The Presence of Apopyruvate Carboxylase and Synthesis of Holopyruvate Carboxylase in Biotin Requiring Baker’s Yeast

Pectin transeliminase from the culture medium of Aspergillus sojae was purified 940-fold by ammonium sulfate fractionation, column chromatography on CM-Cellulose, DEAE-Sephadex and SE-Sephadex and gel filtration. The purified enzyme was free from polygalacturonase, hemicellulase, cellulase and protease, and was almost homogeneous on disc electrophoresis. Using gel filtration a molecular weight of about 32,000 was estimated for the enzyme. The pH optimum of the enzyme for pectin (68% esterified) was 5.5, but was 7.0 for polymethyl polygalacturonate methyl glycoside (98% esterified). The addition of divalent cation to pectin stimulated the activity and shifted the pH optimum to neutral side. This tendency by divalent cation, however, was not observed when polymethyl polygalacturonate methyl glycoside was used as substrate. The enzyme is stable between pH 4 to 7. Heating the enzyme solution at 70°C for 10 min caused complete loss of activity. A marked inhibition of activity was produced by oxidizing reagents such as iodine and N-bromosuccinimide. In contrast, no effect emerged from the action of chelating agents, reducing reagents and those reagents which convert SH-groups in protein into mercaptides. The limits of degradation of pectin and polymethyl polygalacturonate methyl glycoside by the enzyme were 26.3 and 39.8%, respectively.     

Continuous high expression of XBP1 and GRP78 is important for the survival of bone marrow cells in CCl4-treated cirrhotic liver

We have previously shown that infusion of bone marrow cells (BMC) improves CCl₄-induced cirrhosis. However, it is unclear why the injected BMC are resistant to CCl₄ damage and subsequently improve the local microenvironment in damaged liver. To analyze the cellular phenomena involved in this process, we studied the damaged liver using electron microscopy. We found that CCl₄ caused rough endoplasmic reticula to swell in hepatocytes. To analyze the gene expression patterns associated with this process, we conducted PCR-selected suppressive subtractive hybridization. We found that expression levels of HSP84, HSP40, and XBP1 differed markedly between control liver and liver infused with BMC. Immunohistochemical staining revealed that expression levels of HSP84 and HSP40 were markedly higher in the early phase of differentiation immediately after BMC infusion, but decreased over time. XBP1 expression remained high during the late phase, and GRP78 expression increased with XBP1 activation. We also found that GFP-positive BMC expressed XBP1 and GRP78. XBP1 and GRP78 are associated with ER stress. Thus, continuous high XBP1 and GRP78 expression might be essential for the survival and proliferation of BMC in a CCl₄-induced persistent liver damage environment.     

The nucleotide sequence of the yeast PHO5 gene: a putative precursor of repressible acid phosphatase contains a signal peptide.

The nucleotide sequence of the PHO5 gene of the yeast, Saccharomyces cerevisiae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terminal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotic signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PHO5 gene are also discussed.     

Secretion of an active nonglycosylated form of the repressible acid phosphatase of Saccharomyces cerevisiae in the presence of tunicamycin at low temperatures

The role of mannan chains in the formation and secretion of active acid phosphatase of yeast (Saccharomyces cerevisiae), a repressible cell surface mannoprotein, was studied in yeast protoplast systems by using tunicamycin at various temperatures. At 30 degrees C, tunicamycin-treated protoplasts did not produce active acid phosphatase; however, at 25 or 20 degrees C they formed and secreted active enzyme. This form of acid phosphatase gave 59-, 57-, and 55-kDa bands on SDS-PAGE which neither bound to concanavalin A Sepharose, nor changed in molecular weight upon treatment with endoglycosidase H, indicating that the peptides are nonglycosylated. The nonglycosylated form, like its glycosylated counterpart, is a dimer on the basis of gel permeation chromatography. The Km for para-nitrophenyl-phosphate and Ki for inorganic phosphate of both glycosylated and nonglycosylated acid phosphatases were almost the same. These results suggested that 1) the conformation of the nonglycosylated acid phosphatase secreted at low temperatures is probably identical with that of the glycosylated one, and 2) the conformation of acid phosphatase is very important for its secretion. The rate of intracellular transport of nonglycosylated acid phosphatase is about one-fourth that of the glycosylated enzyme, indicating that glycosylation facilitates the transport of acid phosphatase proteins.     

Molecular structure of a yeast gene, PDI1, encoding protein disulfide isomerase that is essential for cell growth.

A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth.     

How does the longevity of <Emphasis Type="Italic">Sasa kurilensis</Emphasis> ramets respond to a light gradient? An analysis of ontogenetic changes to hydraulic resistance and carbon budget within a ramet

We investigated how Sasa kurilensis ramet longevity differs under three light conditions. Ramet longevity is an important factor affecting the abundance of ramet populations and their biomass. The objectives of this study were to clarify: (1) whether ramet longevity varies spatially along a gradient of light conditions; (2) whether physiological functions, including water transport in culms, change with ontogenesis; (3) whether ramet longevity is associated with the most effective turnover for gaining a carbon profit within a ramet following a cost-benefit model or is affected by other factors such as death caused by a decline in physiological function. We analyzed S. kurilensis ramet longevity, hydraulic resistance, photosynthetic rate, and carbon content. We then estimated the ramet carbon budget. The longevity of S. kurilensis ramets was 2.8–4.5 years in Beneath-patch plots, 5.8–8.7 years in Edge-patch plots, and 1.6–2.2 years in Open-patch plots. Although leaf photosynthetic activity was stable, the instantaneous photosynthetic rate decreased during clear days in the open area. This may have been due to increased ramet hydraulic resistance. The rotation period of the most efficient carbon budget quantified by ecophysiological measurements was consistent with ramet longevity in Open-patch (2 years) and Edge-patch (5 years) sites. Meanwhile, the longevity of ramets grown under canopies was inconsistent with the cost-benefit model rules for a carbon budget because the carbon gain was low throughout the ramet life span.     

Expression,purification and characterization of yeast protein disulfide isomerase produced by a recombinant baculovirus-mediated silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>, pupae expression system

Protein disulfide isomerase (PDI) is a multifunctional polypeptide presents in the endoplasmic reticulum of the cell. Silkworm (Bombyx mori) pupae were used as hosts to produce recombinant PDI (rPDI). The concentration-dependent chaperone activity of rPDI was evidenced by the inhibition of the aggregation of rhodanese. Approximately 297 μg rPDI was purified from a single silkworm pupa. Results of rPDI treated with endoglycosidase H and N-glycanase, PNGase F, indicate that non-N-glycosylated rPDI (occupying 90%) and N-glycosylated rPDI are expressed in the silkworm expression system. The difference in glycosylation between silkworm pupae and yeast is discussed.     

Temperature-sensitive forms of large and small invertase in a mutant derived from a SUC1 strain of Saccharomyces cerevisiae.

Mutagenesis of the sucrose-fermenting (SUC1) Saccharomyces cerevisiae strain 4059-358D yielded an invertase-negative mutant (D10). Subsequent mutagenic treatment of D10 gave a sucrose-fermenting revertant (D10-ER1) that contained the same amount of large (mannoprotein) invertase as strain 4059-358D but only trace amounts of the smaller intracellular nonglycosylated enzyme. Limited genetic evidence indicated that the mutations in D10 and D10-ER1 are allelic to the SUC1 gene. The large invertases from D10-ER1 and 4059-358D were purified and compared. The two enzymes have similar specific activity and Km for sucrose, cross-react immunologically, and show the same subunit molecular weight after removal of the carbohydrate with endo-beta-N-acetylglucosaminidae H. They differ in that the large enzyme from the revertant is rapidly inactivated at 55 degrees C, whereas that from the parent is relatively stable at 65 degrees C. The small invertase in extracts of D10-ER1 is also heat sensitive as compared to the small enzyme from the original parent strain. The low level of small invertase in mutant D10-ER1 may reflect increased intracellular degradation of this heat-labile form. In several crosses of D10-ER1 with strains carrying the SUC1 or SUC3 genes, the temperature sensitivity of the large and small invertases and the low cellular level of small invertase appeared to cosegregate. These findings are evidence that SUC1 is a structural gene for invertase and that both large and small forms are encoded by a single gene. A detailed genetic analysis is presented in a companion paper.     

Various types of the pituitary folliculo-stellate cells involving the Siperstein's corticotroph in the normal rats

The differentiation of the folliculo-stellate (F-S) cells was electronmicroscopically investigated in the normal male adult rats from the Wistar, Wistar-Imamichi, Holzmann, Spraque-Dowley and Donryu strains. The F-S cells may be divided into the five types according to the granulation. Each type is, however, provided with the common characteristic features, i.e., the stellate shape due to projecting the cytoplasmic processes and a tendency to embrace an acidophil. The first type is an agranular independent or anastomosing immature cell. It is different in shape and arrangement from the follicular cell, but similar in agranularity and immaturity to it. The second is a slightly differentiated cell, in which scanty small secretory granules 50--100 nm in diameter begin to appear near the plasma membrane. The third is a moderately differentiated cell providing the regularly row arrangement of secretory granules 100--200 nm in diameter along the plasma membrane, corresponding, in fine structure, with a corticotroph. The fourth is a fully differentiated cell with heavy granulation, whose secretory granules 150--250 nm in diameter are accumulated in the whole cytoplasm, suggesting the storing type. It is difficult to determine whether the fourth type coincides with a hypergranulated corticotroph or a stellate thyrotroph. The fifth is a kind of fully differenetiated cell which may refer to the releasing phase of the fourth type, being characterized by the dispersion or loss of minute secretory granules of low density as large as 50 nm in diameter, associated with the cored vesicles. The population densith of the above five types increased in the sequence, 5th leads to 4th leads to 1st leads to 3rd type in the gland. Namely, the 3rd (corticotroph) type and 1st (agranular) type are predominantly distributed, and the 5th (releasing) type and 4th (hypergranulated) type are rarely.