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NF-κB signaling plays a pivotal role in control of the inflammatory response. We investigated how the dynamics and function of NF-κB were affected by temperature within the mammalian physiological range (34 °C to 40 °C). An increase in temperature led to an increase in NF-κB nuclear/cytoplasmic oscillation frequency following Tumor Necrosis Factor alpha (TNFα) stimulation. Mathematical modeling suggested that this temperature sensitivity might be due to an A20-dependent mechanism, and A20 silencing removed the sensitivity to increased temperature. The timing of the early response of a key set of NF-κB target genes showed strong temperature dependence. The cytokine-induced expression of many (but not all) later genes was insensitive to temperature change (suggesting that they might be functionally temperature-compensated). Moreover, a set of temperature- and TNFα-regulated genes were implicated in NF-κB cross-talk with key cell-fate–controlling pathways. In conclusion, NF-κB dynamics and target gene expression are modulated by temperature and can accurately transmit multidimensional information to control inflammation. [ABSTRACT FROM AUTHOR]

Short-chain fatty acids are produced at high concentration in the colonic lumen and stimulate electroneutral Na+ absorption by activating apical Na+/H+ exchange in colonocytes. We used an epithelial cell line derived from a human colon carcinoma (HT29-18-C1) to study activation of apical and basolateral Na+/H+ exchange by a short-chain fatty acid, propionate. Confluent cell monolayers on membrane filters were loaded with 2',7'-bis(2-carboxyethyl)-5 (and 6)-carboxyfluorescein (a fluorescent pH indicator) and intracellular pH was monitored with a digital fluorescence imaging microscope. Cells acidified by transient exposure to NH4Cl demonstrated both apical and basolateral Na+/H+ exchange. In this condition, apical Na+/H+ exchange was 50% of the total Na+/H+ exchange activity. Similar results were obtained when cells were bilaterally perfused with apical and basolateral propionate in an isosmotic medium (130 mM propionate at each membrane surface). However, apical Na+/H+ exchange was a significantly larger fraction (76%) of the total Na+/H+ exchange activity when cells were acidified by exposure to apical propionate alone. Conversely, in cells acidified by basolateral propionate alone, apical Na+/H+ exchange was 21% of the total Na+/H+ exchange activity. The change in relative activity was observed in individual cells which expressed both apical and basolateral Na+/H+ exchange and occurred rapidly (within 7 min). In the presence of transepithelial propionate gradients, all Na(+)-dependent alkalinization was sensitive to 3 microM 5-(N-ethyl-N-isopropyl)amiloride, a potent Na+/H+ exchange inhibitor. These results suggest that transepithelial gradients of short-chain fatty acids, which occur in vivo, can cause preferential activation of apical Na+/H+ exchange.

A specific probe for detecting ecotropic murine leukemia virus sequences was constructed by cloning a 500-base-pair DNA segment, corresponding to a portion of the env region of the AKR ecotropic virus, in a pBR322/Escherichia coli K-12 host/vector system. This probe was used to screen the cellular DNAs of six inbred strains of mice for the presence of ecotropic retroviral DNA sequences by the Southern blot hybridization procedure. Three copies of ecotropic viral DNA were detected in AKR/N (a high-ecotropic virus strain) and two were found in BALB/c (a low-ecotropic virus strain) DNAs. As expected, no sequences reactive with this probe were found in NFS mouse DNA (a virus-negative strain). However, cellular DNA sequences that reacted strongly with the ecotropic-specific DNA probe were detected in certain NZB, C57L, and 129 mice (all virus-negative strains). In contrast to the reactive sequences in AKR and BALB/c, the reactions were chiefly associated with EcoRI segments that were subgenomic in size.

Congenic mouse strains NIH,Akv-1 and NIH,Akv-2 carry the two high ecotropic virus-inducing loci of the AKR mouse on the NIH Swiss genetic background. Progeny tests of animals in three separate congenic families show that both Avk-1 and Akv-2 are stably transmitted as classical mendelian loci in these mice. However, during the process of inbreeding, additional chromosomal viral loci were detected in several NIH.Akv-1 sublines. These loci appeared only in the progeny of virus-positive females. They segregate with mendelian ratios, are unlinked to markers on chromsome 7 near Akv-1, and are phenotypically expressed as high-virus-inducing loci. The generation of new loci for viurs induction, no doubt resulting from the rare germ-line reintegration of the endogenous ectropic provirus, represents a unique form of gene duplication and rearrangement.

The tumorigenicity of DNA from polyoma virus after cleavage with a variety of restriction enzymes was evaluated in suckling hamsters. Cleavage with enzymes that interrupt the region of the genome coding for the large tumor (T) antigen of polyoma virus markedly enhanced the tumorigenicity above that observed with DNA I of the virus. Cell lines established in vitro from tumors induced by polyoma virions, polyoma virus DNA I, or polyoma virus DNA that had been cleaved with restriction endonucleases in the early region all contain the polyoma virus middle and small T antigens but not the large T antigen of polyoma virus is not required for maintenance of the transformed state and probably not for initiation of tumorigenesis by viral DNA.

Recently, a novel class of murine type C virus (MCF), some strains of which are highly oncogenic in the AKR acceleration test, has been isolated from premalignant and malignant thymuses of AKR mice. The biology of these viruses suggested that MCFs are the product of recombination between endogenous ecotropic and xenotropic viruses and, further, that the recombination has taken place within the envelope (env) gene which encodes the surface glycoprotein (gp70) of the virion. We have compared by tryptic peptide analysis, the gp70s of four MCF isolates with the gp70s of various possible parental viruses. In addition, we have compared the tryptic peptides of the gag gene products p30 and p15 from several of these viruses. The results allow the following conclusions: (i) the gp70s of the MCF viruses are not identical to one another and are different from the gp70s of the possible parental viruses tested; (ii) the MCF virus gp70s have tryptic peptides in common with xenotropic virus gp70s as well as with ecotropic virus gp70s; and (iii) the gap region protein, p30, of the MCFs tested is identical to p30 of AKR ecotropic virus (Akv-1 or Akv-2) and distinct from p30 of xenotropic viruses, suggesting that the 5' end of the recombinant viruses is of Akv origin. The findings are discussed with respect to the possible role a recombinant virus might play in leukemogenesis in AKR mice.

Murine leukemia viruses capable of malignant transformation of mink tissue culture cells have been isolated from an AKR thymoma cell line and from a spontaneous reticulum cell sarcoma in an NIH Swiss mouse partially congenic for the AKR ecotropic virus-inducing locus Akv-2. In contrast to the recently described mink cell focus-inducing strains of murine leukemia virus, at least one of the two transforming strains is replication defective. Nonproducer mink cells carrying the genome of the transforming virus of AKR origin have been isolated, and pseudotype transforming viruses generated.

Using a [3H]DNA probe prepared from AKR murine leukemia virus, we determined the number of copies of the AKR virus genome integrated into the cellular DNA after exogenous infection of NIH mouse, AKR mouse, and rat cells in tissue culture. NIH mouse cells, which lack a portion of the viral genome (referred to as Gross-AKR specific sequences), incorporated three to four copies of these sequences per haploid genome. AKR cells, in which the Gross-AKR specific sequences are already present as three to four copies per haploid genome, did not shwo any distinct change in copy number after infection. Rat cells, which lack DNA sequences homologous to murine leukemia virus, incorporated one copy of the viral genome per haploid genome. It is inferred that the presence of viral sequences may affect the efficiency of integration of exogenous provirus, and that there may be a limit to the number of copies that can be inserted.

The proviruses of the N-tropic, ecotropic virus (AKV) of AKR mice (Akv-1, Akv-2) have been studied by the Southern gel--filter transfer technique. These proviruses can be detected by cleavage of cell DNA by BamHI endonuclease, which yields characteristic subgenomic DNA fragments upon cleavage of this type of provirus. Proviruses integrated into different sites in the mouse genome can be resolved with EcoRI endonuclease, which does not cleave the AKV proviruses. Use of congenic and backcrossed mice and a radioactive DNA probe enriched for AKV sequences has allowed identification of the EcoRI fragments carrying the proviruses of the genetically defined Akv-1 and Akv-2 loci. Novel proviruses introduced by superinfection of cultured AKR cells with AKV and present in leukemic cells from AKR mice have also been identified. Comparison of substrains of AKR mice indicates some heterogeneity in their spectra of proviruses.

Studies with a single-stranded DNA probe complementary to the RNA of mouse-tropic AKR murine leukemia virus indicate that the complete genome of the AKR-type murine leukemia virus is present in the DNA of high- and low-virus-yielding mouse strains, while DNA of non-virus-yielding strains contains only a part of the genome. Furthermore, in those strains where the genome is complete, two populations of virus-specific DNA sequences can be identified (more abundant and less abundant species) according to their rate of association with the probe. Low-virus-yielding mouse strains contain fewer copies of the less abundant species and, consequently, fewer complete viral genomes than do high-virus-yielding strains. Thus, in the ten strains tested, there is a good correlation between completeness of the genome of AKR-type murine leukemia virus in cellular DNA and the capacity of the cells to release infectious AKR-type murine leukemia virus. Moreover, the number of complete viral genomes correlates with the frequency of infectious virus production by virus-positive strains. DNA from wild Mus musculus also contained viral sequences, the sample tested showing reassociation kinetics identical to the non-virus-producing strains.

DNA of the AKR mouse contains a set of murine leukemia virus sequences that are not present in DNA of the NIH Swiss mouse. NIH mice partially congenic for the AKR murine-leukemia-virus-inducing locus Akv-1 contain this set of sequences, and, in a three-point cross segregating for Akv-1 on an NIH background, the sequences segregated with Akv-1. It is concluded that the Akv-1 locus contains viral sequences.

While C-type RNA viruses are known to induce leukemias and lymphomas, oncogenic transformation of lymphoid cells by them in vitro has not been reported. In this study, splenocytes from female BALB/c mice were infected in vitro with Abelson virus, a C-type RNA virus that induces nonthymic lymphomas and plasmacytomas in mice. The cells were transplanted into recipients of different karyotype, either male BALB/c mice or hybrid BALB/c x AL/N (CALF1) mice, which bear the Rb(5.19)1Wh translocation. Transplants of eight of the resulting tumors (one plasmacytoma and seven lymphomas) contained cells of donor BALB/c karyotype, indicating that transformation of splenocytes occurred in vitro.

To probe the genetic basis of disease specificity of nondefective murine type C viruses, we are constructing recombinants in vitro between molecular clones of Friend murine leukemia virus (Fr-MuLV) and Moloney murine leukemia virus (Mo-MuLV). Fr-MuLV induces erythroleukemias when injected into newborn NFS mice, whereas Mo-MuLV almost invariably induces T-cell lymphomas. We find that a recombinant whose genome is derived primarily from Fr-MuLV but which has 621 nucleotides of Mo-MuLV information at its 3' end induces almost exclusively thymic lymphomas. The sequences derived from Mo-MuLV include 99 nucleotides encoding the carboxyl terminus of Prp15E, the origin of DNA +-strand synthesis, all of the U3 region, and 36 nucleotides of the R portion of the long terminal repeat. When the segment of Mo-MuLV was removed and replaced with the comparable segment from Fr-MuLV, the virus was again erythroblastosis-inducing. These results, in conjunction with studies from other laboratories [Laimins, L. A., Khoury, G., Gorman, C., Howard, B. & Gruss, P. (1982) Proc. Natl. Acad. Sci. USA 79, 6453-6457], suggest that transcriptional signals in U3 may determine tissue tropism and hence influence disease specificity ("targeting") of murine leukemia viruses.

We recently reported that thymocytes from 6-month-old preleukemic AKR mice express higher levels of murine leukemia virus (MuLV)-related antigens that thymocytes from 2-month-old mice. We have now found that the level of xenotropic MuLV (defined operationally as MuLV able to infect mink cell cultures) is also markedly increased in thymus of 6-month-old AKR mice and that this increase in virus correlates closely with increased MuLV-antigen expression. There is no increase of MuLV antigen or xenotropic virus in spleen or lymph nodes. Production of ecotropic MuLV remains unchanged with age in thymus, lymph nodes, and spleen. Thymic grafts from 6-month-old AKR mice, but not from 2-month-old mice, induce both amplified MuLV-antigen expression and xenotropic virus production in the thymus of young AKR recipients. Experiments with lethally irradiated AKR mice reconstituted with syngeneic bone marrow cells indicate that age-related changes in the thymus rather than in bone marrow precursor cells are responsible for MuLV-antigen amplification.

A new type of murine leukemia virus has been detected in thymuses of leukemic and late preleukemic AKR mice, in lymphomas developing in NIH Swiss mice carrying the AKR ectopic virus-inducing loci Akv-I or Akv-2, and in the thymus of a preleukemic C58 mouse. The viruses induce focal areas of morphologic alteration in a mink lung cell line and are tentatively referred to as "mink cell focus-inducing" (MCF) strains. They have the host range of both xenotropic and N-tropic ecotropic murine leukemia viruses, are neutralized by antisera to both ecotropic and xenotropic viruses, and are interfered with by both viruses. They may represent a particular type of genetic recombinant which emerges during the preleukemic period in high-ecotropic-virus mouse strains, and they may play a significant role in the etiology of spontaneous lymphomas.

Tissue culture cells of a Rous virus-induced rat tumor undergo syncytium formation when placed in contact with mouse embryo cell cultures infected with murine leukemia viruses. This phenomenon can be used as a cytopathic end point for isolating and titrating these viruses in tissue culture. The principle should be applicable to detection of leukemia viruses of other species.

The convulsant agent methionine sulfoximine inhibits brain glutamine synthetase irreversibly and the inhibitor becomes bound to the active site of the enzyme as methionine sulfoximine phosphate. Only one of the four isomers of methionine sulfoximine, L-methionine-S-sulfoximine, inhibits glutamine synthetase. In the present work, D-methionine-SR-sulfoximine, and highly purified preparations of L-methionine-S-sulfoximine and L-methionine-R-sulfoximine were tested in mice for convulsant activity; only L-methionine-S-sulfoximine produced convulsions. The finding that only one of the four optical isomers of methionine sulfoximine induces convulsions, and that only this same isomer inhibits glutamine synthetase, lends support to the conclusion that these two effects of methionine sulfoximine are closely connected.

Genetic information of murine leukemia viruses is known to be present in essentially all mice. Delineation of the biological effects of the expression of this genetic information is complicated by the difficulty in determining whether the presence of a viral antigen in a tissue is the result of gene activation that occurs independently in a large number of cells, proliferation of antigen-positive cells, or of spread of infectious virus from a few cells. This problem is further complicated by the difficulty in determining whether activation of virus genetic material is the cause or result of a biological phenomenon, such as malignancy. Further understanding of the activation of virus genes in normal cells is of great importance in answering these questions. In vitro techniques were used for the study of induction of virus. Virus-negative cell lines were established from embryos of AKR mice, which in vivo produce high titers of infectious murine leukemia virus. It was found that 5-iododeoxyuridine and 5-bromodeoxyuridine were potent inducers of virus synthesis in all clones and subclones of the AKR cells tested. In addition to establishing that the complete viral genome is a heritable component of all cells of the AKR mouse, these studies of virus activation provide novel approaches to a number of important problems in the biology of leukemia viruses.

The AKR mouse has a high titer of murine leukemia virus early in life, and virus-negative cells derived from embryos of this mouse strain can be activated to yield murine leukemia virus by treatment with 5-iododeoxyuridine. In contrast to this high-virus strain, the NIH Swiss mouse has a low incidence of leukemia and no murine leukemia virus has been isolated from it (virus-negative). We have investigated this difference between AKR and NIH mice by examining the sequences specific for murine leukemia virus in nucleic acids of these mice. A single-stranded viral-DNA probe synthesized in vitro using murine-leukemia-virus from the AKR mouse contains at least 87% of the sequences present in the 70S viral RNA; most of these sequences are in proportions similar to their content in the 70S RNA. Using this probe in nucleic acid hybridization experiments, we have shown that NIH-mouse-cell DNA and AKR-mouse-cell DNA differ with respect to sequences specific for AKR murine-leukemia-virus: NIH-mouse-cell DNA lacks some of the virus-specific sequences present in AKR-mouse-cell DNA, and there are two distinct sets of virus-specific sequences in AKR-mouse-cell DNA, whereas there is only one set in NIH-mouse-cell DNA.RNA from virus-negative AKR-mouse cells grown in tissue culture contains some, but not all, virus-specific RNA sequences; however, within 48 hr after initiating treatment of these cells with 5-iododeoxyuridine, the complete viral genome is represented in cellular RNA.