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The hypothesis tested by these studies states that in addition to interendothelial cell tight junction proteins, matrix adhesion by b 1 -integrin receptors expressed by endothelial cells have an important role in maintaining the cerebral microvessel permeability barrier. Primary brain endothelial cells from C57 BL/6 mice were incubated with b 1 -integrin function-blocking antibody (Ha2/5) or isotype control and the impacts on claudin-5 expression and microvessel permeability were quantified. Both flow cytometry and immunofluorescence studies demonstrated that the interendothelial claudin-5 expression by confluent endothelial cells was significantly decreased in a time-dependent manner by Ha2/5 exposure relative to isotype. Furthermore, to assess the barrier properties, transendothelial electrical resistance and permeability measurements of the monolayer, and stereotaxic injection into the striatum of mice were performed. Ha2/5 incubation reduced the resistance of endothelial cell monolayers significantly, and significantly increased permeability to 40 and 150 kDa dextrans. Ha2/5 injection into mouse striatum produced significantly greater IgG extravasation than the isotype or the control injections. This study demonstrates that blockade of b 1 -integrin function changes interendothelial claudin-5 expression and increases microvessel permeability. Hence, endothelial cell-matrix interactions via b 1 -integrin directly affect interendothelial cell tight junction claudin-5 expression and brain microvascular permeability.

Basic fibroblast growth factor (bFGF) has been shown to stimulate wound healing. However, consistent delivery of bFGF has been problematic. We studied the stability of bFGF incorporated into a chitosan film as a delivery vehicle for providing sustained release of bFGF. The therapeutic effect of this system on wound healing in genetically diabetic mice was determined as a model for treating clinically impaired wound healing. A chitosan film was prepared by freeze-drying hydroxypropylchitosan (a water-soluble derivative of chitosan) acetate buffer solution. Growth factor was incorporated into films before drying by mixing bFGF solution with the hydroxypropylchitosan solution. bFGF activity remained stable for 21 days at 5 degrees C, and 86.2% of activity remained with storage at 25 degrees C. Full-thickness wounds were created on the backs of diabetic mice, and chitosan film or bFGF-chitosan film was applied to the wound. The wound was smaller in after 5 days in both groups, but the wound was smaller on day 20 only in the bFGF-chitosan group. Proliferation of fibroblasts and an increase in the number of capillaries were observed in both groups, but granulation tissue was more abundant in the bFGF-chitosan group. These results suggest that chitosan itself facilitates wound repair and that bFGF incorporated into chitosan film is a stabile delivery vehicle for accelerating wound healing.

Hemorrhage and edema accompany evolving brain tissue injury after ischemic stroke. In patients, these events have been associated with metalloproteinase (MMP)-9 in plasma. Both the causes and cellular sources of MMP-9 generation in this setting have not been defined. MMP-2 and MMP-9 in nonhuman primate tissue in regions of plasma leakage, and primary murine microglia and astrocytes, were assayed by immunocytochemistry, zymography, and real-time RT-PCR. Ischemia-related hemorrhage was associated with microglial activation in vivo, and with the leakage of plasma fibronectin and vitronectin into the surrounding tissue. In strict serum-depleted primary cultures, by zymography, pro-MMP-9 was generated by primary murine microglia when exposed to vitronectin and fibronectin. Protease secretion was enhanced by experimental ischemia (oxygen-glucose deprivation, OGD). Primary astrocytes, on each matrix, generated only pro-MMP-2, which decreased during OGD. Microglia—astrocyte contact enhanced pro-MMP-9 generation in a cell density-dependent manner under normoxia and OGD. Compatible with observations in a high quality model of focal cerebral ischemia, microglia, but not astrocytes, respond to vitronectin and fibronectin, found when plasma extravasates into the injured region. Astrocytes alone do not generate pro-MMP-9. These events explain the appearance of MMP-9 antigen in association with ischemia-induced cerebral hemorrhage and edema.

A molecular analysis of betaproteobacterial ammonia oxidizers and a N 2 O isotopomer analysis were conducted to study the sources of N 2 O emissions during the cow manure composting process. Much NO 2 ؊ -N and NO 3 ؊ -N and the Nitrosomonas europaea-like amoA gene were detected at the surface, especially at the top of the composting pile, suggesting that these ammonia-oxidizing bacteria (AOB) significantly contribute to the nitrification which occurs at the surface layer of compost piles. However The very sensitive greenhouse gas nitrous oxide (N 2 O) has a 296 times higher impact than CO 2 (39) and is also responsible for ozone depletion (10). Agricultural activities such as the use of nitrate fertilizers, livestock production, and manure management, including composting, are known to be important sources of N 2 O emissions (18). To devise a strategy to mitigate N 2 O emissions, it is essential to understand its sources in detail. However, the sources of N 2 O emissions during the composting process are still largely unclear.In the composting process, a part of NH 4 ϩ -N is known to be processed through nitrification-denitrification and emitted as N 2 and N 2 O. Nitrous oxide is known to be generated through both the nitrification and denitrification processes as intermediate products or by-products. Nitrous oxide emission is a very complex process because denitrifying bacteria are phylogenetically diverse (60), and nitrifiers are also known to utilize the denitrification process even under aerobic conditions (42). It is thus very difficult to estimate the relative contributions of nitrification and denitrification in actual N 2 O emissions from the environment. Until now, there has been insufficient knowledge about the relative contributions of these processes to N 2 O emissions during the animal manure composting process. Measurement of the actual contributions of N 2 O emissions from compost piles in the field is therefore critical to establishing a strategy of mitigating N 2 O emissions.Recently, a high-precision analytical technique for determining intramolecular 15 N site preference in asymmetric molecules of N 2 O was developed (47). Since N 2 O has two N atoms within the molecule (central and outer N