Nicoletta Gagliano

Actin carbonylation: from a simple marker of protein oxidation to relevant signs of severe functional impairment.

The number of protein-bound carbonyl groups is an established marker of protein oxidation. Recent evidence indicates a significant increase in actin carbonyl content in both Alzheimers disease brains and ischemic hearts. The enhancement of actin carbonylation, causing the disruption of the actin cytoskeleton and the loss of the barrier function, has also been found in human colonic cells after exposure to hypochlorous acid (HOCl). Here, the effects of oxidation induced by HOCl on purified actin are presented. Results show that HOCl causes a rapidly increasing yield of carbonyl groups. However, when carbonylation becomes evident, some Cys and Met residues have been already oxidized. Covalent intermolecular cross-linking as well as some noncovalent aggregation of carbonylated actin have been found. The covalent cross-linking, unaffected by reducing and denaturing agents, parallels an increase in dityrosine fluorescence. Moreover, HOCl-mediated oxidation induces the progressive disruption of actin filaments and the inhibition of F-actin formation. The molar ratios of HOCl to actin that lead to inhibition of actin polymerization seem to have effect only on cysteines and methionines. The process that involves oxidation of amino acid side chains with formation of a carbonyl group would occur at an extent of oxidative insult higher than that causing the oxidation of some critical amino acid residues. Therefore, the increase in actin content of carbonyl groups found in vivo would indicate drastic oxidative modification leading to drastic functional impairments.

Methionine oxidation as a major cause of the functional impairment of oxidized actin

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimers disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.

Mechanisms of Aging and Liver Functions

Background/Aims: Morphofunctional studies suggest that the liver, compared with other organs, ages fairly well. Its success is ascribable to its lasting ability to regenerate, even if the potential of the cells to replicate progressively declines with age. The aim of this study was to analyze some aspects of the early phases of liver regeneration, its capacity to mount a stress response, and the inflammatory response in the early stage of an acute injury. Methods: Rats aged 2, 6, 12 and 19 months received a single intraperitoneal injection of CCl4, and morphological, biochemical and molecular evaluations were done 2 and 24 h later. Results: AST and ALT, starting at age 12 months, were significantly higher than in the younger rats after CCl4. Histological modifications were already detectable after 2 h in rats aged 12 and 19 months, thereafter becoming more diffuse and marked, whereas they become evident only 24 h after the intoxication in rats aged 2 and 6 months. Albumin, c-fos, c-myc, hepatocyte growth factor, transforming growth factor-α and HSP70 mRNA levels decreased 24 h after CCl4 starting at age 12 months. Mast cell density was higher in the young rats than the old ones. Conclusion: Our results point to: (a) a basically preserved regenerative response of the aged liver, although somehow weaker and slower, with reduced ability to counteract agents inducing cell necrosis; (b) a decrease in the HSP70 response suggesting a reduction in homeostatic capacity, and (c) a lower inflammatory response during aging.

Age-dependent expression of fibrosis-related genes and collagen deposition in the rat myocardium

OBJECTIVES We sought to characterize the evolution, during maturational growth and early ageing, of the messenger abundance of four genes involved in cardiac fibrosis regulation (procollagens alpha2(I) and alpha1(III), transforming growth factors beta1, and beta3) and corroborate it with the alterations in collagen deposition in cardiac interstitium and around coronary arteries. METHODS Messenger RNA was quantified in LV and RV of 2-, 6-, 12- and 19-month-old male Sprague-Dawley rats (n = 5 per group) with Northern blot analysis. Collagen deposition was quantified with a semi-automated image analyser on Sirius red-stained sections of LV tissue. RESULTS There was an age-related monotonous decrease of procollagen type I (COL-I) transcript abundance in LV (p < 0.001) but not in RV. Procollagen type III (COL-III) expression decreased rapidly during maturational growth, both in LV and RV. On the other hand, collagen deposition in myocardial interstitium and around coronary arteries was slightly augmented during the maturational period of life (2-12 months), but with a higher rate during early ageing (up to 19 months). This was not accompanied by a significant thickening of the wall of coronary arteries. Transforming growth factor beta1, (TGF-beta1) and transforming growth factor beta3 (TGF-beta3) transcript abundance showed no major variations during ageing. CONCLUSIONS These results reflect a striking ventricular difference regarding the age-dependent expression of COL-I. The expression of TGF-beta(s), pleiotropic factors known to influence collagen pathway at different levels, does not seem to be profoundly altered during ageing. The discrepancy between protein and COL-I and COL-III mRNA levels indicates differences in age-related mRNA stability and/or regulation of collagen translation.

Mechanical Power and Development of Ventilator-induced Lung Injury.

Background:The ventilator works mechanically on the lung parenchyma. The authors set out to obtain the proof of concept that ventilator-induced lung injury (VILI) depends on the mechanical power applied to the lung. Methods:Mechanical power was defined as the function of transpulmonary pressure, tidal volume (TV), and respiratory rate. Three piglets were ventilated with a mechanical power known to be lethal (TV, 38 ml/kg; plateau pressure, 27 cm H2O; and respiratory rate, 15 breaths/min). Other groups (three piglets each) were ventilated with the same TV per kilogram and transpulmonary pressure but at the respiratory rates of 12, 9, 6, and 3 breaths/min. The authors identified a mechanical power threshold for VILI and did nine additional experiments at the respiratory rate of 35 breaths/min and mechanical power below (TV 11 ml/kg) and above (TV 22 ml/kg) the threshold. Results:In the 15 experiments to detect the threshold for VILI, up to a mechanical power of approximately 12 J/min (respiratory rate, 9 breaths/min), the computed tomography scans showed mostly isolated densities, whereas at the mechanical power above approximately 12 J/min, all piglets developed whole-lung edema. In the nine confirmatory experiments, the five piglets ventilated above the power threshold developed VILI, but the four piglets ventilated below did not. By grouping all 24 piglets, the authors found a significant relationship between the mechanical power applied to the lung and the increase in lung weight (r2 = 0.41, P = 0.001) and lung elastance (r2 = 0.33, P < 0.01) and decrease in PaO2/FIO2 (r2 = 0.40, P < 0.001) at the end of the study. Conclusion:In piglets, VILI develops if a mechanical power threshold is exceeded.

S-Glutathiolation in life and death decisions of the cell

Abstract Reversible S-glutathiolation of specific proteins at sensitive cysteines provides a powerful mechanism for the dynamic, post-translational regulation of many cellular processes, including apoptosis. Critical in ascribing any regulatory function to S-glutathiolation is its reversibility, mainly regulated by glutaredoxins. Apoptosis is a controlled form of cell death that plays fundamental roles during embryonic development, tissue homeostasis and some diseases. Much of what happens during the demolition phase of apoptosis is orchestrated primarily by caspases, the final executioners of cell death. Recent findings support an essential role for S-glutathiolation in apoptosis, often at the level of caspases or their inactive precursors, and several studies have demonstrated the importance of glutaredoxins in protecting against apoptosis. These observations have contributed to recent advances in apoptosis research. However, the effective relevance of protein S-glutathiolation and the precise molecular targets in apoptotic signalling remain unresolved and a key challenge for future research.

The potential of resveratrol against human gliomas.

There is growing interest in dietary phytochemicals as potential cancer chemopreventive agents. Resveratrol (3,4′,5-trihydroxy-trans-stilbene), a naturally occurring phytoalexin that is present in grapes, red wine, berries and peanuts, has been studied extensively for its ability to interfere with multistage carcinogenesis. Resveratrol is known to have antioxidant, anti-inflammatory and antiproliferative effects on a variety of cancer cells in vitro and in various animal models. However, the effect(s) of resveratrol in vivo on humans are still controversial. This study discusses current knowledge with regard to the effects of resveratrol in relation to its potential as a chemopreventive and/or chemotherapeutic molecule against human gliomas.

AKAP‐4: a novel cancer testis antigen for multiple myeloma

Purpose Immunotherapy promises to be a more gentle and successful cancer treatment when compared with current standard treatments. Multiple myeloma (MM) is still a fatal hematologic malignancy that represents approximately 1% of all cancers and 2% of all cancer deaths. Approximately 50,000 Americans currently have MM. The research to discover new suitable cancer targets is needed to improve the effects of immunotherapy. The AKAP family9s protein provides an organizing center about which various protein kinases and phosphatases can be assembled to create solid-state signaling devices that can signal, be modulated, and be trafficked within the cell. A member of this family, AKAP-4, is the focus of our study. Human AKAP-4 is a structural protein of the sperm fibrous sheath that also functions to anchor protein kinase A to this structure via the regulatory subunit of the kinase, and seems to be involved in sperm motility. Our aim was to investigate the presence of AKAP-4 as a novel cancer testis antigen target in MM patients. Methods We evaluated the expression of AKAP-4 mRNA in a normal panel of tissues and in 15 MM patients either by PCR and immunocytochemistry. The normal control investigated tissues were kidney, ovary, skeletal muscle, mammary, brain, heart, colon, stomach, liver, lung, pancreas, spleen, trachea, and bone marrow. Summary The analysis of the mRNA expression of AKAP-4 showed that none of the normal tissues produced any positive band signals, whereas 6 of the 15 investigated patients (40%) showed a positive band signal. The immunohistochemical approach to the normal tissue panel showed no staining in any of the evaluated organs, except for the control, the testis. Five of the 15 MM investigated cases (33.3%) showed positive cytoplasmic staining. Conclusion To our knowledge, we established for the first time that AKAP-4 is expressed at the transcriptional level in MM cases, with a rate of 40%, whereas it is not expressed in normal tissues. Immunocytochemical data confirmed the PCR observations even if with a slightly lower percentage rate (33.3%). Since AKAP-4 has yet not been studied in MM, this is the first study that gives evidence of its aberrant expression in MM and suggests its use as possible novel cancer testis antigen target in MM.

Sperm Protein 17 Is Expressed in Human Somatic Ciliated Epithelia

It was once believed that sperm protein 17 (Sp17) was expressed exclusively in the testis and that its sole function was to bind to the oocyte during fertilization. However, immunohistochemistry of the human respiratory airways and reproductive systems show that it is abundant in ciliated cells but not in human cells with stereocilia and microvilli. The high degree of sequence conservation throughout its N-terminal half, and the presence of an A-kinase anchoring protein (AKAP)-binding motif within this region, suggest that Sp17 plays a regulatory role in a PKA-independent AKAP complex in both male germinal and ciliated somatic cells.

Isolation and Characterization of 2 New Human Rotator Cuff and Long Head of Biceps Tendon Cells Possessing Stem Cell–Like Self-Renewal and Multipotential Differentiation Capacity:

Background: Stem cell therapy is expected to offer new alternatives to the traditional therapies of rotator cuff tendon tears. In particular, resident, tissue-specific, adult stem cells seem to have a higher regenerative potential for the tissue where they reside. Hypothesis: Rotator cuff tendon and long head of the biceps tendon possess a resident stem cell population that, when properly stimulated, may be induced to proliferate, thus being potentially usable for tendon regeneration. Study Design: Controlled laboratory study. Methods: Human tendon samples from the supraspinatus and the long head of the biceps were collected during rotator cuff tendon surgeries from 26 patients, washed with phosphate-buffered saline, cut into small pieces, and digested with collagenase type I and dispase. After centrifugation, cell pellets were resuspended in appropriate culture medium and plated. Adherent cells were cultured, phenotypically characterized, and then compared with human bone marrow stromal cells (BMSCs), as an example of adult stem cells, and human dermal fibroblasts, as normal proliferating cells with no stem cell properties. Results: Two new adult stem cell populations from the supraspinatus and long head of the biceps tendons were isolated, characterized, and cultured in vitro. Cells showed adult stem cell characteristics (ie, they were self-renewing in vitro, clonogenic, and multipotent), as they could be induced to differentiate into different cell types—namely, osteoblasts, adipocytes, and skeletal muscle cells. Conclusion: This work demonstrated that human rotator cuff tendon stem cells and human long head of the biceps tendon stem cells can be isolated and possess a high regenerative potential, which is comparable with that of BMSCs. Moreover, comparative analysis of the sphingolipid pattern of isolated cells with that of BMSCs and fibroblasts revealed the possibility of using this class of lipids as new possible markers of the cell differentiation status. Clinical Relevance: Rotator cuff and long head of the biceps tendons contain a stem cell population that can proliferate in vitro and could constitute an easily accessible stem cell source to develop novel therapies for tendon regeneration.