Hepatocyte growth factor/c-MET axis-mediated tropism of cord blood-derived unrestricted somatic stem cells for neuronal injury

An under-agarose chemotaxis assay was used to investigate whether unrestricted somatic stem cells (USSC) that were recently characterized in human cord blood are attracted by neuronal injury in vitro. USSC migrated toward extracts of post-ischemic brain tissue of mice in which stroke had been induced. Moreover, apoptotic neurons secrete factors that strongly attracted USSC, whereas necrotic and healthy neurons did not. Investigating the expression of growth factors and chemokines in lesioned brain tissue and neurons and of their respective receptors in USSC revealed expression of hepatocyte growth factor (HGF) in post-ischemic brain and in apoptotic but not in necrotic neurons and of the HGF receptor c-MET in USSC. Neuronal lesion-triggered migration was observed in vitro and in vivo only when c-MET was expressed at a high level in USSC. Neutralization of the bioactivity of HGF with an antibody inhibited migration of USSC toward neuronal injury. This, together with the finding that human recombinant HGF attracts USSC, document that HGF signaling is necessary for the tropism of USSC for neuronal injury. Our data demonstrate that USSC have the capacity to migrate toward apoptotic neurons and injured brain. Together with their neural differentiation potential, this suggests a neuroregenerative potential of USSC. Moreover, we provide evidence for a hitherto unrecognized pivotal role of the HGF/c-MET axis in guiding stem cells toward brain injury, which may partly account for the capability of HGF to improve function in the diseased central nervous system.     

Can nucleotides prevent Cu-induced oxidative damage?

Cu-induced oxidative damage is associated with cancer, diabetes, neurodegenerative and age related diseases. The quest for Cu-chelators as potential antioxidants spans the past decades. Yet, biocompatible Cu-chelators that do not alter the normal metal-ion homeostasis are still lacking. Here, we explored the potential of natural and synthetic nucleotides and inorganic phosphates as inhibitors of the Cu(I)/(II)-induced ()OH formation via either the Fenton or Haber-Weiss mechanisms. For this purpose, we studied by ESR the modulation of Cu-induced ()OH production, from the decomposition of H(2)O(2), by nucleotides and phosphates. ATP inhibited both Cu(I) and Cu(II) catalyzed reactions (IC(50) 0.11 and 0.04mM, respectively). Likewise, adenosine 5'-beta,gamma-methylene triphosphate (AMP-PCP), adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), ADP and tripolyphosphate were identified as good inhibitors. However, AMP and adenosine were poor inhibitors in the Cu(I)-H(2)O(2) system, IC(50) ca. 1.2mM, and radical enhancers in the Cu(II)-H(2)O(2) system. The best antioxidant was adenosine 5'-[beta,gamma-imino] triphosphate (AMP-PNP) (IC(50) 0.05mM at Cu(I)-H(2)O(2) system) which was 15 times more active than the known antioxidant Trolox. ATP and analogues inhibit Cu-induced ()OH formation through an ion chelation rather than a scavenging mechanism. Two phosphate groups are required for making active Fenton-reaction inhibitors. Nucleotides and phosphates triggered a biphasic modulation of the Haber-Weiss reaction, but a monophasic inhibition of the Fenton reaction. We conclude that nucleotides at sub mM concentrations can prevent Cu-induced OH radical formation from H(2)O(2), and hence may possibly prevent oxidative damage.     

Reactivity of histidine and lysine side-chains with diethylpyrocarbonate -- a method to identify surface exposed residues in proteins

The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.     

Short-term dynamics of bacterial communities in a tidally affected coastal ecosystem

Tidal effects on the composition of free-living (FL) and particle-associated (PA) bacterial communities were studied in a tidal flat ecosystem in the southern North Sea. Denaturing gradient gel electrophoresis targeting the 16S rRNA gene and the 16S rRNA of Bacteria, Bacteroidetes, Alphaproteobacteria and the Roseobacter clade was applied. Despite strong tidal variations in the quantity and, depending on the season, also the quality of suspended matter as well as variations in bacterial activity, the bacterial community composition remained rather stable. FISH showed some variations of the community composition, but these were not related to typical tidal situations. Variations were higher during tidal cycles in May and July compared with November. Bacteroidetes, Alpha- and Gammaproteobacteria constituted the majority of the bacterial communities but relative proportions of the different groups varied considerably. On particles, Betaproteobacteria were also detected to substantial proportions. The Roseobacter clade constituted up to 90% of FL but only 30% of PA Alphaproteobacteria. Banding patterns of the Bacteroidetes-specific amplicons, and in particular those targeting the 16S rRNA, revealed tidally induced effects, as several bands appeared or disappeared at distinct events such as slack water or resuspension. Sequencing of prominent bands revealed predominantly phylotypes reported previously from this ecosystem.     

Eye formation in the absence of retina

Eye development is a complex process that involves the formation of the retina and the lens, collectively called the eyeball, as well as the formation of auxiliary eye structures such as the eyelid, lacrimal gland, cornea and conjunctiva. The developmental requirements for the formation of each individual structure are only partially understood. We have shown previously that the homeobox-containing gene Rx is a key component in eye formation, as retinal structures do not develop and retina-specific gene expression is not observed in Rx-deficient mice. In addition, Rx−/− embryos do not develop any lens structure, despite the fact that Rx is not expressed in the lens. This demonstrates that during normal mammalian development, retina-specific gene expression is necessary for lens formation. In this paper we show that lens formation can be restored in Rx-deficient embryos experimentally, by the elimination of β-catenin expression in the head surface ectoderm. This suggests that β-catenin is involved in lens specification either through Wnt signaling or through its function in cell adhesion. In contrast to lens formation, we demonstrate that the development of auxiliary eye structures does not depend on retina-specific gene expression or retinal morphogenesis. These results point to the existence of two separate developmental processes involved in the formation of the eye and its associated structures. One involved in the formation of the eyeball and the second involved in the formation of the auxiliary eye structures.     

The Socioeconomic Impact of Truffle Cultivation in Rural Spain<Superscript>1</Superscript>

The Socioeconomic Impact of Truffle Cultivation in Rural Spain. Commercial black truffle (Tuber melanosporum) plantations have been promoted in Europe with the intention of benefiting rural economies while conserving biodiversity through the expansion of oak woodlands. In this context, a socioeconomic study was conducted around the town of Sarrión in eastern Spain, where government subsidies have supported oak reforestation and truffle cultivation in unproductive hilly areas since 1987. Currently there are about 4,500 ha of truffle orchards in the surrounding county and 530 members in the local truffle association, which has provided a key forum for truffle cultivators to share technical, financial and administrative experiences. Structured interviews were carried out in 2002 with a number of orchard owners, as well as representatives of financial and governmental institutions. Truffles, which are harvested using trained dogs, typically fetch local cultivators average prices of 220–670 EUR/kg, although retail prices of high-quality specimens may reach twice this amount. In addition to the direct economic impact, an increase in local land prices was also documented, as well as a tendency for continued expansion of truffle orchards, and thus oak reforestation. In conclusion, the promotion of truffle cultivation through autonomous community and provincial government subsidies, in conjunction with support by local banks, a dedicated local truffle association, and growing interest on behalf of local farmers, seems to have achieved the mutual goals of biodiversity conservation and improving the rural economy in this region of Spain.     

Brugia malayi: whole genome amplification for genomic characterization of filarial parasites

Genetic characterization of field isolates and clinical specimens of filarial nematodes is often limited by a shortage of DNA; therefore, we evaluated a multiple displacement amplification (MDA) based whole genome amplification method. The quality of amplified DNA was examined by conventional PCR, real-time PCR, and DNA hybridization. MDA of 5.0 ng of adult Brugia malayi DNA and one-fifteenth of the DNA isolated from a single microfilaria resulted in 6.3 and 4.2 μg of amplified DNA, respectively. Amplified DNA was equivalent to native genomic DNA for hybridization to B. malayi BAC library clones or to an oligonucleotide microarray with approximately 18,000 filarial DNA sequences. MDA is useful for whole genome amplification of filarial DNA from very small amounts of starting material. This technology will permit detailed studies of genetic diversity that were not previously feasible.