Gastropathy and defense mechanisms in common bile duct ligated portal hypertensive rats

Portal hypertensive gastropathy is associated with a broad spectrum of gastric mucosal damage inspite of decreased gastric acid secretion, suggestive of compromised endogenous protective mechanisms. To determine the mechanisms of damage in portal hypertensive gastropathy we measured lipid peroxidation, glutathione, antioxidant and lysosomal enzymes in gastric mucosal homogenates from male Wistar rats with elevated intrasplenic pulp pressure, eighteen days after common bile duct ligation. Thiobarbituric acid-reactive substances and lysosomal enzymes (-glucuronidase and acid phosphatase) were increased in the common bile duct ligated group as compared to the sham-operated group. The levels of antioxidant defense enzymes, superoxide dismutase, glutathione peroxidase, catalase and glutathione were decreased as compared to the sham-operated controls. Pre-operative vitamin E administration decreased mucosal lipid peroxidation increased the levels of antioxidant defense enzymes and lowered the lysosomal enzymes. The plasma vitamin E levels in this group were lower when compared to animals receiving it post-operatively. In conclusion, free radical and lysosomal enzyme mediated damage may play a role in portal hypertensive gastropathy.     

Biosynthesis and therapeutic implications of iridoid glycosides from <Emphasis Type="Italic">Picrorhiza</Emphasis> genus: the road ahead

Picrorhiza genus is emerging as an important paradigm for herbal drug formulations due to its versatile iridoid glycosides exhibition and robustness in the treatment of diverse infections including hepatic amoebiasis, cancer, malaria, ulcerative colitis and cerebral ischemia reperfusion injury. Owing to the superiority of these bioactivities, iridoid glycosides from Picrorhiza have become a hot research area over the years. A metabolic pathway for the formation of iridoid glycosides has been proposed. However, some enzymes and genes of this route are still unidentified and demand the enumeration of facilitating pathways contributing to the biosynthesis of iridoid glycosides. This review summarizes the current knowledge of all naturally occurring iridoid glycosides from Picrorhiza, their biosynthesis and pharmacological capabilities which could provide the insight into metabolic regulation and the basis for the development of new drugs.     

Preventive and curative effects of Achyranthes aspera Linn. extract in experimentally induced nephrolithiasis

The present study was undertaken to evaluate the efficacy of Achyranthes aspera in preventing and reducing the growth of calcium oxalate stones in ethylene glycol induced nephrolithiatic model. Hyperoxaluria was induced in rats using ethylene glycol (EG, 0.4%) and ammonium chloride (1%) for 15 days and was then replaced with EG (0.4%) only. Upon administration of cystone (750 mg/kg body wt.), aqueous extract of A. aspera (500 and 1000 mg/kg body wt.), levels of renal injury markers (lactate dehydrogenase and alkaline phosphatase) were normalized with a decrease in serum urea and serum creatinine. Concurrent treatment reduced changes in the architecture of renal tissue and also decreased the size of crystals thereby helping in quick expulsion of the crystals. The present results indicated that Achyranthes aspera had an ability to maintain renal functioning and reduced renal injury.     

Peeping into Human Renal Calcium Oxalate Stone Matrix: Characterization of Novel Proteins Involved in the Intricate Mechanism of Urolithiasis

Background

The increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal–membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure.

Methods

Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin–Darby Canine Kidney (MDCK) renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS) followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated.

Results

Five proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level.

Conclusions

We identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone formation. Thus, these proteins having potential to modulate calcium oxalate crystallization will throw light on understanding and controlling urolithiasis in humans.     

Interaction of carboxyl-terminal peptides of cytosolic-tail of apactin with PDZ domains of NHERF/EBP50 and PDZK-1/CAP70

The C-terminal PDZ-binding motifs are required for polarized apical/basolateral localization of many membrane proteins. Ezrin–radixin–moesin (ERM) proteins regulate the organization and function of specific cortical structures in polarized epithelial cells by connecting filamentous (F)-actin to plasma membrane proteins through EBP50. Previous work showed that the membrane phosphoprotein apactin (an 80-kDa type I membrane protein derived from pro-Muclin) is associated with the acinar cell apical actin cytoskeleton and that this association is modulated by changes in the phosphorylation state of the apactin cytosolic tail. The carboxyl-terminal amino acids of apactin (–STKL–COOH) are predicted to form a type I PDZ-binding domain, similar to that of CFTR (–DTRL–COOH). Pairwise sequence comparison between NHERF/EBP50 and PDZK1/CAP70 PDZ domains reveals significant identity among the 83 amino-acid residues (12–92) of EBP50 and CAP70 (241–323), which are involved in the interaction with the carboxyl-terminal peptides (STKL–COOH and phosphomimetics) of apactin. Hence, the specificity and affinity of interactions are identical between them, which is corroborated with the two hybrid results. Substitution of all the four-carboxyl-terminal amino acids in the wild type to Ala reduces the interaction. Only the carbonyl oxygen and amide nitrogen of Ala are found to be involved in hydrogen bonding. Further, truncation of the wild carboxyl-terminal peptide to RGQPP–COOH, showed very low affinity of interaction with PDZ1 domain. Only the atom O^ε1 of Gln-2 hydrogen bonds with N^ε2 of His72 of PDZ domain. Ser-3 amino acid in wild type apactin protein (STKL–COOH) is not involved in hydrogen bonding with PDZ1 domain. However, substitution of Ser-3 to Asp-3 in PDTKL–COOH peptide increases the affinity of interaction of PDTKL–COOH with PDZ1 domain. Thus, carboxyl-terminal Asp(D) -3, Thr(T) -2, Lys(K) -1 and Leu(L) 0 are involved in numerous interactions with PDZ1 domains of NHERF/EBP50 and PDZK1/CAP70.     

N-acetylcysteine with apocynin prevents hyperoxaluria-induced mitochondrial protein perturbations in nephrolithiasis

Diminished mitochondrial activities were deemed to play an imperative role in surged oxidative damage perceived in hyperoxaluric renal tissue. Proteomics is particularly valuable to delineate the damaging effects of oxidative stress on mitochondrial proteins. The present study was designed to apply large-scale proteomics to describe systematically how mitochondrial proteins/pathways govern the renal damage and calcium oxalate crystal adhesion in hyperoxaluria. Furthermore, the potential beneficial effects of combinatorial therapy with N-acetylcysteine (NAC) and apocynin were studied to establish its credibility in the modulation of hyperoxaluria-induced alterations in mitochondrial proteins. In an experimental setup with male Wistar rats, five groups were designed for 9?d. At the end of the experiment, 24-h urine was collected and rats were euthanized. Urinary samples were analyzed for kidney injury marker and creatinine clearance. Transmission electron microscopy revealed distorted renal mitochondria in hyperoxaluria but combinatorial therapy restored the normal mitochondrial architecture. Mitochondria were isolated from renal tissue of experimental rats, and mitochondrial membrane potential was analyzed. The two-dimensional electrophoresis (2-DE) based comparative proteomic analysis was performed on proteins isolated from renal mitochondria. The results revealed eight differentially expressed mitochondrial proteins in hyperoxaluric rats, which were identified by Matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) analysis. Identified proteins including those involved in important mitochondrial processes, e.g. antioxidant defense, energy metabolism, and electron transport chain. Therapeutic administration of NAC with apocynin significantly expunged hyperoxaluria-induced discrepancy in the renal mitochondrial proteins, bringing them closer to the controls. The results provide insights to further understand the underlying mechanisms in the development of hyperoxaluria-induced nephrolithiasis and the therapeutic relevance of the combinatorial therapy.     

A Novel Antilithiatic Protein from Tribulus terrestris Having Cytoprotective Potency

Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis.     

Stem cell therapy: A paradigm shift in breast cancer treatment

As per the latest Globocan statistics, the high prevalence rate of breast cancer in low- and middle-income countries has led to it becoming the most common cancer to be diagnosed, hence posing a major public health challenge. As per this data, more than 11.7% of the estimated new cancer cases in 2020 were due to breast cancer. A small but significant subpopulation of cells with self- renewing ability are present in the tumor stroma and have been given the nomenclature of cancer stem cells (CSCs). These cells display a high degree of plasticity owing to their ability to transition from the slowly cycling quiescent phase to the actively proliferating phenotype. This attribute of CSCs allows them to differentiate into various cell types having diverse functions. Breast CSCs have a pivotal role in development, metastasis, treatment resistance and relapse of breast cancers. This review focuses on the pathways regulating breast CSC maintenance and the current strategies that are being explored for directing the development of novel, targeted, therapeutic approaches for limiting and eradicating this aberrant stem cell population.     

Tracking dynamics of enzyme activities and their gene expression in <Emphasis Type="Italic">Picrorhiza kurroa</Emphasis> with respect to picroside accumulation

Picrosides, the terpenoids synthesized by Picrorhiza kurroa, have ample usage in medicine. Identification of the regulatory enzymes involved in picroside biosynthesis needs to be explored for improving the level of these secondary metabolites. Current efforts are based on the analysis of secondary metabolism in picroside biosynthesis but its interpretation is limited by the lack of information on the involvement of primary metabolic pathways. The present study investigated the connection of primary metabolic enzymes with the picrosides levels in P. kurroa. The results showed changes in the catalytic activities as well as in the gene expression profiles of hexokinase, pyruvate kinase, isocitrate dehydrogenase, malate dehydrogenase, and NADP⁺-malic enzyme in congruence with picroside-I content under different conditions of P. kurroa growth, which indicates the role of these enzymes in the accumulation of picrosides. The significant correlation coefficients (p?<?0.05) observed between gene expression and enzyme activity underline the role of integrative studies for a better understanding of connecting links between metabolic pathways leading to picroside biosynthesis. This is apparently the first report on the involvement of glycolytic and TCA cycle enzymes in the accumulation of picrosides in P. kurroa.     

Apactin is involved in remodeling of the actin cytoskeleton during regulated exocytosis

Apactin is an 80-kDa type I membrane glycoprotein derived from pro-Muclin, a precursor that also gives rise to the zymogen granule protein Muclin. Previous work showed that apactin is efficiently removed from the regulated secretory pathway and targeted to the actin-rich apical plasma membrane of the pancreatic acinar cell. The cytosolic tail (C-Tail) of apactin consists of 16 amino acids, has Thr casein kinase II and Ser protein kinase C phosphorylation sites, and a C-terminal PDZ-binding domain. Secretory stimulation of acinar cells causes a decrease in Thr phosphorylation and an increase in Ser phosphorylation of apactin. Fusion peptides of the C-Tail domain pulldown actin, ezrin, and EBP50/NHERF in a phosphorylation-dependent manner. HIV TAT-C-Tail fusion peptides were used as dominant negative constructs on living pancreatic cells to study effects on the actin cytoskeleton. During secretory stimulation, TAT-C-Tail-Thr/Asp phosphomimetic peptide caused an increase in actin-coated zymogen granules at the apical surface, while TAT-C-Tail-S/D phosphomimetic peptide caused a broadening of the actin cytoskeleton. These data indicate that stimulation-mediated Thr dephosphorylation allows decreased association of apactin with EBP50/NHERF and fosters actin remodeling to coat zymogen granules. Stimulation-mediated Ser phosphorylation increases apactin association with the actin cytoskeleton, maintaining tight bundling of actin microfilaments at the apical surface. Thus, apactin is involved in remodeling the apical cytoskeleton during regulated exocytosis in a manner controlled by phosphorylation of the apactin C-Tail.