Spatial analysis of cell death and Hsp70 induction in brain, thymus, and bone marrow of the hyperthermic rat

Heat shock response and programmed cell death are cellular reactions to stressful stimuli. Previous studies have not correlated these responses in vivo at the spatial level in mammalian tissues. This study uses a dual procedure involving immunocytochemistry for Hsp70 localization and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end-labeling (TUNEL) assay for cell death to correlate the pattern of stress-inducible Hsp70 and cell death at the cellular level. After whole-body hyperthermia in the rat, an increase in Hsp70-positive cells and TUNEL-positive cells was noted in brain, thymus, and bone marrow. However, 2 populations of cells were apparent in the tissues examined, those inducing Hsp70 and those triggered into programmed cell death. Cells that were both Hsp70 positive and TUNEL positive were rarely detected. In tissues of the intact mammal, cells that induce Hsp70 after whole-body hyperthermia were not triggered into programmed cell death.     

Mechanism of cell death induction by nitroxide and hyperthermia

Heat stress and nitroxides induce reactive oxygen species (ROS) and proapoptotic effects. The underlying mechanisms remain largely elusive. Here we report that Tempo (2,2,6,6-tetramethylpiperidine-N-oxyl) is a potent thermosensitizer for promoting cell death in human leukemia U937 cells. Treatment with Tempo (10 mM, 37 degrees C/30 min) and hyperthermia (44 degrees C/30 min) induced 30 and 70-80% apoptosis, respectively, through Bax-mediated cytochrome c release and DEVDase activation. The Tempo/heat combination also caused Bax-mediated cytochrome c release, but switched heat-induced apoptosis to the particular pyknotic cell death, resulting in the irreparable inhibition of proliferation. Tempo and heat stress, but not the combination, caused an early transient elevation of H2O2/O2*- and a late induction of only O2*-, respectively. Mitochondrial Ca2+ overloads were indistinguishable after any treatment. Heat stress induced the pan-caspase inhibitor zVAD-fmk-suppressible low-Deltapsi (mitochondrial membrane potential) in 75% of cells as a result of DEVDase activation. In contrast, Tempo yielded low-Deltapsi by deprivation of the mitochondrial H+ gradient. The combined treatment induced 97% zVAD-resistant low-Deltapsi cells through irreversible mitochondrial dysfunction. Together, thus, Tempo or heat stress induced Bax-mediated mitochondrial apoptosis with the possible help of ROS or mitochondrial Ca2+, and Tempo when combined with hyperthermia acts a sensitizer by inducing irreparable pyknotic cell death through irreversible mitochondrial dysfunction.     

The turnover of myelin phospholipids in the adult and developing rat brain

1. Inorganic [(32)P]phosphate, [U-(14)C]glycerol and [2-(14)C]ethanolamine were injected into the lateral ventricles in the brains of adult rats, and the labelling of individual phospholipids was followed over 2-4 months in both a microsomal and a highly purified myelin fraction. 2. All the phospholipids in myelin became appreciably labelled, although initially the specific radioactivities of the microsomal phospholipids were somewhat higher. Eventually the specific radioactivities in microsomal and myelin phospholipids fell rapidly at a rate corresponding to the decline of radioactivity in the acid-soluble pools. 3. Equivalent experiments carried out in developing rats with [(32)P]phosphate administered at the start of myelination showed some persistence of phospholipid labelling in the myelin, but this could partly be attributed to the greater retention of (32)P in the acid-soluble phosphorus pool and recycling. 4. It is concluded that a substantial part of the phospholipid molecules in adult myelin membranes is readily exchangeable, although a small pool of slowly exchangeable material also exists. 5. A slow incorporation into or loss of labelled precursor from myelin phospholipids does not necessarily give a good indication of the rate of renewal of the molecules in the membrane. As presumably such labelled molecules originate by exchange with those in another membrane site (not necessarily where synthesis occurs) it is only possible to calculate the turnover rate in the myelin membrane if the behaviour of the specific radioactivity with time of the phospholipid molecules in the immediate precursor pool is known.     

Subependymal glycosaminoglycan networks in adult and developing rat brain

Summary Histochemical studies of normal adult rat brain indicate two types of glycosaminoglycans in the subependymal region of the lateral ventricle. One network is characterized by an affinity for the cationic dyes alcian blue, aldehyde fuchsin and colloidal iron. These reactions occur at pH 1.0 and at 0.5–0.3 M concentration of MgCl₂, which suggests that this material is chondroitin sulfate. The other system is identified by metachromasia with toluidine blue and a loss of PAS staining following sulfation. These findings are consistent with non-sulfated and non-anionic acid mucopolysaccharides. In developing rat brain the differential development of these networks enhances their separate identity. The metachromatic network is present at least by the 10th postnatal day but the polyanionic electrolytes cannot be identified until the 16th to the 22nd days. The possible functional importance of these systems is discussed.     

Subependymal glycosaminoglycan networks in adult and developing rat brain

Histochemical studies of normal adult rat brain indicate two types of glycosaminoglycans in the subependymal region of the lateral ventricle. One network is characterized by an affinity for the cationic dyes alcian blue, aldehyde fuchsin and colloidal iron. These reactions occur at pH 1.0 and at 0.5-0.3 M concentration of MgCl2, which suggests that this material is chondroitin sulfate. The other system is identified by metachromasia with toluidine blue and a loss of PAS staining following sulfation. These findings are consistent with non-sulfated and non-anionic acid mucopolysaccharides. In developing rat brain the differential development of these networks enhances their separate identity. The metachromatic network is present at least by the 10th postnatal day but the polyanionic electrolytes cannot be identified until the 16th to the 22nd days. The possible functional importance of these systems is discussed.     

Enzymes in intracellular organelles of adult and developing rat brain

Eighty percent of the hexokinase and about a half of the lactate dehydrogenase, pyruvate kinase, and aldolase activities of adult rat cerebral homogenates is particulate, associated to a large extent, with the sediment (P₂) obtained by centrifugation at 17,000g. Centrifugation of P₂ into sucrose gradients shows that all four enzymes are associated with synaptosomes: their peak concentration coincides with that of glutamate decarboxylase rather than with those of mitochondrial enzymes, glutamate dehydrogenase, and aspartate aminotransferase. After hypoosmotic shock and high-speed centrifugation considerable portions of synaptosomal enzymes are recovered in the supernatant phase; the composition of this fluid, as indicated by the higher specific activity of several enzymes, is different from that of the soluble fraction of whole homogenates.The concentration of the seven enzymes studied is considerably lower in fetal than in adult brain and, in general, a larger fraction of the total is soluble. Preferential accumulation with age in the particulate fraction is especially striking in the case of hexokinase. Between fetal and adult life there are changes in the enzymic composition as well as increases in the amount of the total protein attributable to the synaptosomal fraction. Glutamate decarboxylase and lactate dehydrogenase are the synaptosomal enzymes to rise first (before or at birth), followed by hexokinase and, in the third postnatal week, by aldolase and pyruvate kinase. The upsurge of mitochondrial enzymes (that of glutamate dehydrogenase at term and of aspartate aminotransferase 10 days later) is accompanied by insignificant or small increases in the total protein content of the same fraction. The results indicate that the maturation of subcellular organelles involves a stepwise enrichment with various enzymes; some signs of biochemical differentiation precede and others coincide with the development of cerebral functions known to occur in 2- to 4-wk-old rats.     

Immune proteasomes in the developing rat thymus

The age dynamics of the content of the immune proteasome subunits LMP2 and LMP7 in rat thymus during prenatal and early postnatal ontogeny was studied. The LMP2 and LMP7 immune subunits were detected by Western blotting already by the 18th day of embryonic development, their amount increased to the 21st day to the level characteristic of the postnatal state. Double immunofluorescent labeling showed that in the thymus tissue the largest amount of LMP2 and LMP7 is localized in epithelial cells, whereas the level of their expression in thymocytes is lower. The results suggest that the establishment in thymus of selection processes, which depend on activity of immune proteasomes, can take place already in prenatal ontogeny. Analysis of age dynamics of the natural apoptosis level in thymocytes also favors this supposition. The presence of immune proteasomes in thymocytes during perinatal ontogeny suggests that, besides the antigen presentation, immunoproteasomes may possess other important functions.     

The effect of hyperphenylalaninaemia on glycine metabolism in developing rat brain.

The brains of 3--16-day-old rats that were rendered hyperphenylalaninaemic by daily injections of alpha-methylphenylalanine plus phenylalanine were subjected to biochemical analysis. Fluctuations throughout the treatment period in the concentrations of branched-chain amino acids, methionine and serotonin were in agreement with the known interference of excess plasma phenylalanine with transport. The glycine content, however, became abnormal only by day 5, remained so through the treatment, and the elevation was equally apparent at 4, 8 or 24 h after the last daily injections. On the last day of treatment there were small increases in the taurine, glutamate, aspartate and 4-aminobutyrate concentrations, attributable mainly to the diencephalon or brain stem. After day 3 of treatment there were persistent elevations in the specific activity of phosphoserine phosphatase and glycine synthase (but not serine hydroxymethyltransferase) of the brain in each of the regions analysed. The observations indicate that chronic hyperphenylalaninaemia interferes with the normal regulation of intracerebral glycine metabolism during a critical period of early postnatal development, and suggest that the resulting excess in this amino acid (particularly marked in the cortex) contributes to the behavioural abnormalities that these animals exhibit in later life.     

The effects of hyperthermia and hyperthermia plus microwaves on rat brain energy metabolism

The effects of hyperthermia, alone and in conjunction with microwave exposure, on brain energetics were studied in anesthetized male Sprague-Dawley rats. The effect of temperature on adenosine triphosphate concentration [ATP] and creatine phosphate concentration [CP] was determined in the brains of rats that were maintained at 35.6, 37.0, 39.0, and 41.0 degrees C. At 37, 39, and 41 degrees C brain [ATP] and [CP] were down 6.0, 10.8, and 29.2%, and 19.6, 28.7, and 44%, respectively, from the 35.6 degrees C control concentrations. Exposure of the brain to 591-MHz radiation at 13.8 mW/cm2 for 0.5, 1.0, 3.0, and 5.0 min caused further decreases (below those observed for 30 degrees C hyperthermia only) of 16.0, 29.8, 22.5, and 12.3% in brain [ATP], and of 15.6, 25.1, 21.4, and 25.9% in brain [CP] after 0.5, 1.0, 3.0, and 5.0 min, respectively. Recording of brain reduced nicotinamide adenine dinucleotide (NADH) fluorescence before, during, and after microwave exposure showed an increase in NADH fluorescence during microwave exposure that returned to preexposure levels within 1 min postexposure. Continuous recording of brain temperatures during microwave exposures showed that brain temperature varied between -0.1 and +0.05 degrees C. Since the microwave exposures did not induce tissue hyperthermia, it is concluded that direct microwave interaction at the subcellular level is responsible for the observed decrease in [ATP] and [CP].     

O-GlcNAc cycling in the developing,adult and geriatric brain

Hundreds of proteins in the nervous system are modified by the monosaccharide O-GlcNAc. A single protein is often O-GlcNAcylated on several amino acids and the modification of a single site can play a crucial role for the function of the protein. Despite its complexity, only two enzymes add and remove O-GlcNAc from proteins, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Global and local regulation of these enzymes make it possible for O-GlcNAc to coordinate multiple cellular functions at the same time as regulating specific pathways independently from each other. If O-GlcNAcylation is disrupted, metabolic disorder or intellectual disability may ensue, depending on what neurons are affected. O-GlcNAc's promise as a clinical target for developing drugs against neurodegenerative diseases has been recognized for many years. Recent literature puts O-GlcNAc in the forefront among mechanisms that can help us better understand how neuronal circuits integrate diverse incoming stimuli such as fluctuations in nutrient supply, metabolic hormones, neuronal activity and cellular stress. Here the functions of O-GlcNAc in the nervous system are reviewed.     

The effect of hyperthermia on micronucleus induction by mutagens in mice

We administered mitomycin C (0.5 mg/kg) intraperitoneally to hyperthermic-treated mice and examined the effect of hyperthermia on micronucleus induction. Hyperthermia enhanced micronucleus induction. The timing of chemical administration relative to the start of hyperthermic treatment (37 degrees C ambient temperature) influenced micronucleus frequency, and the effect was greatest 2 h after the start of hyperthermic treatment. But the hyperthermic treatment did not change the time course of micronucleus induction. In addition, we investigated the effect of hyperthermia on micronucleus induction by chemicals with different modes of action, i.e., alkylating agents (mitomycin C at 0.1-0.5 mg/kg, cyclophosphamide at 1.25-10 mg/kg), a spindle poison (colchicine at 0.05-1.0 mg/kg), and an antimetabolite (5-fluorouracil at 2.5-50 mg/kg). Hyperthermia enhanced only the clastogenicity of alkylating agents.     

The effect of hyperthermia on micronucleus induction by mutagens in mice

We administered mitomycin C (0.5 mg/kg) intraperitoneally to hyperthermic-treated mice and examined the effect of hyperthermia on micronucleus induction. Hyperthermia enhanced micronucleus induction. The timing of chemical administration relative to the start of hyperthermic treatment (37°C ambient temperature) influenced micronucleus frequency, and the effect was greatest 2 h after the start of hyperthermic treatment. But the hyperthermic treatment did not change the time course of micronucleus induction. In addition, we investigated the effect of hyperthermia on micronucleus induction by chemicals with different modes of action, i.e., alkylating agents (mitomycin C at 0.1–0.5 mg/kg, cyclophosphamide at 1.25–10 mg/kg), a spindle poison (colchicine at 0.05–1.0 mg/kg), and an antimetabolite (5-fluorouracil at 2.5–50 mg/kg). Hyperthermia enhanced only the clastogenicity of alkylating agents.     

Dolichol kinase activity in the developing rat testis

Dolichyl phosphate concentrations, a primary factor in regulating the rate of N-glycosidically linked glycoprotein synthesis, are dependent upon a cytidine triphosphate (CTP)-dependent dolichol kinase. This study examines dolichol kinase in rat testicular microsomes and defines assay conditions. As with dolichol kinases from other tissues, addition of 2-mercaptoethanol increased activity 60%. Inclusion of NaF, an inhibitor of testicular dolichyl phosphate phosphatase activity, also resulted in a 38% increase in activity. Triton X-100 was necessary for phosphorylation of both endogenous and exogenous dolichol; however, concentrations of detergent in excess of 0.25-0.35% were inhibitory. A 2- to 5-fold stimulation of kinase activity was obtained by addition of 50-100 microM exogenous dolichol. The high level of nucleoside triphosphatase activity in testicular microsomes mandated the inclusion of high levels of uridine triphosphate (UTP) to protect the [gamma-32 P] CTP. Increasing UTP concentrations up to 50 mM resulted in increased product formation. A clear requirement for divalent cations was observed; 5 mM ethylenediaminetetraacetate (EDTA) abolished activity. The following order of cation effectiveness was observed: Mn greater than or equal to Ca greater than Cd greater than Zn much greater than Mg. Ten mM optima were established for Ca2+ and Mn2+; the presence of UTP, however, results in significantly reduced concentrations of free Ca2+. Ion combination studies demonstrated interactive inhibitory effects between Ca2+ and other stimulatory divalent cations. Addition of 2 microM brain calmodulin, in the presence of 10 mM Ca2+, resulted in a 75-100% stimulation of activity.(ABSTRACT TRUNCATED AT 250 WORDS)