Proprotein convertases regulate insulin-like growth factor 1-induced membrane-type 1 matrix metalloproteinase in VSMCs via endoproteolytic activation of the insulin-like growth factor-1 receptor

The IGF-1 receptor (IGF-1R) and MT1-MMP are synthesized as larger precursor proproteins, which require endoproteolytic activation by the proprotein convertases (PCs) furin/PC5 to gain full biological activity. The aim of this study was to investigate the contribution of PCs to IGF-1R and/or MT1-MMP activation in vascular smooth muscle cells (VSMCs) as well as VSMC proliferation/migration, which are key elements in vascular remodeling. Furin and PC5 mRNAs and proteins were found in VSMCs. Inhibition of furin-like PCs with the specific pharmacological inhibitor dec-CMK inhibited IGF-1R endoproteolytic activation. Inhibition of IGF-1R maturation abrogated IGF-induced IGF-1R autophosphorylation, PI3-kinase and MAPK induction, as well as VSMC proliferation (p<0.05 vs. controls), whereas it had no effect of PDGF-stimulated signaling pathways or cell growth. Both, IGF-1 and PDGF-BB, induced MT1-MMP expression, but only IGF-1-mediated MT1-MMP induction was inhibited by dec-CMK. Induction of MMP-2 by IGF-1 was inhibited by the PI3-kinase inhibitor wortmannin, but not by the MEK-inhibitor PD98059. Dec-CMK inhibited VSMC chemotaxis comparable to the effects of the MMP-inhibitor GM6001 (both p<0.05 vs. controls), supporting that MMPs are involved. In conclusion, this study demonstrates that targeting furin-like PCs and thus inhibiting IGF-1R activation is a novel target to inhibit IGF-1-mediated signaling and cell functions, such as IGF-1-induced MT1-MMP/MMP-2 in VSMCs.     

Regulation of intracellular Ca2+ by P2Y1 receptors may depend on the developmental stage of cultured rat striatal neurons

Mixed striatal cell cultures containing neurons and glial cells were grown either in neurobasal medium (NBM) or Dulbecco's modified Eagle's medium (DMEM). Whole-cell patch-clamp recordings indicated that, if at all, only a single, low amplitude spike was evoked shortly after starting the injection of a depolarizing current pulse into NBM neurons. In contrast, DMEM neurons fired series of high amplitude action potentials, without apparent spike frequency adaptation. The possible reason for the observed action potential failure in NBM neurons was a low density of Na+ channels per unit of membrane surface area. However, both in NBM and DMEM neurons, ATP did not induce inward current responses via P2X receptor-channels, although GABAA and N-methyl-D-aspartate (NMDA) receptor-channels could be activated by muscimol and NMDA, respectively. Ca2+ imaging experiments by means of the Fura-2 method were utilized to measure intracellular Ca2+ ([Ca2+]i) in neurons and glial cells. NBM, but not DMEM neurons responded to ATP with [Ca2+]i transients; glial cells grown in either culture medium were equally sensitive to ATP. ATP caused an increase of [Ca2+]i by a mechanism only partly dependent on external Ca2+; the residual ATP effect was blocked by cyclopiazonic acid (CPA) and was therefore due to the release of Ca2+ from its intracellular pools. The receptor involved was characterized by P2 receptor antagonists (PPADS, MRS 2179, AR-C69931MX) and was found to belong to the P2Y1 subtype. CPA caused an early [Ca2+]i response due to release from intracellular storage sites, followed by a late [Ca2+]i response due to the influx of this cation from the extracellular space, probably triggered by the opening of store-operated channels (SOCs) in the plasma membrane. It is concluded that in partial analogy with the effect of CPA, ATP releases [Ca2+]i via the Gq/phospholipase C/inositoltrisphosphate (IP3) pathway, thereby opening SOCs. It is hypothesized that this effect of ATP may have an important role for the proliferation and migration of striatal neuronal progenitors.     

Differences in morphology and C/N-balance between clones ofPhragmites australis within a plantation at a degraded fen

The morphology, productivity and C/N-balance of 9 different clones ofPhragmites australis planted in 1997 in a degraded fen area of 40,000 m² was investigated in order to estimate the degree of variation between the genotypes. The planted reed clones showed significant differences in morphology, standing crop and stand structure at the same site. The above-ground biomass of some reed clones was due to high culms and large leaf areas, while among others it was due to high shoot densities and small culms. The productivity of the individual clones also differed. At the end of the 1998 growing season the standing crop of the clones ranged from about 700 to 2,000 g of dry matter per m². Differences were found in the C/N-dynamics as in the standing stock of total nitrogen in the above-ground biomass (ranging from 15 to 50 g N/m²) and the relative nitrogen content of the shoots. Furthermore, seasonal changes in the amount of free amino acids and carbohydrates in the basal internodes of the different clones were compared. The patterns are discussed with respect to the nutritional status of the reed plants. In conclusion, the results suggest high genotypic variation despite the comparable site conditions and thus a strong influence of genetically determined differences in growth and resource exploitation on the characteristics of reed clones.     

Lignin properties in topsoils of a beech/oak forest after 8 years of manipulated litter fall: relevance of altered input and oxidation of lignin

Background and aims

We studied the response of lignin oxidation in soils of a beech/oak forest to changes in litter fall. Additionally we considered possible factors in lignin oxidation, including altered (i) input of fresh organic matter and (ii) fungi-to-bacteria ratios.

Methods

The field-based experiment included (i) doubling and (ii) exclusion of litter fall and (iii) controls with ambient litter fall. Soil (0–20 cm depth) was sampled after 8 years. We analyzed (i) lignin using the CuO oxidation method, (ii) stocks of free and mineral-bound organic carbon (OC), (iii) the response of soil organic matter (SOM) decomposition to addition of labile organic compounds in laboratory incubations, and (iv) ratios of fungal- vs. bacterial-derived amino sugars (F/B ratios).

Results

Litter exclusion increased stocks of free-light fraction OC, F/B ratios, the ability of the microbial community to use labile compounds for SOM decomposition, as well as acid-to-aldehyde ratios of vanillyl-type lignin phenols in A horizons. Litter addition had no such effects. We assume that litter exclusion caused enhanced transport of organic debris from lower forest floor horizons with rainwater into the A horizon. Enhanced input of organic debris might have increased (i) the availability of labile compounds and (ii) F/B ratios. Consequently, lignin oxidation increased.

Conclusions

Enhanced input of organic debris from forest floors can increase lignin oxidation in mineral topsoils of the studied forest. The expected gradual changes in litter fall due to climate change likely will cause no such effects.     

Labeling stem cells with ferumoxytol, an FDA-approved iron oxide nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes. A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo. Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM(-1) sec(-1) strength. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure. Our group has applied this agent in an "off label" use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.     

Feeding ecology and trophic impact of the hydroid Obelia dichotoma in the Kongsfjorden (Spitsbergen, Arctic)

Obelia dichotoma is a thecate hydroid with a worldwide distribution, occurring mainly on shallow water hard substrates. Since the trophic ecology of hydroids in polar waters is badly understood, the aim of the present work was to study qualitatively and quantitatively the diet of these organisms in an Arctic environment and to determine their trophic significance. For this purpose, the density of the hydroid population was documented, and simultaneously, zooplankton was sampled in two different years (1997 and 1998). Prey capture rates were estimated by analysing the gastrovascular content of the polyps in a diurnal cycle. Additionally, the digestion time of O. dichotoma was measured by laboratory feeding experiments using diatoms as food items. The analyses of the gastrovascular cavities of the polyps sampled during the diurnal cycles showed that O. dichotoma fed mainly on faecal pellets, organic matter and microalgae. Zooplankton prey was also observed, but gastrovascular contents and zooplankton abundance did not show any correlation in both years. The consumption rates of the hydroid populations differed between the 2 years. It was almost double (8.9 mg Carbon m^?2) in 1998 compared to 1997 (5.5 mg Carbon m^?2). The significance of the environmental variability in the feeding ecology and population dynamics of hydroids under Arctic conditions is discussed.     

Spatial and temporal trends in yellow stingray abundance: evidence from diver surveys

Recent concerns about changing elasmobranch populations have prompted the need to understand their patterns of distribution and abundance through non-destructive sampling methods. Since scientific divers represent a small portion of the total number of divers worldwide, the use of non-scientific divers could drastically increase the number of observations needed to monitor broad-scale, long-term trends. Here, we use 83,940 surveys collected by trained volunteer divers to examine spatial and temporal trends of the most frequently sighted elasmobranch species in the greater-Caribbean, the yellow stingray (Urobatis jamaicensis). Despite being relatively common and listed as Least Concern on the IUCN Red List, little is known about the status of this species. In total, yellow stingrays were observed on 5,658 surveys (6.7% sighting frequency) with the highest occurrence in the regions surrounding Cuba. Overall, sighting frequency declined from 20.5% in 1994 to 4.7% in 2007—a standardized decline rate of −0.11. However, these trends were not consistent in all regions. The strongest decline occurred in the Florida Keys, the most sampled region, where trends were similar among all areas, habitats and depths. In contrast, sighting frequency significantly increased in Jamaica where large fishes are severely depleted. We discuss possible explanations for these changes including habitat degradation, exploitation and changes in trophic interactions. Our results suggest large-scale changes in yellow stingray abundance that have been unnoticed by the scientific community. Thus, our study highlights the value of non-scientific divers for collecting data that can be used to understand population trends of otherwise poorly studied species.     

The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins

Insulin-like growth factors elicit many responses through activation of phosphoinositide 3-OH kinase (PI3K). The tuberous sclerosis complex (TSC1-2) suppresses cell growth by negatively regulating a protein kinase, p70S6K (S6K1), which generally requires PI3K signals for its activation. Here, we show that TSC1-2 is required for insulin signaling to PI3K. TSC1-2 maintains insulin signaling to PI3K by restraining the activity of S6K, which when activated inactivates insulin receptor substrate (IRS) function, via repression of IRS-1 gene expression and via direct phosphorylation of IRS-1. Our results argue that the low malignant potential of tumors arising from TSC1-2 dysfunction may be explained by the failure of TSC mutant cells to activate PI3K and its downstream effectors.     

Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7

A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5 degrees C was isolated. The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio. By chitinase activity screening of a genomic DNA library of Vibrio sp. strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated. Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa. The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi. The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids. Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases. The chitinase ChiA of isolate Fi:7 was overexpressed in E. coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography. Maximal enzymatic activity was observed at a temperature of 35 degrees C and pH 8. Activity of the chitinase at 5 degrees C was 40% of that observed at 35 degrees C. Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.