Mobilization of Neural Stem Cells and Generation of New Neurons in 6-OHDA�Clesioned Rats by Intracerebroventricular Infusion of Liver Growth Factor

Neural stem cells with self-renewal and multilineage potential persist in the subventricular zone of the adult mammalian forebrain. These cells remain relatively quiescent but, under certain conditions, can be stimulated, giving rise to new neurons. Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and is useful for neuroregenerative therapies. The aim of this study was to investigate the potential neurogenic activity of LGF in the 6-hydroxydopamine rat model of Parkinson''s disease. Proliferation was significantly increased in the subventricular zone and denervated striatum of rats receiving ICV LGF infusions, and 25% of the proliferating cells were doublecortin-positive neurons. Doublecortin-positive cells with the morphology of migrating neuroblasts were also observed in the dorsal and ventral regions of the striatum of LGF-infused animals. Moreover, some newly generated cells were neuronal nuclei-positive mature neurons. LGF also stimulated microglia and induced astrogliosis, both phenomena associated with generation and migration of new neurons in the adult brain. In summary, our study shows that LGF stimulates neurogenesis when applied intraventricularly in 6-hydroxydopamine–lesioned rats. Considering that this factor also promotes neuronal migration into damaged tissue, we propose LGF as a novel factor useful for neuronal replacement in neurodegenerative diseases. (J Histochem Cytochem 57:491–502, 2009)     

Crystal Structure and Catalytic Mechanism of 4-Methylmuconolactone Methylisomerase

When methyl-substituted aromatic compounds are degraded via ortho (intradiol)-cleavage of 4-methylcatechol, the dead-end metabolite 4-methylmuconolactone (4-ML) is formed. Degradation of 4-ML has only been described in few bacterial species, including Pseudomonas reinekei MT1. The isomerization of 4-ML to 3-methylmuconolactone (3-ML) is the first step required for the mineralization of 4-ML and is catalyzed by an enzyme termed 4-methylmuconolactone methylisomerase (MLMI). We identified the gene encoding MLMI in P. reinekei MT1 and solved the crystal structures of MLMI in complex with 3-ML at 1.4-Å resolution, with 4-ML at 1.9-Å resolution and with a MES buffer molecule at 1.45-Å resolution. MLMI exhibits a ferredoxin-like fold and assembles as a tight functional homodimeric complex. We were able to assign the active site clefts of MLMI from P. reinekei MT1 and of the homologous MLMI from Cupriavidus necator JMP134, which has previously been crystallized in a structural genomics project. Kinetic and structural analysis of wild-type MLMI and variants created by site-directed mutagenesis indicate Tyr-39 and His-26 to be the most probable catalytic residues. The previously proposed involvement of Cys-67 in covalent catalysis can now be excluded. Residue His-52 was found to be important for substrate affinity, with only marginal effect on catalytic activity. Based on these results, a novel catalytic mechanism for the isomerization of 4-ML to 3-ML by MLMI, involving a bislactonic intermediate, is proposed. This broadens the knowledge about the diverse group of proteins exhibiting a ferredoxin-like fold.     

Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpo B gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpo B-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpo B-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus , and Shewanella marinus . In contrast to 16S rRNA gene-TTGE, rpo B-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpo B leads to a single band in TTGE, the rpo B gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.     

Is L-methionine a trigger factor for Alzheimer’s-like neurodegeneration?: Changes in Aβ oligomers, <Emphasis Type="Italic">tau</Emphasis> phosphorylation,synaptic proteins, <Emphasis Type="Italic">Wnt</Emphasis> signaling and behavioral impairment in wild-type mice

Background

L-methionine, the principal sulfur-containing amino acid in proteins, plays critical roles in cell physiology as an antioxidant and in the breakdown of fats and heavy metals. Previous studies suggesting the use of L-methionine as a treatment for depression and other diseases indicate that it might also improve memory and propose a role in brain function. However, some evidence indicates that an excess of methionine can be harmful and can increase the risk of developing Type-2 diabetes, heart diseases, certain types of cancer, brain alterations such as schizophrenia, and memory impairment.

Results

Here, we report the effects of an L-methionine-enriched diet in wild-type mice and emphasize changes in brain structure and function. The animals in our studypresented 1) higher levels of phosphorylated tau protein, 2) increased levels of amyloid-β (Aβ)-peptides, including the formation of Aβ oligomers, 3) increased levels of inflammatory response,4) increased oxidative stress, 5) decreased level of synaptic proteins, and 6) memory impairment and loss. We also observed dysfunction of the Wnt signaling pathway.

Conclusion

Taken together, the results of our study indicate that an L-methionine-enriched diet causes neurotoxic effects in vivo and might contribute to the appearance of Alzheimer’s-like neurodegeneration.

     

Caveolin-1-dependent and -independent membrane domains

Lipid rafts defined as cholesterol- and sphingomyelin-rich domains have been isolated from different cell types that vary greatly in their lipid profiles. Here, we investigated the contribution of the structural protein caveolin-1 (Cav1) to the overall lipid composition and domain abundance in mouse embryonic fibroblasts (MEFs) from wild-type (WT) or Cav1-deficient (Cav1^−/−) animals. Our findings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels. However, Cav1^−/−-deficient cells did have a higher proportion of sphingomyelin, decreased abundance of unsaturated phospholipids, and a trend toward shorter fatty acid chains in phosphatidylcholine. We isolated detergent-resistant membranes (DRMs), nondetergent raft domains (NDR), and cholesterol oxidase (CO)-sensitive domains and assessed the abundance of ordered domains in intact cells using the fluorescent dye Laurdan. Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types. The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifically oxidize caveolar cholesterol. In contrast, the abundance of ordered domains in adherent cells is reduced in Cav1^−/− compared with WT MEFs, suggesting that cell architecture is critical in maintaining Cav1-induced lipid rafts.     

The proteins involved in sucrose synthesis in the marine cyanobacterium Synechococcus sp. PCC 7002 are encoded by two genes transcribed from a gene cluster

It has been reported that higher plants and cyanobacteria synthesize sucrose (Suc) by a similar sequential action of sucrose-phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP). In the genome of the marine unicellular cyanobacterium Synechococcus sp. PCC 7002 there is a sequence that was not annotated as a putative SPP encoding gene (sppA), although the sequence was available. In this study, we functionally characterize the sppA gene of that strain and demonstrate that it is cotranscribed with spsA, the SPS encoding gene. This is the first report on the coordination of Suc synthesis gene expression in an oxygenic-photosynthetic organism.     

Analysis of diclofenac and its metabolites by high-performance liquid chromatography: relevance of CYP2C9 genotypes in diclofenac urinary metabolic ratios

In humans, diclofenac is metabolised to 4'-hydroxy (OH), 3'-OH and 5-OH metabolites. The polymorphic CYP2C9 is involved in the metabolism of diclofenac to 4'-OH diclofenac and 3'-OH diclofenac. The aim of the present study was to develop a high-performance liquid chromatographic method to simultaneously measure diclofenac and its metabolites in urine, suitable for metabolic studies. After liquid-liquid extraction the compounds were separated in a reversed-phase column and measured by ultraviolet absorption at 282 nm. For all compounds intra-day and inter-day variations were less than 7%, and the limits of quantitation were 0.25 mg/l. No analytical interference with endogenous compounds was found. The relationship between diclofenac metabolic ratios among different CYP2C9 genotypes is reported. The CYP2C9*3/*3 subject had the highest diclofenac/4'-OH ratios. However no difference was found between CYP2C9*2/*2 and *1/*1 genotypes. The chromatographic method developed was sensitive and reliable for the measurement of diclofenac and its metabolites simultaneously in human urine, and is suitable for use in diclofenac metabolism studies.