Mitochondrial DNA analysis of Exophiala jeanselmei var. lecanii-corni and Exophiala castellanii

A Japanese clinical isolate (KU-A-0094) which was identified by de Hoog et al. as Exophiala jeanselmei var. lecanii-corni with difficulty, was compared with 5 strains including the type cultures of E. jeanselmei var. lecanii-corni, var. jeanselmei and E. castellanii using RFLP (restriction fragment length polymorphism) patterns of mtDNA (mitochondrial DNA). RFLP patterns of KUA-0094 were identical with those of E. jeanselmei var. lecanii-corni and different from those of E. castellanii with restriction enzymes of HaeIII, MspI and hindIII. Therefore, de Hoog et al.'s identification of KU-A-0094 was confirmed. Additionally, mtDNA-RFLP patterns of E. jeanselmei var. lecanii-corni and E. jeanselmei var. jeanselmei were also different from each other. Consequently E. jeanselmei var. lecanii-corni seem to be a species in its own right rather than a variant of E. jeanselmei. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enzyme system catalyzing formation of cis-3-hexenal and n-hexanal from linolenic and linoleic acids in Japanese silver (Farfugium iaponicum Kitamura) leaves

Leaf alcohol (cis-3-hexenol) and leaf aldehyde (trans-2-hexenal)are responsible for the green odor in leaves and fruits. cis-3-Hexenal,a precursor of cis-3-hexenol and trans-2-hexenal, was producedfrom linolenic acid by a homogenate of Farfugium japonicum (Japanesesilver) leaves. n-Hexanal was produced from linoleic acid bya homogenate of the leaves. The enzyme system catalyzing formationof C₆-aldehydes from linolenic and linoleic acids was localizedin chloroplast lamellae, and required oxygen for reaction. C₁₈-unsaturatedfatty acids such as linolenic acid, linoleic acid and -linolenicacid, which have carboxyl groups and cis-1, cis-4-pentadienesystems including a double bond at C-12, acted as substrates,and C₆-aldehydes (cis-3-hexenal or n-hexanal), but not C₉-aldehydes,were produced from them. The properties of the enzyme systemin chloroplasts were as follows: optimal pH 7.0; stable at pH5 to 7; thermolabile and no activity at 50?C. These propertieswere very similar to those of tea chloroplasts. The enzyme systemcould be solubilized from chloroplasts by 2% Triton X-100, butwas very unstable in solubilized form. (Received July 9, 1976; )     

The tyrosine phosphatase CD148 interacts with the p85 regulatory subunit of phosphoinositide 3-kinase

CD148 is a transmembrane tyrosine phosphatase that has been implicated in the regulation of cell growth and transformation. However, the signalling mechanisms of CD148 are incompletely understood. To identify the specific intracellular molecules involved in CD148 signalling, we carried out a modified yeast two-hybrid screening assay. Using the substrate-trapping mutant form of CD148 (CD148 D/A) as bait, we recovered the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase). CD148 D/A, but not catalytically active CD148, interacted with p85 in a phosphorylation-dependent manner in vitro and in intact cells. Growth factor receptor and PI3K activity were also trapped by CD148 D/A via p85 from pervanadate-treated cell lysates. CD148 prominently and specifically dephosphorylated p85 in vitro. Co-expression of CD148 reduced p85 phosphorylation induced by active Src, and attenuated the increases in PI3K activity, yet CD148 did not alter the basal PI3K activity. Finally, CD148 knock-down by siRNA (short interfering RNA) increased PI3K activity on serum stimulation. Taken together, these results demonstrate that CD148 may interact with and dephosphorylate p85 when it is phosphorylated and modulate the magnitude of PI3K activity.     

Rubiscolytics: fate of Rubisco after its enzymatic function in a cell is terminated

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the predominant protein in photosynthesizing plant parts and the most abundant protein on earth. Amino acids deriving from its net degradation during senescence are transported to sinks (e.g. developing leaves, fruits). Rubisco catabolism is not controlled only by the overall sink demand. An accumulation of carbohydrates may also accelerate senescence and Rubisco degradation under certain conditions. Amino acids produced by proteolysis are rapidly redistributed in plants with proper source-sink relationships. In leaves of wheat plants with reduced sink capacity (e.g. sink removal, phloem interruption by steam girdling at the leaf base), Rubisco is degraded and free amino acids accumulate. They may be washed out in the rain during late senescence. In leaves of depodded soybeans, Rubisco is degraded and amino acids can be reutilized in these leaves for the synthesis of special vacuolar proteins in the paraveinal mesophyll (vegetative storage proteins). Nitrogen deriving from Rubisco degradation in older (senescing) leaves of annual crops is integrated to some extent again in newly synthesized Rubisco in younger leaves or photosynthesizing tissues of fruits. Finally, a high percentage of this nitrogen is accumulated in protein bodies (storage proteins). At the subcellular level, Rubisco can be degraded in intact chloroplasts. Reactive oxygen species may directly cleave the large subunit or modify it to become more susceptible to proteolysis. A metalloendopeptidase may play an important role in Rubisco degradation within intact chloroplasts. Additionally, the involvement of vacuolar endopeptidase(s) in Rubisco catabolism (at least under certain conditions) was postulated by various laboratories.     

Involvement of sperm proteases in the binding of sperm to the vitelline envelope in Xenopus laevis

Sperm binding to the vitelline envelope in dejellied Xenopus laevis eggs was effectively inhibited by inhibitors for trypsin (soybean trypsin inhibitor and p-toluenesulfonyl-L-lysine chloroethyl ketone) and aminopeptidase B (o-phenanthroline, bestatin, and arphamenine B). Likewise, synthetic 4-methylcoumaryl-7-amide (MCA) substrates (t-butoxycarbonyl-GlyArgArg-MCA, benzyloxycarbonyl-ArgArg-MCA, and Arg-MCA) inhibited binding. Consistently, when jellied eggs were inseminated in the presence of these substrates or inhibitors for proteases, fertilization was effectively blocked. The medium in which live sperm or the sperm membrane fraction were suspended exhibited hydrolyzing activities against the synthetic substrates mentioned above, and these activities were effectively inhibited by the protease inhibitors. Ultracentrifugal fractionation of the sperm suspension following induction of the acrosome reaction by a calcium ionophore, A23187, indicated that a considerable amount of the total tryptic and aminopeptidase B activity was released into the medium. On this occasion, part of the tryptic and aminopeptidase B activity was definitely estimated to be discharged in association with a vesiculated membrane, supporting the notion that the proteases involved in binding to the vitelline envelope are present on the sperm plasma membrane.     

Formation of Aldehyde Flavor (n-hexanal, 3Z-nonenal and 2E-nonenal) in the Brown Alga, Laminaria Angustata

2E-Nonenal and n-hexanal are the major and minor flavor compounds in the edible brown alga, Laminaria angustata, respectively. They are believed to characterize the flavor of this alga. However the metabolism of the two compounds is not precisely known. The pathways were clarified by elucidation of the intermediate structure through purification of the intermediate compounds from an enzymatic reaction and identification using HPLC and GC-MS techniques. Formation of n-hexanal, 3Z-nonenal and 2E-nonenal are proposed to be via two cascades from unsaturated fatty acids. They are C18:2(n-6), linoleic acid cascade and C20:4(n-6), arachidonic acid cascade through their hydroperoxides as intermediates by the lipoxygenase/fatty acid hydroperoxide lyase pathway.