Inhibition of <Emphasis Type="Italic">E. coli</Emphasis> DNA Polymerase II by Ara-CTP

THE potent antileukaemic agent, 1-β-D-arabinofuranosylcytosine (ara-C), specifically inhibits DNA synthesis in bacterial and animal cells^1,2. Although the exact mechanism of inhibition has been in doubt, it seems likely that it occurs at the DNA polymerization reaction itself^2–5. We describe here the effects of ara-CTP, which is the most prominent form of ara-C in the cell, on in vitro replicative systems^6–11 of E. coli and on isolated DNA polymerases II and III^12–15.     

Lack of Anti-macrophage Species Specificity in Anti-lymphocytic Serum

THE mode of action of anti-lymphocytic serum (ALS) in suppressing cellular and humoral immune responses remains uncertain^1,2 although there is evidence that it acts preferentially on a sub-population of thymus derived small lymphocytes^3–5. Its action, however, is not confined to lymphocytes but it also affects neutrophils⁶, epithelial cells⁷ and macrophages^8–14. On the other hand, specific anti-macrophagic activity of ALS has been either questioned¹⁵ or refuted^16–18. Anti-neutrophil activity of ALS may be responsible for the anti-inflammatory properties of ALS¹⁹ which have been shown to be separate from its immuno-suppressive activity and not to prolong graft survival²⁰. Macrophages and monocytes, however, play an important role in the immune response²¹ and anti-macro phage activity might account for much of the immuno-suppressive capacity of ALS. Previous reports indicated that ALS was species but not strain specific with respect to both anti-lymphocytic⁸ and anti-macrophagic activity¹³. Species specificity of anti-lymphocytic activity is, however, not absolute^22,33,34; and this report demonstrates that the same is true for anti-macrophage activity, particularly between related species.     

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.     

Specific Collaboration between T and B Lymphocytes across a Cell Impermeable Membrane <Emphasis Type="Italic">in vitro</Emphasis>

ANTIBODY production to many antigens including heterologous erythrocytes^1–3, serum proteins^4,5 and hapten protein conjugates^6,7, occurs as a result of an interaction between antigen and thymus-derived (T) and non-thymus-derived (B) lymphocytes. Although the specificity of the antibody response is determined by T as well as B cells^8–10, T cells do not actively secrete any of the known classes of immunoglobulin molecules¹¹. Their function seems rather to initiate the sequence of events whereby antigen is presented to B cells in an immunogenic form capable of stimulating antibody synthesis¹².     

Relation between Alkaline Phosphatase and Ca<Superscript>2+</Superscript>-ATPase in Calcium Transport

SEVERAL recent studies suggest that at least part of the effect of vitamin D on intestinal calcium transport may be mediated by a calcium-stimulated ATPase (Ca ATPase) situated in the brush borders of intestinal mucosal cells¹. Intestinal brush borders are known to contain several other enzymes which may be involved in digestion and transport, but the relationship between them is poorly understood². In the case of the disaccharidases, the question of the number of enzymes responsible for the various activities has been raised³. Alkaline phosphatase is also present in brush borders but its function is unknown. During studies of the effect of various agents On intestinal calcium transport we noted a close correlation between the activities of alkaline phosphatase and Ca ATPase. We therefore wondered whether the activities might be due to a single enzyme species. Haussler et al.⁴ and Norman et al.⁵ have also noted a close relationship between the two activities under a variety of conditions but Holdsworth⁶ thinks the two activities are not related. We report here further evidence, based on denaturation by heat and on various dietary manipulations in the rat, which suggest that the two enzymes may be part of a single enzyme complex.     

Macrophage Functional Heterogeneity in the <Emphasis Type="Italic">in vitro</Emphasis> Induced Immune Response

RNA from antigenically stimulated peritoneal macrophages is immunogenic in vitro^1–4. The studies of Fishman and Adler^5–6 suggest that the peritoneal macrophage population consists of at least three functionally distinct subpopulations. Although most peritoneal macrophages act as scavenger cells⁷, a second population—possibly less than one cell per 1,000—consists of cells that produce but do not necessarily secrete antibody^8,9 and respond to antigen by synthesizing informational RNA. On transfer to normal lymphoid cells, this RNA elicits IgM antibody with the allotypic specificity of the macrophage donor¹⁰. A third type of macrophage gives rise to the RNA-antigen complex responsible for the in vitro synthesis of IgG antibody with the allotypic specificity of the lymphocyte donor¹⁰.     

Binding of <Superscript>3</Superscript>H-Concanavalin A by Normal and Transformed Cells

CERTAIN plant lectins selectively agglutinate tissue culture cells transformed by oncogenic viruses and chemical carcinogens^1–7. Agglutination of transformed cells is inhibited by certain small carbohydrates which are thought to be sterically similar to the lectin-binding sites on the cell surface. Agglutination induced by a protein from wheatgerm is inhibited by N-acetyl-glucosamine^2,3 and that induced by concanavalin A (Con A) is inhibited by α-methyl-D-glucopyranoside (α-MG⁴). Normal cells are thought also to have lectin-binding sites but in a “cryptic” form, for mild protease treatment renders them agglutinable by wheatgerm agglutinin and Con A^4,6. Transformed cells are thought to bind more lectin than untransformed cells⁵. This study was designed to test this hypothesis for jackbean lectin, Con A.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Autoradiographic Localization of <Superscript>3</Superscript>H-GABA in Rat Retina

γ-AMINOBUTYRIC acid (GABA) is present in all layers of vertebrate retinae^1–3: in the rabbit retina it seems to be most concentrated in the ganglion cell layers² while in the frog it is concentrated primarily in cell layers which are rich in amacrine cells¹. Recent autoradiographic studies of the distribution of ³H-GABA in rat brain slices after incubation in vitro suggest that the labelled amino-acid is selectively concentrated by certain neural elements^4,5. In a study of the distribution of ³H-GABA in rabbit retina after injection of the labelled amino-acid into the eye, Ehinger⁶ found that radioactivity was accumulated principally in the inner plexiform, inner nuclear and ganglion cell and nerve fibre layers. Labelling was also concentrated in some cells occupying the same position as amacrine cells and in some nerve cells of the ganglion cell layer.     

Cellular Interactions and the Secondary Response <Emphasis Type="Italic">in vitro</Emphasis>

ALTHOUGH the role of cellular cooperation in the induction of the immune response has become firmly established only recently¹, morphological evidence suggesting that such cooperation takes place is quite old. Reports² of the aggregation of lymphoid cells around macrophages³ have been confirmed: “islets”, “rosettes” or “clusters” in cultures of cells (derived from humans⁴, guinea-pigs⁵, rabbits⁶ or mice⁷) stimulated with antigen or PHA⁸ were reported. We have investigated cluster formation to ascertain its relationship, if any, to the antigen-induced stimulation of sensitized cells⁹. We used peripheral blood leucocytes from rabbits immunized to bovine serum albumin (BSA) or to human red blood cells (HRBC). The BSA was given in complete Freund's adjuvant (three intramuscular injections of 7.5 mg BSA each, into the hind legs at weekly intervals). HRBC (1 ml.) was given once into the ear vein, as a 20% suspension in saline. Cell cultures and ³H-thymidine incorporation were measured as before¹⁰. To prepare cell smears, cells were washed three times and suspended in one drop of normal rabbit serum and 1 µl. of the suspension was spread on a microscope slide. This ensured a reasonably constant number of cells per slide and made possible comparisons between different experiments. Smears were fixed with methanol and stained with Giemsa.     

Infection of Tumour Cells by Fowl Plague Virus in the Presence of Actinomycin D

INFLUENZA virus is one of the few viruses in which replication is inhibited by the antimetabolite actinomycin D (AM-D)^1–4, which inhibits DNA-dependent RNA synthesis in mammalian cells⁵. It has been reported that the growth of fowl plague virus (FPV) in virus-transformed hamster cells is less sensitive to AM-D^6,7. We have examined the sensitivity of FPV to AM-D to see whether it is related to differences in the oncogenic properties of tumour cells. We found that in cells transformed by polyoma virus (PV) and also in cells transformed by methylcholanthrene, although no infectious virus was produced the cells synthesized viral haemagglutinin (HA). It was only the cell-associated HA, however, that was affected by AM-D and not that released by the cells.     

Lymphoid Cell Lines: Activation of a Latent Herpes-like Virus Particle?

SINCE the discovery¹ of the herpes-like virus particle (EBV) in cell cultures of Burkitt's lymphoma, the virus has been found in many (but not all) lymphoid cell cultures derived from patients with Burkitt's lymphoma² and other lymphoid neoplasms³ or infectious mononucleosis⁴ as well as from normal individuals⁵. The virus has also been found in some biopsies of Burkitt tumours⁶. Serological evidence has implicated it as a causal or associated agent in heterophile-positive infectious mononucleosis^{7, 8}, although its relationship to Burkitt's lymphoma is less clear. Antigenic studies suggest a relationship between the herpes-like virus and antigens detected by indirect immunofluorescence (IF) techniques in fixed cells⁹, surface antigens on viable cells¹⁰ and complement-fixing (CF) antigens in extracts of Burkitt cell cultures¹¹.     

End-to-End Association of <Emphasis Type="Italic">X</Emphasis> and <Emphasis Type="Italic">Y</Emphasis> Chromosomes in Mouse Meiosis

ACCORDING to the hypothesis of Crew and Koller¹ and Koller and Darlington², there are homologous segments in the X and Y chromosomes of the mouse and other mammals. The homologous regions in the mouse were believed to be localized in the extremely short arms proximal to the kinetochores. The end-to-end association at meiosis was thought to be the result of the formation of a chiasma between these homologous regions³. Electron microscopy revealed a short synaptonemal complex in mouse meiotic cells⁴. However, partial sex linkage has never been demonstrated in the mouse⁵ and other authors^6–10 believe that the X and Y chromosomes associate only by connexion between the chromosome ends furthest from the centromeres.     

Subcellular Mouse Alloantigens: Cytotoxic Immune Responses and Specific Blocking <Emphasis Type="Italic">in vitro</Emphasis>

CELL-MEDIATED allograft responses (CMI) in dissociated lymphoid cell cultures can be used to investigate the interaction of alloantigen with surface receptors of immunocompetent cells^1–4. The generation of cytotoxic lymphocytes (CL) in vitro has been shown to be a function of thymus and thymus-derived (T) lymphocytes^5,6. In vitro systems used to induce a CMI involved cell-bound alloantigens^1–6 and therefore were unsuitable for detailed studies of the interactions occurring on the surface of reacting T lymphocytes. Here we describe the induction of a primary cytotoxic allograft response in vitro by subcellular alloantigens. Moreover, evidence is given that subcellular alloantigens block specifically the cytotoxic activity of CL suggesting that the specificity of the cytotoxic action is dictated by surface receptors.     

DIATOM MINERALIZATION OF SILICIC ACID IV. KINETICS OF SOLUBLE Si POOL FORMATION IN EXPONENTIALLY GROWING AND SYNCHRONIZED NAVICULA PELLICULOSA1

Silicic acid taken up from the growth medium by Navicula pelliculosa (Bréb.) Hilse was shown to enter at least two compartments: i) soluble pools; ii) insoluble fraction comprised predominantly of the silica frustule. Soluble Si pools were extracted by a variety of agents from cells uniformly labeled for ten generations in medium containing ⁶⁸Ge-Si(OH)₄. 100 C water soluble and 0 C perchloric acid (PCA) soluble Si pools of 680 mM Si·l^?1 and 490 mM Si·l^?1 cell water represented 13 and 9%, respectively, of total, cell Si in exponential growth phase cells. Uniformly labeled cells synchronized by the combined synchronization technique accumulate at the cell cycle stage where silica frustule development is initiated. These cells contain water and PCA soluble pools of 10 nmol Si·10⁶ cells^?1 and-8.8 nmol Si·10⁶ cells^?1, respectively. On addition of Si(OH)₄, a rapid uptake ensues allowing the Si pool to expand 2.5-fold, apparently to provide precursors of the silica frustule.     

Difference in the Cyclic Adenosine 3′,5′-Monophosphate Levels in Normal and Transformed Cells

CYCLIC 3′5′-adenosine monophosphate (cyclic AMP) regulates many physiological phenomena^1,2. Cellular morphology changes when the dibutyryl derivative of cyclic AMP is added in vitro to the nutrient media of cultured mammalian cells^3–6. Dibutyryl cyclic AMP has also been shown to restore controlled growth to transformed cells³, change the cell's surface architecture^3,7 and induce axon formation⁸ with an accompanied increase in acetylcholinesterase activity⁹ in neuroblastoma cells growing in culture. These effects suggest that the cyclic AMP moiety may have some basic regulatory action on cell growth and cell specialization.     

Immunological Potential of the in vitro Mixed Skin Cell-Leukocyte Reaction

THE mixed skin cell-leukocyte reaction (MSLR) is an in vitro reaction between dissociated skin cells and allogeneic peripheral blood leukocytes. It is similar in its immunological implications to the reaction between allogeneic leukocytes, commonly known as the mixed leukocyte reaction (MLR) or the mixed lymphocyte interaction (MLI). In the latter, immunological recognition between lymphocytes from genetically disparate donors can be detected and measured either by blastoid transformation, enhanced mitotic rate or induction of DNA synthesis^1–4. This system has been useful in the study of the transfer of transplantation immunity in vivo by lymphocytes sensitized in vitro⁵ and also in the identification of tissue incompatibility between donor and recipient^{6, 7}. In studies with inbred strains of rats the proliferative phase of the MLR has been shown to possess characteristics required for an immunological response; that is, selectivity and specificity^{8, 9}. Furthermore, mouse peripheral lymphocytes sensitized in mixed culture with allogeneic lymphocytes have been shown to engender immunospecific destruction of target cells¹⁰, an event which is directly associated with allograft rejection.