Construction and Characterization of Isogenic Series of Saccharomyces cerevisiae Polyploid Strains

Tetraploid cells of Saccharomyces cerevisiae are generated spontaneously in a homothallic MATa/MATα diploid population at low frequency (approximately 10⁻⁶ per cell) through the homozygosity of mating-type alleles by mitotic recombination followed by homothallic switching of the mating-type alleles. To isolate tetraploid clones more effectively, a selection method was developed that used a dye plate containing 40 mg each of eosin Y and amaranth in synthetic nutrient agar per liter. It was possible to isolate tetraploid clones on the dye plate at a frequency of 1 to 3% among the colonies colored dark red in contrast to the light red of the original diploid colonies. Isogenic series of haploid to tetraploid clones with homozygous or heterozygous genomic configurations were easily constructed with the tetraploid strains. No significant differences in specific growth rate or fermentative rate were observed corresponding to differences in ploidy, although the haploid clones showed a higher frequency of spontaneous respiratory-deficient cells than did the others. However, a significant increment in the fermentative rate in glucose nutrient medium was observed in the hybrid strains constructed with two independent homozygous cell lines. These observations strongly suggest that the polyploid strains favored by the brewing and baking industries perform well not because of the physical increment of the cellular volume by polyploidy but because of the genetic complexity or heterosis by heterozygosity of the genome in the hybrid polyploid cells.     

Evidence for the presence and structure of asparagine-linked oligosaccharide units in the core protein of proteodermatan sulphate.

Hormonally produced changes in the synthesis and secretion of the serum copper-containing protein caeruloplasmin were studied in primary cultures of rat liver parenchymal cells isolated by the collagenase-perfusion technique. A rabbit antibody directed against rat caeruloplasmin was used to immunoprecipitate labelled caeruloplasmin. Isolated liver cells synthesized and secreted caeruloplasmin over a period of 3 days. Synthesis and secretion of this protein was enhanced when cells were treated with dexamethasone. The accumulation of copper was also moderately enhanced with glucocorticoid treatment. Inclusion of adrenaline in the culture medium resulted in elevated incorporation of copper into newly synthesized caeruloplasmin as well as an increase in 64Cu-labelled caeruloplasmin in the culture medium. However, adrenaline did not seem to increase the secretion of 3H-labelled protein, despite the elevation in secreted 64Cu-caeruloplasmin. This may be due to a large increase in the intracellular pool of 64Cu caused by enhanced accumulation of this metal when adrenaline is included in the incubation medium. Enhanced copper accumulation was also seen when cells were treated with glucagon. Adrenaline-stimulated accumulation of 64Cu could be inhibited by including phenoxybenzamine, an alpha-adrenergic blocker, in the culture medium. Elevation of extracellular copper caused enhancement in the detection of labelled caeruloplasmin in the medium of cultured cells, probably owing to the ability of this metal to stabilize the protein.     

Sialidase-like Enzymes Produced by Group A, B, C, G, and L Streptococci and by Streptococcus sanguis

A group of enzymes were prepared from the culture fluids of streptococci belonging to groups A, B, C, G, and L, and from a strain of Streptococcus sanguis. These streptococcal enzymes (designated St-sialidases) released a substance shown to belong to the sialic acid group from the specific substrate BSM-St, a sialomucoid prepared from bovine submaxillary gland. They were inactive on N-acetylneuramin lactose prepared from bovine colustrum and on a sialomucoid prepared from bovine submaxillary mucin, whereas these substances are susceptible to sialidases produced by group K streptococci and by Vibrio cholerae. Some of the St-sialidases were markedly activated by divalent cations, but others showed little response. The heat stability of the enzymes produced by the different strains varied. The optimal pH was between 5.5 and 6.5 with acetate buffer and was about 7 with phosphate buffer. K(m) values were determined for the St-sialidases with BSM-St as substrate.     

Mode of antitumor action of recombinant human tumor necrosis factor on the sarcoma Meth A transplanted in the mouse

The mode of antitumor action of rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation with respect to time course, dose-response relationships and selectivity of the effects. The maximal cytotoxic effect on tumor cells revealed by inhibition of DNA synthesis and maximal lesional effect on tumor vasculature revealed by change in blood pool-size in the tissue were detected at 30 min and I h after administration of rHu-TNF, respectively. The dose-response relationship between cytotoxic and tumoricidal effects of rHu-TNF was irrespective of administration route. ED₅₀s of these antitumor effects afteri.v. administration of rHu-TNF were about 50 times as high as ED₅₀s afteri.t. administration. ED₅₀ ofi.t. given rHu-TNF for vascular effect was about 20 times as high as that for cytotoxicity while ED₅₀ ofi.v. rHu-TNF for vascular effect was only 2–3 times as high as that for cytotoxicity. The whole body autoradiographies with [¹²⁵I] HSA giveni.v. to see the blood influx into tumor tissue and [¹⁴C]thymidine given i.v. to see DNA synthesis in the whole body after administration of rHu-TNF revealed that the distribution of radioactivity was markedly changed in the tumor alone without any detectable change in other whole body tissues.In conclusion, thein vivo antitumor effect of rHu-TNF giveni.t. ori.v., appears to be exerted through the direct action on Meth A sarcoma rather than indirectly on tumor vasculature. Under present conditions, the effect of rHu-TNF in the whole body tissues seems rather selective on cells and vasculature of the tumor.     

Base sequence specificity of a monoclonal antibody binding to (6-4)photoproducts.

The DNA base sequence specificity of the 64M-1 monoclonal antibody, which recognizes ultraviolet (UV)-induced (6-4)photoproducts, was characterized. The 64M-1 antibody strongly bound to UV-poly(dU) as well as to UV-poly(dT), and weakly to UV-poly(dC), UV-poly(me5dC) and UV-poly(rU). A competitive inhibition assay using UV-oligo(dT)8, UV-oligo(dTdC)4, UV-oligo(dC)8, UV-PvuI linker (GCGATCGC) and UV-PvuII linker (GCAGCTGC) indicated that the main (6-4)photoproducts detected by the 64M-1 antibody in UV-irradiated DNA are TT(6-4)photoproducts and TC(6-4)photoproducts. Comparison between dTpdT(6-4)photoproduct and dTpdC(6-4)photoproduct showed that the affinity of the 64M-1 antibody for dTpdT(6-4)photoproduct was about 5 times higher than that for dTpdC(6-4)photoproduct. The antibody also binds to isolated TT(6-4)photoproducts.     

Two Types of Apoptotic Cell Death of Rat Central Nervous System-Derived Neuroblastoma B50 and B104 Cells: Apoptosis Induced During Proliferation and After Differentiation

Abstract: We describe here two types of apoptotic cell death observed in the rat CNS-derived neuroblastoma B50 and B104 cells. One type was induced by dibutyryl cyclic AMP (DBcAMP) after differentiation, and the other was induced by treatment of proliferating cells with cycloheximide. When B50 and B104 cells were treated with 1 m M DBcAMP in the presence of 0.5% fetal calf serum, they began to extend neurites within 12 h and differentiated into neurons at 24 h, as reported previously. However, further cultivation with DBcAMP for up to 72 h led to flotation and, finally, death. Death was by apoptosis as shown by chromatin condensation and DNA fragmentation. Addition of a protein kinase A inhibitor or removal of DBcAMP after differentiation suppressed apoptosis, indicating the involvement of cyclic AMP and protein kinase A in apoptotic cell death. Cell death was also induced in proliferating cells without neurite outgrowth by treatment with cycloheximide. The death was also judged to be by apoptosis based on chromatin condensation and apoptotic body formation, although DNA fragmentation into small sizes was not detected. Both types of cell death showed similar responses to inhibitors for protein kinases and protein phosphatases.     

Transient Expression of CD20 Antigen (Pan B Cell Marker) in Activated Lymph Node T Cells

In contrast to the case of peripheral T cells, the surface expression of CD20 antigen and the expression of CD20 mRNA in monkey lymph node (LN) T cells underwent a noticeable increase when they were cultured with mitogen and interleukin-2 (IL-2). To confirm in vivo regulation of CD20 expression during the activation of LN T cells, we examined LNs derived from monkeys experimentally inoculated with simian immunodeficiency virus (SIV). Significant expression of CD20 antigen was detected in the T cells of the LNs at the stage of lymphadenopathy. These findings suggest that lymphocyte activation in the LNs induced expression of the CD20 molecule in some T cells.     

Sulfated extracellular polysaccharide production by the halophilic cyanobacterium Aphanocapsa halophytia immobilized on light-diffusing optical fibers

 The cyanobacterium, Aphanocapsa halo-phytia MN-11, was immobilized in calcium alginate gel and coated on light-diffusing optical fibers (LDOF) for sulfated extracellular polysaccharide production. Results indicated that sulfated extracellular polysaccharide production depends on the number of immobilized cells and the light intensity. In addition, the production rate reached 116.0 mg (mg dry cells)^-1 day^-1 when the cells that were immobilized on LDOF were incubated under a light intensity of 1380 cd sr m^-2 at a cell concentration of 1.0×10⁸ cells/cm³ gel. Cells immobilized on LDOF produced about ten times more sulfated extracellular polysaccharide than those immobilized in calcium alginate beads only (11.7 mg(mg dry cells)^-1day^-1). Received: 31 March 1995/Revised last revision 12 June 1995/Accepted 26 July 1995