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Metallothioneins (MTs) are noncatalytic peptides involved in storage of essential ions, detoxification of nonessential metals, and scavenging of oxyradicals. They exhibit an unusual primary sequence and unique 3D arrangement. Whereas vertebrate MTs are characterized by the well-known dumbbell shape, with a β domain that binds three bivalent metal ions and an α domain that binds four ions, molluscan MT structure is still poorly understood. For this reason we compared two MTs from aquatic organisms that differ markedly in primary structure: MT 10 from the invertebrate Mytilus galloprovincialis and MT A from Oncorhyncus mykiss. Both proteins were overexpressed in Escherichia coli as glutathione S-transferase fusion proteins, and the MT moiety was recovered after protease cleavage. The MTs were analyzed by gel electrophoresis and tested for their differential reactivity with alkylating and reducing agents. Although they show an identical cadmium content and a similar metal-binding ability, spectropolarimetric analysis disclosed significant differences in the Cd7-MT secondary conformation. These structural differences reflect the thermal stability and metal transport of the two proteins. When metal transfer from Cd7-MT to 4-(2-pyridylazo)resorcinol was measured, the mussel MT was more reactive than the fish protein. This confirms that the differences in the primary sequence of MT 10 give rise to peculiar secondary conformation, which in turn reflects its reactivity and stability. The functional differences between the two MTs are due to specific structural properties and may be related to the different lifestyles of the two organisms.

A novel mutant of the catalytic alpha subunit of human protein kinase CK2 (CK2 alpha) was designed in an attempt to clarify the role of the carboxylic-terminal segment characteristic of vertebrates, excluding fish. Starting from the sequence alignments, we constructed a phylogenetic tree of the primary structure of CK2 alpha. On this basis, we substituted two distal prolines with alanines (PA 382-384). Theoretical calculations and spectropolarimetry measurements, performed both on native and mutant subunits, indicate an increased content of alpha-helix after this double amino acidic substitution. In order to clarify the structure/function relationship of the C-terminal region, we verified if the structural change affects the catalytic activity of CK2 alpha. The mutant exhibits slightly increased phosphorylation efficiency, but reduced ability to transfer phosphate in comparison with the native subunit. At last, we compared the thermal stability of the mutant with respect to the native subunit and we tested the proteolytic degradability. The observation that the PA 382-384 mutant exhibits an increased thermal and proteolytic stability suggests that this mutant could be employed to solve the three-dimensional (3D) structure of human CK2 alpha and to overcome difficulties in crystallizing the native form.

The recombinant catalytic subunit of human protein kinase CK2 bas been mutagenised at the C-terminal region in an attempt to induce this tail to fold. We suppose in fact that this unstructured C-terminus just might be responsible for the high degradability of the human enzyme. On the basis of theoretical calculations we choose to substitute two distal prolines with alanines (PA 382-384). The mutant bas been purified to the electrophoretic homogeneity by means of three chromatographic steps. By circular dichroism Spectroscopy we verified if the double amino acids substitution reflected on the secondary structure of the recombinant α subunit. According to our theoretical predictions, we observed that the α-helix content of the protein increased when the two distal prolines were substituted by alanines. Moreover the mutant catalytic subunit shows a reduced ability to bind a classical inhibitor such as heparin.

Chromatin of Trypanosoma cruzi is known to be organized in classical nucleosomal filaments, but surprisingly, these filaments do not fold in visible chromosomes and the nuclear envelope is preserved during cell division. Our hypothesis about the role of chromatin structure in regulating gene expression and, more generally, cell functioning, pressed us to verify if chromatin organization is modulated during the parasite life-cycle. To this end, we analyzed in situ the fine structural organization of T. cruzi chromatin by means of an integrated biophysical approach, using differential scanning calorimetry and fluorescence microscopy. We observed that logarithmic forms exhibit a less condensed chromatin with respect to the stationary ones. Thermal analysis revealed that parasite chromatin is organized in three main levels of condensation, barring from the polynucleosomal filament till to superstructured fibers. Besides, the fluorescence images of nuclei showed a characteristic chromatin distribution, with defined domains localized near to the nuclear envelope. While in stationary parasites, these regions are highly condensed, in logarithmic forms they unfold by extending themselves toward the center of nucleus. These observations suggest that, in comparison with higher eukaryotes, in T. cruzi the nuclear envelope plays an unusual and pivotal role in interphase and in mitosis.

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Tetrabromobisphenol A (2,2-bis(3,5-dibromo-4-hydroxyphenyl propane-TBBPA) is the most produced brominated flame retardant, detected in the environment and in biological samples. TBBPA shares structural similarities with thyroid hormones (THs), and it has been shown to interfere with different aspects of TH physiology, this raising concern on its possible effects as an endocrine disruptor in humans and wildlife. THs play a major role in lipid metabolism, with the liver representing one of their main target tissues. At the cellular level, THs act through interactions with TH receptors (TRs), as well as through TR-independent mechanisms. Rat hepatoma FaO cells (a liver cell line defective for functional TRs) overloaded with lipids have been utilized as a model to investigate the anti-steatotic effects of THs in the hepatocyte. In this work, the possible effects of TBBPA in steatotic FaO cells were investigated. Exposure to TBBPA for 24 h reduced triglyceride (TAG) content and the size of lipid droplets (LDs); similar effects were obtained with equimolar doses (10(-6) M) of T3 (3,3',5-L-triiodothyronine). TBBPA and T3 showed common effects on transcription of genes involved in lipid homeostasis. In particular, TBBPA mainly up-regulated mRNA levels for LD-associated oxidative tissue-enriched PAT protein (OXPAT), peroxisome proliferator-activated receptor (PPAR) isoform β/δ, and the mitochondrial uncoupling protein 2 (UCP2). The results demonstrate that TBBPA can decrease lipid accumulation in steatotic cells through stimulation of oxidative pathways. These data identify novel thyromimetic actions of TBBPA at the cellular level.

Bone is a topic of great interest for researchers, such as biologists or engineers, both interested in understanding the structure- related properties of bone and how they are affected by aging, disease and therapies. In particular, a topic of common interest between medicine and engineering is the fracture behavior of bone. Indeed, a thorough understanding of the mechanical behavior of bone is helpful to predict the fracture risk, but it can also provide the basis for the design of de novo biomimetic materials. In this paper, we show the initial results of an experimental study of the mechanical behavior of bovine bone, with a special focus on fracture toughness. The latter is evaluated under tensile and bending loading, by following the ASTM adopted for metals. Finally, we perform microscopic observations to better understand the fracture behavior and correlate it with the microscopic structure.

Excess fat accumulation renders the liver more vulnerable to ethanol, but it is still unclear how alcohol enhances lipid dysmetabolism and oxidative stress in a pre-existing steatosis condition. The effects produced by binge ethanol consumption in the liver of male Wistar rats fed a standard (Ctrl) or a high-fat diet HFD were compared. The liver status was checked through tissue histology and standard serum parameters. Alteration of hepatic lipid homeostasis and consequent oxidative unbalance were assessed by quantifying the mRNA expression of the lipid-regulated peroxisome proliferator-activated receptors (PPARs), of the cytochromes CYP2E1 and CYP4A1, and of some antioxidant molecules such as the metallothionein isoforms MT1 and MT2 and the enzymes catalase and superoxide dismutase. The number of adipose differentiation-related protein (ADRP)-positive lipid droplets (LDs) was evaluated by immunohistochemical staining. As a response to the double insult of diet and ethanol the rat liver showed: (1) a larger increase in fat accumulation within ADRP-positive LDs; (2) stimulation of lipid oxidation in the attempt to limit excess fat accumulation; (3) induction of antioxidant proteins (MT2, in particular) to protect the liver from the ethanol-induced overproduction of oxygen radicals. The data indicate an increased susceptibility of fatty liver to ethanol and suggest that the synergistic effect of diet and ethanol on lipid dysmetabolism might be mediated, at least in part, by PPARs and cytochromes CYP4A1 and CYP2E1.

Amyotrophic lateral sclerosis (ALS) is a late-onset fatal neurodegenerative disease reflecting degeneration of upper and lower motoneurons (MNs). The cause of ALS and the mechanisms of neuronal death are still largely obscure, thus impairing the establishment of efficacious therapies. Glutamate (Glu)-mediated excitotoxicity plays a major role in MN degeneration in ALS. We recently demonstrated that the activation of Group I metabotropic Glu autoreceptors, belonging to both type 1 and type 5 receptors (mGluR1 and mGluR5), at glutamatergic spinal cord nerve terminals, produces excessive Glu release in mice over-expressing human superoxide-dismutase carrying the G93A point mutation (SOD1(G93A)), a widely used animal model of human ALS. To establish whether these receptors are implicated in ALS, we generated mice expressing half dosage of mGluR1 in the SOD1(G93A) background (SOD1(G93A)Grm1(crv4/+)), by crossing the SOD1(G93A) mutant mouse with the Grm1(crv4/+) mouse, lacking mGluR1 because of a spontaneous recessive mutation. SOD1(G93A)Grm1(crv4/+) mice showed prolonged survival probability, delayed pathology onset, slower disease progression and improved motor performances compared to SOD1(G93A) mice. These effects were associated to reduction of mGluR5 expression, enhanced number of MNs, decreased astrocyte and microglia activation, normalization of metallothionein and catalase mRNA expression, reduced mitochondrial damage, and decrease of abnormal Glu release in spinal cord of SOD1(G93A)Grm1(crv4/+)compared to SOD1(G93A) mice. These results demonstrate that a lower constitutive level of mGluR1 has a significant positive impact on mice with experimental ALS, thus providing the rationale for future pharmacological approaches to ALS by selectively blocking Group I metabotropic Glu receptors.

Background/Aims: Fatty acids are the main energy stores and the major membrane components of the cells. In the hepatocyte, fatty acids are esterified to triacylglycerols (TAGs) and stored in lipid droplets (LDs). The lipid lowering action of 3,5-diiodo-L-thyronine (T 2 ) on an in vitro model of hepatosteatosis was investigated in terms of fatty acid and protein content of LDs, lipid oxidation and secretion. Methods: FaO cells were exposed to oleate/ palmitate, then treated with T 2 . Results: T 2 reduced number and size of LDs, and modified their acyl composition by decreasing the content of saturated (SFA) vs monounsaturated (MUFA) fatty acids thus reversing the SFA/MUFA ratio. The expression of the LD-associated proteins adipose differentiation-related protein (ADRP), oxidative tissue-enriched PAT protein (OXPAT), and adipose triglyceride lipase (ATGL) was increased in ‘steatotic’ cells and further up-regulated by T 2 . Moreover, T 2 stimulated the mitochondrial oxidation by up-regulating carnitine-palmitoyl-transferase (CPT1), uncoupling protein 2 (UCP2) and very long-chain acylcoenzyme A dehydrogenase (VLCAD). Conclusions: T 2 leads to mobilization of TAGs from LDs and stimulates mitochondrial oxidative metabolism of fatty acids, in particular of SFAs, and thus enriches of MUFAs the LDs. This action may protect the hepatocyte from excess of SFAs that are more toxic than MUFAs.

The investigated material is a quenched&tempered steel for hydrogen applications. Understanding of hydrogen embrittlement and its effect on the mechanical characteristics of steel becomes fundamental. It is, in fact, well known that the atomic hydrogen can cause embrittlement and degradation of the mechanical characteristics of the steels. Fracture mechanics parameters are usually considered to define the embrittlement effect of hydrogen. In the present work AISI 4130 is experimentally investigated. Several specimens were hydrogen charged by an electrochemical method, that allows to obtain a controlled amount of hydrogen into the specimens. Toughness and fatigue crack growth tests have been carried out on uncharged and hydrogen charged CT specimens. The effect of hydrogen is evident: the propagation curves are shifted and the toughness values are reduced.

Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.

The most recent trend in power transmission design considers the damage-tolerant approach as one of the methods to obtain safe, reliable and light systems. This means that components containing cracks must be considered and analysed to understand the conditions that cause critical cracks and defects and their dimensions. In this paper a cracked tooth of an automotive gearwheel is considered. A numerical procedure (based on the slice synthesis weight function method) to calculate the stress intensity factors of curved cracks due to bending loads is illustrated. The results are compared with those obtained by expensive finite element calculations. The agreement is satisfactory and the proposed technique proves to be very attractive from the point of view of time saving. One example of an application to fatigue design practice is provided, namely the analysis of fatigue crack propagation in surface-treated gears. The results show the role played by residual stresses induced by carburizing and shot peening.

The aim of this work is a review of scientific results in the literature, related to the application of thermographic techniques to composite materials. Thermography is the analysis of the surface temperature of a body by infrared rays detection via a thermal-camera. The use of this technique is mainly based on the modification of the surface temperature of a material, when it is stimulated by means of a thermal or mechanical external source. The presence of defects, in fact, induces a localized variation in its temperature distribution and, then, the measured values of the surface temperature can be used to localize and evaluate the dimensions and the evolution of defects. In the past, many applications of thermography were proposed on homogeneous materials, but only recently this technique has also been extended to composites. In this work several applications of thermography to fibres reinforced plastics are presented. Thermographic measurements are performed on the surface of the specimens, while undergoing static and dynamic tensile loading. The joint analysis of thermal and mechanical data allows one to assess the damage evolution and to study the damage phenomenon from both mechanical and energetic viewpoints. In particular, one of the main issues is to obtain information about the fatigue behaviour of composite materials, by following an approach successfully applied to homogenous materials. This approach is based on the application of infrared thermography on specimens subjected to static or stepwise dynamic loadings and on the definition of a damage stress, ?D, that is correlated to the fatigue strength of the material. A wide series of experimental fatigue tests has been carried out to verify if the value of the damage stress, ?D, is correlated with the fatigue strength of the material. The agreement between the different values is good, showing the reliability of the presented thermographic techniques, to the study of composite damage and their fatigue behaviour.

Modifications of the higher-order chromatin structure induced by polyamines have been quantitatively investigated in situ through a non-invasive biophysical approach using Differential Scanning Calorimetry and Quantitative Fluorescence Microscopy. Calorimetric and intensitometric profiles have been acquired for samples of native thymocytes, alternatively suspended in buffers, with or without natural polyamines (spermine and spermidine). The results here reported show that the structure and distribution of nuclear chromatin in situ considerably change upon the ionic composition of the environment. A quantitative analysis of this data and a comparison with previous results obtained from isolated chromatin fibers was carried out. Finally, an inverse relationship between chromatin condensation and nuclear volume was observed.

This work investigates the structure of native calf thymus chromatin as a function of fiber length and isolation procedures by using X-ray small angle scattering technique. Two methods of chromatin isolation have been compared in order to better understand the differences reported by various authors in terms of chromatin high order structure. In addition to these experimental results the effects of shearing have also been studied. In order to explain the differences among these chromatin preparations we built several models of chromatin fibers (represented as a chain of spherical subunits) assuming increasing level of condensation at increasing salt concentrations. For all these fiber models the corresponding theoretical X-ray scattering curves have been calculated and these results have been used to explain the influence of fiber length on the scattering profiles of chromatin. The comparison between experimental and theoretical curves confirms that the high molecular weight chromatin-DNA prepared by hypotonic swelling of nuclei (without enzymatic digestion) displays a partially folded structure even at low ionic strength, whereas the low molecular weight chromatin-DNA prepared by a brief nuclease digestion appears very weakly folded at the same ionic conditions.

The effect of histone acetylation was monitored on CHO chromatin structure, following the addition of 7 mM Na-butyrate to the cell culture medium. The properties of both control and hyperacetylated chromatins and nuclei were investigated by circular dichroism, ethidium bromide intercalation, differential scanning calorimetry, and affinity chromatography. Our results are compatible with modest but significant alterations in the various levels of chromatin organization, as a result of the charge neutralization of some lysine residues within the N-terminal region of the histonic octamer. Namely, large statistically significant differences do exist in the heat capacity thermograms of native nuclei, where unfolding into single nucleofilament of the highly packed native chromatin superfiber appears associated with acetylation; at the same time CD, EB, and affinity chromatography point to modest but consistent differences in the compactness of isolated nucleosomes and polynucleosomes.

Flanged joints arefundamental components of pipelines and they must be optimized to avoid leaks,bolts overstress and flanged joints damage. Overcoming standards indications asover-torqueing, and combination of different parameters, as difference in hardnessbetween flanges and gasket, can cause groove impressions. Aim of this study isto provide an efficient tool to optimize the choice of characteristicparameters of flanged joints, by numerical simulations. Geometries of a realtest case are simulated by FE models, considering different values of flangeand gasket hardness, as well as tightening torques, in order to cover possibleranges. From the analyses, it is possible to highlight that a particularcombination of hardness difference and over-torque is responsible for permanentflange impression.

The present paper deals with the development of a bench for testing prostheses for legs. The bench includes a fake leg (from femur to foot) and it allows rotations of femur (up to 40°) and of tibia with respect to femur (up to 40°). Lockable gas springs allow stopping tibia and femur rotations. An hydraulic actuator applies a load to the hip, while a sled driven by an electric motor moves the foot simulating walking. Attachment between foot and sled has been designed to allow load transfer from heel to forefoot. Once maximum rotations for tibia and femur are reached, the leg is lifted and swings. As a first application, the test bench will be used to test transtibial prosthetics, including the knee socket, but it has been designed so that it could allow testing, with a limited number of adjustments, also transfemoral, foot and ankle prosthetics.

Composite materials are largely used for structural applications, thanks to their high strength-to-weight ratios. However, it is difficult to make accurate estimations on their mechanical behavior, as it is affected by several factors, involved both in the manufacturing process and in the experimental testing. In this study, GFRP laminates, with different stacking sequences, are tested under static loading conditions. During testing, thermal analyses are also performed by means of a thermal camera, obtaining an energetic parameter (i.e. the temperature) useful for the evaluation of damage. The thermographic method allows both qualitative and quantitative analyses to be performed in a relatively short time. Besides thermal analyses, damage is also assessed by means of static tests, interrupted at different load levels, and followed by stiffness reduction measurements and microscopic analyses, allowing for a comparison of the obtained results.

When the material yields local stress and strain behaviour changes, especially if there is a notch, determination of the local strain value can be difficult. Therefore it is not easy to predict the life of mechanical components in the low-cycle region. In the present work pressure vessels are considered and fatigue tests carried out. The most stressed zones, which are at the connections between the nozzles and the vessel, are subjected to cyclic loads with subsequent repeated plastic strains that cause nucleation and propagation of fatigue cracks. The life of such a mechanical component under low cycle fatigue can be predicted by utilizing the life strain curves e − N f or the energy approach (plastic strain and total strain energy density). In the present work, life prediction is performed by considering several different approaches. The results are compared with the experimental results obtained by fatigue testing pressure vessel nozzles. The material considered is Fe 510 B UNI 7070-72 steel.

In this study, keyhole and smooth specimens, made from a low alloy pressure vessel steel (ASTM A-533 grade B), were subjected to monoaxial fatigue tests. The results show the influence of the stress concentration factor, K[sub t], on the number of cycles to failure, N[sub f]. Total strain energy per cycle, [Delta]W[sub t] = [Delta]W[sub p] + [Delta]W[sub e], was proved to be a good parameter for predicting the life of notched components. Elasto-plastic FEM analysis, utilizing the cyclic and monotonic curve of the material, showed close agreement with the experimental values.

In the presence of H2S, metallic materials, such as carbon and low alloy steels, may suffer hydrogen damage and hydrogen embrittlement. Sour service materials must be used in compliance with international standards when fluids are classified as sour. Gas transporting pipes in low temperature environment, during the shutdown and the subsequent re-starting operations, are exposed to very low temperatures (T = −40 °C). In presence of high H2S content in the gas the risk of brittle failure can be increased due to the effect of hydrogen on steel toughness. In this paper the influence of hydrogen and low temperature on mechanical properties of two pipeline materials, X65 micro-alloyed and F22 low alloy steels, is studied. An electrochemical hydrogen charging method has been setup, avoiding any critical condition from the point of view of preparation, safety and disposal. Diffusible hydrogen content of steels is in the range 0.6 to 2 ppm. Charpy and J–R curve tests were carried out in the range from room temperature to T = −120 °C. Hydrogen affects mechanical properties of the tested materials, mainly reducing fracture toughness in J integral tests, while little influence has been observed in Charpy tests. Fracture surface examination confirms the results of mechanical testing.

Bisphenol A (BPA), used in the manufacture of polycarbonate plastic and epoxy resin, is one of the most abundant endocrine disruptors in the environment, considered as a xenoestrogen. BPA has recently become of additional public health concern because of increasing evidence of deleterious effects on metabolism. Dietary intake seems the most important route for BPA exposure, followed by rapid biotransformation in the gut and liver and elimination in the urine. Although hepatocytes can represent a significant target for this compound, little is known on the direct effects and mechanisms of action of BPA on lipid homeostasis at the cellular level. In this work, the effects of BPA (0.3–3–30–300 ng mL −1 , 24 h) were investigated in rat FaO hepatoma, a well differentiated liver cell line. At both 30 and 300 ng mL −1 , BPA significantly increased intracellular triglyceride (TAG) content and lipid accumulation in lipid droplets (LDs), without affecting cell viability. The effects of BPA were associated with decreased mRNA levels of the transcription factors Peroxisome Proliferator-Activated Receptor (PPAR) isoforms α and βδ, as well as of their downstream genes acyl-CoA oxidase (AOX) and carnitine palmitoyl transferase (CPT1) involved in lipid oxidation. No increase in transcription of lipogenic genes was observed. BPA also decreased mRNA levels of ApolipoproteinB (apoB) and the extracellular TAG content, indicating alterations in lipid secretion. FaO cells did not express Estrogen Receptor α (ERα and showed a very low expression of ERβ compared to rat liver. All the effects of BPA were prevented by cell pretreatment with Wortmannin, indicating the involvement of phosphatidyl inositol-3 kinase activation. The results demonstrate a direct action of BPA on lipid homeostasis in FaO cells through interference with lipid oxidation and secretion, and add further information on the cellular pathways that can be perturbed by this compound.

Oxidative stress is considered to be one of the main causes of neural damage after injury. However, little is known about the changes in mRNA expression of antioxidant molecules that occur after injury and regeneration of the peripheral nerve. In the present study, the rat median nerve was transected, and transcriptional changes were studied at day 6 and day 12 after injury in both the proximal and the distal stumps, in the absence or presence of microsurgical repair. The expression profiles of the following genes were investigated: three metallothionein isoforms (MT-1, MT-2, and MT-3), the main antioxidant enzymes (catalase, superoxide dismutase, and glutathione-S-transferase), and the marker of cellular damage poly(ADP-ribose) polymerase-1 (PARP-1). The results showed that, in the proximal nerve stump, MT-3 mRNA expression was significantly and markedly up-regulated in the absence of surgical repair, whereas MT-1 and MT-2 showed significant down-regulation. In the distal nerve portion, mRNA expression of all MT isoforms decreased significantly in the absence of microsurgical reconstruction, whereas, after repair, MT-3 mRNA expression alone was up-regulated. Expression of all the antioxidant enzymes decreased significantly after repair in the proximal nerve portion, but a significant general increase in their mRNA expression was revealed in the distal nerve stump. PARP-1 expression was significantly up-regulated in the proximal nerve portion without repair but dramatically reduced after reconstruction. In contrast, PARP-1 expression increased markedly in the distal stump after surgical repair. Taken together, these findings indicate that antioxidant molecules are differentially modulated and might, therefore, play an important role in peripheral nerve injury and regeneration.

Mesenchymal stem cells (MSC) display a remarkable ability to modulate the immune response and protect the central nervous system mainly through the release of soluble factors in a paracrine fashion, affecting the functional behavior of cells in the tissues. Here we investigated the effect of the interaction between MSC and microglia in vitro, and we dissected the molecular and cellular mechanisms of this crosstalk. We demonstrated that MSC impair microglia activation by inflammatory cues through the inhibition of the expression and release of inflammatory molecules and stress-associated proteins. We showed that MSC significantly increase microglial expression and release of molecules associated with a neuroprotective phenotype such as CX3CR1, nuclear receptor 4 family, CD200 receptor, and insulin growth factor 1. Interestingly, MSC can enhance functional changes on microglia as depicted by the increase of intracellular calcium concentration and phagocytic activity. This last event is associated with an increased expression of triggering receptor expressed on myeloid cells-2, an innate immune receptor involved in phagocytosis in the absence of inflammation. The observed effects on CX3CR1-expressing microglia are due to the release of CX3CL1 by MSC, driven by inflammatory signals, as demonstrated by the reversal of the observed results when CX3CL1 expression was silenced in MSC or its release was blocked. Finally, we showed that exogenous CX3CL1 induce phenotypic and functional changes of microglia similar to those induced by MSC. These findings demonstrate that MSC instruct, through the release of CX3CL1, microglia responsiveness to proinflammatory signals by modulating constitutive “calming” receptors, typically expressed by “steady-state microglia” thus switching microglia from a detrimental phenotype to a neuroprotective one.

Native chromatin has been characterized at different ionic strengths, according to different methods of preparation, by means of circular dichroism and ethidium bromide intercalation. For a more precise interpretation of these results, a series of analogous experiments on phagic DNA, as a function of its circularization, and on mononucleosomes have been performed. Results are discussed here in terms of high order chromatin structure, DNA supercoiling, and topological constraints.

Object of the present study is an ankle prosthesis for transtibial amputees, designed for children. Standards for prostheses compliance available in literature are all related to adults, while very few studies are present for children. In the first part of the paper a discussion on the modification of the standard in terms of loads and distances is presented, to adapt the available data to the test case, a boy of 12 years and 45 kg. In the second part, experimental results are shown, considering four different types of tests: static proof and ultimate tests, fatigue tests and static torsion tests. Results were positive for the designed and tested ankle prosthesis.

In the presence of H2S, metallic materials, such as carbon and low alloy steels, may suffer hydrogen damage and hydrogen embrittlement. Gas transporting pipes in low temperature environment, during the shutdown and the subsequent re-starting operations, are exposed to very low temperatures (T=-40 °C). In the presence of high H2S content in the gas, the risk of brittle failure can be increased due to the effect of hydrogen on steel toughness. In this paper the influence of hydrogen and low temperature on mechanical properties of two pipeline materials, F22 low alloy and X65 micro-alloyed steels, is studied. Steels have been hydrogen charged by means of an electrochemical method: diffusible hydrogen content of steels is in the range 0.6 to 2 ppm. Charpy and J-R curves tests were carried out in the range from room temperature to T=-120 °C. Hydrogen affects mechanical properties of the tested materials, mainly reducing fracture toughness in J integral tests, while little influence has been observed in CV tests. Fracture surface examination confirms the results of mechanical testing.

Aim of this work is to test and understand the mechanical behaviour, in multi-axial stress conditions, of a composite material (E-glass reinforce fibres and vinyl-ester matrix) used to build a structural component: a corner beam of bus cabin. A series of experimental tests is performed on specimens cut out from this beam. Different unidirectional (UD) fibres orientations with respect to the direction of load application are considered: longitudinal (0°), normal (90°), inclined at 30° and 45°. Static and fatigue characterization are carried out to identify the mechanical behaviour in terms of σ – N curves. Obtained fatigue data are then interpolated by fitting the parameters required for multi-axial models of fatigue life estimation. A final comparison with the literature data is included, proposing a simple tool to evaluate the damage evolution of E-glass reinforced composites during fatigue life.

Iodothyronines influence lipid metabolism and energy homeostasis. Previous studies demonstrated that 3,5-l-diiodothyronine (T2), as well as 3,3′,5-l-triiodothyronine (T3), was able to both prevent and reverse hepatic steatosis in rats fed a high-fat diet, and this effect depends on a direct action of iodothyronines on the hepatocyte. However, the involvement of thyroid hormone receptors (TRs) in mediating the lipid-lowering effect of iodothyronines was not elucidated. In this study, we investigated the ability of T2 and T3 to reduce the lipid overloading using the rat hepatoma FaO cells defective for functional TRs. The absence of constitutive mRNA expression of both TRα1 and TRβ1 in FaO cells was verified by RT-qPCR. To mimic the fatty liver condition, FaO cells were treated with a fatty acid mixture and then exposed to pharmacological doses of T2 or T3 for 24 h. Lipid accumulation, mRNA expression of the peroxisome proliferator-activated receptors (PPAR-α, -γ, -δ) the acyl-CoA oxidase (AOX), and the stearoyl CoA desaturase (SCD1), as well as fuel-stimulated O2 consumption in intact cells, were evaluated. Lipid accumulation was associated with an increase in triacylglycerol content, PPARγ mRNA expression, and a decrease in PPARδ and SCD1 mRNA expression. The addition of T2 or T3 to lipid-overloaded cells resulted in i) reduction in lipid content; ii) downregulation of PPARα, PPARγ, and AOX expression; iii) increase in PPARδ expression; and iv) stimulation of mitochondrial uncoupling. These data demonstrate, for the first time, that in the hepatocyte, the lipid-lowering actions of both T2 and T3 are not mediated by TRs.

Background & Aims Previous studies have demonstrated that 3,5-l-diiodothyronine (T 2 ) is able to prevent lipid accumulation in the liver of rats fed a high-fat diet. Whether this effect is due to a direct action of T 2 on the liver has not been elucidated. In this study, we investigated the ability of T 2 to reduce the excess lipids in isolated hepatocytes treated with fatty acids (FFAs). The effects of T 2 were compared with those elicited by 3,3′,5-l-triiodothyronine (T 3 ). Methods To mimic the fatty liver condition, primary cultures of rat hepatocytes were overloaded with lipids, by exposure to FFAs ("fatty hepatocytes"), and then treated with T 2 or T 3 . Lipid content, morphometry of lipid droplets (LDs), and expression of the adipocyte differentiation-related protein (ADRP) and the peroxisome proliferator-activated receptors (PPAR-α, -γ, -δ) were evaluated. Activities of the lipolytic enzyme acyl CoA oxidase – AOX and the antioxidant enzymes superoxide dismutase – SOD and catalase – CAT were also determined. Results FFA-induced lipid accumulation was associated with an increase in both number/size of LDs and expression of ADRP , PPAR-γ, and PPAR-δ/β mRNAs, as well as in the activities of AOX, SOD, and CAT. The addition of T 2 or T 3 to "fatty hepatocytes" resulted in a reduction in: (i) lipid content and LD diameter; (ii) PPAR-γ and PPAR-δ expression; (iii) activities of AOX and antioxidant enzymes. Conclusions These data demonstrate, for the first time, a direct action of both T 2 and T 3 in reducing the excess fat in cultured hepatocytes.

Excess energy is stored as neutral lipids in lipid droplets (LDs) whose surface is coated by PAT proteins, each playing a distinct cellular function. The adipocyte differentiation-related protein (ADRP) and tail-interacting protein (TIP47) are expressed almost ubiquitously, whereas the oxidative tissue-enriched PAT protein (OXPAT) is expressed in specific tissues, such as the liver. In rat liver, only ADRP expression has been documented. This study was aimed at identifying OXPAT and TIP47 transcripts in rat hepatocytes, and investigating how their expression is modulated by excess lipids, using fat-enriched hepatocytes to mimic different degrees of steatosis. Primary rat hepatocytes were exposed to fatty acids (FFAs) for 12, 24 and 36 h. Lipid accumulation was estimated by spectrophotometric quantification of triacylglycerol. Expression of PAT proteins as well as of PPARgamma was evaluated by real-time RT-PCR. Hepatocytes exposed to FFAs showed progressive lipid accumulation. The increase in lipid content was associated with the induction of PAT protein expression. At 12 h, OXPAT and TIP47 mRNA expression was up-regulated. At longer times, the level of OXPAT transcripts remained high, whereas that of TIP47 slowly declined. Conversely, ADRP expression showed a time-dependent increase with exposure to FFAs. This study demonstrates, for the first time, the presence of OXPAT and TIP47 transcripts in rat hepatocytes, as well as their up-regulation with lipid accumulation. The distinct time courses observed for the three PAT proteins during FFA exposure might reflect the different roles played by each protein in lipid metabolism in the hepatocyte. Up-regulation of TIP47 and OXPAT might represent an early response to excess lipids, while, in correspondence with a lipid overload, up-regulation of ADRP could address lipids towards storage.

Aim of this work is to test and understand the mechanical behavior of a composite material used to build a structural component, in particular a corner beam of a bus cabin. This component is obtained by means of the pultrusion technique and presents random and longitudinal E-glass fibers as reinforce, while the matrix is a vinyl-ester resin. A series of experimental tests was performed on specimens cut out from this beam. Different fibers orientation with respect to the direction of load application were considered: longitudinal (0°), normal (90°) and inclined (45°), thus both in uni-axial and multi-axial loading conditions. A static and fatigue characterization was carried out to identify the mechanical behaviors and the failure modes in these directions. Obtained fatigue data are then interpolated by fitting parameters required for models of fatigue life prediction.

The aim of this work is the fatigue design of a structural component manufactured in composite materials. The use of composite materials in structural applications implies that the reliability and safety requirements are met, in particular from a point of view of the fatigue life. A bus component is considered: the composite material is obtained by means of the pultrusion technique: the longitudinal reinforcement is glass fibre, while the matrix is polyester resin. At first, a static and fatigue characterization of the pultruded composite is performed to identify the mechanical behaviour along the fibres and in the normal direction, following an extensive testing programme on specimens. During the fatigue tests, the variation of the elastic modulus and the residual strength are monitored to characterize the damage; further experimental tests are performed to fit parameters required for models of fatigue life prevision. Fatigue bending tests are performed on the component, showing crack propagation through the section: a check at the SEM is performed to identify the main internal type of damage. A final comparison with a numerical simulation is proposed: this numerical model will be useful for the optimal fatigue design of the section.

Synchronized CHO-K1 cells and their dibutyryl c-AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules. In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI-stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen-stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase. In the early part of G1, the drastic alteration in anti-β tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide. The known 1–2 h lengthening of the G1 period after reverse-transformation appears to correlate with a similar delay in the abrupt chromatin decondensation. The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast-like cells. © 1992 Wiley-Liss, Inc.

The interaction of direct electric current (dc) and proteins is a little explored topic. We had reported that exposure of Crotalus atrox venom to dc caused irreversible inactivation of phospholipase A2 and metalloprotease and that the eukaryote adenylate kinases (AK) precipitate in nondenaturing gel electrophoresis. AK1 displays an elevated percent difference of CHarged versus POlar amino acid content (CH-PO 14). Commercial AK1 and other 17 enzymes with various CH-PO values were exposed in solution to dc (0–0.7 mA) from low voltage (0–10 V), then enzymatic activity was assayed. The enzymes with CH-PO higher than 10.0 were irreversibly inactivated by current exposure; those with CH-PO between +3 and −5 were not. Inactivation was dependent on the ionic strength of the medium and not on the net charge of the protein. Circular dichroic spectroscopy showed a structural modification in some of the inactivated enzymes. CH-PO could be a crucial, although rough, parameter for predicting protein inactivation by low-voltage exposure. The observed effect seems due to the current density. Enzymatic activity maybe a more accurate sensor of conformational changes than circular dichroism spectroscopy. A better understanding of efficacy of many electrical devices utilized in medical practice may follow. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:309–317, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20293

This title is not available to purchase from Royal Society of Chemistry. Please visit www.bioinorganic-chemistry.org/mils for title information. These sulfur-rich chelators, being important in metal ion homeostasis, find increasing attention. MILS-5, written by 30 internationally recognized experts, focuses on this hot topic. The reader is supported by about 20 tables, more than 80 illustrations and nearly 2000 references. This book is an essential resource for scientists working in a wide range of disciplines from environmental toxicology and inorganic biochemistry all the way through to physiology and medicine.

Interference between heavy metals and growth hormone (GH) on cell signaling has been previously demonstrated in fish cells. This study was aimed at assessing their effects on expression of the metallothionein isoforms MT-A and MT-B. The results indicate that all heavy metals induce MT-A more markedly than MT-B, but differences appeared when metals were combined with GH. For MT-B induction, a positive interference between metals and GH was observed for Zn(2+)/GH and Cd(2+)/GH, a negative interference for Hg(2+)/GH. With regards to MT-A, no interference was observed for Zn(2+)/GH and Hg(2+)/GH, while a negative interference occurred with Cu(2+)/GH and a positive interference with Cd(2+)/GH. The possible mechanisms underlying the differential regulation of metallothioneins include different signaling pathways. The results show that STAT5 and ERKs responded differently to different combinations, and Zn(2+)/GH and Cd(2+)/GH exerted a slight positive interference on ERK activation. On the other hand, a synergic rise in [Ca(2+)](i) occurred for all combinations except for Cu(2+)/GH. Our data suggest that the cross-talk between heavy metals and GH resulting in MT transcription modulation does not strictly depend on Ca(2+) signalling; (ii)ERK activation may represent the point of cross-talk between Zn(2+) or Cd(2+) and GH, converging on MT-B transcription, probably through a differential recruitment of transcription factors.

Nucleosomes, chromatin and nuclei, extracted from rat hepatocytes, were studied by an experimental configuration which measures circular intensity differential scattering (CIDS) and other elements of the polarized light scattering matrix. The Mueller matrix elements, S/sub 14/ and S/sub 34/, that are related to the geometric parameters of the superhelical arrangement of polynucleosomes point to the existence of a quarternary structure at low ionic strength for chromatin prepared by the cold-water method, which is lost by shearing, and is not found in the soluble chromatin prepared through the nuclease method. Only the addition of salt to produce a final concentration of 5 mM MgCl/sub 2/, 150 mM NaCl and 10 mM Tris HCl (pH 7) yields a sizable (S/sub 14/+S/sub 34/) signal in the latter chromatin. This signal is however still different from the corresponding signal of native nuclei and of cold-water chromatin. The (S/sub 14/+S/sub 34/) signal from isolated nucleosomes in consistently very low (nearly zero) as predicted by multiple dipole simulation within the framework of classical electrodynamics. Results are discussed in terms of the topological constraints present in the native long chromatin fiber, which are lost after limited nuclease digestion and after shearing. >

The wheel–rail interface is fundamental for the comfort and the safety of the train. For this reason the wheel and the rail surface condition are checked with periodic non-destructive controls. The presence of a crack can be tolerated if we are able to predict its evolution by using fracture mechanics concepts. The problem is complicated by the fact that surface cracks propagate in mixed mode conditions. In this paper a photoelastic technique is presented to experimentally determine the stress intensity factors in a model of cracked rail under contact loads. The first results are critically discussed.

In rats fed a high fat diet (HFD), long-term administration of 3,5-diiodo-L-thyronine (T2), a naturally occurring iodothyronine, was shown to reduce body-weight gain, fat mass, and hepatic lipid accumulation. This work was aimed at investigating the mechanisms of T2 action in the liver of HFD rats. The results show that HFD induces liver lipid peroxidation and stimulates the activity of enzymes involved in hydrogen peroxide (H2O2) metabolism, catalase in particular. Moreover, quantitative RT-PCR revealed HFD-induced upregulation of the transcription factor PPARα, as well as of metallothionein isoforms (MT-1 and MT-2). T2 administration prevented the HDF-induced lipid peroxidation, as well as the increase in H2O2 metabolism, and reduced the upregulation of both PPARα and MT-2. These data demonstrate that in the liver of HFD rats, T2 prevents both lipid accumulation and oxidative stress associated with increased fat metabolism.