Re-examination ofPyrenomonas andRhodomonas (class cryptophyceae) through ultrastructural survey of red pigmented cryptomonads

Several taxa of cryptomonads, including species of marineChroomonas, Cryptomonas and freshwaterRhodomonas were examined using transmission electron microscopy. They have cellular structures fundamentally in common: a single bilobed chlorplast, a single pyrenoid between the chloroplast lobes, and a nuclemorph embedded within a cleft of the pyrenoidal matrix. These features are in accordance with the taxonomic characteristics of the recently established genusPyrenomonas. The algae also have similar pigmentation to that ofRhodomonas andPyrenomonas which is red or reddish-brown. On the basis of these observations, the genusRhodomonas Karsten (1898) is redescribed in this paper and the genusPyrenomonas Santore is considered to be synonymous withRhodomonas.     

Biochemical and clinical analysis of accumulated glycolipids in symptomatic heterozygotes of angiokeratoma corporis diffusum (Fabry's disease) in comparison with hemizygotes

Angiokeratoma corporis diffusum (Fabry's disease) is an X-linked disorder of glycosphingolipid catabolism. Heterozygous females, although usually asymptomatic, are occasionally as severely afflicted as hemizygous males; recently we identified a heterozygous patient with cardiomyopathy and severe pain in the extremities. In order to elucidate the difference of the clinical features, we analyzed the glycolipid composition of the heart, liver, and kidney obtained from the patient and from a hemizygote. Gas-liquid chromatography revealed that globotriaosylceramide (Gb3) was markedly increased in the heart (32.4 times higher than control) and increased to a lesser extent in the liver and kidney (3.74 and 6.79 times, respectively). The pattern of Gb3 accumulation in the heterozygote, where the highest increases were seen in the heart, was distinct from that in the hemizygote, where elevated levels of Gb3 and Ga2 were found in the kidney. Furthermore, the alpha-galactosidase activity in the heart, liver, and kidney of the heterozygote was 17%, 26%, and 36%, respectively, of normal controls, which correlated well with the accumulation of glycosphingolipid in the heart and with the disease's clinical manifestations. Two other hemizygotic patients, who were identified by low alpha-galactosidase activities, demonstrated the cardiac involvement.     

Metabolism of exogenous galactosylceramide in the twitcher mouse brain

The in vivo metabolism of galactosylceramide (gal-cer) in normal mice and in twitcher mice, a model of human GLD, was examined following intracerebral administration of gal-cer containing [1-¹⁴C]stearic acid. In normal mice, gal-cer was hydrolyzed to ceramide within 6 hours and ceramide was hydrolyzed to sphingosine and fatty acid. Most of the released fatty acid was immediately incorporated into other lipids. About 75% of injected gal-cer was hydrolyzed 80 hours after the injection, while in the twitcher mouse, only 17% of gal-cer was hydrolyzed. These results show that degradation of gal-cer is impaired in the twitcher mouse brain, but contradict to the fact that there was no evidence of any accumulation of gal-cer in the brain. This discrepancy may be due to the different sorting routes of biosynthesized and exogenously-administered gal-cer in the mouse brain. Most of the biosynthesized gal-cer is incorporated into myelin, while the injected gal-cer is incorporated into lysosomes.     

Collection and Chemical Composition of Pure Phloem Sap from Zea mays L.

Pure phloem sap was collected from leaf sheaths of Zea maysL. by the insect laser technique, and its chemical compositionwas analyzed. Sucrose was the only sugar detected. The predominantinorganic ions were K⁺ and Cl^–. The adenylate energy chargeof phloem sap was between 0.72 and 0.86. (Received October 18, 1989; Accepted May 11, 1990)     

Characterization of Cysteine Proteases Functioning in Degradation of Dynorphin in Neuroblastoma Cells: Evidence for the Presence of a Novel Enzyme with Strict Specificity Toward Paired Basic Residues

Two dynorphin-degrading cysteine proteases, I and II, were extracted with Triton X-100 from neuroblastoma cell membrane, isolated from accompanying dynorphin-degrading trypsin-like enzyme by affinity chromatography on columns of soybean trypsin inhibitor-immobilized Sepharose and p-mercuribenzoate-Sepharose, and separated by ion-exchange chromatography on diethylaminoethyl (DEAE)-cellulose and TSK gel DEAE-5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes, were inhibited by p-chloromercuribenzoate, N-ethylmaleimide, and high-molecular-weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E-64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1-17) at the Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1-17) at the Lys11-Leu12 bond and the Leu12-Lys13 bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg-Arg doublet, when susceptibilities of various peptides containing paired basic residues were examined as substrates for the enzyme.     

Production of a herbicide, 5-aminolevulinic acid,by Rhodobacter sphaeroides using the effluent of swine waste from an anaerobic digestor

Summary For the production of a herbicide, 5-amino-levulinic acid (ALA), from anaerobic digestion liquor, the utilization of the photosynthetic bacterium, Rhodobacter sphaeroides was examined. This bacterium could produce ALA extracelularly from this liquor with the addition of levulinic acid (LA), an inhibitor of ALA dehydratase (ALAD), and glycine, a precursor of ALA biosynthesis in the Shemin pathway. Succinate (another precursor) addition was unnecessary for ALA production. When repeated additions of LA were made together with glycine ALA production was significantly enhanced. However, above three additions of LA, ALA production was not further enhanced. The maximum value of ALA production attained was 4.2 mM (0.63 g/ 1), which was over double that of other ALA producers such as Chlorella vulgaris. Propionic acid was predominantly utilized compared with other lower fatty acids, suggesting that this might be converted to ALA via succinyl-coenzyme A (CoA) in the methylmalonyl-CoA pathway.Offprint requests to: Y. Nishizawa     

Chinese hamster ovary cells continuously secrete a cysteine endopeptidase

Summary The protease activity in serum-free conditioned medium of chinese hamster ovary (CHO) cells was measured using peptidyl (or aminoacyl)-4-methylcoumaryl-7-amides (MCAs) as the substrates. Aminopeptidase increased in level as amounts of nonviable cells increased during cultivation in serum-free medium, indicating that the activity seems to be originated from intracellular proteases. The activity toward Boc-Leu-Arg-Arg-MCA, which was strongly inhibited by p-chloromercuribenzonate and N-ethylmaleimide, was the strongest among those toward peptidyl-MCAs in the conditioned medium within 48 h-cultivation in serum-free medium. In contrast to the case of aminopeptidase activity, the endopeptidase activity decreased in level after 48 h-cultivation although amounts of nonviable cells increases. Thus, CHO cells continuously secrete the cysteine proteases. This work was supported by the management of the Research Association for Biotechnology as a part of the R&D of Basic Technology for Future Industries sponsored by NEDO (New Energy and Industrial Technology Development Organization).