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The sequestration of iron in case of infection, termed nutritional immunity, is an established strategy of host defense. However, the interaction between pathogens and the mammalian iron storage protein ferritin is hitherto not completely understood. To better characterize the function of ferritin in Gram-negative infections, we incubated iron-starved cultures of Salmonella Typhimurium and knockout mutant strains defective for major iron uptake pathways or Escherichia coli with horse spleen ferritin or ionic iron as the sole iron source. Additionally, we added bovine superoxide dismutase and protease inhibitors to the growth medium to assess the effect of superoxide and bacterial proteases, respectively, on Salmonella proliferation and reductive iron release. Compared to free ionic iron, ferritin-bound iron was less available to Salmonella, but was still sufficient to significantly enhance the growth of the bacteria. In the absence of various iron acquisition genes, the availability of ferritin iron further decreased. Supplementation with superoxide dismutase significantly reduced the growth of the ΔentC knockout strain with holoferritin as the sole iron source in comparison with ionic ferrous iron. In contrast, this difference was not observed in the wildtype strain, suggesting that superoxide dismutase undermines bacterial iron uptake from ferritin by siderophore-independent mechanisms. Ferritin seems to diminish iron availability for bacteria in comparison to ionic iron, and its iron sequestering effect could possibly be enhanced by host superoxide dismutase activity.

Background The amount of intracranial blood is a strong predictor of poor outcome after subarachnoid hemorrhage (SAH). Here, we aimed to measure iron concentrations in the cerebral white matter, using the cerebral microdialysis (CMD) technique, and to associate iron levels with the local metabolic profile, complications, and functional outcome. Methods For the observational cohort study, 36 patients with consecutive poor grade SAH (Hunt & Hess grade of 4 or 5, Glasgow Coma Scale Score ≤ 8) undergoing multimodal neuromonitoring were analyzed for brain metabolic changes, including CMD iron levels quantified by graphite furnace atomic absorption spectrometry. The study time encompassed 14 days after admission. Statistical analysis was performed using generalized estimating equations. Results Patients were admitted in a poor clinical grade (n = 26, 72%) or deteriorated within 24 h (n = 10, 28%). The median blood volume in the subarachnoid space was high (SAH sum score = 26, interquartile range 20–28). Initial CMD iron was 44 µg/L (25–65 µg/L), which significantly decreased to a level of 25 µg/L (14–30 µg/L) at day 4 and then constantly increased over the remaining neuromonitoring days (p p = 0.04) but not with the hemorrhage load in the subarachnoid space (p = 0.8). In patients developing vasospasm, the CMD iron load was higher, compared with patients without vasospasm (Wald-statistic = 4.1, degree of freedom = 1, p = 0.04), which was not true for delayed cerebral infarction (p = 0.4). Higher iron concentrations in the brain extracellular fluid (34 µg/L, 36–56 µg/L vs. 23 µg/L, 15–37 µg/L) were associated with mitochondrial dysfunction (CMD lactate to pyruvate ratio > 30 and CMD-pyruvate > 70 µM/L, p p > 0.5). Conclusions This study suggests that iron accumulates in the cerebral white matter in patients with poor grade SAH. These findings may support trials aiming to scavenger brain extracellular iron based on the hypothesis that iron-mediated neurotoxicity may contribute to acute and secondary brain injury following SAH.

Molecular imaging using magnetic resonance imaging (MRI) is expected to play a crucial future role in oncological diagnosis and in monitoring of therapeutic progress. Targeted nanoparticle contrast media (CM) with high relaxivities are required in order to obtain adequate signal-to-noise ratios as well as visualization of a desired pathologic area of the human body. The aims of this study were to synthesize and define certain physicochemical and enhancement properties of new doubly derivatized polylactic acid–bovine serum albumin (PLA-BSA) nanoparticles (NPs) modified by the covalent coupling of glutaraldehyde as a crosslinking agent. An additional functionalization with endothelial cells (ECs) targeting groups (tomato lectins; LEA) and signal-emitting moieties (DTPA-Gd) enables its use as a macromolecular, biodegradable contrast agent for MRI. The NPs were characterized by different spectroscopies, size exclusion chromatography, and scanning and transmission electron microscopy. In a human vein model, the dynamics of the nanoparticle interactions with the vein wall were examined in MRI, with correlative imaging in electron microscopy. In vitro studies were conducted to show endothelial binding and persistent enhancement at the apical EC surface. NPs with a diameter between 55 and 75 nm, able to carry simultaneous signal emitting, and targeting motifs on a single construct were successfully prepared. A high Gd payload and endothelial binding to blood vessel walls were observed. The binding affinity and specificity of LEA was preserved, and a strong enhancement at the endothelium was achieved. The stabilized core–shell structure of PLA-NP might allow for further encapsulation of lipophilic drugs or for attachment of other targeting molecules, such as antibodies. Graphical abstract

This multidisciplinary study examined the pharmacokinetics of nanoparticles based on albumin-DTPA-gadolinium chelates, testing the hypothesis that these nanoparticles create a stronger vessel signal than conventional gadolinium-based contrast agents and exploring if they are safe for clinical use. Nanoparticles based on human serum albumin, bearing gadolinium and designed for use in magnetic resonance imaging, were used to generate magnet resonance images (MRI) of the vascular system in rats (“blood pool imaging”). At the low nanoparticle doses used for radionuclide imaging, nanoparticle-associated metals were cleared from the blood into the liver during the first 4 h after nanoparticle application. At the higher doses required for MRI, the liver became saturated and kidney and spleen acted as additional sinks for the metals, and accounted for most processing of the nanoparticles. The multiple components of the nanoparticles were cleared independently of one another. Albumin was detected in liver, spleen, and kidneys for up to 2 days after intravenous injection. Gadolinium was retained in the liver, kidneys, and spleen in significant concentrations for much longer. Gadolinium was present as significant fractions of initial dose for longer than 2 weeks after application, and gadolinium clearance was only complete after 6 weeks. Our analysis could not account quantitatively for the full dose of gadolinium that was applied, but numerous organs were found to contain gadolinium in the collagen of their connective tissues. Multiple lines of evidence indicated intracellular processing opening the DTPA chelates and leading to gadolinium long-term storage, in particular inside lysosomes. Turnover of the stored gadolinium was found to occur in soluble form in the kidneys, the liver, and the colon for up to 3 weeks after application. Gadolinium overload poses a significant hazard due to the high toxicity of free gadolinium ions. We discuss the relevance of our findings to gadolinium-deposition diseases.

Numerous in vivo, in vitro and clinical studies report on beneficial effects of strontium with respect to increased bone growth. Based on this knowledge the aim of this study was to evaluate early and late osseointegration stages of functionalized titanium implants showing sustained release of strontium (Sr) and further investigate its potential systemic effect. Strontium functionalized (Ti-Sr-O) and Grade 4 (Control) titanium implants were inserted in the femoral condyle of New Zealand White rabbits. The Ti-Sr-O coating was characterized using Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectrometry (EDX) for structure, coating thickness and chemical composition. Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) was used to evaluate released strontium in vitro while Atomic Absorption Spectrometry (AAS) was utilized to monitor serum levels of strontium and calcium. Additionally, histological and tomographic analysis of bone-to-implant contact (BIC%) and bone formation (BF%) was performed, following implantation periods of two or twelve weeks, respectively. Median values for BIC% for Ti-Sr-O revealed significant differences within the two-and twelve-week observation periods, while exceeding BF% was discovered especially after twelve weeks when performing the histological evaluation. The results from the micro-computed tomography (mu-CT) showed no significant differences, when comparing the experimental groups. AAS measurements did not indicate a systemic effect by the local strontium release. Within the limitations of the study, it was shown that a Ti-Sr-O coating with sustained release characteristics of strontium, accelerates bone apposition and represents a potential potent surface modification for endosseous medical implant devices.Statement of SignificanceThis study presents first data with respect to early and late in vivo response on a strontium functionalized titanium surface comprising a nanotopography manufactured by a magnetron sputtering process. We investigated different osseointegration stages of screw-shaped implants with dental implant geometries in a rabbit femur model observing beneficial effects of the functionalized surface on bone-to-implant contact and bone formation caused by tailored release of the bone anabolic strontium. Histomorphometrical data revealed that a functionalized titanium surface with controlled liberation of strontium accelerates osseointegration while spectrometry measurements did not indicate a potential systemic effect of this osteoinductive agent and could thus have impact on modifications of medical implant devices. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND.

Background Previous studies on breast cancer (BC), underarm cosmetic products (UCP) and aluminum salts have shown conflicting results. We conducted a 1:1 age-matched case-control study to investigate the risk for BC in relation to self-reported UCP application. Methods Self-reported history of UCP use was compared between 209 female BC patients (cases) and 209 healthy controls. Aluminum concentration in breast tissue was measured in 100 cases and 52 controls. Multivariable conditional logistic regression analysis was performed to estimate odds ratios (ORs) with 95% confidence intervals (CIs), adjusting for established BC risk factors. Findings Use of UCP was significantly associated with risk of BC (p = 0.036). The risk for BC increased by an OR of 3.88 (95% CI 1.03–14.66) in women who reported using UCP's several times daily starting at an age earlier than 30 years. Aluminum in breast tissue was found in both cases and controls and was significantly associated to self-reported UCP use (p = 0.009). Median (interquartile) aluminum concentrations were significantly higher (p = 0.001) in cases than in controls (5.8, 2.3–12.9 versus 3.8, 2.5–5.8 nmol/g). Interpretation Frequent use of UCPs may lead to an accumulation of aluminum in breast tissue. More than daily use of UCPs at younger ages may increase the risk of BC., Highlights • Frequent use of underarm cosmetic products may be related to incorporated aluminum concentration in breast tissue. • Use of underarm cosmetic products several times a day at younger ages may increase the risk of breast cancer. Previous studies regarding breast cancer (BC) risk and underarm cosmetic products (UCPs) with aluminum salts have shown conflicting results. Here we provide comprehensive information about the use of UCPs and aluminum measurements in breast cancer patients and healthy individuals. The findings suggest that the frequent use of UCPs lead to an accumulation of aluminum in breast tissue. We observed an increased risk for BC in women who reported to use UCPs more than once daily starting at an age

Macrophages are central for the immune control of intracellular microbes. Heme oxygenase 1 (HO-1, hmox) is the first and rate limiting enzyme in the breakdown of heme originating from degraded senescent erythrocytes and heme-proteins, yielding equal amounts of iron, carbon monoxide and biliverdin. HO-1 is strongly up-regulated in macrophages in response to inflammatory signals, including bacterial endotoxin. In view of the essential role of iron for the growth and proliferation of intracellular bacteria along with known effects of the metal on innate immune function, we examined whether HO-1 plays a role in the control of infection with the intracellular bacterium Salmonella Typhimurium. We studied the course of infection in stably-transfected murine macrophages (RAW264.7) bearing a tetracycline-inducible plasmid producing hmox shRNA and in primary HO-1 knockout macrophages. While uptake of bacteria into macrophages was not affected, a significantly reduced survival of intracellular Salmonella was observed upon hmox knockdown or pharmacological hmox inhibition, which was independent of Nramp1 functionality. This could be traced to limitation of iron availability for intramacrophage bacteria along with enhanced stimulation of innate immune effector pathways, including the formation of reactive oxygen and nitrogen species and increased TNF-α expression. Mechanistically, these latter effects result from intracellular iron limitation with subsequent activation of NF-κB and further inos, tnfa and p47phox transcription along with reduced formation of the anti-inflammatory and radical scavenging molecules, CO and biliverdin as a consequence of HO-1 silencing. Taken together our data provide novel evidence that the infection-driven induction of HO-1 exerts detrimental effects in the early control of Salmonella infection, whereas hmox inhibition can favourably modulate anti-bacterial immune effector pathways of macrophages and promote bacterial elimination.

Escherichia coli-induced hemolytic uremic syndrome (eHUS) is a life-threatening complication of infection with Shiga toxin (Stx), in particular Stx2a-producing Escherichia coli. Enhanced coagulation activation with formation of microthrombi seems to be a key event in development of eHUS. Platelet activation has been postulated as a possible, but controversially debated mechanism. The present study investigated the effect of Stx2a on plasmatic coagulation and platelets. Binding studies were initially performed with ELISA and co-immunoprecipitation and supported by quartz crystal microbalance with dissipation monitoring (QCM-D). Antithrombin (AT) activity was measured using the automated BCS XP® system. ROTEM® was used for functional coagulation testing. Platelet binding and activation was studied with FACS and light-transmission aggregometry. We found binding of Stx2a to AT, an important inhibitor of blood coagulation, but only a mild albeit significant reduction of AT activity against FXa in the presence of Stx2a. QCM-D analysis also showed binding of Stx2a to heparin and an impaired binding of AT to Stx2a-bound heparin. ROTEM® using Stx2a-treated platelet-poor plasma revealed a significant, but only moderate shortening of clotting time. Neither binding nor activation of platelets by Stx2a could be demonstrated. In summary, data of this study suggest that Stx2a binds to AT, but does not induce major effects on plasmatic coagulation. In addition, no interaction with platelets occurred. The well-known non-beneficial administration of heparin in eHUS patients could be explained by the interaction of Stx2a with heparin.

Zinc sequestration by macrophages is considered a crucial host defense strategy against infection by the intracellular bacterium Salmonella enterica serovar Typhimurium. However, the underlying mechanisms remain elusive. In this study, we found that zinc favors pathogen survival within macrophages. Salmonella-hosting macrophages contained higher free zinc levels than did uninfected macrophages and cells that successfully eliminated bacteria, which was paralleled by the impaired production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in bacterium-harboring cells. A profound, zinc-mediated inhibition of NF-κB p65 transcriptional activity affecting the expression of the ROS- and RNS-forming enzymes phos47 and inducible nitric oxide synthase (iNOS) provided a mechanistic explanation for this phenomenon. Macrophages responded to infection by enhancing the expression of zinc-scavenging metallothioneins 1 and 2, whose genetic deletion caused increased free zinc levels, reduced ROS and RNS production, and increased the survival of Salmonella. Our data suggest that Salmonella invasion of macrophages results in a bacterium-driven increase in the intracellular zinc level, which weakens antimicrobial defense and the ability of macrophages to eradicate the pathogen. Thus, limitation of cytoplasmic zinc levels may help to control infection by intracellular bacteria.

Lipocalin-2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron-laden bacterial siderophores, chemo-attracts neutrophils and has immunomodulatory and apoptosis-regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella-infected macrophages, which reduces cellular iron content and enhances the generation of pro-inflammatory cytokines. Lcn2 represses IL-10 production while augmenting Nos2, TNF-α, and IL-6 expression. Lcn2(-/-) macrophages have elevated IL-10 levels as a consequence of increased iron content. The crucial role of Lcn-2/IL-10 interactions was further demonstrated by the greater ability of Lcn2(-/-) IL-10(-/-) macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2(-/-) counterparts. Overexpression of the iron exporter ferroportin-1 in Lcn2(-/-) macrophages represses IL-10 and restores TNF-α and IL-6 production to the levels found in wild-type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL-10 production, and that both functions are linked to its ability to shuttle iron from macrophages.

NOS2-derived nitric oxide drives ferroportin-1–mediated iron export in Salmonella-infected macrophages, thus limiting bacterial growth., Nitric oxide (NO) generated by inducible NO synthase 2 (NOS2) affects cellular iron homeostasis, but the underlying molecular mechanisms and implications for NOS2-dependent pathogen control are incompletely understood. In this study, we found that NO up-regulated the expression of ferroportin-1 (Fpn1), the major cellular iron exporter, in mouse and human cells. Nos2−/− macrophages displayed increased iron content due to reduced Fpn1 expression and allowed for an enhanced iron acquisition by the intracellular bacterium Salmonella typhimurium. Nos2 gene disruption or inhibition of NOS2 activity led to an accumulation of iron in the spleen and splenic macrophages. Lack of NO formation resulted in impaired nuclear factor erythroid 2-related factor-2 (Nrf2) expression, resulting in reduced Fpn1 transcription and diminished cellular iron egress. After infection of Nos2−/− macrophages or mice with S. typhimurium, the increased iron accumulation was paralleled by a reduced cytokine (TNF, IL-12, and IFN-γ) expression and impaired pathogen control, all of which were restored upon administration of the iron chelator deferasirox or hyperexpression of Fpn1 or Nrf2. Thus, the accumulation of iron in Nos2−/− macrophages counteracts a proinflammatory host immune response, and the protective effect of NO appears to partially result from its ability to prevent iron overload in macrophages

Background. Iron overload can adversely influence the course of infection by increasing microbial replication and suppressing antimicrobial immune effector pathways. Recently, we have shown that the calcium channel blocker nifedipine can mobilize tissue iron in mouse models of iron overload. We therefore investigated whether nifedipine treatment affects the course of infection with intracellular bacteria via modulation of iron homeostasis. Methods. The effect of nifedipine on intramacrophage replication of bacteria and modulation of cellular iron homeostasis was investigated in the murine macrophage cell line RAW264.7, and the impact of nifedipine treatment on the course of systemic infection was investigated in C57BL/6 mice in vivo. Results. In RAW264.7 cells, nifedipine treatment significantly reduced intracellular bacterial survival of Salmonella enterica serovar Typhimurium and Chlamydophila pneumoniae. This could be attributed to the induction of the iron exporter ferroportin 1, which limited the availability of iron for intracellular Salmonella. When C57BL/6 mice were infected intraperitoneally with Salmonella and subsequently injected with nifedipine for 3 consecutive days, bacterial counts in livers and spleens were significantly reduced and survival of the mice significantly was prolonged compared with solvent-treated littermates. Nifedipine treatment increased expression of ferroportin 1 in the spleen, whereas splenic levels of the iron storage protein ferritin and serum iron concentrations were reduced. Conclusions. Our data provide evidence for a novel mechanism whereby nifedipine enhances host resistance to intracellular pathogens via limitation of iron availability.

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.

Altered mitochondrial metabolism plays a pivotal role in the development and progression of various diseases, including cancer. Cell lines are frequently used as models to study mitochondrial (dys)function, but little is known about their mitochondrial respiration and metabolic properties in comparison to the primary tissue of origin. We have developed a method for assessment of oxidative phosphorylation in prostate tissue samples of only 2 mg wet weight using high-resolution respirometry. Reliable protocols were established to investigate the respiratory activity of different segments of the mitochondrial electron transfer system (ETS) in mechanically permeabilized tissue biopsies. Additionally, the widely used immortalized prostate epithelial and fibroblast cell lines, RWPE1 and NAF, representing the major cell types in prostate tissue, were analyzed and compared to the tissue of origin. Our results show that mechanical treatment without chemical permeabilization agents or sample processing constitutes a reliable preparation method for OXPHOS analysis in small amounts of prostatic tissue typically obtained by prostate biopsy. The cell lines represented the bioenergetic properties of fresh tissue to a limited extent only. Particularly, tissue showed a higher oxidative capacity with succinate and glutamate, whereas pyruvate was a substrate supporting significantly higher respiratory activities in cell lines. Several fold higher zinc levels measured in tissue compared to cells confirmed the role of aconitase for prostate-specific metabolism in agreement with observed respiratory properties. In conclusion, combining the flexibility of cell culture models and tissue samples for respirometric analysis are powerful tools for investigation of mitochondrial function and tissue-specific metabolism.

Lipocalin-2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron-laden bacterial siderophores, chemo-attracts neutrophils and has immunomodulatory and apoptosis-regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella-infected macrophages, which reduces cellular iron content and enhances the generation of pro-inflammatory cytokines. Lcn2 represses IL-10 production while augmenting Nos2, TNF-α, and IL-6 expression. Lcn2(-/-) macrophages have elevated IL-10 levels as a consequence of increased iron content. The crucial role of Lcn-2/IL-10 interactions was further demonstrated by the greater ability of Lcn2(-/-) IL-10(-/-) macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2(-/-) counterparts. Overexpression of the iron exporter ferroportin-1 in Lcn2(-/-) macrophages represses IL-10 and restores TNF-α and IL-6 production to the levels found in wild-type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL-10 production, and that both functions are linked to its ability to shuttle iron from macrophages.

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.

Purified histones in solution, purified nuclei, or whole endothelial cells in cell culture were used to study the reactivity of histones with various sugars. The sugar incubation of purified histones produced nonenzymatic glycation and formation of histone cross-links showing disappearance of individual histone molecules and appearance of dimers and polymers in SDS-PAGE. In solution, core histones react considerably faster with sugars as compared to H1 histones. In sugar-incubated nuclei where histones are nucleosomally organized, H1 histones, which are located at the periphery of the nucleosome, and H2A-H2B dimers, which are associated with the central H3(2)-H4(2) tetramer, are more reactive as compared to H3 and H4 histones, which are most protected from the glycation reaction. Our in vivo experiments using endothelial cells show that high concentrations of ribose are able to generate protein cross-links paralleled by apoptotic cell death. High concentrations of glucose or fructose do not increase histone glycation or cell death, even after 60 days of incubation of endothelial cells. In long-time glucose- or fructose-treated cells, under nondenaturing and nonreducing SDS-PAGE conditions part of the H3 histones shifted away from their normal location. Because it is known that the mitochondrial production of reactive oxygen species (ROS) increases after hyperglycaemia, we hypothesize that ROS could be responsible for the formation of a disulphide bridge between the side chain of the cysteine residues of H3 molecules.

The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra- and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of ΔargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in ΔargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants ΔargEF and ΔargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.

Gemcitabine (2',2'difluoro-2'deoxycytidine, dFdC) is a synthetic antimetabolite of the cellular pyrimidine nucleotide metabolism. In a first series of in vitro experiments, the drug showed a strong effect on the proliferation and colony formation of the human androgen-sensitive tumor cell line LNCaP and the androgen-insensitive cell lines PC-3 and DU-145. Maximal inhibition occurred at a dFdC concentration as low as 30 nM. In contrast to the cell lines which were derived from metastatic lesions of prostate cancer patients, no inhibitory effects were found in normal primary prostatic epithelial cells at concentrations up to 100 nM. The effect of gemcitabine was reversed by co-administration of 10-100 microM of its natural analogue deoxycytidine. In view of a future clinical application of this anti-tumor drug in advanced prostatic carcinoma, we have compared the effect of gemcitabine on prostatic tumor cells with that on bone marrow granulopoietic-macrophage progenitor cells, because neutropenia is a common side effect of gemcitabine treatment. The time course of action on the two kinds of cells was markedly different. Colony formation of tumor cells was inhibited by two thirds at a gemcitabine concentration of about 3.5 nM. The same effect on granulopoietic-macrophagic progenitor cells required a concentration of 9 nM. Co-administration of deoxycytidine to gemcitabine-treated tumor cell cultures completely antagonized the effect of gemcitabine whereas addition of deoxycytidine after 48 hr of gemcitabine treatment could not prevent gemcitabine action on the tumor cells. In contrast, more than half of the granulopoietic-macrophagic progenitor cells could still be rescued by deoxycytidine administration after 48 hr. These findings and the marked difference in the susceptibility of neoplastic and normal prostatic cells suggest that gemcitabine is a promising substance which should be further evaluated as to its efficacy in the treatment of advanced prostatic carcinoma.

Neutrophil gelatinase-associated lipocalin (NGAL/Lipocalin-2/Lcn-2) is a 25 kDa protein which is involved in host defence against certain Gram negative bacteria upon binding of iron loaded bacterial siderophores thereby limiting the availability of this essential nutrient to bacteria resulting in inhibition of their growth and pathogenicity. As iron is important for the growth of the intracellular bacterium Chlamydia pneumoniae we questioned whether Lcn-2 affects the course of this infection. We employed primary peritoneal macrophages obtained from wildtype and Lcn-2 −/− mice and RAW 264.7 cells which were infected with C. pneumoniae. In addition, we studied C. pneumoniae multiplication in vivo in mice receiving diets with varying iron contents. We analyzed C. pneumoniae numbers by immunohistochemistry and RT-PCR and studied the expression of iron metabolism and cytokine genes by RT-PCR, Western blot or ELISA. Infection with Chlamydiae ex vivo and in vivo revealed a significantly higher bacterial growth in peritoneal macrophages of Lcn-2 −/− than of wildtype mice. These differences were significantly more pronounced upon iron challenge, which stimulated bacterial growth. Accordingly, treatment with an anti-Lnc-2 antibody increased whereas addition of recombinant Lcn-2 reduced bacterial growth in infected macrophages. When investigating the underlying mechanisms we observed partly different expression of several iron metabolism genes between Lcn-2 +/+ and Lcn-2 −/− macrophages and most strikingly an increased formation of the anti-inflammatory cytokine IL-10 by Lcn-2 −/− macrophages. Upon treatment with an anti-IL10 antibody we experienced a significant increase of Chlamydial growth within Lcn-2 −/− macrophages along with a reduction of the major iron storage protein ferritin. Herein we provide first time evidence that Lcn-2 is involved in host defence against Chlamydia presumably by limiting the availability of iron to the pathogen. In the absence of Lcn-2, increased formation of IL-10 exerts protective effects by increasing the intracellular formation of ferritin, thereby reducing the access of iron for bacteria.

Generalized pruritus of unknown origin (PUO) is a highly distressing condition that is unrelated to any underlying dermatologic or systemic disorder (e.g. cholestasis). Little is known about the potential contribution of elevated total serum bile acid (TSBA) levels to PUO. Our aim in the present study was to investigate the role of elevated TSBA levels in patients with PUO and the efficacy of ursodeoxycholic acid (UDCA) and cholestyramine therapy.Retrospective study comprising 117 patients with chronic pruritic conditions (PUO, atopic disease, asteatotic eczema, latent cholestasis, etc.); 99 patients with available TSBA levels were included and compared with healthy controls.Elevated TSBA levels were detected more frequently in patients with chronic pruritic diseases than in the control population (28.28% vs 6%; P0.001) with significantly higher pathological absolute levels (mean 17.45±34.46 µmol/L vs 6.02±4.73 µmol/L; P=0.001). Patients with PUO (n=18) showed the second-highest prevalence of pathological bile acid level elevation (83.3%; control population 6%; P0.001), after patients with subclinical cholestasis and presented with particularly high TSBA serum values (mean 37.79±53.38 µmol/L; P0.001). Cholestyramine (n=9) and UDCA (n=8) therapy were both effective in lowering TSBA levels and lead to substantial improvement of pruritus in patients with elevated TSBA levels.Total serum bile acid levels are elevated in a high proportion of patients with PUO. These results provide evidence of a potential involvement of subclinical cholestasis in the pathogenesis of PUO. We suggest that evaluation of TSBA levels should be included in the diagnostic work-up of patients with chronic unexplained pruritus.

High-performance capillary electrophoresis (HPCE) was used to separate successfully distinct phosphorylated derivatives of individual histone HI variants. With an untreated capillary (50 cm x 75 μm I.D.) the electrophoresis was performed in about 15 min. Inconvenient interactions of these highly basic proteins with the capillary wall were eliminated by using 0.1 M sodium phosphate buffer (pH 2.0) containing 0.03% hydroxypropylmethylcellulose. Under these experimental conditions the histone H1 variants Hlb and Hlc obtained from mitotic enriched NIH 3T3 fibroblasts and isolated by reversed-phase high-performance liquid chromatography were clearly separated in their non-phosphorylated and different phosphorylated forms. This result was confirmed by acid-urea gel electrophoresis, comparison with non-phosphorylated histones H1b and H1c, isolated from quiescent NIH 3T3 cells, and incubation of multi-phosphorylated histone H1b with alkaline phosphatase and subsequent acid-urea and capillary electrophoresis. The results illustrate that the application of HPCE to the analysis of histone modifications provides a new alternative to traditional gel electrophoresis.

This study focused on the investigation of the permeation enhancing effects of a stomach targeted, nanoparticulate drug delivery system. The polyacrylic acid-cysteine/polyvinylpyrrolidon nanoparticles were loaded with the magnetic resonance imaging (MRI) contrast agent diethylenetriaminepentaacetic acid gadolinium(III)dihydrogen salt (Gd-DTPA). Average particle size was determined to be 130 nm and the optimum for stability was found to be below a pH of 4.5. In vitro permeation studies were performed on rat gastric mucosa and revealed an eightfold increase in Gd-DTPA uptake when incorporated in the nanoparticles compared to evaluation in the presence of unformulated polyacrylic acid-cysteine. In vivo investigations with rats were performed via the noninvasive MRI method in order to track the nanoparticles way through the gastrointestinal tract. When Gd-DTPA was administered orally as nanoparticulate suspension, an increased MRI signal in the urinary bladder was detected after 34 min, providing evidence for systemic uptake and renal elimination of the contrast agent. As control experiments with Gd-DTPA only or in combination with unformulated polyacrylic acid-cysteine revealed no MRI signal increase at all, the significant permeation enhancing effect could be identified based on the nanoparticulate formulation.

The cell cycle-associated phosphorylation of histone H1.5 is manifested as three discrete phosphorylated forms, occurring exclusively on Ser(17), Ser(172), and Ser(188) during interphase. During late G2 and mitosis the up-phosphorylation occurs exclusively on threonine at either Thr(137) or Thr(154) to build the tetraphosphorylated forms of H1.5, whereas the pentaphosphorylated forms result from phosphorylation at Thr(10). To determine the kinetic and spatial distribution of histone H1 phosphorylation within the nucleus of synchronized Hela cells we localized three distinct phosphorylation sites of histone subtype H1.5 using affinity-purified polyclonal antibodies generated against phosphorylated Ser(17), Ser(172), and Thr(10). Immunofluorescence labeling of synchronized HeLa cells using the specific antibodies revealed that phosphorylation of H1.5 Ser(17) appeared early in G1 at discrete speckles followed by phosphorylation of Ser(172). Thr(10) phosphorylation started during prophase, showed highest phosphorylation levels during metaphase, and disappeared clearly before chromatin decondensation occurred. Experiments using the kinase inhibitor staurosporine indicate the involvement of different kinases at the various phospho-sites. Colocalization studies revealed that Ser(172) phosphorylation of H1.5 and H1.2 does colocalize to DNA replication and transcription sites. These results favor the idea that the various site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different functions during the cell cycle.

Recent identification of genetic variants in iron storage disease has changed the classification system and diagnostic algorithms for hemochromatosis. Clinical diagnosis of the disease requires phenotypic evidence of iron overload because the commonly disease-associated HFE genotypes have an incomplete penetrance. Furthermore, approximately 20% of patients with a clinical diagnosis of hemochromatosis have no disease-associated genotype, which underlines the importance of clear phenotypic criteria of hemochromatosis. A diagnosis of hemochromatosis cannot be made even in patients with liver cirrhosis simply on the basis of genetic testing that indicates that iron overload is the cause of the disease and not its consequence. Proper diagnosis requires integration of clinical presentation, family history, and the results of biochemical and histopathologic tests. Here we propose a rational diagnostic algorithm for hepatic iron overload syndromes and illustrate potential pitfalls by presenting a family study in a pedigree with rare HFE variants (H63D and E168Q), in cis on the same chromosome. Although the clinical suspicion of hemochromatosis was confirmed by histology, chemical analysis of liver tissue revealed a normal hepatic iron concentration, which is compatible with the genetic finding of 1 normal and 1 doubly mutated allele. In conclusion, clinical suspicion of hemochromatosis and elevated serum iron parameters should prompt HFE genotyping for C282Y and H63D. Should they be uninformative, further genetic tests should be recommended only if iron overload in liver tissue has been confirmed chemically.

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.

In stimulating effector functions of mononuclear phagocytes, IFN-gamma is of pivotal importance in host defense against intramacrophage pathogens including salmonellae. As the activity of IFN-gamma is modulated by iron and since a sufficient availability of iron is essential for the growth of pathogens, we investigated the regulatory effects of IFN-gamma on iron homeostasis and immune function in murine macrophages infected with Salmonella typhimurium. In Salmonella-infected phagocytes, IFN-gamma caused a significant reduction of iron uptake via transferrin receptor 1 and resulted in an increased iron efflux caused by an enhanced expression of the iron exporter ferroportin 1. Moreover, the expression of haem oxygenase 1 and of the siderophore-capturing antimicrobial peptide lipocalin 2 was markedly elevated following bacterial invasion, with IFN-gamma exerting a super-inducing effect. This observed regulatory impact of IFN-gamma reduced the intracellular iron pools within infected phagocytes, thus restricting the acquisition of iron by engulfed Salmonella typhimurium while concomitantly promoting NO and TNF-alpha production. Our data suggest that the modulation of crucial pathways of macrophage iron metabolism in response to IFN-gamma concordantly aims at withdrawing iron from intracellular Salmonella and at strengthening macrophage immune response functions. These regulations are thus consistent with the principles of nutritional immunity.

The effect of alkylating agents on histone acetyltransferase (EC 2.3.1.48) activity and thymidine incorporation was investigated in benign and malignant proliferating rat liver tissue and compared with the effect in normal non-proliferating rat liver tissue. In both, benign and malignant proliferating tissue, but not in quiescent tissue, the histone acetylation is depressed by alkylating agents and this depression correlates with the inhibition of the thymidine incorporation. This effect suggests that the depression of the replication associated histone acetylation may be an important factor for the antiproliferative activity of alkylating agents.

Hepcidin, a master regulator of iron homeostasis, is produced in small amounts by inflammatory monocytes/macrophages. Chronic immune activation leads to iron retention within monocytes/macrophages and the development of anemia of chronic disease (ACD). We questioned whether monocyte-derived hepcidin exerts autocrine regulation toward cellular iron metabolism. Monocyte hepcidin mRNA expression was significantly induced within 3 hours after stimulation with LPS or IL-6, and hepcidin mRNA expression was significantly higher in monocytes of ACD patients than in controls. In ACD patients, monocyte hepcidin mRNA levels were significantly correlated to serum IL-6 concentrations, and increased monocyte hepcidin mRNA levels were associated with decreased expression of the iron exporter ferroportin and iron retention in these cells. Transient transfection experiments using a ferroportin/EmGFP fusion protein construct demonstrated that LPS inducible hepcidin expression in THP-1 monocytes resulted in internalization and degradation of ferroportin. Transfection of monocytes with siRNA directed against hepcidin almost fully reversed this lipopolysaccharide-mediated effect. Using ferroportin mutation constructs, we found that ferroportin is mainly targeted by hepcidin when expressed on the cell surface. Our results suggest that ferroportin expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin, thus contributing to iron sequestration within monocytes as found in ACD.

BACKGROUND As a component of the enzyme glutathione peroxidase, the essential trace element selenium contributes to the reduction of peroxides. Disturbed selenium availability may relate to an activated immune response. In humans, immune activation is reflected by increased neopterin production and accelerated tryptophan degradation, expressed as the kynurenine to tryptophan ratio (kyn/trp). Th1-type cytokine interferon-gamma induces both these immunobiological events in human macrophages and they are often activated in patients with cardiac disorders. The aim of this study was to determine the relationship between serum selenium concentrations and neopterin production and tryptophan degradation in patients with cardiac disorders. METHODS In 56 patients (28 females) with cardiac disorders, serum selenium concentrations were determined by graphite-furnace atomic absorption spectrometry. Serum neopterin concentration was measured by ELISA and tryptophan degradation was examined by HPLC. RESULTS Selenium concentrations were in the range 0.41-1.90 micromol/L (median 1.02) and were well within the local normal range. Approximately two-thirds of patients presented with higher neopterin concentrations (median 16.4 nmol/L) and tryptophan degradation (median 57 micromol/mmol kyn/trp). There was an inverse correlation between serum selenium and kyn/trp (Spearman's rank correlation, r(s)=-0.431; p

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.

Methylation and acetylation of position-specific lysine residues in the N-terminal tail of histones H3 and H4 play an important role in regulating chromatin structure and function. In the case of H3-Lys(4), H3-Lys(9), H3-Lys(27), and H4-Lys(20), the degree of methylation was variable from the mono- to the di- or trimethylated state, each of which was presumed to be involved in the organization of chromatin and the activation or repression of genes. Here we investigated the interplay between histone H4-Lys(20) mono- and trim-ethylation and H4 acetylation at induced (beta-major/beta-minor glo-bin), repressed (c-myc), and silent (embryonic beta-globin) genes during in vitro differentiation of mouse erythroleukemia cells. By using chromatin immunoprecipitation, we found that the beta-major and beta-minor promoter and the beta-globin coding regions as well as the promoter and the transcribed exon 2 regions of the highly expressed c-myc gene were hyperacetylated and monomethylated at H4-Lys(20). Although activation of the beta-globin gene resulted in an increase in hyperacetylated, monomethylated H4, down-regulation of the c-myc gene did not cause a decrease in hyperacetylated, monomethylated H4-Lys(20), thus showing a stable pattern of histone modifications. Immunofluorescence microscopy studies revealed that monomethylated H4-Lys(20) mainly overlaps with RNA pol II-stained euchromatic regions, thus indicating an association with transcriptionally engaged chromatin. Our chromatin immunoprecipitation results demonstrated that in contrast to trimethylated H4-Lys(20), which was found to inversely correlate with H4 hyper-acetylation, H4-Lys(20) monomethylation is compatible with histone H4 hyperacetylation and correlates with the transcriptionally active or competent chromatin state.

The physical and chemical parameters involved in the design and synthesis of biospecifically targeted nanoparticulate contrast media for magnetic resonance molecular imaging (MRMI) were explored in this pilot investigation. Latex nanoparticles 100, 400 and 900 nm in diameter were doubly derivatised, first with tomato lectin and then with gadolinium(III)-diethylenetriamine pentaacetic acid (Gd-chelates) to target them to epithelial and endothelial glycocalyceal N-glycans and to generate contrast enhancement in magnetic resonance imaging (MRI). After intravenous injection into mice, human placental cotyledons or human Vena saphena magna, contrasty images of the vascular structures were obtained in 1.5 T MRI with spatial resolution 0.1 mm in the imaging plane and 0.6 mm in the z axis, persisting >60 min and resistant to washing out by buffer rinses. Ultrastructural analysis of the nanoparticles revealed the targeting groups at the nanoparticle surfaces and the distribution of the Gd-chelates within the nanoparticles and enabled counts for use in determining relaxivity. The relaxivity values revealed were extremely high, accounting for the strong MR signals observed. Occasionally, nanoparticles larger than 100 nm were seen in close spatial association with disrupted regions of cell membrane or of collagen fibrils in the extracellular matrix. The data suggest that 100-nm nanoparticles generate adequate contrast for MRMI and cause least disruption to endothelial cell surfaces.

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-Lys(20)) trimethylation. An increase in H4-Lys(20) trimethylation, however, to a clearly lesser extent, was also found when cells accumulated in the stationary phase. Since we show that trimethylated H4-Lys(20) is localized to heterochromatin, the increase in H4-Lys(20) 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-Lys(20) trimethylation, but not mono- or dimethylation, and that the nonacetylated unmethylated H4-Lys(20) is therefore the most suitable substrate for H4-Lys(20) trimethylase. Summarizing, histone H4-Lys(20) hypotrimethylation correlates with H4 hyperacetylation and H4-Lys(20) hypertrimethylation correlates with H4 hypoacetylation. The results provide a model for how transcriptionally active euchromatin might be converted to the compacted, transcriptionally silent heterochromatin.

Concentrations of creatine kinase (CK) MB mass and cardiac troponin T were measured in serial peripheral venous blood samples from 21 patients who underwent percutaneous transluminal coronary angioplasty (PTCA). Angiography showed side-branch occlusion during PTCA without clinical signs of myocardial injury in 5 patients. After PTCA, CKMB mass concentrations were substantially higher than normal in all 5 patients with side-branch occlusion, and troponin T concentrations were high in 3. By contrast, only 2 patients and 1 patient, respectively, without side-branch occlusion had slight rises in CKMB and troponin T. Release of the contractile protein troponin T reflects more severe damage to myocytes than simple leakage of CKMB. Therefore, myocardial damage induced by side-branch occlusion can be graded by measurement of troponin T in plasma.

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFalpha-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.

The binding of all known linker histones, named H1a through H1e, including H1(0) and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1(0), completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270-360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.

The purposes of the present study were to evaluate cardiac troponin 1 (cTnl) in the diagnosis of percutaneous transluminal coronary angioplasty (PTCA)-related myocardial injury in comparison with cardiac troponin T (cTnT) and creatine kinase (CK) MB mass concentration, and to investigate the frequency of myocardial injury, as indicated by myocardial protein release, after clinically symptomless side-branch occlusion (SBO) which may occur in the proximity of the attempted stenosis. The final study population comprised 80 patients undergoing elective, single vessel PTCA. Blood samples were drawn before, 6, 24 and 48 h after PTCA. cTnI, cTnT and CKMB mass baseline values were within the reference intervals in all patients (cTnI0.1 microgram/l, cTnT0.2 microgram/l, CKMB5 micrograms/l). Two patients presented with primary failure of PTCA, and visually successful PTCA was performed in all remaining patients. Seven patients (four with SBO) subsequently developed acute myocardial infarction (AMI). Symptomless SBO occurred in 16 patients. In controls (n = 55) there were no significant increases in cTnI, cTnT, or CKMB concentrations compared with baseline values, and all markers stayed within their reference intervals. In half the patients with symptomless SBO (n = 8) all markers were slightly to moderately increased, in two additional patients only CKMB was elevated (cTnI: 0.1-1.0 microgram/l; cTnT: 0.25-0.81 microgram/l and CKMB: 7.9-25.6 micrograms/l). In the majority of patients with primary failure or AMI we found pronounced increases in all tested markers (cTnI: 0.2-12.0 micrograms/l; cTnT: 0.44-12.10 micrograms/l; CKMB: 19.2-423.0 micrograms/l). The results of this study indicate that cTnI is comparably useful to cTnT or CKMB mass for diagnosing myocardial injury in PTCA patients. From our results a preference for one of the tested parameters cannot be clearly derived. Post-procedural cTnI, cTnT, and CKMB mass values are not higher than baseline values in uncomplicated cases, whereas AMI after PTCA leads to pronounced marker increases. SBO, even when symptomless, leads frequently (in about half the patients) to slight marker increases.

In vivo phosphorylation of the five histone H1 variants H1a-H1e including H1(0) in NIH 3T3 mouse fibroblasts was examined during the cell cycle by using a combination of HPLC techniques and conventional AU gel electrophoresis. Phosphorylation starts during the late G1 phase and increases throughout the S phase. In the late S phase, the H1 variants exist as a combination of molecules containing 0 or 1 (H1a, H1c), 0-2 (H1d), or 0-3 (H1b, H1e) phosphate groups with a share of unphosphorylated protein ranging between 35% and 75%, according to the particular subtype. Pulse-chase experiments show that phosphorylation during the S phase is a dynamic phosphorylation process with a limited phosphorylation maximum. In most H1 subtypes, phosphorylation occurs very rapidly at the G2/M transition with only small amounts of intermediate phosphorylated molecules. Phosphorylation of mouse H1c, however, occurs stepwise during this transition. Phosphorylated mouse histone subtypes from cells in mitosis contain four phosphate groups in the case of H1a, H1c, and H1e and five in the case of H1b and H1d. Comparison of the mouse phosphorylation pattern to that in rat C-6 glioma cells showed differences for H1e and H1d. By comparing the different phosphorylation patterns of the individual H1 variants during the cell cycle, we were able to classify the H1 histones into subtypes with low (H1a, H1c, H1(0)) and high (H1b, H1d, H1e) phosphorylation levels.

Cardiac troponin T (cTnT), a new marker of myocardial tissue damage, was investigated in 32 consecutive multiply injured patients. cTnT, creatine kinase (CK) and CK isoenzyme MB (CK-MB) mass concentrations were measured immediately after admission, 12 and 24 h later and daily thereafter for 4 days. We found a moderate increase in cTnT in 22 patients (72 per cent; peaks; 0.6–5.1μg/l). In only four of these 22 patients did the CK-MB mass/CK index indicate myocardial injury. ST-T alterations and arrhythmias did not occur significantly more frequently in patients with increased cTnT plasma concentrations or positive CK-MB mass/CK index. We found a moderate increase in cTnT in 72 per cent of all patients with multiple injuries, but we found no association between an increase in cTnT and the occurence of electrocardiographic changes and arrhythmias.

The rates of synthesis of histone H1 subtypes in synchronized mouse NIH 3T3 fibroblasts were compared with those of rat C6 glioma cells during the G0, G1, and S phases by using a combination of HPLC techniques and conventional gel electrophoresis. In the mouse cell line, all H1 subtypes, H1a-H1e including histone H1(0), were detectable. In the rat cell line, however, no histone H1a was found. H1c and H1e from both cell lines show in the quiescent state a relatively high specific activity comparable with that of H1(0). After release from the G0/G1 block, the synthesis of H1(0) and likewise that of H1c and H1e increase for a short period. All H1 subtypes have their maximum specific activity at the same time after stimulation. The percentage of total H1 specific activity of H1a, H1b, and H1d increases, those of H1c and H1e remain relatively constant, and that of H1(0) decreases while cells cycle from the G0/G1 to the S phase. These findings support our assumption that H1 subtypes could be classified into three groups with common metabolic characteristics: one consists of H1a, H1b, and H1d; another of H1c and H1e; and a third of H1(0) histone. Moreover, the corresponding H1 subtypes from two different species seem to have similar specific activities during the G1 and S phases.