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Suppression of tumorigenicity in temperature-sensitive mutants of embryonal carcinoma cells by induction of cell differentiation at non-permissive temperature.

The temperature-sensitive (ts) mutants of cell differentiation derived from the mouse teratocarcinoma cell line F9, when exposed to non-permissive temperature, undergo stem-cell differentiation concomitant with a transient retardation of cell-cycle progression. By incubating these mutant strains at non-permissive temperature, we were able to study the relationship between cell differentiation and tumorigenicity. Upon exposure to non-permissive temperature, the mutant cells undergo extensive differentiation and lose their ability to initiate tumors in vivo, but retain their in vitro proliferative potential. Our data suggest that the loss of tumorigenicity is not caused by altered proliferative potential, but rather by cell differentiation. We therefore suggest that the loss of proliferative potential and the onset of cell differentiation in teratocarcinoma F9 cells are 2 independent events which can be genetically dissected, and that there is (a) crucial step(s) in the differentiation pathway at which these ts mutant cells lose their tumorigenicity.

Alkaline Phosphatase↗

The development of theoretical relationships between some handling parameters (setting time and setting temperature), composition (relative amounts of initiator and activator) and ambient temperature for acrylic bone cement.

Commercially available acrylic bone cements are two-component systems based on the polymerization of methyl methacrylate around poly(methyl methacrylate) particles. When benzoyl peroxide (BPO), which is the initiator, in the powder component meets accelerator (N,N-dimethyl-p-toluidine (DMpT)) in the liquid component, radicals are produced, initiating the polymerization. This solidifies the cement. In this work, kinetic expressions have been developed that describe the relationship between bone cement setting time on the one hand, and BPO and DMpT concentrations on the other. Changes in setting time with ambient temperature follow a complex relationship, because both the polymerization process (initiation, propagation, and termination) and the swelling and dissolution of the polymer particles contribute to setting. The contribution of polymer swelling and dissolution to the setting process was determined by developing a relationship between the doughing time, which is substantially independent of DMpT or BPO concentrations, and ambient temperature. A value of 64 kJ mol(-1) was found for the activation energy for this process. An activation energy for the overall setting process of 68 kJ mol(-1) was determined from setting-time measurements over several ambient temperatures. This indicates that the sensitivity of setting time to temperature depends more on swelling and dissolution than on the polymerization process.

Benzoyl Peroxide↗

Temperature-dependent transmembrane potential changes in cells infected with a temperature-sensitive Moloney sarcoma virus.

Normal rat kidney cells (NRK) infected with the temperature-sensitive (ts) transformation mutant of Moloney murine sarcoma virus yielded a clone of cells, 6m2, that exhibited a transformed morphology at 33 degrees C and a normal morphology at 39 degrees C. Transmembrane potential (Em) was measured fluorometrically using a cyanine dye diS-C3-(5). Fluorescence was inversely correlated with Em. Cells at 33 degrees C had lower Em. Em changes were recorded within 15 minutes of temperature shift from 33 degrees C to 39 degrees C in both directions, increasing in the 33 degrees C to 39 degrees C direction and decreasing in the 39 degrees C to 33 degrees C direction. Uninfected NRK cells when shifted under the same condition exhibited small fluorescence changes in the 33 degrees C to 39 degrees C direction. Shifting from 39 degrees C to 33 degrees C resulted in Em changes similar to those in 6m2 cells. Also studied was a cell line infected with a spontaneous revertant of the ts mutant, designated 54-5A4; it was transformed at both temperatures. Shifting from 33 degrees C to 39 degrees C in both directions yielded small changes. Transmembrane potential changes in 6m2 cells precede other transformation-specific changes that occur after a temperature shift.

Animals↗

Solvent and temperature gradients in separation of synthetic oxyethylene-oxypropylene block (co)polymers using high-temperature liquid chromatography.

Chromatographic behavior of synthetic block (co)oligomer samples (EO)n(PO)m(EO)n and (PO)n(EO)m(PO)n with different distribution of propylene oxide (PO) and ethylene oxide (EO) monomer units was investigated on three types of stationary phases on zirconium dioxide support: Zr-PS (polystyrene), Zr-carbon, and Zr-carbon C18. The effects of the distribution and sequence of the oxyethylene and oxypropylene monomer units on the chromatographic retention depend on the type of the stationary phase, but are strongly affected by the organic modifier (methanol or ACN) in aqueous-organic mobile phase. Special attention was focused on the influence of the mobile-phase composition on the separation according to the EO and PO distribution. Zirconia-based columns are stable at elevated temperatures and can be used in high-temperature LC (HTLC); hence, we investigated the temperature effects on the chromatographic behavior up to 90 degrees C. The applications of solvent and temperature gradients were compared on the zirconia stationary phases in the RP mode.

Carbon↗

Apoptosis is induced at nonpermissive temperature by a transient increase in p53 in cell lines immortalized with temperature-sensitive SV40 large T-antigen gene.

Hepatocyte (TLR2) and kidney tubule (TKC2) cell lines established from temperature-sensitive (ts) SV40 T-antigen gene transgenic mice not only were arrested in growth, but also exhibited cell death at nonpermissive temperature (39 degrees C). The cell death was determined to be caused by apoptosis from observations of nuclear fragmentation and DNA fragmentation. These cell lines contained relatively high levels of wild-type p53 which formed complexes with T-antigen at permissive temperature (33 degrees C), but after shift to a nonpermissive temperature, the inactivation of T-antigens led to the liberation of an abundance of p53 proteins from the complexes, apparently inducing apoptosis.

Animals↗

Susceptibility of three different strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at two different temperatures to Vibrio anguillarum and temperature effect on antibody response.

Three geographically distinct-reared strains (Canadian, Icelandic, Norwegian) of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at optimal and super-optimal growth temperatures (12 and 18 degrees C respectively), were challenged with a virulent isolate of Vibrio anguillarum by injection. The halibut were injected intraperitoneally with 100 microl of the bacterial suspension (1 x 10(6) cells per fish). After challenge, temperature and strain-related differences in survival were observed. Canadian and Icelandic halibut cultured at the super-optimal temperature of 18 degrees C were significantly more susceptible to infection than those strains cultured at 12 degrees C. Total mortality at 18 degrees C for the Canadian and Icelandic strains was 56.4 and 61.85% respectively, compared to 32 and 26.6% respectively at 12 degrees C. Norwegian halibut were significantly more resistant to infection with V. anguillarum at 18 degrees C compared to the other strains, with total mortality of 13.3%. There was no significant difference in total mortality of Norwegian halibut at 18 or 12 degrees C (13.3, 25% respectively). The specificity of the antibodies in sera from challenged halibut cultured at 18 degrees C was primarily to LPS. Immunoblots showed the presence of antibodies against O-side chain antigens. This reaction was strongest in sera from the Norwegian halibut strain compared with the Canadian and Icelandic halibut, which suggests that the difference in resistance to challenge may be ascribable to the presence of antibodies to LPS. Specific antibody levels, as measured by ELISA, increased with increasing temperature and strain differences were apparent, however these did not relate to disease resistance.

Animals↗

Effects of CNS temperature on generation and transmission of temperature signals in homeotherms. A common concept for mammalian and avian thermoregulation.

Neurophysiological studies on avian hypothalamic thermosensitivity have presented evidence for a higher Q10 of cold than of warm signal transmission in the CNS of birds. An identical temperature dependence of central cold and warm signal transmission in mammals is suggested by considerations on the phylogeny of temperature regulation. By taking into account the experimental evidence for the existence of thermosensory afferents in the CNS of mammals and birds, being differently developed in the various sections of the neural axis and exerting quantitatively different influences on the various thermoregulatory effectors, a common concept of homeothermic thermoregulation is proposed resting on the same basic assumptions for mammals and birds. The great diversity of negative as well as positive feedback effects of CNS temperature displacements on homeothermic thermoregulation, which is particularly expressed in avian autonomic and behavioral thermoregulation and, further, certain pathophysiological conditions of disturbed thermoregulation could be accounted for by assuming quantitatively different contributions of the central thermosensory inputs of thermoregulatory effector control, but maintaining the Q10 values of hypothalamic warn and cold signal transmission constant. The proposed model, while basically additive in its mathematical design, meets a number of properties described by multiplicative models of thermoregulation. In additionally generalizes these models of predicting that changes of hypothalamic temperature modify the sensitivities with which any thermoregulatory effector responds to any thermosensory input.

Animals↗

Comparative studies on the neurovirulence of temperature-sensitive and temperature-resistant viruses of enterovirus 71 in monkeys.

The neurovirulence of strain BrCr of enterovirus 71 (E71) was compared in monkeys between temperature-sensitive (ts) and temperature-resistant (tr) viruses. Comparisons are made relative to clinical disease, pathologic findings, serum neutralizing antibody titers, CNS virus replication as measured by infectivity titrations and immunofluorescence. Clinically, ts virus did not produce a clinical disease. The tr virus, however, produced paralysis. Pathologically, little or no nerve cell damage was found in the CNS of monkeys inoculated with ts virus, although mild to moderate interstitial changes occurred. In monkeys inoculated with tr virus, marked nerve cell damage and inflammatory reaction were found in the CNS. Serum neutralizing antibody titers in monkeys inoculated with ts virus rose on day 25. No virus was detected in the CNS of monkeys inoculated with ts virus, while a high virus titer was detected in the CNS of monkeys inoculated with tr virus. No specific immunofluorescence was detected in the nerve cells of the CNS in monkeys inoculated with ts virus, but specific fluorescence was detected in the nerve cells of the CNS in monkeys inoculated with tr virus. Virus growth in the CNS correlated well with the severity of clinical and pathologic findings, and immunofluorescent studies. The results show that ts virus was much less neurovirulent than tr virus, indicating that ts virus resembles the attenuated poliovirus which could not grow at a higher temperature. It is inferred that the genetic factors which influence the reproductive capacity of E71 at a higher temperature are very closely correlated with the neurovirulence.

Animals↗

Isolation of temperature-sensitive CHO-K1 cell mutants exhibiting chromosomal instability and reduced DNA synthesis at nonpermissive temperature.

Twenty-five temperature-sensitive (ts) mutants were isolated from Chinese hamster CHO-K1 cells after mutagenization with N-methyl-N'-nitro-N-nitrosoguanidine. Of 13 complementation groups identified, nine exhibited chromosomal instability at a nonpermissive temperature. They were classified into three major classes according to inducibility of sister chromatid exchange (SCE) and/or chromosomal aberration (CA): class 1 resulted in predominant SCEs, class 2 manifested both SCEs and CAs, and class 3 exhibited higher induction of CAs. Flow cytometric analysis of the mutants exhibiting chromosomal instability indicated that many of the mutants were arrested in the S or S to G2 phases of the cell cycle at the nonpermissive temperature, accompanied by a decrease in the rate of DNA synthesis. These results imply that ts defects are related to some points in DNA replication and might be responsible for the induction of SCEs and/or CAs at the nonpermissive temperature.

Animals↗

Effect of temperature on nuclear membranes and nucleo-cytoplasmic RNA-transport in Tetrahymena grown at different temperatures.

The effect of temperature on the nuclear envelope structure and the transport of total RNA and ribosomal subunits from nucleus to cytoplasm was examined in Tetrahymena cells propagated at two different temperatures. Freeze-etch electron microscopy of cells grown at 23 and 18 degrees C detects the emergence of smooth areas on the fracture faces of the nuclear membranes upon lowering the temperature below approximately 15 and approximately 12 degrees C, respectively. Coincident with these freeze-etch changes, a discontinuous decrease is observed in the nucleocytoplasmic RNA-transport; this is probably not due to a cease in RNA-synthesis. Below the thermotropic discontinuity observed in the transport of total RNA in 18 degrees-cells the nucleocytoplasmic transport of the small and large ribosomal subunits is equally retarded. Recent temperature studies on the endoplasmic reticulum membranes of Tetrahymena suggest that the freeze-etch changes in the nuclear membranes are induced by a thermotropic clustering of the membrane lipids. We conclude that this lipid clustering induces the permanent protein constituents in the nuclear envelope pore complexes to change from a relatively "open" into a relatively "closed" state thus causing the observed decrease in RNA-transport.

Biological Transport↗

Effect of 3,4-methylenedioxymethamphetamine ("ecstasy") on body temperature and liver antioxidant status in mice: influence of ambient temperature.

The consumption of 3,4-methylenedioxymethamphetamine (MDMA; ecstasy) is known to cause severe hyperthermia and liver damage in humans. The thermogenic response induced by MDMA is complex and partially determined by the prevailing ambient temperature (AT). This is of extreme importance since ecstasy is often consumed at "rave" parties, where dancing takes place in a warm environment, which may exacerbate the effect of MDMA on thermoregulation. In view of the fact that hyperthermia is a well-known pro-oxidant aggressive condition, its potential role in ecstasy-induced hepatocellular toxicity should be further studied. Thus, the present study was performed in order to evaluate the influence of AT on the effects of single administration of MDMA on body temperature and liver toxicity in Charles River mice. Animals were given an acute intraperitoneal dose of MDMA (5, 10 or 20 mg/kg) and placed in AT of 20+/-2 degrees C or 30+/-2 degrees C for 24 h. Body temperature was measured during the study using implanted transponders and a temperature probe reading device. Plasma and liver samples were used for biochemical analysis. Liver sections were also taken for histological examination. The parameters evaluated were (1) plasma levels of transaminases and alkaline phosphatase, (2) hepatic glutathione (GSH), (3) hepatic lipid peroxidation, (4) activity of hepatic antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase, glutathione- S-transferase, copper/zinc superoxide dismutase and manganese superoxide dismutase), and (5) liver histology. The hyperthermic response elicited by MDMA was clearly dose-related and potentiated by high AT. Administration of MDMA produced some evidence of oxidative stress, expressed as GSH depletion at both ATs studied, as well as by lipid peroxidation and decreased catalase activity at high AT. High AT, by itself, decreased glutathione peroxidase activity. Histological examination of the liver revealed abnormalities of a dose- and AT-dependent nature. These changes included vacuolation of the hepatocytes, presence of blood clots and loss of typical hepatic cord organisation. The results obtained in the present study suggest that oxidative stress plays a part in the first stage of MDMA-induced liver damage and that liver antioxidant status is aggravated by increased AT. Thus, these findings are in accordance with the hypothesis that high AT may potentiate ecstasy-induced hepatotoxicity by increasing body hyperthermia.

Alanine Transaminase↗

Predictions of onset of crystallization from experimental relaxation times I-correlation of molecular mobility from temperatures above the glass transition to temperatures below the glass transition.

PURPOSE: Predicting onsets of crystallization at temperatures below T (g), from data above T (g), would require that the correlation between crystallization onset and mobility is same above and below T (g), and the techniques being used to measure mobility above and below T (g) are measuring essentially the same kind of mobility. The aim of this work is to determine if the relaxation times obtained using different techniques (DSC, TAM) below T (g) correlate with relaxation time obtained above T (g) using dielectric spectroscopy. METHODS: Model compounds for this work were chosen based on their varied DeltaH (f), DeltaC (p)(T (g)) and H-bonding in crystalline state vs. amorphous state. Relaxation times above T (g) were determined by the simultaneous fit of real and imaginary permittivity to the Cole-Davidson model. Tau and beta below T (g) were determined using isothermal microcalorimetry (TAM) or MDSC. MDSC was used to calculate Kauzmann temperature and strength of the glass using established relationships. RESULTS: Indomethacin, nifedipine and flopropione showed Arrhenius temperature dependence throughout the entire temperature range and extrapolation of tau ( beta ) measured above T (g) by dielectric relaxation agreed with tau ( beta ) measured below T (g) by TAM/MDSC. Ketoconazole, however, showed the expected VTF behavior. For at least two compounds compared (indomethacin and ketoconazole), relaxation times measured by TAM and MDSC did not agree, with TAM giving significantly lower values of tau ( beta ), but TAM and MDSC relaxation times appeared to extrapolate to a common value at T (g). CONCLUSIONS: It was found that, for all cases studied, relaxation time constants determined above and below T (g) did appear to extrapolate to the same value around T (g) indicating that molecular mobility measured above and below T (g) using different techniques is highly correlated.

Calorimetry↗

Characterization of DNA polymerase alpha activity from a mouse DNA temperature-sensitive mutant, strain tsFT20, which shows a defect in DNA polymerase alpha activity at restrictive temperatures.

tsFT20 cells derived from mouse FM3A cells are DNA temperature-sensitive mutants, which have heat-labile DNA polymerase alpha activity. When tsFT20 cells were incubated at restrictive temperatures, intracellular levels of DNA polymerase alpha activity changed biphasically, showing an initial fast decrease (phase I) and a subsequent slow decrease (phase II). The activity of DNA polymerase alpha from tsFT20 cells cultured at a permissive temperature (33 degrees C) was greatly increased by the addition of glycerol or ethylene glycol to the reaction mixture, while little increase in enzyme activity was observed at any concentration of glycerol or ethylene glycol tested with the enzyme from the cells cultured at a restrictive temperature (39 degrees C) for 8 h (phase II). The activity of DNA polymerase alpha from wild-type cells was also increased by the addition of glycerol but the increase was much less than that in the tsFT20 cells. An in vitro preincubation experiment showed that DNA polymerase alpha from tsFT20 cells cultured at 33 degrees C very rapidly lost its ability to be stimulated by glycerol. Furthermore, the experiment using the extracts prepared from tsFT20 cells cultured at 39 degrees C for various periods showed that the ability to be stimulated by glycerol decreased with the duration of incubation time at 39 degrees C. DNA polymerase alpha from the revertants, which can grow at 39 degrees C and exhibit a partial recovery in heat stability of DNA polymerase alpha activity, showed an intermediate response to glycerol, between those of DNA polymerase alpha from tsFT20 and from the wild-type cells. Finally, it was observed that the level of enzyme activity that can be stimulated by glycerol correlated well with the DNA synthesizing ability of tsFT20 cells.

Animals↗

Effect of different ambient temperature and lipopolysaccharide administration on the circadian rhythm of rectal temperature and hypothalamic PGE2 production in aged male rats.

Old and young male rats (22 and 7 months respectively) were exposed to ambient temperatures of 4, 22, 27 and 35 degrees C. The rats' rectal temperatures (RTs) were measured periodically, after exposure to the varying temperatures at different hours during the day. The mean circadian value of RTs in the aged rats was different from that of the young rats. Whereas exposure to low temperatures caused a decrease of 2.0 degrees C in the RTs of the old rats, exposure to heat (35 degrees C) caused an increase of 1 degree C in their RTs. An injection of 200 micrograms (intraperitoneally) of E. coli lipopolysaccharide caused them to experience a long period of hypothermia. Elevation in the RTs after the hypothermic period ended was significantly lower in the old rats. However no significant differences in hypothalamic PGE2 production were to be found between the old and young groups 24 h after pyrogen administration.

Aging↗

Temperature-mediated processes in teleost immunity: in vitro immunosuppression induced by in vivo low temperature in channel catfish.

In an attempt to understand the interrelationships between environmental temperature and immune competence, channel catfish in the laboratory were subjected to a rapid change in water temperature in order to mimic conditions which might be encountered in commercial ponds during the winter months and subsequently examined for a variety of immune parameters. The results indicated that lowering the water temperature from 23 to 11 degrees C over a 24 h period suppressed both B and T cell functions for 3-5 weeks as assessed by in vitro responses. Furthermore, this form of suppression was not a typical stress-induced response, i.e. blood serum chemistry and lymphocyte and neutrophil compositions did not change in a manner reminiscent of transport-induced stress. Collectively these results indicate that channel catfish are probably immunocompromised during the winter months and consequently it seems plausible that many of the fish losses associated with the syndrome termed "winter kill" may be attributable, at least in part, to a low temperature-induced immuno-deficient state.

Adaptation, Biological↗

Establishment of latency in vitro with herpes simplex virus temperature-sensitive mutants at nonpermissive temperature.

This report describes a latency model using human embryo lung cells that were infected with herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutants and cultivated at nonpermissive temperature (40.5 degrees C). ts mutants tsG8 (parental strain HSV-1 KOS) and tsG5 (parental strain HSV-1 13) could be maintained in a latent state at 40.5 degrees C for at least 40 days without exhibiting virus infectivity. During this time, viable virus could be reactivated by reducing the incubation temperature to the permissive level (34 degrees C). Virus replication could be detected 2 to 6 days after temperature reduction and the virus reactivated from the latent state seemed to retain the same ts phenotype as the input virus for at least 14 days.

Cell Line↗

The effect of growth temperature on the biosynthesis of Yersinia enterocolitica O:3 lipopolysaccharide: temperature regulates the transcription of the rfb but not of the rfa region.

The rfb region of Yersinia enterocolitica O:3 (YeO3) that determines the synthesis of the O-side chain of the lipopolysaccharide was cloned and expressed in Escherichia coli K12 previously. The clone did not show the in vitro temperature variation in O-side chain expression known for YeO3, instead analogous O-side-chain was produced at 25 degrees C and at 37 degrees C and both were similar to that produced by YeO3 at 25 degrees C. In Northern blot analysis marked reduction in the amount of rfb-specific mRNA was observed from YeO3 grown at 37 degrees C when compared with those grown at 25 degrees C. On the other hand, equal amounts of rfb-specific mRNA were detected in the E. coli clone at both growth temperatures. This indicates that the transcription of YeO3 rfb region is dramatically repressed at 37 degrees C. The repressor gene is located outside the rfb region with no analogous locus in E. coli chromosome. We cloned 18.2 kilobase pairs of YeO3 chromosomal DNA that rendered E. coli K12 reactive with 2B5, a YeO3 core-specific monoclonal antibody, in colony blotting, indirect immunofluorescence and immunoblotting. Hence this clone contains the YeO3 rfa region, encompassing the genes involved in the core oligosaccharide biosynthesis. No apparent difference, in the aforementioned tests, was noticed in the expression of the 2B5 epitope at different growth temperatures either in YeO3 or in E. coli. In Northern blot analysis comparable amounts of rfa-specific mRNA were detected at 25 degrees and 37 degrees C. This argues that in YeO3 the core oligosaccharide biosynthesis is not temperature-regulated.

Blotting, Northern↗

Genes involved in nonpermissive temperature-induced cell differentiation in Sertoli TTE3 cells bearing temperature-sensitive simian virus 40 large T-antigen.

Sertoli TTE3 cells, derived from transgenic mice bearing temperature-sensitive simian virus 40 large T (tsSV40LT)-antigen, proliferated continuously at a permissive temperature (33 degrees C) whereas inactivation of the large T-antigen by a nonpermissive temperature (39 degrees C) led to differentiation as judged by elevation of transferrin. To clarify the detailed mechanisms of differentiation, we investigated the time course of changes in gene expression using cDNA microarrays. Of the 865 genes analyzed, 14 genes showed increased levels of expression. Real-time quantitative PCR revealed that the mRNA levels of p21(waf1), milk fat globule membrane protein E8, heat-responsive protein 12, and selenoprotein P were markedly elevated. Moreover, the differentiated condition induced by the nonpermissive temperature significantly increased mRNA levels of these four genes in several cell lines from the transgenic mice bearing the oncogene. The present results regarding changes in gene expression will provide a basis for a further understanding of molecular mechanisms of differentiation in both Sertoli cells and cell lines transformed by tsSV40LT-antigen.

Animals↗