Occupational scenarios and exposure assessment to formaldehyde: A systematic review.

The objectives of the systematic review were to: identify the work sectors at risk for exposure to formaldehyde; investigate the procedures applied to assess occupational exposure; evaluate the reported exposure levels among the different settings. An electronic search of Pubmed, Scopus, Web of Science and ToxNet was carried out for collecting all the articles on the investigated issue published from January 1, 2004 to September 30, 2019. Forty-three papers were included in the review, and evidenced a great number of occupational scenarios at risk for formaldehyde exposure. All the included studies collected data on formaldehyde exposure levels by a similar approach: environmental and personal sampling followed by chromatographic analyses. Results ranged from not detectable values until to some mg m-3 of airborne formaldehyde. The riskiest occupational settings for formaldehyde exposure were the gross anatomy and pathology laboratories, the hairdressing salons and some specific productive settings, such as wooden furniture factories, dairy facilities and fish hatcheries. Notice that formaldehyde, a well-known carcinogen, was recovered in air at levels higher than outdoor in almost all the studied scenarios/activities; thus, when formaldehyde cannot be removed or substituted, targeted strategies for exposure elimination or mitigation must be adopted.

The Puzzling Problem of Cardiolipin Membrane-Cytochrome c Interactions: A Combined Infrared and Fluorescence Study.

The interaction of cytochrome c (cyt c) with natural and synthetic membranes is known to be a complex phenomenon, involving both protein and lipid conformational changes. In this paper, we combined infrared and fluorescence spectroscopy to study the structural transformation occurring to the lipid network of cardiolipin-containing large unilamellar vesicles (LUVs). The data, collected at increasing protein/lipid ratio, demonstrate the existence of a multi-phase process, which is characterized by: (i) the interaction of cyt c with the lipid polar heads; (ii) the lipid anchorage of the protein on the membrane surface; and (iii) a long-distance order/disorder transition of the cardiolipin acyl chains. Such effects have been quantitatively interpreted introducing specific order parameters and discussed in the frame of the models on cyt c activity reported in literature.

Cytotoxicity and internalization analysis of silicon nanowires in Buffalo Green Monkey cells: a preliminary study to evaluate the possibility of carrying viruses inside the cells.

Silicon nanowires (SiNWs) are attractive functional nanomaterials for biomedical applications. The ability to easily tune their size and density, potential biocompatibility, and knowledge of the chemical activation of SiNWs surface make them natural tools to interact with biological materials. We evaluated the possibility of exploiting SiNWs as carriers to introduce organic compounds into cells. The cellular toxicity and the internalization capacity of free-standing and label-free SiNWs were tested on Buffalo Green Monkey cells (BGM). Confocal fluorescent observation of SiNWs conjugated with fluorescein-polyethylene imine (PEI) confirmed the internalization of the NWs into the Buffalo Green Monkey Cells (BGM).

Studying the TRAF2 binding to model membranes: The role of subunits dissociation.

The ability of a C-terminal truncated form of TRAF2 to bind synthetic vesicles has been quantitatively studied by steady-state fluorescence energy transfer from the protein to large unilamellar vesicles (LUVs) prepared with different lipid mixtures. The dissociation constants, the free energy of binding, and the average number of phospholipids interacting with truncated TRAF2 have been evaluated from the corresponding binding curves. The results indicate that the protein strongly interacts with the lipid bilayer, preferentially in the monomeric state. These findings have been discussed in terms of their possible role in the activity of TRAF2 in vivo.

Unravelling the Non-Native Low-Spin State of the Cytochrome c-Cardiolipin Complex: Evidence of the Formation of a His-Ligated Species Only.

The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH- ion.

The key role played by charge in the interaction of cytochrome c with cardiolipin.

Cytochrome c undergoes structural variations upon binding of cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several mechanisms governing cytochrome c/cardiolipin (cyt c/CL) recognition have been proposed, the interpretation of the process remains, at least in part, unknown. To better define the steps characterizing the cyt c-CL interaction, the role of Lys72 and Lys73, two residues thought to be important in the protein/lipid binding interaction, were recently investigated by mutagenesis. The substitution of the two (positively charged) Lys residues with Asn revealed that such mutations cancel the CL-dependent peroxidase activity of cyt c; furthermore, CL does not interact with the Lys72Asn mutant. In the present paper, we extend our study to the Lys â†’ Arg mutants to investigate the influence exerted by the charge possessed by the residues located at positions 72 and 73 on the cyt c/CL interaction. On the basis of the present work a number of overall conclusions can be drawn: (i) position 72 must be occupied by a positively charged residue to assure cyt c/CL recognition; (ii) the Arg residues located at positions 72 and 73 permit cyt c to react with CL; (iii) the replacement of Lys72 with Arg weakens the second (low-affinity) binding transition; (iv) the Lys73Arg mutation strongly increases the peroxidase activity of the CL-bound protein.

The nitrite reductase activity of horse heart carboxymethylated-cytochrome c is modulated by cardiolipin.

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO2 (-)-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k on = (7.3 ± 0.7) × 10(-2) M(-1) s(-1); at pH 7.4], whereas the value of k on for NO2 (-) reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M(-1) s(-1) (at pH 7.4). CL facilitates the NO2 (-)-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k on for the NO2 (-)-mediated conversion of CL-CM-cytc-Fe(II) to CL-CM-cytc-Fe(II)-NO (5.6 ± 0.6 M(-1) s(-1); at pH 7.4) being slightly higher than that for the NO2 (-)-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO (2.6 ± 0.3 M(-1) s(-1); at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10(-6) M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k on versus pH are -1.05 ± 0.07 and -1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH(-). These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL-CM-cytc.

Cardiolipin-cytochrome c complex: Switching cytochrome c from an electron-transfer shuttle to a myoglobin- and a peroxidase-like heme-protein.

Cytochrome c (cytc) is a small heme-protein located in the space between the inner and the outer membrane of the mitochondrion that transfers electrons from cytc-reductase to cytc-oxidase. The hexa-coordinated heme-Fe atom of cytc displays a very low reactivity toward ligands and does not exhibit significant catalytic properties. However, upon cardiolipin (CL) binding, cytc achieves ligand binding and catalytic properties reminiscent of those of myoglobin and peroxidase. In particular, the peroxidase activity of the cardiolipin-cytochrome c complex (CL-cytc) is critical for the redistribution of CL from the inner to the outer mitochondrial membranes and is essential for the execution and completion of the apoptotic program. On the other hand, the capability of CL-cytc to bind NO and CO and the heme-Fe-based scavenging of reactive nitrogen and oxygen species may affect apoptosis. Here, the ligand binding and catalytic properties of CL-cytc are analyzed in parallel with those of CL-free cytc, myoglobin, and peroxidase to dissect the potential mechanisms of CL in modulating the pro- and anti-apoptotic actions of cytc.

Human cytochrome C natural variants: Studying the membrane binding properties of G41S and Y48H by fluorescence energy transfer and molecular dynamics.

Cytochrome C (cyt C), the protein involved in oxidative phosphorylation, plays several other crucial roles necessary for both cell life and death. Studying natural variants of cyt C offers the possibility to better characterize the structure-to-function relationship that modulates the different activities of this protein. Naturally mutations in human cyt C (G41S and Y48H) occur in the protein central Ω-loop and cause thrombocytopenia 4. In this study, we have investigated the binding of such variants and of wild type (wt) cyt C to synthetic cardiolipin-containing vesicles. The mutants have a lower propensity in membrane binding, displaying higher dissociation constants with respect to the wt protein. Compressibility measurements reveal that both variants are more flexible than the wt, suggesting that the native central Ω-loop is important for the interaction with membranes. Such hypothesis is supported by molecular dynamics simulations. A minimal distance analysis indicates that in the presence of cardiolipin the central Ω-loop of the mutants is no more in contact with the membrane, as it happens instead in the case of wt cyt C. Such finding might provide a hint for the reduced membrane binding capacity of the variants and their enhanced peroxidase activity in vivo.

Role of lysines in cytochrome c-cardiolipin interaction.

Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c-cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c-cardiolipin interaction, as these side chains appear to be critical for cytochrome c-cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet-visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c-cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.

Risk of upper extremity biomechanical overload in automotive facility.

OBJECTIVE: To assess the risk factors for upper extremity-work-related musculoskeletal disorders (UE-WMSD) on 13 production lines in an airbag factory using the threshold limit values-American conference of industrial hygienists- hand activity level (TLV-ACGIH-HAL) method and introduce the ergonomic improvement to reduce the repetitiveness and the peak force (Pf). METHODS: Professional exposure level on 13 production lines in a automobile factory was measured using the TLV-ACGIH-HAL method and a further risk was assessed according to the ergonomic improvement. RESULTS: The first assessment of 9 production lines showed that the professional exposure level was above the TLV or HAL limit. The second assessment showed that the professional exposure level was below the AL limit on all production lines except 1, in which the professional exposure level was between TLV and HAL. CONCLUSION: The assessment of UE-WMSD-related risk can identify the riskiest emplacements and evaluate the reduction of risk in professional exposure through interventions of structural- organizational type.

Conversion of cytochrome c into a peroxidase: inhibitory mechanisms and implication for neurodegenerative diseases.

A further function of cytochrome c (cyt c), beyond respiration, is realized outside mitochondria in the apoptotic program. In the early events of apoptosis, the interaction of cyt c with a mitochondrion-specific phospholipid, cardiolipin (CL), brings about a conformational transition of the protein and acquirement of peroxidase activity. The hallmark of cyt c with peroxidase activity is its partial unfolding accompanied by loosening of the Fe sixth axial bond and an enhanced access of the heme catalytic site to small molecules like H2O2. To investigate the peroxidase activity of non-native cyt c, different forms of the protein were analyzed with the aim to correlate their structural features with the acquired enzymatic activity and apoptogenic properties (wt cyt c/CL complex and two single cyt c variants, H26Y and Y67H, free and bound to CL). The results suggest that cyt c may respond to different environments by changing its fold thus favouring the exertion of different biological functions in different pathophysiological cell conditions. Transitions among different conformations are regulated by endogenous molecules such as ATP and may be affected by synthetic molecules such as minocycline, thus suggesting a mechanism explaining its use as therapeutic agent impacting on disease-associated oxidative and apoptotic mechanisms.

[Alienation to burn-out. Psyche and the Universe of Technology]. / Dall'Alienazione al Burn-out. la Psiche e l'Universo della Tecnica.

The new D. Lgs. N 81, 2008 Article 28 paragraph 1 sanctions that the risk assessment must involve all the possible risks to safety and health of workers, including the work-related stress factors. Stressors at work may vary as to: quantity of work assigned, whether excessive or inadequate; lack of recognition or reward for good job performance; degree of responsibility; precariousness of work; emotional pressures exerted on workers; violence and harassment of psychological nature, poor balance between work and private life. The need man has to understand the causes of his psycho-physical and social disease are old. Only the words we use when dealing with the topic has changed over the time: once it was Alienation now it is Burn-out. The concept of alienation, which has been very important over the time, has many different aspects and has had countless interpretations (which have followed one another), the psycho-analytical, the sociological analysis and the Marxist one, Burnout is actually a syndrome characterized by three interrelated dimensions: exhaustion, cynicism and inefficacy. Therefore it is important to prevent, eliminate or reduce problems related to occupational stress. Among preventive measures, the Europe Agreement identified in the management and in the communication the information necessary to define the goals of the company and the role each employee has. Moreover information and formation are considered the necessary elements to increase awareness and understanding of the problem, its potential causes and possible ways of approading it. Our research group, has developed targeted questionnaires, biological indicators and medical instrumental examinations the occupational doctors can make use of to assess these issues.

Peroxynitrite detoxification by horse heart carboxymethylated cytochrome c is allosterically modulated by cardiolipin.

Carboxymethylation of equine heart cytochrome c (cytc) changes its tertiary structure by disrupting the heme-Fe-Met80 distal bond, such that carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) on peroxynitrite isomerization by ferric CM-cytc (CM-cytc-Fe(III)) is reported. Unlike native ferric cytc (cytc-Fe(III)), CM-cytc-Fe(III) catalyzes peroxynitrite isomerization, the value of the second order rate constant (k(on)) is 6.8 × 10(4)M(-1)s(-1). However, CM-cytc-Fe(III) is less effective in peroxynitrite isomerization than CL-bound cytc-Fe(III) (CL-cytc-Fe(III); k(on)=3.2 × 10(5)M(-1)s(-1)). Moreover, CL binding to CM-cytc-Fe(III) facilitates peroxynitrite isomerization (k(on)=5.3 × 10(5)M(-1)s(-1)). Furthermore, the value of the dissociation equilibrium constant for CL binding to CM-cytc-Fe(III) (K=1.8 × 10(-5)M) is lower than that reported for CL-cytc-Fe(III) complex formation (K=5.1 × 10(-5)M). Although CM-cytc-Fe(III) and CL-cytc-Fe(III) display a different heme distal geometry and heme-Fe(III) reactivity, the heme pocket and the CL cleft are allosterically linked.

Cardiolipin modulates allosterically peroxynitrite detoxification by horse heart cytochrome c.

Upon interaction with bovine heart cardiolipin (CL), horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential out of the range required for its physiological role, binds CO and NO with high affinity, and displays peroxidase activity. Here, the effect of CL on peroxynitrite isomerization by ferric cytc (cytc-Fe(III)) is reported. In the absence of CL, hexa-coordinated cytc does not catalyze peroxynitrite isomerization. In contrast, CL facilitates cytc-Fe(III)-mediated isomerization of peroxynitrite in a dose-dependent fashion inducing the penta-coordination of the heme-Fe(III)-atom. The value of the second order rate constant for CL-cytc-Fe(III)-mediated isomerization of peroxynitrite (k(on)) is (3.2±0.4)×10(5) M(-1) s(-1). The apparent dissociation equilibrium constant for CL binding to cytc-Fe(III) is (5.1±0.8)×10(-5) M. These results suggest that CL-cytc could play either pro-apoptotic or anti-apoptotic effects facilitating lipid peroxidation and scavenging of reactive nitrogen species, such as peroxynitrite, respectively.

The peculiar heme pocket of the 2/2 hemoglobin of cold-adapted Pseudoalteromonas haloplanktis TAC125.

The genome of the cold-adapted bacterium Pseudoalteromonas haloplanktis TAC125 contains multiple genes encoding three distinct monomeric hemoglobins exhibiting a 2/2 α-helical fold. In the present work, one of these hemoglobins is studied by resonance Raman, electronic absorption and electronic paramagnetic resonance spectroscopies, kinetic measurements, and different bioinformatic approaches. It is the first cold-adapted bacterial hemoglobin to be characterized. The results indicate that this protein belongs to the 2/2 hemoglobin family, Group II, characterized by the presence of a tryptophanyl residue on the bottom of the heme distal pocket in position G8 and two tyrosyl residues (TyrCD1 and TyrB10). However, unlike other bacterial hemoglobins, the ferric state, in addition to the aquo hexacoordinated high-spin form, shows multiple hexacoordinated low-spin forms, where either TyrCD1 or TyrB10 can likely coordinate the iron. This is the first example in which both TyrCD1 and TyrB10 are proposed to be the residues that are alternatively involved in heme hexacoordination by endogenous ligands.

Misfolded proteins and neurodegeneration: role of non-native cytochrome c in cell death.

Intermediates play a relevant role in the protein-folding process, because the onset of diseases of genetic nature is usually coupled with protein misfolding and the formation of stable intermediate species. This article describes and briefly discusses the mechanisms considered responsible, at molecular level, for a number of neurodegenerative diseases. In particular, interest is focused on the newly discovered role of cytochrome c in programmed cell death (apoptosis), consisting of acquisition of powerful cardiolipin-specific peroxidase action. Cardiolipin oxidation induces cytochrome c detachment from the mitochondrial membrane and favors the accumulation of products releasing proapoptotic factors. Cytochrome c showing peroxidase activity has non-native structure, and shows enhanced access of the heme catalytic site to small molecules, such as H(2)O(2). The strict correlation linking cytochrome c with the onset of neurodegenerative disorders is described and the therapeutic approach discussed.

Extended cardiolipin anchorage to cytochrome c: a model for protein-mitochondrial membrane binding.

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c-CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c-CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c-CL complex.

ATP acts as a regulatory effector in modulating structural transitions of cytochrome c: implications for apoptotic activity.

The binding of lipids (free fatty acids as well as acidic phospholipids) to cytochrome c (cyt c) induces conformational changes and partial unfolding of the protein, strongly influencing cyt c oxidase/peroxidase activity. ATP is unique among the nucleotides in being able to turn non-native states of cyt c back to the native conformation. The peroxidase activity acquired by lipid-bound cyt c turns out to be very critical in the early stages of apoptosis. Nucleotide specificity is observed for apoptosome formation and caspase activation, the cleavage occurring only in the presence of dATP or ATP. In this study, we demonstrate the connection between peroxidase activity and oleic acid-induced conformational transitions of cyt c and show how ATP is capable of modulating such interplay. By NMR measurement, we have demonstrated that ATP interacts with a site (S1) formed by K88, R91, and E62 and such interaction was weakened by mutation of E62, suggesting the selective role in the interaction played by the base moiety. Interestingly, the interactions of ATP and GTP with cyt c are significantly different at low nucleotide concentrations, with GTP being less effective in perturbing the S1 site and in eliciting apoptotic activity. To gain insights into the structural features of cyt c required for its pro-apoptotic activity and to demonstrate a regulatory role for ATP (compared to the effect of GTP), we have performed experiments on cell lysates by using cyt c proteins mutated on amino acid residues that, as suggested by NMR measurements, belong to S1. Thus, we provide evidence that ATP acts as an allosteric effector, regulating structural transitions among different conformations and different oxidation states of cyt c, which are endowed with apoptotic activity or not. On this basis, we suggest a previously unrecognized role for ATP binding to cyt c at low millimolar concentrations in the cytosol, beyond the known regulatory role during the oxidative phosphorylation in mitochondria.

Cytochrome c: An extreme multifunctional protein with a key role in cell fate.

Cytochrome c, a protein that belongs to class 1 of the c-type cytochrome family, exerts different functions depending on its cellular localization and the conditions in which it operates; therefore, it can be defined as 'extreme multifunctional' protein. It mediates electron-transfer in the respiratory chain and acts as a detoxifying agent to dispose of ROS. In addition, cytochrome c plays a role in cell apoptosis. After its release into the cytosol, the protein binds to APAF-1, activates pro-caspase 9, and triggers an enzymatic cascade leading to cell death. The interaction with cardiolipin, one of the phospholipids making up the mitochondrial membrane, is essential to start apoptosis; the binding partially unfolds cytochrome c, alters the heme pocket region, and facilitates detachment of Met80 from the sixth coordination position of the heme iron. These events change the function of cytochrome c from an electron-transfer shuttle to a peroxidase-like hemoprotein, capable to trigger the process that leads to cell death. This review provides an overview of the key role played by the cytochrome c-cardiolipin interaction in apoptosis. This is not only important per se, it provides interesting perspectives for applications in clinical diagnostics that use the protein as a biomarker.