Immunofluorescent detection of the activation of the small GTPase Rac1 in mouse skeletal muscle fibers

The small GTPase Rac1 acts as a molecular switch of intracellular signaling in mammals. For understanding the regulatory mechanism, it is important to identify subcellular locations in which Rac1 is activated following multiple extracellular stimuli. However, it is difficult to detect Rac1 activation in situ in animal tissues, and thus a novel method is highly desirable. Here, we report a simple method to visualize the activation of endogenous Rac1 in mouse skeletal muscle fibers. In this assay, specific interaction between activated Rac1 and a binding polypeptide is detected by immunofluorescent microscopy. This approach is readily applicable to other small GTPases.     

Free Cyanide Sorption on Freshwater Sediment and Model Components

The sorption of free cyanide (HCN) on mineral components of sediment, activated carbon, and a freshwater sediment was studied via batch experiments in synthetic freshwater at pH 6.4–7.6. It was found that free cyanide did not sorb to any significant extent on sediment mineral components, but did sorb strongly to activated carbon and moderately to a freshwater sediment. Results of experiments with 100 and 150 μ g/L free cyanide spike amounts resulted in no observed sorption to kaolin clay, Ottawa sand, or alumina in the synthetic freshwater. Extensive removal of free cyanide from the aqueous phase was observed in the partitioning experiments with the powdered activated carbon. Results with whole sediment indicated up to 46% removal of free cyanide from the aqueous phase in experiments with 20 and 50 μ g/L free cyanide. The organic-carbon-normalized distribution coefficient K_oc (= C_s/C_wf_oc) for free cyanide sorption on the activated carbon (f_oc = 1.0), 4.2 L/g_s, was similar to the K_oc value for free cyanide sorption on the freshwater sediment (f_oc = 0.0031), approximately 12.9 L/g_s. The results indicate that free cyanide can sorb to sediments with organic carbon content under freshwater conditions, primarily through interaction of HCN with organic carbon in the sediment.     

Implication of anti-inflammatory macrophages in regenerative moto-neuritogenesis: Promotion of myoblast migration and neural chemorepellent semaphorin 3A expression in injured muscle

Regenerative mechanisms that regulate intramuscular motor innervation are thought to reside in the spatiotemporal expression of axon-guidance molecules. Our previous studies proposed a heretofore unexplored role of resident myogenic stem cell (satellite cell)-derived myoblasts as a key presenter of a secreted neural chemorepellent semaphorin 3A (Sema3A); hepatocyte growth factor (HGF) triggered its expression exclusively at the early-differentiation phase. In order to verify this concept, the present study was designed to clarify a paracrine source of HGF release. In vitro experiments demonstrated that activated anti-inflammatory macrophages (CD206-positive M2) produce HGF and thereby promote myoblast chemoattraction and Sema3A expression. Media from pro-inflammatory macrophage cultures (M1) did not show any significant effect. M2 also enhanced the expression of myoblast-differentiation markers in culture, and infiltrated predominantly at the early-differentiation phase (3–5 days post-injury); M2 were confirmed to produce HGF as monitored by in vivo/ex vivo immunocytochemistry of CD11b/CD206/HGF-positive cells and by HGF in situ hybridization of cardiotoxin- or crush-injured tibialis anterior muscle, respectively. These studies advance our understanding of the stage-specific activation of Sema3A expression signaling. Findings, therefore, encourage the idea that M2 contribute to spatiotemporal up-regulation of extracellular Sema3A concentrations by producing HGF that, in turn, stimulates a burst of Sema3A secretion by myoblasts that are recruited to site of injury. This model may ensure a coordinated delay in re-attachment of motoneuron terminals onto damaged fibers early in muscle regeneration, and thus synchronize the recovery of muscle-fiber integrity and the early resolution of inflammation after injury.     

EST-library construction using spore RNA of the arbuscular mycorrhizal fungus Gigaspora rosea

RNA was extracted from activated spores of the arbuscular mycorrhizal fungus Gigaspora rosea. Double-stranded cDNA was synthesised, digested and cloned into the vector lambda-ZAP express. Of the 1,500 clones obtained, 1.5% carried inserts of the rRNA gene cluster. After excision, inserts from 50 randomly selected clones were sequenced. Database searches revealed that 62% of the clones had similarities to already known sequences. These mainly code for proteins involved in translation and protein processing, replication and the cell cycle and cell signal transduction. One fragment probably belonged to a metallothionein-encoding gene which may be involved in heavy-metal binding. The method presented is an easy and rapid way to obtain short fragments of coding regions for expressed sequence tag libraries. Accepted: 28 November 2000     

How adaptation and mass transfer control the biodegradation of linear alkylbenzene sulfonate by activated sludge

We use a nonsteady-state model to evaluate the effects of community adaptation and sorption kinetics on the fate of linear alkylbenzene sulfonate (LAS) in batch experiments conducted with activated sludge that was continuously fed different concentrations of LAS. We observed a sharp decrease in the biodegradation rate between 30 and 60 minutes and the presence of an LAS residual at the end of the batch experiments. The modeling analysis indicates that these phenomena were caused by relatively slow inter-phase mass transport of LAS. The modeling analyses also showed that the amount of LAS-degrading biomass increased when the continuous activated sludge was fed a higher LAS concentration. Although community adaptation to LAS involved accumulation of more LAS degraders, the increase was not proportional to the feed concentration of LAS, which supports the concept that LAS degraders also utilized portions of the general biochemical oxygen demand (BOD) fed to the continuous activated sludge systems.     

Operation of partial nitrification to nitrite of landfill leachate and its performance with respect to different oxygen conditions

The coupled system of partial nitrification and anaerobic ammonium oxidation (Anammox) is efficient in nitrogen removal from wastewater. In this study, the effect of different oxygen concentrations on partial nitrification performance with a sequencing batch reactor (SBR) was investigated. Results indicate that, partial nitrification of landfill leachate could be successfully achieved under the 1.0–2.0 mg L⁻¹ dissolved oxygen (DO) condition after 118 d long-term operation, and that the effluent is suitable for an Anammox reactor. Further decreasing or increasing the DO concentration, however, would lead to a decay of nitrification performance. Additionally, the MLSS concentration in the reactor increased with increasing DO concentration. Respirometric assays suggest that low DO conditions (<2 mg L⁻¹) favor the ammonia-oxidizing bacteria (AOB) and significantly inhibit nitrite oxidizing bacteria (NOB) and aerobic heterotrophic bacteria (AHB); whereas high DO conditions (>3 mg L⁻¹) allow AHB to dominate and significantly inhibit AOB. Therefore, the optimal condition for partial nitrification of landfill leachate is 1.0–2.0 mg L⁻¹ DO concentration.