Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets

Extracellular matrix molecules--including chondroitin sulfate proteoglycans, hyaluronan, and tenascin-R--are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type-dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice-like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin-expressing interneurons, which developed after 2-3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN-expressing neurons revealed that these are large fast-spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity-dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca2+ permeable AMPA subtype of glutamate receptors or L-type Ca2+ voltage-gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca2+ influx via AMPA receptors and L-type channels is necessary for activity-dependent formation of PNs. To study functions of chondroitin sulfate-rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity.     

The stereochemistry of benzo[a]pyrene-2'-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases

The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation of (+)-trans-B[a]P-N(2)-dG and, to a lesser extent, (+)-cis-B[a]P-N(2)-dG adducts. Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively. The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N(2)-dG and (+)-trans-B[a]P-N(2)-dG adducts in complexes with MTases are enhanced, but to different extents, indicating that aromatic B[a]P residues are positioned in different microenvironments in the DNA-protein complexes. We have previously shown that the (+)-trans-isomeric adduct inhibits both the binding and methylating efficiencies (k(cat)) of both MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F, Bonala R, et al. (2006) Biochemistry45, 6142-6159]. Here we show that the stereoisomeric (+)-cis-B[a]P-N(2)-dG lesion has only a minimal effect on the binding of these MTases and on k(cat). The minor-groove (+)-trans adduct interferes with the formation of the normal DNA minor-groove contacts with the catalytic loop of the MTases. However, the intercalated base-displaced (+)-cis adduct does not interfere with the minor-groove DNA-catalytic loop contacts, allowing near-normal binding of the MTases and undiminished k(cat) values.     

Pathophysiology of ageing, longevity and age related diseases

On April 18, 2007 an international meeting on Pathophysiology of Ageing, Longevity and Age-Related Diseases was held in Palermo, Italy. Several interesting topics on Cancer, Immunosenescence, Age-related inflammatory diseases and longevity were discussed. In this report we summarize the most important issues. However, ageing must be considered an unavoidable end point of the life history of each individual, nevertheless the increasing knowledge on ageing mechanisms, allows envisaging many different strategies to cope with, and delay it. So, a better understanding of pathophysiology of ageing and age-related disease is essential for giving everybody a reasonable chance for living a long and enjoyable final part of the life.     

Stress impacts telomere dynamics

Telomeres are DNA-protein complexes at the ends of chromosomes that control genomic integrity but appear to become shorter with age and stress. To test whether stress causes telomere attrition, we exposed the offspring of wild-caught house mice (Mus musculus) to stressful conditions and examined the changes in telomere length over six months. We found that females exposed to males and reproductive stress (either with or without crowding) had significantly shorter telomeres than controls, and males exposed to crowding stress had shorter telomeres than males that were not crowded. Our results indicate that stress alters telomere dynamics, causing attrition and hindering restoration, and these effects are sex dependent. Telomeres may thus provide a biomarker for assessing an individual's cumulative exposure or ability to cope with stressful conditions.     

NMR assignments of 1H, 13C and 15N spectra of methionine sulfoxide reductase B1 from Mus musculus

Isotopically labeled, ¹⁵N and ¹⁵N/¹³C forms of recombinant methionine-r-sulfoxide reductase 1 (MsrB1, SelR) from Mus musculus were produced, in which catalytic selenocysteine was replaced with cysteine. We report here the ¹H, ¹⁵N and ¹³C NMR assignment of the reduced form of this mammalian protein.     

Subunit-dependent cadmium and nickel inhibition of acid-sensing ion channels

Acid-sensing ion channels (ASIC) are ligand-gated cation channels that are highly expressed in peripheral sensory and central neurons. ASIC are transiently activated by decreases in extracellular pH and are thought to play important roles in sensory perception, neuronal transmission, and excitability, and in the pathology of neurological conditions, such as brain ischemia. We demonstrate here that the heavy metals Ni(2+) and Cd(2+) dose-dependently inhibit ASIC currents in hippocampus CA1 neurons and in Chinese hamster ovary (CHO) cells heterologously expressing these channels. The effects of both Ni(2+) and Cd(2+) were voltage-independent, fast, and reversible. Neither metal affected activation and desensitization kinetics but rather decreased pH-sensitivity. Moreover, distinct ASIC isoforms were differentially inhibited by Ni(2+) and Cd(2+). External application of 1 mM Ni(2+) rapidly inhibited homomeric ASIC1a and heteromeric ASIC1a/2a channels without affecting ASIC1b, 2a, and ASIC3 homomeric channels and ASIC1a/3 and 2a/3 heteromeric channels. In contrast, external Cd(+) (1 mM) inhibited ASIC2a and ASIC3 homomeric channels and ASIC1a/2a, 1a/3, and 2a/3 heteromeric channels but not ASIC1a homomeric channels. The acid-sensing current in isolated rat hippocampus CA1 neurons, thought to be carried primarily by ASIC1a and 1a/2a, was inhibited by 1 mM Ni(2+). The current study identifies ASIC as a novel target for the neurotoxic heavy metals Cd(2+) and Ni(2+).     

Analysis of creatinine in mouse and rat serum by ion exchange high performance liquid chromatography for in vivo studies of renal function

An ion exchange high performance liquid chromatography method was developed for determining creatinine levels in both mouse and rat serum samples. Separation of creatinine from other serum components was achieved in 10 min using a 100 x 4.1-mm, 10 microm strong cation exchange column following acetonitrile precipitation of serum proteins. Incorporation of a guard cartridge placed in-line prior to the analytical column was employed to prevent interference from compounds used in renal disease animal trials. Creatinine levels in normal and diseased animals were accurately determined in the 0.01-10 mg/dL range, and average recovery of the method was approximately 85% for both mouse and rat serum. Addition of 0.5-1.0% acetic acid to the acetonitrile used for protein precipitation significantly improved creatinine recovery to above 97% in mouse serum. The method was used for routine preclinical diagnosis of rat and mouse model renal function, and for the evaluation of renal disease treatment efficacy.     

Proposed minimum reporting standards for chemical analysis

There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly, the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation, experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line discussion forum at or . Further, community input related to this document can also be provided via this electronic forum. The contents of this paper do not necessarily reflect any position of the Government or the opinion of the Food and Drug Administration Sponsor: Metabolomics Society http://www.metabolomicssociety.org/ Reference: http://msi-workgroups.sourceforge.net/bio-metadata/reporting/pbc/ http://msi-workgroups.sourceforge.net/chemical-analysis/ Version: Revision: 5.1 Date: 09 January, 2007     

Antiamoebin I in methanol solution: rapid exchange between right-handed and left-handed 3(10)-helical conformations

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe(1)-Aib-Aib-Aib-Iva-Gly-Leu-Aib(8)-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl(16). By using the uniformly (13)C,(15)N-labeled sample of Aam-I, the set of conformationally dependent J couplings and (3h)J(NC) couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the (13)C(beta) nuclei (Deltadelta((13)C(beta))) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe(1)-Gly(6)) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 3(10)-helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib(8) residue. The (15)N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib(8) residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic 'propensity' possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism.