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.     

Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis.

Summary From a collection of 8,000 transposon-insertion mutants of Escherichia coli K12 we identified two mutations, trg-1::Tn5 and trg-2::Tn10, that simultaneously eliminate chemotactic response to ribose and galactose, two attractants recognized by independent receptors. We show that these transposon-insertions confer a Trg phenotype, indicating that this specific pattern of tactic defects is a null phenotype. The two mutation sites are cotransductionally linked to an extend consistent with placement in the same gene. The Trg phenotype of a family of deletion mutants produced by curing trg-2::Tn10 implies that trg is a single gene. Experiments with appropriate F-primes and Hfr's locate the trg locus at approximately 31 min on the linkage map, with a marker order: pyrF-rac-(P.O. 43)-trg-man.We also found one trg mutant whose Trg phenotype was not linked to a transposon-insertion but is probably the result of a mutator activity in the parent strain. Selection of transposon-insertions near, but not in trg allowed demonstration of a very close linkage between the spontaneous trg-3 and the transposon-generated trg's, indicating all three mutations are probably in the same gene. In our manipulations of transposon-insertions we found that Tn5 had a tendency to translocate from its initial site of insertion while Tn10 was relatively stable.The trg-product is probably a chemotactic signal transducer, which interacts directly with two independent receptor proteins and transmits information to the central chemotactic machinery.     

The roles of ethanol and of acid in the production of gastric mucosal erosions in rats

This study was undertaken to differentiate between the morphological changes produced in chambered rat gastric mucosae by 40% ethanol and by 50 mM HCl. 40% ethanol produced both focal mucosal hyperemia and widespread exfoliation of the surface epithelium. Massive release of mucus accompanied both events. In the absence of acid the released mucus was stabilized by a network of fibrin, and epithelial continuity was re-established over non-hyperemic regions by migration of epithelial (and parietal) cells from the gastric pits. Hemorrhagic erosions occurred only in the presence of acid, but were limited to the hyperemic regions. Acid had the following effects: (1) platelet thrombi were destroyed, thus promoting hemorrhage; (2) destruction of the fibrin network by acid caused dissipation of the adherent mucous coat; (3) vulnerable cells which had previously shown only ischemic damage were irreversibly damaged by acid; (4) exposed basal lamina was destroyed, thus removing the substratum necessary for orderly epithelial re-establishment.     

Cross-reactivity between human and murine lymphocyte antigens

The anti-H-2 alloantiserum D-32 [(BlO.A(2R) × C3H.SW) anti-C3H] is cytolytic to human lymphocytes. Fab₂ blocking assays, indirect immunoprecipitation and sequential immunoprecipitation experiments showed that the anti-H-2 alloantiserum D-32 recognizes antigenic determinants which are expressed on the heavy chain of subpopulations of HLA-A, B antigens. These determinants are different from those defining the serological polymorphism of the HLA-A, B, C system, are the same as or spatially close to those recognized by the anti-HLA-A, B monoclonal antibody Q6/64 and are expressed on rabbit, rat or guinea pig lymphocytes.     

Modelling the rate of transfer of uranyl ions onto microbial cells

It has been observed that microbial cells can adsorb uranium ions from dilute aqueous solutions. Data collected from such experiments can be used to estimate correlative mass transfer coefficients. Physical observations bear out several inadequacies, however, of using an adsorption mass transfer model with a constant transfer coefficient relating the rate of transfer to the concentration gradient. By itself, the mass transfer model contains no provision to include (1) the initial transient, (2) the curvature in the later time rate curve, and (3) the non-linear curve relating initial levels of uranium concentration in solution to final residual uranium concentration for a set of batch experiments. It is found that a better match to observed data can be achieved by utilizing an intermediate state adsorption model analogous to a kinetic model based on an enzyme - substrate coupling scheme.     

Oxygen consumption during the fenton-type reaction between hydrogen peroxide and a ferrous-chelate (Fe2+-DTPA)

The Fenton-type reaction between ferrous diethylenetriamine pentaacetic acid (Fe²⁺-DTPA, 50–200 μM) and H₂O₂ (20–1000 μM) in phosphate buffer at pH 7.0 results in consumption of dissolved oxygen. This observation differs from many prior reports that oxygen is liberated when more concentrated solutions of H₂O₂ are decomposed by iron salts. The rate and total quantity of oxygen consumed were dependent upon the concentrations of ferrous chelate, H₂O₂, and excess DTPA. Evidence is provided that both the ferrous-DTPA chelate and free DTPA can participate in the oxygen-consuming reactions. Oxygen was also consumed during the Fenton reaction between ferrous ions and H₂O₂ when DTPA and phosphate buffer were omitted. Under these conditions, oxygen evolution was observed at higher H₂O₂ concentrations (e.g., 400 μM). The consumption of oxygen during the Fenton-type reaction of an iron chelate at neutral pH may be relavant to events that take place in biologic systems.     

Determination of indoles and catechols in rat brain and pineal using liquid chromatography with fluorometric and amperometric detection

Tryptophan, serotonin, 5-hydroxyindoleacetic acid, and homovanillic acid were determined in rat brain by the direct injection of a centrifuged tissue homogenate into a liquid chromatographic—fluorometric/amperometric system. The above indoles, along with melatonin, were also determined in single rat pineal glands. The utility of the system in determining several additional catechols and indoles in brain was examined.     

The hypothalamo-choroidal tract

Summary Light and electron microscopic examination of choroid plexuses from lateral ventricles of water-deprived and subcutaneously or intravenously vasopressin administered rats reveal morphologic changes typical for vasopressin responsive fluid transporting epithelia during hormonal stimulation. Ultrastructural changes noted in both dehydrated and vasopressin treated animals included: the frequent occurrence of choroidal dark cells, dilatation of the lateral and basal intercellular spaces, increased vacuolization of the apical cytoplasm, and a change in microvillar conformation from the normal clavate type to those with a filiform shape. On the basis of the ultrastructural changes observed it is proposed that the choroid plexus be regarded as a target tissue for vasopressin. These findings indicated that a vasopressinmediated transchoroidal cerebrospinal fluid absorption capability exists.Supported by U.S.P.H.S. Grant HD-08867This work submitted as partial requirement for the Master of Science degree in the Department of Anatomy, Colorado State University