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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. In this manuscript, we describe the application of a dynamic-SILAC approach to study the turnover rate and the abundance of proteins in a cellular model of diabetic nephropathy. We estimated protein half-lives and relative abundance for thousands of proteins, several of which are characterized by either an altered turnover rate or altered abundance between diabetic nephropathic subjects and diabetic controls. Many of these proteins were previously shown to be related to diabetic complications and represent therefore, possible biomarkers or therapeutic targets. Beside the aspects strictly related to the pathological condition, our data also represent a consistent compendium of protein half-lives in human fibroblasts and a rich source of important information related to basic cell biology.

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. Objective To investigate whole body methionine and homocysteine kinetics and the effects of insulin in essential hypertension. Design and Setting Eight hypertensive male subjects and six male normotensive controls were infused with l-[methyl-2H3,1-13C]methionine for 6 hours. In the last 3 hours a euglycemic, hyperinsulinemic clamp was performed. Steady-state methionine and homocysteine kinetics were determined in postabsorptive and hyperinsulinemic conditions. Results Postabsorptive hypertensive subjects had elevated homocysteine concentrations (+30%, P = 0.035) and slightly (by 15% to 20%) but insignificantly lower methionine rates of appearance (Ras) (P = 0.07 to P = 0.05) and utilization for protein synthesis (P = 0.06) than postabsorptive normotensive controls. Hyperinsulinemia suppressed methionine Ra and protein synthesis, whereas it increased homocysteine trans-sulfuration, clearance, and methionine transmethylation (the latter only in the normotensive subjects). However, in the hypertensive subjects trans-sulfuration was significantly lower (P < 0.05) and increased ~50% less [by +1.59 ± 0.34 vs +3.45 ± 0.52 µmol/kg lean body mass (LBM) per hour, P < 0.005] than in normotensive controls. Homocysteine clearance through trans-sulfuration was ~50% lower in hypertensive than in normotensive subjects (P < 0.005). In the hypertensive subjects, insulin-mediated glucose disposal was ~45% lower (460 ± 44 vs 792 ± 67 mg/kg LBM per hour, P < 0.0005) than in normotensive controls and was positively correlated with the increase of trans-sulfuration (P < 0.0015). Conclusions In subjects with essential hypertension, hyperhomocysteinemia is associated with decreased homocysteine trans-sulfuration and probably represents a feature of insulin resistance.

We tested the effects of insulin on production of nitrous oxide (NO)-related substances (nitrites and nitrates [NOx]) after 15N-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). A total of 26 male subjects (age 23–71 years, BMI 23–33 kg/m2), some of whom were affected by mixed pathologic features, were enrolled. NOx fractional synthesis rate (FSR) was lower in elderly (P < 0.015) and T2DM subjects (P < 0.03) than in matched control subjects. Hyperinsulinemia generally increased both NOx FSR and absolute synthesis rate (ASR) and reduced NOx, ADMA, and SDMA concentrations. Insulin sensitivity was impaired only in T2DM. With use of simple linear regression analysis across all subjects, age was inversely correlated with both NOx FSR (R2 = 0.23, P < 0.015) and ASR (R2 = 0.21, P < 0.02). NOx FSR inversely correlated with both ADMA and SDMA. With use of multiple regression analysis and various models, NOx FSR remained inversely associated with age and ADMA, whereas ASR was inversely associated with age and diabetes. No association with insulin sensitivity was found. We conclude that whole-body NOx production is decreased in aging and T2DM. Age, ADMA concentration, and T2DM, but not insulin resistance, appear as negative regulators of whole-body NOx production.

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). Suspensions of S. aureus cells were incubated with selected T4 concentrations and irradiated with doses of blue light that reduced the survival to about 60% or 1%. Proteomics analyses of a membrane proteins enriched fraction revealed that these sub-lethal PDT treatments affected the expression of several functional classes of proteins, and that this damage is selective. Most of these proteins were found to be involved in metabolic activities, in oxidative stress response, in cell division and in the uptake of sugar. Subsequent analyses revealed that PDT treatments delayed the growth and considerably reduced the glucose consumption capacity of S. aureus cells. This investigation provides new insights towards the characterization of PDT induced damage and mechanism of bacterial killing using, for the first time, a proteomic approach.

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. However, whether the decreased NOx excretion in type 2 diabetes is caused by a defective NOx production from arginine in response to hyperinsulinemia has never been studied. RESEARCH DESIGN AND METHODS We measured NOx fractional (FSR) and absolute (ASR) synthesis rates in type 2 diabetic patients with diabetic nephropathy and in control subjects, after l-[15N2-guanidino]-arginine infusion, and use of precursor–product relationships. The study was conducted both before and after an euglycemic hyperinsulinemic (∼1,000–1,200 pmol/l) clamp. RESULTS In type 2 diabetes, NOx FSR was reduced both under basal (19.3 ± 3.9% per day, vs. 22.9 ± 4.5% per day in control subjects) and hyperinsulinemic states (24.0 ± 5.6% per day, vs. 37.9 ± 6.4% per day in control subjects; P < 0.03 by ANOVA). Similarly, in type 2 diabetes, NOx ASR was lower than in control subjects under both conditions (basal, 0.32 ± 0.06 vs. 0.89 ± 0.34 mol per day; hyperinsulinemia, 0.35 ± 0.07 vs. 1.15 ± 0.38 mol per day; P = 0.01 by ANOVA). In type 2 diabetes, the ability of insulin to stimulate both the FSR (4.7 ± 3.2% per day) and the ASR (0.03 ± 0.04 mol per day) of NOx was several-fold lower than that in control subjects (15.0 ± 2.9% per day and 0.25 ± 0.07 mol per day, P < 0.03 and P < 0.02, respectively). Also the fraction of arginine flux converted to NOx (basal, 0.22 ± 0.05% vs. 0.65 ± 0.25%; hyperinsulinemia, 0.32 ± 0.06% vs. 1.03 ± 0.33%) was sharply reduced in the patients (P < 0.01 by ANOVA). CONCLUSIONS In type 2 diabetic patients with nephropathy, intravascular NOx synthesis from arginine is decreased under both basal and hyperinsulinemic states. This defect extends the concept of insulin resistance to NO metabolism.

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. We have compared this method with a 35S-thiolactone radioactive binding assay. Incubations with and without dithiothreitol allowed measurement of the amide-linked and disulfide-linked thiolactone-protein adducts, respectively. A good correspondence in time- and dose-dependent protein-thiolactone formation was observed between the two methods. A maximum of 9 to 12 thiolactone residues were bound to each albumin molecule. The 35S-thiolactone bound albumin tightly, particularly at the lowest concentrations, with approximately 70% of the binding amide-linked. Although the results of the two methods were rather similar, the radioactive method appears to be more sensitive than the MALDI-MS technique.

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. In T1DM with DN, compared to the other two groups, quantitative analyses revealed an altered expression of 17 spots (p

Malaria is one of the diseases for which even today not many suitable drugs are available. The rapid spread of resistance toward current drugs encourages the study for new active molecules. Ethnobotanical research can be of help to find new leads. Traditional remedies have always been a source of important antimalarial drugs and continue to provide novel and effective treatments, both where pharmaceuticals are not available and also where the disease is highly resistant to commonly prescribed drugs. This article provides a comparative compilation of various studies reported between 1995 and 2001 on natural compounds with antiplasmodial activity, with the goal of providing a basis for further in vitro and in vivo studies, as well as for clinical trials for the development of new antimalarial medicines. Referee: Dr. Robert Verpoorte, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, PO Box 9502, 2300 RA Leiden, The Netherlands

Subjects with type 2 diabetes mellitus (T2DM) and diabetic nephropathy (DN) often exhibit hypertriglyceridemia. The mechanism(s) of such an increase are poorly known.We investigated very low-density lipoprotein (VLDL)-Apo B 100 kinetics in T2DM subjects with and without DN, and in healthy controls.Stable isotope (13)C-leucine infusion and modeling analysis of tracer-to-tracee ratio dynamics in the protein product pool in the 6-8-h period following tracer infusion were employed.Male subjects affected by T2DM, either with (n = 9) or without (n = 5) DN, and healthy male controls (n = 6), were studied under spontaneous glycemic levels in the post-absorptive state.In the T2DM patients with DN, plasma triglyceride (TG) (mean ± SD; 2.2 ± 0.8 mmol/L) and VLDL-Apo B 100 (17.4 ± 10.4 mg/dL) concentrations, and VLDL-Apo B 100 pool (0.56 ± 0.29 g), were ∼60-80% greater (P.05 or less) than those of the T2DM subjects without DN (TG, 1.4 ± 0.5 mmol/L; VLDL-Apo B 100, 9.9 ± 2.5 mg/dL; VLDL-Apo B 100 pool, 0.36 ± 0.09 g), and ∼80-110% greater (P.04 or less) than those of nondiabetic controls (TG, 1.2 ± 0.4 mmol/L; VLDL-Apo B 100, 8.2 ± 1.7 mg/dL; VLDL-Apo B 100, 0.32 ± 0.09 g). In sharp contrast however, in the subjects with T2DM and DN, VLDL-Apo B 100 fractional synthesis rate was ≥50% lower (4.8 ± 2.2 pools/d) than that of either the T2DM subjects without DN (9.9 ± 4.3 pools/d; P.025) or the control subjects (12.5 ± 9.1 pools/d; P.04).The hypertriglyceridemia of T2DM patients with DN is not due to hepatic VLDL-Apo B 100 overproduction, which is decreased, but it should be attributed to decreased apolipoprotein removal.

A new protocol to obtain an embryogenic cell line from cultured seedling explants of Catharanthus roseus is described. In order to assess the relationship between tissue differentiation and secondary metabolite biosynthesis, the biosynthetic capabilities (alkaloid production) of an embryogenic cell line and two non-embryogenic C. roseus strains were comparatively examined. Faster cell growth rate was associated with higher alkaloid production in the embryogenic cell line. The kinetics of ajmalicine and serpentine production by the three cell lines is also reported.

A peroxidase from spent medium of shoot cultures from Haplophyllum patavinum (L.) G. Don catalyzes the biotransformation of a synthetic dibenzybutanolide into a podophyllotoxin analogue and a novel compound, derived by the opening of the lactone ring.

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. barely 7-cm in 2-PE, an equivalent resolving power is found. Moreover, due to the possibility of running up to seven Immobiline strips in the radial gel format, the reproducibility of spot position is seen to be higher, this resulting in a 20% higher matching efficiency. As an extra bonus, strings of “isobaric” spots (i.e. polypeptides of identical mass with different pI values) are more resolved in the radial gel format, especially in the 10 to 30 kDa region, where the gel area fans out leaving extra space for spot resolution. In conclusion, this novel gel format in the second dimension of 2D gels is seen as an important improvement of this technique, still one of the most popular in proteome analysis.

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. Our aim is to make a critical overview of the studies involving primary cultures of skin fibroblasts as tools to investigate the pathophysiology of diabetic nephropathy performed until now in this area. Cultured skin fibroblasts could be useful not only for the identification of patients at risk of developing diabetic renal disease, but also for a better understanding of the complex multifactorial mechanisms leading to the long-term complications in diabetes. Copyright © 2011 John Wiley & Sons, Ltd.

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. The response to insulin of plasma protein synthesis (albumin and fibrinogen) is also normal. However, some metabolic steps of amino acids related to vascular complications (methionine and arginine) exhibit a defective response to insulin in T2DM subjects with nephropathy. In summary, although gross alterations in the response of whole-body protein turnover are not evident in T2DM, specific investigations reveal subtle abnormalities in metabolic steps of selected amino acids. Furthermore, the effects of interaction between diabetes (with the associated insulin resistance) and older age in the pathogenesis of sarcopenia in the elderly deserve more specific studies.

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. Hence, we analyzed caldesmon gene and protein expression in cultured fibroblasts from T1DM patients with and without nephropathy and from control subjects. Caldesmon gene was studied in cells cultured under normal glucose levels by quantitative real-time RT-PCR. Caldesmon protein isoforms were quantified both under normal and high glucose conditions by two-dimensional electrophoresis. Caldesmon gene was over-expressed in fibroblasts from diabetic patients with nephropathy, in comparison to both those from diabetic patients without nephropathy and those from controls. We quantified six caldesmon protein isoforms, two of them were increased whereas another one was decreased only in fibroblasts from diabetic patients with nephropathy. None of these isoforms showed any difference in their relative abundance in response to high glucose. Variable results in response to high glucose were observed in the expression of other proteins in the three experimental groups. Our data lend further support to an involvement of caldesmon in the susceptibility to diabetic nephropathy in type 1 diabetes, independently from environmental glucose levels.

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. The overall variability which affects 2-DE includes biological, experimental, and post-experimental (software-related) variance. It is important to highlight how much of the total variability of this technique is due to post-experimental variability, which, so far, has been largely neglected. In this short review, we have focused on this topic and explained that post-experimental variability and source of error can be further divided into those which are software-dependent and those which are operator-dependent. We discuss these issues in detail, offering suggestions for reducing errors that may affect the quality of results, summarizing the advantages and drawbacks of each approach.

A new arylnaphthalene lignan glycoside, patavine (1), together with five known lignans, justicidin B (2), diphyllin (3), tuberculatin (4), majidine (5), and arabelline (6) were isolated from shoot cultures of Haplophyllum patavinum. The structure of the new compound was elucidated by extensive one-dimensional (1D) and two-dimensional (2D) NMR experiments and mass spectrometry. The cytotoxicity of compounds 1, and 3-6 against LoVo human colon carcinoma cells was investigated.

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. Experimental variability depends on physical and chemical parameters, whereas post-experimental variability arises when gels are analyzed by different software packages, particularly when different workflows are followed. In this paper, we compared the analysis performance of two software packages, Delta2D and Proteomweaver, using both standard and experimental gel images. Using standard gel images, the false negative spot count was 50% lower, the false positive count was 77% lower, the true positive count was 19% higher and spot matching was 4% higher in Delta2D when compared to Proteomeweaver. Using experimental gel images, we found that the total amount of time taken to complete the analysis with Delta2D was 30% that of the time needed with Proteomweaver and required fewer user interventions. The differences between ease of use and workflow strategy of these programs is discussed.

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.Five subjects were studied after 6 days of HCl-, CaCl(2)-, and NH(4)Cl-induced acidosis; 8 subjects served as controls. Blood samples were collected from the radial artery and the hepatic veins. Measurements were performed on plasma and whole-blood samples.Based on plasma measurements, subjects who had undergone CMA had lower rates of splanchnic proteolysis (-35%) and protein synthesis (-50%; P.05) than controls, as well as a negative leucine kinetic balance (-6.81 +/- 2.48 micromol/kg/min/1.73 m(2) body surface [BS](-1)), compared with the neutral balance in control plasma samples (0.76 +/- 2.11 micromol/kg/min/1.73; P.05 between groups). Based on measurements from whole blood, splanchnic proteolysis and protein synthesis did not differ significantly between CMA and control samples, and the net leucine kinetic balance was neutral in both groups (CMA, -0.69 +/- 1.57; controls, -0.74 +/- 3.45 micromol/kg/min/1.73). In CMA whole-blood measurements, splanchnic oxygen consumption (44.8 +/- 4.3 mL/min/1.73 m(2) BS) was slightly lower than in controls (57.5 +/- 8.4 mL/min/1.73 m(2) BS; P = NS). Splanchnic protein synthesis correlated with oxygen consumption (r = 0.82; P.001).CMA reduces splanchnic protein turnover and results in a negative leucine balance--an effect that apparently is offset by the contribution of blood cells to organ leucine (and protein) dynamics. Protein synthesis is a major contributor (about 67%) to energy expenditure in splanchnic organs.

Background Although hyperfibrinogenemia and insulin resistance are common in obesity and diabetes mellitus, the impact of obesity per se on fibrinogen turnover and the insulin effects on fibrinogen and protein kinetics is unknown. Methods We measured fibrinogen and albumin fractional (FSR) and absolute (ASR) synthesis rates, as well as protein turnover, in non-diabetic, obese and in control male subjects both before and following an euglycemic, euaminoacidemic, hyperinsulinemic clamp, using L-[2H3]-Leucine isotope infusion. Results In the obese, basal fibrinogen concentrations was ≈ 25% greater (p < 0.035), and fibrinogen pool ≈ 45% greater (p < 0.005), than in controls. Both FSR and ASR of fibrinogen were similar to control values. With hyperinsulinemia, although fibrinogen FSR and ASR were not significantly modified with respect to baseline in either group, fibrinogen ASR resulted to be ≈ 50% greater in the obese than in controls (p < 0.015). Hyperinsulinemia equally stimulated albumin synthesis and suppressed leucine appearance from endogenous proteolysis in both groups. Amino acid clearance was also similar. In the obese, the insulin-mediated glucose disposal was ≈ 50% lower (p < 0.03) than in controls, and it was inversely correlated with fibrinogen ASR during the clamp in both groups (r = − 0.58). Conclusions In obese, non-diabetic males, post absorptive fibrinogen production is normal. Whole-body amino acid disposal, basal and insulin-responsive protein degradation, and albumin synthesis are also normal. However, the greater fibrinogen ASR in the obese with hyperinsulinemia, and the inverse relationship between insulin sensitivity and clamp fibrinogen production, suggest a role for hyperinsulinemia and/or insulin resistance on fibrinogen production in obesity. Copyright © 2009 John Wiley & Sons, Ltd.

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 (20.8 +/- 1.6%/day) nor ASR (1.8 +/- 0.1 g/day), or concentration (274 +/- 15 mg/dl), were different from those of the younger group (FSR: 20.2 +/- 1.4; ASR: 1.7 +/- 0.2; concentration: 265 +/- 8, respectively). Leucine Ra, an index of endogenous proteolysis, was approximately 20% lower in the older than in the younger group (P0.02). Thus, middle age in males is not associated with increased fibrinogen concentration and turnover, whereas endogenous protein breakdown in decreased. Factor(s) different from age per se are likely to be involved in the dysfibrinogenemia possibly occurring with ageing. Protein turnover is already reduced in middle-age males.

Purpose of review Phenylalanine conversion to tyrosine (i.e., 'hydroxylation') is the first irreversible step in phenylalanine catabolism and a source of circulating tyrosine. The purpose of the present review is both to examine hydroxylation from a biochemical standpoint and to report data measured in vivo under physiological conditions, as well as in liver and kidney disease. Recent findings The simultaneous infusion of phenylalanine and tyrosine tracers in humans allows us to determine the hydroxylation rate in vivo. Hydroxylation accounts for a minor ( approximately 10-20%) although significant portion of tyrosine flux. The liver and the kidney are the key organs accounting for virtually the whole-body hydroxylation rates. It is regulated by substrate availability, being acutely stimulated by mixed meal ingestion and by dietary adaptation to high phenylalanine intakes. Theoretically, it may be impaired in advanced liver and kidney disease. Nevertheless, in compensated liver cirrhosis, hydroxylation as well as tyrosine flux are not decreased but rather increased. Only in end stage liver disease hydroxylation may be impaired and is corrected by transplantation. Hydroxylation is also reduced in end stage renal disease. Summary Phenylalanine hydroxylation in vivo appears to represent a regulatory step of phenylalanine disposal and tyrosine production under acute and/or extreme conditions.

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. We have compared this method with a S-35-thiolactone radioactive binding assay. Incubations with and without dithiothreitol allowed measurement of the amide-linked and disulfide-linked thiolactone-protein adducts, respectively. A good correspondence in time- and dose-dependent protein-thiolactone formation was observed between the two methods. A maximum of 9 to 12 thiolactone residues were bound to each albumin molecule. The S-35-thiolactone bound albumin tightly, particularly at the lowest concentrations, with approximate to 70% of the binding amide-linked. Although the results of the two methods were rather similar, the radioactive method appears to be more sensitive than the MALDI-MS technique. Copyright (C) 2009 John Wiley & Sons, Ltd.

In plasma and serum, the presence of high-abundance proteins can overwhelm the signals of low-abundance proteins, which then become undetectable either by two-dimensional gels or chromatographic techniques. Therefore, depletion of abundant proteins is a prerequisite to detect low-abundance components. Furthermore, the regeneration of pre-purification tools could be money-saving. We applied an affinity chromatography kit to remove albumin and the immunoglobulin chains from plasma and propose a simple and effective technical procedure for the regeneration of these affinity columns.

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. The [DN+] T1DM patients, compared with the two other groups, exhibited increased abundance of a high-molecular weight isoform of protein disulphide-isomerase A3 and a decrease of two low-molecular weight isoforms. They also had increased levels of heat shock protein (HSP) 60 kDa isoform #A4, of HSP71 kDa isoform #A30, and of HSP27 kDa isoform #6, whereas the HSP27 kDa isoforms #A90 and #A71 were decreased. Cathepsin beta-2 (#40), the cation-independent mannose 6-phosphate receptor binding protein 1 (CIMPR) (#A27), and annexin 2 (#A9) were also decreased in the [DN+] T1DM patients, whereas the RNA-binding protein regulatory subunity (#38) and the translationally-controlled tumor protein (TCTP) (#A45) were increased. These changes of chaperone-like proteins in fibroblasts may highlight those of the kidney and be patho-physiologically related to the development of nephropathy in T1DM.

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.Design: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry.Results: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and α-actinin 4 and a decrease of tubulin-β2 and splice isoform 2 of tropomyosin β-chain were detected.Conclusions: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.

OBJECTIVE—Insulin stimulates albumin synthesis but inhibits that of fibrinogen in both type 1 diabetic and healthy subjects. In type 2 diabetes, fibrinogen production is increased both in the postabsorptive state and in response to hyperinsulinemia. No data exist on the rate of albumin synthesis and its response to insulin in type 2 diabetes. RESEARCH DESIGN AND METHODS—We measured fractional synthesis rates (FSRs) and absolute synthesis rates (ASRs) of both albumin and fibrinogen in postabsorptive normoalbuminuric type 2 diabetic patients at their spontaneous glucose levels (study A), as well as albumin FSR and ASR before and after a hyperinsulinemic-euglycemic euaminoacidemic clamp (study B), using leucine isotope methods. RESULTS—In postabsorptive type 2 diabetes (study A), albumin FSR (11.2 ± 0.9%/day) and albumin ASR (15.4 ± 1.2 g/day) were not different from control values (albumin FSR: 9.4 ± 0.7%/day; albumin ASR: 13.8 ± 1.2 g/day, P > 0.1 for both). In contrast, in the type 2 diabetic subjects, both fibrinogen FSR (24.9 ± 2.1%/day) and ASR (2.4 ± 0.2 g/day) were greater (P < 0.025 and P < 0.007, respectively) compared with the control subjects (FSR: 18.6 ± 1.51%/day; ASR: 1.6 ± 0.2 g/day). Worse metabolic control in the type 2 diabetic patients was associated with hyperfibrinogenemia and increased leucine rate of appearance, whereas neither the (increased) fibrinogen ASR nor the (normal) albumin production was affected. In study B, after hyperinsulinemia (raised to ∼860 nmol/l), albumin FSR and ASR increased by ∼25% versus basal (P < 0.04) and to the same extent in both type 2 diabetic and control subjects. CONCLUSIONS—In normoalbuminuric type 2 diabetic patients, postabsorptive albumin synthesis and its response to insulin were normal, whereas fibrinogen synthesis was increased, irrespective of metabolic control. Furthermore, in normoalbuminuric type 2 diabetic patients, a normal insulin sensitivity with respect to albumin production but a selective hepatic dysregulation of fibrinogen metabolism were present.

A widespread occurrence of melatonin in plant kingdom has been reported. The circadian rhythm in the level of melatonin observed in both unicellular algae and higher plants, suggests a role in regulation of photoperiodic and rhythmic phenomena, i.e. a similar function for melatonin in both plants and animals. Evidence has been obtained for a role of melatonin in plant morphogenesis, but more research is needed to ascertain other suggested physiological roles in higher plants (seed dormancy regulation, radical scavenger activity, interaction with calmodulin) as well the ecological significance of the high melatonin levels recorded in alpine plants. Setting-up more reliable analytical methods for melatonin detection and quantification is a basic requirement to get more insight into melatonin roles in plant physiology and ecology.

Fruit's decoctions of Passiflora edulis and P. foetida var. albiflora were evaluated for the inhibition of activity of gelatinase MMP-2 and MMP-9, two metallo-proteases involved in the tumour invasion, metastasis and angiogenesis. Both water extracts, at different concentrations, inhibited the enzymes.

A widespread occurrence of melatonin in plant kingdom has been reported. The circadian rhythm in the level of melatonin observed in both unicellular algae and higher plants, suggests a role in regulation of photoperiodic and rhythmic phenomena, i.e. a similar function for melatonin in both plants and animals. Evidence has been obtained for a role of melatonin in plant morphogenesis, but more research is needed to ascertain other suggested physiological roles in higher plants (seed dormancy regulation, radical scavenger activity, interaction with calmodulin) as well the ecological significance of the high melatonin levels recorded in alpine plants. Setting-up more reliable analytical methods for melatonin detection and quantification is a basic requirement to get more insight into melatonin roles in plant physiology and ecology.

A new arylnaphthalene lignan glycoside, patavine (1), together with five known lignans, justicidin B (2), diphyllin (3), tuberculatin (4), majidine (5), and arabelline (6) were isolated from shoot cultures of Haplophyllum patavinum. The structure of the new compound was elucidated by extensive one-dimensional (1D) and two-dimensional (2D) NMR experiments and mass spectrometry. The cytotoxicity of compounds 1, and 3-6 against LoVo human colon carcinoma cells was investigated.

A protocol for shoot induction from callus of Haplophyllum patavinum was established. Two known furoquinoline (skimmianine and haplopine), and three quinolone (edulinine, ribalinine and isoplatydesmine) alkaloids were isolated for the first time from plant material, callus and shoot cultures of this species. The structures of these compounds have been characterised on the basis of spectroscopic evidence.

Three known arylnaphthalene lignans (justicidin B, diphyllin and tuberculatin), and one dibenzylbutyrolactone (arctigenin) were isolated from native plants, callus and suspension cultures of Haplophyllum patavinum. The present paper represents the first report on arylnaphthalene lignan production by in vitro cultures; in vitro, the biogenetic potential is strictly dependent on the cell strain.

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. By employing a precursor-product tracer method in humans, we have directly estimated the acute effect of insulin on intravascular NOx(i.e., the NO oxidation products) fractional (FSR) and absolute (ASR) synthesis rates in vivo. Nine healthy male volunteers were infused iv with l-[15N2-guanidino]arginine ([15N2]arginine) for 6 h. Timed measurements of15NOxand [15N2]arginine enrichments in whole blood were performed in the first 3 h in the fasting state and then following a 3-h euglycemic-hyperinsulinemic clamp (with plasma insulin raised to ≈1,000 pmol/l). In the last 60 min of each experimental period, at ≈steady-state arginine enrichment, a linear increase of15NOxenrichment (mean r = 0.9) was detected in both experimental periods. In the fasting state, NOxFSR was 27.4 ± 4.3%/day, whereas ASR was 0.97 ± 0.36 mmol/day, accounting for 0.69 ± 0.27% of arginine flux. Following hyperinsulinemia, both FSR and ASR of NOxincreased (FSR by ≈50%, to 42.4 ± 6.7%/day, P < 0.005; ASR by ≈25%, to 1.22 ± 0.41 mmol/day, P = 0.002), despite a ≈20–30% decrease of arginine flux and concentration. The fraction of arginine flux used for NOxsynthesis was doubled, to 1.13 ± 0.35% ( P < 0.003). In conclusion, whole body NOxsynthesis can be directly measured over a short observation time with stable isotope methods in humans. Insulin acutely stimulates NOxsynthesis from arginine.