Antiangiogenic properties of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin: an orally bioavailable heat shock protein 90 modulator.

PURPOSE: The purpose of this study was to investigate the antiangiogenic properties of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; NSC707545), a water-soluble benzoquinone ansamycin. EXPERIMENTAL DESIGN: The activity of 17-DMAG, in vivo, was evaluated for inhibition of fibroblast growth factor (FGF)-2-induced angiogenesis in s.c. implanted Matrigel in mice. In vitro, the activity of 17-DMAG on endothelial cells (human umbilical vein endothelial cells; HUVEC) was tested in FGF-2; and vascular endothelial growth factor (VEGF)-induced proliferation and apoptosis, motility, and extracellular matrix invasion; and on the alignment of capillary like structures in Matrigel. The protein level of heat shock protein (Hsp)90 and client proteins was examined by Western blot in FGF-2 and VEGF-stimulated HUVEC. RESULTS: Daily oral administration of 17-DMAG affected the angiogenic response in Matrigel in a dose-dependent manner. The hemoglobin content in the Matrigel implants was significantly inhibited, and the histological analysis confirmed a decrease of CD31(+) endothelial cells and of structures organized in cord and erythrocyte-containing vessels. In vitro, the compound inhibited dose-dependently the migration and the extracellular matrix-invasiveness of HUVEC and their capacity to form capillary like structures in Matrigel. 17-DMAG treatment also inhibited FGF-2 and VEGF-induced HUVEC proliferation and resulted in apoptosis. Accordingly, the expression of Hsp90 direct client proteins (pAkt and c-Raf-1) or their downstream substrates including pERK was also affected. 17-DMAG consistently increased the expression of Hsp70. Throughout the study similar results were obtained with 17-allylamino-17-demethoxygeldanamycin (17-AAG; NSC330507), the analog compound currently undergoing clinical trials. CONCLUSIONS: We show that the Hsp90 targeting agents 17-DMAG and 17-AAG inhibit angiogenesis. The strong effects on endothelial cell functions, in vitro, indicate that the antiangiogenic activity of 17-DMAG/17-AAG could also be due to a direct effect on endothelial cells. The oral bioavailability of 17-DMAG might be of advantage in investigating the potential of this compound in clinical trials with antiangiogenic as well as antiproliferative endpoints.     

Balancing orbital volume reduction and redistribution for a tailored surgical treatment in Graves’ ophthalmopathy

Graefe's Archive for Clinical and Experimental Ophthalmology - The purpose of this study is to share our experience on the use of different orbital decompression techniques, as well as the...     

Naphthylphthalamic acid associates with and inhibits PIN auxin transporters

N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.

Many aspects of plant growth are controlled by the hormone auxin. A distinct feature of auxin is that its hormonal action requires it to be actively transported between cells and ultimately throughout the whole plant in a controlled directional or polarized manner, a process known as polar auxin transport (PAT). The ability of plants to perform PAT is ascribed to the auxin export activity of PIN transporters (1). Plasma membrane PINs can be restricted to a specific side of cells (2), and when this polarity is maintained in continuous plant cell files, the combined activity of identically localized PINs results in auxin flowing in that direction (3). This lays the vectorial foundations for PAT to create local auxin gradients and plant-wide PAT streams that are critical for auxin action and normal plant growth (4, 5).Synthetic PAT inhibitors such as N-1-naphthylphthalamic acid (NPA) were initially developed as herbicides and then subsequently exploited by researchers to identify and characterize the unique PAT-based mechanisms that drive plant development (6). Having been used for over six decades, the question as to how NPA actually inhibits PAT has been keenly pursued. Several putative modes of action have been proposed, but the topic remains to date not fully or satisfactorily resolved (6).Early studies established NPA binding with high affinity to membrane-integral components of plant membranes (7–10). With the later discovery of pin1 mutants bearing their distinct bare inflorescences reminiscent of NPA-treated plants (11), followed by identification of the PIN gene family and gradual confirmation that PINs were NPA-sensitive auxin transporters that mediated PAT (1–5), it was apparent that the physiological and genetic evidence overwhelmingly linked NPA to inhibition of PIN activity (6). However, direct molecular association of NPA with PINs has never been reported (6). Instead, a substantial body of data has accumulated suggesting that the NPA target is not PIN itself, but rather other proteins or complexes that either actively coparticipate in PAT or are indirectly involved in control of PAT components (6, 12). Members of the B-family of ABC transporters, such as ABCB1 and ABCB19, showed high-affinity NPA binding and NPA-sensitive auxin export (1, 12–15), thus leading to proposals that they may either physically interact with PINs, or functionally interact such that their nonpolar auxin export activity contributes to PAT and/or to regulation of PINs (12, 16). In these scenarios, PIN/PAT would be rendered vulnerable to the NPA sensitivity of ABCB. However, these schemes are not yet fully resolved, are not fully consistent with key genetic and physiological data (6), and are particularly obfuscated by ABCB1/19 functioning both interactively and independently from PINs (1, 12, 15–20), with ABCB-PIN interaction occurring in an as-yet-unclarified manner (15, 18).A further twist in assigning ABCBs as the main NPA target is their regulation by their chaperone TWD1/FKBP42 (14, 16), with TWD1 itself also being an NPA-binding protein (14, 17). NPA interferes with this regulation and affects TWD1-ABCB interaction, but curiously NPA cannot bind stably to the ABCB-TWD1 complex (14, 17). As TWD1 has also been implicated in NPA-sensitive actin-based PIN trafficking (17), this has led to a model proposing that TWD1 could mediate the NPA sensitivities of both ABCB and PINs, thus presenting TWD1 as a modulator of PAT (17, 21). In an analogous scheme in some plant species, CYPA immunophilins such as tomato DGT, which are functionally similar to TWD1/FKBP42, are suggested to replace TWD1 in modulating auxin transporters and transducing NPA effects to PINs (12, 21).Similar to TWD1, BIG/TIR3 has also been associated with NPA and PIN trafficking (22). Given the undisputed role of trafficking in controlling PIN polarity (5), these reported effects warrant attention, although they are inconsistent with other reports that NPA perturbs neither vesicular trafficking nor actin dynamics in conditions where auxin transport is inhibited (23, 24). Together with trafficking, phosphorylation is another key modulator of PIN polarity as well as activity (5), so it is not surprising to find hypotheses suggesting that NPA could interfere with critical phosphorylation events (6), particularly as PID, a kinase crucial for PIN trafficking and activation, has also been connected to ABCB function and TWD1/ABCB/NPA interactions (25). Others propose that NPA may mimic natural compounds in their capacity as endogenous regulators of PAT, with plant flavonoids being suspected candidates (6, 26). Since flavonoids can compete with or inhibit ATP-binding in mammalian kinases and ABC transporters (27, 28), and as flavonoids can bind to and inhibit PID (25), a phosphorylation-based NPA mode of action would overlap with this hypothesis and poses the question whether NPA acts similarly as an ATP mimic.With these many potential NPA-affected pathways, there is a need to distinguish between low- and high-affinity NPA targets and possible secondary effects due to prolonged PAT inhibition. Current consensus is that low concentrations of NPA (<10 µM) cause direct inhibition of auxin transporters in PAT (21) and the consequent physiological effects seen in planta (IC₅₀ 0.1 to 10 µM) (7, 9, 19, 23, 29). This is associated with high-affinity binding to membranes (K_d 0.01 to 0.1 µM) (7, 8) and the inhibition of PIN/ABCB activity in short-term auxin transport assays (1, 14, 18, 20, 23). In contrast, NPA is thought to affect trafficking (21, 30) and other non-PAT processes (31) when used at higher doses (50 to 200 µM NPA), presumably via binding to its lower-affinity targets, although excessive NPA exposure may also have fast-acting toxic side effects (23). As the in vitro affinity of TWD1 for NPA is surprisingly low (K_d ∼100 µM) (17), the TWD1-mediated NPA effects on PIN/PAT are thought to be of the low-affinity type and linked to trafficking perturbations (17, 21). However, as NPA is always externally applied to plants or cells, it is not clear how or where the drug distributes or accumulates, and thus there may be discrepancies between actual and reported/apparent effective concentrations, as might be the case for TWD1 (17). Finally, NPA also binds with low affinity to inhibit APM1, an aminopeptidase implicated in auxin-related plant growth, but as with trafficking effects, this low-affinity NPA interaction is not connected to direct regulation of PAT (31).Thus, the available data proffer various indirect mechanisms that could lead to NPA inhibition of PIN-mediated PAT, but the proposed schemes have complicating aspects and struggle at times to satisfactorily explain the prime effects of NPA. Here we propose an alternative simpler scenario involving a more direct link between NPA and PINs that would resolve some of these currently outstanding issues. We present evidence from heterologous transport assays, classical in situ membrane binding, and oligomerization studies which collectively suggest that NPA can interact directly in a high-affinity manner with PINs, leading to conformational or structural effects and inhibition of auxin export activity.     

Dissociation and Alterations in Brain Function and Structure: Implications for Borderline Personality Disorder

Dissociation involves disruptions of usually integrated functions of consciousness, perception, memory, identity, and affect (e.g., depersonalization, derealization, numbing, amnesia, and analgesia). While the precise neurobiological underpinnings of dissociation remain elusive, neuroimaging studies in disorders, characterized by high dissociation (e.g., depersonalization/derealization disorder (DDD), dissociative identity disorder (DID), dissociative subtype of posttraumatic stress disorder (D-PTSD)), have provided valuable insight into brain alterations possibly underlying dissociation. Neuroimaging studies in borderline personality disorder (BPD), investigating links between altered brain function/structure and dissociation, are still relatively rare. In this article, we provide an overview of neurobiological models of dissociation, primarily based on research in DDD, DID, and D-PTSD. Based on this background, we review recent neuroimaging studies on associations between dissociation and altered brain function and structure in BPD. These studies are discussed in the context of earlier findings regarding methodological differences and limitations and concerning possible implications for future research and the clinical setting.     

Clinical manifestations,treatment, and outcomes of children and adolescents with eosinophilic esophagitis

ObjectiveThis study aimed to describe the clinical, endoscopic, and histologic characteristics, as well as the response to conventional treatment of pediatric patients with the classical form of eosinophilic esophagitis (EoE).MethodsStudy of clinical, laboratory, endoscopic, and histologic data and response to conventional treatment of 43 previously followed pediatric patients with the classical form of EoE.ResultsA total of 43 patients diagnosed with EoE were included in the study, of which 37 were males (86%), with a mean age of 8.4 years. The most common symptoms were: nausea, vomiting, and abdominal pain (100%) in children younger than 7 years, and loss of appetite (60%), heartburn (52%), and food impaction (48%) in children older than 7 years and adolescents. Regarding the endoscopic findings, 12 (28%) patients had whitish plaques on the esophageal lining, 8 (18.5%) had longitudinal grooves, 2 (4.5%) had concentric rings, 3 (7%) had longitudinal grooves and whitish plaques, and the remaining 18 (42%) had esophageal mucosa with normal appearance. Despite the initial favorable response, 76.7% of patients required more than one course of corticosteroid therapy (systemic or aerosol) and diet (exclusion or elimination of food or elementary allergens). Persistence of eosinophil infiltration was found in some patients despite favorable clinical response.ConclusionsThe classic form of EoE typically shows different symptoms according age range. A significant number of patients required more than one treatment cycle to show clinical remission. Endoscopic and histologic improvement was observed; however, eosinophilic infiltration persisted in some patients.     

Synthesis of novel cyclosiloxane monomers containing push–pull moieties and their anionic ring opening polymerization

The synthesis of three novel tetracyclosiloxane monomers modified either with a nitroaniline (NA) or with a Disperse Red 1 (DR1) push–pull group and their ring opening polymerization reaction in the presence of tetramethylammonium hydroxide are presented. The prepared monomers and polymers were characterized by different spectral methods and gel permeation chromatography. For the crystalline monomers, the structures were further proven by single crystal X-ray diffraction. Dynamic scanning calorimetry shows that the polymers that carry NA groups have a glass transition temperature (T_g) well below room temperature (RT), while the one that carries DR1 groups melts at 55 °C. The transition temperatures have a strong effect on permittivity as indicated by broadband impedance spectroscopy measurements conducted at different temperatures and frequencies. The polymers modified with NA groups have a high permittivity (maximum value of 17.3) at RT, suggesting the polar groups to be mobile and orientation polarization to be effective. However, the polar groups of the polymer modified with DR1 are frozen and thus cannot contribute to the permittivity via orientation polarization. Consequently, the permittivity is only 8.8 at RT, but increases to 22 above the melting temperature, where the dipoles are mobile. Because of the high dielectric permittivity and rather low T_g, the polymers modified with NA are attractive as active dielectric materials in actuators, capacitors, and stretchable electronics, whereas the polymer modified with DR1 may be of interest in nonlinear optical devices.

The synthesis of polysiloxanes modified with a nitroaniline or with a Disperse Red 1 push–pull group and their dielectric properties are reported.     

Temporomandibular Disorders in Burning Mouth Syndrome Patients: An Observational Study

BACKGROUND: Burning Mouth Syndrome (BMS) is a chronic disease characterized by absence of any lesions and burning of the oral mucosa associated to a sensation of dry mouth and/or taste alterations. The purpose of our study is to estimate signs and symptoms of Temporomandibular Disorders (TMD) in patients with BMS and to investigate for the existence of an association between BMS and TMD.MATERIALS AND METHODS: Forty-four BMS patients were enrolled; BMS subtype was established according to the classification of Lamey. After a gnathological evaluation, according to the protocol of the European Academy of Craniomandibular Disorders, patients were classified by RDC/TMD criteria. The data were compared and analyzed using a chi-square test to describe the existence of an association between BMS and TMD.RESULTS: 65.9% the BMS patients showed disorders classified as primary signs and symptoms of TMD according to RDC / TMD criteria, and 72.7% showed parafunctional habits. The chi-square test revealed a statistically significant association (p = 0.035) between BMS and TMD.CONCLUSION: The data suggest that there is a possible relationship not yet well understood between BMS and TMD, may be for neurophatic alterations assumed for BMS that could be also engaged in TMD pathogenesis.     

Expression of hepcidin is down-regulated in TfR2 mutant mice manifesting a phenotype of hereditary hemochromatosis

Transferrin receptor 2 (TfR2) is a membrane glycoprotein that mediates cellular iron uptake from holotransferrin. Homozygous mutations of this gene cause one form of hereditary hemochromatosis in humans. We recently reported that homozygous TfR2(Y245X) mutant mice, which correspond to the TfR2(Y250X) mutation in humans, showed a phenotype similar to hereditary hemochromatosis. In this study, we further analyzed the phenotype as well as iron-related gene expression in these mice by comparing the TfR2-mutant and wild-type siblings. Northern blot analyses showed that the levels of expression of hepcidin mRNA in the liver were generally lower, whereas those of duodenal DMT1, the main transporter for uptake of dietary iron, were higher in the TfR2-mutant mice as compared to the wild-type siblings. Expression of hepcidin mRNA in the TfR2 mutant mice remained low even after intraperitoneal iron loading. In isolated hepatocytes from both wild-type and TfR2 mutant mice, interleukin-6 and lipopolysaccharide each induced expression of hepcidin mRNA. These results suggest that up-regulation of hepcidin expression by inflammatory stimuli is independent of TfR2 and that TfR2 is upstream of hepcidin in the regulatory pathway of body iron homeostasis.     

Soluble factors produced by activated CD4+ T cells modulate EBV latency

Following infection with Epstein-Barr virus (EBV), the virus is carried for life in the memory B-cell compartment in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCLs) (latency III) that are generated after infection. It is of fundamental significance to identify how the different EBV expression patterns are established in the latently infected cell. In view of the prompt activatability of CD4(+) T cells in primary EBV infection, and their role in B-cell differentiation, we studied the involvement of CD4(+) T cells in the regulation of EBV latency. Lymphoblastoid cell lines (LCLs) were cocultured with autologous or allogeneic CD4(+) T cells. Activated T cells influenced the expression of two key viral proteins that determine the fate of the infected B cell. EBNA2 was down-regulated, whereas LMP1 was unregulated and the cells proliferated less. This was paralleled by the down-regulation of the latency III promoter (Cp). Experiments performed in the transwell system showed that this change does not require cell contact, but it is mediated by soluble factors. Neutralizing experiments proved that the up-regulation of LMP1 is, to some extent, mediated by IL21, but this cytokine was not responsible for EBNA2 down-regulation. This effect was partly mediated by soluble CD40L. We detected similar regulatory functions of T cells in in vitro-infected lymphocyte populations. In conclusion, our results revealed an additional mechanism by which CD4(+) T cells can control the EBV-induced B-cell proliferation.