Germinal vesicle contents are required for the cytoplasmic cycle during meiotic division of starfish oocytes

Enucleated oocytes of starfish still show cyclic changes in cortical tension with a temporal pattern similar to that exhibited by intact oocytes during meiotic division, provided that the enucleation is performed a certain time after the breakdown of the germinal vesicle (K. Yamamoto and M. Yoneda, Dev. Biol. 96, 166-172, 1983). If an oocyte is bisected immediately after germinal vesicle breakdown, the resulting nonnucleate fragment shows some change in tension, but the pattern of change is much less regular than that seen in intact oocytes, suggesting that the dispersion of germinal vesicle (GV) contents into cytoplasm is required for the establishment of the cytoplasmic cycle. In order to demonstrate the role of GV contents directly, nonnucleate fragments derived from immature oocytes were injected with GV contents taken from other immature oocytes. On treatment with 1-methyladenine (1-MA) these fragments showed two rounds of increase in tension as is characteristic of intact maturing oocytes. The first rise in tension was always observed 50-70 min after the treatment with 1-MA, similar to the time of first polar body formation in intact oocytes, regardless of the time of injection of GV contents. Even when GV contents were injected into nonnucleate fragments which had been already treated with 1-MA, these fragments showed two rounds of change in tension. The timing of the first rise in tension was found to be 38 +/- 7 min after injection, irrespective of the time of the foregoing treatment with 1-MA. These results prove the indispensability of GV contents for inducing the cytoplasm of the maturing starfish oocyte to initiate its own cyclic activity, and suggest that the normal process of cytoplasmic maturation may consist of two phases, i.e., (1) a GV-independent phase initiated by 1-MA treatment, and (2) a second phase initiated by mixing of GV contents with cytoplasm.     

Human breast milk stimulates prostaglandin synthesis in cultured human skin fibroblasts

Effect of human breast milk or its fractions on prostaglandin synthesis was investigated in cultured human skin fibroblasts. Prostaglandins released into the media were measured by radioimmunoassay. Incorporation of breast milk (2% level) into 10% fetal calf serum media (for 48 hours) stimulated the synthesis of 6-keto-PGF1 alpha (stable product of prostacyclin) by 800%. This stimulating effect of milk persisted after cold acetone extraction to remove phospholipids and potentiated further after dialysis. Stimulation by one of the commercial formulas (Similac) was less than 50% of the milk effect. Milk also stimulated PGE2 synthesis, although to a much lesser degree. These studies show for the first time that a) human breast milk contains potent factor(s) capable of influencing prostaglandin synthesis and suggest that b) these factors might have a role in the development of lipid synthetic pathways during early life.     

Antileukemic effect of coral-prostanoids clavulones from the stolonifer Clavularia viridis on human myeloid leukemia (HL-60) cells

To elucidate the biological activities of coral-prostanoids, clavulones, discovered from the Japanese stolonifer Clavularia viridis, we examined the effect of clavulone on the cell growth of human cancer (human promyelocytic leukemia (HL-60) cells and HeLa cells) and normal (Chang liver cells and lung fibroblasts) cells in vitro. Clavulone showed strong antiproliferative and cytotoxic activities in the human cells and it had some selectivity to leukemic (HL-60) cells over other HeLa cells or normal cells on the basis of the IC50 values and cytotoxic effect of the cells. The IC50 value of clavulone in the HL-60 cells was about 0.4 microM (0.2 micrograms/ml). Over 1.0 microM (0.5 micrograms/ml), clavulone showed a significant cytotoxic activity on the HL-60 cells. The data on DNA synthesis and flow cytometric analysis revealed that clavulone arrests the cells in the G1-phase and inhibits the cell growth of HL-60 cells by inhibiting S-phase DNA synthesis. These results suggest that clavulone has a potent antileukemic effect on HL-60 cells.     

Suppressor T lymphocyte induction by a factor released from cultured blastocysts

For the analysis of immunologic escape mechanisms of embryos during the implantation period in mice, the effects of culture supernatant of blastocysts on in vitro responsiveness to alloantigen of mice was investigated. Blastocyst-cultured conditioned medium was prepared by culturing late blastocysts of outbred ICR mice for 5 days. The addition of culture supernatant containing four or eight blastocysts to allogeneic mixed lymphocyte culture inhibited both the MLR responses and the generation of cytotoxic T lymphocytes (CTL). Preincubation of the culture supernatant with lymphocytes syngeneic to the responder cells of MLR induced potent suppressor cell activity in the MLR. The supernatant did not inhibit the activity of CTL at the effector phase, but preinduced suppressor cells obtained by incubation of splenocytes with the supernatant showed almost complete suppression of CTL activity at the effector phase. Both of the suppressor cells, active on MLR and at the generation phase of CTL as well as active at the effector phase, had a surface phenotype of Thy-1+ and Ig-. The suppressive material could be extracted from the eight-cell stage of fertilized ova or blastocysts but not from unfertilized ova, indicating that the production of the factor(s) is dependent on the stages of early embryogenesis. These results suggest that the active induction of suppressor T lymphocytes by the factor(s) released from implanted embryos is one of the protective mechanisms from maternal immunologic attack.     

One gene encodes two distinct Ia-associated invariant chains

Murine immune response region-encoded Ia antigens are associated not only with invariant (Ii) chain but also with several other polypeptides, most notably a 41K protein. The present report demonstrates a striking similarity between Ii and 41K. These polypeptides were found to be serologically cross-reactive, and they also display a similar peptide composition on partial enzymatic digestion. Both polypeptides are derived from distinct unglycosylated precursors, as demonstrated by tunicamycin treatment and cellfree translation. Hybrid selection of mRNA, as well as Northern blotting with an Ii cDNA, shows the presence of two mRNA coding for the Ii chain and the 41K protein. Rat fibroblasts transfected with a mouse genomic Ii clone synthesize both the murine Ii chain and the 41K protein. These observations demonstrate that a single Ii gene encodes two distinct but closely related proteins.     

Location of the SH group of the alkali light chain on the myosin head as revealed by electron microscopy

The location of the single cysteinyl residue of the alkali light chain on the myosin head was determined by electron microscopy. The cysteinyl residue of isolated alkali light chain 2 was biotinylated and the light chain was exchanged with that of heavy meromyosin in 4.7 M-NH4Cl. Avidin was attached to the biotin in the heavy meromyosin and the complex was rotary shadowed and observed in the electron microscope. The distance from the head-rod junction to the centre of avidin was 8(+/- 3) nm (mean value +/- standard deviation: n = 105).     

The Effect of Ageing on Bud Differentiation in the Moss, Physcomitrium sphaericum

In the moss Physcomitrium sphaericum, we examined the numberof buds per filament, the position of buds, and the ratio ofbud-differentiated filaments when treated with cytokinin, inrelation to the increase in the number of cells per filament. When filaments of a young protonema were treated with cytokinin,many filaments did not differentiate buds. As the number ofcells in a filament increased, both the mean number of budsper filament and the ratio of bud-differentiated filaments increased.However, the position of bud differentiation was unaffectedby application of cytokinin. A higher concentration of cytokininincreased the mean number of buds per filament and the ratioof bud-differentiated filaments. The relationship between cytokinin, ageing of filaments andthe ability to differentiate buds is discussed. (Received June 17, 1985; Accepted September 9, 1985)     

Spinach nitrate reductase: purification, molecular weight, and subunit composition

Nitrate reductase was purified about 3,000-fold from spinach leaves by chromatography on butyl Toyopearl 650-M, hydroxyapatite-brushite, and blue Sepharose CL-6B columns. The purified enzyme yielded a single protein band upon polyacrylamide gel electrophoresis under nondenaturing conditions. This band also gave a positive stain for reduced methylviologen-nitrate reductase activity. The specific NADH-nitrate reductase activities of the purified preparations varied from 80 to 130 units per milligram protein. Sucrose density gradient centrifugation and gel filtration experiments gave a sedimentation coefficient of 10.5 S and a Stokes radius of 6.3 nanometers, respectively. From these values, a molecular weight of 270,000 ± 40,000 was estimated for the native reductase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured enzyme yielded a subunit band having a molecular weight of 114,000 together with a very faint band possessing a somewhat smaller molecular weight. It is concluded that spinach nitrate reductase is composed of two identical subunits possessing a molecular weight of 110,000 to 120,000.     

Characteristics of a Urate-Degrading Diamine Oxidase-Peroxidase Enzyme System in Soybean Radicles

The cadaverine content of soybean radicles showed a maximumpeak 3–4 days after planting. The variation coincidedwith radicle uricase activity during seed germination. The uricase activity could not be fractionate when the bufferpH for the extraction was at 6.0. The addition of 1 M KCl orNaCl to the buffer allowed the extraction of the uricase activity,but an addition of 1 M MgCl₂ or BaCl₂ inhibited this enzyme'sactivity. The urate-degrading enzyme system was purified 248-fold permilligram of protein from soybean radicles. The respective K_mvalues of the diamine oxidase activity for cadaverine and ofthe urate-degrading activity for hydrogen peroxide and uratewere 1.25, 2.93 and 50.3 µM. Analysis by gel electrophoresisof the partially purified enzyme fraction revealed that theurate-degrading enzyme system consisted of a peroxidase thatdegrades urate with hydrogen peroxide and a diamine oxidasethat releases hydrogen peroxide. These data are evidence that a urate-degrading diamine oxidaseand peroxidase system exists in soybean radicles and that thereaction rate of urate-degradation is controlled by the concentrationof cadaverine. (Received November 28, 1984; Accepted April 8, 1985)