Distinctive sphingolipid patterns in chronic multiple sclerosis lesions.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Sp1 mediates phorbol ester (PMA)-induced expression of membrane-bound guanylyl cyclase GC-A in human monocytic THP-1 cells.

Cyclic guanosine monophosphate (cGMP) is synthesized by two types of enzymes: particulate (membrane-bound) guanylyl cyclases (pGCs) and soluble (cytosolic) guanylyl cyclases (sGCs). sGCs are primarily activated by binding of nitric oxide to their prosthetic heme group while pGCs are activated by binding of peptide ligands to their extracellular domains. One of them, pGC type A (GC-A) is activated by atrial and brain natriuretic peptides (ANP and BNP, respectively). Human monocytes isolated from peripheral blood mononuclear cells have been found to display sGC expression without concomitant expression of GC-A. However, GC-A activity appears in monocytes under certain conditions but a molecular mechanism of GC-A expression is still poorly understood. In this report we show that phorbol ester (PMA) induces transcription of a gene encoding GC-A in human monocytic THP-1 cells. Moreover, we find that PMA-treated THP-1 cells raise cGMP content following treatment with ANP. Studies using pharmacological inhibitors of protein kinases suggest involvement of protein kinase C (PKC), mitogen extracellular kinases (MEK1/2), and extracellular signal-regulated kinases (ERK1/2) in PMA-induced expression of the GC-A encoding gene in THP-1 cells. Finally, we show that PMA stimulates binding of Sp1 transcription factor to GC-rich DNA sequences and mithramycin A (a selective Sp1 inhibitor) inhibits expression of the GC-A mRNA in PMA-treated THP-1 cells. Taken together, our findings suggest that the PMA-stimulated PKC and MEK/ERK signaling pathways induce Sp1-mediated transcription of the GC-A encoding gene in human monocytic THP-1 cells.

The novel compound PBT434 prevents iron mediated neurodegeneration and alpha-synuclein toxicity in multiple models of Parkinson's disease.

Elevated iron in the SNpc may play a key role in Parkinson's disease (PD) neurodegeneration since drug candidates with high iron affinity rescue PD animal models, and one candidate, deferirpone, has shown efficacy recently in a phase two clinical trial. However, strong iron chelators may perturb essential iron metabolism, and it is not yet known whether the damage associated with iron is mediated by a tightly bound (eg ferritin) or lower-affinity, labile, iron pool. Here we report the preclinical characterization of PBT434, a novel quinazolinone compound bearing a moderate affinity metal-binding motif, which is in development for Parkinsonian conditions. In vitro, PBT434 was far less potent than deferiprone or deferoxamine at lowering cellular iron levels, yet was found to inhibit iron-mediated redox activity and iron-mediated aggregation of α-synuclein, a protein that aggregates in the neuropathology. In vivo, PBT434 did not deplete tissue iron stores in normal rodents, yet prevented loss of substantia nigra pars compacta neurons (SNpc), lowered nigral α-synuclein accumulation, and rescued motor performance in mice exposed to the Parkinsonian toxins 6-OHDA and MPTP, and in a transgenic animal model (hA53T α-synuclein) of PD. These improvements were associated with reduced markers of oxidative damage, and increased levels of ferroportin (an iron exporter) and DJ-1. We conclude that compounds designed to target a pool of pathological iron that is not held in high-affinity complexes in the tissue can maintain the survival of SNpc neurons and could be disease-modifying in PD.

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line.

Th1 pro-inflammatory cytokines, i.e., TNF-α and IFN-γ, in combination are known to induce cell death in several cell types, including oligodendrocytes, but the mechanism of their synergistic cytotoxicity is unclear. Although ceramide (Cer) has been implicated in cytokine- and stress-induced cell death, its intracellular levels alone cannot explain cytokine synergy. We considered the possibility that Cer released as part of extracellular vesicles may contribute to cytokine-induced synergistic cell death. Using a human oligodendroglioma (HOG) cell line as a model, here we show that exosomes derived from TNF-α-treated "donor" cells, while being mildly toxic to fresh cultures (similar to individual cytokines), induce enhanced cell death when added to IFN-γ-primed target cultures in a fashion resembling the effect of cytokine combination. Further, the sphingolipid profiles of secreted exosomes, as determined by HPLC-MS/MS, revealed that the treatment with the cytokines time-dependently induced the formation and exosomal release, in particular of C16-, C24-, and C24:1-Cer species; C16-, C24-, and C24:1-dihydroCer species; and C16-, C24-, and C24:1-SM species. Finally, exogenous C6-Cer or C16-Cer mimicked and enhanced the cytotoxic effects of the cytokines upon HOG cells, thereby supporting the cell death-signaling role of extracellular Cer.

SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF NEW SULFONAMIDE ISOXAZOLO[5,4-b]PYRIDINE DERIVATIVES.

A series of novel sulfonamide isoxazolo[5,4-b]pyridines were synthesized. The substrates for their synthesis were 3-aminoisoxazolo[5,4-b]pyridine and selected aryl sulfonic chlorides, chlorosulfonic acid and selected amines. Reactions were carried out using the classical and microwave methods. Selected compounds were tested towards antibacterial and antiproliferative activity. The structure of the obtained new derivatives was determined by elemental analysis and acquired IR and 1H NMR spectra. Among the tested compounds: N- isoxazolo[5,4-b]pyridine-3-yl-benzenesulfonamide (2) and N-isoxazolo[5,4-b]pyridine-3-yl-4-methylbenzene-sulfonamide (5) showed antimicrobial activity towards Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) at doses: 125, 250 and 500 µg. Both compounds showed a 50% inhibition of proliferation of breast carcinoma cell line MCF7 at concentrations of 152.56 µg/mL and 160 161.08 µg/mL, respectively.

Establishment of a cellular model to study TrkC-dependent neuritogenesis.

The rat PC12 cell line has become a widely used research tool for many aspects of neurobiology. Nerve growth factor (NGF)-responsive PC12 cells were engineered to drive expression of doxycycline (Dox)-induced gene of interest in the Tet-On expression system that resulted in obtaining PC12-Tet-On cells. TrkA and TrkC are neurotrophin receptors derived from the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases. TrkA receptor binds and is activated mainly by NGF, while TrkC receptor binds and is activated by neurotrophin 3 (NT3). The purpose of this research was to design and describe PC12-based neuronal cell model to study TrkC-triggered versus TrkA-triggered neurite outgrowth. The second-generation tetracycline-responsive promoter (P tight) was used in order to provide low basal expression in the absence of Dox and high-level Dox-induced expression of TrkC. The main advantage of presented model system is dependence of TrkC level on Dox concentration. It also allows to compare activation of intracellular signaling proteins and neurite outgrowth following activation of TrkA and TrkC receptors by NGF and NT3, respectively, in the context of the same quality and quantity of intracellular adaptor proteins, Ras proteins, protein kinases and phosphatases, and phospholipase Cγ1, as a difference in the activation of intracellular signaling network by these two distinct although related receptor tyrosine kinases is expected. The results of our studies suggest that despite slightly weaker activation of ERK1/2 mitogen-activated protein kinases, NT3-triggered TrkC seems to provide apparently stronger than NGF-triggered TrkA signal for neurite elongation in differentiating PC12 cells.

Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

[Effect water intake on body weight]. / Wplyw spozycia wody na mase ciala.

Water is essential for life. There wouldn't be the proper functioning of body processes without it. An inadequate water intake relative to recommendation contributes to the decline in physical capacity and adversely effects on cognitive function and mood. On the other hand, an adequate water intake helps maintain the balance between total energy intake and daily energy expenditure and determines the correct rate of fat oxidation. This might be useful and commonly used in weight reduction and thus might favorably affect on body composition in overweight and obese people by increasing the total body water and lean muscle mass and might contribute to a decrease in body fat. Research results indicate clearly that drinking water instead of caloric beverages might be an effective way to reduce daily total energy consumption and in this way might may contribute to the reduction of weight, body circumferences and body fat.

Revised coordination model and stability constants of Cu(II) complexes of tris buffer.

2-Amino-2-hydroxymethyl-propane-1,3-diol, or tris(hydroxymethyl)aminomethane (Tris), is probably the most common biochemical buffer used alone or in combination with other buffers because it is stable, unreactive, and compatible with most proteins and other biomolecules. Being nontoxic, it has even found applications in medicine. Tris is known, however, to coordinate transition metal ions, Cu(II) among them. Although often ignored, this feature affects interactions of Cu(II) ions with biomolecules, as Tris is usually used in high molar excess. Therefore, it is important to have precise knowledge on the stoichiometry, stability, and reactivity of cupric Tris complexes. The literature data are incoherent in this respect. We reinvestigated the complex formation in the Tris-Cu(II) system by potentiometry, UV-vis, ESI-MS, and EPR at a broad range of concentrations and ratios. We found, contrary to several previous papers, that the maximum stoichiometry of Tris to Cu(II) is 2 and at neutral pH, dimeric complexes are formed. The apparent affinity of Tris buffer for Cu(II), determined by the competitivity index (CI) approach [Krezel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. Chem. Commun. 2003, 6, 704-705] at pH 7.4 varies between 2 × 10(6) and 4 × 10(4) M(-1), depending on the Tris and Cu(II) concentrations and molar ratio.

Ternary complex formation and competition quench fluorescence of ZnAF family zinc sensors.

Our current understanding of the intracellular thermodynamics and kinetics of Zn(ii) ions is largely based on the application of fluorescent sensor molecules, used to study and visualize the concentration, distribution and transport of Zn(ii) ions in real time. Such agents are designed for high selectivity for zinc in respect to other biological metal ions. However, the issue of their sensitivity to physiological levels of low molecular weight Zn(ii) ligands (LMWLs) has not been addressed. We followed the effects of eight such compounds on the fluorescence of ZnAF-1 and ZnAF-2F, two representatives of the ZnAF family of fluorescein-based zinc sensors containing the N,N-bis(2-pyridylmethyl)ethylenediamine chelating unit. Fluorescence titrations of equimolar Zn(ii)-ZnAF-1 and Zn(ii)-ZnAF-2F solutions with acetate, phosphate, citrate, glycine, glutamic acid, histidine, ATP and GSH demonstrated strong fluorescence quenching. These results are interpreted in terms of an interplay of the formation of the [ZnAF-Zn(ii)-LMWL] ternary complexes and the competition for Zn(ii) between ZnAF and LMWLs. UV-vis spectroscopic titrations revealed the existence of supramolecular interactions between the fluorescein moiety of ZnAF-1 and ATP and His, which, however, did not contribute to fluorescence quenching. Therefore, the obtained results show that the ZnAF sensors, other currently used zinc sensors containing the N,N-bis(2-pyridylmethyl)ethylenediamine unit, and, in general, all sensors that do not saturate the Zn(ii) coordination sphere may co-report cellular metabolites and Zn(ii) ions, leading to misrepresentations of the concentrations and fluxes of biological zinc.

Binding of transition metal ions to albumin: sites, affinities and rates.

BACKGROUND: Serum albumin is the most abundant protein in the blood and cerebrospinal fluid and plays a fundamental role in the distribution of essential transition metal ions in the human body. Human serum albumin (HSA) is an important physiological transporter of the essential metal ions Cu(2+), and Zn(2+) in the bloodstream. Its binding of metals like Ni(2+), Co(2+), or Cd(2+) can occur in vivo, but is only of toxicological relevance. Moreover, HSA is one of the main targets and hence most studied binding protein for metallodrugs based on complexes with Au, Pt and V. SCOPE OF REVIEW: We discuss i) the four metal-binding sites so far described on HSA, their localization and metal preference, ii) the binding of the metal ions mentioned above, i.e. their stability constants and association/dissociation rates, their coordination chemistry and their selectivity versus the four binding sites iii) the methodology applied to study issues of items i and ii and iv) oligopeptide models of the N-terminal binding site. MAJOR CONCLUSIONS: Albumin has four partially selective metal binding sites with well-defined metal preferences. It is an important regulator of the blood transport of physiological Cu(II) and Zn(II) and toxic Ni(II) and Cd(II). It is also an important target for metal-based drugs containing Pt(II), V(IV)O, and Au(I). GENERAL SIGNIFICANCE: The thorough understanding of metal binding properties of serum albumin, including the competition of various metal ions for specific binding sites is important for biomedical issues, such as new disease markers and design of metal-based drugs. This article is part of a Special Issue entitled Serum Albumin.

Myelin recovery in multiple sclerosis: the challenge of remyelination.

Multiple sclerosis (MS) is the most common demyelinating and an autoimmune disease of the central nervous system characterized by immune-mediated myelin and axonal damage, and chronic axonal loss attributable to the absence of myelin sheaths. T cell subsets (Th1, Th2, Th17, CD8+, NKT, CD4+CD25+ T regulatory cells) and B cells are involved in this disorder, thus new MS therapies seek damage prevention by resetting multiple components of the immune system. The currently approved therapies are immunoregulatory and reduce the number and rate of lesion formation but are only partially effective. This review summarizes current understanding of the processes at issue: myelination, demyelination and remyelination-with emphasis upon myelin composition/ architecture and oligodendrocyte maturation and differentiation. The translational options target oligodendrocyte protection and myelin repair in animal models and assess their relevance in human. Remyelination may be enhanced by signals that promote myelin formation and repair. The crucial question of why remyelination fails is approached is several ways by examining the role in remyelination of available MS medications and avenues being actively pursued to promote remyelination including: (i) cytokine-based immune-intervention (targeting calpain inhibition), (ii) antigen-based immunomodulation (targeting glycolipid-reactive iNKT cells and sphingoid mediated inflammation) and (iii) recombinant monoclonal antibodies-induced remyelination.

The final frontier of pH and the undiscovered country beyond.

The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, K(w) = [H(+)]×[OH(-)].We used K(w) [in a reversed way] to calculate the number of undissociated H(2)O molecules required by this equilibrium constant to yield at least one of its daughter ions, H(+) or OH(-) at a given pH. In this way we obtained a formula that relates pH to the minimal volume V(pH) required to provide a physical meaning to K(w), V(pH)=10(pH-pK(w/2) x 10(pK(w/2)/N(A) (where N(A) is Avogadro's number). For example, at pH 7 (neutral at 25°C) V(pH) =16.6 aL. Any deviation from neutral pH results in a larger V(pH) value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H(+) ions at equilibrium, thus the definition of pH based on K(w) is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H(+) ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H(+) ions as a general source for biochemical reactions. Consequences of this finding are discussed.

The C2H2 zinc finger transcription factors are likely targets for Ni(II) toxicity.

Ni(II) ions are able to hydrolyze Naa-(Ser/Thr) peptide bonds in Naa-(Ser/Thr)-Xaa-His-Zaa sequences. We found that various human transcription factors contain such nickel hydrolytic patterns within C2H2 zinc finger (ZF) domains. We demonstrated the hydrolysis on two peptide models, the 3rd ZF of the Sp1 transcription factor and the 1st ZF of the ZNF302 transcription factor. The experimentally studied reaction rates indicate that the hydrolysis reaction is likely to be an element of intracellular nickel toxicity.

Salivary histatin-5, a physiologically relevant ligand for Ni(II) ions.

Histatins are a family of human salivary antimicrobial peptides. Histatin-5 (Hst-5, DSHAKRHHGYKRKFHEKHHSHRGY), a prominent member of this family contains an albumin-like, N-terminal Asp-Ser-His sequence, known to bind a Ni(II) ion in a square-planar geometry. Nickel is a strong allergen, and oral exposure to Ni(II) ions can elicit allergic reaction in sensitized persons. In contrast, prior oral exposure to nickel in non-sensitized persons can prevent sensitization. The fate of Ni(II) ions in saliva is obviously important for these processes, yet little is known about it. Using potentiometry, UV-visible titrations and circular dichroism, we determined stability constants for Ni(II) complexes of Hst-5 and two truncated analogs, 5Hst-5 (DSHAK) and 10Hst-5 (DSHAKRHHGY). The conditional binding constant at pH 7.4 for Hst-5 was 10(7.5±0.2), compared to the corresponding value for albumin, 10(6.8±0.3) (M. Sokolowska, A. Krezel, M. Dyba, Z. Szewczuk, W. Bal, Eur. J. Biochem. 269 (2002) 1323-1331). These values indicate that Hst-5 binds Ni(II) five times stronger than HSA. The simulated competition for Ni(II) between Hst-5 and albumin shows that significant amounts of Ni(II) ions may be carried by Hst-5 in vivo. Therefore, Hst-5 and other histatins should be considered as factors in nickel allergy and other forms of nickel toxicity.

Different effects of soluble and particulate guanylyl cyclases on expression of inflammatory cytokines in rat peripheral blood mononuclear cells.

Inflammation involves the cooperation of various cells and biologically active molecules. An important intracellular messenger molecule participating in the regulation of the process is cyclic GMP (cGMP), which is synthesized by guanylyl cyclases (GCs). The GC family comprises cytosolic (soluble) and membrane-bound (particulate) enzymes. The aim of this study was to determine whether and how the synthesis of cGMP by various forms of GC affects the expression of inflammatory cytokines depending on the activity of the transcription factors NF-κB (nuclear factor-κB) and AP-1 (activator protein-1). We established that in rat peripheral blood mononuclear cells (PBMCs), synthesis of cGMP was elevated by sodium nitroprusside (SNP), the activator of soluble GC, and by atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), the activators of particulate GC-A and GC-B, respectively. Stimulation of various GCs differently affected the expressions of the cytokines IL-1ß, IL-6, and TNF-α in control cells and in cells activated by bacterial endotoxin (LPS). In control PBMCs their expression was elevated by stimulation of soluble, but not particulate, GC. SNP caused an increase in NF-κB activity, but had no influence on the activity of AP-1. The cells treated with LPS decreased the expressions of IL-1ß, IL-6, and TNF-α in response to stimulation of particulate GC-A, but not other guanylyl cyclases. This inhibitory effect was a result of suppression of the activities of NF-κB and AP-1. Both effects that of SNP and of ANP, were cGMP dependent, as shown using its membrane-permeable analog 8-Br-cGMP. The implementation of specific inhibitors showed that the stimulatory effect of SNP was mediated by soluble GC and cGMP-dependent protein kinase (PKG-I). However, PKG-I was not involved in the inhibition of NF-κB and AP-1 activities by ANP in LPS-activated cells. Taken together, these results for the first time indicate that various GCs and various cGMP-dependent signaling pathways can modulate the activity of AP-1 and/or NF-κB and thus affect the expressions of IL-1ß, IL-6, and TNF-α, which play important roles in the development of inflammation.

Structures of unique globoside elongation products present in erythrocytes with a rare NOR phenotype.

Rare polyagglutinable erythrocytes of NOR phenotype were found to contain two unique glycosphingolipids (designated NOR1 and NOR2). These components (not detected in normal erythrocytes) were reactive with Griffonia simplicifolia isolectin IB4 (GSL-IB4) and commonly present human anti-NOR antibodies. The NOR1 component has been reported to be a globoside containing a single galactose residue linked alpha1,4 to the terminal N-acetylgalactosamine. Here, we report the structural studies on a second glycolipid, NOR2, and a third novel component migrating in high-performance thin-layer chromatography (HPTLC) between NOR1 and NOR2. The structures were determined by a combination of ion trap sequential mass spectrometry (MALDI-QIT-TOF) and step-wise treatment with glycosidases, followed by identification of products on HPTLC plates with lectins and mouse monoclonal anti-NOR antibody. The NOR2 component was found to be a disaccharide extension of NOR1 with the following structure: Galalpha1-4GalNAcbeta1-3Galalpha1-4GalNAcbeta1-3Galalpha1-4Galbeta1-4Glcbeta1-Cer. Treatment of NOR2 with alpha-galactosidase gave a glycolipid migrating between NOR1 and NOR2, which did not react with either GSL-IB4 or anti-NOR antibodies but did react with GalNAc-specific soybean agglutinin. This intermediate glycolipid (now designated NOR(int)) was identified as a relatively abundant component of a neutral glycolipid fraction from NOR erythrocytes, suggesting its presence as a precursor to NOR2. The structure of NOR(int) was also confirmed by sequential mass spectrometry studies. These results indicate that polyagglutination in NOR subjects is due to unique erythrocyte glycolipids that are synthesized by sequential addition of Galalpha1,4 and GalNAcbeta1,3 to globoside.

Detection of serum antibodies to S-antigen by surface plasmon resonance (SPR).

Serum autoantibodies to visual arrestin, also termed S-antigen, have been shown to accompany several autoimmune-related diseases. However, they were also detected in sera of healthy individuals; there is lack of a sensitive and fast method for evaluation of putative differences between those two groups of antibodies. We show that, using biosensor technology based on surface plasmon resonance (SPR), it was possible to characterize real-time interactions of immune sera with immobilized arrestin. Binding characteristics revealed different interaction kinetics of antiarrestin antibodies present in two distinct rabbit sera and, thus, broadened results of immunoblotting analysis. Therefore, we suggest that SPR-based biosensor technology might be a valuable method for monitoring and evaluation of antiarrestin antibodies in patients' sera.