Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

Proteins from bovine tissues and biological fluids: defining a reference electrophoresis map for liver,kidney, muscle,plasma and red blood cells

A number of high resolution two-dimensional electrophoresis (2-DE) reference maps for bovine tissues and biological fluids have been determined for animals in basal state. Among the 1863 distinct protein features detected in samples of liver, kidney, muscle, plasma and red blood cells, 509 species were identified and associated to 209 different genes. Difficulties in the identification were related to the poorly characterized Bos taurus genome and were solved by a combined matrix-assisted laser desorption/ionisation-mass spectrometry and liquid chromatography-electrospray ionization tandem mass spectrometry approach. The experimental output allowed us to establish a 2-DE database accessible through the World Wide Web network at the URL address (http://www.iabbam.na.cnr.it/Biochem). These reference maps may serve as a tool in future veterinary medical studies aimed at the evaluation of changes in protein repertoire for altered animal physiological conditions and infectious diseases, to the definition of molecular markers for novel diagnostic kits and vaccines, as well as the characterization of protein modifications in bovine materials following technological processes used in the food industry.     

Melusin,a muscle-specific integrin beta1-interacting protein,is required to prevent cardiac failure in response to chronic pressure overload

Cardiac hypertrophy is an adaptive response to a variety of mechanical and hormonal stimuli, and represents an early event in the clinical course leading to heart failure. By gene inactivation, we demonstrate here a crucial role of melusin, a muscle-specific protein that interacts with the integrin beta1 cytoplasmic domain, in the hypertrophic response to mechanical overload. Melusin-null mice showed normal cardiac structure and function in physiological conditions, but when subjected to pressure overload--a condition that induces a hypertrophic response in wild-type controls--they developed an abnormal cardiac remodeling that evolved into dilated cardiomyopathy and contractile dysfunction. In contrast, the hypertrophic response was identical in wild-type and melusin-null mice after chronic administration of angiotensin II or phenylephrine at doses that do not increase blood pressure--that is, in the absence of cardiac biomechanical stress. Analysis of intracellular signaling events induced by pressure overload indicated that phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) was specifically blunted in melusin-null hearts. Thus, melusin prevents cardiac dilation during chronic pressure overload by specifically sensing mechanical stress.     

Left ventricular end-diastolic pressure-volume relationship in septic rats with open thorax

To estimate changes in compliance, we evaluated the effects of sepsis on the end-diastolic pressure-volume relationship (EDPVR) in the left ventricle of rats that had undergone an open thorax procedure. Sepsis was induced in male Wistar Hannover rats (n = 7; 240 to 270 g) by intraperitoneal administration of a slurry of cecal contents; control rats (n = 7) were given 5% dextrose only. On the third day after induction of sepsis, left ventricular (LV) pressure and LV dimensions were recorded simultaneously in animals of both groups. Using a micromanometer and ultrasonic crystals, measurements were obtained at baseline and during the increase of afterload. Blood samples were taken for determination of complete blood count, white blood cell differential count, and lactate concentration, and for bacteriologic examination. Septic rats lost weight, and developed changes in body temperature, ascites, and abscesses in the abdominal and thoracic cavities, gram-negative bacteremia, and increase in heart rate. On the third day after induction of sepsis, LV EDPVR decreased, compared with that in the control rats (regression coefficients: control group, 8.41 to 23.95; sepsis group, 3.94 to 7.92). Myocardial compliance in the left ventricle increased on the third day of sepsis in the open-thorax rat model, as evidenced by the downward shift of LV EDPVR in rats with sepsis, compared with controls.     

Antiendotoxin activity of protegrin analog IB-367 alone or in combination with piperacillin in different animal models of septic shock

The therapeutic efficacy of protegrin peptide IB-367 was investigated in three rat models of septic shock: (i) rats injected intraperitoneally with 1mg Escherichia coli 0111:B4 lipopolysaccharide, (ii) rats given an intraperitoneal injection of 2 X 10(10) CFU of E. coli ATCC 25922, and (iii) rats in which intra-abdominal sepsis was induced via cecal ligation and puncture. All animals were randomized to receive parenterally isotonic sodium chloride solution, 1mg/kg of IB-367, 60mg/kg piperacillin and 1mg/kg of IB-367 plus 60mg/kg piperacillin. The peptide demonstrated lower level of antimicrobial activity than piperacillin, nevertheless it exhibited the dual properties of antimicrobial and antiendotoxin agent. Finally IB-367 and piperacillin association showed to be the most effective therapeutic approach.     

Ca2+-independent protein kinase C signalling in mouse eggs during the early phases of fertilization

Protein kinase C (PKC), an enzyme playing a central role in signal transduction pathways, is activated in fertilized mouse eggs downstream of the fertilization Ca2+ signal, to regulate different aspects of egg activation. Given the presence of Ca2+-independent PKC isoforms within the egg, we investigated whether fertilization triggers PKC stimulation in mouse eggs by activating Ca2+-independent signalling pathways. An increase in PKC activity was detected as early as 10 min after the beginning of insemination, when about 90% of eggs had fused with sperm and the first Ca2+ rise was evident in most of the eggs. A similar level of activity was found 20 min later, when about 60% of eggs had resumed meiosis. When the Ca2+ increase was buffered by an intracellular Ca2+ chelating agent, PKC stimulation was not blocked but only slightly reduced. Confocal microscopy analysis revealed that the increase in PKC activity at fertilization coincided with the translocation of PKCdelta, a Ca2+-independent and diacylglycerol-dependent PKC isoform, to the meiotic spindle. When, in the absence of the Ca2+ signal, metaphase-anaphase transition was inhibited, PKCdelta moved to the meiotic spindle but still maintained a sustained cytoplasmic distribution. In summary, our results indicate that: 1) PKC activation is an early event of egg activation; 2) both Ca2+-dependent and Ca2+-independent pathways contribute to increased PKC activity at fertilization; 3) PKCdelta is one of the isoforms participating in this signalling process.     

Anorexia nervosa and refusal of naso-gastric treatment: a response to Heather Draper

Imposing artificial feeding on people with anorexia nervosa may be unethical. This seems to be Heather Draper's suggestion in her article, 'Anorexia Nervosa and Respecting a Refusal of Life-Prolonging Therapy: A Limited Justification.' Although this is an important point, I shall show that the arguments supporting this point are flawed. Draper should have made a brave claim: she should have claimed that people with anorexia nervosa, who competently decide not to be artificially fed, should be respected because everybody is entitled to exercise their autonomy, not only 'in the middle' of their life, but also at the end of it, or when their own life is at stake, because autonomy also extends to the most difficult moments of our life, and, ultimately, 'stretches [.*T*.*T*.] far out into the distance' at the end of it. I explain why Draper should have made the brave claim, and why she has not made it. I conclude that a defence of people's entitlement to competently refuse artificial feeding cannot rest upon the arguments developed by Draper. Whether or not we should respect competent refusal of artificial feeding depends on the normative strength that we are ready to ascribe to the principle of autonomy, to the moral relevance that we ascribe to the circumstances in which a person's autonomy is exercised, and, perhaps, eventually, on our sense of compassion.     

Stable analogues of geranylgeranyl diphosphate possessing improved geranylgeranyl versus farnesyl protein transferase inhibitory selectivity

Phosphonoacetamido(oxy) groups have proven to be good mimics of the diphosphate portion in geranylgeranyl protein transferase I (GGTase I) inhibitors. The introduction of small alkyl groups (Me, Et) into the diphosphate mimic moiety caused a further decrease in collateral farnesyl protein transferase (FTase) inhibitory activity, thereby improving GGTase I over FTase selectivity.     

Secretin, glucagon, gastric inhibitory polypeptide, parathyroid hormone, and related peptides in the regulation of the hypothalamus- pituitary-adrenal axis

Secretin, glucagon, gastric inhibitory polypeptide (GIP), and parathyroid hormone (PTH) belong, together with vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase (AC)-activating polypeptide, to a family of peptides (the VIP-secretin-glucagon family), which also includes growth hormone-releasing hormone and exendins. All the members of this peptide family possess a remarkable amino-acid sequence homology, and bind to G-protein-coupled receptors, whose signaling mechanism primarily involves AC/protein kinase A and phospholipase C/protein kinase C cascades. VIP and pituitary AC-activating polypeptide play a role in the regulation of the hypothalamus-pituitary-adrenal (HPA) axis, and in this review we survey findings that also other members of the VIP-secretin-glucagon family may have the same function. Secretin and secretin receptors are expressed in the hypothalamus and pituitary gland, and secretin inhibits adrenocorticotropic hormone (ACTH) release. No evidence is available for the presence of secretin receptors in adrenal glands, but secretin selectively depresses the glucocorticoid response to ACTH of dispersed zona fasciculata-reticularis (ZF/R) cells. Glucagon and glucagon-like peptide-1 are contained in the hypothalamus, and all the components of the HPA axis are provided with glucagon and glucagons-like-1 receptors. These peptides exert a short-term inhibitory effect on stress-induced pituitary ACTH release and depress the ZF/R cell response to ACTH by inhibiting the AC/protein kinase A cascade; they also stimulate hypothalamic arginine-vasopressin release. GIP receptors are present in the ZF/R of the normal adrenals, and are particularly abundant in some types of adrenocortical adenomas and hyperplasias. GIP, through the activation of the AC/protein kinase A cascade, evokes a sizeable glucocorticoid secretagogue effect, leading to the identification of a food/GIP-dependent Cushing's syndrome. PTH and PTH-related protein are expressed in the hypothalamus and pituitary gland, and PTH and PTH-related protein receptors in all the components of the HPA axis. Both peptides enhance ACTH and arginine-vasopressin release, as well as stimulate aldosterone and glucocorticoid secretion of dispersed zona glomerulosa and ZF/R cells, respectively. The involvement of growth hormone-releasing hormone and exendins in the functional regulation of the HPA axis has not yet been extensively investigated.