Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction

For determination of the physiological role and mechanism of vacuolar proteolysis in the yeast Saccharomyces cerevisiae, mutant cells lacking proteinase A, B, and carboxypeptidase Y were transferred from a nutrient medium to a synthetic medium devoid of various nutrients and morphological changes of their vacuoles were investigated. After incubation for 1 h in nutrient-deficient media, a few spherical bodies appeared in the vacuoles and moved actively by Brownian movement. These bodies gradually increased in number and after 3 h they filled the vacuoles almost completely. During their accumulation, the volume of the vacuolar compartment also increased. Electron microscopic examination showed that these bodies were surrounded by a unit membrane which appeared thinner than any other intracellular membrane. The contents of the bodies were morphologically indistinguishable from the cytosol; these bodies contained cytoplasmic ribosomes, RER, mitochondria, lipid granules and glycogen granules, and the density of the cytoplasmic ribosomes in the bodies was almost the same as that of ribosomes in the cytosol. The diameter of the bodies ranged from 400 to 900 nm. Vacuoles that had accumulated these bodies were prepared by a modification of the method of Ohsumi and Anraku (Ohsumi, Y., and Y. Anraku. 1981. J. Biol. Chem. 256:2079-2082). The isolated vacuoles contained ribosomes and showed latent activity of the cytosolic enzyme glucose-6-phosphate dehydrogenase. These results suggest that these bodies sequestered the cytosol in the vacuoles. We named these spherical bodies "autophagic bodies." Accumulation of autophagic bodies in the vacuoles was induced not only by nitrogen starvation, but also by depletion of nutrients such as carbon and single amino acids that caused cessation of the cell cycle. Genetic analysis revealed that the accumulation of autophagic bodies in the vacuoles was the result of lack of the PRB1 product proteinase B, and disruption of the PRB1 gene confirmed this result. In the presence of PMSF, wild-type cells accumulated autophagic bodies in the vacuoles under nutrient-deficient conditions in the same manner as did multiple protease-deficient mutants or cells with a disrupted PRB1 gene. As the autophagic bodies disappeared rapidly after removal of PMSF from cultures of normal cells, they must be an intermediate in the normal autophagic process. This is the first report that nutrient-deficient conditions induce extensive autophagic degradation of cytosolic components in the vacuoles of yeast cells.     

Expression of activities of two 20 alpha-hydroxysteroid dehydrogenase isozymes in rat corpora lutea.

The rat ovary contains two isozymes of 20 alpha-hydroxysteroid dehydrogenase (HSD-1 and HSD-2). In this study, the expression of activity of each isozyme was investigated in ovaries that contained a single generation of corpora lutea during pseudopregnancy. This condition was induced by cervical stimulation in rats that had been rendered anovulatory by housing them in a continuously lit environment. The total activity of cytosolic 20 alpha-HSD was lower in the ovaries of these pseudopregnant rats than in ovaries containing multiple generations of corpora lutea. In normal pseudopregnancy, HSD-1 activity was low on days 5 and 9 and increased markedly on day 15, whereas HSD-2 was lower than HSD-1 and did not vary throughout pseudopregnancy. However, on days 5 and 9 of continuous-light pseudopregnancy, low activity of HSD-1 only was detected; by day 15, HSD-1 activity had increased sixfold and HSD-2 activity could be detected. Immunohistochemical methods using a specific antibody recognizing both HSD-1 and HSD-2 revealed that the number of 20 alpha-HSD-positive luteal cells increased by day 15. Thus, the increase in total enzyme activity and appearance of HSD-2 activity observed at late pseudopregnancy was accompanied by an increase in the number of 20 alpha-HSD-positive luteal cells.     

Effect of visual light on in vitro embryonic development in the hamster

The effect of light exposure during collection and culture of hamster embryos on their subsequent development in vitro was examined. When embryos were collected under dark conditions (70 lux) within 10 minutes and then cultured in a HECM-1 medium in 5% CO(2) in air, the developmental rates of 1-cell embryos to the 4- and 8-cell stages were 88.6% (93 105 ) and 66.7% (70 105 ), respectively. These rates were significantly higher than those under light conditions (1600 lux): 51.9% (56 108 ) and 34.3% (37 108 ). Light irradiation during the culture of 1-cell embryos suppressed subsequent development. The degree of suppression correlated inversely with duration of light irradiation, and light irradiation of 30 minutes or more completely blocked development to the 2-cell stage. When 1-cell embryos were irradiated through a yellow filter, cutting the light wavelengths to less than 500 nm, embryonic development was still suppressed. However, the degree of the suppression varied and 45.7% (53 116 ), 6.0% (7 116 ), and 0.9% (1 116 ) of the embryos developed to the 2-, 4-, and 8-cell stages, respectively, under 30 minute light irradiation. Inhibitory effects of light irradiation on the development of 2- and 8-cell embryos were also observed, showing an inverse correlation with duration; the developmental rates of 2-cell embryos to the 8-cell stage under 0, 10, and 30 minutes of irradiation were 85.6% (107 125 ), 1.6% (2 122 ), and 0% (0 129 ), respectively, and those of 8-cell embryos to the blastocyst stage were 79.8% (91 114 ), 74.8% (86 115 ), and 0% (0 110 ), respectively. These findings indicate that early-stage embryos are sensitive to light exposure; however, severe light exposure adversely affects the development of embryos at any stage. Thus, the protection of embryos from light exposure at all stages of embryo manipulation, from collection to culture, is essential.     

Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis.

We determined the nucleotide sequence of a 1.9-kilobase fragment of Pseudomonas paucimobilis SYK6 chromosomal DNA that included genes encoding protocatechuate 4,5-dioxygenase, the enzyme responsible for the aromatic ring fission of protocatechuate. Two open reading frames of 417 and 906 base pairs were found that had no homology with previously reported sequences, including those encoding protocatechuate 3,4-dioxygenase. Since both open reading frames were indispensable for the enzyme activity, they should encode the subunits of protocatechuate 4,5-dioxygenase. We named these genes ligA and ligB. Protocatechuate 4,5-dioxygenase was efficiently expressed in Escherichia coli with the aid of the lac promoter, and the polypeptides of the ligA and ligB gene products were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid sequencing.     

Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation

Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.     

Lipid synthesis in germinating safflower seeds and protoplasts

Galactolipids and phospholipids rapidly accumulated in a whole seed between 2 and 4 days after germination. However, the rate of incorporation of [¹⁴C] acetate into galactolipids was very low. The predominant fatty acid of galactolipids was linolenic acid, while those of phospholipids were linoleic and palmitic acids. Fatty acids of monogalactosyldiacylglycerol in germinating safflower seeds were randomly distributed between the 1 - and 2-positions of the glycerol molecule and the distribution in digalactosyldiacylglycerol was slightly non-random, while fatty acids of galactolipids in mature safflower leaves were non-randomly distributed. Triacylglycerol was synthesized in the cotyledon tissue of the germinating seeds simultaneously with its rapid degradation. In addition, lipid biosynthesis in protoplasts is described.     

Induction of L-lysine-2-oxoglutarate reductase by glucagon and glucocorticoid in developing and adult rats in vivo and in vitro studies

L-Lysine-2-oxoglutarate reductase (EC 1.5.1.8, NADP⁺) in the liver of adult rats increased 4–5-times when the animals were treated with alloxan. In diabetic rats injection of insulin or adrenalectomy prevented the increase in enzyme activity. The activity of the similar enzyme in kidney was not changed by these treatments. The enzyme activity in primary cultured adult rat hepatocytes was also induced by addition of dexamethasone and glucagon together, and glucagon could be replaced by dibutyryl cyclic AMP. Insulin inhibited the induction. The hormonal induction was also inhibited by actinomycin D and by cycloheximide. During development of rats, fetal liver showed very low activity, but the activity appeared on day 1 after birth and then increased rapidly, reaching the adult level by day 5. The activity of the kidney enzyme increased more slowly and reached the adult level 1 month after birth. Intra-uterine injection of glucagon caused precocious induction of the liver enzyme in fetuses. These results indicate that the activity of L-lysine-2-oxoglutarate reductase in the adult liver and in part in neonatal liver also, is controlled by both glucagon and glucocorticoid.     

Hormonal regulation of serine dehydratase activity in primary cultures of adult rat hepatocytes

The capacity of the following peptides to stimulate steroidogenesis in suspensions of capsule (largely glomerulosa) and fasciculata/reticularis cells from rat adrenals was studied: ACTH_1–24, ACTH_1–13-amide, α-MSH, γ₁-MSH, γ-MSH precursor, ACTH_4–10, CLIP, and ovine and human β-lipotropin. Only α-MSH and ACTH_1–13-amide stimulated glomerulosa cells alone, without effect on fasciculata/reticularis cells. Like ACTH_1–24 the two samples of β-lipotropin stimulated both capsule and inner zone cell types in a similar manner. Their activity is attributable to slight ACTH_1–39 contamination, as shown by HPLC fractionation. The other peptides lacked any activity. It is likely that the predicted specific glomerulosa stimulant from the pituitary closely resembles α-MSH.     

Mitochondrial GTP-AMP phosphotransferase. 1. Purification and properties.

GTP-AMP phosphotransferase has been purified 116-fold with a yield of 24% from beef heart mitochondria using freeze-thawing, alkali and acid treatment and successive column chromatography on phosphocellulose, Sephadex G-100 and blue-dextran--Sepharose. It has crystallized from poly-(ethylene glycol) and is essential homogeneous by sodium dodecylsulfate electrophoresis and isoelectrofocusing. The specific activity of the crystalline preparation was 290 U/mg. The molecular weight was found to be 26000 and the isoelectric point to be 9.8. Amino acid analysis showed 21 aspartic acid or asparagine, 19 threonine, 12 serine, 26 glutamic acid or glutamine, 15 proline, 16 glycine, 14 alanine, 15 valine, 4 methionine, 12 isoleucine, 28 leucine, 7 tyrosine, 7 phenylalanine, 5 histidine, 14 lysine, 16 arginine, 2 tryptophan, no --SS-- bonds or free --SH. Guanosine(5')pentaphospho(5')adenosine is a very strong inhibitor similar to adenosine(5')pentaphospho(5')adenosine as an inhibitor of cytosolic adenylate kinase.     

Asymmetrical radical formation in D- and L-alanines irradiated with yttrium-90β-rays

Radical formation in^{9 0}Y--irradiated D- and L-alanines was studied using ESR. It was observed that the relative radical concentration by -irradiation was distinguishably (13.9–21.5%) more in D-alanine than in L-alanine. Discussion was made on the possible mechanisms for the observed results.