Protective Role of Proton-Sensing TDAG8 in Lipopolysaccharide-Induced Acute Lung Injury

Acute lung injury is characterized by the infiltration of neutrophils into lungs and the subsequent impairment of lung function. Here we explored the role of TDAG8 in lung injury induced by lipopolysaccharide (LPS) administrated intratracheally. In this model, cytokines and chemokines released from resident macrophages are shown to cause neutrophilic inflammation in the lungs. We found that LPS treatment increased TDAG8 expression in the lungs and confirmed its expression in resident macrophages in bronchoalveolar lavage (BAL) fluids. LPS administration remarkably increased neutrophil accumulation without appreciable change in the resident macrophages, which was associated with increased penetration of blood proteins into BAL fluids, interstitial accumulation of inflammatory cells, and damage of the alveolar architecture. The LPS-induced neutrophil accumulation and the associated lung damage were enhanced in TDAG8-deficient mice as compared with those in wild-type mice. LPS also increased several mRNA and protein expressions of inflammatory cytokines and chemokines in the lungs or BAL fluids. Among these inflammatory mediators, mRNA and protein expression of KC (also known as CXCL1), a chemokine of neutrophils, were significantly enhanced by TDAG8 deficiency. We conclude that TDAG8 is a negative regulator for lung neutrophilic inflammation and injury, in part, through the inhibition of chemokine production.     

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzymeat a low temperature was applied to isolate cold-adapted mutantsof a thermophilic enzyme. Four mutant enzymes with enhancedspecific activities (up to 4.1-fold at 40°C) at a moderatetemperature were isolated from randomly mutated Thermus thermophilus3-isopropylmalate dehydrogenase. Kinetic analysis revealed twotypes of cold-adapted mutants, i.e. k_cat-improved and K_m-improvedtypes. The k_cat-improved mutants showed less temperature-dependentcatalytic properties, resulting in improvement of k_cat (up to7.5-fold at 40°C) at lower temperatures with increased K_mvalues mainly for NAD. The K_m-improved enzyme showed higheraffinities toward the substrate and the coenzyme without significantchange in k_cat at the temperatures investigated (30–70°C).In k_cat-improved mutants, replacement of a residue was foundnear the binding pocket for the adenine portion of NAD. Twoof the mutants retained thermal stability indistinguishablefrom the wild-type enzyme. Extreme thermal stability of thethermophilic enzyme is not necessarily decreased to improvethe catalytic function at lower temperatures. The present strategyprovides a powerful tool for obtaining active mutant enzymesat lower temperatures. The results also indicate that it ispossible to obtain cold-adapted mutant enzymes with high thermalstability.     

Effects of Shrunken and Other Mutations on the Properties of Rice Endosperm Starch

Starch granules were isolated from rice endosperms of twenty mutants, included two kinds of shrunken (shr) mutant, and two cultivars of their normal counterparts. The shr mutants, EM-20 and Kenkei 2088, showed opposite effects on the amylose content, that is, EM-20 had 5% higher than its normal counterpart and Kenkei 2088 had 3% lower than its one. The amylopectin of EM-20 was composed of slightly shorter chains on the average than its normal one. The gelatinization temperature and heat of gelatinization were affected by the mutation of shr, EM-20. The amylose extender (ae) mutant, Kenkei 2064, had a comparable amylose content with its normal counterpart but its amylopectin had higher content of long chain than the normal one. Twelve kinds of dull (du) mutant reduced the amylose content which varied from 2% to 16%.     

The Effect of Environmental Temperature on Distribution of Unit Chains of Rice Amylopectin

The effects of environmental temperature during the early development of seeds on the structural characteristics of the endosperm starch were investigated using near‐isogenic lines of rice plants (Taichung 65, waxy), grown under temperature controlled conditions. High performance gel permeation chromatography (HPLC) demonstrated that Pseudomonas isoamylase‐debranched amylopectins of rice plants grown at lower temperature (25°C) contain increased amounts of short chains and decreased amounts of long chains as compared with amylopectins obtained from rice plants grown at higher temperature (30°C). By high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) of isoamylase‐debranched amylopectins it was detected that the amount of unit chains with degree of polymerization (DP) 6 and 11‐13, in the amylopectin of rice plants grown at lower temperature (25°C) had significantly increased and the amount of unit chains with DP 8, 22‐24 and 29 had significantly decreased, as compared with the amylopectin of rice plants grown at higher temperature (30°C). It was confirmed that the environmental temperature between 5 and 10 d after pollination strongly influenced the structure characteristics of the endosperm starch of rice plants.