Inhibition of the Equilibrative Nucleoside Transporter 1 and Activation of A2A Adenosine Receptors by 8-(4-Chlorophenylthio)-modified cAMP Analogs and Their Hydrolytic Products

Cyclic AMP analogs containing hydrophobic modification of C⁸ at the adenine ring such as 8-(4-chlorophenylthio)-cAMP (8-pCPT-cAMP) and 8-(4-chlorophenylthio)-2′-O-methyl-cAMP (8-pCPT-2′-O-methyl-cAMP) can penetrate membranes due to their high lipophilicity and directly activate intracellular cAMP effectors. Therefore, these cAMP analogs have been used in numerous studies, assuming that their effects reflect the consequences of direct activation of cAMP effectors. The present study provides evidence that 8-pCPT-modified cAMP analogs and their corresponding putative hydrolysis products (8-(4-chlorophenylthio)-adenosine (8-pCPT-ado) and 8-(4-chlorophenylthio)-2′-O-methyl-adenosine (8-pCPT-2′-O-methyl-ado)) inhibit the equilibrative nucleoside transporter 1 (ENT1). In PC12 cells, in which nucleoside transport strongly depended on ENT1, 8-pCPT-ado, 8-pCPT-2′-O-methyl-ado, and, to a smaller extent, 8-pCPT-2′-O-methyl-cAMP caused an increase of protein kinase A substrate motif phosphorylation and anti-apoptotic effect by an A_2A adenosine receptor (A_2AR)-dependent mechanism. In contrast, the effects of 8-pCPT-cAMP were mainly A_2AR-independent. In HEK 293 showing little endogenous ENT1-dependent nucleoside transport, transfection of ENT1 conferred A_2AR-dependent increase in protein kinase A substrate motif phosphorylation. Together, the data of the present study indicate that inhibition of ENT1 and activation of adenosine receptors have to be considered when interpreting the effects of 8-pCPT-substituted cAMP/adenosine analogs.     

Effects of climate change and horticultural use on the spread of naturalized alien garden plants in Europe

Climate warming is supposed to enlarge the area climatically suitable to the naturalization of alien garden plants in temperate regions. However, the effects of a changing climate on the spread of naturalized ornamentals have not been evaluated by spatially and temporarily explicit range modelling at larger scales so far. Here, we assess how climate change and the frequency of cultivation interactively determine the spread of 15 ornamental plants over the 21st century in Europe. We coupled species distribution modelling with simulations of demography and dispersal to predict range dynamics of these species in annual steps across a 250 × 250 m raster of the study area. Models were run under four scenarios of climate warming and six levels of cultivation intensity. Cultivation frequency was implemented as size of the area used for planting a species. Although the area climatically suitable to the 15 species increases, on average, the area predicted to be occupied by them in 2090 shrinks under two of the three climate change scenarios. This contradiction obviously arises from dispersal limitations that were pronounced although we assumed that cultivation is spatially adapting to the changing climate. Cultivation frequency had a much stronger effect on species spread than climate change, and this effect was non‐linear. The area occupied increased sharply from low to moderate levels of cultivation intensity, but levelled off afterwards. Our simulations suggest that climate warming will not necessarily foster the spread of alien garden plants in Europe over the next decades. However, climatically suitable areas do increase and hence an invasion debt is likely accumulating. Restricting cultivation of species can be effective in preventing species spread, irrespective of how the climate develops. However, for being successful, they depend on high levels of compliance to keep propagule pressure at a low level.     

HIV Shedding from Male Circumcision Wounds in HIV-Infected Men: A Prospective Cohort Study

BackgroundA randomized trial of voluntary medical male circumcision (MC) of HIV—infected men reported increased HIV transmission to female partners among men who resumed sexual intercourse prior to wound healing. We conducted a prospective observational study to assess penile HIV shedding after MC.ConclusionPenile HIV shedding is significantly reduced after healing of MC wounds. Lower plasma VL is associated with decreased frequency and quantity of HIV shedding from MC wounds. Starting ART prior to MC should be considered to reduce male-to-female HIV transmission risk. Research is needed to assess the time on ART required to decrease shedding, and the acceptability and feasibility of initiating ART at the time of MC.     

Effects of pregabalin on central sensitization in patients with chronic pancreatitis in a randomized, controlled trial

Background

Intense abdominal pain is the dominant feature of chronic pancreatitis. During the disease changes in central pain processing, e.g. central sensitization manifest as spreading hyperalgesia, can result from ongoing nociceptive input. The aim of the present study is to evaluate the effect of pregabalin on pain processing in chronic pancreatitis as assessed by quantitative sensory testing (QST).

Methods

This randomized, double-blind, placebo-controlled trial evaluated effects of pregabalin on pain processing. QST was used to quantify pain processing by measuring thresholds to painful electrical and pressure stimulation in six body dermatomes. Descending endogenous pain modulation was quantified using the conditioned pain modulation (CPM) paradigm to elicit a DNIC (diffuse noxious inhibitory controls) response. The main effect parameter was the change in the sum of all body pain threshold values after three weeks of study treatment versus baseline values between both treatment groups.

Results

64 patients were analyzed. No differences in change in sum of pain thresholds were present for pregabalin vs. placebo after three weeks of treatment. For individual dermatomes, change vs. baseline pain thresholds was significantly greater in pregabalin vs. placebo patients for electric pain detection threshold in C5 (P = 0.005), electric pain tolerance threshold in C5 (P = 0.04) and L1 (P = 0.05), and pressure pain tolerance threshold in T4 (P = 0.004). No differences were observed between pregabalin and placebo regarding conditioned pain modulation.

Conclusion

Our study provides first evidence that pregabalin has moderate inhibitory effects on central sensitization manifest as spreading hyperalgesia in chronic pancreatitis patients. These findings suggest that QST can be of clinical use for monitoring pain treatments in the context of chronic pain.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00755573","term_id":"NCT00755573"}}NCT00755573     

Fibrin gel-immobilized VEGF and bFGF efficiently stimulate angiogenesis in the AV loop model

The modulation of angiogenic processes in matrices is of great interest in tissue engineering. We assessed the angiogenic effects of fibrin-immobilized VEGF and bFGF in an arteriovenous loop (AVL) model in 22 AVLs created between the femoral artery and vein in rats. The loops were placed in isolation chambers and were embedded in 500 microL fibrin gel (FG) (group A) or in 500 microL FG loaded with 0.1 ng/microL VEGF and 0.1 ng/microL bFGF (group B). After two and four weeks specimens were explanted and investigated using histological, morphometrical, and ultramorphological [scanning electron microscope (SEM) of vascular corrosion replicas] techniques. In both groups, the AVL induced formation of densely vascularized connective tissue with differentiated and functional vessels inside the fibrin matrix. VEGF and bFGF induced significantly higher absolute and relative vascular density and a faster resorption of the fibrin matrix. SEM analysis in both groups revealed characteristics of an immature vascular bed, with a higher vascular density in group B. VEGF and bFGF efficiently stimulated sprouting of blood vessels in the AVL model. The implantation of vascular carriers into given growth factor-loaded matrix volumes may eventually allow efficient generation of axially vascularized, tissue-engineered composites.     

GPCR-induced migration of breast carcinoma cells depends on both EGFR signal transactivation and EGFR-independent pathways

The epidermal growth factor receptor (EGFR) plays a key role in the regulation of important cellular processes under normal and pathophysiological conditions such as cancer. In human mammary carcinomas the EGFR is involved in regulating cell growth, survival, migration and metastasis and its activation correlates with the lack of response in hormone therapy. Here, we demonstrate in oestrogen receptor-positive and -negative human breast cancer cells and primary mammary epithelial cells a cross-communication between G protein-coupled receptors (GPCRs) and the EGFR. We present evidence that specific inhibition of ADAM15 or TACE blocks GPCR-induced and proHB-EGF-mediated EGFR tyrosine phosphorylation, downstream mitogenic signalling and cell migration. Notably, activation of the PI3K downstream mediator PKB/Akt by GPCR ligands involves the activity of sphingosine kinase (SPHK) and is independent of EGFR signal transactivation. We conclude that GPCR-induced chemotaxis of breast cancer cells is mediated by EGFR-dependent and -independent signalling pathways, with both parallel pathways having to act in concert to achieve a complete migratory response.     

Modulation of the immunological synapse: a key to HIV-1 pathogenesis?

AIDS is the result of a constant struggle between the lentivirus HIV and the immune system. Infection with HIV interferes directly with the function of CD4(+) T cells and manipulates the host immune response to the virus. Recent studies indicate that the viral protein Nef, a central player in HIV pathogenesis, impairs the ability of infected lymphocytes to form immunological synapses with antigen-presenting cells and affects T-cell-receptor-mediated stimulation. An integrative picture of the abnormal behaviour of HIV-infected lymphocytes is therefore emerging. We propose that modulating lymphocyte signalling, apoptosis and intracellular trafficking ensures efficient spread of the virus in the hostile environment of the immune system.     

The effect of sample handling on cross sectional HIV incidence testing results

Objective(s)

To determine if mishandling prior to testing would make a sample from a chronically infected subject appear recently infected when tested by cross-sectional HIV incidence assays.

Methods

Serum samples from 31 subjects with chronic HIV infection were tested. Samples were subjected to different handling conditions, including incubation at 4°C, 25°C and 37°C, for 1, 3, 7 or 15 days prior to testing. Samples were also subjected to 1,3, 7 and 15 freeze-thaw cycles prior to testing. Samples were tested using the BED capture enzyme immuno assay (BED-CEIA), Vironostika-less sensitive (V-LS), and an avidity assay using the Genetic Systems HIV-1/HIV-2 plus O EIA (avidity assay).

Results

Compared to the sample that was not subjected to any mishandling conditions, for the BED-CEIA, V-LS and avidity assay, there was no significant change in test results for samples incubated at 4°C or 25°C prior to testing. No impact on test results occurred after 15 freeze-thaw cycles. A decrease in assay results was observed when samples were held for 3 days or longer at 37°C prior to testing.

Conclusions

Samples can be subjected up to 15 freeze-thaw cycles without affecting the results the BED-CEIA, Vironostika-LS, or avidity assays. Storing samples at 4°C or 25°C for up to fifteen days prior to testing had no impact on test results. However, storing samples at 37°C for three or more days did affect results obtained with these assays.     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.