Identification of residues of the IFNAR1 chain of the type I human interferon receptor critical for ligand binding and biological activity

The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.     

Frailty modelling of testicular cancer incidence using Scandinavian data

The incidence of testicular cancer is highest among young men, and then decreases sharply with age. This points towards a frailty effect, where some men have a much greater risk of testicular cancer than the majority of the male population. Those with the highest risk get cancer, drain the group of individuals at risk, and leave a healthy male population which has approximately zero risk of testicular cancer. This leads to the observed decrease in incidence. We discuss a frailty model, where the frailty is compound-Poisson-distributed. This allows for a non-susceptible group (of zero frailty). The model is successfully applied to incidence data from the Danish and Norwegian registries. It is indicated that there was a decrease in incidence for males born during World War II in both countries. Bootstrap analysis is used to find the degree of variation in the estimates. In the Armitage-Doll multistage model, the estimated number of transitions needed for a cell to become malignant is close to 3 for non-seminomas and 4 for seminomas in both the Danish and Norwegian data. This paper demonstrates that a model including a frailty effect fits the incidence data well and gives interesting results and interpretations, although this is no proof of the effect's truth.     

Rapid determination of chloramphenicol and its glucuronide in food products by liquid chromatography-electrospray negative ionization tandem mass spectrometry

Chloramphenicol (CAP) is subjected to monitoring in food products, with a minimum required performance level set at 0.3 ng/g. CAP was isolated from chicken meat and seafood by very simple solvent extraction procedure. For honey, a fast SPE procedure was applied. CAP-D5 was used as internal standard. HPLC separation was done on RP18 123 mm x 3 mm column in acetonitrile-ammonium formate 10 mM, pH 3.0 (40:60) at flow rate of 0.3 ml/min. A TSQ Quantum instrument with ESI source has been used in negative ionization mode. A MRM procedure has been applied and following transitions were monitored: m/z 321 > 152 (quantifier), 321 > 194, 321 > 257(qualifiers), 326 > 157 (IS). CAP peak was eluted at around 5 min; the total run time was 7 min. LOD was around 0.1 ng/g meat or 0.05 ng/g honey. Matrix effects were studied for all materials used, involving injection of blank extracts with post-column infusion of CAP, as well as checking the influence of the co-injected blank extracts on the signal intensity of CAP. No influence of matrix on the results of CAP determination were observed. The method allows analyzing up to 30 duplicate samples per day, including all calibration standards. Additionally, the method for determination of CAP glucuronide (CAP-G) was established, using urine from rats that were given this drug as a source of the metabolite. Full validation of the metabolite was not possible, due to the unavailability of reference standard.     

Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters

Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (β-carotene, crocin, retinoic acid, canthaxanthin, and fucoxanthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytometry using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they themselves also function as substrates. Fucoxanthin and canthaxanthin (50-100μM) produced a 3-5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC(50) values between 100 and 200μM. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxicity in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respectively as compared to untreated control levels (p<0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy.     

Season of diagnosis is a predictor of cancer survival. Sun-induced vitamin D may be involved: a possible role of sun-induced Vitamin D

The calcidiol level in a group of Norwegians (14,000 individuals, age range 16–80) was found to be highest in late summer. The seasonal variation was larger for young than for old persons. The calcitriol concentration was practically constant throughout the year. Younger persons had less calcidiol and more calcitriol than older persons, indicating that the conversion of calcidiol to calcitriol is more efficient in younger persons.

A seasonal variation of prognosis of cancer (colon-, breast-, prostate- cancer and Hodgkin lymphoma) was found. The survival is highest for summer and autumn diagnosis, corresponding to maximal calcidiol levels. Thus, calcidiol may act synergistically with traditional treatment modalities.

In view of these calcitriol and calcidiol data, the seasonal variation of cancer survival may be related to the calcidiol gradient, indicating that this Vitamin D metabolite may be more important than believed so far.     

Demographic determinants and effect of pre-operative angiotensin converting enzyme inhibitors and angiotensin receptor blockers on the occurrence of atrial fibrillation after CABG surgery

Background

Alport syndrome (AS) is a rare inherited disorder characterized by an inflammation of the kidneys and damage to the glomerular capillaries, ultimately leading to renal failure at an early age. To date, rare reports of cardiac involvement in AS have been described, due in the majority of cases to the higher risk of heart conduction abnormalities in these patients, at times requiring implantation of a transcutaneous pacemaker. An increased risk of hypertension is likewise commonly featured.

Case presentation

We report the case of a 17-year-old female affected by a very severe early form of AS. A previously unreported association of the syndrome with congenital heart disease (CHD), (in this case membranous ventricular septal defect), is also reported. A possible pathophysiological mechanism underlying the concomitant manifestation of these two disorders is suggested. Complications implicated in surgical treatment of CHD are described. Clinical and therapeutic management of AS with cardiovascular involvement are discussed, and a short literature review performed.

Conclusions

This first report of a cardiovascular association highlights the possible involvement of collagen mutations in the two pathologies. Even when drug-resistance appears to be responsible for the failure to control secondary hypertension in AS, clonidine may represent a safe, effective option in the normalization of high blood pressure.