates SMC proliferation (neointimal hyperplasia), with concomitant activation of the p42/44 MAP kinase cascade and upregulation of cyclin D1. Importantly, our current study is the first to investigate the role of Cav-1 in SMC proliferation in the vascular system using Cav-1 -/- mice.     

Satellite glial cells in the trigeminal ganglion as a determinant of orofacial neuropathic pain

Satellite glial cells (SGCs) tightly envelop the perikarya of primary sensory neurons in peripheral ganglion and are identified by their morphology and the presence of proteins not found in ganglion neurons. These SGC-unique proteins include the inwardly rectifying K(+) channel Kir4.1, the connexin-43 (Cx43) subunit of gap junctions, the purinergic receptor P2Y4 and soluble guanylate cyclase. We also present evidence that the small-conductance Ca(2+)-activated K(+) channel SK3 is present only in SGCs and that SGCs divide following nerve injury. All the above proteins are involved, either directly or indirectly, in potassium ion (K(+)) buffering and, thus, can influence the level of neuronal excitability, which, in turn, has been associated with neuropathic pain conditions. We used in vivo RNA interference to reduce the expression of Cx43 (present only in SGCs) in the rat trigeminal ganglion and show that this results in the development of spontaneous pain behavior. The pain behavior is present only when Cx43 is reduced and returns to normal when Cx43 concentrations are restored. This finding shows that perturbation of a single SGC-specific protein is sufficient to induce pain responses and demonstrates the importance of PNS glial cell activity in the pathophysiology of neuropathic pain.     

Auxin triggers transient local signaling for cell specification in Arabidopsis embryogenesis

The Arabidopsis embryonic root meristem is initiated by the specification of a single cell, the hypophysis. This event critically requires the antagonistic auxin response regulators MONOPTEROS and BODENLOS, but their mechanism of action is unknown. We show that these proteins interact and transiently act in a small subdomain of the proembryo adjacent to the future hypophysis. Here they promote transport of auxin, which then elicits a second response in the hypophysis itself. Our results suggest that hypophysis specification is not the direct result of a primary auxin response but rather depends on cell-to-cell signaling triggered by auxin in adjacent cells.