Purification and properties of O-acetyl-L-serine sulphydrylase from wheat leaves

O-Acetyl-L-serine sulphydrylase (OASS), the enzyme which produces L-cysteine from O-acetyl-L-serine (OAS) and sulphide, is     

Ubiquitylation by the Ltn1 E3 ligase protects 60S ribosomes from starvation-induced selective autophagy

Autophagy, the process by which proteins or organelles are engulfed by autophagosomes and delivered for vacuolar/lysosomal degradation, is induced to ensure survival under starvation and other stresses. A selective autophagic pathway for 60S ribosomal subunits elicited by nitrogen starvation in yeast—ribophagy—was recently described and requires the Ubp3-Bre5 deubiquitylating enzyme. This discovery implied that an E3 ligases act upstream, whether inhibiting the process or providing an initial required signal. In this paper, we show that Ltn1/Rkr1, a 60S ribosome-associated E3 implicated in translational surveillance, acts as an inhibitor of 60S ribosomal subunit ribophagy and is antagonized by Ubp3. The ribosomal protein Rpl25 is a relevant target. Its ubiquitylation is Ltn1 dependent and Ubp3 reversed, and mutation of its ubiquitylation site rendered ribophagy less dependent on Ubp3. Consistently, the expression of Ltn1—but not Ubp3—rapidly decreased after starvation, presumably to allow ribophagy to proceed. Thus, Ltn1 and Ubp3-Bre5 likely contribute to adapt ribophagy activity to both nutrient supply and protein translation.     

Galactodendritic Phthalocyanine Targets Carbohydrate-Binding Proteins Enhancing Photodynamic Therapy

Photosensitizers (PSs) are of crucial importance in the effectiveness of photodynamic therapy (PDT) for cancer. Due to their high reactive oxygen species production and strong absorption in the wavelength range between 650 and 850 nm, where tissue light penetration is rather high, phthalocyanines (Pcs) have been studied as PSs of excellence. In this work, we report the evaluation of a phthalocyanine surrounded by a carbohydrate shell of sixteen galactose units distributed in a dendritic manner (PcGal₁₆) as a new and efficient third generation PSs for PDT against two bladder cancer cell lines, HT-1376 and UM-UC-3. Here, we define the role of galacto-dendritic units in promoting the uptake of a Pc through interaction with GLUT1 and galectin-1. The photoactivation of PcGal₁₆ induces cell death by generating oxidative stress. Although PDT with PcGal₁₆ induces an increase on the activity of antioxidant enzymes immediately after PDT, bladder cancer cells are unable to recover from the PDT-induced damage effects for at least 72 h after treatment. PcGal₁₆ co-localization with galectin-1 and GLUT1 and/or generation of oxidative stress after PcGal₁₆ photoactivation induces changes in the levels of these proteins. Knockdown of galectin-1 and GLUT1, via small interfering RNA (siRNA), in bladder cancer cells decreases intracellular uptake and phototoxicity of PcGal₁₆. The results reported herein show PcGal₁₆ as a promising therapeutic agent for the treatment of bladder cancer, which is the fifth most common type of cancer with the highest rate of recurrence of any cancer.     

Propulsion Phase of the Single Leg Triple Hop Test in Women with Patellofemoral Pain Syndrome: A Biomechanical Study

Asymmetry in the alignment of the lower limbs during weight-bearing activities is associated with patellofemoral pain syndrome (PFPS), caused by an increase in patellofemoral (PF) joint stress. High neuromuscular demands are placed on the lower limb during the propulsion phase of the single leg triple hop test (SLTHT), which may influence biomechanical behavior. The aim of the present cross-sectional study was to compare kinematic, kinetic and muscle activity in the trunk and lower limb during propulsion in the SLTHT using women with PFPS and pain free controls. The following measurements were made using 20 women with PFPS and 20 controls during propulsion in the SLTHT: kinematics of the trunk, pelvis, hip, and knee; kinetics of the hip, knee and ankle; and muscle activation of the gluteus maximus (GM), gluteus medius (GMed), biceps femoris (BF) and vastus lateralis (VL). Differences between groups were calculated using three separate sets of multivariate analysis of variance for kinematics, kinetics, and electromyographic data. Women with PFPS exhibited ipsilateral trunk lean; greater trunk flexion; greater contralateral pelvic drop; greater hip adduction and internal rotation; greater ankle pronation; greater internal hip abductor and ankle supinator moments; lower internal hip, knee and ankle extensor moments; and greater GM, GMed, BL, and VL muscle activity. The results of the present study are related to abnormal movement patterns in women with PFPS. We speculated that these findings constitute strategies to control a deficient dynamic alignment of the trunk and lower limb and to avoid PF pain. However, the greater BF and VL activity and the extensor pattern found for the hip, knee, and ankle of women with PFPS may contribute to increased PF stress.     

Integrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model

Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.     

Development and Validation of the Self-Acceptance Scale for Persons with Early Blindness: The SAS-EB

Investigations of self-acceptance are critical to understanding the development and maintenance of psychological health. However, valid and reliable instruments for measuring self-acceptance in persons with early blindness have yet to be developed. The current research describes three studies designed to develop and validate the Self-acceptance Scale for Persons with Early Blindness (SAS-EB). In Study 1, we developed the initial item pool. Thirty-three items were generated, based on data from specialized literature and from 2 focus groups. Items were organized in a three-factor structure, theoretically predicted for SAS-EB - (1) body acceptance, (2) self-protection from social stigmas, and (3) feeling and believing in one''s capacities. In Study 2, information obtained from a panel of 9 experts and 22 persons with early blindness representing the target population was used to refine the initial item pool, generating a new pool of 27 items. In Study 3, 318 persons with early blindness (141 women and 177 men), between 18 and 60 years of age (M = 37.74 years, SD = 12.37) answered the new pool of 27 items. After the elimination of 9 items using confirmatory factor analysis, we confirmed the theoretical three-factor structure of the SAS-EB. Study 3 also provided support for the scale''s internal consistency and construct validity. Finally, the psychometric properties of the SAS-EB, its utility, and its limitations are discussed along with considerations for future research.     

Pioglitazone Improves Fat Distribution,the Adipokine Profile and Hepatic Insulin Sensitivity in Non-Diabetic End-Stage Renal Disease Subjects on Maintenance Dialysis: A Randomized Cross-Over Pilot Study

Background

Fat redistribution, increased inflammation and insulin resistance are prevalent in non-diabetic subjects treated with maintenance dialysis. The aim of this study was to test whether pioglitazone, a powerful insulin sensitizer, alters body fat distribution and adipokine secretion in these subjects and whether it is associated with improved insulin sensitivity.

Trial Design

This was a double blind cross-over study with 16 weeks of pioglitazone 45 mg vs placebo involving 12 subjects.

Methods

At the end of each phase, body composition (anthropometric measurements, dual energy X-ray absorptometry (DEXA), abdominal CT), hepatic and muscle insulin sensitivity (2-step hyperinsulinemic euglycemic clamp with 2H2-glucose) were measured and fasting blood adipokines and cardiometabolic risk markers were monitored.

Results

Four months treatment with pioglitazone had no effect on total body weight or total fat but decreased the visceral/sub-cutaneous adipose tissue ratio by 16% and decreased the leptin/adiponectin (L/A) ratio from 3.63×10⁻³ to 0.76×10⁻³. This was associated with a 20% increase in hepatic insulin sensitivity without changes in muscle insulin sensitivity, a 12% increase in HDL cholesterol and a 50% decrease in CRP.

Conclusions/Limitations

Pioglitazone significantly changes the visceral-subcutaneous fat distribution and plasma L/A ratio in non diabetic subjects on maintenance dialysis. This was associated with improved hepatic insulin sensitivity and a reduction of cardio-metabolic risk markers. Whether these effects may improve the outcome of non diabetic end-stage renal disease subjects on maintenance dialysis still needs further evaluation.

Trial Registration

ClinicalTrial.gov {"type":"clinical-trial","attrs":{"text":"NCT01253928","term_id":"NCT01253928"}}NCT01253928     

Glucolipotoxicity Impairs Ceramide Flow from the Endoplasmic Reticulum to the Golgi Apparatus in INS-1 β-Cells

Accumulating evidence suggests that glucolipotoxicity, arising from the combined actions of elevated glucose and free fatty acid levels, acts as a key pathogenic component in type II diabetes, contributing to β-cell dysfunction and death. Endoplasmic reticulum (ER) stress is among the molecular pathways and regulators involved in these negative effects, and ceramide accumulation due to glucolipotoxicity can be associated with the induction of ER stress. Increased levels of ceramide in ER may be due to enhanced ceramide biosynthesis and/or decreased ceramide utilization. Here, we studied the effect of glucolipotoxic conditions on ceramide traffic in INS-1 cells in order to gain insights into the molecular mechanism(s) of glucolipotoxicity. We showed that glucolipotoxicity inhibited ceramide utilization for complex sphingolipid biosynthesis, thereby reducing the flow of ceramide from the ER to Golgi. Glucolipotoxicity impaired both vesicular- and CERT-mediated ceramide transport through (1) the decreasing of phospho-Akt levels which in turn possibly inhibits vesicular traffic, and (2) the reducing of the amount of active CERT mainly due to a lower protein levels and increased protein phosphorylation to prevent its localization to the Golgi. In conclusion, our findings provide evidence that glucolipotoxicity-induced ceramide overload in the ER, arising from a defect in ceramide trafficking may be a mechanism that contributes to dysfunction and/or death of β-cells exposed to glucolipotoxicity.