High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor

Interleukin-1 beta (IL-1 beta) is derived from an inactive precursor by proteolytic cleavage. To study IL-1 beta processing, we expressed the precursor in Escherichia coli, partially purified it, and used it as a substrate for various potentially relevant protease preparations. The precursor alone was virtually inactive, but incubation with membranes from human monocytes or myeloid cell lines yielded a 500-fold increase in IL-1 bioactivity. Western blot analysis of the incubated material showed that the 31,000-Da precursor is broken down to three major products, ranging from 17,400 to about 19,000 Da. The most active of these products is the smallest one, and it co-migrates during electrophoresis with mature IL-1 beta. Four purified known proteases were also tested for their effect on precursor IL-1 beta, and none of these products co-migrated with the mature protein. Chymotrypsin and Staphylococcus aureus protease yielded slightly larger products, which were highly active. Elastase and trypsin yielded substantially larger products, and these had little IL-1 activity. The products of three of the known proteases were identified by NH2-terminal sequencing. These results show conclusively that proteolysis of precursor IL-1 beta generates biological activity and that the cleavage must occur close to the mature NH2 terminus.     

Regression of line-10 hepatocellular carcinomas following treatment with water-soluble,microbial extracts combined with trehalose or arabinose mycolates

Summary Intratumor injections of the aqueous phase of phenol-water extracts of Re mutant Salmonella typhimurium (Re glycolipid) in combination with trehalose dimycolate at dose levels of 150 to 15 g were consistently and highly effective (65–93%) in producing regression of line-10 tumors in strain-2 guinea pigs. We observed that the rate of regression was more rapid than that seen after treatment with cell walls from Mycobacterium bovis strain Bacillus of Calmette and Guèrin (BCG). Arabinose mycolate could be substituted for trehalose dimycolate in the Re glycolipid-mycolate mixture without appreciably compromising antitumor activity, providing that the level of arabinose mycolate was not reduced below 15 g. In addition to the Re glycolipid preparation, similarly prepared aqueous extracts from Mycobacterium bovis strain BCG and strain AN5 in combination with trehalose dimycolate also possessed tumor-regressive activity. The activity of these last extracts was reduced when the arabinose mycolate was substituted for the trehalose dimycolate. The aqueous extract of a rickettsia, Coxiella burnetii, in combination with either trehalose dimycolate or arabinose mycolate was also active (50 and 80% tumor regression rates, respectively). Intracutaneous administration of Re glycolipid or aqueous extracts from BCG in combination with trehalose or arabinose mycolates did not produce life-threatening, clinical signs of toxicity in young mice. If additional toxicity studies demonstrate that adverse side effects can be satisfactorily controlled, these watersoluble extracts may prove beneficial in the treatment of spontaneous tumors of humans and other animals.     

Further studies on the structural requirements of agents for immunotherapy of the guinea pig line-10 tumor

Summary We made a comparative study of the in vivo binding of immunoglobulins (Ig) to a polyoma virus-induced ascitic tumor propagated in syngeneic or allogeneic mice. The Ig coat was found to appear more rapidly and to be denser in H 2-incompatible than in H 2-compatible mice. This suggests that antibodies were fixed specifically on strong normal transplantation antigens (H-2) recognized as non-self by allogeneic mice. Experiments with mice in which immunosuppression had been achieved by means of X-irradiation confirmed that the Ig fixed on SEWA cells are actively bound antibodies. The only mice that could fix Ig on tumor cells were those that had been specifically immunized against cell surface antigens shared by SEWA cells before irradiation, while mice hyperimmunized against nonrelated antigens could not.In partial fulfilment of doctorate thesis requirements     

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

Different structural requirements of endotoxic glycolipid for tumor regression and endotoxic activity

Endotoxic glycolipid extracted from the heptose-less mutant of $\text{Salmonella typhimurium}$ was treated with alkali and acid reagents. The glycolipid freed of all O-ester linked fatty acids by hydroxylamine had lost tumor regression activity and toxicity, whereas a partial removal of O-ester linked fatty acids by mild alkali did not impair with these activities. The glycolipid retained both activities after removal of 2-keto-3-deoxyotonate by sodium acetate (pH 4.5) but was rendered nontoxic while retaining antitumor activity when hydrolyzed by 0.1N HCl whereby 2-keto-3-deoxyoctonate and glycosidic phosphate was split off the glycolipid molecule. Nontoxic and tumor regressive fractions were separated by means of preparative thin layer chromatography of glycolipid hydrolyzed by mild acid. Thus, it was concluded that glycosidic bound phosphate and at least a portion of fatty acids of the lipid A moiety were essential for toxicity, but that this phosphate is not essential for tumor regression activity.     

CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.

T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.     

Activation and mechanism of action of suppressor macrophages

Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro. CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.