70-kDa heat shock protein expression in cultured rat astrocytes after hypoxia: regulatory effect of almitrine

Induction of heat shock proteins (Hsps), especially the 70-kDa family, is well observed in nervous tissues in response to various stressful conditions. By using rat astrocytes in primary culture, the expression of the inducible (Hsp70) and the constitutive (Hsc70) 70-kDa Hsps immunoreactivity of cells exposed to hypoxic conditions has been investigated. We observed that exposure of astroglial cells to an hypoxic-normoxic sequence induces a significant decrease of Hsc70 immunoreactivity. The presence of the heat inducible stress protein Hsp70 is never observed in hypoxic cells not in control. Hsc 70 lowering is associated with ultrastructural alterations characterized by mitochondria swelling, formation of vacuoles and accumulation of dense material in the cell cytoplasm. The effects of addition of almitrine to the culture medium before and during hypoxia on Hsps immunoreactivity have been examined. The presence of the drug prevents the decrease of Hsc 70 immunoreactivity induced by hypoxia. Furthermore, some ultrastructural improvement is observed in astroglial cells treated with almitrine suggesting some protecting role of Hsc70 on cell damage induced by hypoxia.     

Different expression time of the 105-kDa protein and 90-kDa heat-shock protein in rat testis

To understand the physiological functions of the 105-kDa protein which is testis-specific and HSP90 (90-kDa heat-shock protein) related protein, the appearance of it in the testis has been followed during the development of rat. On immunoblotting analysis, the 105-kDa protein did not appear until after the age of five weeks, while HSP90 could be detected at three weeks. In the spermatozoa, the 105-kDa protein was much abundant but not in the LC-540 cells (a cell line from Leydig cell tumor in rat testis) cytosol. This finding has attracted much attention to the relationship between this protein and sperm functions.     

The major heat-shock protein hsp 68 is not induced by stress in mouse erythroleukemia cell lines

Most mammalian cells respond to brief incubation at elevated temperatures by enhanced or new synthesis of a set of heat-shock proteins (hsp). In mouse cells, as determined by SDS--one-dimensional gel electrophoresis, the most prominent hsps have molecular masses of approximately 89,000, 70,000, and 68,000 Da. When the heat-shock response of the mouse erythroleukemia cell line D1B, or two other DBA/2 cell lines (707C1 and 745C2), was examined by [35S]methionine labelling, following heat shocks of 10 min at 42 or 44 degrees C, or 1 h at 45 degrees C, no protein band corresponding to hsp 68 was observed. However, the synthesis of both hsp 89 and hsp 70 was enhanced. Northern blot analysis of cytoplasmic RNA extracted from control and stressed cells indicated that hsp 68 mRNA was absent, even after stresses of up to 1 h at 45 degrees C. Differentiation induced by dimethyl sulphoxide (DMSO) (monitored by the induction of globin synthesis) had no effect on hsp 68 expression in D1B cells; also, hsp 68 could not be induced at various stages of differentiation (0-72 h). Southern blot analysis showed that all three hsp-68 genes were present and not rearranged, and apparently did not carry any deletion in their 5' ends. To determine whether methylation could be involved in maintaining the genes in their silent state, we treated cells with 10 microM 5-azacytidine for 48 h. No hsp 68 expression was observed following such treatment in either undifferentiated or DMSO-induced differentiated D1B cells. Furthermore, Southern blot analysis of MspI/HpaII-digested genomic D1B DNA did not display any differences in methylation patterns around the promoter region of the probed gene compared with control cells, indicating that methylation is not involved in hsp-68 repression. When chimeric plasmids carrying the bacterial chloramphenicol acetyl transferase gene under regulation of the mouse hsp-68 or Drosophila hsp-70 promoters were transfected into D1B cells, minimal (2-fold) or no induction was observed, in contrast with the 60-fold induction seen in a control myeloma cell line. These results suggest a trans-acting mechanism of hsp-68 repression in erythroleukemia cells.     

Prenatal expression of the 65-kDa heat-shock protein homologue in pig tissues

A monoclonal antibody (ML30) recognizing the 65-kDa heat-shock protein of mycobacteria and reacting with homologous human protein was found to stain various porcine tissues. Development of this reactivity was studied. The first ML30-positive cells were embryonic hepatocytes. The protein reacting with the ML30 antibody was localized predominantly in the Golgi area and mitochondria of hepatocytes. Cell membranes of some peripheral blood lymphocytes were also found to bind ML30.     

Induction of four heat-shock proteins and their mRNAs in rat after whole-body hyperthermia

When the body temperature of rats was brought to 42 degrees C, four heat-shock proteins, with molecular weights of 70,000, 71,000, 85,000, and 100,000 (hsp 70, hsp 71, hsp 85, and hsp 100, respectively), were induced in various tissues of the rats. The hsp 70 was strongly induced by hyperthermia, and its accumulation was detected by Coomassie blue staining. The hsp 71 was abundant in various tissues of rats that were not heat-shocked. Analysis of translation products of liver mRNAs from heat-shocked rats also showed increased synthesis of the four heat-shock proteins, indicating that these hsp-mRNAs were induced after hyperthermia. Induction of the hsp-mRNAs was transient after hyperthermia. The four heat-shock proteins produced in various tissues after hyperthermia may be involved in homeostatic control in the heat-shock response of the rat.     

Induction of the mitochondrial permeability transition in cultured astrocytes by glutamine

Ammonia is a toxin that has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and astrocytes appear to be the principal target of ammonia toxicity. Glutamine, a byproduct of ammonia metabolism, has been implicated in some of the deleterious effects of ammonia on the CNS. We have recently shown that ammonia induces the mitochondrial permeability transition (MPT) in cultured astrocytes, but not in neurons. We therefore determined whether glutamine is also capable of inducing the MPT in cultured astrocytes. Astrocytes were treated with glutamine (4.5 mM) for various time periods and the MPT was assessed by changes in 2-deoxyglucose (2-DG) mitochondrial permeability, calcein fluorescence assay, and by changes in cyclosporin A (CsA)-sensitive inner mitochondrial membrane potential (deltapsi(m)) using the potentiometric dye, JC-1. Astrocytes treated with glutamine significantly increased 2-DG permeability (120%, P<0.01), decreased mitochondrial calcein fluorescence, and concomitantly dissipated the deltapsi(m). All of these effects were blocked by CsA. These data indicate that glutamine induces the MPT in cultured astrocytes. The induction of the MPT by glutamine in astrocytes, and the subsequent development of mitochondrial dysfunction, may partially explain the deleterious affects of glutamine on the CNS in the setting of hyperammonemia.     

Induction of a 60-kDa heat shock protein in rat pancreas by water-immersion stress

• 1.1. In this study, expression of a 60-kDa heat shock protein in rat pancreas was investigated before and after water-immersion stress, which has been known as an exacerbation factor of caerulein-induced pancreatitis in rats, by Western blot.
• 2.2. A 60-kDa heat shock protein increased after water-immersion stress in both soluble and insoluble fractions of the pancreas.
• 3.3. Serum amylase level and pancreas weight did not increase after water-immersion.
• 4.4. No pathologic alteration was observed in the pancreas after water-immersion.

     

Induction of cyclooxygenase-2 expression by manganese in cultured astrocytes

Inflammatory and oxidative events are present in neurodegenerative disorders and appear to contribute to initiation and/or progression of the disease. Within the brain, redox-active metals, such as manganese, play an important role as components of proteins essential for neural function. However, increasing evidence implies its participation in neurodegenerative diseases involving immune modulation. Prostaglandins (PGs) are lipid mediators that participate in the regulation of physiological and pathophysiological processes, particularly during brain inflammation. In this study, we investigated whether the immune modulating action of manganese involved regulation of PGE₂ production in cortical astrocytes. Within non-toxic concentrations, manganese caused an elevation in the expression of cyclooxygenase-2 (COX-2) mRNA and protein and increased PGE₂ release. Manganese potentiated COX-2 expression and PGE₂ generation by lipopolysaccharide/interferon-γ-activated astrocytes. The inductive action of manganese was accompanied by generation of oxidative stress, activation of mitogen-activated protein kinases (MAPKs), AKT, and protein kinase C- (PKC-), and increased NF-κB and AP-1 DNA binding activities. The generation of reactive oxygen species (ROS) was critical to manganese-induced changes in astrocytes, including MAPKs, PKC-, NF-κB, AP-1, and COX-2 expression but not AKT. Collectively, these data indicate that manganese might cause changes in neural activity through the modulation of oxidative and inflammatory events in astrocytes.     

Dynamic changes of intracellular pH in individual lactic acid bacterium cells in response to a rapid drop in extracellular pH

We describe the dynamics of changes in the intracellular pH (pH(i)) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pH(ex)). Strains of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis were investigated. Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pH(i) within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pH(ex) from 7.0 to 5.0. Under these conditions, the pH(i) of L. innocua remained neutral (between 7 and 8). In contrast, the pH(i) values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pH(ex) was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pH(i) at pH(ex) 7.0, while different kinetics of pH(i) regulation were observed in different species and also in different strains of S. thermophilus.     

Effects of calcium buffering on the synthesis of the 26-kDa heat-shock protein family

We have reported on the effect of heat in C127 cells having various basal levels of the Ca(2+)-binding proteins calmodulin (CaM) or parvalbumin [Evans, Simonette, Rasmussen, Means, and Tomasovic, J. Cell. Physiol. 142, 615-627 (1990)]. These studies suggested that induction of the synthesis of 26-kDa heat-shock protein (hsp-26) depended on increased intracellular free Ca2+ [Ca2+]i and that induction was abrogated by increased Ca(2+)-binding capacity. To evaluate further the role of [Ca2+]i in mediating the response to hyperthermia and the potential for Ca(2+)-buffering to affect these processes, we loaded C127 parental cells with the Ca2+ chelators BAPTA or quin-2 (5 microM for 60 min) and then immediately heated the cells (30 min at 43 degrees C) and labeled them (3 h at 37 degrees C) with [3H]leucine. Measurements of [Ca2+]i with quin-2 and fura-2 showed that an increase in [Ca2+]i occurred with this heat dose, but that the quin-2 buffered that increase. Two-dimensional gels showed that cells loaded with BAPTA and quin-2 had a reduced rate of synthesis of the most basic (nonphosphorylated) hsp-26a isoform. The apparent synthesis of the more acidic isoforms (hsp-26b, hsp-26c) was less affected, but labeling studies with 32P showed this reflected continued accumulation of these phosphorylated isoforms, especially the most highly phosphorylated hsp-26c. Although it reduced hsp-26a synthesis, the temporary buffering of [Ca2+]i did not alter the subsequent expression of heat killing or the extent of thermotolerance significantly, possibly because phosphorylated hsp-26 was still generated. These data support the hypothesis that perturbations of [Ca2+]i directly modulate induction of hsp-26a synthesis.     

Functional expression of the norepinephrine transporter in cultured rat astrocytes

We assessed the functional expression of the norepinephrine (NE) transporter (NET) in cultured rat cortical astrocytes. Specific [3H]NE uptake increased in a time-dependent manner, and this uptake involves temperature- and Na+-sensitive mechanisms. The Na+-dependent [3H]NE uptake was saturable, and the Km for the process was 539.3 +/- 55.4 nm and the Vmax was 1.41 +/- 0.03 pmol/mg protein/min. Ouabain, a Na+-K+ ATPase inhibitor, significantly inhibited Na+-dependent [3H]NE uptake. The selective NE uptake inhibitor nisoxetine, the tricyclic antidepressants desipramine and imipramine, and the serotonin and NE reuptake inhibitor (SNRI) milnacipran very potently inhibited Na+-dependent [3H]NE uptake. On the other hand, GBR-12935 (a selective dopamine uptake inhibitor), fluvoxamine (a selective serotonin reuptake inhibitor), venlafaxine (a SNRI) and cocaine had weaker inhibitory activities. RT-PCR demonstrated that astrocytes expressed mRNA for the cloned NET protein, which was characterized as neuronal NET. Western blots indicated that anti-NET polyclonal antibody recognized a major band of 80 kDa in astrocytes. These data indicate that the neuronal NET is functionally expressed in cultured rat astrocytes. Glial cells may exert significant control of noradrenergic activity by inactivating NE that escapes neuronal re-uptake in sites distant from terminals, and are thus cellular targets for antidepressant drugs that inhibit NE uptake.     

Serum deprivation increases the expression of low density lipoprotein receptor-related protein in primary cultured rat astrocytes

Scanning immunoelectron microscopy was applied to human endometrial epithelium for the first time to simultaneously determine epitope localisation and cellular architecture. The method was established using HMFG1, an antibody to a glycoform of the MUC1 mucin. This was chosen because of the potential importance of MUC1 in connection with endometrial receptivity. Biopsies of mid-secretory phase endometrium were labelled using HMFG1 and silver-enhanced, gold-conjugated secondary antibody was then visualised by back-scattered electron imaging. The method provided a highly specific localisation of the HMFG1 epitope to the ciliated and "ciliogenic" cells of the endometrial surface. In contrast, no reactivity was evident on the microvillous cells and endometrial pinopodes. The potential to integrate the study of the molecular and ultrastructural changes that occur in the endometrium by using scanning immunoelectron microscopy offers a powerful means of expanding our understanding of the adaptation of the endometrium in preparation for embryo implantation.     

Effect of glucose on the major testosterone-maintained protein in the cultured rat ventral prostate

The effect of glucose on the androgen-maintained protein synthesis was studied in the cultured rat ventral prostate. The expiants were cultivated for 5 days in the glucose-free medium containing 10% fetal calf serum with or without 10 mM glucose and 10⁻⁷M testosterone. In some experiments tunicamycin, a specific inhibitor of protein glycosylation was added to the glucose-containing medium. The morphological integrity of the tissue was maintained in all the mediums used. At the end of the culture, the expiants were incubated with [³⁵S]methionine. Soluble radioactive proteins were separated by the SDS-polyacrylamide gel electrophoresis and analyzed further by the fluorography. Glucose was necessary for the testosterone-maintained accumulation of three components (M_r less than 14,000) of the major prostatic secretory protein. The electrophoretic migration, glycosylation pattern and immunological data (not shown) indicated that it was the well-known prostatic binding protein. On the other hand, two prominent polypeptides (M_r 70,000 and 100,000) appeared in the absence of glucose. Glucose starvation and the inhibition of glycosylation with tunicamycin caused similar effects on the labelling of the newly-synthesized soluble proteins. The mechanisms of glucose maintenance of the major prostatic protein and suppression of two high molecular weight proteins seemed to be different, although glycosylation was probably involved in both glucose effects.     

Expression and cellular localization of the mRNA for the 25-kDa heat-shock protein in the mouse

The 25-kDa heat-shock protein (Hsp25) is a member of the small heat-shock protein family but its function remains largely unknown. In the present study we examined the expression and cellular localization of Hsp25 mRNA in mice under physiological, unstressed conditions using Northern blot and in situ hybridization analyses with specific oligonucleotide probes. At the organ level, high amounts of Hsp25 mRNA were detected in the oesophagus, skin,eye, stomach, lung and urinary bladder, with moderate amounts in the heart, skeletal muscle, aorta, adrenal gland, ovary, testis, uterus, large intestine, and thymus. At the cellular level, intense to moderate signals for Hsp25 mRNA were localized in the muscle cells of smooth, heart and skeletal types, in the epithelial cells of stratified squamous and transitional types and of the oviduct, in the steroid endocrine cells of the adrenal cortex and corpus luteum, as well as in the spermatocytes of the testis. In contrast, the signal was scarcely detectable in the nervous tissues, lymphatic tissues, the columnar epithelial cells of the digestive tract, or the parenchymal cells of the liver, pancreas and kidney. These results suggest some significant role for Hsp25 in distinct populations of mouse cells under physiological conditions.     

The relationship of the rat brain 68 kDa microtubule-associated protein with synaptosomal plasma membranes and with the Drosophila 70 kDa heat-shock protein.

A protein of molecular mass 68 kDa and pI5.6 is a major translation product of rat brain mRNA [Hall, Mahadevan, Whatley, Biswas & Lim (1984) Biochem. J. 219, 751-761]. In the rat brain this protein was associated with microtubule preparations and was present together with tubulin as a component of the synaptosomal plasma membranes, synaptic vesicles and post-synaptic structures. The brain mRNA for this protein was found to hybridize specifically to the Drosophila gene for the 70 kDa heat-shock protein, thus enabling its rapid isolation.