“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Failure of Neuraminidase to unmask Histocompatibility Antigens on Trophoblast

DURING outbred pregnancy the mother is exposed to genetically foreign tissue because the offspring inherits transplantation antigens from the father. The survival of the foetus is ensured by the intervention of the trophoblast which does not express transplantation antigens between mother and foetus: mouse trophoblast is not rejected even when transplanted into immune recipients^1,3. The mechanism of this failure to express histocompatibility antigens is not understood^1–4, but Kirby et al. have suggested that the extracellular fibrinoid surrounding trophoblast cells is involved^5,6. Currie has suggested that the thick sialomucinous glycocalyx of the trophoblast cell might “mask” the histocompatibility antigens on the trophoblast^7,8 and has demonstrated that neuraminidase unmasked these antigens⁸. Our experiments, however, show that trophoblast incubated with neuraminidase cannot sensitize allogeneic mice to donor histocompatibility antigens. Furthermore, pretreatment of trophoblastic implants with neuraminidase did not interfere with their proliferation and growth in highly immune allogeneic recipients.     

Substituted pyrazolones require N2 hydrogen bond donating ability to protect against cytotoxicity from protein aggregation of mutant superoxide dismutase 1

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal neurodegenerative disease. Although the cause remains unknown, misfolded protein aggregates are seen in neurons of sporadic ALS patients, and familial ALS mutations, including mutations in superoxide dismutase 1 (SOD1), produce proteins with an increased propensity to misfold and aggregate. A structure activity relationship of a lead scaffold exhibiting neuroprotective activity in a G93A-SOD1 mouse model for ALS has been further investigated in a model PC12 cellular assay. Synthesis of biotinylated probes at the N¹ nitrogen of the pyrazolone ring gave compounds (5d–e) that retained activity within 10-fold of the proton-bearing lead compound (5a) and were equipotent with a sterically less cumbersome N¹-methyl substituted analogue (5b). However, when methyl substitution was introduced at N¹ and N² of the pyrazolone ring, the compound was inactive (5c). These data led us to investigate further the pharmacophoric nature of the pyrazolone unit. A range of N¹ substitutions were tolerated, leading to the identification of an N¹-benzyl substituted pyrazolone (5m), equipotent with 5a. Substitution at N² or excision of N², however, removed all activity. Therefore, the hydrogen bond donating ability of the N²–H of the pyrazolone ring appears to be a critical part of the structure, which will influence further analogue synthesis.     

Effect of Bicuculline on the GABA Receptor of the Crayfish Neuromuscular Junction

SINCE it was reported that bicuculline is a specific antagonist of γ-aminobutyric acid (GABA)^1–3, much attention has been paid to the action of this alkaloid on cortical neurones^4–6. Thus it seemed worthwhile to test the mode of action of bicuculline on the inhibitory neuromuscular junction of the crayfish, where the action of GABA has been well documented^7,8.     

Mechanism of Antiviral Activity <Emphasis Type="Italic">in vivo</Emphasis> of Polycarboxylates which induce Interferon Production

SYNTHETIC polycarboxylates have been reported to impart resistance to viral infection to experimental animals^1–8. Injection of these polyanions induces interferon^1–3,5–9, to which it therefore seemed logical to attribute the antiviral effect. The high degree and long duration of protection, however, are not in accord with the low and transitory levels of interferon induced, suggesting that mechanisms other than interferon are involved. Certain polyanions have been found directly to inactivate virus or to inhibit its adsorption to cells¹⁰in vitro. This may delay the development of viral infection in vivo. Stimulation of reticuloendothelial cell activity, as demonstrated by increased phagocytosis induced by pyran copolymer¹¹, may deviate virus from its target cells.     

Effect of Streptomycin on Ribosome Interconversion,a Possible Basis for the Action of the Antibiotic

STREPTOMYCIN affects bacterial protein synthesis in vitro by interfering with ribosomal functions^1,2. For example, it inhibits polypeptide synthesis directed by natural mRNA^3–5 or by synthetic polynucleotides^4,6–10, and, in the latter case, causes extensive misreading of the genetic code^10–15. The drug also induces breakdown of polysomes^5,14,15 and the release of fMet-tRNA from its complex with the 70S ribosome^16–18. It has been proposed that streptomycin causes these effects by distorting the ribosomal binding sites for tRNA derivatives^14–18. The nature of the primary effect of streptomycin on the ribosome is, however, still not fully understood. We present evidence that might provide an insight into the basic mechanism underlying the mode of action of the antibiotic.     

Separation of α- and β-Globin Messenger RNAs

THE 10S RNA fraction of reticulocytes from various species contains the haemoglobin messenger RNA^1–4. When this 10S RNA fraction is added to a cell-free system derived from reticulocytes or Krebs II ascites cells, it directs the synthesis of α and β chains of haemoglobin^5–8. The α and β messenger RNA molecules contained in this fraction, however, have not yet been separated and identified. When reticulocyte. RNA of mouse is subjected to electrophoresis on 6% polyacrylamide gels, the 10S fraction contains two major bands and three minor bands⁹, suggesting that the major lOS RNA bands contain the messenger RNAs for the α- and β-globin chains.     

Distribution of N<Superscript>6</Superscript>-Methyladenine in DNA of T2 Phage and its Host <Emphasis Type="Italic">Escherichia coli</Emphasis> B

N⁶-METHYLADENINE (6-MeAde) and 5-methylcytosine occur as minor bases in bacterial and phage DNA^1–7 and seem to result from the selective methylation of adenine and cytosine residues by specific DNA methylases⁸. Methylation is the final stage in DNA synthesis and is essential for the phenomenon of host modification of phages^9–11; it is one of the mechanisms controlling DNA replication in the cell^{12, 13}. A study of the distribution of minor bases in DNA is therefore important not only for the elucidation of the specificity and mechanism of action of DNA methylases but also for an understanding of the purpose of this methylation. We believe that in Escherichia coli, DNA methylase exerts its action on adenine residues in chain terminating triplets: 6-MeAde may serve as a signal for gene termination in this system.     

Involvement of <Emphasis Type="Italic">H</Emphasis>-2 Locus in a Multigenically-determined Immune Response

IT has been known for a number of years that specific immune responses can be under genetic control. In some cases the immunological phenotype is controlled by a single Mendelian genetic factor^1–3, while in other cases a more complex mode of inheritance determines the animal's ability to mount a specific immune response^4–7. An intriguing feature of this line of investigation is that in many cases a gene controlling a specific immune response is genetically linked to a histocompatibility locus^3,8–13. We now report that a specific immune response which has been shown to be under complex genetic control⁷ can be explained by the segregation of two genetic loci, one of which is closely linked or identical to the H-2 locus.     

Inhibitors acting on Nucleic Acid Synthesis in an Oncogenic RNA Virus

IN infection with an oncogenic RNA virus, synthesis of viral RNA seems to be catalysed by an RNA dependent DNA polymerase in the host cell^1–4. Several specific inhibitors of viral DNA polymerases have been found^5–7 and Spiegelman⁸ has shown that the activity of viral enzymes depends strongly on the chemical composition of the template. We report here first a new highly specific poison of the Rauscher murine leukaemia virus (RMLV) DNA polymerases; second, several inactivators of the RNA and DNA template involved in the RMLV enzyme systems; and third, the action of actinomycin D on viral DNA polymerases and on host DNA/RNA polymerase. The results are discussed with respect to the influence of actinomycin D on virus multiplication.     

Cytotoxic Activity <Emphasis Type="Italic">in vitro</Emphasis> of Thymus-derived Lymphocytes Sensitized to Xenograft Antigens

THE importance of the participation of thymus-derived lymphocytes in both humoral antibody production^1,2 and cell-mediated anti-allograft immunity³ has been clearly demonstrated. Evidence for the involvement of thymus-derived cells in the rejection of xenografts is, however, largely indirect. In mice, antilymphocyte serum (ALS) prolongs the survival of xeno-grafted skin⁴ and promotes the growth of tumour xenograft⁵. This can be taken to imply that thymus-derived cells are involved in the rejection process because ALS is known to deplete preferentially the thymus-dependent areas of lymphoid tissue⁶. Raff⁷ has also shown that in ALS treated mice the percentage of theta (θ) positive cells in lymphoid organs is decreased.     

Photoaffinity labeling of the (Ca+Mg)ATPase of skeletal and cardiac sarcoplasmic reticulum with [γ-32P]-8-azido-ATP

8-azido-ATP, when used in the 0.2–5 μM concentration range, fulfills the criteria for a specific photoaffinity label for the (Ca+Mg)ATPase of sarcoplasmic reticulum. It is a substrate for the enzyme. It is a mixed inhibitor of ATPase activity. When photolyzed at 0° it is an inhibitor of ATPase activity. The photoinduced binding of 8-azido-ATP to the (Ca+Mg)ATPase is promoted by Ca²⁺. The dependence of the labeling of the (Ca+Mg)ATPase on 8-azido-ATP, Ca²⁺ and Mg²⁺ concentrations strongly suggests that 2 classes of sites are labeled. When 10–60 μM 8-azido-ATP was used to label sarcoplasmic reticulum, proteins in addition to the (Ca+Mg)ATPase were labeled.     

Production of Anti-glucagon Antibodies in Poly-L-lysine “Responder” Guinea-pigs

THE availability of anti-glucagon antiserum would greatly facilitate studies on the action of the pancreatic peptide hormone glucagon (molecular weight 3,48s)¹; this peptide is poorly immunogenic^2–8. Grey et al.⁹ report that immunization of rabbits with a glucagon-haemocyanin conjugate yielded antibodies of high affinity. We now describe a method of producing and assaying antibodies to glucagon in which glucagon is coupled to poly-L-lysine (PLL). Immunization of guinea-pigs with glucagon-PLL conjugate yields large amounts of high affinity anti-glucagon antibody; the immune response to PLL and hapten-PLL conjugates is determined by a dominant autosomal genetic factor¹⁰.     

Direct Action of Insulin on Plasma Membrane ATPase Activity in Human Lymphocytes

LITTLE is known about the effects of insulin on lymphocytes. Helmreich and Eisen¹ concluded that it has insignificant effects, but others^2–5 have made a case for a role in inflammatory and immunological responses. We^6,7 have demonstrated that noradrenaline enhances the uptake of both glucose and potassium by lymphocytes, as does insulin in several tissues. We have associated this action of noradrenaline with a direct effect on membrane adenosine triphosphatase (ATPase) activity⁷. The observation⁸ that insulin bound to ‘Sepharose’ polymers enhances glucose transport while in contact only with the plasma membrane indicated that insulin might have a direct action similar to that of noradrenaline on membrane ATPase. The observations reported here show that insulin stimulates ATPase activity and glucose uptake in the lymphocyte and suggest a relationship between membrane ATPase activity and glucose transport.     

RNA of Australia Antigen

ALTHOUGH the exact nature of Australia (Au) antigen is not resolved, increasing evidence suggests that it is the causal agent of viral hepatitis. This supposition is based chiefly on the frequent occurrence of Au antigen in the sera of patients with viral hepatitis^1–4 and on its virus-like appearance under the electron microscope^5–7. Biochemical studies have shown that Au antigen consists largely of protein, with a minor lipid moiety^{8, 9}. So far, however, no genetic material has been detected in the Au antigen and it has been suggested that the Au antigen might be “a unique infectious particle with little or no nucleic acid”¹⁰. We wish to present evidence, however, that RNA is an essential component of Au antigen.     

Site of Nitrogenase Activity in the Blue-Green Alga <Emphasis Type="Italic">Anabaena</Emphasis> sp. L-31

MANY blue-green algae fix nitrogen, assimilate carbon dioxide and evolve oxygen and as algal nitrogenase is inhibited^1–3 by high oxygen pressure, enhanced nitrogen fixation accompanying photosynthesis is surprising. Heterocysts do not contain⁴ or have comparatively less amounts^4–7 of photosystem II (PS II) pigments, which are responsible for the evolution of oxygen. This tends to favour the suggestion of Fay et al.⁸ that these cells are the sites of nitrogenase activity. Until now, however, attempts at obtaining unequivocal evidence for heterocysts as principal loci for nitrogenase activity have yielded conflicting results. Stewart et al.⁷ first demonstrated nitrogenase activity in heterocysts incubated aerobically, a finding confirmed by Wolk and Wojciuch⁹ and Van Gorkom and Donze¹⁰. By contrast, Smith and Evans^3,11 and Kurz and La Rue¹² reported results favouring vegetative cells as the major site of nitrogenase activity. Other evidence^2,13 showed high nitrogenase activity in cell-free preparations of Anabaena cylindrica and the non-heterocystous alga Plectonema boryanum strain 594.     

Characterization of early transient accumulation of PrP in immune cells

PrP^Sc is known to elicit no specific immune response and the immune cells are suspected to support its accumulation. In the present study, we investigated the response of some immune cell types to PrP^Sc to characterize an observed early transient accumulation of PrP^Sc. After cells were treated with PrP^Sc-brain homogenate, PrP^Sc was transiently accumulated for the first 8–12 h post-exposure then completely cleared by the 5th day of the experiment. The accumulated PrP^Sc was not a de novo product of the cell PrP^C. Further investigation of this phenomenon revealed some potential factors influencing it. These factors included cholesterol homeostasis, temperature, the degradation power of the cell and the availability of sufficient PrP^C. Our in vitro results suggest that immune cells, especially macrophages are potential risk factors for the accumulation and intercellular spread of PrP^Sc if the complete clearance of PrP^Sc were not fulfilled.     

Gene expression of foreign metaphase chromosomes introduced into cultured mammalian cells?

Transfer of genetic information from isolated hamster chromosomes to mouse cells is described. Metaphase chromosomes isolated from Chinese hamster diploid cells were incubated with mouse Cl. 1-d cells deficient in thymidine kinase activity. Two viable colonies appeared from the treated mouse cells after HAT selection with a frequency of about 10⁻⁸. The first colony isolated (Cl. 1) failed to grow, however. The second colony isolated (Cl. 2) grew well in HAT medium and was subcultured for more than 70 generations. Cl. 2 cells possessed an elevated tetrahydrofolate dehydrogenase activity of molecular species resembling that of Chinese hamster cells, as shown by disc electrophoresis. The cell line also expressed surface antigen(s) specific to hamster species, as shown by mixed hemadsorption test and immune cell electrophoresis. This latter phenotype disappeared after prolonged cultivation (59 generations) of the cells in non-selective medium. The karyotype of Cl. 2 cells corresponded to that of the mouse species and was quite different from that of hamster cells. Hamster chromosomes could not be identified in any of the cell clones by detailed analysis by the banding method (Q- and C-band). Not one revertant cell was obtained among 4.2×10⁸ Cl. 1-d cells in the control.