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Cadmium is a widespread environmental pollutant which induces severe toxic alterations, including osteomalacia and osteoporosis, likely by estrogen receptor-dependent mechanisms. Indeed, cadmium has been described to act as an endocrine disruptor and its toxicity is exerted both in vivo and in vitro through induction of apoptosis and/or necrosis by not fully clarified intracellular mechanism(s) of action. Aim of the present study was to further investigate the molecular mechanism by which cadmium might alter homeostasis of estrogen target cells, such as osteoblast homeostasis, inducing cell apoptosis and/or necrosis. Human osteoblastic cells (hFOB 1.19) in culture were used as an in vitro model to characterize the intracellular mechanisms induced by this heavy metal. Cells were incubated in the presence/ absence of 10-50 μM cadmium chloride at different times and DNA fragmentation and activation of procaspases- 8 and -3 were induced upon CdCl(2) treatment triggering apoptotic and necrotic pathways. Addition of caspase-8 and -3 inhibitors (Z-IETD-FMK and Z-DQMD-FMK) partially blocked these effects. No activation of procaspase-9 was observed. To determine the role of mitogen-activated protein kinases (MAPK) in these events, we investigated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated protein kinase (ERK1/2) phosphorylation which were activated by 10 μM CdCl(2). Chemical inhibitors of JNK, p38, and ERK1/2, SP600125, SB202190, and PD98059, significantly reduced the phosphorylation of the kinases and blunted apoptosis. In contrast, caspase inhibitors did not reduce the cadmium-induced MAPK phosphorylation, suggesting an independent activation of these pathways. In conclusion, at least 2 pathways appear activated by cadmium in osteoblasts: a direct induction of caspase-8 followed by activation of caspase-3 and an indirect induction by phosphorylation of ERK1/2, p38, and JNK MAPK triggering activation of caspase-8 and -3.

Increased use of titanium and titanium-base alloys in medicine is occurring due to their relatively low elasticity modulus and enhanced corrosion resistance as compared to more convenient alloys intended for implantation into the human body [1]. However, the properties of oxides present in the near-surface region of Tibase materials deserve special attention. The titanium oxides from TiO to Ti3O5 can exist in a stable state at the harsh conditions of the body fluids surrounding the implant. Tribo-chemical reactions during use can modify the oxidized surface of the implant producing wear debris accumulation, which results in an adverse cellular response and implant loosening. To improve bone response and reliability of implanted device, the surface modification by coating of implants with various substances can be used. In particular, titanium carbide on titanium can provide protection against oxidation, excellent corrosion resistance, enhanced hardness, and superior wear resistance of the implant. To cover a substrate with a thin carbide film, different methods are commonly used, e.g., chemical vapor phase deposition, pulsed laser ablation deposition (PLAD), magnetron sputtering [2–4]. The present study is aimed at the study of the hardness of electron beam deposited (EBD) titanium carbide films onto Ti substrates. To prepare the targets for EBD, titanium carbide powder (Aldrich, 98% pure) was pressed into 18 mm diameter pellets. The pellets were placed into a crucible made of titanium diboride/boron nitride composite (Advanced Ceramics Corp. Europe, UK). The crucible was then inserted into a water-cooled electron beam gun (EVI-8, Ferrotec, Germany), which has been positioned into a stainless-steel chamber evacuated by a turbomolecular pump supported by a rotation pump. The electron beam gun was controlled by a joypad that permits a complete control of the accelerating voltage in the range between −3.05 and −10 kV, the shape, pattern and position of the beam. The maximum operation power was 5 kW. The gun has a magnetic lenses system that allows a 270 ◦ deflection of the beam for avoiding contamination of the evaporating material with tungsten of the emitting filament. The substrates were heated under vacuum of 5.10−2 mbar in the chamber with a high-power halogen lamp. The deposition process was performed at the accelerating voltage −3.5 kV and the emission current of 130 to 200 mA. The pattern of electron beam was circular and slow, to ensure uniform consumption of the evaporating material. The deposition was performed at the substrate pre-heating temperature 200 or 800 ◦C for several minutes. After the cooling and venting with N2, the samples were of metallic luster and dark grey colored. The thickness of film was evaluated by scanning electron microscopy observation of the cross-sections of the samples (a LEO 1530 SEM apparatus, Carl Zeiss, Germany). An absolute error of the thickness measurement was ±10 nm. The hardness was measured with the use of a Leica VMHT apparatus (Leica GmbH, Germany) equipped with a standard Vickers pyramidal indenter (square-based diamond pyramid of face angle 136 ◦). On each sample indentations were made with 5 to 7 loads ranging from 0.01 to 5 N. Both diagonals were measured to diminish the effects of asymmetry on the imprint. Standard deviation of the diagonal measurements was about 5 to 9% of the diagonal length. The measured hardness was that of the film-substrate composite system. To separate the hardness of the film-substrate system on its constituents from the film and the substrate, a model based on an area “law-of-mixtures” approach was applied [5], where the composite hardness Hc of the film-substrate system is expressed as

N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornitine and N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine phenyl and p-nitrophenyl esters (7-10) were synthesized and tested as inhibitors of trypsin, chymotrypsin and thrombin. The N,N-dimethylcarbamoyl group was chosen to decrease the tendency of acylcarbazates to cyclization into 1,3,4-oxadiazol-2(3H)-ones. Only the p-nitrophenyl alpha-azaornithine derivative 8 was inactivated rapidly by intramolecular acylation of the terminal amino group, rather than by cyclization to oxadiazolone, in aqueous solution at pH 8. The corresponding alpha-azalysine derivative 10 is completely unaffected under the same conditions. Rapid inactivation of thrombin and trypsin only was observed for all alpha-azapeptide esters 7-10 at 0.5 mM inhibitor concentration. No proteolytic activity was restored after 24 h following 2,000 fold dilution of the inhibitor concentration suggesting formation of very stable acylenzymes.

Fifteen 1-peptidyl-2-haloacetyl hydrazines, which can be considered halometanes of azapeptides containing Phe in P2 and alpha-aza-Ala or alpha-aza-Gly in P1, were synthesized and tested as models of cysteine-proteases inhibitors. By use of kinetic methods, they proved to irreversibly inactivate papain and cathepsin B via a reversible enzyme-inhibitor intermediate. Second-order rate constants of inactivation in the range 26-23000 M-1s-1 were observed for papain and 2000-39600 M-1s-1 for cathepsin B. KI for the reversible EI adducts ranged from 230 to 0.16 microM for papain and from 11 to 0.37 microM for cathepsin B. Structure of possible reversible EI complex is proposed and used to discuss the effects of structural variation of the inhibitors on the kinetic parameters of inactivation. Title compounds proved to be selective for cysteine-proteases, since no inhibiting activity could be detected toward trypsin, chymotrypsin and porcine pancreatic elastase at 0.1 mM concentration, after 6 h incubation. Relatively low aspecific alkylating properties were also verified in tests using glutathione as the nucleophile.

A brief review on the methods for vitamin B12 determination is presented. The most appropriate utilization of each method based on its sensitivity, on the availability of the sample and on practical difficulties has been indicated. The methods have been divided into: a) physico-chemical methods, where the vitamin is identified spectrophotometrically; b) microbiological methods, where the vitamin is determined quantitatively by means of the growth of cobalamine dependent bacterial strains; c) kinetic methods, based on the different rate of conversion of the different cobalamines into dicyanocobalamine in the presence of an alkaline solution of cyanide; d) enzymatic methods, where activation of apodioldehydrase is used for the quantitative determination of adenosylcobalamine; e) chromatographic methods, where the separation of the cobalamines is obtained by chromatography and the quantitative determination is obtained by means of the growth of cobalamine-dependent bacterial strains (bioautography); f) radioisotopic methods where total cobalamines are determined quantitatively by measuring radioactivity after addition of (57Co)-CNCbl and a specific binder.

Mitochondrial and cytoplasmic isozymes of aspartate transaminase are separated from beef kidney homogenates by ammonium sulfate fractionation. The mitochondrial isozyme is purified essentially as described earlier (Eur. J. Biochem., 1972, 26, 196-206) with slight modification in order to increase the yield. The cytoplasmic isozyme is purified by heat treatment followed by ion exchange cellulose chromatography and gel chromatography. The enzyme is pure in the ultracentrifuge and in polyacrylamide gel electrophoresis; it shows only one anionic band and no subforms. It has a molecular weight of 93,000 +/- 2000 and is composed of two subunits of 46,000 M.W. The enzyme has a specific activity of 49 micronmoles of oxalacetate x min-1 x mg-1. It contains 5 SH groups per subunit; three are directly titratable with p-mercuribenzoate and the other two only after addition of 0.2% SDS; there is no evidence of S-S groups. Km values for aspartate, glutamate, alpha-ketoglutarate and oxalacetate are in the order 1.25, 3.2, 0.06 and 0.41 mM in the cytoplasmic isozyme and 0.7, 5.0, 1.25 and 0.12 mM in the mitochondrial one.

In vivo production of taurine can not proceed from cysteine via the action of cysteinsulphinic acid-decarboxylase in those mammalian organs where this enzymatic activity is absent, such as heart. The possibility of existence of another metabolic way is strengthened by the experimental finding that, in vitro, cysteamine is obtained from pantothenylcysteine-4'-phosphate through the combined action of two mammalian enzymes, i.e. pantothenylcysteine-4'-phosphate decarboxylase and pantetheinase, used in partially purified forms. This new route should be related to the known biosynthetic pathway of CoA.

Sodium azide is known to produce alterations in mammalian copper proteins, thus rendering them unable to bind exogenous metal, which remains in the "labile pool" condition. Continuous administration of sodium azide at LD50 for 30 days causes copper accumulation in several tissues and even in the nervous system, with characteristic changes in neurones and glial cells, very much resembling the alterations observed in Wilson's disease. Dietary copper administration, on the contrary, though raising the level of tissue-bound metal, does not produce cellular damage. These findings allow us to suppose that sodium azide may alterate the coppper chelating proteins in the tissues, especially in the nervous system, thus causing the storage of cell-toxic "labile pool" metal. The pathogenesis of Wilson's disease and the problem of "pathoclisis" in the nervous system are debated.

l-glutamate-phenylpyruvate aminotransferase was extracted from beef kidney mitochondria. The purification procedure included lysis of mitochondria in n-butanol, ammonium sulphate fractionation between 55% and 80% saturation, gel filtration on G-100 Sephadex and fractionation on DEAE-Sephadex. The optimum pH of the enzyme is around 8. The enzyme is homogeneous to starch gel electrophoresis at various pH but shows two components when submitted to ultracentrifugal analyses. A possible dissociation of the enzyme into subunits dependent on concentration is discussed.

A simple, rapid, and sensitive method is described for the assay of phenylalanine aminotransferase activity, based on the conversion in alkaline media of the keto form of the phenyl pyruvate to its enol form, which absorbs at 318 mμ and is sufficiently stable to permit a quantitative determination of the keto acid under the experimental conditions described in the text.

Each new era brings with it new technologies and new challenges to civilization. The impetuous technological development is changing the economy and work more and more every day; to the point of making the passage from agrarian society to industrial society seem like a trifle. Nevertheless, if there is widespread awareness of the general issues of society, there is definitely less on the way in which anthropomorphic robots (increasingly in an emotional and cognitive relationship with human beings) are radically modifying intimate and exclusive spheres such as sexuality and love. An epochal passage is being announced from an organic sexuality (driven by desire and pleasure) to an inorganic, artificial sexuality, suspended in an abstract excitement, always at hand and disengaged from categories such as beauty, forms and age. In this paper, we will discuss the plausibility of an emotional relationship between robots and human beings and its ethical implications. It is probable, in fact, that soon there will be self-aware robots, capable of experiencing feelings such as love, joy, suffering, pain and pleasure. If this scenario is realized, will we continue to think that they are only machines, or will we recognize the moral relevance that they do not have today?

For over a century, it has been accepted that there exists a remote psychic space that influences our way of thinking, perception, decision-making and so on. This space, defined by Freud as the ‘unconscious’, embodies the psychic element that we are unaware of. It is a space that is an extension and a wider representation of the complex and sophisticated metapsychological apparatus he conceived. With respect to the conscious sphere (whose related anatomical function concerns the encephalic trunk, diencephalon and associative cortical areas), this unconscious dimension relates to the limbic lobe and specific areas of the frontal, parietal and temporal lobes. This complex neurophysiological system connects and coordinates the sensory, emotional, cognitive and behavioural systems. Its sophisticated adaptive functions, implied and unaware, allow the prefrontal cortex to transform a huge amount of information into explicit behaviour, thus affecting our behaviour in terms of executive functions, decision-making, moral judgments and so on. In this paper, we advance the hypothesis that these subcortical components constitute interconnected modal matrices that intervene under certain circumstances to respond to environmental requirements. In this space of functional integration, they act as an intermediary between the frontal cortex, the limbic system and the basal ganglia and are a key player in the planning, selection and decision to carry out appropriate actions. Due to their generativity and intramodal and extramodal connections, it is plausible to assume that they also play a role in mediation between unconscious and conscious thought. © Springer Nature Singapore Pte Ltd. 2020.

This study is concerned with the structural characterization in solution of the glutamate dehydrogenase from the Archaeon Sulfolobus solfataricus. At neutral pH both alpha-helix and beta-sheet constitute the secondary structure of this enzyme, on the basis of circular dichroism. A complex, temperature dependent self-association equilibrium regulates the formation of the enzyme quaternary structure, which seems to be accompanied by a reversible structural change. At 25 degrees C the enzyme is mostly represented by monomeric subunits at concentrations lower than 0.02 mg/ml, while oligomers are predominant at concentrations higher than 0.12 mg/ml. The mid-point of the association curve shifts from 0.05 mg/ml at 25 degrees C to about 0.1 mg/ml at 45 degrees C. Only the oligomeric form appears to be temperature resistant. Monomeric and oligomeric enzyme show distinct behaviour on guanidine hydrochloride perturbation at neutral pH. The monomer denaturation, although complex, is reversible. Two fluorescent tryptophan classes are detectable in the monomer, monitoring the independent unfolding of two regions through a multistate transition. Instead, the oligomeric protein shows a complex denaturation pattern with the tendency to aggregate irreversibly at high denaturant concentration.

The first thermophilic eubacterial glutamate dehydrogenase was purified to homogeneity from Bacillus acidocaldarius to compare its molecular properties with those of the glutamate dehydrogenase from thermophilic archaea. Glutamate dehydrogenase represents 2% of the total soluble proteins of B. acidocaldarius, an amount that may suggest an important role for this enzyme in nitrogen metabolism of the thermophilic eubacterium. The protein is a hexamer (subunit mass 48 kDa) and undergoes dissociation during gel filtration analysis. Isoelectric focusing of the purified enzyme indicated a pI of 4.5. The enzyme is strictly specific for NAD, 2-oxoglutarate, and l-glutamate. The thermal stability of B. acidocaldarius glutamate dehydrogenase is dependent on protein concentration.

The hyperthermophilic archaebacterium Pyrococcus furiosus contains high levels of NAD(P)-dependent glutamate dehydrogenase activity. The enzyme could be involved in the first step of nitrogen metabolism, catalyzing the conversion of 2-oxoglutarate and ammonia to glutamate. The enzyme, purified to homogeneity, is a hexamer of 290 kDa (subunit mass 48 kDa). Isoelectric-focusing analysis of the purified enzyme showed a pI of 4.5. The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate but utilizes both NADH and NADPH as cofactors. The purified enzyme reveals an outstanding thermal stability (the half-life for thermal inactivation at 100 degrees C was 12 h), totally independent of enzyme concentration. P. furiosus glutamate dehydrogenase represents 20% of the total protein; this elevated concentration raises questions about the roles of this enzyme in the metabolism of P. furiosus.