Role of serum concentration of VEGFR1 and TIMP2 on clinical outcome in primary cervical cancer: Results of a companion protocol of the randomized,NOGGO–AGO phase III adjuvant trial of simultaneous cisplatin-based radiochemotherapy vs. carboplatin and paclitaxel containing sequential radiotherapy

ObjectiveAim of the present study was to analyze the expression-profile of IGF1, IGFBP3, sICAM1, sVCAM1, MMP2, MMP9, TIMP2, VEGFA, VEGFD, VEGFC and VEGFR1 in patients with high-risk FIGO-stage Ib-IIb cervical cancer.MethodsSerum from 68 cervical cancer patients treated within a phase-III-trial with either simultaneous cisplatin radiochemotherapy or sequential systemic carboplatin and paclitaxel followed by percutaneous irradiation was analyzed by ELISA. Both target expression and correlation with important clinicopathological factors were analyzed following standard statistic procedures.ResultsAll 68 patients underwent a primary radical hysterectomy with pelvic and/or paraaortic lymphadenectomy. 85.3% of the extirpated tumors had clear surgical margins (R0). Increased levels of VEGFR1, TIMP2 and MMP2 were significantly associated with positive surgical margins (p = 0.004, p = 0.018 and p = 0.004, respectively). High concentration of MMP2 and TIMP2 correlated additionally with an advanced age at time of diagnosis (p = 0.001 and p = 0.007, respectively). For the cut-off value of 100 pg/ml, an increased VEGFR1 was significantly associated with poor overall (OS) and progression-free (PFS) survival (p = 0.017 and p = 0.015, respectively). A TIMP2 concentration of lower than 90 ng/ml was significantly associated with poorer OS and PFS (p = 0.009 and p = 0.043, respectively). In the multivariate analysis, TIMP2 expression in serum was the only independent prognostic factor for OS (p = 0.032, HR = 6.51, 95% CI = 1.17–36.01).ConclusionsExpression-profile of specific biomarkers associated with tumor invasion, cell migration and angiogenesis seems to be of prognostic value for both OS and PFS in patients undergoing surgery due to primary cervical cancer. Further analyses are warranted to allow an implementation of such markers into clinical practice.     

Aminomethylpyrimidines as novel DPP-IV inhibitors: a 10(5)-fold activity increase by optimization of aromatic substituents

The influence of aromatic substitution on a newly discovered class of inhibitors of dipeptidyl peptidase IV was investigated. A 10(5)-fold increase in potency was achieved by the optimization of aromatic substituents in a parallel chemistry program. The observed SAR could be explained by an X-ray structure of the protein-ligand complex.     

Free fatty acid palmitate impairs the vitality and function of cultured human bladder smooth muscle cells

Background

Incidence of urinary tract infections is elevated in patients with diabetes mellitus. Those patients show increased levels of the saturated free fatty acid palmitate. As recently shown metabolic alterations induced by palmitate include production and secretion of the pro-inflammatory cytokine interleukine-6 (IL-6) in cultured human bladder smooth muscle cells (hBSMC). Here we studied the influence of palmitate on vital cell properties, for example, regulation of cell proliferation, mitochondrial enzyme activity and antioxidant capacity in hBSMC, and analyzed the involvement of major cytokine signaling pathways.

Methodology/Principal Findings

HBSMC cultures were set up from bladder tissue of patients undergoing cystectomy and stimulated with palmitate. We analyzed cell proliferation, mitochondrial enzyme activity, and antioxidant capacity by ELISA and confocal immunofluorescence. In signal transduction inhibition experiments we evaluated the involvement of NF-κB, JAK/STAT, MEK1, PI3K, and JNK in major cytokine signaling pathway regulation. We found: (i) palmitate decreased cell proliferation, increased mitochondrial enzyme activity and antioxidant capacity; (ii) direct inhibition of cytokine receptor by AG490 even more strongly suppressed cell proliferation in palmitate-stimulated cells, while counteracting palmitate-induced increase of antioxidant capacity; (iii) in contrast knockdown of the STAT3 inhibitor SOCS3 increased cell proliferation and antioxidant capacity; (iv) further downstream JAK/STAT3 signaling cascade the inhibition of PI3K or JNK enhanced palmitate induced suppression of cell proliferation; (v) increase of mitochondrial enzyme activity by palmitate was enhanced by inhibition of PI3K but counteracted by inhibition of MEK1.

Conclusions/Significance

Saturated free fatty acids (e.g., palmitate) cause massive alterations in vital cell functions of cultured hBSMC involving distinct major cytokine signaling pathways. Thereby, certain cytokines might counteract the palmitate-induced downregulation of cell proliferation and vitality. This could be an important link to clinical findings of increased risk of metabolic related bladder diseases such as overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC).     

"Let There be Light!" pigeon eggs are regularly exposed to light during breeding

Light stimulation before hatching initiates the emergence of avian visual lateralisation. Since several studies show that birds benefit from being lateralised, we can conjecture that their clutch is being exposed to light during breeding. We tested this assumption in pigeons with a semi-natural setup where the animals were systematically recorded using a movement detection system throughout their breeding period. The results show that pigeon pairs perform their relieves in a regular way by abandoning their clutch for a mean of about 55 s at approximately every 43 min. Thus, the developing visual pathways are repetitively stimulated by light for cumulatively over 3h before the breeding period ends. It becomes apparent that both the duration as well as the repetitions of light stimulation play a crucial role in the onset of visual asymmetry.     

Selective mobility and sensitivity to SNAREs is exhibited by the Arabidopsis KAT1 K+ channel at the plasma membrane

Recent findings indicate that proteins in the SNARE superfamily are essential for cell signaling, in addition to facilitating vesicle traffic in plant cell homeostasis, growth, and development. We previously identified SNAREs SYP121/Syr1 from tobacco (Nicotiana tabacum) and the Arabidopsis thaliana homolog SYP121 associated with abscisic acid and drought stress. Disrupting tobacco SYP121 function by expressing a dominant-negative Sp2 fragment had severe effects on growth, development, and traffic to the plasma membrane, and it blocked K(+) and Cl(-) channel responses to abscisic acid in guard cells. These observations raise questions about SNARE control in exocytosis and endocytosis of ion channel proteins and their organization within the plane of the membrane. We have used a dual, in vivo tagging strategy with a photoactivatable green fluorescent protein and externally exposed hemagglutinin epitopes to monitor the distribution and trafficking dynamics of the KAT1 K(+) channel transiently expressed in tobacco leaves. KAT1 is localized to the plasma membrane within positionally stable microdomains of approximately 0.5 microm in diameter; delivery of the K(+) channel, but not of the PMA2 H(+)-ATPase, to the plasma membrane is suppressed by Sp2 fragments of tobacco and Arabidopsis SYP121, and Sp2 expression leads to profound changes in KAT1 distribution and mobility within the plane of the plasma membrane. These results offer direct evidence for SNARE-mediated traffic of the K(+) channel and a role in its distribution within subdomains of the plasma membrane, and they implicate a role for SNAREs in positional anchoring of the K(+) channel protein.     

Serotonylation of small GTPases is a signal transduction pathway that triggers platelet alpha-granule release

Serotonin is a neurotransmitter in the central nervous system. In the periphery, serotonin functions as a ubiquitous hormone involved in vasoconstriction and platelet function. Serotonin is synthesized independently in peripheral tissues and neurons by two different rate-limiting tryptophan hydroxylase (TPH) isoenzymes. Here, we show that mice selectively deficient in peripheral TPH and serotonin exhibit impaired hemostasis, resulting in a reduced risk of thrombosis and thromboembolism, although the ultrastructure of the platelets is not affected. While the aggregation of serotonin-deficient platelets in vitro is apparently normal, their adhesion in vivo is reduced due to a blunted secretion of adhesive alpha-granular proteins. In elucidating the mechanism further, we demonstrate that serotonin is transamidated to small GTPases by transglutaminases during activation and aggregation of platelets, rendering these GTPases constitutively active. Our data provides evidence for a receptor-independent signaling mechanism, termed herein as "serotonylation," which leads to alpha-granule exocytosis from platelets.     

Pyrido pyrimidinones as selective agonists of the high affinity niacin receptor GPR109A: Optimization of in vitro activity

Pyrido pyrimidinones are selective agonists of the human high affinity niacin receptor GPR109A (HM74A). They show no activity on the highly homologous low affinity receptor GPR109B (HM74). Starting from a high throughput screening hit the in vitro activity of the pyrido pyrimidinones was significantly improved providing lead compounds suitable for further optimization.     

An aminomethylpyrimidine DPP-IV inhibitor with improved properties

A recently identified DPP-IV inhibitor (1) was found to induce phospholipidosis and to inhibit CYP3A4. A small series of less lipophilic and less amphiphilic analogues was synthesized in an effort to overcome these issues. One compound from this series was equipotent to 1, did not induce phospholipidosis and showed a reduced CYP3A4 inhibition.     

ATP enhances spontaneous calcium activity in cultured suburothelial myofibroblasts of the human bladder

Background

Suburothelial myofibroblasts (sMF) are located underneath the urothelium in close proximity to afferent nerves. They express purinergic receptors and show calcium transients in response to ATP. Therefore they are supposed to be involved in afferent signaling of the bladder fullness. Since ATP concentration is likely to be very low during the initial filling phase, we hypothesized that sMF Ca²⁺ activity is affected even at very low ATP concentrations. We investigated ATP induced modulation of spontaneous activity, intracellular calcium response and purinergic signaling in cultured sMF.

Methodology/Principal Findings

Myofibroblast cultures, established from cystectomies, were challenged by exogenous ATP in presence or absence of purinergic antagonist. Fura-2 calcium imaging was used to monitor ATP (10⁻¹⁶ to 10⁻⁴ mol/l) induced alterations of calcium activity. Purinergic receptors (P2X1, P2X2, P2X3) were analysed by confocal immunofluorescence. We found spontaneous calcium activity in 55.18%±1.65 of the sMF (N = 48 experiments). ATP significantly increased calcium activity even at 10⁻¹⁶ mol/l. The calcium transients were partially attenuated by subtype selective antagonist (TNP-ATP, 1 µM; A-317491, 1 µM), and were mimicked by the P2X1, P2X3 selective agonist α,β-methylene ATP. The expression of purinergic receptor subtypes in sMF was confirmed by immunofluorescence.

Conclusions/Significance

Our experiments demonstrate for the first time that ATP can modulate spontaneous activity and induce intracellular Ca²⁺ response in cultured sMF at very low concentrations, most likely involving P2X receptors. These findings support the notion that sMF are able to register bladder fullness very sensitively, which predestines them for the modulation of the afferent bladder signaling in normal and pathological conditions.