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Rapid inactivation of antimicrobial test agents after exact incubation times with microorganisms is required in time-kill assays. Sodium thiosulphate and a combination of methionine and histidine were compared for neutralisation of active halogen compounds. Test oxidants were mixed with surplus sodium thiosulphate (3%–6%) or histidine/methionine (1% each) in phosphate-buffered saline and incubated for different times, followed by addition of Staphylococcus aureus , Escherichia coli , or Pseudomonas aeruginosa at 1000 CFU/ml. After further incubation, quantitative cultures were performed. Thiosulphate did not sufficiently inactivate chlorine and bromine compounds, indicated by a 10-fold ( S. aureus ) up to > 100-fold ( E. coli , P. aeruginosa ) reduction of CFU. This was particularly true for high concentrations of the oxidants of about 50 mM, for highly reactive agents (HOCl and bromamine T) more than for chloramine T and N -chlorotaurine, and for short pre-incubation times before addition of the bacteria. By contrast, histidine/methionine proved to be suitable for chloramines and bromamine T and for low concentrations of HOCl (0.07%). HOCl at 0.7% could neither be inactivated completely by thiosulphate nor by histidine/methionine. In contrast to chlorine and bromine compounds, iodine was neutralized by thiosulphate, but not by histidine/methionine. Histidine/methionine is superior to inactivate chlorine and bromine and should replace sodium thiosulphate at least in killing tests with high concentrations of these disinfectants. Inclusion of a short reaction time (maximum one minute) of test oxidant and neutralising substance before addition of bacteria is decisive in inactivation tests to obtain reliable results. [ABSTRACT FROM AUTHOR]

During normal spermatogenesis, the testis-specific linker histone Hit appears at pachytene stage becomes phosphorylated in early spermatids and disappears in late spermatids, Using reversed-phase and hydrophilic interaction liquid chromatography, Hit from rat and mouse testes was isolated, subjected to enzymatic digestion, and analyzed by mass spectrometry. We observed different phosphorylated states of Hit (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 Hit 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 Hit phosphorylation starts first by phosphorylation of the non- consensus 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 Hit 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 Hit. 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 Hit by transition proteins and protamines. [ABSTRACT FROM AUTHOR]

H1 histones, isolated from logarithmically growing and mitotically enriched human lymphoblastic T-cells (CCRF-CEM), were fractionated by reversed phase and hydrophilic interaction liquid chromatography, subjected to enzymatic digestion, and analyzed by amino acid sequencing and mass spectrometry. During interphase the four H1 subtypes present in these cells differ in their maximum phosphorylation levels: histone H1.5 is tri-, H1.4 di-, and H1.3 and H1.2, only monophosphorylated. The phosphorylation is site-specific and occurs exclusively on serine residues of SP(K/A)K motifs. The phosphorylation sites of histone H1.5 from mitotically enriched cells were also examined. In contrast to the situation in interphase, at mitosis there were additional phosphorylations, exclusively at threonine residues. Whereas the tetraphosphorylated H1.5 arises from the triphosphosphorylated form by phosphorylation of one of two TPKK motifs in the C-terminal domain, namely Thr137 and Thr154, the pentaphosphorylated H1.5 was the result of phosphorylation of one of the tetraphosphorylated forms at a novel nonconsensus motif at Thr10 in the N-terminal tail. Despite the fact that histone H1.5 has five (S/T)P(K/A)K motifs, all of these motifs were never found to be phosphorylated simultaneously. Our data suggest that phosphorylation of human H1 variants occurs nonrandomly during both interphase and mitosis and that distinct serine- or threonine-specific kinases are involved in different cell cycle phases. The order of increased phosphorylation and the position of modification might be necessary for regulated chromatin decondensation, thus facilitating processes of replication and transcription as well as of mitotic chromosome condensation. [ABSTRACT FROM AUTHOR]

Posttranslational modification of histones is a common means of regulating chromatin structure and thus diverse nuclear processes. Using a hydrophilic interaction liquid chromatographic separation method in combination with mass spectrometric analysis, the present study investigated the alterations in histone H4 methylation/acetylation status and the interplay between H4 methylation and acetylation during in vitro differentiation of mouse erythroleukemia cells and how these modifications affect the chromatin structure. Independently of the type of inducer used (dimethyl sulfoxide, hexamethylenebisacetamide, butyrate, and trichostatin A), we observed a strong increase in non- and monoacetylated H4 lysine 20 (H4-Lys20) trimethylation. An increase in H4-Lys20 trimethylation, however, to a clearly lesser extent, was also found when cells accumulated in the stationary phase. Since we show that trimethylated H4Lys20 is localized to heterochromatin, the increase in H4-Lys20 trimethylation observed indicates an accumulation of chromatin-dense and transcriptionally silent regions during differentiation and during the accumulation of control cells in the stationary phase, respectively. When using the deacetylase inhibitors butyrate or trichostatin A, we found that H4 hyperacetylation prevents H4-Lys20 trimethylation, but not mono- or dimethylation, and that the nonacetylated unmethylated H4-Lys20 is therefore the most suitable substrate for H4-Lys20 trimethylase. Summarizing, histone H4-Lys20 hypotrimethylatlon correlates with H4 hyperacetylation and H4-Lys20 hypertrimethylation correlates with H4 hypoacetylation. The results provide a model for how transcriptionally active euchromatin might be converted to the compacted, transcriptionally silent heterochromatin. [ABSTRACT FROM AUTHOR]

Protein C inhibitor (PCI) is a serpin with broad protease reactivity. It binds glycosaminoglycans and certain phospholipids that can modulate its inhibitory activity. PCI can penetrate through cellular membranes via binding to phosphatidylethanolamine. The exact mechanism of PCI internalization and the intracellular role of the serpin are not well understood. Here we showed that testisin, a glycosylphosphatidylinositol-anchored serine protease, cleaved human PCI and mouse PCI (mPCI) at their reactive sites as well as at sites close to their N terminus. This cleavage was observed not only with testisin in solution but also with cell membrane-anchored testisin on U937 cells. The cleavage close to the N terminus released peptides rich in basic amino acids. Synthetic peptides corresponding to the released peptides of human PCI (His¹-Arg11) and mPCI (Arg¹-Ala18) functioned as cell-penetrating peptides. Because intact mPCI but not testisin-cleaved mPCI was internalized by Jurkat T cells, a truncated mPCI mimicking testisin-cleaved mPCI was created. The truncated mPCI lacking 18 amino acids at the N terminus was not taken up by Jurkat T cells. Therefore our model suggests that testisin or other proteases could regulate the internalization of PCI by removing its N terminus. This may represent one of the mechanisms regulating the intracellular functions of PCI. [ABSTRACT FROM AUTHOR]

Abstract: Escape from the proper control of the cell cycle by up-regulation of cyclins or aberrant activation of cyclin-dependent kinases (CDKs) as well as by inactivation of cellular inhibitors of CDKs (CKI) leads to malignant transformation. Loss of cellular CKIs in cancers provided a rationale for development of pharmacological inhibitors of CDKs. Recently synthesized CKIs, e.g., purine derivatives such as olomoucine (OLO) and roscovitine (ROSC) are non-genotoxic and exhibit increased selectivity towards CDK2 and CDK7/9. Interestingly, both drugs induce additional effects. Recently, a new, unexpected action of OLO on normal human cells was observed. OLO strongly up-regulates CLIMP-63, a 65kD protein that mediates the anchoring of the ER to microtubules. Moreover, ROSC induces in human MCF-7 cells phosphorylation of p53 protein at Ser-46 which in turn initiates caspase-independent apoptosis. In the present contribution we raised the question whether both CKIs would be able to block cell cycle progression and to reactivate p53 protein in human HPV-positive HeLa cervix carcinoma cells. We also addressed the question whether exponentially growing cancer cells are more susceptible to the inhibitory action of CKIs than normal cells. Our results show that HeLa cells are much more sensitive to ROSC than normal fibroblasts. ROSC induces G2 arrest and apoptosis in HeLa cells. It also reactivates and stabilizes wt p53 protein. The increase of p53 protein coincides with down-regulation of E6 oncoprotein. Thus, the biological action of substituted purines is not restricted to the inhibition of CDKs and open new perspectives for their therapeutic applications. [Copyright &y& Elsevier]

Abstract: Biomarkers that predict response to therapy with inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase remain largely uncharacterized. In order to define proteins involved in potential resistance mechanisms, we examined the effect of gefitinib (ZD1839, Iressa) in the EGFR-positive colon cancer cell lines Caco-2, DiFi, HRT-18 and HT-29. None of them exhibited an activating mutation in exons 19 or 21 of EGFR. Proteome profiling with two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry revealed 12 proteins differentially expressed in responsive and non-responsive cells. These proteins are involved in metabolic pathways, partially relevant in malignant growth and four of them are known to interact with the EGFR signalling pathway. Ubiquitin carboxyl-terminated hydrolase isozyme L1 (UCH-L1) and galectin-3 are overexpressed in the responsive cell line Caco-2, whereas fatty acid-binding protein (E-FABP) and heat shock protein (hsp) 27 are expressed more in the resistant cell lines HRT-18 and HT-29 suggesting a role in non-responsiveness of cells to gefitinib. [Copyright &y& Elsevier]

Abstract: The Transient Receptor Potential Vanilloid 1 (TRPV1, vanilloid receptor 1) ion channel plays a key role in the perception of thermal and inflammatory pain, however, its molecular environment in dorsal root ganglia (DRG) is largely unexplored. Utilizing a panel of sequence-directed antibodies against TRPV1 protein and mouse DRG membranes, the channel complex from mouse DRG was detergent-solubilized, isolated by immunoprecipitation and subsequently analyzed by mass spectrometry. A number of potential TRPV1 interaction partners were identified, among them cytoskeletal proteins, signal transduction molecules, and established ion channel subunits. Based on stringent specificity criteria, the voltage-gated K+ channel beta 2 subunit (Kvβ2), an accessory subunit of voltage-gated K+ channels, was identified of being associated with native TRPV1 channels. Reverse co-immunoprecipitation and antibody co-staining experiments confirmed TRPV1/Kvβ2 association. Biotinylation assays in the presence of Kvβ2 demonstrated increased cell surface expression levels of TRPV1, while patch-clamp experiments resulted in a significant increase of TRPV1 sensitivity to capsaicin. Our work shows, for the first time, the association of a Kvβ subunit with TRPV1 channels, and suggests that such interaction may play a role in TRPV1 channel trafficking to the plasma membrane. [Copyright &y& Elsevier]

Abstract: RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [3H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca2+ oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca2+ dynamics the in neurons. [Copyright &y& Elsevier]

ICln is a multifunctional protein involved in regulatory mechanisms as different as membrane ion transport and RNA splicing. The protein is water-soluble, and during regulatory volume decrease after cell swelling, it is able to migrate from the cytosol to the cell membrane. Purified, water-soluble ICln is able to insert into lipid bilayers to form ion channels. Here, we show that ICln159, a truncated ICln mutant, which is also able to form ion channels in lipid bilayers, belongs to the pleckstrin homology (PH) domain superfold family of proteins. The ICln PH domain shows unusual properties as it lacks the electrostatic surface polarization seen in classical PH domains. However, similar to many classical PH domain-containing proteins, ICln interacts with protein kinase C, and in addition, interacts with cAMP-dependent protein kinase and cGMP-dependent protein kinase type II but not cGMP-dependent protein kinase type Iβ. A major phosphorylation site for all three kinases is Ser-45 within the ICln PH domain. Furthermore, ICln159 interacts with LSm4, a protein involved in splicing and mRNA degradation, suggesting that the ICln159 PH domain may serve as a protein-protein interaction platform. [ABSTRACT FROM AUTHOR]

Abstract: An extracellular protease from Pedobacter cryoconitis, isolated from alpine cryoconite on glacier ice, was purified and characterized. Despite high cell densities at a temperature range of 1–25 °C, the optimum temperature for protease production was 15 °C. Maximum enzyme production was achieved when the strain was grown in a pH-neutral medium containing soybean meal, wheat flour and citrate over 72 h. The 27-kDa enzyme was a metalloprotease (sensitive to EDTA, EGTA and phenanthroline) and showed maximal activity towards azocasein at 40 °C and pH 8. The protease was stable for 60 min at 20–30 °C, lost 50% of activity after 30 min at 40 °C, and was inactivated at 50 °C, but was resistant to repeated freezing and thawing. Calcium ions had no protective effect against thermal denaturation. More than 80% of the maximum activity were retained at a pH in the range of 7–10. No activity loss was detected after 1 h at pH 7–9 and 20 °C, nor after 1 h of incubation with 3 M urea or 0.1% perborate. [Copyright &y& Elsevier]