Important role of matrix metalloproteinase 9 in epileptogenesis

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling-induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.     

Contribution of the mevalonate and methylerythritol phosphate pathways to the biosynthesis of dolichols in plants

Plant isoprenoids are derived from two biosynthetic pathways, the cytoplasmic mevalonate (MVA) and the plastidial methylerythritol phosphate (MEP) pathway. In this study their respective contributions toward formation of dolichols in Coluria geoides hairy root culture were estimated using in vivo labeling with (13)C-labeled glucose as a general precursor. NMR and mass spectrometry showed that both the MVA and MEP pathways were the sources of isopentenyl diphosphate incorporated into polyisoprenoid chains. The involvement of the MEP pathway was found to be substantial at the initiation stage of dolichol chain synthesis, but it was virtually nil at the terminal steps; statistically, 6-8 isoprene units within the dolichol molecule (i.e. 40-50% of the total) were derived from the MEP pathway. These results were further verified by incorporation of [5-(2)H]mevalonate or [5,5-(2)H(2)]deoxyxylulose into dolichols as well as by the observed decreased accumulation of dolichols upon treatment with mevinolin or fosmidomycin, selective inhibitors of either pathway. The presented data indicate that the synthesis of dolichols in C. geoides roots involves a continuous exchange of intermediates between the MVA and MEP pathways. According to our model, oligoprenyl diphosphate chains of a length not exceeding 13 isoprene units are synthesized in plastids from isopentenyl diphosphate derived from both the MEP and MVA pathways, and then are completed in the cytoplasm with several units derived solely from the MVA pathway. This study also illustrates an innovative application of mass spectrometry for qualitative and quantitative evaluation of the contribution of individual metabolic pathways to the biosynthesis of natural products.     

Nuclear microsatellite markers reveal the low genetic structure of <Emphasis Type="Italic">Pinus mugo</Emphasis> Turra (dwarf mountain pine) populations in Europe

Twenty-one populations (555 individuals) covering the entire native range of Pinus mugo Turra (dwarf mountain pine) were investigated for genetic variation scored at 13 nuclear microsatellite markers (nSSRs). The main objective of the present study was to determine the genetic structure across the present distribution of the species and locate populations of different genetic compositions. Most of the genetic variation was observed within the populations (95%). The assignment of populations based on Bayesian clustering methods revealed that the Sudeten populations of P. mugo form a separate genetic cluster. These stands have likely been established through the founder effects of Alpine migrants. The distribution and level of SSR polymorphisms, along with no evidence of isolation by distance or phylogeographic structure, indicate that the present populations of P. mugo have diverged relatively recently and originate from a larger glacial distribution of the species. One peripheral stand from Italy had the lowest values of most calculated genetic variation indices. This stand could therefore be more susceptible to genetic drift and a negative impact of predicted environmental changes. We discuss our findings with respect to previously published results on the genetic and morphological variation of P. mugo and with consideration for the conservation genetics of the species.     

<Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of mammalian Y145Stop prion protein amyloid fibrils

The Y145Stop prion protein (PrP23-144), which has been linked to the development of a heritable prionopathy in humans, is a valuable in vitro model for elucidating the structural and molecular basis of amyloid seeding specificities. Here we report the sequential backbone and side-chain ¹³C and ¹⁵N assignments of mouse and Syrian hamster PrP23-144 amyloid fibrils determined by using 2D and 3D magic-angle spinning solid-state NMR. The assigned chemical shifts were used to predict the secondary structures for the core regions of the mouse and Syrian hamster PrP23-144 amyloids, and the results compared to those for human PrP23-144 amyloid, which has previously been analyzed by solid-state NMR techniques.     

Hypotensive effects of hemopressin and bradykinin in rabbits, rats and mice. A comparative study

Hemopressin is a novel vasoactive nonapeptide derived from hemoglobin's alpha-chain as recently reported by Rioli et al. [Rioli V, Gozzo FC, Heimann AS, Linardi A, Krieger JE, Shida CS, et al. Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme. J Biol Chem 2003;278(10):8547-55]. In anesthetized male Wistar rats, this peptide exhibited hypotensive actions similar to those of bradykinin (BK) when administered intravenously (i.v.), and was found to be metabolized both in vitro and in vivo by several peptidases, including the angiotensin-converting enzyme (ACE). In this study, these findings were expanded upon by examining: (i) the degradation kinetics following incubation with ACE purified from rabbit lung and (ii) the blood pressure lowering effects of HP and BK injected i.v. or intra-arterially (i.a.) in male rabbits, rats, and mice. Our findings demonstrate that, in vitro, HP and BK are both degraded by ACE, but at different velocity rates. Furthermore, both HP and BK induced transient hypotension in all animals tested, although the responses to HP relative to the administration sites were significantly lower (by 10-100-fold) on an equimolar basis compared to those of BK. In rabbits, the decrease of blood pressure induced by HP (10-100 nmol/kg) did not differ whether it was administered i.v. or i.a., suggesting an absence of pulmonary/cardiac inactivation in contrast to BK (0.1-1 nmol/kg). The in vivo effect of HP was significantly potentiated in rabbits immunostimulated with bacterial lipopolysaccharide (LPS), but was unaffected by both the B2 receptor antagonist HOE 140 (0.1 micromol/kg) and captopril (100 microg/kg), contrary to BK. Therefore, HP acts as a weak hypotensive mediator, which does not activate kinin B2 receptors, but uses a functional site and/or signaling paths appearing to be up-regulated by LPS.     

Bacterial flora from bronchoalveolar lavage and occurrence of class IgG and IgA antibodies to Chlamydia pneumoniae in patients with chronic obstructive pulmonary disease (COPD)

Bronchoalveolar lavage taken from 46 patients (ranging in age from 21 to 71 years, mean 50.6 +/- 13.9) was examined for aerobic and anaerobic bacterial flora. Sera taken from 39 of patients as well as sera taken from 25 healthy blood donors of similar age (P = 0.99) were examined to determine IgG and IgA antibodies to C. pneumoniae. Bacterial flora was routinely cultured and determined using ATB computer system (bioMérieux,). IgG and IgA antibodies were tested by the enzyme immunoassays (Labsystems, Finland, Helsinki). Sera containing anti -C. pneumoniae IgG antibodies with titers of 45 EIU or higher and IgA with titers of 12 EIU or higher were considered positive. 143 of aerobic and 74 of anaerobic bacterial strains were cultured. Streptococci group viridans, pneumococci, enteric bacilli, Haemophilus spp., Prevotella spp., Actinomyces spp., Bifidobacterium spp. and Veilonella spp. were most often cultured. 66.6% of patients had IgG or IgA antibodies, in contrast, to the control group in which 60.0% and 44.0% of examined blood donors had IgG and IgA antibodies respectively. COPD patients were more frequently positive for specific anti-C. pneumoniae antibodies than the healthy donors (p = 0.003). The difference in a seropositivity rate of specific IgA and IgG antibodies was significant (p = 0.00002 and p = 0.003 respectively). Bronchoalveolar lavage of patients suffering from COPD can be contaminated with high number of aerobic and anaerobic bacterial species, and immunological status of the patients indicated persistent infection caused by C. pneumoniae more often than in controls.     

Crystal structures of the semireduced and inhibitor-bound forms of cyclic nucleotide phosphodiesterase from Arabidopsis thaliana.

The crystal structure of the semireduced form of cyclic nucleotide phosphodiesterase (CPDase) from Arabidopsis thaliana has been solved by molecular replacement and refined at the resolution of 1.8 A. We have previously reported the crystal structure of the native form of this enzyme, whose main target is ADP-ribose 1",2"-cyclic phosphate, a product of the tRNA splicing reaction. CPDase possesses six cysteine residues, four of which are involved in forming two intra-molecular disulfide bridges. One of these bridges, between Cys-104 and Cys-110, is opened in the semireduced CPDase, whereas the other remains intact. This change of the redox state leads to a conformational rearrangement in the loop covering the active site of the protein. While the native structure shows this partially disordered loop in a coil conformation, in the semireduced enzyme the N-terminal lobe of this loop winds up and elongates the preceding alpha-helix. The semireduced state of CPDase also enabled co-crystallization with a putative inhibitor of its enzymatic activity, 2',3'-cyclic uridine vanadate. The ligand is bound within the active site, and the mode of binding is in agreement with the previously proposed enzymatic mechanism. Selected biophysical properties of the oxidized and the semireduced CPDase are also discussed.