Hyperbaric and normobaric reoxygenation of hypoxic rat brain slices--impact on purine nucleotides and cell viability

Hyperbaric oxygen treatment has been suggested as able to reduce hypoxia induced neuronal damage. The aim of the study was to compare the impact of different reoxygenation strategies on early metabolical (purine nucleotide content determined by HPLC) and morphological changes (index of cell injury after celestine blue/acid fuchsin staining) of hypoxically damaged rat neocortical brain slices. For this purpose slices (300 μm and 900 μm) were subjected to either 5 or 30 min of hypoxia by gassing the incubation medium with nitrogen. During the following reoxygenation period treatment groups were administered either 100% oxygen (O) or room air (A) at normobaric (1 atm absolute, NB-O; NB-A) or hyperbaric (2.5 atm absolute, HB-O; HB-A) conditions. After 5 min of hypoxia, both HB-O and NB-O led to a complete nucleotide status restoration (ATP/ADP; GTP/GDP) in 300 μm slices. However, reoxygenation after 30 min of hypoxia was less effective, irrespective of the oxygen pressure. Furthermore, administering hyperbaric room air resulted in no significant posthypoxic nucleotide recovery. In 900 μm slices, both control incubation as well as 30 min of hypoxia resulted in significantly lower trinucleotide and higher dinucleotide levels compared to 300 μm slices. While there was no significant difference between HB-O and NB-O on the nucleotide status, morphological evaluation revealed a better recovery of the index of cell injury (profoundly injured/intact cell-ratio) in the HB-O group. Conclusively, the posthypoxic recovery of metabolical characteristics was dependent on the duration of hypoxia and slice thickness, but not on the reoxygenation pressure. A clear restorative effect on purine nucleotides was found only in early-administered HB-O as well as NB-O in contrast to room air treated slices. However, these pressure independent metabolic changes were morphologically accompanied by a significantly improved index of cell injury, indicating a possible neuroprotective role of HB-O in early posthypoxic reoxygenation.     

Chemical identification of<Emphasis Type="Italic"> N</Emphasis>-acyl homoserine lactone quorum-sensing signals produced by<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> strains in defined medium

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.     

Cryptic MBP epitope 1-20 is inducing autoimmune anterior uveitis without EAE in Lewis rats

Lewis rats immunized with myelin basic protein (MBP) developed experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Although several cryptic epitopes of MBP have strong encephalitogenic and uveitogenic properties, the peptide corresponding to the MBP residues 1-20 was uniquely capable of inducing AU without EAE. In this study, we showed that acetylation of the N-terminal amino acid did not produce encephalitogenicity, did not enhance uveitogenicity, and did not improve T cell proliferation in Lewis rats. The cytokine production profile induced by MBP(1-20) immunization was consistent with a Th1 response. In MBP-injected rats and in peptide-injected rats, the frequency of the IFN-gamma-secreting cells in MBP(69-89)-stimulated T cells was significantly higher than the frequency of IFN-gamma-secreting cells in MBP(1-20)-stimulated T cells. However, similar numbers of IFN-gamma-producing specific cells were found in the eyes of MBP(69-89) and MBP(1-20) immunized rats. In these rats, the iris-infiltrating cells consisted of a much higher percentage of CD4(+) T cells expressing L-selectin (CD62L) than did those cells found in the spinal cord. The results demonstrate that MBP(1-20) is immunogenic and uveitogenic, although it induced only weak proliferation and weak Th1 reaction. The fact that T cells with the same specificity have different effects on target organs suggested that, in the eye and spinal cord, a distinct mechanism might mediate the recruitment of cells to these organs.     

Characterization of the Sinorhizobium meliloti sinR/sinI locus and the production of novel N-acyl homoserine lactones

Sinorhizobium meliloti is a soil bacterium which can establish a nitrogen-fixing symbiosis with the legume Medicago sativa. Recent work has identified a pair of genes, sinR and sinI, which represent a potential quorum-sensing system and are responsible for the production of N-acyl homoserine lactones (AHLs) in two S. meliloti strains, Rm1021 and Rm41. In this work, we characterize the sinRI locus and show that these genes are responsible for the synthesis of several long-chain AHLs ranging from 12 to 18 carbons in length. Four of these, 3-oxotetradecanoyl HL, 3-oxohexadecenoyl HL, hexadecenoyl HL, and octadecanoyl HL, have novel structures. This is the first report of AHLs having acyl chains longer than 14 carbons. We show that a disruption in sinI eliminates these AHLs and that a sinR disruption results in only basal levels of the AHLs. Moreover, the same sinI and sinR mutations also lead to a decrease in the number of pink nodules during nodulation assays, as well as a slight delay in the appearance of pink nodules, indicating a role for quorum sensing in symbiosis. We also show that sinI and sinR mutants are still capable of producing several short-chain AHLs, one of which was identified as octanoyl HL. We believe that these short-chain AHLs are evidence of a second quorum-sensing system in Rm1021, which we refer to here as the mel system, for "S. meliloti."     

Lipid peroxidation in neurodegeneration: new insights into Alzheimer's disease

Imbalances of oxidative homeostasis and lipid peroxidation have been revealed as important factors involved in neurodegenerative disorders such as Alzheimer's disease. The brains of patients with Alzheimer's disease contain increased levels of lipid-peroxidation products such as 4-hydroxy-2-nonenal or acrolein, and enhanced lipid peroxidation can also be detected in cerebrospinal fluid and plasma from such patients. Recent research revealed that the interplay of transition metals, amyloid-beta peptide and lipid peroxidation might be responsible for increased oxidative stress and cell damage in this disease. In particular, the contrasting roles of amyloid-beta peptide, as a possible transition metal-chelating antioxidant for lipoproteins and a pro-oxidant when aggregated in brain tissue, has been the focus of discussion recently. In this context, lipid peroxidation has to be seen as an important part of the pathophysiological cascade in Alzheimer's disease, and its measurement in body fluids might serve as a therapy control for Alzheimer's disease and other neurodegenerative diseases.     

Stability of protein-bound conformations of bioactive peptides: the folded conformation of an epidermal growth factor-like thrombomodulin fragment is similar to that recognized by thrombin

The relationship between the free and bound conformations of bioactive peptides is explored using the epidermal growth factor (EGF)-like thrombomodulin fragment hTM409-426 as a model system. The hTM409-426 peptide has a sequence of C(409)PEGYILDDGFIC(421)TDIDE (with a disulfide bond between Cys409 and Cys421) and is a selective inhibitor of thrombin. Upon binding to thrombin, hTM409-426 adopts a well-defined conformation-namely, a beta-turn followed by an antiparallel beta-sheet, similar to those found in all other EGF-like protein repeats (Hrabal et al., Protein Science, 1996, Vol. 5, 195-203). Here we demonstrate that, at pH 6.8 and at 25 degrees C, the hTM409-426 peptide in the free state is very flexible, but still populates a type II beta-turn over residues Pro410-Glu411-Gly412-Tyr413 and the clustering of some hydrophobic side chains, both of which are present in the thrombin-bound conformation. At a lower temperature of 5 degrees C, significant conformational shifts of the C alpha H proton resonances and extensive medium- and long-range NOEs are observed, indicating the presence of folded conformations with unique backbone-backbone and side-chain interactions. A comparison of the NOE patterns in the free state with transferred NOEs shows that the free-state folded and the thrombin-bound conformations of the hTM409-426 peptide are very similar, particularly over residues Pro410-Ile424. The folded conformation of hTM409-426 appears to be stabilized by two hydrophobic clusters, one formed by the side chains of residues Pro410, Tyr413, Leu415, and Phe419 and the Cys409-Cys421 disulfide bond, the second involving residues Ile414 and Ile424. These results indicate that the overall topology of the thrombin-bound conformation of the hTM409-426 peptide is prefolded in the free state and the primary sequence (including the disulfide bond) may be selective for an ensemble of conformations similar to that recognized by thrombin.     

Alzheimer''s Amyloid-β as a Preventive Antioxidant for Brain Lipoproteins

1. Increased production of A in a form of lipoprotein antioxidant under the action of increased oxidative stress in aging with subsequent chelation of transition metal ions by A, accumulation of toxic A–metal lipoprotein complexes, production of reactive oxygen species, and neurotoxicity are reviewed and postulated to form the temporal sequence of events in the development of Alzheimer's disease (AD).2. Since (i) A binds copper stronger than iron and other transition metals, and (ii) copper is a more efficient catalyst of oxidation than other transition metals, chelation of copper by A is proposed to be a most important part of this pathway.3. Whereas this amyloid-binds-copper (ABC) model does not remove A peptide from its central place in our current thinking of AD, it places additional factors in the center of discussion.4. Most importantly, they embrace pathological mechanisms known to develop in aging (which is the most important risk factor for AD), such as increased production of reactive oxygen species by mitochondria, that can be positioned upstream relative to the generation of A.     

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).     

Neurotransmission: emerging roles of endocannabinoids

Postsynaptic release of endocannabinoids can inhibit presynaptic neurotransmitter release on short and long timescales. This retrograde inhibition occurs at both excitatory and inhibitory synapses and may provide a mechanism for synaptic gain control, short-term associative plasticity, reduction of synaptic crosstalk, and metaplasticity.