Production of 9-hydroperoxy-γ-linolenic acid by soybean lipoxygenase in a two-phase system

9-Hydroperoxy-γ-linolenic acid (9-GOOH) was produced selectively by soybean lipoxygenase (LG) from γ-linolenic acid (GLA) using a two-phase (borate buffer, pH 6.5/hexane) system at a low temperature (10°C) with some anionic surfactants that showed little inhibitory effect on the enzyme at pH 6.5. The system avoided the inhibitory effect of a higher substrate concentration and of hydroperoxide as well as low substrate solubility in an aqueous system. Not lipoxygenase-2 (LG2) but lipoxygenase-1 (LG1) was indicated to be responsible for the production of 9-GOOH. Among the anionic surfactants examined, acetate and sarcosinate were shown to be suitable, but phosphate was not. Ca²⁺ increased the 9-GOOH productivity. The LG1 fraction gave the maximum yield of 35% with 0.5 mM Ca²⁺, ECT-3N (anionic surfactant, acetate) at 10°C and at 4.8 mM GLA in an emulsion.     

Oral motor patterns during feeding in severely physically disabled children

Surface electrogastrography was performed in 18 patients with esophageal atresia (EA) and 10 normal controls to investigate the possible role of a congenital enteric nerve defect as a cause of gastroesophageal reflux (GER), which is common after repair of EA. The means of the dominant frequencies and ranges of the frequency distribution were compared. The dominant frequencies (0.047+/-0.007 Hz) in the EA group did not differ significantly from those of the controls (0.050+/-0.007 Hz, P >0.1), although 2 patients had bradygastria and 2 had tachygastria in the EA group. The range of the frequency distribution was significantly wider in the EA group compared with normal children (P = 0.002). The wide frequency distribution in children with EA suggests disturbed electrical activity of the stomach, which could be associated with poor electromechanical coupling and, hence abnormal gastric contraction.     

The pliability of wood and its application to molding

The feasibility of using wood as a substitute for plastics was examined. Wood is primarily processed by cutting and gluing, but these processes are often complicated and the shapes of products and productivity are limited. Productivity would improve dramatically if efficient molding methods, such as extrusion and injection molding, could be applied to the processing of wood. In this study, the effect of moisture content on the pliability of wood, anisotropy, and the effect of anisotropy on formability were examined. The pliability improved as moisture content increased. This is considered to be due to the softening of hemicellulose and lignin in the presence of water. The deformation perpendicular to the fiber orientation was dominated by the disassociation of the fibers at the interface between fibers and required relatively low pressure. Conversely, deformation in the fiber orientation required cutting of the fiber cells and higher pressure. Backward extrusion was performed to examine the influence of the fiber orientation of raw materials on formability and fiber orientation was observed to have a major influence on formability. It was possible to improve the quality of the molded product and decrease the load required by controlling the fiber orientation of the raw materials.     

Clinical profiles of subjects with subcortical leukomalacia and border-zone infarction revealed by MR

Clinical profiles of 13 children with subcortical leukomalacia and border-zone infarction revealed by MR were analysed. The causes of brain damage were neonatal asphyxia, hypoglycaemia, circulatory disturbance associated with congenital heart diseases, and other perinatal events in five, three, two and three, respectively. Severe, moderate and mild mental retardation were present in three, five and five, respectively. Of the seven children who could walk alone, six had ataxia and one spastic diplegia; the remaining six manifested truncal instability. The ataxia was deduced to have resulted from cerebral lesions.     

Possible function of human brown adipose tissue as suggested by observation on perirenal brown fats from necropsy cases of variable age groups

In 134 out of 180 perirenal fat samples (74%) derived from Japanese necropsy cases aged from 1 month to 86 years, the brown fat tissue persisted in variable amounts. Brown fat cells were classified into 6 types: Type 1 cells are fat-depleted cells filled with granular cytoplasm and are believed to be produced after oxidation of fat for heat production. Type 2 cells are small-locular cells suitable for rapid oxidation of fat droplets. Type 3 (middle-locular) and 4 (large-locular) represent fat-storage cells containing large amounts of fat. Type 5 cells are thought to be transitional forms between multilocular brown fat cells and monolocular white fat cells. Type 6 (cytoplasm-rich multilocular) cells, usually found together with Type 1 cells, are thought to be fat-depleting or -consuming cells, since in them fat droplets are reduced in number and size probably in consequence of oxidation of fat, but by contrast granular cytoplasm is increased in amount separating the individual fat droplets by thick cytoplasmic septa. The occurrence of Types 1 and/or 6 cells that has been revealed in 65 out of the total 180 samples (36%), suggests that the oxidation of fat for the thermogenesis proceeds in the brown fat tissue and that brown fat cells partially undergo fat depletion. In the present study, the thermogenesis of human brown fat tissue was suggested chiefly with regard to the occurrence of Types 1 and/or 6 cells. In the majority of perirenal fat samples from infants (1-11 months) relatively numerous Types 1 and 6 cells were frequently revealed together with Type 2 cells, suggesting rapid and active heat production in support of the view that in human infants the brown fat tissue may be thermogenetically active to maintain body temperature. In the same manner, marked ability to produce a considerable amount of heat was evidenced in brown fat tissue of children and teenagers. In younger and elderly adults the frequency of occurrence and the amount of the perirenal brown fat tissue were decreased but Types 1 and/or 6 cells could be found in 17-40% of them, infrequently together, with Type 2 cells, suggesting persistence of the thermogenic activity with occasional large heat production especially in younger adults (20-39 years). Thus, the results obtained in this study have clarified that the human brown fat tissue can respond to stimuli given to the body by oxidation of stored fat even in the latest decades of life. In cases of death from burning, drowning, bleeding, drug poisoning etc., numerous Types 1 and/or 6 cells were found, suggesting that an active fat oxidation would take place in brown fat tissue assumedly as the result of the raised noradrenalin level in this tissue. The so-called small cytoplasmic cells found in perirenal fats from cases of death from liver cirrhosis and other causes were assumed to be atrophic fat-depleted brown fat cells.     

Studies on the production of lipids in fungi XIII. Changes of amount and composition of lipids in fungi in species of the genusPellicularia from cellulose by cultural conditions

The influence of growth temperature, carbon to nitrogen (C/N) ratio of the medium and the nitrogen source on the cell and lipid formation from cellulose by the speciesfilamentosa andpraticola of the genusPellicularia were investigated. The strains of thePellicularia genus fungi can be grown well utilizing powdery cellulose and sugar cane bagasse as the carbon source. The amount of lipids accumulated in the mycelium varied considerably depending on the difference in the cell growth associated with the cultivation condition, and the difference in the strain, C/N ratio and nitrogen source. The maximum accumulation of lipids in the mycelium (256 mg/400 ml of the medium) from cellulose was observed at 20 C with a C/N ratio of 5.7 using potassium nitrate as the nitrogen source forPellicularia filamentosa var.solani IFO 5879. Protein formation in the liquid medium is at its peak when the cell growth is at its maximum. The fatty acid compositions of the neutral and polar lipid fractions also were determined. Linoleic acid is the major fatty acid component of both fractions. The change in the total lipid content is less than 10% under any cuitivation condition.     

An evaluation tool for FKBP12-dependent and -independent mTOR inhibitors using a combination of FKBP-mTOR fusion protein, DSC and NMR

Mammalian target of rapamycin (mTOR), a large multidomain protein kinase, regulates cell growth and metabolism in response to environmental signals. The FKBP rapamycin-binding (FRB) domain of mTOR is a validated therapeutic target for the development of immunosuppressant and anticancer drugs but is labile and insoluble. Here we designed a fusion protein between FKBP12 and the FRB domain of mTOR. The fusion protein was successfully expressed in Escherichia coli as a soluble form, and was purified by a simple two-step chromatographic procedure. The fusion protein exhibited increased solubility and stability compared with the isolated FRB domain, and facilitated the analysis of rapamycin and FK506 binding using differential scanning calorimetry (DSC) and solution nuclear magnetic resonance (NMR). DSC enabled the rapid observation of protein-drug interactions at the domain level, while NMR gave insights into the protein-drug interactions at the residue level. The use of the FKBP12-FRB fusion protein combined with DSC and NMR provides a useful tool for the efficient screening of FKBP12-dependent as well as -independent inhibitors of the mTOR FRB domain.     

Modulation of fatty acid incorporation and desaturation by trifluoperazine in fungi

The effects of trifluoperazine (TFP) on [1-¹⁴C]fatty acid incorporation into the lipids ofMortierella ramanniana var.angulispora were studied. TFP decreased [1-¹⁴C]-fatty acid incorporation into phosphatidylcholine, phosphatidylethanolamine and triacylglycerol, but greatly increased¹⁴C-labeling in phosphatidic acid. These changes in [1-¹⁴C]fatty acid incorporation induced by TFP were accompanied by a decrease in desaturation of some [1-¹⁴C]fatty acids taken up by the fungal cells. When [1-¹⁴C]lioleic acid (LA) was incubated with the fungal cells, total γ-linolenic acid (GLA) formation from incorporated [1-¹⁴C]LA decreased, but the¹⁴C-labeled GLA conent in individual lipid classes was essentially unchanged. This suggests that the site of the TFP effect on GLA formation from [1-¹⁴C]LA taken up from the medium is not the desaturase acting on LA linked to complex lipids. On the other hand, GLA formation from [1-¹⁴C]oleic acid was much less susceptile to TFP, which suggests that in this fungus Δ6 desaturation to GLA has at least two different pathways with different degrees of susceptibility to TFP.     

Incorporation of linoleic acid and its conversion to λ-linolenic acid in fungi

The incorporation of [1-¹⁴C]linoleic acid (LA) into lipids ofMortierella ramanniana var.angulispora was studied to determine which lipid classes participated in the δ6-desaturation of [1-¹⁴C]LA. [1-¹⁴C]LA was rapidly taken up into fungal cells and esterified into various lipids. Comparison of the profile of [1-¹⁴C]LA incorporation between fungal cells at the exponential growth phase and the stationary growth phase showed that [1-¹⁴C]LA incorporation into most lipids—except for triacylglycerol (TG) and phosphatidylcholine (PC)—were greatly reduced at the stationary growth phase. Desaturation of [1-¹⁴C]LA into λ-linolenic acid (GLA) readily occurred at the exponential growth phase, but was greatly decreased at the stationary growth phase. Moreover, pulse-chase experiments revealed that the radiolabel incorporated into phosphatidylserine (PS) and PC rapidly turned over, while that in TG and diacylglycerol (DG) accumulated after the 4 hr chase. In addition to the change of the radiolabel in individual lipids, the content of radiolabeled GLA converted from [1-¹⁴C]LA varied with individual lipids. In phospholipids such as PC, phosphatidylethanolamine (PE) and PS, radiolabeled GLA rapidly increased after 1 hr and then decreased after 4 hr. On the other hand, a gradual increase in radiolabeled GLA until 4 hr was observed in TG. These results suggest that LA, which has been esterified into phospholipids such as PC, PE and PS, is readily desaturated to GLA, which is then transferred to TG. These differences in the fate of GLA derived from LA between phospholipids and neutral lipids may be reflected in the GLA content in the individual lipids.