Purification of natural human interferon-gamma by antibody affinity chromatography: analysis of constituent protein species in the dimers

A simple procedure for purifying human interferon-gamma from leukocytes was established, based on monoclonal antibody affinity chromatography. The recovery of interferon activity was essentially quantitative, and the specific activity of the product was (4-12) x 10(7) international units/mg protein. SDS-polyacrylamide gel electrophoresis reproducibly revealed four components associated with interferon activity (and no other proteins): two major ones with molecular weights (MW) of 24,000-25,000 (25K) and 19,000-20,000 (20K), a minor one with MW 14,000-15,000 (15K) (these three bands were doublets), and a still less prominent one(s) with MV 40,000-48,000. Gel filtration in neutral solution indicated that all the 25K, 20K, and 15K species exist as oligomers, probably dimers. By means of experiments using a cleavable crosslinking reagent, the dimers were shown to comprise both homo-and heterodimers. Gel filtration in alkali (the condition used during purification) indicated that the molecules are largely in a monomeric state. Thus, the molecules once dissociated in alkali appear to reassociate at random upon neutralization; this process takes place without being accompanied by inactivation.     

Interactions of permeant cations with sodium channels of squid axon membranes.

To determine how the permeant cations interact with the sodium channel, the instantaneous current-voltage (I-V) relationship, conductance-ion concentration relationship, and cation selectivity of sodium channels were studied with internally perfused, voltage clamped squid giant axons in the presence of different permeant cations in the external solution. In Na-containing media, the instantaneous I-V curve was almost linear between +60 and -20 mV, but deviated from the linearity in the direction to decrease the current at more negative potentials. The linearity of instantaneous I-V curve extended to more negative potentials with lowering the external Ca concentration. The I-V curve in Li solution was almost the same as that in Na solution. The linearity of the I-V curve improved in NH4 solution exhibiting only saturation at -100 mV with no sign of further decrease in current at more negative potentials. Guanidine and formamidine further linearized the instantaneous I-V curve. The conductance of the sodium channels as measured from the tail current saturated at high concentrations of permeant cations. The apparent dissociation constants determined from the conductance-ion concentration curve at -60 mV were as follows: Na, 378 mM; Li, 247 mM; NH4, 174 mM; guanidine, 111 mM; formamidine, 103 mM. The ratio of the test cation permeability to the sodium permeability as measured from the reversal potentials of tail currents varied with the test cation concentration and/or the membrane potential. These observations are incompatible with the independence principle, and can be explained on the basis of the Eyring's rate theory. It is suggested that the slope of the instantaneous I-V curve is determined by the relative affinity of permeant cations and blocking ions (Ca) for the binding site in the sodium channel. The ionic selectivity of the channel depends on the energy barrier profile of the channel.     

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.