Effect of methionine feeding on oxidative stress, intracellular calcium and contractility in cardiomyocytes isolated from male and female rats

Homocysteine (Hcy) is a breakdown product of methionine metabolism. The risk of cardiovascular disease (CVD) correlates with an increase in plasma Hcy levels. The aim of this study was to investigate whether 1% methionine supplementation of adult rats altered intracellular reactive oxygen species (ROS) generation, intracellular Ca2+ content, and contractile activity in freshly isolated cardiomyocytes. This was measured under normal conditions and during oxidative stress in freshly isolated cardiomyocytes. Single rat cardiomyocytes from both sexes were isolated by enzymatic and mechanical dispersion techniques. Fluorescence microscopy was used to measure ROS production and intracellular Ca2+ concentration. Cell contraction was measured using a video camera. During exposure to 200 μM, H2O2 female cardiomyocytes produced significantly fewer ROS and had a higher intracellular Ca2+ concentration compared to male cardiomyocytes in control and methionine-fed conditions. The contractility of cardiomyocytes isolated from male rats was insignificantly decreased after methionine feeding compared to control, while the contractility of cardiomyocytes from female rats insignificantly reduced after methionine feeding and acute exposure to oxidative stress. These findings provide evidence that during exposure to 200 μM H2O2, cardiomyocytes from female rats produce less ROS and have higher intracellular Ca2+ levels. There were no significant effects on contractility in cardiomyocytes from either gender and under any of the different conditions

Effect of ageing and hypertension on the expression and activity of PEPT2 in normal and hypertrophic hearts

Some dipeptides have been implicated in myocardial protection, but little is known about their membrane transporter PEPT2. The aim of this study was to determine whether the expression and activity of the cardiac-type PEPT2 cotransporter could be affected by ageing and/or hypertension. Sarcolemmal vesicles (SV) were isolated from the hearts of all rat groups using a standard procedure to investigate the transport activity and protein abundance by fluorescence spectroscopy and Western blot, respectively. SLC15A2 "PEPT2" gene expression was relatively quantified by RT-qPCR. In the Wistar rat groups, the protein and gene expression of PEPT2 were upregulated with ageing. These changes were accompanied by corresponding increases in the competitive inhibition and the transport rate (Vmax) of β-Ala-Lys (AMCA) into SV isolated from middle-aged hearts. Although, the transport rate of β-Ala-Lys (AMCA) into SV isolated from old hearts was significantly the lowest compared to middle-aged and young adult hearts, the inhibition percentage of β-Ala-Lys (AMCA) transport by Gly-Gln was the highest. In the WKY and SHR rat groups, Y-SHR hypertrophied hearts showed an increase in PEPT2 gene expression accompanied by a significant decrease in protein expression and activity. With advanced age, however, M-SHR hypertrophied hearts revealed significantly lower gene expression, but higher protein expression and activity than Y-SHR hearts. These findings suggest that increased expression of PEPT2 cotransporter in all types of middle-aged hearts could be exploited to facilitate di-and tripeptide transport by PEPT2 in these hearts, which subsequently could result in improved myocardial protection in these populations

Hypertension alters the function and expression profile of the peptide cotransporters PEPT1 and PEPT2 in the rodent renal proximal tubule

Hypertension is a major risk factor for kidney and cardiovascular disease. The treatment of hypertensive individuals by selected ACE inhibitors and certain di-and tripeptides halts the progression of renal deterioration and extends life-span. Renal reabsorption of these low molecular weight substrates are mediated by the PEPT1 and PEPT2 cotransporters. This study aims to investigate whether hypertension and ageing affects renal PEPT cotransporters at gene, protein expression and distribution as well as function in the superficial cortex and the outer medulla of the kidney. Membrane vesicles from the brush border (BBMV) and outer medulla (OMMV) were isolated from the kidneys of young Wistar Kyoto (Y-WKY), young spontaneously hypertensive (Y-SHR), and middle aged SHR (M-SHR) rats. Transport activity was measured using the substrate, β-Ala-Lys (AMCA). Gene expression levels of PEPT genes were assessed with qRT-PCR while renal localisation of PEPT cotransporters was examined by immunohistochemistry with Western Blot validation. The Km and Vmax of renal PEPT1 were decreased significantly in SHR compared to WKY BBMV, whilst the Vmax of PEPT2 showed differences between SHR and WKY. By contrast to the reported cortical distribution of PEPT1, PEPT1-staining was detected in the outer medulla, whilst PEPT2 was expressed primarily in the cortex of all SHR; PEPT1 was significantly upregulated in the cortex of Y-SHR. These outcomes are indicative of a redistribution of PEPT1 and PEPT2 in the kidney proximal tubule under hypertensive conditions that has potential repercussions for nutrient handling and the therapeutic use of ACE inhibitors in hypertensive individuals

The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex.

The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a chromatin body with unique biochemical activities at the inner centromere

The evolution of multiple active site configurations in a designed enzyme

Developments in computational chemistry, bioinformatics, and laboratory evolution have facilitated the de novo design and catalytic optimization of enzymes. Besides creating useful catalysts, the generation and iterative improvement of designed enzymes can provide valuable insight into the interplay between the many phenomena that have been suggested to contribute to catalysis. In this work, we follow changes in conformational sampling, electrostatic preorganization, and quantum tunneling along the evolutionary trajectory of a designed Kemp eliminase. We observe that in the Kemp Eliminase KE07, instability of the designed active site leads to the emergence of two additional active site configurations. Evolutionary conformational selection then gradually stabilizes the most efficient configuration, leading to an improved enzyme. This work exemplifies the link between conformational plasticity and evolvability and demonstrates that residues remote from the active sites of enzymes play crucial roles in controlling and shaping the active site for efficient catalysis

A new use of β-Ala-Lys (AMCA) as a transport reporter for PEPT1 and PEPT2 in renal brush border membrane vesicles from the outer cortex and outer medulla.

Integral membrane proteins PEPT1 and PEPT2 are essential for reabsorbing almost all hydrolysed or filtered di- and tripeptides alongside a wide range of peptidomimetic drugs in the kidney. The aim of this study was to investigate the potential use of the fluorophore-conjugated dipeptide β-Ala-Lys (AMCA) as a biosensor for measuring peptide transport activity in brush border membrane vesicles isolated from the outer cortex (BBMV-OC) and outer medulla (BBMV-OM) (representing PEPT1 and PEPT2 respectively). The vesicles were isolated using a dual magnesium precipitation and centrifugation technique. Intravesicular fluorescence accumulation was measured after incubating extra-vesicular media at pH6.6 and different concentrations of β-Ala-Lys (AMCA) with vesicles pre-equilibrated at pH7.4. Both BBMV-OC and BMMV-OM showed accumulation of an intravesicular fluorescence signal after 20min incubation. Changing the extra-vesicular pH to 7.4 caused a significant reduction in the β-Ala-Lys (AMCA) uptake into BBMV-OC at concentrations >100μM. When different concentrations of dipeptide, Gly-Gln was added, there was a significant inhibition of 100μM β-Ala-Lys (AMCA) uptake into BBMV-OC and BMMV-OM, reaching 69% and 80%, respectively. Kinetic analysis of β-Ala-Lys (AMCA) at 20min showed that the Kmand Vmaxwere 783.7±115.7μM and 2191.2±133.9ΔF/min/mg for BBMV-OC, while BMMV-OM showed significantly higher affinity, but lower capacity at Km=93.6±21.9μM and Vmax=935.8±50.2ΔF/min/mg. These findings demonstrate the applicability of β-Ala-Lys (AMCA) as a biosensor to measure the transport activity of the renal-type PEPT1 and PEPT2 in BBMV-OC and BMMV-OM respectively

Towards a better control of the wastewater treatment process: excitation-emission matrix fluorescence spectroscopy of dissolved organic matter as a predictive tool of soluble BOD5 in influents of six Parisian wastewater treatment plants

The online monitoring of dissolved organic matter (DOM) in raw sewage water is expected to better control wastewater treatment processes. Fluorescence spectroscopy offers one possibility for both the online and real-time monitoring of DOM, especially as regards the DOM biodegradability assessment. In this study, three-dimensional fluorescence spectroscopy combined with a parallel factor analysis (PARAFAC) has been investigated as a predictive tool of the soluble biological oxygen demand in 5 days (BOD5) for raw sewage water. Six PARAFAC components were highlighted in 69 raw sewage water samples: C2, C5, and C6 related to humic-like compounds, along with C1, C3, and C4 related to protein-like compounds. Since the PARAFAC methodology is not available for online monitoring, a peak-picking approach based on maximum excitation-emission (Ex-Em) localization of the PARAFAC components identified in this study has been used. A good predictive model of soluble BOD5 using fluorescence spectroscopy parameters was obtained (r (2) = 0.846, adjusted r (2) = 0.839, p < 0.0001). This model is quite straightforward, easy to automate, and applicable to the operational field of wastewater treatment for online monitoring purposes