Ameliorative Effect of Fisetin on Cisplatin-Induced Nephrotoxicity in Rats via Modulation of NF-κB Activation and Antioxidant Defence

Nephrotoxicity is a dose-dependent side effect of cisplatin limiting its clinical usage in the field of cancer chemotherapy. Fisetin is a bioactive flavonoid with recognized antioxidant and anti-inflammatory properties. In the present study, we investigated the potential renoprotective effect and underlying mechanism of fisetin using rat model of cisplatin-induced nephrotoxicity. The elevation in serum biomarkers of renal damage (blood urea nitrogen and creatinine); degree of histopathological alterations and oxidative stress were significantly restored towards normal in fisetin treated, cisplatin challenged animals. Fisetin treatment also significantly attenuated the cisplatin-induced IκBα degradation and phosphorylation and blocked the NF-κB (p65) nuclear translocation, with subsequent elevation of pro-inflammatory cytokine, TNF-α, protein expression of iNOS and myeloperoxidase activities. Furthermore, fisetin markedly attenuated the translocation of cytochrome c protein from the mitochondria to the cytosol; decreased the expression of pro-apoptotic proteins including Bax, cleaved caspase-3, cleaved caspase-9 and p53; and prevented the decline of anti-apoptotic protein, Bcl-2. The cisplatin-induced mRNA expression of NOX2/gp91phox and NOX4/RENOX and the NADPH oxidase enzyme activity were also significantly lowered by fisetin treatment. Moreover, the evaluated mitochondrial respiratory enzyme activities and mitochondrial antioxidants were restored by fisetin treatment. Estimation of platinum concentration in kidney tissues revealed that fisetin treatment along with cisplatin did not alter the cisplatin uptake in kidney tissues. In conclusion, these findings suggest that fisetin may be used as a promising adjunct candidate for cisplatin use.     

Low plasma albumin levels are associated with increased plasma protein glycation and HbA1c in diabetes

Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.     

Inhibition by diethylstilboestrol of proliferative potential of follicles of different sizes in immature rat ovaries

Intact immature female rats were treated with 1, 2, 3 or 4 subcutaneous injections of 2 mg diethylstilboestrol (DES)/rat at intervals of 24 h and then killed. Ovaries were collected, cleaned, enzymically digested and serially filtered through Teflon sieves to yield follicles of diameter less than 200 microns (small), 200-400 microns (medium) and greater than 400 microns (large). Follicular supernatant was collected and granulosa cells were extracted from these isolated follicles. There was a general increase in [3H]thymidine incorporation in all sizes of follicles after 1 or 2 DES injections, the increase in the medium and large follicles being significant after 2 doses. With 3 and 4 injections of DES, there was a sudden decrease in the rates of [3H]thymidine incorporation, particularly in the medium-sized follicles, which also had higher concentrations of follicular supernatant protein. Protein contents in small and large follicles did not change significantly. The follicular supernatant protein had a specific and dose-dependent inhibitory effect on [3H]thymidine incorporation when added to cultures of rapidly dividing granulosa cells. Addition of the same amounts of bovine serum albumin (BSA) to the cultures had no effect. Heat-denaturing did not abolish the inhibition by the protein. Removal of the protein from the cultures after the first 48 h resulted in a rebound increase in [3H]thymidine incorporation during the following 48 h, showing that the inhibitory effects were reversible. Though aromatase activity after 1 or 2 DES injections abruptly decreased after 3 and 4 injections, follicular supernatant protein had no effect on steroidogenesis in cultured granulosa cells. Taken together, these findings suggest that oestrogen can inhibit follicular development, depending on the duration of exposure. We propose that the inhibitory effects of DES on cell proliferation are mediated via the synthesis of a specific peptide factor which is produced in high amounts in the medium-sized follicles only, on prolonged exposure to the oestrogen. This factor may be autocrine or paracrine, serving as an in-built autoregulatory control mechanism for follicle development, particularly at pro-oestrus, when oestrogen concentrations are highest.     

Decoding the trans-histone crosstalk: methods to analyze H2B ubiquitination, H3 methylation and their regulatory factors

Regulation of histone H3 lysine 4 and 79 methylation by histone H2B lysine 123 monoubiquitination is an evolutionarily conserved trans-histone crosstalk mechanism, which demonstrates a functional role for histone ubiquitination within the cell. The regulatory enzymes, factors and processes involved in the establishment and dynamic modulation of these modifications and their genome-wide distribution patterns have been determined in many model systems. Rapid progress in understanding this trans-histone crosstalk has been made using the standard experimental tools of chromatin biology in budding yeast (Saccharomyces cerevisiae), a highly tractable model organism. Here, we provide a set of modified and refined experimental procedures that can be used to gain further insights into the underlying mechanisms that govern this crosstalk in budding yeast. Importantly, the improved procedures and their underlying principles can also be applied to other model organisms. Methods presented here provide a rapid and efficient means to prepare enriched protein extracts to better preserve and assess the steady state levels of histones, non-histone proteins and their modifications. Improved chromatin immunoprecipitation and double immunoprecipitation protocols are provided to measure the occupancy and distribution of proteins and their modified forms at specific chromatin regions or loci. A quick and easy method to measure overall protein abundance and changes in protein-protein and protein-DNA interactions on native chromatin is also described.     

Comparative Genomic Analysis of Buffalo (Bubalus bubalis) NOD1 and NOD2 Receptors and Their Functional Role in In-Vitro Cellular Immune Response

Nucleotide binding and oligomerization domain (NOD)-like receptors (NLRs) are innate immune receptors that recognize bacterial cell wall components and initiate host immune response. Structure and function of NLRs have been well studied in human and mice, but little information exists on genetic composition and role of these receptors in innate immune system of water buffalo—a species known for its exceptional disease resistance. Here, a comparative study on the functional domains of NOD1 and NOD2 was performed across different species. The NOD mediated in-vitro cellular responses were studied in buffalo peripheral blood mononuclear cells, resident macrophages, mammary epithelial, and fibroblast cells. Buffalo NOD1 (buNOD1) and buNOD2 showed conserved domain architectures as found in other mammals. The domains of buNOD1 and buNOD2 showed analogy in secondary and tertiary conformations. Constitutive expressions of NODs were ubiquitous in different tissues. Following treatment with NOD agonists, peripheral lymphocytes showed an IFN-γ response along-with production of pro-inflammatory cytokines. Alveolar macrophages and mammary epithelial cells showed NOD mediated in-vitro immune response through NF-κB dependent pathway. Fibroblasts showed pro-inflammatory cytokine response following agonist treatment. Our study demonstrates that both immune and non-immune cells could generate NOD-mediated responses to pathogens though the type and magnitude of response depend on the cell types. The structural basis of ligand recognition by buffalo NODs and knowledge of immune response by different cell types could be useful for development of non-infective innate immune modulators and next generation anti-inflammatory compounds.     

Free Fatty Acid Effects on the Atrial Myocardium: Membrane Ionic Currents Are Remodeled by the Disruption of T-Tubular Architecture

Background

Epicardial adiposity and plasma levels of free fatty acids (FFAs) are elevated in atrial fibrillation, heart failure and obesity, with potentially detrimental effects on myocardial function. As major components of epicardial fat, FFAs may be abnormally regulated, with a potential to detrimentally modulate electro-mechanical function. The cellular mechanisms underlying such effects of FFAs are unknown.

Objective

To determine the mechanisms underlying electrophysiological effects of palmitic (PA), stearic (SA) and oleic (OA) FFAs on sheep atrial myocytes.

Methods

We used electrophysiological techniques, numerical simulations, biochemistry and optical imaging to examine the effects of acutely (≤ 15 min), short-term (4–6 hour) or 24-hour application of individual FFAs (10 μM) on isolated ovine left atrial myocytes (LAMs).

Results

Acute and short-term incubation in FFAs resulted in no differences in passive or active properties of isolated left atrial myocytes (LAMs). 24-hour application had differential effects depending on the FFA. PA did not affect cellular passive properties but shortened (p<0.05) action potential duration at 30% repolarization (APD₃₀). APD₅₀ and APD₈₀ were unchanged. SA had no effect on resting membrane potential but reduced membrane capacitance by 15% (p<0.05), and abbreviated APD at all values measured (p≤0.001). OA did not significantly affect passive or active properties of LAMs. Measurement of the major voltage-gated ion channels in SA treated LAMs showed a ~60% reduction (p<0.01) of the L-type calcium current (I_Ca-L) and ~30% reduction (p<0.05) in the transient outward potassium current (I_TO). A human atrial cell model recapitulated SA effects on APD. Optical imaging showed that SA incubated for 24 hours altered t-tubular structure in isolated cells (p<0.0001).

Conclusions

SA disrupts t-tubular architecture and remodels properties of membrane ionic currents in sheep atrial myocytes, with potential implications in arrhythmogenesis.     

Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare's serum gonadotropin-primed immature rat and the adult rat

The role of progesterone in the regulation of the preovulatory surge in gonadotropins and ovulation was examined in this study by use of a potent antagonist of progesterone, RU 486 (17 beta-hydroxy-11 beta-[4-dimethyl-aminophenyl]-17 alpha- [prop-1-ynyl]estra-4,9-diene-3-one). The immature rat primed with pregnant mare's serum gonadotropin (PMSG) and the cycling adult animal were the models used to verify the role of progesterone. When RU 486 (200 micrograms/rat) was given as a single dose on the morning of proestrus, there was a significant reduction in the preovulatory surge levels of gonadotropins and ovulation in both animal models. Serum progesterone levels in both models at the time of death on the evening of proestrus were unaltered upon treatment with RU 486. RU 486 did not have any effect on gonadotropin levels in immature rats 7 days after castration. These results show that the actin of RU 486 on the preovulatory gonadotropin surge is due to an antagonism of the action of progesterone on the hypothalamic-pituitary axis. Thus, a role for progesterone in modulating the preovulatory surge of gonadotropins and, consequently, ovulation is strongly suggested.     

Novel approaches to manipulating foetal cells in the maternal circulation for non-invasive prenatal diagnosis of the unborn child

Due to the risks to the foetus with invasive prenatal diagnosis, non-invasive prenatal diagnosis (NIPD) is gaining tremendous interest but no reliable method that can be widely used has been developed to date. Manipulation of foetal cells and foetal cell-free genetic material in the maternal blood are two promising approaches being researched. The manipulation of foetal cells in the maternal circulation is more popular as it can provide complete genetic information of the foetus particularly the diagnosis of aneuploidies. However, the foetal cell numbers in the maternal circulation are small and their enrichment and ex vivo culture remain two major challenges for NIPD. Primitive foetal erythroblasts (pFEs) have been considered as a good potential candidate for early first trimester NIPD but their nature, properties and manipulation to provide adequate cell numbers remain a challenging task and several approaches need to be meticulously evaluated. In this review we describe the current status of NIPD and suggest some novel approaches in manipulating pFEs for future clinical application of NIPD. These novel approaches include (1) understanding the pFE enucleation process, (2) enriching pFE numbers by individual pick-up of pFEs from maternal blood using micromanipulation and microdroplet culture, (3) expansion of pFEs using mitogens and (4) decondensation of the pFE nucleus with histone deacetylase (HDAC) inhibitors followed by reprogramming using gene delivery protocols with/without small reprogramming molecules to improve reprogrammed pFE proliferation rates for successful NIPD.     

Regulation of estrogen receptor protein and messenger ribonucleic acid by estradiol and progesterone in rat uterus

The objective of this study was to examine the mechanisms of estrogen receptor (ER) processing and replenishment in the uterus of ovariectomized rats after estradiol and progesterone treatment. Uterine ER binding activity, ER protein and ER mRNA were measured by receptor binding exchange assay, Western blot and slot blot, respectively. The regulation of ER levels in rat uterus by estradiol and progesterone was very dramatic. Changes in ER protein were faithfully reflected by changes in binding activity. Estradiol caused receptor “processing” within 4 h of administration followed by recovery or “replenishment” of ER levels to the initial level by 20 h. The term “processing” has previously been used to describe the loss of ER binding activity in the early phase of estradiol-action, but it was never clear whether the ligand binding site was inactivated by processing or if the receptor molecule actually disappeared. This study shows that receptor “processing” constitutes disappearance of receptor protein and the later “replenishment” phase represents new ER protein rather than recycling of “processed” receptor. Progesterone-action, on the other hand, influenced only the “replenishment” phase by blocking recovery of ER protein. ER mRNA was suppressed by estradiol at 8 h, after the receptor was “processed” and “replenishment” already initiated. Progesterone, on the other hand, did not alter the steady state level of the message. Other mechanisms, such as regulation of translation rate of existing mRNA and changes in the rate of degradation of ER proteins are more likely involved in acute regulation of ER by these ovarian steroid hormones.     

Influence of bioprocess variables on the production of extracellular chitinase under submerged fermentation by Streptomyces pratensis strain KLSL55

Chitinases are the enzymes which are capable of hydrolyzing chitin to its monomer N-acetyl glucosamine (GlcNac). Present study emphasizes on the impact of critical process variables on the production of chitinase from Streptomyces pratensis strain KLSL55. Initially the isolate was noticed to produce 84.67?IU chitinase in basal production medium. At optimization of bioprocess variables, the physical parameters pH of 8.00, 40?°C of incubation temperature, agitation speed of 160?rpm and 1.25?mL of spore suspension were found optimum for improved production of chitinase. Further, formulated production medium with 1.5% colloidal chitin, 1.25% fructose greatly influenced the chitinase production. At all described optimum conditions with formulated production media, a total of 14.30-fold increment was achieved in the chitinase production with final activity of 1210.67?IU when compared to the initial fermentation conditions in basal production medium.