Ascorbic acid oxidase is dynamically regulated by light and oxygen. A tool for oxygen management in plants?

Ascorbic acid oxidase (AAO) is a plant blue-copper protein catalyzing dioxygen reduction to water using ascorbic acid as the electron donor. In spite of extensive molecular characterization the physiological role of AAO is still uncertain. Abundant mRNA, protein and activity of AAO were observed in illuminated leaves of Cucurbita pepo. AAO activity was found to be proportional to light intensity. The light effect was rapidly reversed in dark and activity remained low throughout the dark period. Activity was elicited in dark by increased oxygen concentration. AAO activity increased in the facultative CAM Kalancho? blossfeldiana upon induction of the CAM cycle and decreased during germination of C. pepo and Zea mays under hypoxic conditions. These results strongly suggest that AAO activity could be part of a dynamic system for oxygen management in plants.     

Transmission of Atherosclerosis Susceptibility with Gut Microbial Transplantation

Recent studies indicate both clinical and mechanistic links between atherosclerotic heart disease and intestinal microbial metabolism of certain dietary nutrients producing trimethylamine N-oxide (TMAO). Here we test the hypothesis that gut microbial transplantation can transmit choline diet-induced TMAO production and atherosclerosis susceptibility. First, a strong association was noted between atherosclerotic plaque and plasma TMAO levels in a mouse diversity panel (n = 22 strains, r = 0.38; p = 0.0001). An atherosclerosis-prone and high TMAO-producing strain, C57BL/6J, and an atherosclerosis-resistant and low TMAO-producing strain, NZW/LacJ, were selected as donors for cecal microbial transplantation into apolipoprotein e null mice in which resident intestinal microbes were first suppressed with antibiotics. Trimethylamine (TMA) and TMAO levels were initially higher in recipients on choline diet that received cecal microbes from C57BL/6J inbred mice; however, durability of choline diet-dependent differences in TMA/TMAO levels was not maintained to the end of the study. Mice receiving C57BL/6J cecal microbes demonstrated choline diet-dependent enhancement in atherosclerotic plaque burden as compared with recipients of NZW/LacJ microbes. Microbial DNA analyses in feces and cecum revealed transplantation of donor microbial community features into recipients with differences in taxa proportions between donor strains that were transmissible to recipients and that tended to show coincident proportions with TMAO levels. Proportions of specific taxa were also identified that correlated with plasma TMAO levels in donors and recipients and with atherosclerotic lesion area in recipients. Atherosclerosis susceptibility may be transmitted via transplantation of gut microbiota. Gut microbes may thus represent a novel therapeutic target for modulating atherosclerosis susceptibility.     

Two Fatty Acid Desaturases,STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 and FATTY ACID DESATURASE3, Are Involved in Drought and Hypoxia Stress Signaling in Arabidopsis Crown Galls

Agrobacterium tumefaciens-derived crown galls of Arabidopsis (Arabidopsis thaliana) contain elevated levels of unsaturated fatty acids and strongly express two fatty acid desaturase genes, ω3 FATTY ACID DESATURASE3 (FAD3) and STEAROYL-ACYL CARRIER PROTEIN Δ⁹-DESATURASE6 (SAD6). The fad3-2 mutant with impaired α-linolenic acid synthesis developed significantly smaller crown galls under normal, but not under high, relative humidity. This strongly suggests that FAD3 plays a role in increasing drought stress tolerance of crown galls. SAD6 is a member of the SAD family of as yet unknown function. Expression of the SAD6 gene is limited to hypoxia, a physiological condition found in crown galls. As no sad6 mutant exists and to link the function of SAD6 with fatty acid desaturation in crown galls, the lipid pattern was analyzed of plants with constitutive SAD6 overexpression (SAD6-OE). SAD6-OE plants contained lower stearic acid and higher oleic acid levels, which upon reduction of SAD6 overexpression by RNA interference (SAD6-OE-RNAi) regained wild-type-like levels. The development of crown galls was not affected either in SAD6-OE or SAD6-OE-RNAi or by RNA interference in crown galls. Since biochemical analysis of SAD6 in yeast (Saccharomyces cerevisiae) and Escherichia coli failed, SAD6 was ectopically expressed in the background of the well-known suppressor of salicylic acid-insensitive2 (ssi2-2) mutant to confirm the desaturase function of SAD6. All known ssi2-2 phenotypes were rescued, including the high stearic acid level. Thus, our findings suggest that SAD6 functions as a Δ⁹-desaturase, and together with FAD3 it increases the levels of unsaturated fatty acids in crown galls under hypoxia and drought stress conditions.Plant tumors, also referred to as crown galls, develop upon infection of susceptible plants with Agrobacterium tumefaciens. A DNA fragment, the transfer DNA (T-DNA) of the tumor-inducing plasmid of virulent A. tumefaciens strains, is randomly integrated into the genome of a host plant (Thomashow et al., 1980; Kim et al., 2007; Pitzschke and Hirt 2010). Expression of the T-DNA-encoded oncogenes drives increased production of auxin and cytokinin, thereby promoting cell proliferation. Plant tumor growth causes disruption of the epidermal cell layer that is covered by a cuticle and thus is permanently endangered to lose water (Schurr et al., 1996). In order to prevent desiccation and wilting, rescue processes appear to be constitutively activated (Veselov et al., 2003). Thereby, ethylene and abscisic acid trigger the expression of drought stress-responsive genes, the accumulation of osmoprotectants, and suberization of the surface cell layers (Efetova et al., 2007). In addition, cell membrane lipids are the major targets of environmental stresses, and tolerance to drought stress is dependent on high levels of polyunsaturated fatty acids (PUFAs) and the ability to maintain fatty acid (FA) desaturation activity (Berberich et al., 1998; Mikami and Murata, 2003; Torres-Franklin et al., 2009). In Arabidopsis (Arabidopsis thaliana) crown gall tumors, 27% of the genes involved in lipid metabolism are differentially regulated (Deeken et al., 2006). Under drought stress, Arabidopsis increases the ratio of digalactosyl diglyceride (DGDG) to monogalactosyl diglyceride (MGDG) and FA unsaturation (Gigon et al., 2004). An increase in α-linolenic acid levels (18:3, where x:y denotes an FA with x carbons and y double bonds) by overexpression of the ω3 fatty acid desaturases FAD3 and FAD7 has been shown to enhance tolerance to drought stress in Nicotiana tabacum cells (Zhang et al., 2005), whereas nontolerant plants decline their fraction of 18:3 (Monteiro de Paula et al., 1993; Dakhma et al., 1995; Upchurch, 2008).Developing crown gall tumors also face permanent oxidative stress. Reactive oxygen species (ROS) are produced in tumors, and glutathione S-transferases and peroxidases are strongly up-regulated (Jia et al., 1996; Lee et al., 2009). Due to a reduced respiratory and photosynthetic capacity in crown gall tumors, ATP production is predominantly derived from glycolysis and alcoholic fermentation (Deeken et al., 2006). In other words, the hypoxia physiology of the Arabidopsis tumor is governed by heterotrophic metabolism (Deeken et al., 2006). Since dioxygen is a cofactor of desaturases, its depletion limits the de novo synthesis of unsaturated FAs and thus membrane lipids (Brown and Beevers, 1987). In addition, hypoxia appears to be associated with ROS production, peroxidation of PUFAs, and finally, loss of membrane integrity (Blokhina et al., 2003; Upchurch, 2008).The biosynthesis of PUFAs is initiated by introduction of the first double bond into stearic acid (18:0) by STEAROYL-ACYL CARRIER PROTEIN Δ⁹-DESATURASE (SAD). SAD genes exhibit a tissue-specific expression profile, and the encoding enzymes regulate the pools of oleic acid (18:1), a monounsaturated fatty acid (MUFA; Shanklin and Somerville 1991; Thompson et al., 1991; Cahoon et al., 1996, 1998; Whittle et al., 2005). In Arabidopsis, five out of seven members of the SAD gene family (SAD1, SAD3, SAD4, SAD5, and SUPPRESSOR OF SALICYLIC ACID INSENSITIVE2 [SSI2]) are capable of desaturating 18:0 and contribute to the 18:1 pool (Kachroo et al., 2007). A mutation in the Arabidopsis ssi2 locus results in the accumulation of 18:0, whereas the 18:1 content is reduced. Furthermore, the salicylic acid-mediated defense signaling pathway is constitutively active, resulting in lesion formation and increased expression of the PATHOGENESIS-RELATED (PR) genes. The 18:1 MUFAs are incorporated into membrane lipids by two glycerolipid biosynthesis pathways. Phospholipids and galactolipids of photosynthetic membranes in plastids are synthesized by the prokaryotic pathway, while lipids of extraplastidic membranes are produced in the endoplasmic reticulum (ER) by the eukaryotic pathway (Ohlrogge and Browse, 1995). MUFAs are further desaturated to PUFAs by two sets of membrane-bound FADs. These enzymes are either located in plastids or the ER (Ohlrogge and Browse, 1995). In the ER, conversion of the unsaturated phospholipid FAs 18:1 to 18:2 and of 18:2 to 18:3 is carried out by the ω6 desaturase FAD2 and the ω3 desaturase FAD3, respectively (Browse et al., 1993; Okuley et al., 1994; Los and Murata, 1998).This study focuses on the role of desaturases in Arabidopsis crown galls in the context of drought and hypoxia stress. We document that crown galls produce increased levels of α-linolenic acid and strongly express the two FAD genes FAD3 and SAD6. In contrast to the well-known ω3 desaturase FAD3, the function of SAD6, a putative SAD, is unknown, and mutants for this gene are not available. However, the ability of SAD6 to replace the well-characterized SSI2 functionally in the ssi2-2 mutant suggests that SAD6 is a functional SAD. Overexpression of SAD6 decreased stearic acid and increased oleic acid levels. Down-regulation of SAD6 overexpression by RNA interference (RNAi) restored wild-type-like FA levels. The ability of SAD6 to influence the oleic acid levels together with the finding that SAD6 gene expression is restricted to hypoxia suggest that SAD6 catalyzes FA desaturation under hypoxic conditions. Unlike SAD6, the results obtained with the fad3-2 mutant impaired in α-linolenic acid biosynthesis indicate a role of FAD3 in increasing the drought stress tolerance of crown galls. Thus, both desaturases shape the pool of unsaturated FAs in drought stress- and oxidative stress-challenged Arabidopsis crown gall tumors.     

Distribution of Mesenchymal Stem Cells and Effects on Neuronal Survival and Axon Regeneration after Optic Nerve Crush and Cell Therapy

Bone marrow-derived cells have been used in different animal models of neurological diseases. We investigated the therapeutic potential of mesenchymal stem cells (MSC) injected into the vitreous body in a model of optic nerve injury. Adult (3–5 months old) Lister Hooded rats underwent unilateral optic nerve crush followed by injection of MSC or the vehicle into the vitreous body. Before they were injected, MSC were labeled with a fluorescent dye or with superparamagnetic iron oxide nanoparticles, which allowed us to track the cells in vivo by magnetic resonance imaging. Sixteen and 28 days after injury, the survival of retinal ganglion cells was evaluated by assessing the number of Tuj1- or Brn3a-positive cells in flat-mounted retinas, and optic nerve regeneration was investigated after anterograde labeling of the optic axons with cholera toxin B conjugated to Alexa 488. Transplanted MSC remained in the vitreous body and were found in the eye for several weeks. Cell therapy significantly increased the number of Tuj1- and Brn3a-positive cells in the retina and the number of axons distal to the crush site at 16 and 28 days after optic nerve crush, although the RGC number decreased over time. MSC therapy was associated with an increase in the FGF-2 expression in the retinal ganglion cells layer, suggesting a beneficial outcome mediated by trophic factors. Interleukin-1β expression was also increased by MSC transplantation. In summary, MSC protected RGC and stimulated axon regeneration after optic nerve crush. The long period when the transplanted cells remained in the eye may account for the effect observed. However, further studies are needed to overcome eventually undesirable consequences of MSC transplantation and to potentiate the beneficial ones in order to sustain the neuroprotective effect overtime.     

The tempered polymerization of human neuroserpin

Neuroserpin, a member of the serpin protein superfamily, is an inhibitor of proteolytic activity that is involved in pathologies such as ischemia, Alzheimer's disease, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). The latter belongs to a class of conformational diseases, known as serpinopathies, which are related to the aberrant polymerization of serpin mutants. Neuroserpin is known to polymerize, even in its wild type form, under thermal stress. Here, we study the mechanism of neuroserpin polymerization over a wide range of temperatures by different techniques. Our experiments show how the onset of polymerization is dependent on the formation of an intermediate monomeric conformer, which then associates with a native monomer to yield a dimeric species. After the formation of small polymers, the aggregation proceeds via monomer addition as well as polymer-polymer association. No further secondary mechanism takes place up to very high temperatures, thus resulting in the formation of neuroserpin linear polymeric chains. Most interesting, the overall aggregation is tuned by the co-occurrence of monomer inactivation (i.e. the formation of latent neuroserpin) and by a mechanism of fragmentation. The polymerization kinetics exhibit a unique modulation of the average mass and size of polymers, which might suggest synchronization among the different processes involved. Thus, fragmentation would control and temper the aggregation process, instead of enhancing it, as typically observed (e.g.) for amyloid fibrillation.     

A chronological framework for a long and persistent archaeological record: Melka Kunture, Ethiopia

New ⁴⁰Ar/³⁹Ar geochronological data for several volcanic ash horizons from Melka Kunture, Ethiopia, allow for significantly more precise age constraints to be placed upon the lithostratigraphy, archaeology and paleontology from this long record. Ashes from the Melka Kunture Formation at Gombore yielded the most reliable age constraints, from 1.393 ± 0.162 Ma² (millions of years ago) near the base of the section to 0.709 ± 0.013 Ma near the top. Dating the Garba section proved more problematic, but the base of the section, which contains numerous Oldowan obsidian artifacts, may be >1.719 ± 0.199 Ma, while the top is securely dated to 0.869 ± 0.020 Ma. The large ignimbrite from the Kella Formation at Kella and Melka Garba is dated to 1.262 ± 0.034 Ma and pre-dates Acheulean artifacts in the area. The Gombore II site, which has yielded two Homo skull fragments, ‘twisted bifaces,’ and a preserved butchery site, is now constrained between 0.875 ± 0.010 Ma and 0.709 ± 0.013 Ma. Additional ashes from these and other sites further constrain the timing of deposition throughout the section.Integration with previously published magnetostratigraphy has allowed for the first time a relatively complete, reliable timeline for the deposition of sediments, environmental changes, archaeology, and paleontology at Melka Kunture.     

Bone marrow-derived fibroblast growth factor-2 induces glial cell proliferation in the regenerating peripheral nervous system

ABSTRACT: BACKGROUND: Among the essential biological roles of bone marrow-derived cells, secretion of many soluble factors is included and these small molecules can act upon specific receptors present in many tissues including the nervous system. Some of the released molecules can induce proliferation of Schwann cells (SC), satellite cells and lumbar spinal cord astrocytes during early steps of regeneration in a rat model of sciatic nerve transection. These are the major glial cell types that support neuronal survival and axonal growth following peripheral nerve injury. Fibroblast growth factor-2 (FGF-2) is the main mitogenic factor for SCs and is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS). RESULTS: Here we show that bone marrow-derived cell treatment induce an increase in the expression of FGF-2 in the sciatic nerve, dorsal root ganglia and the dorsolateral (DL) region of the lumbar spinal cord (LSC) in a model of sciatic nerve transection and connection into a hollow tube. SCs in culture in the presence of bone marrow derived conditioned media (CM) resulted in increased proliferation and migration. This effect was reduced when FGF-2 was neutralized by pretreating BMMC or CM with a specific antibody. The increased expression of FGF-2 was validated by RT-PCR and immunocytochemistry in co-cultures of bone marrow derived cells with sciatic nerve explants and regenerating nerve tissue respectivelly. CONCLUSION: We conclude that FGF-2 secreted by BMMC strongly increases early glial proliferation, which can potentially improve PNS regeneration.     

Immunohistochemical localization of polypeptides in peripheral autonomic nerves using whole mount preparations

Summary A method is described for the immunohistochemical localization of peptides in whole-mount preparations. Tissue is fixed as laminae with a picric acid/formaldehyde mixture and then dehydrated, cleared and rehydrated before exposure to antibodies. This procedure ensures adequate penetration of the antibody molecules without the need to freeze and thaw the tissue or to use detergents, preserves antigenicity and lowers non-specific background staining. The laminae are incubated with the primary antisera for 16 h at room temperature and, after washing, with a second, fluorescent tagged, antiserum. This can be followed by a peroxidase-anti-peroxidase localization of the second antiserum, which acts as a bridge. The method gives a precise and reproducible localization of immunoreactive peptides, with good penetration and low background even in thick preparations. Large areas can be scanned and neuroeffector relationships studied more easily than in sections.     

Parafollicular cells of rabbit thyroid store both calcitonin and somatostatin and resemble gut D cells ultrastructurally

Summary Both calcitonin and somatostatin have been detected immunohistochemically in rabbit parafollicular cells; only calcitonin has been found in the same cells of the dog, guinea-pig and man. Large amounts of a peptide radioimmunochemically identical with synthetic somatostatin have been detected in extracts of rabbit thyroid. The ultrastructural and staining features of rabbit parafollicular cells differ from those of parafollicular cells in other species, while resembling in part those of somatostatin D cells scattered in the rabbit stomach.Supported in part by grants from the Italian Ministero della Pubblica Istruzione and Consiglio Nazionale delle Ricerche