Isozymes of S-adenosylmethionine synthetase from rat liver: isolation and characterization

S-Adenosylmethionine synthetase exists in at least two distinct forms, alpha- and beta-forms, in adult liver. The beta-form was purified to homogeneity from the soluble fraction of rat liver with a yield of about 10%. An antiserum directed against the purified beta-form from rat liver was prepared by injecting the purified enzyme into a rabbit. Ouchterlony double diffusion analysis and immunochemical titrations revealed that the isozymes, alpha- and beta-forms, are identical. Thus, the alpha-form was isolated from rat liver as a single protein using immunoaffinity chromatography against the beta-form. The molecular weights of the beta- and alpha-forms were determined to be 48,000 each by sodium dodecyl sulfate disc gel electrophoresis, and about 100,000, and 200,000, respectively, by Sephacryl S-200 gel filtration. These results indicate that the beta-form consisted of two subunits of 48,000 daltons and the alpha-form of four subunits of 48,000 daltons. The sedimentation coefficient was calculated to be 5.5S for the beta-form and 8.0S for the alpha-form.     

Effect of ethrel treatment on the postharvest changes in the turnover of ascorbic acid and reducing sugar inAchras sapota fruits

There was a definite relationship between growth and ascorbic acid content inAchras sapota. Increase in ethrel concentration from 250 ppm to 500 ppm hastened early ripening and increased the amount of reducing sugars but depleted the ascorbic acid content. Other aspects of ascorbic acid turnover viz. ascorbigen, bound form of ascorbic acid, ascorbic acid utilization, net ascorbic acid bound and ascorbic acid oxidase were also studied.     

Cell salvage using the continuous autotransfusion device CATSmart – an observational bicenter technical evaluation

Background

The use of cell salvage and autologous blood transfusion has become an important method of blood conservation. So far, there are no clinical data about the performance of the continuous autotransfusion device CATSmart.

Methods

In total, 74 patients undergoing either cardiac or orthopedic surgery were included in this prospective, bicenter and observational technical evaluation to validate red cell separation process and washout quality of CATSmart. The target of red cell separation process was defined as a hematocrit value in the packed red cell unit of 55–75% and of washout quality of 80–100% removal ratio.

Results

Hematocrit values measured by CATSmart and laboratory analysis were 78.5% [71.3%; 84.0%] and 73.7% [67.5%; 75.5%], respectively. Removal ratios for platelets 94.7% [88.2%; 96.7%], free hemoglobin 89.3% [85.2%; 94.9%], albumin 97.9% [96.6%; 98.5%], heparin 99.9% [99.9%; 100.0%], and potassium 92.5% [90.8%; 95.0%] were within the target range while removal of white blood cells was slightly worse 72.4% [57.9%; 87.3%].

Conclusion

The new autotransfusion device enables sufficient red cell separation and washout quality.

     

Synthesis of S-adenosylmethionine synthetase isozymes from rat and mouse livers. Immunochemical studies

The alpha- and beta-forms of S-adenosylmethionine synthetase in rat liver were completely fractionated by chromatography on a hydrophobic resin, phenyl-Sepharose. The alpha-form was eluted in low-ionic strength buffer, and the beta-form was eluted with 50% dimethylsulfoxide. The alpha-form is less sensitive to dimethylsulfoxide, whereas the beta-form is strikingly stimulated by dimethylsulfoxide, after removal of the dimethylsulfoxide. The levels of the alpha-form activity in rat liver after treatment with ethionine and adenine for 2 consecutive days, and those of the beta-form activity in mouse liver on the 12th day after transplantation of Ehrlich ascites tumor cells, were increased several fold compared to normal liver. Immunochemical titrations with specific antibody against the beta-form as well as kinetic studies indicated that the observed increase in the levels of each activity from the S-adenosylmethionine synthetase isozymes is due to an increase in the cellular content of the enzyme.     

A Nondimensional Model Reveals Alterations in Nuclear Mechanics upon Hepatitis C Virus Replication

Morphology of the nucleus is an important regulator of gene expression. Nuclear morphology is in turn a function of the forces acting on it and the mechanical properties of the nuclear envelope. Here, we present a two-parameter, nondimensional mechanical model of the nucleus that reveals a relationship among nuclear shape parameters, such as projected area, surface area, and volume. Our model fits the morphology of individual nuclei and predicts the ratio between forces and modulus in each nucleus. We analyzed the changes in nuclear morphology of liver cells due to hepatitis C virus (HCV) infection using this model. The model predicted a decrease in the elastic modulus of the nuclear envelope and an increase in the pre-tension in cortical actin as the causes for the change in nuclear morphology. These predictions were validated biomechanically by showing that liver cells expressing HCV proteins possessed enhanced cellular stiffness and reduced nuclear stiffness. Concomitantly, cells expressing HCV proteins showed downregulation of lamin-A,C and upregulation of β-actin, corroborating the predictions of the model. Our modeling assumptions are broadly applicable to adherent, monolayer cell cultures, making the model amenable to investigate changes in nuclear mechanics due to other stimuli by merely measuring nuclear morphology. Toward this, we present two techniques, graphical and numerical, to use our model for predicting physical changes in the nucleus.     

Functional rescue of a defective angiotensin II AT1 receptor mutant by the Mas protooncogene

Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas. Cells single-expressing Mas or C18F/K20A did not show any binding for AngII. The co-expression of the wild-type AT(1) receptor and Mas showed a binding profile similar to that observed for the wild-type AT(1) expressed alone. Surprisingly, the co-expression of the double mutant C18F/K20A and Mas evoked a total recovery of the binding affinity for AngII to a level similar to that obtained for the wild-type AT(1). Functional measurements using inositol phosphate and extracellular acidification rate assays also showed a clear recovery of activity for AngII on cells co-expressing the mutant C18F/K20A and Mas. In addition, immunofluorescence analysis localized the AT(1) receptor mainly at the plasma membrane and the mutant C18F-K20A exclusively inside the cells. However, the co-expression of C18F-K20A mutant with the Mas changed the distribution pattern of the mutant, with intense signals at the plasma membrane, comparable to those observed in cells expressing the wild-type AT(1) receptor. These results support the hypothesis that Mas is able to rescue binding and functionality of the defective C18F-K20A mutant by dimerization.     

Species discrimination through DNA barcoding in the genus Salacia of the Western Ghats in India

The genus Salacia (Celastraceae) is a source of many important pharmaceutical chemicals used in the Ayurvedic system of medicine in India. Owing to morphological similarities between species, the taxonomy of Salacia is complex and not fully settled. To ensure quality and assured therapeutic effects in the raw drugs from the genus, proper identification at the species level is critical. The main objective of this study was to find suitable DNA barcodes that can accurately and efficiently identify the potential medicinal species of the genus. Among the barcode loci analyzed, ITS2 exhibited the highest interspecific divergence, followed by trnH‐psbA, matK and rbcL. A clear barcoding gap was evident for the ITS2 barcode region whereas it was less conspicuous for trnH‐psbA and matK. The ITS2 barcode could discriminate all the eight analyzed Salacia species with 100% accuracy. We therefore propose barcoding with ITS2 to confirm the taxonomic identity of the raw drugs in the market. Further, the ITS2 region can be recommended for biosystematic studies in the genus.     

The tyrosine-kinase inhibitor Nintedanib ameliorates autosomal-dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and is characterized by progressive growth of fluid-filled cysts. Growth factors binding to receptor tyrosine kinases (RTKs) stimulate cell proliferation and cyst growth in PKD. Nintedanib, a triple RTK inhibitor, targets the vascular endothelial growth-factor receptor (VEGFR), platelet-derived growth-factor receptor (PDGFR), and fibroblast growth-factor receptor (FGFR), and is an approved drug for the treatment of non-small-cell lung carcinoma and idiopathic lung fibrosis. To determine if RTK inhibition using nintedanib can slow ADPKD progression, we tested its effect on human ADPKD renal cyst epithelial cells and myofibroblasts in vitro, and on Pkd1^f/fPkhd1^Cre and Pkd1^RC/RC, orthologous mouse models of ADPKD. Nintedanib significantly inhibited cell proliferation and in vitro cyst growth of human ADPKD renal cyst epithelial cells, and cell viability and migration of human ADPKD renal myofibroblasts. Consistently, nintedanib treatment significantly reduced kidney-to-body-weight ratio, renal cystic index, cystic epithelial cell proliferation, and blood-urea nitrogen levels in both the Pkd1^f/fPkhd1^Cre and Pkd1^RC/RC mice. There was a corresponding reduction in ERK, AKT, STAT3, and mTOR activity and expression of proproliferative factors, including Yes-associated protein (YAP), c-Myc, and Cyclin D1. Nintedanib treatment significantly reduced fibrosis in Pkd1^RC/RC mice, but did not affect renal fibrosis in Pkd1^f/fPkhd1^Cre mice. Overall, these results suggest that nintedanib may be repurposed to effectively slow cyst growth in ADPKD.Subject terms: Growth factor signalling, Polycystic kidney disease