Modeling SILAC Data to Assess Protein Turnover in a Cellular Model of Diabetic Nephropathy.

Protein turnover rate is finely regulated through intracellular mechanisms and signals that are still incompletely understood but that are essential for the correct function of cellular processes. Indeed, a dysfunctional proteostasis often impacts the cell's ability to remove unfolded, misfolded, degraded, non-functional, or damaged proteins. Thus, altered cellular mechanisms controlling protein turnover impinge on the pathophysiology of many diseases, making the study of protein synthesis and degradation rates an important step for a more comprehensive understanding of these pathologies.

Decreased Homocysteine Trans-Sulfuration in Hypertension With Hyperhomocysteinemia: Relationship With Insulin Resistance.

Context: Homocysteine is an independent cardiovascular risk factor and is elevated in essential hypertension. Insulin stimulates homocysteine catabolism in healthy individuals. However, the mechanisms of hyperhomocysteinemia and its relationship with insulin resistance in essential hypertension are unknown.

Decreased VLDL-Apo B 100 Fractional Synthesis Rate Despite Hypertriglyceridemia in Subjects With Type 2 Diabetes and Nephropathy.

Context: Subjects with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) often exhibit hypertriglyceridemia. The mechanism(s) of such an increase are poorly known.

Objective: We investigated very low-density lipoprotein (VLDL)-Apo B 100 kinetics in T2DM subjects with and without DN, and in healthy controls.

Roles of insulin, age, and asymmetric dimethylarginine on nitric oxide synthesis in vivo.

We tested the effects of insulin on production of nitrous oxide (NO)-related substances (nitrites and nitrates [NOx]) after (15)N-arginine intravenous infusion and on asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) concentrations in conditions reportedly associated with altered NO availability, i.e., aging, hypertension, hypercholesterolemia, and type 2 diabetes mellitus (T2DM).

Molecular targets of antimicrobial photodynamic therapy identified by a proteomic approach.

Antimicrobial photodynamic therapy (PDT) is a promising tool to combat antibiotic-resistant bacterial infections. During PDT, bacteria are killed by reactive oxygen species generated by a visible light absorbing photosensitizer (PS). We used a classical proteomic approach that included two-dimensional gel electrophoresis and mass spectrometry analysis, to identify some proteins of Staphylococcus aureus that are damaged during PDT with the cationic PS meso-tetra-4-N-methyl pyridyl porphine (T4).

Polar electrophoresis: shape of two-dimensional maps is as important as size.

The performance of two-dimensional electrophoresis in conventional gels in Cartesian coordinates (2-DE) vs. polar coordinates (2-PE) is here evaluated. Although 2-DE is performed in much longer Immobiline gels in the first dimension (17 cm) vs.

Skin fibroblasts as a tool for identifying the risk of nephropathy in the type 1 diabetic population.

Human fibroblasts in culture have been employed as an in vitro system to investigate some pathophysiological mechanisms of diabetes mellitus also associated with the development of diabetic nephropathy. In fact, there is increasing evidence that genetic factors either convey the risk of, or protect from, diabetic nephropathy and that the expression profiles and/or the behaviour of the cultured skin fibroblasts from type 1 diabetic patients could reflect these genetic influences. On the other hand, alterations could be attributable not only to changes in DNA sequence, but also to epigenetic factors.

High abundance proteins depletion vs low abundance proteins enrichment: comparison of methods to reduce the plasma proteome complexity.

Background: To date, the complexity of the plasma proteome exceeds the analytical capacity of conventional approaches to isolate lower abundance proteins that may prove to be informative biomarkers. Only complex multistep separation strategies have been able to detect a substantial number of low abundance proteins (<100 ng/ml). The first step of these protocols is generally the depletion of high abundance proteins by the use of immunoaffinity columns or, alternatively, the enrichment of by the use of solid phase hexapeptides ligand libraries.

Insulin resistance of amino acid and protein metabolism in type 2 diabetes.

Although insulin resistance in T2DM (type 2 diabetes mellitus) is usually referred to glucose and lipid metabolism, the question whether such a resistance affects also amino acid and protein metabolism is both relevant and not easy to be answered. Available data indicate a reduced response to insulin in the inhibition of proteolysis at low, near basal hormone levels, whereas such a response appears to be normal at high physiological doses. In most studies in T2DM subjects the stimulation of whole-body protein synthesis in the presence of hyperinsulinemia and euaminoacidemia appears to be normal, although one single study reported lower rates in male T2DM subjects with obesity.

Operator- and software-related post-experimental variability and source of error in 2-DE analysis.

In the field of proteomics, several approaches have been developed for separating proteins and analyzing their differential relative abundance. One of the oldest, yet still widely used, is 2-DE. Despite the continuous advance of new methods, which are less demanding from a technical standpoint, 2-DE is still compelling and has a lot of potential for improvement.

Improved instrumentation for large-size two-dimensional protein maps.

Novel instrumentation for performing large-size (>25 cm) 2-D maps is reported here. To perform the first dimension, we developed a power supply that can deliver a voltage of up to 15,000 V and allows regulation of current (up to 200 μA) onto each individual focusing IPG strip. The IEF strip tray can accommodate up to 12 IPG strips and the electrodes slide on a ruler, thus permitting running strips of any length up to 45 cm.

Caldesmon over-expression in type 1 diabetic nephropathy.

Substantial evidence supports a genetic susceptibility to develop nephropathy in type 1 diabetes and a key pathogenic role of actin cytoskeleton dysfunction in this complication. We previously reported that many cytoskeletal proteins were either up- or down-regulated in fibroblast cells from type 1 diabetic (T1DM) patients with nephropathy. The gene of one of these proteins, caldesmon, lies in a chromosomal region linked to nephropathy and its promoter region contains a single nucleotide polymorphism that is associated with nephropathy.

Nitric oxide synthesis is reduced in subjects with type 2 diabetes and nephropathy.

Objective: Nitric oxide (NO) is a key metabolic and vascular regulator. Its production is stimulated by insulin. A reduced urinary excretion of NO products (NOx) is frequently found in type 2 diabetes, particularly in association with nephropathy.

Delta2D and Proteomweaver: Performance evaluation of two different approaches for 2-DE analysis.

2-DE is a fundamental technology used in proteomics research. However, despite its high capacity to simultaneously separate several proteins for subsequent identification and quantitative comparison studies, a drawback for this technique is its limited reproducibility, especially when comparing data from different laboratories. 2-DE-related variability can be broadly divided into two categories: experimental and post-experimental.

SDS-PAGE and two-dimensional maps in a radial gel format.

A novel method for performing 2-D map analysis is here reported, consisting in a modification of the second dimension run, which is performed not in a conventional square- or rectangular-size gel, but in a radial surface. This has the advantage of permitting resolution of closely adjacent bands, representing strings of isoforms of similar or identical mass but of closely spaced isoelectric points. When used in a mono-dimensional, SDS-PAGE format, this system allows the simultaneous running of 62 sample tracks.

Effects of chronic metabolic acidosis on splanchnic protein turnover and oxygen consumption in human beings.

Background & Aims: Although metabolic acidosis stimulates protein catabolism, its effects on splanchnic protein turnover and energy expenditure have not been measured in human beings. We investigated the effects of chronic metabolic acidosis (CMA) on splanchnic protein dynamics and oxygen consumption in human beings by using a leucine tracer and mass-balance techniques.

Methods: Five subjects were studied after 6 days of HCl-, CaCl(2)-, and NH(4)Cl-induced acidosis; 8 subjects served as controls.

The effects of rosiglitazone and high glucose on protein expression in endothelial cells.

Rosiglitazone is a thiazolidinedione used to treat insulin resistance in diabetes. Although thiazolidinediones may also exert cardiovascular effects, contrasting results were reported. Favorable effects were shown for pioglitazone, whereas adverse reactions were suspected for rosiglitazone.

Kinetics of albumin homocysteinylation measured with matrix-assisted laser/desorption ionization mass spectrometry versus with a radioactive tracer.

Homocysteinylation is a post-translational protein modification which involves homocysteine-thiolactone and may be responsible for many pathophysiological changes secondary to hyperhomocysteinemia. Therefore, methods to measure protein homocysteinylation in intact biological samples are required. We tested whether matrix assisted-laser/desorption ionization mass spectrometry (MALDI-MS) can detect time- and dose-dependent changes in in vitro homocysteine-thiolactone binding to human serum albumin.

Effect of liver cirrhosis on phenylalanine and tyrosine metabolism.

Purpose Of Review: Phenylalanine conversion to tyrosine (i.e., 'hydroxylation') is the first irreversible step in phenylalanine catabolism and a source of circulating tyrosine.

Middle age is not associated with altered fibrinogen concentration and production in males.

Whether ageing is associated with increased fibrinogen concentration and production remains unclear. We measured fibrinogen fractional (FSR) and absolute synthesis (ASR) rates in male volunteers, of either young (mean age: 28 years, range: 22-34) or middle age (mean age: 57 years, range: 38-72), using a leucine-tracer isotope dilution technique. In the middle-age group, neither fibrinogen FSR (20.

Glycolytic enzyme expression and pyruvate kinase activity in cultured fibroblasts from type 1 diabetic patients with and without nephropathy.

Since type 1 diabetes mellitus (T1DM) patients with nephropathy (DN+) are insulin-resistant, we aimed to identify (new) potential molecular sites involved in the alterations of glucose metabolism in these patients. We examined the expression of glycolytic enzymes in cultured fibroblasts from T1DM(DN+) patients as compared to those from T1DM patients without nephropathy (DN-) and from controls. Pyruvate kinase (PK) activity was also determined.

Abnormal cytoskeletal protein expression in cultured skin fibroblasts from type 1 diabetes mellitus patients with nephropathy: A proteomic approach.

Diabetic nephropathy (DN) develops in about 40% of insulin-dependent type 1 diabetes mellitus (T1DM) patients, and is associated not only with diabetes duration and metabolic control, but also with a genetic predisposition. Constitutive alterations of cytoskeletal proteins may play a role in the development of DN. We investigated the expression of these proteins in cultured skin fibroblasts, obtained from long-term T1DM patients with and without DN but comparable metabolic control, and from matched healthy subjects, by means of 2-DE electrophoresis and MS-MALDI analyses.

Acute effect of insulin on nitric oxide synthesis in humans: a precursor-product isotopic study.

Nitric oxide (NO) is a key regulatory molecule with wide vascular, cellular, and metabolic effects. Insulin affects NO synthesis in vitro. No data exist on the acute effect of insulin on NO kinetics in vivo.

Altered chaperone and protein turnover regulators expression in cultured skin fibroblasts from type 1 diabetes mellitus with nephropathy.

In type-1 diabetes mellitus (T1DM) with diabetic nephropathy (DN), accumulation of abnormal proteins in the kidney and other tissues may derive from constitutive alterations of intracellular protein recognition, assembly, and turnover. We characterized the proteins involved in these functions in cultured skin fibroblasts from long-term T1DM patients with [DN+] or without [DN-] nephropathy but similar metabolic control, and from matched healthy subjects. 2-D gel electrophoresis and MS-MALDI analysis were employed.

Proteome analysis of cultured fibroblasts from type 1 diabetic patients and normal subjects.

Context: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes.

Objective: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls.