Separation of multiple forms of acidic glutathione S-transferase isozymes in a susceptible and a resistant strain of house fly,Musca domestica (L.)

The acidic glutathione S-transferases from a CSMA (susceptible) strain and a Cornell-R (resistant) strain of houseflies were purified and separated utilizing affinity chromatography followed by chromatofocusing. Nine fractions were isolated from each house fly strain. Fraction 1 had the highest 1-chloro-2,4-dinitrobenzene vs. 1,2-dichloro-4-nitrobenzene ratio (CDNB/DCNB ratio) in both strains and the ratio of all the other fractions tended to decrease as the isoelectrical points decreased except for fractions 4 and 9. Most fractions from the CSMA strain had higher CDNB conjugation activities than the fractions from the Cornell-R strain, but all the fractions from the CSMA strain had lower DCNB conjugation activities than fractions from the Cornell-R strain. Steady-state kinetics of all the fractions were examined. The Km values obtained from both strains ranged from 0.36 to 1.12 mM, while the Vmax value ranged from 3.0 to 32.6 μmol/min/mg. In the 100,000 g supernatant, the CDNB specific activities in the CSMA strain was about 1/3 of the activity in the Cornell-R strain but it was about 1.5-fold following affinity chromatography. The specific activity for DCNB measured in the CSMA strain was only 1/5 of the activities of the Cornell-R strain in the 100,000 g supernatant, but was about the same after affinity chromatography. The difference was due to the selectivity of the affinity column used in the current study. © 1995 Wiley-Liss, Inc.     

Role of lamellar inclusions in surfactant production: studies on phospholipid composition and biosynthesis in rat and rabbit lung subcellular fractions.

Lamellar inclusion bodies in the type II alveolar epithelial cell are believed to be involved in pulmonary surfactant production. However, it is not clear whether their role is that of synthesis, storage, or secretion. We have examined the phospholipid composition and fatty acid content of rabbit lung wash, lamellar bodies, mitochondria, and microsomes. Phosphatidylcholine and phosphatidylglycerol, the surface-active components of pulmonary surfactant, accounted for over 80% of the total phospholipid in lung wash and lamellar bodies but for only about 50% in mitochondria and microsomes. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin accounted for over 40% of the total in mitochondria and microsomes but for only 6% in lung wash and 15% in lamellar bodies. The fatty acid composition of lamellar body phosphatidylcholine was similar to that of lung wash, but different from that of mitochondria and microsomes, in containing palmitic acid as a major component with little stearic acid and few fatty acids of chain length greater than 18 carbon atoms. The biosynthesis of phosphatidylcholine and phosphatidylglycerol was examined in the mitochondrial, microsomal, and lamellar body fractions from rat lung. Cholinephosphotransferase was largely microsomal. The activity in the lamellar body fraction could be attributed to microsomal contamination. The activity of glycerolphosphate phosphatidyltransferase, however, was high in the lamellar body fraction, although it was highest in the mitochondria and was also active in the microsomes. These data suggest that the lamellar bodies are involved both in the storage of the lipid components of surfactant and in the synthesis of at least one of those components, phosphatidylglycerol.     

Unfolding of CBP21, a lytic polysaccharide monooxygenase,without dissociation of its copper ion cofactor

Chitin-binding protein 21 (CBP21) from Serratia marcescens is a lytic polysaccharide monooxygenase that contains a copper ion as a cofactor. We aimed to elucidate the unfolding mechanism of CBP21 and the effects of Cu²⁺ on its structural stability at pH 5.0. Thermal unfolding of both apo- and holoCBP21 was reversible. ApoCBP21 unfolded in a simple two-state transition manner. The peak temperature of the DSC curve, t_p, for holoCBP21 (74.4°C) was about nine degrees higher than that for apoCBP21 (65.6°C). The value of t_p in the presence of excess Cu²⁺ was around 75°C, indicating that Cu²⁺ does not dissociate from the protein molecule during unfolding. The unfolding mechanism of holoCBP21 was considered to be as follows: N∙Cu²⁺ ⇌ U∙Cu²⁺, where N and U represent the native and unfolded states, respectively. Urea-induced equilibrium unfolding analysis showed that holoCBP21 was stabilized by 35 kJ mol⁻¹ in terms of the Gibbs energy change for unfolding (pH 5.0, 25°C), compared with apoCBP21. The increased stability of holoCBP21 was considered to result from the structural stabilization of the protein-Cu²⁺ complex itself.     

Kallikrein-related Peptidase 5 Functions in Proteolytic Processing of Profilaggrin in Cultured Human Keratinocytes

Filaggrin protein is synthesized in the stratum granulosum of the skin and contributes to the formation of the human skin barrier. Profilaggrin is cleaved by proteolytic enzymes and converted to functional filaggrin, but its processing mechanism remains not fully elucidated. Kallikrein-related peptidase 5 (KLK5) is a major serine protease found in the skin, which is secreted from lamellar granules following its expression in the stratum granulosum and activated in the extracellular space of the stratum corneum. Here, we searched for profilaggrin-processing protease(s) by partial purification of epidermal extracts and found KLK5 as a possible candidate. We used high performance liquid chromatography coupled with electrospray tandem mass spectrometry to show that KLK5 cleaves profilaggrin. Furthermore, based on a proximity ligation assay, immunohistochemistry, and immunoelectron microscopy analysis, we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis. KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage.     

A paradoxical method for NAD(+)/NADH accumulation on an electrode surface using a hydrophobic ionic liquid

In this communication, we describe a novel and facile method for the immobilization of NAD(+)/NADH on an electrode surface using a hydrophobic ionic liquid, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([C4mim][Tf(2)N]). By taking advantage of the insolubility of NAD(+)/NADH in hydrophobic ionic liquids, it is expected that NAD(+)/NADH can be retained on the electrode's surface. Alcohol dehydrogenase (ADH) and NAD(+)/NADH were immobilized with a gelatin hydrogel on an electrode that was modified with an electropolymerized ruthenium complex containing 5-amino-1,10-phenanthroline (pAPRu) as a mediator for NADH oxidation. The (ADH, NAD(+))/pAPRu-immobilized electrode exhibited the electrocatalytic oxidation of ethanol in [C4mim][Tf(2)N]. The obtained catalytic current in [C4mim][Tf(2)N] was comparable to that in buffer solution containing NAD(+). It was confirmed by UV-vis spectroscopy that NAD(+) did not dissolve in the [C4mim][Tf(2)N] and was retained on the electrode's surface. Furthermore, we succeeded in constructing an ethanol/O(2) biofuel cell comprised of an (ADH, NAD(+))/pAPRu anode and a bilirubin oxidase cathode using [C4mim][Tf(2)N] as an electrolyte.     

Yellow Pigments of Aspergillus niger and Asp. awamori

From mycelia of Asp. niger and Asp. awamori aurasperones A, B and C along with related two yellow pigments have been isolated.

Aurasperone A, C₃₂H₂₆O₁₀, is obtained in yellow prisms; m.p. 207°C; [α]_d —136°; gives the diacetate and the dimethyl ether and is assumed to be a dimeric 2-methyl-5- hydroxy-6,8-dimethoxy-4H-naphtho [2,3-b] pyran-4-one (IV). Aurasperone B, [α]_D +46.3°, is the main yellow metabolite, m.p. 186°C, and affords aurasperone A on hydrochloric acid-treatment. It has molecular formula C₃₂H₃₀O₁₂ and is supposed to have the structure (V). The other yellow pigments have been found to be also congeners of aurasperone A.     

Studies on the Hypertrophic Disease of Cherry (Genus Prunus), So-called “Witch’s Broom” Caused by Taphrina wiesneri

Metabolites of Taphrina wiesneri (Rath.) Mix. were examined. Brassicasterol, stearic acid, and p-hydroxyphenylacetic acid were isolated in crystalline form. p-Hydroxybenzoic acid and vanillic acid were identified by paper chromatography and UV measurement. Palmitic acid was identified by gas-chromatography. The fungus produced usually these compounds on any one of four kinds of medium used. p-Hydroxyphenylacetic acid promoted germination of rape seeds at the concentration of 20 ppm in water and showed inhibition at 250 ppm.

Phenolic acids and their related compounds in Japanese flowering cherry leaves infected by Taphrina wiesneri were examined. In the acidic and neutral extracts of infected cherry leaves (I), eighteen compounds positive to diazotized sulfanilic acid and two fluorescent compounds were detected by paper chromatography. Of these compounds, coumarin, 3, 4-dihydrocoumarin, melilotic acid, o- and p-coumaric acids, p-hydroxybenzoic melilotic acid, ferulic acid and caffeic acid were identified. Melilotic acid and coumarin were obtained in crystalline form. The amount of melilotic acid in I was higher than that in healthy leaves independent of sample source, although increased with the growth of cherry leaves.