Effect of ischemia on localization of heat shock protein 25 in kidney

Effect of ischemia on localization of heat shock protein 25 in kidney. The effects of renal ischemia on the intracellular distribution of the low-molecular weight heat shock protein (HSP)25 were examined using immunofluorescence microscopy. In all kidney zones, ischemia decreased HSP25 in the supernatant of the tissue homogenates and increased it in the pellet fraction (containing mainly nuclei and cytoskeletal components). This was associated with disappearance of HSP25 staining from the brush border of proximal convoluted tubule (PCT) cells. Because no nuclear staining of cortical tubule cells was apparent either in control or ischemic kidneys, ischemia seems to cause a closer association of HSP25 with cytoskeletal components. HSP25 probably participates in the postischemic restructuring of the cytoskeleton of PCT cells

CFTR mRNA and its truncated splice variant (TRN-CFTR) are differentially expressed during collecting duct ontogeny

AbstractThe collecting duct epithelium originates from the embryonic ureter by branching morphogenesis. Ontogeny-dependent changes of CFTR mRNA expression were assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in primary monolayer cultures of rat ureteric buds (UB) and cortical collecting ducts, microdissected at different embryonic and postnatal developmental stages. The amount of wild-type CFTR-specific PCR product in UB declined to 20% of the initial value between embryonic gestational day E15 and postnatal day P1. After birth the CFTR product increased transiently between P1 and P7 by a factor of 10 and decreased towards day P14. PCR products specific for TRN-CFTR, a truncated splice variant, however, were low in early embryonic cells, increased markedly between day E17 and P2, and reached a plateau postnatally. Therefore, mRNA encoding TRN-CFTR does not appear to have a specific embryonic-morphogenetic function. By contrast, such function is suggested for wild-type CFTR mRNA as its abundance was high in early embryonic nephrogenesis, as well as during a postnatal period shortly before branching morphogenesis is completed

Enzyme-functionalized biomimetic apatites: concept and perspectives in view of innovative medical approaches

Biomimetic nanocrystalline calcium-deficient apatite compounds are particularly attractive for the setup of bioactive bone-repair scaffolds due to their high similarity to bone mineral in terms of chemical composition, structural and substructural features. As such, along with the increasingly appealing development of moderate temperature engineered routes for sample processing, they have widened the armamentarium of orthopedic and maxillofacial surgeons in the field of bone tissue engineering. This was made possible by exploiting the exceptional surface reactivity of biomimetic apatite nanocrystals, capable of easily exchanging ions or adsorbing (bio)molecules, thus leading to highly-versatile drug delivery systems. In this contribution we focus on the preparation of hybrid materials combining biomimetic nanocrystalline apatites and enzymes (lysozyme and subtilisin). This paper reports physico-chemical data as well as cytotoxicity evaluations towards Cal-72 osteoblast-like cells and finally antimicrobial assessments towards selected strains of interest in bone surgery. Biomimetic apatite/enzyme hybrids could be prepared in varying buffers. They were found to be non-cytotoxic toward osteoblastic cells and the enzymes retained their biological activity (e.g. bond cleavage or antibacterial properties) despite the immobilization and drying processes. Release properties were also examined. Beyond these illustrative examples, the concept of biomimetic apatites functionalized with enzymes is thus shown to be useable in practice, e.g. for antimicrobial purposes, thus widening possible therapeutic perspectives

Human-based immune responsive in vitro infection models for validation of novel TLR4 antagonists identified by computational discovery

Infectious diseases are still a major problem worldwide. This includes microbial infections, with a constant increase in resistance to the current anti-infectives employed. Toll-like receptors (TLRs) perform a fundamental role in pathogen recognition and activation of the innate immune response. Promising new approaches to combat infections and inflammatory diseases involve modulation of the host immune system via TLR4. TLR4 and its co-receptors MD2 and CD14 are required for immune response to fungal and bacterial infection by recognition of microbial cell wall components, making it a prime target for drug development. To evaluate the efficacy of anti-infective compounds early on, we have developed a series of human-based immune responsive infection models, including immune responsive 3D-skin infection models for modeling fungal infections. By using computational methods: pharmacophore modeling and molecular docking, we identified a set of 46 potential modulators of TLR4, which were screened in several tests systems of increasing complexity, including immune responsive 3D-skin infection models. We could show a strong suppression of cytokine and chemokine response induced by lipopolysacharide (LPS) and Candida albicans for individual compounds. The development of human-based immune responsive assays provides a more accurate and reliable basis for development of new anti-inflammatory or immune-modulating drugs.Fraunhofer ICON grantInternational Cooperation and Networking grantYissu

Low-Energy Electron Irradiation Efficiently Inactivates the Gram-Negative Pathogen Rodentibacter pneumotropicus—A New Method for the Generation of Bacterial Vaccines with Increased Efficacy

Bacterial pathogens cause severe infections worldwide in livestock and in humans, and antibiotic resistance further increases the importance of prophylactic vaccines. Inactivated bacterial vaccines (bacterins) are usually produced via incubation of the pathogen with chemicals such as formaldehyde, which is time consuming and may cause loss of immunogenicity due to the modification of structural components. We evaluated low-energy electron irradiation (LEEI) as an alternative method to generate a bacterin. Rodentibacter pneumotropicus, an invasive Gram-negative murine pathogen, was inactivated with LEEI and formaldehyde. LEEI resulted in high antigen conservation, and LPS activity was significantly better maintained when compared with formaldehyde treatment. Immunization of mice with LEEI-inactivated R. pneumotropicus elicited a strong immune response with no detectable bacterial burden upon sublethal challenge. The results of this study suggest the inactivation of bacteria with LEEI as an alternative, fast and efficient method to generate bacterial vaccines with increased efficacy

An In Vitro HSV-1 Reactivation Model Containing Quiescently Infected PC12 Cells

Advances in the understanding of the infection and reactivation process of herpes simplex type 1 (HSV-1) are generally gained by monolayer cultures or extensive and cost-intensive animal models. So far, no reliable in vitro skin model exists either to investigate the molecular mechanisms involved in controlling latency and virus reactivation or to test pharmaceuticals. Here we demonstrate the first in vitro HSV-1 reactivation model generated by using the human keratinocyte cell line HaCaT grown on a collagen substrate containing primary human fibroblasts. We integrated the unique feature of a quiescently infected neuronal cell line, the rat pheochromocytoma line PC12, within the dermal layer of the three-dimensional skin equivalent. Transmission electron microscopy, a cell-based TCID50 assay, and polymerase chain reaction analysis were used to verify cell latency. Thereby viral DNA could be detected, whereas extracellular as well as intracellular virus activity could not be found. Further, the infected PC12 cells show no spontaneous reactivation within the in vitro skin equivalent. In order to simulate a physiologically comparable HSV-1 infection, we achieved a specific and pointed reactivation of quiescently HSV-1 infected PC12 cells by UVB irradiation at 1000 mJ/cm2

Binding of JAB1/CSN5 to MIF is mediated by the MPN domain but is in dependent of the JAMM motif

AbstractMacrophage migration inhibitory factor (MIF) binds to c-Jun activation domain binding protein-1 (JAB1)/subunit 5 of COP9 signalosome (CSN5) and modulates cell signaling and the cell cycle through JAB1. The binding domain of JAB1 responsible for binding to MIF is unknown. We hypothesized that the conserved Mpr1p Pad1p N-terminal (MPN) domain of JAB1 may mediate binding to MIF. In fact, yeast two hybrid (YTH) and in vitro translation/coimmunoprecipitation (CoIP) analysis showed that a core MPN domain, which did not cover the functional JAB1/MPN/Mov34 metalloenzyme (JAMM) deneddylase sequence, binds to MIF comparable to full-length JAB1. YTH and pull-down analysis in conjunction with nanobead affinity matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry demonstrated that MIF(50–65) and MPN are sufficient to mediate MIF–JAB1 interaction, respectively. Finally, endogenous CoIP of MIF–CSN6 complexes from mammalian cells demonstrated that MPN is responsible for MIF–JAB1 binding in vivo, and, as CSN6 does not contain a functional JAMM motif, confirmed that the interaction does not require JAMM

Phenomenological investigation of the cytotoxic activity of fucoidan isolated from Fucus vesiculosus

The development of natural-based anti-tumor medicaments has acquired a great interest especially in the last few decades. Hence, cytotoxic activity of different fractions of fucoidan was evaluated. The fractions, produced from the total crude extract of the brown alga Fucus vesiculosus and purified by the recently-developed immobilized cationic dyes at different conditions, had different physicochemical properties and named fucoidan_1, fucoidan_6 and fucoidan_PDD. The activity of these fractions was studied in vitro against different kinds of cancerous mammalian cell lines including MCF-7 and Caco-2 and compared to their effects against skin primary fibroblasts. The results indicated a potent cytotoxic activity with regard to MCF-7 cells, while negligible (>1500 μg mL−1) towards primary fibroblasts. Moreover, higher general toxicity of crude fucoidan indicated that purification process succeeded to remove extraneous, co-extracted, cytotoxic compounds (e.g., polyphenols), which has a strong activity and possible interference in previously-published studies. Furthermore, a correlation was made between the cytotoxic activity and physico-chemical properties of fucoidan fractions, such as the sulfation degree and molecular weight. These findings reflected a real picture and expected low side effects regarding the cytotoxic activity of fucoidan purified by affinity chromatography

Characterization and Cytotoxic Activity of Microwave-Assisted Extracted Crude Fucoidans from Different Brown Seaweeds

Microwave-assisted extraction (MAE) is recognized as a green method for extraction of natural products. The current research aimed to explore the MAE for fucoidans extraction from different brown seaweeds, including Fucus vesiculosus, F. spiralis, and Laminaria saccharina. Following several solvent-extraction pre-treatment steps and MAE optimization, the algal biomasses were extracted in a ratio of 1:25 in 0.1 M HCl containing 2 M CaCl2 for 1.0 min. The results showed that L. saccharina’s extract was different from the others, regarding the highest sugar content reached 0.47 mg glucose equivalent/mg extract being confirmed by monosaccharide composition analysis and the lowest fucoidan content and sulfation degree at 0.09 mg/mg extract and 0.13, respectively. Moreover, these findings were confirmed by tentative structural elucidation based on Fourier-transform infrared spectrometry which also showed a different spectrum. However, the MAE enhanced melanoidins formation in products, which was confirmed by the intense band at 1420 cm−1. Interestingly, the results of monomeric composition showed that fucoidan extract by MAE from F. vesiculosus belonged to sulfated galactofucans which are known for their potential bioactivities. Furthermore, the cytotoxic activity of the four fucoidans in concentrations ranging from 4.9 µg/mL to 2500 µg/mL was investigated and correlated with the chemical characterization showing that F. vesiculosus_MAE fucoidan was the most potent and safest. The current research revealed the chemical heterogeneity of fucoidans regarding taxonomical class and used greener extraction method of fucoidans toward the achievement of the UN sustainability goals