Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the n-Alkane-Assimilating Yeast Candida tropicalis

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1aΔ/t1bΔ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C₁₀ to C₁₃). All thiolase activities in this yeast disappeared with the ct-t1aΔ/t1bΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C₄ substrate butyrate.     

A genetic method to enhance the accumulation of S-adenosylmethionine in yeast

S-Adenosylmethionine (SAM) is a key component of sulphur amino acid metabolism in living organisms and is synthesised from methionine and adenosine triphosphate by methionine adenosyltransferase. This molecule serves as the main biological methyl donor due to its active methylthio ether group. Notably, SAM has shown beneficial effects in clinical trials for the treatment of alcoholic liver disease, depression and joint pain. Due to the high potential value of SAM, current research efforts are attempting to develop a more rapid, cost-effective and higher yielding SAM production method than the conventional production system. In this mini-review, we describe the previously reported yeast gene that contributes to SAM accumulation by overexpression, mutation or deletion and summarise the genetic approach for the production of SAM in large industrial quantities.

     

Immunochemically distinct NADP-linked isocitrate dehydrogenase isozymes in mitochondria and peroxisomes of Candida tropicalis

Although peroxisomal localization of NADP-linked isocitrate dehydrogenase (Idp) was first demonstrated in Candida tropicalis, the mitochondrial isozyme has not been found in this yeast. Here we report that the presence of mitochondrial Idp in the yeast was demonstrated by screening for its gene with a DNA probe containing conserved sequences of Idps from various organisms. The nucleotide sequence of the gene (CtIDP1) revealed a 1,290-bp open reading frame corresponding to a 430-amino-acid protein with a high similarity to previously reported Idps. Overexpression of CtIDP1 in Saccharomyces cerevisiae gave a high intracellular Idp activity, and the purified recombinant Idp was shown to be a homodimer with a subunit molecular mass of approximately 44 kDa, different from that of peroxisomal Idp (45 kDa) previously purified from C. tropicalis. Western blot analysis of the subcellular fractions from acetate-grown C. tropicalis with polyclonal antibodies raised against the recombinant CtIdp1 showed that the CtIdp1 in C. tropicalis was localized in mitochondria but not in peroxisomes. Similar levels of CtIDP1 mRNA and its protein product were detected in cells grown on glucose, acetate, and n-alkane, although a slight decrease was observed in n-alkane-grown cells. From these results, CtIdp1 was demonstrated to be mitochondrial Idp. The properties of mitochondrial Idp and peroxisomal Idp isozymes were proven to be similar, but they were immunochemically distinct, suggesting the presence of another gene responsible for peroxisomal Idp in C. tropicalis. Received: 11 March 1997 / Accepted: 24 June 1997     

Effects of space flight on the histological characteristics of the aortic depressor nerve in the adult rat: electron microscopic analysis.

The effects of microgravity on the histological characteristics of the aortic depressor nerve, which is the afferent of the aortic baroreflex arc, were determined in 10 female adult rats. The rats were assigned for nursing neonates in the Space Shuttle Columbia or in the animal facility on the ground (NASA Neurolab, STS-90), and were housed for 16 days under microgravity in space (microg, n=5) or under one force of gravity on Earth (one-g, n=5). In the Schwann cell unit in which the axons of unmyelinated fibers are surrounded by one Schwann cell, the average number of axons per unit in the microg group was 2.1 +/- 1.6 (mean +/- SD, n=312) and significantly less than that in the one-g group (3.0 +/- 2.9, n=397, p<0.05). The proportion of unmyelinated fibers in the aortic depressor nerve in the microg group was 64.5 +/- 4.4% and significantly less than that in the one-g group (74.0 +/- 7.3%, p<0.05). These results show that there is a decrease in the number of high-threshold unmyelinated fibers in the aortic depressor nerve in adult rats flown on the Shuttle Orbiter, suggesting that the aortic baroreflex is depressed under microgravity during space flight.     

Peroxide-Scavenging Systems during Cold Acclimation of Apple Callus in Culture

The relationship between peroxide-scavenging systems and coldacclimation was studied in apple callus in culture during acclimationunder artificial conditions. Unacclimated callus did not survivefreezing at –10?C, whereas callus acclimated at 0?C exhibitedgradually increased resistance to freezing and, after acclimationfor 20 days, it survived at temperatures as low as –15–C.During acclimation of callus, there was an immediate and abruptincrease in the activities of ascorbate peroxidase (EC 1.11.1.11 [EC] ),peroxidase (EC 1.11.1.7 [EC] ) and catalase (EC 1.11.1.6 [EC] ), which reachedmaximum values after acclimation for 10 days, at the same timeas the very beginning of the increase in cold hardiness wasobserved. An increase in the activity of glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12 [EC] ) occurred during the first 5 daysof cold treatment. The activities of glucose-6-phosphate dehydrogenase(EC 1.1.1.49 [EC] ), hexokinase (EC 2.7.1.1 [EC] ), glutathione reductase(EC 1.6.4.2 [EC] ), glutathione peroxidase (EC 1.11.1.9 [EC] ) and dehydro-ascorbatereductase (EC 1.8.5.1 [EC] ) increased gradually during the cold treatment.In contrast, the activity of glucosephosphate isomerase (EC5.3.1.9 [EC] ) decreased gradually during acclimation. Furthermore,during acclimation, the levels of glucose-6-phosphate, fructose-6-phosphateand glucose-1-phosphate increased slowly and steadily, and thelevels of GSH and ascorbate remained at consistently higherlevels. In addition, acclimation caused marked cytological changes.The most striking of these changes was the microvacuolationand thickening of the cell wall. These results indicate thatthe enhancement of peroxide-scavenging systems at the time ofcold acclimation proceeds in two stages: during the first stage,the enzymatic activities involved in the degradation of peroxides(i.e., the activities of ascorbate peroxidase, peroxidase andcatalase) increase; and, in the second stage, an alternativeenzymatic system develops for detoxification of peroxides, coupledwith the pentose phosphate cycle. (Received July 20, 1990; Accepted April 16, 1991)     

Scanning electron microscopy of vascular architecture in the gastric mucosa of the golden hamster

Summary The three-dimensional architectures and the regional differences of the vascular system in the mucosa of the hamster stomach were revealed by scanning electron microscopy of corrosion casts. In the forestomach, the vascular network spreads two-dimensionally in a thin lamina propria. In the corpus and the antrum, the capillaries in the thick lamina propria are well developed, extending three-dimensionally along the gastric pits and glands. In the corpus, the submucosal arteries enter the lamina propria to become ascending capillaries, which project toward the top of the lamina propria and anastomose to create a capillary network beneath the mucosal epithelium. A subepithelial capillary is much wider in diameter than an ascending capillary and is, therefore, a sinusoid capillary. Subepithelial capillaries join descending venules, which are less numerous than the ascending capillaries. Near the gastric lumen, the capillaries in the corpus can be classified into two types: arched type in the cephalic (upper) region and honeycomb type in the caudal (lower) region. In the antrum, the submucosal arterial plexus is less well developed than that in the corpus. The mucosal aspect of the corrosion cast shows many clumps, formed by a unit of capillary network. Functional significances of different vascular architectures in the gastric mucosa of the forestomach, corpus, and antrum are discussed.This study was supported in part by grants from the Research Fund of the Ministry of Education, Science, and Culture, Japan     

Release of ecdysteroid-phosphates from egg yolk granules and their dephosphorylation during early embryonic development in silkworm, Bombyx mori

Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.     

Complete Genome Sequence of Desulfurococcus fermentans,a Hyperthermophilic Cellulolytic Crenarchaeon Isolated from a Freshwater Hot Spring in Kamchatka,Russia

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.