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One of the greatest challenges worldwide is containing the spread of problematic microorganisms. A promising approach is the use of antimicrobial coatings (AMCs). The antimicrobial potential of certain metals, including copper and zinc, has already been verified. In this study, polyethylene terephthalate and aluminum (PET-Al) foils were coated with copper, zinc, and a combination of these two metals, known as core-shell particles, respectively. The resistance of the three different types of coatings to mechanical and chemical exposure was evaluated in various ways. Further, the bacteria Staphylococcus aureus and the bacteriophage ϕ6 were used to assess the antimicrobial efficacy of the coatings. The best efficacy was achieved with the pure copper coating, which was not convincing in the abrasion tests. The result was a considerable loss of copper particles on the surfaces and reduced effectiveness against the microorganisms. The core-shell particles demonstrated better adhesion to the surfaces after abrasion tests and against most chemical agents. In addition, the antimicrobial efficiency remained more stable after the washability treatment. Thus, the core-shell particles had several benefits over the pure copper and zinc coatings. In addition, the best core-shell loading for durability and efficacy was determined in this study.

The versatility of sol-gel systems makes them ideal for functional coatings in industry. However, existing coatings are either too thin or take too long to cure. To address these issues, this paper proposes using an atmospheric pressure plasma source to fully cure and functionalize thicker sol-gel coatings in a single step. The study explores coating various substrates with sol-gel layers to make them scratch-resistant, antibacterial, and antiadhesive. Microparticles like copper, zinc, or copper flakes are added to achieve antibacterial effects. The sol-gel system can be sprayed on and quickly functionalized on the substrate. The study focuses on introducing and anchoring particles in the sol-gel layer to achieve an excellent antibacterial effect by changing the penetration depth. Overall, this method offers a more efficient and effective approach to sol-gel coatings for industrial applications. In order to achieve a layer thickness of more than 100 µm, the second part of the study proposes a multilayer system comprising 15 to 30 µm thick monolayers that can be modified by introducing fillers (such as TiO 2 ) or scratch-resistant chemicals like titanium isopropoxide. This system also allows for individual plasma functionalization of each sol-gel layer. For instance, the top layer can be introduced with antibacterial particles, while another layer can be enhanced with fillers to increase wear resistance. The study reveals the varying antibacterial effects of spherical particles versus flat flakes and the different scratch hardnesses induced by changes in pH, number of layers, and particle introduction.

During normal spermatogenesis, the testis-specific linker histone H1t appears at pachytene stage becomes phosphorylated in early spermatids and disappears in late spermatids. Using reversed-phase and hydrophilic interaction liquid chromatography, H1t from rat and mouse testes was isolated, subjected to enzymatic digestion, and analyzed by mass spectrometry. We observed different phosphorylated states of H1t (mono-, di-, and triphosphorylated) as well as the unphosphorylated protein. Tandem mass spectrometry and immobilized metal ion affinity chromatography experiments with MS/MS/MS and multistage activation were utilized to identify five phosphorylation sites on H1t from rats. Phosphorylation occurs on both serine and threonine residues, whereas only two of these sites were located on peptides containing the CDK consensus motif (S/T)PXZ. Rat H1t phosphorylation starts first by phosphorylation of the nonconsensus motif SPKS in the COOH-terminal domain, namely at Ser-140 and to a smaller degree at a further nonconsensus motif at Ser-186. This is followed by phosphorylation of Ser-177 and Thr-155, both located in CDK consensus motifs. A single phosphorylation site at Ser-8 in the NH2-terminal tail was also found. Mouse H1t lacks Ser-186 and is phosphorylated at up to four sites. In contrast to somatic linker histones, no strict order of increasing phosphorylation could be detected in H1t. Thus, it appears that not the order of up-phosphorylation but the number of the phosphate groups is necessary for regulated chromatin decondensation, thus facilitating the substitution of H1t by transition proteins and protamines.

The metabolic coupling of insulin secretion by pancreatic beta cells is mediated by membrane depolarization due to increased glucose-driven ATP production and closure of K(ATP) channels. Alternative pathways may involve the activation of anion channels by cell swelling upon glucose uptake. In INS-1E insulinoma cells superfusion with an isotonic solution containing 20 mM glucose or a 30% hypotonic solution leads to the activation of a chloride conductance with biophysical and pharmacological properties of anion currents activated in many other cell types during regulatory volume decrease (RVD), i.e. outward rectification, inactivation at positive membrane potentials and block by anion channel inhibitors like NPPB, DIDS, 4-hydroxytamoxifen and extracellular ATP. The current is not inhibited by tolbutamide and remains activated for at least 10 min when reducing the extracellular glucose concentration from 20 mM to 5 mM, but inactivates back to control levels when cells are exposed to a 20% hypertonic extracellular solution containing 20 mM glucose. This chloride current can likewise be induced by 20 mM 3-Omethylglucose, which is taken up but not metabolized by the cells, suggesting that cellular sugar uptake is involved in current activation. Fluorescence resonance energy transfer (FRET) experiments show that chloride current activation by 20 mM glucose and glucose-induced cell swelling are accompanied by a significant, transient redistribution of the membrane associated fraction of ICln, a multifunctional 'connector hub' protein involved in cell volume regulation and generation of RVD currents.

How can a large number of different phenotypes be generated by a limited number of genotypes? Promiscuity between different, structurally related and/or unrelated proteins seems to provide a plausible explanation to this pertinent question. Strategies able to predict such functional interrelations between different proteins are important to restrict the number of putative candidate proteins, which can then be subjected to time-consuming functional tests. Here we describe the use of the operon structure of the nematode genome to identify partner proteins in human cells. In this work we focus on ion channels proteins, which build an interface between the cell and the outside world and are responsible for a growing number of diseases in humans. However, the proposed strategy for the partner protein quest is not restricted to this scientific area but can be adopted in virtually every field of human biology where protein-protein interactions are assumed.

ICln is a multifunctional protein that is essential for cell volume regulation. It can be found in the cytosol and is associated with the cell membrane. Besides its role in the splicing process, ICln is critically involved in the generation of ion currents activated during regulatory volume decrease after cell swelling (RVDC). If reconstituted in artificial bilayers, ICln can form ion channels with biophysical properties related to RVDC. We investigated (i) the cytosol versus cell membrane distribution of ICln in rat kidney tubules, NIH 3T3 fibroblasts, Madin-Darby canine kidney (MDCK) cells, and LLC-PK1 epithelial cells, (ii) fluorescence resonance energy transfer (FRET) in living fibroblasts between fluorescently tagged ICln and fluorochromes in the cell membrane, and (iii) possible functional consequences of an enhanced ICln presence at the cell membrane. We demonstrate that ICln distribution in rat kidneys depends on the parenchymal localization and functional state of the tubules and that cell swelling causes ICln redistribution from the cytosol to the cell membrane in NIH 3T3 fibroblasts and LLC-PK1 cells. The addition of purified ICln protein to the extracellular solution or overexpression of farnesylated ICln leads to an increased anion permeability in NIH 3T3 fibroblasts. The swelling-induced redistribution of ICln correlates to altered kinetics of RVDC in NIH 3T3 fibroblasts, LLC-PK1 cells, and MDCK cells. In these cells, RVDC develops more rapidly, and in MDCK cells the rate of swelling-induced depolarization is accelerated if cells are swollen for a second time. This coincides with an enhanced ICln association with the cell membrane.

Cell volume alterations are involved in numerous cellular events like epithelial transport, metabolic processes, hormone secretion, cell migration, proliferation and apoptosis. Above all it is a need for every cell to counteract osmotic cell swelling in order to avoid cell damage. The defence against excess cell swelling is accomplished by a reduction of the intracellular osmolarity by release of organic- or inorganic osmolytes from the cell or by synthesis of osmotically less active macromolecules from their specific subunits. De-spite the large amount of experimental data that has accumulated, the intracellular mechanisms underlying the sensing of cell volume perturbations and the activation of volume compensatory processes, commonly summarized as regulatory volume decrease (RVD), are still only partly revealed. Moving into this field opens a complex scenario of molecular rearrangements and interactions involving intracellular messengers such as calcium, phosphoinositides and inositolphosphates as well as phosphoryla-tion/dephosphorylation processes and cytoskeletal reorganization with marked cell type- and tissue specific variations. Even in one and the same cell type significant differences regarding the activated pathways during RVD may be evident. This makes it virtually im-possible to unambigously define common sensing- and sinaling pathways used by differ-ent cells to readjust their celll volume, even if all these pathways converge to the activa-tion of comparatively few sets of effectors serving for osmolyte extrusion, including ion channels and transporters. This review is aimed at providing an insight into the manifold cellular mechanisms and alterations occuring during cell swelling and RVD., (Copyright 2002 S. Karger AG, Basel)

In previous studies we have shown that the expression of the transforming Ha-ras oncogene in NIH 3T3 fibroblasts stimulates cellular calcium entry, which triggers oscillatory calcium induced calcium release from internal stores. The intracellular calcium oscillations lead to cytoskeletal remodeling by actin stress fiber depolymerization and activation of the Na(+)/H(+) exchanger thus mediating cell swelling and intracellular alkalosis, both important mitogenic signals. This is evidenced by abrogation of Ha-ras induced growth factor independent cell proliferation by interference with any of these events, i.e. by inhibition of cellular calcium entry or inhibition of the Na(+)/H(+) exchanger. As shown in this study, simvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme for cholesterol biosynthesis, is able to prevent these events following the expression of the transforming Ha-ras oncogene. We show, that simvastatin inhibits farnesylation dependent membrane translocation of a CAAX motive bearing yellow fluorescent protein and suppresses Ha-ras stimulated cellular calcium influx, which can be identified as capacitative calcium entry. In addition simvastatin is able to block regulatory volume decrease channels and to suppress the cytoskeletal remodeling, intracellular alkalinization, increase in cell volume and growth factor independent cell proliferation induced by the oncogene. Thus simvastatin is able to prevent crucial cellular events following expression of the transforming Ha-ras oncogene., (Copyright 2002 S. Karger AG, Basel)

The Na(+)/H(+) exchangers (NHEs) are among the major ion transporters involved in cell volume regulation. NHE activation leads to a cellular influx of Na(+) ions and extrusion of H(+) ions, which are readily replenished from intracellular buffers. This will result in a net import of Na(+). In many systems NHE operates in parallel to Cl(-)/ HCO3(-) exchange, resulting in cellular uptake of NaCl. The influx of osmotically obliged water will consequently lead to cell swelling. This makes NHEs suitable to serve as powerful mechanisms for increasing cell volume (CV). The low volume threshold for NHE activation enables the cells to respond to very minute reductions of the CV. By the coupling to the export of H(+) ions cell volume regulatory NHE activation may lead to changes in intracellular pH. On the other hand NHEs are activated by a broad variety of ligands and by intracellular acidosis, which, in turn, may consequently lead to cell swelling. In addition, NHEs are linked to other intracellular proteins and structures, like e.g. the cytoskeleton, which themelves are involved in the regulation of numerous cellular processes. Therefore NHEs link CV regulation to a diversity of cellular functions, both in physiological and pathophysiological conditions. Six isoforms of the Na(+)/H(+) exchanger, termed NHE1--6, have been cloned so far. NHE 1--5 are located in the plasma membrane, whereas NHE6 is sorted to the mitochondrial membrane. NHE1 and NHE6 are the ubiquitously expressed isoforms. The expression of the isoforms NHE2 to NHE5 is restricted to specific tissues and the pattern of their expression, as well as their subcellular localization indicate that they fulfill specialized functions. Cell shrinkage induced activation has been shown for NHE1,2 and 4. In contrast, NHE3 is inhibited by cell shrinkage. In many cells several isoforms are present and assigned to specific membrane domains where they may serve a functional crosstalk between the different ion transporters.

Normal function of organs and cells is tightly linked to the cytoarchitecture. Control of the cell volume is therefore vital for the organism. A widely established strategy of cells to counteract swelling is the activation of chloride and potassium channels, which leads to a net efflux of salt followed by water - a process termed regulatory volume decrease. Since there is evidence for swelling-dependent chloride channels (IClswell) being activated also during pathological processes, the identification of the molecular entity underlying IClswell is of utmost importance. Several proteins are discussed as the channel forming IClswell, i.e. phospholemman, p-glycoprotein, CLC-3 and ICln. In this review we would like to focus on the properties of ICln, a protein cloned from a Madin Darby canine kidney (MDCK) cell library whose expression in Xenopus laevis oocytes resulted in a nucleotide sensitive outwardly rectifying chloride current closely resembling the biophysical properties of IClswell., (Copyright 2000 S. Karger AG, Basel.)