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J R Subjeck

Publications and source records attributed to J R Subjeck.

At least 37 records · Page 2Linked to original sources
PubMed1989

Involvement of rRNA synthesis in the enhanced survival and recovery of protein synthesis seen in thermotolerance.

Although acquired thermotolerance has been linked to the induction of heat shock proteins, the molecular mechanism(s) by which cells become resistant to heat is unknown. The present study shows a strong correlation between the survival of cells following heat shock and the rate of recovery of protein, total RNA, and rRNA synthesis. Increasing exposure of CHO cells to 45 degrees C was found to decrease survival and cause a lengthening delay in these synthetic processes. The same reciprocal correlation was seen in thermotolerant cells. As thermotolerance develops, more cells survive a heat challenge and the delay in synthesis decreases. These data argue that enhanced recovery of protein and RNA synthesis is one factor which plays a key role in thermotolerance. The involvement of rRNA synthesis was further investigated by using actinomycin D at 0.1 microgram m1(-1), a concentration at which rRNA synthesis is selectively inhibited. When the drug was present during the recovery from a challenge heat treatment, the survival of thermotolerant cells was approximately 3-fold lower than expected from the mild toxicity of the drug. As this could not be accounted for by an interaction of the drug with the response of cells to single heat treatments, it is concluded that the drug inhibits the expression of thermotolerance in cells which would otherwise express a full degree of thermotolerance. The time and concentration dependence of this effect indicates that the drug acts though inhibition of rRNA synthesis. Therefore, enhanced recovery of RNA synthesis, presumably rRNA synthesis, is identified as one of the mechanisms responsible for enhanced survival of thermotolerant cells following heat shock.

Animals↗
PubMed1989

Glucose deprivation results in the induction of glucose-regulated proteins and domes in MDCK monolayers in hormonally defined serum-free medium.

Madin-Darby canine kidney (MDCK) cells spontaneously form dome-like structures in vitro, a phenomenon which has been proposed to be indicative of cellular differentiation. This study indicates the existence of a correlation between the induction of domes and of glucose-regulated proteins during glucose starvation. When MDCK monolayers were glucose deprived, domes appeared very rapidly. After only 3 h of glucose deprivation domes appeared in 69% of the microscope fields. The level of expression of glucose-regulated proteins (grps) as well as domes was examined over a 6-h time interval of glucose deprivation. Both grp 76 and 97 were induced over this time interval, with grp 76 being the more readily detectable. The level of induction of grp 76 as a function of time was quantitated by means of densitometry measurements. The induction of domes was examined in parallel with the induction of grp 76. The results indicated that the induction of grp 76 and domes occurs with a similar time course. The effect of glucose deprivation on the initial rate of ouabain-sensitive Rb+ uptake was also examined. Within the first 4 h of glucose deprivation, the initial rate of ouabain-sensitive Rb+ uptake did not differ significantly in glucose-deprived and control MDCK monolayers. These observations indicate that unlike the case with other methods of dome induction (e.g., treatment with either prostaglandin E1 (PGE1) or hexamethylene bis-acetamide (HMBA] glucose deprivation does not affect the Na+K+ ATPase activity of MDCK monolayers. These observations suggest that PGE1, HMBA, and glucose deprivation affect dome formation in MDCK monolayers by means of distinct mechanisms.

Acetamides↗
PubMed1989

Depletion of topoisomerase II in isolated nuclei during a glucose-regulated stress response.

Conditions, such as anoxia or glucose starvation, which induce the glucose-regulated set of stress proteins also lead to resistance to adriamycin (J. Shen, C. Hughes, C. Chao, J. Cai, C. Bartels, T. Gessner, and J. Subjeck, Proc. Natl. Acad. Sci. USA 84:3278-3282, 1987) and etoposide. We report here that chronic anoxia, glucose starvation, 2-deoxyglucose, the calcium ionophore A23187, glucosamine, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and tunicamycin (all specific inducers of the glucose regulated system) lead to a rapid and selective depletion of topoisomerase II from isolated nuclei of Chinese hamster ovary cells. This effect precedes a decline in tritiated thymidine incorporation and a redistribution of cells from S into G1/G0. The depletion of the enzyme is not accompanied by a decline in mRNA levels. We have also examined the mutant Chinese hamster K12 cell line which is temperature sensitive for expression of glucose-regulated proteins. When nuclei were isolated from K12 cells incubated at the nonpermissive temperature, a loss of topoisomerase II was again observed in congruence with the expression of stress proteins and cellular resistance to etoposide. These changes were not obtained in parental Wg1A cells incubated at the same temperature. These studies indicate that topoisomerase II is highly sensitive to glucose-regulated stresses and that its depletion from the nucleus, with the associated changes in cell cycle parameters, may represent general characteristics of the glucose-regulated state. Since anoxia and glucose starvation can occur during tumor development, this pathway for expression of drug resistance may have clinical ramifications.

Animals↗
PubMed1987

Effects of hyperthermia on spectrin expression patterns of murine lymphocytes.

In this study the influence of whole-body hyperthermia on the distribution of spectrin in murine lymphocytes isolated from various lymphoid tissues is examined. Lymphocytes normally vary in terms of the pattern of spectrin distribution within the cell. In certain populations of lymphocytes, spectrin is distributed into a dense submembranous aggregate that can be easily identified by immunofluorescence microscopy. In these lymphocytes, little or no spectrin is seen at the plasma membrane region in the rest of the cell. Other lymphocytes have no such cytoplasmic aggregates, and the protein is seen at the region of the plasma membrane. Following whole-body hyperthermia (40.5 degrees C for 90 min) there is a 100% increase in cells exhibiting polar spectrin aggregates in the spleen, while lymphocytes from the thymus show no alteration in the number of cells showing such aggregates. The increase in the percentage of splenic cells that express aggregated spectrin is a result of increases occurring in both T- and B-cell subsets. This increase gradually returns to control levels by 48 h post-heating. During recovery to control levels this phenomenon is resistant to additional changes when a second heat treatment is applied. The effects described above are not observed when the experiments are performed in vitro; therefore, it is likely that the in vivo heat-induced alteration in the splenic lymphocyte population reflects the physiological response of lymphocytes to stimuli during a natural fever. The role that spectrin may play in the modulation of lymphocyte membrane properties is discussed.

Animals↗
PubMed1986

Stress protein systems of mammalian cells.

Living organisms are known to react to a heat stress by the selective induction in the synthesis of several polypeptides. In this review we list the major stress proteins of mammalian cells that are induced by heat shock and other environments and categorize these proteins into specific subgroups: the major heat shock proteins, the glucose-regulated proteins, and the low-molecular-weight heat shock proteins. Characteristics of the localization and expression of proteins in each of these subgroups are presented. Specifically, the nuclear/nucleolar locale of certain of the major heat shock proteins is considered with respect to their association with RNA and the recovery of cells after a heat exposure. The induction of these major heat shock proteins and the repression of the glucose-regulated proteins as a result of reoxygenation of anoxic cells or by the addition of glucose to glucose-deprived cultures is described. Changes in the expression of these protein systems during embryogenesis and differentiation in mammalian and nonmammalian systems is summarized, and the protective role that some of these proteins appear to play in protecting the animal against the lethal effects of a severe heat treatment and against teratogenesis is critically examined.

Animals↗
PubMed1986

Effect of growth state and heat shock on nucleolar localization of the 110,000-Da heat shock protein in mouse embryo fibroblasts.

We have shown previously that the mammalian 110,000-Da heat shock protein (hsp110) associates with nucleoli in several cell types and that in 2-day postconfluent mouse 10T1/2 cells, a segregation of the antigen from the nucleolar phase-dense body is seen (J.R. Subjeck, T. Shyy, J. Shen, and R.J. Johnson. J. Cell Biol. 97: 1389-1395, 1983). Here we further characterize the nucleolar segmentation of hsp110 in mouse 10T1/2 and 3T3 cells with respect to the formation of this structure in dense cultures and investigate the behavior of this protein following conditions (serum deprivation, actinomycin D, and heat shock) known to affect the functional and morphological integrity of the nucleolus. It is shown that in addition to its nucleolar locale, an affinity of hsp110 for the nonnucleolar, nuclear compartment in actively proliferating cells is also observed. When proliferating cells are treated with actinomycin D (1 microgram/ml) for 8 h, hsp110 separates from the nucleolar phase-dense body to form a fluorescent nucleolar cap which resembles that seen in confluent cultures. This drug also results in a disappearance of hsp110 from the nucleoplasm. Incubation of cells for 24 h in media without serum also results in the nucleolar segmentation of hsp110 and a reduction in nucleoplasmic staining. A moderate nonlethal heat treatment does not lead to segmentation of hsp110 in proliferating cells but conversely results in a transient reversal of segmentation in confluent cultures. Examination of segmented nucleoli of postconfluent cells by immunoelectron microscopy reveals that hsp110 is associated with the fibrillar component of these nucleoli, the site of ribosomal DNA.

Animals↗
PubMed1985

Analysis of the expression of the two major proteins of the 70 kilodalton mammalian heat shock family.

This study compares the expression after heat shock of the two major variants of the mammalian 70 kilodalton heat shock family in three separate systems. The ability of wild type and temperature sensitive mutant (ts85) FM3A cells to elicit a heat shock response following a 45 degrees C, 12 min exposure was examined. The ts85 cells were found to be both significantly more thermosensitive than parent FM3A cells and to induce a 66kDa heat shock protein (hsp66) not visibly synthesized in the parent line by this exposure. However, a constitutive (synthesized at 37 degrees C) 68kDa heat shock protein (hsp68) is comparably induced in both cell lines after heat. A relationship between the severity of the heat exposure as seen by the cell and hsp66 expression is suggested and tested in Chinese hamster ovary cells. In CHO cells a brief 45 degrees C heat shock induces the constitutive hsp68 (but not hsp66), while longer and more severe exposures are required for the expression of hsp66. The induction of these two proteins is also examined in situ in mouse skeletal muscle. In this case both hsp66 and hsp68 are induced following comparatively mild heat treatments, and the 'threshold' for hsp66 induction observed in cultured cells either does not occur or is greatly reduced. However, once again, hsp68 is naturally synthesized at 37 degrees C while hsp66 appears to be de novo synthesized after heat shock.

Acclimatization↗
PubMed1984

The effect of nitroimidazoles on the oxygen consumption rate and respiratory control ratio of beef heart mitochondria.

The neurotoxic effect of the nitroimidazole radiosensitizers misonidazole (MISO) and desmethylmisonidazole (DMM) has seriously compromised their clinical effectiveness. We compare here the effect of MISO and DMM on oxygen consumption in purified beef heart mitochondria. MISO has been found to significantly increase the oxygen consumption rate and decrease the respiratory control ratio in isolated mitochondria when incubated in the presence of the NAD+ dependent substrate, beta-hydroxybutyrate. DMM has a similar but less pronounced effect than MISO on these respiratory parameters. When mitochondria were incubated in the presence of these radiosensitizers for 8, 15, 30, 45, and 60 minutes, the oxygen consumption rate was decreased when succinate, a FAD dependent substrate, was added following the incubation. This decrease, which is both time and dosage dependent, is equivalent for MISO and DMM.

Animals↗
PubMed1984

Induction of glucose-regulated proteins during anaerobic exposure and of heat-shock proteins after reoxygenation.

In this report we examine the effects of chronic anaerobic exposure and subsequent reoxygenation on protein synthesis patterns in Chinese hamster ovary cells. It is observed by two-dimensional gel electrophoresis (isoelectric focusing/NaDodSO4/PAGE) that the transition from an atmospheric environment to an anaerobic state transiently induces the major heat-shock proteins (at 68 and 89 kDa). As the period of anaerobiosis increases, this heat-shock induction disappears and a new set of proteins (at 76 and 97 kDa) is induced. By two-dimensional gel electrophoresis and partial proteolytic mapping, these new proteins, which are induced by anaerobic exposures exceeding 12 hr, are identical to 76 and 97 kDa (p76 and p97, respectively) proteins induced by extended periods of glucose deprivation (greater than 14 hr) when oxygen is present. Furthermore, the induction of these proteins under anoxia occurs in the presence of glucose, and increasing the glucose content of the starting media does not affect the induction. When anaerobic p76 and p97 induced cells are returned to atmospheric oxygen, p76 and p97 are repressed, while the heat-shock proteins are again transiently induced. This work further suggests the importance of deprivation and release environments in controlling the expression of these two stress protein systems. It is suggested that their natural expression may be determined by comparable circumstances.

Anaerobiosis↗
PubMed1984

Heat shock proteins and protection of proliferation and translation in mammalian cells.

The characteristics of heat shock protein(s) (HSP) and thermotolerance induction were examined in Chinese hamster ovary cells following two diverse nonlethal heat shocks: a continuous 41 degree exposure and a 45 degree, 5-min exposure. While induction of both HSP and thermotolerance were observed to initially develop simultaneously, a significant period was observed during which HSP induction was not accompanied by any further increase in the cells' ability to survive a thermal stress. Conversely, the achievement of the full tolerant state, as measured by colony survival, correlates in these instances with the moment at which development of the induction of HSP either ceased or began to be repressed. When a 99% lethal 45 degree, 22-min heat shock was examined, HSP and a thermal resistance were again observed to develop synchronously, despite the fact that the tolerance was measured in only 1% of the cell population. In this instance, a new protein of molecular weight 66,000 was observed which was not visibly induced by either of the two nonlethal treatments. Finally, the ability of heat-shocked thermotolerant cells to translate proteins following a second heat challenge (protection of translation) was investigated as an alternative measure of thermotolerance. While a protection of translation which encompassed several normal cellular proteins was observed, the phenomenon paralleled the induction and repression phases of HSP synthesis and was therefore not related to thermotolerance.

Animals↗
PubMed1984

The effect of nitroimidazoles on glucose utilization and lactate accumulation in mouse brain.

The radiation sensitizers misonidazole (MISO) and desmethylmisonidazole (DMM) can produce central and peripheral neuropathy in patients and laboratory animals. Behavioral and pathological investigations have indicated that in the central nervous system this primarily involves the cochlear and vestibular systems. Nitroimidazoles can also interfere with glycolysis in vitro under aerobic and anaerobic conditions. In the present work we have studied the effect of MISO or DMM on lactate production and glucose utilization in mouse brain. It is observed that these compounds result in a 25% inhibition of lactate production in brain slices relative to the control at a 10 mM level. Additionally, MISO (1.0 mg/g/day) or DMM (1.4 mg/g/day) were administered daily (oral) for 1, 4, 7, or 14 days to examine the effect of these two drugs on the regional glucose utilization in C3Hf mouse brain. Five microcuries of 2-deoxy[14C]glucose was given following the last drug dose and autoradiographs of serial brain sections were made and analyzed by a densitometer. Following a single dose of either MISO or DMM, no significant differences in glucose uptake were observed when compared with controls. However, following 4, 7, and 14 doses the rate of glucose utilization was significantly reduced in the intoxicated animals. Larger reductions were measured in specific regions including the posterior colliculus, cochlear nuclei, vestibular nuclei, and pons with increasing effects observed at later stages. These results share a degree of correspondence with the regional brain pathology produced by these nitroimidazoles.

Animals↗
PubMed1983

The effects of glucose on protein synthesis and thermosensitivity in Chinese hamster ovary cells.

Glucose deprivation induces the major glucose regulated proteins (GRPs) in Chinese hamster ovary cells. When these cells are then returned to a glucose containing environment, GRP synthesis is repressed while concurrently other proteins, identified as heat shock proteins, are induced. The induction of the GRPs is found to mark precisely the onset of a decline in the cell's ability to survive a thermal stress while the expression of heat shock proteins, when glucose is restored, is paralleled by significant increases in survival protection or thermotolerance.

Animals↗
PubMed1983

Association between the mammalian 110,000-dalton heat-shock protein and nucleoli.

A rabbit antiserum has been prepared using as antigen the 110,000-dalton mammalian heat-shock protein. This protein was purified for injection by two-dimensional PAGE of heat-shocked Chinese hamster ovary cells. Characterization by immunoautoradiography and immunoprecipitation reveals that the antiserum is specific for the 110,000-dalton protein. Both techniques also reveal that the protein against which the antiserum is directed is induced by heat shock. Indirect immunofluorescence shows that the antigen is primarily localized at or near the nucleolus in cultured cells and numerous murine tissues. Treatment of cultured cells with deoxyribonuclease destroys the organization of staining within the nucleus while ribonuclease appears to completely release the antigen from the nucleus. A binding of the antiserum to cytoplasmic structures is also observed by immunofluorescence. This association with nucleoli may have implications in the regulatory aspects of the heat-shock response.

Animals↗
PubMed1983

Behavioural and ultrastructural studies of desmethylmisonidazole-induced neurotoxicity in mice.

The hypoxic cell radiosensitiser desmethylmisonidazole (DMM, Ro-05-9963) was administered orally to C3Hf female mice at three different dose levels (1.4, 1.6 and 1.8 mg g-1 day-1). Behavioural and morphological end-points were used to evaluate the neurotoxicity of this compound at these doses. The behavioural changes present at all three dose levels appeared when the total dose approached approximately 19 mg g-1. The behavioural changes initially appeared and developed to a severe state rapidly (24 to 48 h). A pattern of haemorrhagic necrosis was observed in the brain stem and cerebellum, but no pathology was observed in the cerebrum. Peripheral nerve degeneration was also evident and appeared concurrent with the central pathology. DMM appears to affect both the central and peripheral nervous systems at the same time at these dose levels. This is in some degree different from previous experience with misonidazole where peripheral damage distinctly precedes central effects. The development of the disorder appeared to be more rapid and severe when compared with earlier experience with misonidazole.

Animals↗
PubMed1982

Heat shock proteins and thermotolerance; a comparison of induction kinetics.

The hypothesis that the expression of heat shock proteins following a preliminary hyperthermic treatment is responsible for subsequent thermotolerance to a second heat treatment is examined. CHO cells were given a 12 min, 45 degrees C pretreatment and then incubated for varying intervals at 37 degrees C. The synthesis of certain intracellular proteins was monitored as a function of time post-incubation by using 35S-methionine incorporation as determined in SDS polyacrylamide gel electrophoresis. Cell survival was concurrently measured by challenging the cells with a second heat treatment (45 degrees C/27 min). Major heat shock proteins were observed at 68 000, 89 000 and 110 000 daltons. The synthesis of these proteins was significantly reduced in the presence of cyclohexamide. The total 35S-methionine incorporation into these proteins correlated well with the induction of survival resistance (thermotolerance). An approximate exponential relationship between survival and the amount of each of these proteins may occur. These and other heat shock proteins were also present, in a significantly reduced degree, in control (non-heat shocked) cells maintained under normal culture conditions at 37 degrees C. It is possible that heat shock proteins are responsible for the phenomenon of thermotolerance.

Cell Line↗
PubMed1982

Heat shock proteins and biological response to hyperthermia.

Thermotolerance is of significant interest because the resistance of pre-heated cells greatly surpasses the resistance of control cells. The induction of protein synthesis following a temperature shock (12 min, 45 degrees C) which produces thermotolerance is examined using SDS polyacrylamide gel electrophoresis. It was observed that the synthetic rates of certain proteins following heat shock significantly exceeded their synthetic rates in control (non-heat shocked) cells. The most strongly induced bands were observed at 68,000, 89,000 and 110,000 daltons. The induction of these proteins was blocked by inhibitors of transcription (actinomycin D) or translation (cycloheximide). This phenomenon strongly resembles the heat shock induction of gene transcription well studied in Drosophila melanogaster. The strong induction of gene transcription well studied in Drosophila melanogaster. The strong correlation between the kinetics of the induction of these heat shock proteins and the kinetics of thermotolerance induction is discussed.

Animals↗
PubMed1982

The effect of high intensity microwave exposure on enucleation of murine erythroid cells in vitro.

We have examined the effects of microwave vs sham exposure on the enucleation phase of murine erythroid cells in vitro. While enucleation occurs rapidly in vivo, it occurs somewhat slower in vitro and can be quantitated in terms of rate. Exposure to 915 MHz electromagnetic radiation is found to significantly reduce the rate of enucleation (P less than 0.001). Exposure is carried out in a TE10 mode energized water-filled temperature-controlled waveguide. It is hypothesized that the effects of the exposure are on cytoplasmic or plasma membrane-associated structures since the nucleus at this stage of maturation is completely condensed and inactive. This assay is of direct interest in itself as well as providing a tool for the investigation of biological response to microwave exposure.

Animals↗