Bone marrow stromal cells stimulate an angiogenic program that requires endothelial MT1‐MMP

Bone marrow‐derived stromal/stem cells (BMSCs) have recently been characterized as mediators of tissue regeneration after injury. In addition to preventing fibrosis at the wound site, BMSCs elicit an angiogenic response within the fibrin matrix. The mechanistic interactions between BMSCs and invading endothelial cells (ECs) during this process are not fully understood. Using a three‐dimensional, fibrin‐based angiogenesis model, we sought to investigate the proteolytic mechanisms by which BMSCs promote vessel morphogenesis. We find that BMSC‐mediated vessel formation depends on the proteolytic ability of membrane type 1‐matrix metalloproteinase (MT1‐MMP). Knockdown of the protease results in a small network of vessels with enlarged lumens. Contrastingly, vessel morphogenesis is unaffected by the knockdown of MMP‐2 and MMP‐9. Furthermore, we find that BMSC‐mediated vessel morphogenesis in vivo follows mechanisms similar to what we observe in vitro. Subcutaneous, cellular fibrin implants in C.B‐17/SCID mice form aberrant vasculature when MMPs are inhibited with a broad‐spectrum chemical inhibitor, and a very minimal amount of vessels when MT1‐MMP proteolytic activity is interrupted in ECs. Other studies have debated the necessity of MT1‐MMP in the context of vessel invasion in fibrin, but this study clearly demonstrates its requirement in BMSC‐mediated angiogenesis. J. Cell. Physiol. 227: 3546–3555, 2012. © 2012 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92409/1/24056_ftp.pd

Two-Phase Anaerobic Digestion of Corn Steep Liquor in Pilot Scale Biogas Plant with Automatic Control System with Simultaneous Hydrogen and Methane Production

Experimental studies of two-phase anaerobic digestion of corn steep liquor in semi-continuous automatic and semi-automatic modes of operation of a cascade of two anaerobic bioreactors with monitoring and control systems were performed. Corn steep liquor—a waste product from the process of treating corn grain for starch extraction—was used as a substrate in the process of anaerobic digestion with simultaneous hydrogen and methane production. The daily yields of biohydrogen in bioreactor 1 of the cascade (with a working volume of 8 dm3) are variable. In good operation, they are in the range of 0.7 to 1.0 L of biogas from a 1 dm3 working volume of the bioreactor, and the optimal pH is in the range of 5.0–5.5. The concentration of hydrogen in the biogas from the hydrogen bioreactor 1 is in the range of 14–34.7%. The daily yields of biomethane in bioreactor 2 of the cascade (with a working volume of 80 dm3) vary in the range 0.4 to 0.85 L of biogas from a 1 dm3 working volume of the bioreactor, and the concentration of methane in the biogas from bioreactor 2 is high and remains practically constant (in the range 65–69%). At a dilution rate of 0.4 day−1 and an organic loading rate of 20 gL for bioreactor 1, respectively, and a dilution rate of 0.05 day−1 for bioreactor 2, the best results were obtained. The computer control system is presented. Some energetical considerations were discussed

Safe Sialidase Production by the Saprophyte <i>Oerskovia paurometabola</i>: Gene Sequence and Enzyme Purification

Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed β-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application

Temperature downshift induces antioxidantresponse in fungi isolated from Antarctica

Although investigators have been studying the cold-shock response in a variety of organisms for the last two decades or more, comparatively little is known about the difference between antioxidant cell response to cold stress in Antarctic and temperate microorganisms. The change of environmental temperature, which is one of the most common stresses, could be crucial for their use in the biotechnological industry and in ecological research. We compared the effect of short-term temperature downshift on antioxidant cell response in Antarctic and temperate fungi belonging to the genus Penicillium. Our study showed that downshift from an optimal temperature to 15° or 6°C led to a cell response typical of oxidative stress: significant reduction of biomass production; increase in the levels of oxidative damaged proteins and accumulation of storage carbohydrates (glycogen and trehalose) in comparison to growth at optimal temperature. Cell response against cold stress includes also increase in the activities of SOD and CAT, which are key enzymes for directly scavenging reactive oxygen species. This response is more species-dependent than dependent on the degree of cold-shock. Antarctic psychrotolerant strain Penicillium olsonii p14 that is adapted to life in extremely cold conditions demonstrated enhanced tolerance to temperature downshift in comparison with both mesophilic strains (Antarctic Penicillium waksmanii m12 and temperate Penicillium sp. t35)

Structural and functional characterization of cold-active sialidase isolated from Antarctic fungus Penicillium griseofulvum P29

The fungal strain, Penicillium griseofulvum P29, isolated from a soil sample taken from Terra Nova Bay, Antarctica, was found to be a good producer of sialidase (P29). The present study was focused on the purification and structural characterization of the enzyme. P29 enzyme was purified using a Q-Sepharose column and fast performance liquid chromatography separation on a Mono Q column. The determined molecular mass of the purified enzyme of 40 kDa by SDS-PAGE and 39924.40 Da by matrix desorption/ionization mass spectrometry (MALDI-TOF/MS) analysis correlated well with the calculated mass (39903.75 kDa) from the amino acid sequence of the enzyme. P29 sialidase shows a temperature optimum of 37 °C and low-temperature stability, confirming its cold-active nature. The enzyme is more active towards α(2 → 3) sialyl linkages than those containing α(2 → 6) linkages.Based on the determined amino acid sequence and 3D structural modeling, a 3D model of P29 sialidase was presented, and the properties of the enzyme were explained. The conformational stability of the enzyme was followed by fluorescence spectroscopy, and the new enzyme was found to be conformationally stable in the neutral pH range of pH 6 to pH 9. In addition, the enzyme was more stable in an alkaline environment than in an acidic environment. The purified cold-active enzyme is the only sialidase produced and characterized from Antarctic fungi to date