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One of the primary risk factors for implant failure is thought to be implant-related infections during the early healing phase. Developing coatings with cell stimulatory behaviour and bacterial adhesion control is still difficult for bone implants. This study proposes an approach for one-step deposition of biocompatible and antimicrobial Cu-doped TiO 2 coatings via glow-discharge sputtering of a mosaic target. During the deposition, the bias of the Ti6Al4V substrates was changed. Structure examination, phase analysis, and surface morphology were carried out using X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). The hardness values and hydrophilic and corrosion performance were also evaluated together with cytocompatible and antibacterial examinations against E. coli and S. aureus . The results show great chemical and phase control of the bias identifying rutile, anatase, CuO, or ternary oxide phases. It was found that by increasing the substrate bias from 0 to -50 V the Cu content increased from 15.3 up to 20.7 at% while at a high bias of -100 V, the copper content reduced to 3 at%. Simultaneously, apart from the Cu 2+ state, Cu 1+ is also found in the biased samples. Compared with the bare alloy, the hardness, the water contact angle and corrosion resistance of the biased coatings increased. According to an assessment of in vitro cytocompatibility, all coatings were found to be nontoxic to MG-63 osteoblast cells over the time studied. Copper release and cell-surface interactions generated an antibacterial effect against E. coli and S. aureus strains. The -50 V biased coating combined the most successful results in inhibiting bacterial growth and eliciting the proper responses from osteoblastic cells because of its phase composition, electrochemical stability, hydrophilicity, improved substrate adhesion, and surface roughness. Using this novel surface modification approach, we achieved multifunctionality through controlled copper content and oxide phase composition in the sputtered films.

To fix the bone in orthopedics, it is almost always necessary to use implants. Metals provide the needed physical and mechanical properties for load-bearing applications. Although widely used as biomedical materials for the replacement of hard tissue, metallic implants still confront challenges, among which the foremost is their low biocompatibility. Some of them also suffer from excessive wear, low corrosion resistance, infections and shielding stress. To address these issues, various coatings have been applied to enhance their in vitro and in vivo performance. When merged with the beneficial properties of various bio-ceramic or polymer coatings remarkable bioactive, osteogenic, antibacterial, or biodegradable composite implants can be created. In this review, bioactive and high-performance coatings for metallic bone implants are systematically reviewed and their biocompatibility is discussed. Updates in coating materials and formulations for metallic implants, as well as their production routes, have been provided. The ways of improving the bioactive coating performance by incorporating bioactive moieties such as growth factors, osteogenic factors, immunomodulatory factors, antibiotics, or other drugs that are locally released in a controlled manner have also been addressed.

In this work, the micro-arc oxidation method is used to fabricate surface-modified complex-structured titanium implant coatings to improve biocompatibility. Depending on the utilized electrolyte solution and micro-arc oxidation process parameters, three different types of coatings (one of them-oxide, another two-calcium phosphates) were obtained, differing in their coating thickness, crystallite phase composition and, thus, with a significantly different biocompatibility. An analytical approach based on X-ray computed tomography utilizing software-aided coating recognition is employed in this work to reveal their structural uniformity. Electrochemical studies prove that the coatings exhibit varying levels of corrosion protection. In vitro and in vivo experiments of the three different micro-arc oxidation coatings prove high biocompatibility towards adult stem cells (investigation of cell adhesion, proliferation and osteogenic differentiation), as well as in vivo biocompatibility (including histological analysis). These results demonstrate superior biological properties compared to unmodified titanium surfaces. The ratio of calcium and phosphorus in coatings, as well as their phase composition, have a great influence on the biological response of the coatings.

Expectations for safer and sustainable chemicals and products are growing to comply with the United Nations and European strategies for sustainability. The application of Safe(r) by Design (SbD) in nanotechnology implies an iterative process where functionality, human health and safety, environmental and economic impact and cost are assessed and balanced as early as possible in the innovation process and updated at each step. The EU H2020 NanoReg2 project was the first European project to implement SbD in six companies handling and/or manufacturing nanomaterials (NMs) and nano-enabled products (NEP). The results from this experience have been used to develop these guidelines on the practical application of SbD. The SbD approach foresees the identification, estimation, and reduction of human and environmental risks as early as possible in the development of a NM or NEP, and it is based on three pillars: (i) safer NMs and NEP; (ii) safer use and end of life and (iii) safer industrial production. The presented guidelines include a set of information and tools that will help deciding at each step of the innovation process whether to continue, apply SbD measures or carry out further tests to reduce uncertainty. It does not intend to be a prescriptive protocol where all suggested steps have to be followed to achieve a SbD NM/NEP or process. Rather, the guidelines are designed to identify risks at an early state and information to be considered to identify those risks. Each company adapts the approach to its specific needs and circumstances as company decisions influence the way forward., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Here, we describe the synthesis, characterization, and biological activities of a series of 26 new styryl-2(3H)-benzothiazolone analogs of combretastatin-A4 (CA-4). The cytotoxic activities of these compounds were tested in several cell lines (EA.hy926, A549, BEAS-2B, MDA-MB-231, HT-29, MCF-7, and MCF-10A), and the relations between structure and cytotoxicity are discussed. From the series, compound ( Z )-3-methyl-6-(3,4,5-trimethoxystyryl)-2(3 H )-benzothiazolone ( 26Z ) exhibits the most potent cytotoxic activity (IC 50 0.13 ± 0.01 µM) against EA.hy926 cells. 26Z not only inhibits vasculogenesis but also disrupts pre-existing vasculature. 26Z is a microtubule-modulating agent and inhibits a spectrum of angiogenic events in EA.hy926 cells by interfering with endothelial cell invasion, migration, and proliferation. 26Z also shows anti-proliferative activity in CA-4 resistant cells with the following IC 50 values: HT-29 (0.008 ± 0.001 µM), MDA-MB-231 (1.35 ± 0.42 µM), and MCF-7 (2.42 ± 0.48 µM). Cell-cycle phase-specific experiments show that 26Z treatment results in G2/M arrest and mitotic spindle multipolarity, suggesting that drug-induced centrosome amplification could promote cell death. Some 26Z -treated adherent cells undergo aberrant cytokinesis, resulting in aneuploidy that perhaps contributes to drug-induced cell death. These data indicate that spindle multipolarity induction by 26Z has an exciting chemotherapeutic potential that merits further investigation.

Successful osseointegration, i.e. the fully functional connection of patient's bone and artificial implant depends on the response of the cells to the direct contact with the surface of the implant. The surface properties of the implant which trigger cell responses leading to its integration into the surrounding bone can be tailored by surface modifications or coating with thin layers. One potential material for such applications is ultrananocrystalline diamond (UNCD). It combines the exceptional mechanical properties of diamond with good biocompatibility and possibility of coating as thin uniform films on different substrates of biological interest. In the current work we firstly deposited UNCD films on titanium-coated substrates and applied oxygen or ammonia plasma to modify their surface properties. The as-grown and modified UNCD exhibited relatively smooth surfaces with topography dominated by rounded features. The modifications induced oxygen- or amino-terminated surfaces with increased hydrophilicity. In addition, the UNCD coatings exhibited very low coefficient of friction when diamond was used as a counterpart. As-grown and modified UNCD samples were applied to study the responses of human osteoblast MG63 cells triggered by surfaces with various terminations assessed by proteomic analysis. The results revealed that the coating of Ti with UNCD as well as the plasma modifications resulting in O- or NH 2 -terminated UNCD induced upregulation of proteins specific for cytoskeleton, cell membrane, and extracellular matrix (ECM) involved in the cell-ECM-surface interactions. Proteins from each of these groups, namely, vimentin, cadherin and fibronectin were further studied immunocytochemically and the results confirmed their increased abundance leading to improved cell-to-surface adhesion and cell-to-cell interactions. These findings demonstrate the potential of implant coating with UNCD and its surface modifications for better osseointegration and bone formation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Vacuum cathodic arc TiN coatings with overlaying TiO 2 film were deposited on polished and surface roughened by electron beam modification (EBM) Ti6Al4V alloy. The substrate microtopography consisted of long grooves formed by the liner scan of the electron beam with appropriate frequencies (500 (AR500) and 850 (AR850) Hz). EBM transformed the α + β Ti6Al4V mixed structure into a single α'-martensite phase. Тhe gradient TiN/TiO 2 films deposited on mechanically polished (AR) and EBM (AR500 and AR850) alloys share the same surface chemistry and composition (almost stoichiometric TiN, anatase and rutile in different ratios) but exhibit different topographies (S a equal to approximately 0.62, 1.73, and 1.08 μm, respectively) over areas of 50 × 50 μm. Although the nanohardness of the coatings on AR500 and AR850 alloy (approximately 10.45 and 9.02 GPa, respectively) was lower than that measured on the film deposited on AR alloy (about 13.05 GPa), the hybrid surface treatment offered improvement in critical adhesive loads, coefficient of friction, and wear-resistance of the surface. In phosphate buffer saline, all coated samples showed low corrosion potentials and passivation current densities, confirming their good corrosion protection. The coated EBM samples cultured with human osteoblast-like MG63 cells demonstrated increased cell attachment, viability, and bone mineralization activity especially for the AR500-coated alloy, compared to uncoated polished alloy. The results underline the synergetic effect between the sub-micron structure and composition of TiN/TiO 2 coating and microarchitecture obtained by EBM.

Nanotechnology is a key enabling technology with billions of euros in global investment from public funding, which include large collaborative projects that have investigated environmental and health safety aspects of nanomaterials, but the reuse of accumulated data is clearly lagging behind. Here we summarize challenges and provide recommendations for the efficient reuse of nanosafety data, in line with the recently established FAIR (findable, accessible, interoperable and reusable) guiding principles. We describe the FAIR-aligned Nanosafety Data Interface, with an aggregated findability, accessibility and interoperability across physicochemical, bio-nano interaction, human toxicity, omics, ecotoxicological and exposure data. Overall, we illustrate a much-needed path towards standards for the optimized use of existing data, which avoids duplication of efforts, and provides a multitude of options to promote safe and sustainable nanotechnology.

This paper presents a comprehensive review of European Union (EU) legislation addressing the safety of chemical substances, and possibilities within each piece of legislation for applying grouping and read-across approaches for the assessment of nanomaterials (NMs). Hence, this review considers both the overarching regulation of chemical substances under REACH (Regulation (EC) No 1907/2006 on registration, evaluation, authorization, and restriction of chemicals) and CLP (Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures) and the sector-specific pieces of legislation for cosmetic, plant protection and biocidal products, and legislation addressing food, novel food, and food contact materials. The relevant supporting documents (e.g. guidance documents) regarding each piece of legislation were identified and reviewed, considering the relevant technical and scientific literature. Prospective regulatory needs for implementing grouping in the assessment of NMs were identified, and the question whether each particular piece of legislation permits the use of grouping and read-across to address information gaps was answered.

In order to design and synthesize a new class of heterocyclic analogues of natural combretastatin A-4 and its synthetic derivative AVE8062, the benzoxazolone ring was selected as a scaffold for a bioisosteric replacement of the ring B of both molecules. A library of 28 cis- and trans-styrylbenzoxazolones was obtained by a modified Wittig reaction under Boden's conditions. Structures of the newly synthesized compounds bearing the 3,4,5-trimethoxy-, 3,4-dimethoxy-, 3,5-dimethoxy-, and 4-methoxystyryl fragment at position 4, 5, 6 or 7 of benzoxazolone core were determined on the basis of spectral and X ray data. The in vitro cytotoxicity of styrylbenzoxazolones against different cell lines was examined. Stilbene derivative 16Z, (Z)-3-methyl-6-(3,4,5-trimethoxystyryl)-2(3H)-benzoxazolone, showed highest antiproliferative potential of the series, with IC50 of 0.25 μM against combretastatin resistant cell line HT-29, 0.19 μM against HepG2, 0.28 μM against EA.hy926 and 0.73 μM against K562 cells. Furthermore, the results of flow cytometric analysis confirmed that 16Z induced cell cycle arrest in G2/M phase in the cell lines like combretastatin A-4. This arrest is followed by an abnormal exit of cells from mitosis without cytokinesis into a pseudo G1-like multinucleate state leading to late apoptosis and cell death. Accordingly, synthetic analogue 16Z was identified as the most promising potential anticancer agent in present study, and was selected as lead compound for further detailed investigations., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

A novel approach for the preparation of nano- and microcapsules in aqueous solutions by using thermoresponsive polymer (TRP) templates (mesoglobules) is described. The method comprised three steps: formation of mesoglobules, coating the templates by seeded radical copolymerization, followed by core dissolution and core removal upon cooling. When mesoglobule entraps biomacromolecules during the process of their formation, it makes it possible to load a controlled amount of bioactive compounds without covalent attachment. Special attention is paid to the mesoglobule dissolution upon cooling, as well as their loading efficiency. Details on the outer shell formation and the possibilities for targeting ligands incorporation and control of the shell porosity are discussed. Finally, the seeded radical copolymerization was used for covering DNA complexes with cationic copolymers bearing TRP blocks. This Review is an attempt to convince researchers of the promising perspectives for using mesoglobules as potential reservoirs, carriers, and transferring agents for biologically active substances.

Stable, nanosized polyelectrolyte complexes between rationally designed thermally sensitive block copolymers and plasmid DNA (polyplexes) were formed and their in vitro transfection efficiency was tested. The polyplexes were further stabilized through encapsulation into a biodegradable polymer shell. Although reduced as compared to that of the corresponding polyplexes, the encapsulated systems still show acceptable transfection efficiency. That opens the possibility to tune the balance between the safe transport and efficient delivery of DNA into the cells., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The paper reports on a simple method of synthesizing [PEO(PCL)(2)] triarm star-shaped copolymers by a combination of Michael-addition type reaction and ring-opening polymerization. A Michael-addition reaction yielded a PEO end-capped by two hydroxyl groups-a [PEO(OH)(2)] macroinitiator-which was used for sequential building of PCL blocks. The macroinitiator and copolymers were analyzed by FTIR, (1)H NMR spectroscopy and SEC. The self-assembly behavior of the copolymers in aqueous media was studied by UV-Vis spectroscopy. The size and morphology of the obtained micelles were determined by TEM. None of the polymers had cytotoxic effects in vitro. Cellular uptake studies showed the accumulation of neutral red loaded micelles in the perinuclear area of human hepatocellular carcinoma cells revealing a cellular uptake associated with macropinocytosis and caveolae mediated endocytosis. The accumulation had a sustained effect over 3 days pointing at the potential application of the copolymers micelles as a drug delivery system.

EXT1/EXT2-CDG (Multiple cartilagineous exostoses, hereditary multiple osteochondroma (MO); OMIM 133700/133701) are common defects of O-xylosylglycan glycosylation. The diagnostic criteria are at least two osteochondromas of the juxta-epiphyseal region of long bones with in the majority of cases a positive family history and/or mutation in one of the EXT genes. The authors report data on clinical symptoms and complications of 23 patients (from 16 families), discussing the family history, age of diagnosis, new clinical and molecular data. Fifteen mutations and large deletions, of which nine are new, were detected in the EXT1 and EXT2 gene by sequence analysis, FISH and MLPA analysis.

Aim: To study differences and prognostic effect of some factors on brain (B-type) natriuretic peptide (BNP) levels at discharge of patients with chronic heart failure (CHF) treated in accord with current treatment guidelines., Patients and Methods: Eighty-five consecutive patients hospitalized for CHF were recruited into the study. A standardized study protocol was used for them including collection of blood samples for measurement of electrolytes, creatinine at baseline and BNP at discharge. High BNP levels were determined at values above the threshold value for the highest BNP quartile (> or = 463 pg/ml). Linear regression analyses were performed using the SPSS 16.0., Results: High BNP levels at discharge were measured in 21 (24.7%) of the patients. Patients with persisting high BNP levels, despite the administered correct therapy, had significantly more frequently worse clinical and instrumental characteristics: pulmonary congestion (76.2% vs. 40.6%), IV NYHA functional class (23.8% vs. 4.7%), atrial fibrillation (AF) (71.4% vs. 35.9%) and ischemic etiology of HF (47.6% vs. 15.6%). Multivariate linear regression analysis (F = 7.1, p < 0.001) identified systolic blood pressure (SBP), AF and instrumental data for pulmonary congestion as significant and independent predictors of high BNP at discharge., Conclusions: There were statistically significant differences in the distribution and prognostic effect of the studied factors across the BNP levels. CHF patients with hypotension, AF and instrumental evidence for pulmonary congestion at admission are at higher risk of high BNP levels at discharge and require special care and clinical approach.

In the title compound, C(7)H(8)BNO(3), the mol-ecule lies on an inversion center leading to a statistical disorder of the B(OH)(2) and CONH(2) groups. In the crystal structure, mol-ecules are linked by N-H⋯O and O-H⋯O hydrogen bonds, forming sheets parallel to the bc plane. The B(OH)(2) and CONH(2) groups are twisted out of the mean plane of the benzene ring by 23.9 (5) and 24.6 (6)°, respectively.

1 The effect of short-term exposure to cupric ions (Cu2+) on electric field-stimulated (EFS) or agonist-induced contractions of guinea-pig isolated ileum was studied. 2 EFS elicited tetrodotoxin- and atropine-sensitive contractions that were concentration dependently inhibited by Cu2+ (IC50 = 14.7 +/- 4.2 microm). Maximal inhibition (90.4 +/- 3.1% of baseline contractions) was attained with 30 microm Cu2+. 3 Carbachol induced concentration-dependent contractions (EC50 = 0.021 +/- 0.004 microm) that were inhibited by 0.3 microm atropine to a non-competitive manner (decreased maximal response, EC50 value = 0.26 +/- 0.04 microm, K(e) = 0.026 microm). Cu2+ (15 microm) potentiated contractions induced by carbachol, such that the maximum response was increased by 30.3 +/- 10.4%. 4 Histamine induced concentration-dependent contractions of the longitudinal muscle (EC50 = 0.11 +/- 0.03 microm). Dyphenhydramine (0.1 microm) decreased the maximum response to histamine and shifted the curve to the right (EC50 value = 4.71 +/- 0.35 microm, K(e) = 0.0024 microm). Cu2+ (15 microm) caused a rightward shift of the histamine concentration-response curve (EC50 = 0.61 +/- 0.1 microm) without changing the maximum response. Serotonin induced concentration-dependent contractions at concentrations higher than 10 nM (EC50 value of 0.34 +/- 0.12 microm) were not significantly affected by 15 microm Cu2+. 5 Our results suggest that in ileal longitudinal muscle, Cu2+ inhibits cholinergic neurotransmission but also facilitates postsynaptic muscarinic receptor responses.

Cadmium (Cd2+) is known to cause a selective disruption of the filamentous actin cytoskeleton in the smooth muscle-like renal mesangial cell. We examined the effect of Cd2+ on the distribution of the actin-severing protein, gelsolin. Over 8 h, CdCl2 (10 microM) caused a progressive shift of gelsolin from a diffuse perinuclear and cytoplasmic distribution to a pattern decorating F-actin filaments. Over this time filaments were decreased in number in many cells, and membrane ruffling was initiated. Western blotting and 125I-F-actin gel overlays demonstrated an increase in actin-binding gelsolin activity in the cytoskeletal fraction of cell extracts following Cd2+ treatment. In in vitro polymerization assays, gelsolin acted as a nucleating factor and increased the rate of polymerization. Cytosolic extracts also increased the polymerization rate. Addition of Cd2+ together with gelsolin further increased the rate of polymerization. Gelsolin enhanced depolymerization of purified actin, and Cd2+ partially suppressed this effect. However, cytoskeletal extracts from Cd2+-treated cells also markedly increased depolymerization, suggesting further that Cd2+ may activate cellular component(s) such as gelsolin for actin binding. We conclude that a major effect of Cd2+ on the mesangial cell cytoskeleton is manifest through activating the association of gelsolin with actin, with gelsolin's severing properties predominating under conditions found in Cd2+-treated cells.

During the last 2 decades it was proposed that atherogenesis was closely related to the homeostasis of homocysteine (hCys) and/or copper. We hypothesized that the physiological action of hCys may be connected with its ability to form complexes with Cu. Our results showed the presence of two different Cu-hCys complexes. At a molar ratio Cu:hCys 1:1, a blue complex most probably consistent with a tentative dimeric Cu(II)(2)(hCys)(2)(H(2)O)(2) formula was formed, with tetrahedral Cu coordination and anti-ferromagnetic properties. The redox processes between Cu(II) and hCys, in a molar ratio > or =1:3 led to formation of a second yellow Cu(I)hCys complex. Both Cu-hCys complexes affected the metabolism of extracellular thiols more than hCys alone and inhibited glutathione peroxidase-1 activity and mRNA abundance. The biological action of hCys and Cu-hCys complexes involved remodeling and phosphorylation of focal adhesion complexes and paxillin. The adhesive interactions of monocytes with an endothelial monolayer led to the redistribution of both paxillin and F-actin after all treatments, but the diapedesis of monocytes through endothelial cell monolayer was both greater and faster in the presence of the tentative Cu(II)(2)(hCys)(2)(H(2)O)(2) complex. Together, these observations suggest that Cu-hCys complexes actively participate in the biochemical responses of endothelial cells that are involved in the aethiopathogenesis of atherosclerosis.

Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.

Vascular endothelial cells are constantly exposed to oxidative stress and must be protected by physiological responses. In diabetes mellitus, endothelial cell permeability is impaired and may be increased by high extracellular glucose concentrations. It has been postulated that metallothionein (MT) can protect endothelial cells from oxidative stress with its increased expression by cytokines, thrombin, and endothelin (ET)-1. In this study, we demonstrate that high glucose concentration can induce MT expression in endothelial cells through a distinct ET-dependent pathway. Exposure of human umbilical vein endothelial cells (HUVEC) to increasing concentrations of glucose resulted in a rapid dose-dependent increase in MT-2 and ET-1 mRNA expression. MT expression may be further augmented with addition of ET-1. Preincubation of the cells with the specific ET(B) antagonist BQ-788 blocked MT-2 mRNA expression more effectively than the ET(A) inhibitor TBC-11251. High glucose also increased immunoreactive MT protein expression and induced translocation of MT into the perinuclear area. Perinuclear localization of MT was related to high-glucose-induced reorganization of F-actin filaments. These results demonstrate that an increase in extracellular glucose in HUVEC can lead to a rapid dose-dependent increase in MT-2 mRNA expression and to perinuclear localization of MT protein with changes to the cytoskeleton. These effects are mediated via the ET receptor-dependent pathway.

Alteration of endothelins (ET) and/or their receptors may be important in mediating vascular dysfunction in diabetes. We investigated mechanisms regulating ET-1 expression in human umbilical vein endothelial cells (HUVEC) in response to glucose and the functional significance of these mechanisms. Permeability across HUVEC, grown in medium containing either low (5 mmol/l) or high (25 mmol/l) D-glucose were investigated. L-glucose was used as a control. ET-1, ET(A), and ET(B) mRNA were assessed by semiquantitative RT-PCR. ET-1 immunoreactivity and F-actin microfilament assembly were investigated using confocal microscopy. Increased transendothelial permeability was noted in cells cultured in high glucose or when the cells grown in low (physiologic) glucose were incubated with ET-1, vascular endothelial growth factor (VEGF), or N (G) -nitro-L-arginine methyl ester but not when they were incubated with ET-3, N(G)-nitro-D-arginine methyl ester, or L-glucose. Increased permeability was associated with increased ET-1, ET(A), and ET(B) mRNA expression and augmented ET-1 immunoreactivity. High glucose induced increased permeability, increased ET-1, ET(A), and ET(B) mRNA expression. ET-1 immunoreactivity was blocked by the protein kinase C (PKC) inhibitor chelerythrine, the specific PKC isoform inhibitor 379196, VEGF-neutralizing antibody, or the ET(A) blocker TBC11251, but was not blocked by the specific ET(B) blocker BQ788 or by a VEGF-non-neutralizing antibody. Increased permeability was also associated with deranged F-actin assembly in the endothelial cells and by derangement of endothelial cell junctions as assessed by electron microscopy. Data from this study suggest that high glucose-induced increased permeability may be induced through increased ET-1 expression and disorganization of F-actin assembly. ET-1 expression and increased permeability may occur secondary to PKC isoform activation and may be modulated by VEGF and nitric oxide.

The transient nuclear localization of metallothionein during cell growth and differentiation may be related to the increased requirement of zinc for DNA synthesis, activation of metalloenzymes, and transcription factors. Treatment of 3T3-L1 fibroblasts with aphidicolin, an inhibitor of nuclear DNA synthesis, caused a cell-cycle block at G1/S phase and a delay in the onset of M phase. This also resulted in the accumulation of both zinc and metallothionein in the nucleus. After removal of aphidicolin, the cells rapidly reentered S phase, and during the G2/M phase of cell cycle both zinc and metallothionein began to relocate to the cytoplasm. Delaying the onset of M phase in 3T3-L1 cells could prevent the cytoplasmic relocation of metallothionein. The nuclear translocation of both zinc and metallothionein during the cell cycle can be considered as a normal process and this may be a general mechanism in response to mitogenic signals., (Copyright 2000 Wiley-Liss, Inc.)

The cell viability, lipid peroxidation (LPO) and hydrogen peroxide (H(2)O(2)) generation were measured in cultured primary astrocytes, from metallothionein (MT)-I isoform overexpressing transgenic (MT-I*), MT-I/MT-II null and control mice after exposure to tert-butylhydroperoxide (tBH). Astrocytes from MT-I* mice have high basal levels of both MT-I mRNA and MT protein, whereas there is only MT-III isoform in astrocytes from MT-I/MT-II null mice. The results showed that (1) cultured astrocytes from MT-I* mice were most resistant to the cytotoxicity of tBH and those from MT-I/MT-II null mice were most sensitive to the cytotoxicity of tBH; (2) LPO after exposure to tBH were increased in all cells, but the levels were the highest in astrocytes from MT-I/MT-II null mice, while those in MT-I* mice were the lowest; (3) the levels of H(2)O(2) in cultured astrocytes from MT-I* mice were the lowest, while those in astrocytes from MT-I/MT-II null mice were the highest. These results support the hypothesis that MT can scavenge free radicals and protect astrocytes from oxidative stress.

Although MT is detected as a cytoplasmic protein in hepatocytes of adult liver, it can be localized in the hepatocyte nuclei in human fetal liver bound to zinc and copper. Both nuclear and cytoplasmic localization of MT have been observed in several human tumours, especially in regions of high proliferation. Transient co-localization of zinc and MT has been shown in differentiating myoblast and 3T3-L1 fibroblasts, and during the G1-/S-phase progression in cell cycle. Several mechanisms have been proposed for the import and retention of MT in the nucleus, including signal transduction pathways. The high levels of MT in the nucleus of cells under certain conditions may be related to the increased requirement for zinc for several metallo-enzymes and transcription factors during rapid growth. The function of nuclear MT may be to protect the cell from DNA damage and apoptosis, and also to regulate gene expression during certain stages of cell cycle.

The changes in subcellular localization of metallothionein during differentiation were studied in two myoblast cell lines, L6 and H9C2. Addition of insulin like growth factor-I or lowering foetal bovine serum to 1% can induce differentiation of myoblasts to myotubes. Metallothionein and zinc were localized mainly in the cytoplasm in myoblasts but were translocated into the nucleus of newly formed myotubes during early differentiation. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. Addition of an inhibitor of mitogen-activated protein kinase, PD 98059, did not affect differentiation but blocked nuclear translocation of metallothionein. LY 294092, an inhibitor of PI3 kinase, and rapamycin, an inhibitor of p70S6 serine/threonine kinase, abolished insulin-like growth factor-I induced differentiation of myoblasts, retained metallothionein in the cytoplasm, and decreased metallothionein content. These results demonstrate that the cytoplasmic-nuclear translocation of metallothionein occurs during the early stage of differentiation of myoblasts to myotubes and can be blocked by inhibition of certain signal transduction pathways. The transient nuclear localization of metallothionein and zinc may be related to a high requirement for zinc for metabolic activities during the early stage of differentiation.

The changes in subcellular localization of metallothionein (MT) during differentiation were studied in two muscle cell lines, L6 and H9C2, myoblasts in order to understand the nuclear presence of MT and its antiapoptotic property. In myoblasts, MT and zinc were localized mainly in the cytoplasm but were translocated into the nucleus of newly formed myotubes during early stage of differentiation, which was initiated by lowering FBS from 10% to 1%. In fully differentiated myotubes, metallothionein content was decreased with a cytoplasmic localization. These changes in subcellular localization of MT and Zn were accompanied by increased apoptosis in myotubes. The changes in the apoptosis at different stages of differentiation were measured by both DNA ladder formation and TUNEL technique. The results also show that the apoptosis may be initiated by free radical generation and may be accompanied by p53 expression. The H9C2 cells contained high levels of MT, differentiated slowly, and had low incidence of apoptotic bodies compared to L6 cell line., (Copyright 1999 Academic Press.)

In order to clarify a possible protective role of metallothionein (MT) in the development of streptozotocin (STZ)-caused insulin-dependent diabetes mellitus (IDDM) and its mechanisms, we studied whether MT is effective for protection against STZ-caused IDDM by utilizing MT-null (isoforms MT-I and II) transgenic mice. It was found that Zn pretreatment (I mg/kg body weight as ZnSO4) has a unique inhibitory effect on IDDM development in MT-null mice in contrast to no marked effect in control (C57BL/6J) mice, suggesting that Zn ions free from MT molecules exerted this protective effect. The highest Zn dose (10 mg/kg body weight) fully suppressed development of hyperglycaemia in both types of mice. Pretreatment with Zn partially led to recovery of superoxide dismutase activities in the liver and pancreas in which STZ administration suppressed superoxide dismutase activity in both types of mice. The present study suggests that Zn plays an important role in the pathogenesis of IDDM, although a possible involvement of MT in the protection of STZ-caused IDDM cannot be completely negated.

Using transgenic mice in which metallothionein (MT)-I and MT-II genes, we have studied a putative role of MT as a free radical scavenger against paraquat, a free radical generator. Male mice were injected s.c. with paraquat (PQ) at a single dose of 40 or 60 mg/kg of body weight (b.w.). Two of the six MT-null mice died within 16 h at the dose of 60 mg PQ/kg. b. w. PQ administration increased hepatic MT concentration in the normal mice (C57BL/6J), but not in the MT-null mice. The lipid peroxidation (LP) determined by thiobarbituric acid-reactive substance formation was increased by PQ in the liver of normal and MT-null mice, and the enhanced level was greater in the MT-null mice than in the C57BL/6J mice. Administration of PQ significantly increased blood urea nitrogen only in the MT-null mice, indicating renal damage. Without paraquat administration, the hepatic concentration of non-protein sulphydryl compounds was less in the MT-null mice than in the C57BL/6J mice, and the basal level of LP was higher in the MT-null mice than in the C57BL/6J mice. The present results support the notion that MT plays an antioxidative role against paraquat insult under physiological conditions.