Lattice Shrinkage by Incorporation of Recombinant Starmaker-Like Protein within Bioinspired Calcium Carbonate Crystals.

The biological mediation of mineral formation (biomineralization) is realized through diverse organic macromolecules that guide this process in a spatial and temporal manner. Although the role of these molecules in biomineralization is being gradually revealed, the molecular basis of their regulatory function is still poorly understood. In this study, the incorporation and distribution of the model intrinsically disordered starmaker-like (Stm-l) protein, which is active in fish otoliths biomineralization, within calcium carbonate crystals, is revealed. Stm-l promotes crystal nucleation and anisotropic tailoring of crystal morphology. Intracrystalline incorporation of Stm-l protein unexpectedly results in shrinkage (and not expansion, as commonly described in biomineral and bioinspired crystals) of the crystal lattice volume, which is described herein, for the first time, for bioinspired mineralization. A ring pattern was observed in crystals grown for 48 h; this was composed of a protein-enriched region flanked by protein-depleted regions. It can be explained as a result of the Ostwald-like ripening process and intrinsic properties of Stm-l, and bears some analogy to the daily growth layers of the otolith.

Involvement of cell surface 90 kDa heat shock protein (HSP90) in pattern recognition by human monocyte-derived macrophages.

Heat shock proteins (HSPs) are typical intracellular chaperones which also appear on the cell surface and in extracellular milieu. HSP90, which chaperones many proteins involved in signal transduction, is also a regular component of LPS-signaling complexes on Mϕ. As LPS is a prototypical PAMP, we speculated that HSP90 is engaged in pattern recognition by professional phagocytes. In this report, we provide the first evidence, to our knowledge, of the geldanamycin (Ge)-inhibitable HSP90 on the surface of live monocyte-derived Mϕs (hMDMs). Using cytometry and specific Abs, we showed both HSP90 isoforms (α and ß) on the surface of human monocytes and hMDMs. The cell-surface HSP90 pool was also labeled with cell-impermeable Ge derivatives. Confocal analysis of hMDMs revealed that HSP90-inhibitor complexes were rapidly clustered on the cell surface and recycled through the endosomal compartment. This finding suggests that the N-terminal (ATPase) domain of HSP90 is exposed and accessible from the extracellular space. To study the role of cell-surface HSP90 in pattern recognition, we used pathogen (PAMPs)- or apoptotic cell-associated molecular patterns (ACAMPs). We showed that blocking the cell-surface HSP90 pool leads to a dramatic decrease in TNF production by monocytes and hMDMs exposed to soluble (TLRs-specific ligands) and particulate [bacteria Staphylococcus aureus (SA) and Porphyromonas gingivalis (PG)] PAMPs. Surprisingly, in hMDMs the functional cell-surface HSP90 was not necessary for the engulfment of either apoptotic neutrophils or bacteria. The presented data suggest that the cell-surface HSP90 is a "signaling complex chaperone," with activity that is essential for cytokine response but not for target engulfment by Mϕ.

Low level phosphorylation of histone H2AX on serine 139 (γH2AX) is not associated with DNA double-strand breaks.

Phosphorylation of histone H2AX on serine 139 (γH2AX) is an early step in cellular response to a DNA double-strand break (DSB). γH2AX foci are generally regarded as markers of DSBs. A growing body of evidence demonstrates, however, that while induction of DSBs always brings about phosphorylation of histone H2AX, the reverse is not true - the presence of γH2AX foci should not be considered an unequivocal marker of DNA double-strand breaks. We studied DNA damage induced in A549 human lung adenocarcinoma cells by topoisomerase type I and II inhibitors (0.2 µM camptothecin, 10 µM etoposide or 0.2 µM mitoxantrone for 1 h), and using 3D high resolution quantitative confocal microscopy, assessed the number, size and the integrated intensity of immunofluorescence signals of individual γH2AX foci induced by these drugs. Also, investigated was spatial association between γH2AX foci and foci of 53BP1, the protein involved in DSB repair, both in relation to DNA replication sites (factories) as revealed by labeling nascent DNA with EdU. Extensive 3D and correlation data analysis demonstrated that γH2AX foci exhibit a wide range of sizes and levels of H2AX phosphorylation, and correlate differently with 53BP1 and DNA replication. This is the first report showing lack of a link between low level phosphorylation γH2AX sites and double-strand DNA breaks in cells exposed to topoisomerase I or II inhibitors. The data are discussed in terms of mechanisms that may be involved in formation of γH2AX sites of different sizes and intensities.

The cellular internalization of liposome encapsulated protoporphyrin IX by HeLa cells.

The proper lipid composition of liposomes designed to carry drugs determines their surface properties ensuring their accumulation within selected tissue. The electrostatic potential and surface topology of liposomes affect the internalization by single cells. The high-resolution imaging of cancer cells and the distribution of protoporphyrin-loaded liposomes within the cytoplasm and its dependence on the liposome surface properties are presented. In the paper, HeLa cells were used to investigate the uptake of porphyrin-loaded liposomes and liposomes alone by means of confocal and differential interference contrast microscopies. The effect of liposomes surface electrostatic potential and surface topology on their intracellular distribution was evaluated. The time evolution of the intracellular distribution of liposomes labelled with Rhodamine-PE was examined on HeLa cells. These studies allow for the identification of the liposome lipid composition so the efficient delivery of the active substance to cancer cells will be achieved. The obtained results showed that neutral PC-liposomes are the most efficiently internalized by HeLa cells. Moreover, results showed that properties of liposomes affect not only the internalization efficiency of the photosensitizer but also its distribution within the cells, as revealed by colocalization measurements.

The nucleocapsid protein of human coronavirus NL63.

Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.

Fenofibrate enhances barrier function of endothelial continuum within the metastatic niche of prostate cancer cells.

OBJECTIVE: Extravasation of circulating cancer cells is an important step of the metastatic cascade and a potential target for anti-cancer strategies based on vasoprotective drugs. Reports on anti-cancer effects of fenofibrate (FF) prompted us to analyze its influence on the endothelial barrier function during prostate cancer cell diapedesis. RESEARCH DESIGN AND METHODS: In vitro co-cultures of endothelial cells with cancer cells imitate the 'metastatic niche' in vivo. We qualitatively and quantitatively estimated the effect of 25 µM FF on the events which accompany prostate carcinoma cell diapedesis, with the special emphasis on endothelial cell mobilization. RESULTS: Fenofibrate attenuated cancer cell diapedesis via augmenting endothelial cell adhesion to the substratum rather than through the effect on intercellular communication networks within the metastatic niche. The inhibition of endothelial cell motility was accompanied by the activation of PPARα-dependent and PPARα-independent reactive oxygen species signaling, Akt and focal adhesion kinase (FAK) phosphorylation, in the absence of cytotoxic effects in endothelial cells. CONCLUSIONS: Fenofibrate reduces endothelial cell susceptibility to the paracrine signals received from prostate carcinoma cells, thus inhibiting endothelial cell mobilization and reducing paracellular permeability of endothelium in the metastatic niche. Our data provide a mechanistic rationale for extending the clinical use of FF and for the combination of this well tolerated vasoactive drug with the existing multidrug regimens used in prostate cancer therapy.

Blue-light-activated phototropin2 trafficking from the cytoplasm to Golgi/post-Golgi vesicles.

Phototropins are plasma membrane-localized UVA/blue light photoreceptors which mediate phototropism, inhibition of primary hypocotyl elongation, leaf positioning, chloroplast movements, and stomatal opening. Blue light irradiation activates the C-terminal serine/threonine kinase domain of phototropin which autophosphorylates the receptor. Arabidopsis thaliana encodes two phototropins, phot1 and phot2. In response to blue light, phot1 moves from the plasma membrane into the cytosol and phot2 translocates to the Golgi complex. In this study the molecular mechanism and route of blue-light-induced phot2 trafficking are demonstrated. It is shown that Atphot2 behaves in a similar manner when expressed transiently under 35S or its native promoter. The phot2 kinase domain but not blue-light-mediated autophosphorylation is required for the receptor translocation. Using co-localization and western blotting, the receptor was shown to move from the cytoplasm to the Golgi complex, and then to the post-Golgi structures. The results were confirmed by brefeldin A (an inhibitor of the secretory pathway) which disrupted phot2 trafficking. An association was observed between phot2 and the light chain2 of clathrin via bimolecular fluorescence complementation. The fluorescence was observed at the plasma membrane. The results were confirmed using co-immunoprecipitation. However, tyrphostin23 (an inhibitor of clathrin-mediated endocytosis) and wortmannin (a suppressor of receptor endocytosis) were not able to block phot2 trafficking, indicating no involvement of receptor endocytosis in the formation of phot2 punctuate structures. Protein turnover studies indicated that the receptor was continuously degraded in both darkness and blue light. The degradation of phot2 proceeded via a transport route different from translocation to the Golgi complex.

Analysis of spatial correlations between patterns of DNA damage response and DNA replication in nuclei of cells subjected to replication stress or oxidative damage.

Sites of DNA replication (EdU incorporation) and DNA damage signaling (γH2AX) induced by camptothecin (Cpt) or hydrogen peroxide (H2O2) form characteristic patterns of foci in cell nuclei. The overlap between these patterns is a function of the number of DNA double strand breaks (DSBs) formed in replication sites. The goal of this study was to optimize a method of quantitative assessment of a degree of correlation between these two patterns. Such a correlation can be used to estimate a probability of inducing damage in sections of replicating DNA. The damage and replication foci are imaged in 3D with confocal microscopy and their respective positions within nuclei are determined with adaptive image segmentation. Using correlation functions spatial proximity of the resultant point patterns is quantified over the range of distances in cells in early-, mid- and late S-phase. As the numbers (and nuclear densities) of γH2AX and replication foci differ significantly in the subsequent substages of S phase, the detected association values were corrected for the expected random overlap between both classes of foci. Thus, the probability of their nonrandom association was estimated. Moreover, self association (clustering) of DNA replication sites in different stages of S-phase of the cell cycle was detected and accounted for. While the analysis revealed a strong correlation between the γH2AX foci and the sites of DNA replication in cells treated with Cpt, only a low correlation was apparent in cells exposed to H2O2. © 2013 International Society for Advancement of Cytometry.

Sequences that direct subcellular traffic of the Drosophila methoprene-tolerant protein (MET) are located predominantly in the PAS domains.

Methoprene-tolerant protein (MET) is a key mediator of antimetamorphic signaling in insects. MET belongs to the family of bHLH-PAS transcription factors which regulate gene expression and determine essential physiological and developmental processes. The ability of many bHLH-PAS proteins to carry out their functions is related to the patterns of intracellular trafficking, which are determined by specific sequences and indicate that a nuclear localization signal (NLS) or a nuclear export signal (NES) is present and active. Therefore, the identification of NLS and NES signals is fundamental in order to understand the intracellular signaling role of MET. Nevertheless, data on the intracellular trafficking of MET are inconsistent, and until now there hasn't been any data on potential NLS and NES sequences. To analyze the trafficking of MET we designed a number of expression vectors encoding full-length MET, as well as various derivatives, that were fused to yellow fluorescent protein (YFP). Confocal microscopy analysis of the subcellular distribution of YFP-MET indicated that while this protein was localized mainly in the nucleus, it was also observed in the cytoplasm. This suggested the presence of both an NLS and NES in MET. Our work has shown that each of the two PAS domains of MET (PAS-A and PAS-B, respectively) contain one NLS and one NES sequence. Additional NES activity was present in the C-terminal fragment. The NLS activity located in PAS-B was dependent on the presence of juvenile hormone (JH), the potential ligand for MET. In contrast to this, JH didn't seem to be required for the NLS in PAS-A to be active. However, on the basis of current knowledge about the function of PAS-A in other bHLH-PAS proteins, we suggest there might be other proteins that control the activity of the NLS and possibly the NES located in the PAS-A of MET. Thus, the intracellular trafficking of MET seems to be regulated by a rather complicated network of different factors.

Light-induced isomerization of the LHCII-bound xanthophyll neoxanthin: possible implications for photoprotection in plants.

Light-harvesting pigment-protein complex of Photosystem II (LHCII) is the largest photosynthetic antenna complex of plants and the most abundant membrane protein in the biosphere. Plant fitness and productivity depend directly on a balance between excitations in the photosynthetic apparatus, generated by captured light quanta, and the rate of photochemical processes. Excess excitation energy leads to oxidative damage of the photosynthetic apparatus and entire organism and therefore the balance between the excitation density and photosynthesis requires precise and efficient regulation, operating also at the level of antenna complexes. We show that illumination of the isolated LHCII leads to isomerization of the protein-bound neoxanthin from conformation 9'-cis to 9',13- and 9',13'-dicis forms. At the same time light-driven excitation quenching is observed, manifested by a decrease in chlorophyll a fluorescence intensity and shortened fluorescence lifetimes. Both processes, the neoxanthin isomerization and the chlorophyll excitation quenching, are reversible in dim light. The results of the 77K florescence measurements of LHCII show that illumination is associated with appearance of the low-energy states, which can serve as energy traps in the pigment-protein complex subjected to excess excitation. Possible sequence of the molecular events is proposed, leading to a protective excess excitation energy quenching: neoxanthin photo-isomerization→formation of LHCII supramolecular structures which potentiate creation of energy traps→excitation quenching.

Interaction of human peripheral blood monocytes with apoptotic polymorphonuclear cells.

Macrophages have the potential to recognize apoptotic neutrophils and phagocytose them while the same function for monocytes is uncertain. In fact, early findings indicated that monocytes started to phagocytose neutrophils on the third day of differentiation to macrophages. Here we show, using flow cytometry and confocal microscopy, that peripheral blood monocytes phagocytose apoptotic but not freshly isolated granulocytes. Recognition of apoptotic cells is predominantly connected with CD16(+) monocytes (CD14(high) CD16(+) and CD14(dim) CD16(+)) and requires CD36. Clearance of apoptotic polymorphonuclear leucocytes appears to be independent of the CD14 mechanism. Uptake of apoptotic Jurkat T cells by monocytes is CD14 and CD36 dependent. Liposomes containing phosphatidyl-l-serine reduce binding of apoptotic polymorphonuclear leucocytes. Lipopolysaccharide-activated subpopulations of monocytes while in contact with apoptotic cells produce more anti-inflammatory cytokine interleukin-10 whereas the production of pro-inflammatory cytokines, tumour necrosis factor-alpha and interleukin-1beta is reduced.

Studies on the role of the receptor protein motifs possibly involved in electrostatic interactions on the dopamine D1 and D2 receptor oligomerization.

We investigated the influence of an epitope from the third intracellular loop (ic3) of the dopamine D(2) receptor, which contains adjacent arginine residues (217RRRRKR222), and an acidic epitope from the C-terminus of the dopamine D(1) receptor (404EE405) on the receptors' localization and their interaction. We studied receptor dimer formation using fluorescence resonance energy transfer. Receptor proteins were tagged with fluorescence proteins and expressed in HEK293 cells. The degree of D(1)-D(2) receptor heterodimerization strongly depended on the number of Arg residues replaced by Ala in the ic3 of D(2)R, which may suggest that the indicated region of ic3 in D(2)R might be involved in interactions between two dopamine receptors. In addition, the subcellular localization of these receptors in cells expressing both receptors D(1)-cyan fluorescent protein, D(2)-yellow fluorescent protein, and various mutants was examined by confocal microscopy. Genetic manipulations of the Arg-rich epitope induced alterations in the localization of the resulting receptor proteins, leading to the conclusion that this epitope is responsible for the cellular localization of the receptor. The lack of energy transfer between the genetic variants of yellow fluorescent protein-tagged D(2)R and cyan fluorescent protein-tagged D(1)R may result from differing localization of these proteins in the cell rather than from the possible role of the D(2)R basic domain in the mechanism of D(1)-D(2) receptor heterodimerization. However, we find that the acidic epitope from the C-terminus of the dopamine D(1) receptor is engaged in the heterodimerization process.

Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll-protein complexes in bean thylakoid membranes.

Changes in chloroplast structure and rearrangement of chlorophyll-protein (CP) complexes were investigated in detached leaves of bean (Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 degrees C and subsequent 3-h photoactivation under white light (200 mumol photons m(-2) s(-1)) at 22 degrees C. Although, no change in chlorophyll (Chl) content and Chl a/b ratio in all samples was observed, overall fluorescence intensity of fluorescence emission and excitation spectra of thylakoid membranes isolated from dark-chilled leaves decreased to about 50%, and remained after photoactivation at 70% of that of the control sample. Concomitantly, the ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120 K increased 1.5-fold upon chilling, and was fully reversed after photoactivation. Moreover, chilling stress seems to induce a decrease of the relative contribution of LHCII fluorescence to the thylakoid emission spectra at 120 K, and an increase of that from LHCI and PSI, correlated with a decrease of stability of LHCI-PSI and LHCII trimers, shown by mild-denaturing electrophoresis. These effects were reversed to a large extent after photoactivation, with the exception of LHCII, which remained partly in the aggregated form. In view of these data, it is likely that dark-chilling stress induces partial disassembly of CP complexes, not completely restorable upon photoactivation. These data are further supported by confocal laser scanning fluorescence microscopy, which showed that regular grana arrangement observed in chloroplasts isolated from control leaves was destroyed by dark-chilling stress, and was partially reconstructed after photoactivation. In line with this, Chl a fluorescence spectra of leaf discs demonstrated that dark-chilling caused a decrease of the quantum yield PSII photochemistry (F(v)/F(m)) by almost 40% in 5 days. Complete restoration of the photochemical activity of PSII required 9 h post-chilling photoactivation, while only 3 h were needed to reconstruct thylakoid membrane organization and chloroplast structure. The latter demonstrated that the long-term dark-chilled bean leaves started to suffer from photoinhibition after transfer to moderate irradiance and temperature conditions, delaying the recovery of PSII photochemistry, independently of photo-induced reconstruction of PSII complexes.

Large-conductance K+ channel openers NS1619 and NS004 as inhibitors of mitochondrial function in glioma cells.

Recently, it has been reported that large-conductance Ca(2+)-activated potassium channels, also known as BK(Ca)-type potassium channels, are present in the inner mitochondrial membrane of the human glioma LN229 cell line. Hence, in the present study, we have investigated whether BK(Ca)-channel openers (BK(Ca)COs), such as the benzimidazolone derivatives NS004 (5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one) and NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one), affect the functioning of LN229 glioma cell mitochondria in situ. We examined the effect of BK(Ca)COs on mitochondrial membrane potential, mitochondrial respiration and plasma membrane potassium current in human glioma cell line LN229. We found that BK(Ca)COs decrease the mitochondrial membrane potential with an EC(50) value of 3.6+/-0.4 microM for NS1619 and 5.4+/-0.8 microM for NS004. This mitochondrial depolarization was accompanied by an inhibition of the mitochondrial respiratory chain. Both BK(Ca)COs induced whole-cell potassium current blocked by charybdotoxin, as measured by the patch-clamp technique. The BK(Ca)COs had no effect on membrane bilayer conductance. Moreover, the inhibition of mitochondrial function by NS004 and NS1619 was without effect on cell survival, as measured by lactate dehydrogenase release from the cells.

Three-dimensional visualization of connexin 43 on the human cardiomyocytes.

Gap junctions created by a family of connexin proteins play an important role in the development of human heart. It has been previously shown that the abnormalities of right ventricular outflow tract are related to an altered level of expression of connexin 43. The right ventricular outflow tract narrowing, stenosis, or atresia of the main pulmonary artery and hypertrophy of the right ventricle are observed in tetralogy of Fallot. The aim of the current study was to determine the distribution of connexin 43 on the surface of human cardiomyocytes obtained during reparative surgery for tetralogy of Fallot. Connexin 43 distribution in these cells was compared with distribution of connexin 43 in cardiomyocytes obtained from patients without right ventricular outflow tract pathology. Cardiomyocytes isolated from tissue biopsy were cultured on collagen substratum, fixed with paraformaldehyde, and incubated with goat antihuman connexin 43 antibodies and secondary donkey antigoat antibodies conjugated with fluorescent indocarbocyanine. Z-series of optical sections were recorded using a laser scanning confocal microscope. Three-dimensional data stacks were visualized using volume-rendering techniques. Images of connexin 43 fluorescence revealed a pattern of three-dimensional distribution of connexin on the surface of an individual cardiomyocyte. Cardiomyocytes from tetralogy of Fallot and hearts with normal right ventricular outflow tract differ in the organization of connexin 43. Cardiomyocytes from tetralogy of Fallot hearts revealed disturbed distribution of connexin 43. The protein is located irregularly on the entire surface of the cell. In the controls, connexin 43 can be visualized within the intercalated disks only. These disturbances may influence heart maturation, cause hypertrophy of the right ventricle, and induce severe arrhythmias in children with tetralogy of Fallot.