The K-Segments of the Wheat Dehydrin DHN-5 are Essential for the Protection of Lactate Dehydrogenase and β-Glucosidase Activities In Vitro

The wheat dehydrin DHN-5 has been previously shown to exhibit heat protecting effect on enzymatic activities. In order to understand the molecular mechanism by which DHN-5 exerts its protective function, we performed an approach to dissect the functional domains of DHN-5 responsible for this feature. In two distinct enzymatic assays, we found that the truncated forms of DHN-5 containing only one K- or two K-segments are able to protect albeit to less extent than the wild type protein, lactate dehydrogenase and β-glucosidase against damage induced by various stresses in vitro. However, the YS- and Φ-segments alone have no protective effects on these enzymes. Therefore, our study provides the evidence that the protective function of DHN-5 seems to be directly linked to its K-segments which through their amphipatic α-helical structure, may act to prevent protein aggregation.     

Molecular characterization of larval anisakid nematodes from marine fishes off the Moroccan and Mauritanian coasts

A total of 242 larval forms of Anisakis collected from marine fishes at different sites off the Moroccan and Mauritanian coasts, recognised as belonging to Type I and Type II larvae, were identified by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms) of the ITS (Internal Transcribed Spacers) region (ITS-1, 5.8 subunit rRNA gene and ITS-2), using a previously established molecular key. The Type I larvae were found with a frequency of 98.34% and were identified as belonging to the following species: A. simplex s.str., A. pegreffii, A. simplex s.str/A. pegreffii heterozygote genotypes, A. typica, A. ziphidarum and Anisakis sp. A. The Type II larvae were found to belong to A. physeteris, with the frequency of 1.65%. The results reported in the present study provide further epizootiological and biological data on the Anisakis spp. in marine fishes off the Moroccan and Mauritanian coasts, improving the picture of the occurrence of these species in the central Atlantic coasts.     

Segmental vitiligo as the possible expression of cutaneous somatic mosaicism: implications for common non-segmental vitiligo

Clinical findings in vitiligo challenge the widely accepted organ specific autoimmune pathomechanisms. We draw the attention to the fact that the distribution of segmental vitiligo (SV) fits in at least a subset of patients a pattern usually associated with cutaneous mosaicism. The association of SV to non-segmental vitiligo (NSV) now confirmed by several observations indicates a continuum between the two subsets with shared predisposing genetic factors, including genes operating specifically in the skin. Some pedigrees associating SV and NSV further suggest a mechanism of loss of heterozygosity for a dominant gene controlling part of the cutaneous phenotype. The mosaic hypothesis applies only to SV and to the rare SV-NSV association, but suggests that predisposing genetic factors in common NSV should also be searched directly in the skin. SV would be a good candidate disease to explore as a proof of principle of a new gene discovery strategy useful for multigenic disorders with organ specificity, applicable in priority to chronic inflammatory skin disorders.     

Lactobacillus rhamnosus GG conditioned media modulates acute reactive oxygen species and nitric oxide in J774 murine macrophages

Phagocytes such as macrophages are capable of detecting and killing pathogenic bacteria by producing reactive oxygen and nitrogen species. Formation of free radicals in macrophages may be regulated by probiotics or by factors released by probiotics but yet to be identified. Thus, studies were carried out to determine whether cell-free conditioned medium obtained from cultures of Lactobacillus rhamnosus GG (LGG-CM) regulate production of reactive oxygen species (ROS) and/or nitric oxide (NO) in macrophages. J774 macrophages in culture were loaded with either H₂DCFDA for monitoring ROS or with DAFFM-DA for NO detection. Free radical production was measured on a fluorescence microplate reader and changes were analysed by Cumulative sum (CuSum) calculations. Low concentration of LGG-CM (10% LGG-CM) or LPS did not cause any significant change in basal levels of ROS or NO production. In contrast, high concentration of LGG-CM (75% and 100%) significantly enhanced ROS generation but also significantly reduced NO level. These findings are novel and suggest for the first time that probiotics may release factors in culture which enhance ROS production and may additionally reduce deleterious effects associated with excessive nitrogen species by suppressing NO level. These events may account, in part, for the beneficial bactericidal and anti-inflammatory actions ascribed to probiotics and may be of clinical relevance.     

Radiation dose verification of an X-ray based blood irradiator using EBT3 radiochromic films calibrated using Gamma Knife machine

AimBlood irradiators (BI) initial acceptance testing and routine annual dosimetry checks require radiation dose measurements in order to comply with regulatory requirements.BackgroundTraditionally thermo-luminescence dosimeters (TLD) have been used to measure the dose. The EBT3 film is reported to be a better dosimeter for low energy X-rays than its predecessors EBT2 and EBT. To the best of our knowledge, the use of EBT3 films to perform dosimetry on X-ray based BI has not been reported yet.Materials and methodsWe performed routine radiation dosimetry checks using EBT3 films on a new X-ray based BI and compared the results with TLD dosimetry. Calibration films were irradiated with radiation beam from a Co-60 Gamma Knife (GK) radiosurgery machine and, alternatively, using an Ir-192 high dose rate (HDR) brachytherapy device. The films were calibrated to cover a wide dose range from 1 to 40 Gy. Such a wide dose range has not been reported yet in BI film dosimetry.ResultsWe obtained a relative difference of about 6.6% between doses measured using TLD and those measured using EBT3 films. Both irradiation methods using GK or HDR were found to be adequate for the calibration of the EBT3 Gafchromic films.ConclusionsWe recommend the use of EBT3 films in routine X-ray based BI dosimetry checks. The presented method takes advantage of available radiotherapy equipment that can be efficiently used for EBT3 films calibration. The method is fast, reproducible and saves valuable medical physicist's time.     

Sestrin 2 and AMPK Connect Hyperglycemia to Nox4-Dependent Endothelial Nitric Oxide Synthase Uncoupling and Matrix Protein Expression

Mesangial matrix accumulation is an early feature of glomerular pathology in diabetes. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. Here, we demonstrate that, in glomerular mesangial cells (MCs), endothelial nitric oxide synthase (eNOS) is uncoupled upon exposure to high glucose (HG), with enhanced generation of reactive oxygen species (ROS) and decreased production of nitric oxide. Peroxynitrite mediates the effects of HG on eNOS dysfunction. HG upregulates Nox4 protein, and inhibition of Nox4 abrogates the increase in ROS and peroxynitrite generation, as well as the eNOS uncoupling triggered by HG, demonstrating that Nox4 functions upstream from eNOS. Importantly, this pathway contributes to HG-induced MC fibronectin accumulation. Nox4-mediated eNOS dysfunction was confirmed in glomeruli of a rat model of type 1 diabetes. Sestrin 2-dependent AMP-activated protein kinase (AMPK) activation attenuates HG-induced MC fibronectin synthesis through blockade of Nox4-dependent ROS and peroxynitrite generation, with subsequent eNOS uncoupling. We also find that HG negatively regulates sestrin 2 and AMPK, thereby promoting Nox4-mediated eNOS dysfunction and increased fibronectin. These data identify a protective function for sestrin 2/AMPK and potential targets for intervention to prevent fibrotic injury in diabetes.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.     

Synthesis and bioevaluation of N-(arylalkyl)-homospermidine conjugates

N1-(Arylalkyl)homospermidines (1c-1f) and terminally piperazine-substituted homospermidine conjugates (2a-2e) were synthesized and evaluated for cytotoxicity in mouse leukemia L1210, alpha-difluoromethylornithine (DFMO)-treated L1210, melanoma B16, spermidine (SPD)-treated B16, and HeLa cell lines. Results demonstrated that homospermidine was a more effective vector than piperazine-substituted homospermidine in ferrying diverse arenes into cells via the polyamine transporter. The leading compound, 9-anthracenemethyl-homospermidine (1a), was shown to induce apoptosis in B16 cells and IL-3 dependent FL5.12A pro-B cells. The novel conjugate 4-biphenylmethyl-homospermidine (1e) could also induce apoptosis. However, it exhibited different effect on the cell cycle of B16 cells compared to 1a.