DISAGGREGATION OF POLYSOMES IN FETAL ORGANS AND MATERNAL BRAIN AFTER ADMINISTRATION OF d-LYSERGIC ACID DIETHYLAMIDE IN VIVO

The intravenous administration of d-lysergic acid diethylamide (LSD) to pregnant female rabbits induced hyperthermia as well as disaggregation of polysomes in fetal organs and maternal brain. The LSD-induced polysome shift in maternal brain and fetal organs was not due to an activation of ribonuclease or associated with alterations in the levels of free amino acids. Pretreatment with the receptor blocking agents haloperidol and pizotyline blocked both LSD-induced polysome shift in maternal brain and fetal organs and LSD-induced hyperthermia. Fine dissection of adult rabbit brain showed that the extent of LSD-induced disaggregation of polysomes does not exhibit any marked regional variation.     

Establishment of a C3Hf Mammary Tumor Cell Line Expressing Endogenous Mouse Mammary Tumor Virus: Antigenic and Genetic Relationships of This Virus with Highly Oncogenic Mouse Mammary Tumor Viruses

A single-cell clone of C3Hf mammary tumor cells (clone 14) was developed into a continuous cell line expressing high levels of endogenous mouse mammary tumor virus (MMTV) with less than 0.1% murine leukemia virus expression. Comparison of the C3Hf MMTV protein profile on sodium dodecyl sulfatepolyacrylamide gel electrophoresis with that of C3H MMTV revealed that the protein content of the two viruses was quite similar. However, oligonucleotide fingerprints obtained of MMTV 70S RNA revealed that approximately 20% of the large oligonucleotides examined were unique to each virus. The oligonucleotide fingerprint indicated that although the viruses were similar, they differed in their genetic content. The differences in the two viruses extended to immunological differences in the major envelope glycoprotein, gp52. C3Hf MMTV competed only partially in a homologous radioimmunoassay for gp52 of C3H MMTV, whereas C3H MMTV gave complete competition, indicating that gp52 of C3H MMTV contained type-specific determinants not present on gp52 of C3Hf MMTV. Comparison of C3Hf MMTV with highly oncogenic C3H, GR, and RIII MMTVs in a homologous C3H MMTV gp52 assay gave two patterns of reactivity: complete competition by GR and C3H MMTV and incomplete competition by C3Hf and RIII MMTV. Absorption of anti-C3H MMTV serum by either C3Hf MMTV or RIII MMTV removed all antibodies against both viruses but not against GR and C3H MMTVs. These results indicate that C3H and GR MMTVs are more closely related to each other than to RIII and C3Hf MMTVs.     

Mice with Spontaneous Mammary Tumors Develop Type-Specific Neutralizing and Cytotoxic Antibodies Against the Mouse Mammary Tumor Virus Envelope Protein gp52

Sera from C3H mammary tumor-bearing mice contain cytotoxic antibodies for mouse mammary tumor virus (MMTV)-producing cells, based on (51)Cr release in a complement-dependent serum cytotoxicity assay. The cytotoxic antibodies could be absorbed by purified C3H MMTV gp52 and C3H MMTV-infected cat cells (C3H [MMTV] CrFK) containing cell surface MMTV gp52. However, purified MMTV p27 and uninfected CrFK cat cells were negative. Absorption of the sera with GR (MMTV) CrFK cells also removed all of the cytotoxicity, whereas absorption with RIII (MMTV) CrFK cells was negative, even though all three infected cat cells contained equivalent amounts of gp52. The same C3H cytotoxic sera also neutralized the focus-forming capacity of a C3H MMTV pseudotype of Kirsten sarcoma virus containing MMTV gp52. In contrast, sera from mammary tumor-bearing GR and RIII mice did not neutralize the pseudotype. Furthermore, neutralization could be achieved only by anti-gp52 but not by anti-gp36, -p27, -p14, or -p10 C3H MMTV sera. The gp52's of C3H, GR, and RIII MMTV could also be distinguished by using a type-specific competition radioimmunoassay employing (125)I-gp52 of C3H MMTV and a hyperimmune rabbit anti-C3H MMTV serum. To demonstrate these differences directly, we studied the primary structure of gp52 on the surface of the C3H, GR, and RIII (MMTV) CrFK cells. Two-dimensional tryptic peptide maps of the cell surface lactoper-oxidase-catalyzed iodinated gp52's revealed a greater number of peptides common to the gp52's of C3H and GR MMTVs than to RIII MMTV gp52. These results demonstrate that gp52 is a major target antigen for both cytotoxic and neutralizing antibodies, that the cell surface and virion-associated gp52's of C3H, GR, and RIII MMTV contain both group- and type-specific determinants, and that C3H and GR MMTV gp52's are antigenically more related to each other than to RIII MMTV gp52. Furthermore, C3H mammary tumor-bearing mice develop type-specific antibodies capable of recognizing unique gp52 determinants and, therefore, are able to distinguish the gp52 of C3H MMTV from the gp52's of GR and RIII MMTV.     

Effect of carbon dioxide on pigment and membrane content in Synechococcus lividus

The effect of carbon dioxide on pigment and membrane content in Synechococcus lividus was studied by depriving cells of CO₂ and examining cell populations biochemically and by electron microscopy. After 120 h of CO₂ deprivation, S. lividus lost all detectable chlorophyll a and C-phycocyanin. Such bleached cultures were mustard yellow, the result of approximately 1.8 times more carotenoid per cell than green control cultures.Although cells from beached cultures appeared morphologically identical to control green cells when examined by light microscopy, electron microscopic examination revealed them to be devoid of detectable thylakoid membrane. Thylakoid membrane could not be recovered by physical isolation or revealed by freeze etching of bleached S. lividus. In addition, inclusion bodies characteristically found in S. lividus were also absent.Reintroduction of CO₂ into bleached cultures resulted in a rapid resynthesis of both chlorophyll a and C-phycocyanin. Electron microscopic examination of these regreening cultures revealed that thylakoid membrane was also rapidly resynthesized. Growth of regreened cultures did not occur until there was the synthesis of a full complement of chlorophyll a, C-phycocyanin, and thylakoid membrane.A time course study of the cytological events occurring during bleaching and regreening is presented.     

Defluorination of fluoroacetate in the rat

Rats given 5 ppm F as FAc (equivalent to 26 ppm of NaFac) in the drinking water for approximately four months deposited as much fluoride in the skeletal system as did rats receiving 5 ppm F as NaF in the water. Little evidence could be found for the presence of organically bound fluoride in bone after ingesting FAc, though an appreciable proportion of skeletal fluoride deposited when NaF was ingested was shown not to respond to the fluoride ion electrode. The daily urinary excretion of total fluoride after FAc was somewhat greater than after NaF; about two thirds of this fluoride responded to the electrode, whereas more than 90 percent of the total fluoride after NaF was ionic in nature. The data are interpreted as showing that the rat is capable of splitting the C-F bond in FAc and/or in its fluoride-containing metabolites, with subsequent skeletal storage and renal excretion of the released fluoride ion. The chronic administration of this low level of FAc caused an early but temporary retardation of growth. The Krebs cycle was interfered with, as evidenced by increased concentrations of citrate in the kidney and urine. At termination of the experiment, histological examination of the testes showed that the FAc had induced severe damage characterized by massive disorganization of the tubules, nearly total loss of functional cells, absence of sperm, and damage to the Sertoli cells.     

Correlation of cytokinins and abscisic acid with monocarpic senescence in soybeans

The involvement of cytokinins and abscisic acid (ABA) in themonocarpic senescence (foliar yellowing following fruit development)of soybeans was examined. Foliar sprays of cytokinin (10^–4M zeatin or 10^–5 M benzyladenine), begun when the plantsfirst set fruit and repeated every other day, significantlydelayed, but did not prevent, monocarpic senescence. Foliarsprays of 10^–4 M ABA, applied in the same manner, significantlyhastened senescence of fruiting soybeans but apparently hadno effect on depodded plants. Leaf and stem material from pre-senescentand senescent plants was extracted, chromatographed, and bioassayedfor cytokinin-lilce activity (Amaranthus betacyanin productionassay) and ABA-like activity (oat coleoptile straight growthassay for inhibitors). ABA-like activity increased, and cytokinin-likeactivity decreased in shoot tissue before the plants began tosenesce. Cytokinin-like activity in the fruit also declinedduring this period. These results implicate a decrease in cytokininsand an increase in ABA-like inhibitors in the control of monocarpicsenescence of soybeans, but neither alone is causal. ¹ Supported in part by Research Grant 416-15-79 from the USDACooperative State Research Service under PL 89–106. ² Present address: Biology Dept., College of St. Benedict'sSt. Joseph, Minn. 56374, U.S.A. (Received February 4, 1978; )     

Isolation and characterization of kinetoplast DNA and RNA of Phytomonas davidi

Kinetoplast DNA networks were isolated from stationary-phase culture forms of Phytomonas davidi. The networks banded in CsCl at a density of 1.699 g/ml and consisted of covalently closed circular molecules. The networks were sensitive to shear forces and exhibited several discrete sedimenting components in neutral and alkaline sucrose. Closed monomeric minicircles were isolated from sonicated networks by alkaline band sedimentation. Closed monomers showed a heterogeneous banding pattern on electrophoresis in acrylamide-agarose gels and had sedimentation coefficients of 20.5 S in alkaline sucrose and 11 S in neutral sucrose. The mean minicircle molecular weight as measured by cospreading with φXRF II was 0.70 × 10⁶ or 1064 nucleotide pairs. Minicircles exhibited a sequence microheterogeneity as evidenced by restriction enzyme analysis, melting analysis, and renaturation kinetics. Network maxicircles were evidenced by the appearance of high molecular weight fragments after restriction with several enzymes and by the existence of supertwisted “edge loops” extending out from the periphery of networks. The maxicircle molecular weight was estimated to be approximately 24 × 10⁶. A purified kinetoplast-mitochondrion fraction was found to contain 9 and 12 S RNA species that comigrated with L. tarentolae 9 and 12 S kinetoplast RNAs.     

Neutrophil aggregation is beta 2-integrin- and L-selectin-dependent in blood and isolated cells.

Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.