Interferon-γ- and lipopolysaccharide-induced tumor necrosis factor-α is required for nitric oxide production: Tumor necrosis factor-α and nitric oxide are independently involved in the killing ofMycobacterium microti in interferon-γ-and lipopolysaccharide-treated J774A.1 cells

A comparative study was done using J774A.1 and J774A. 1-derived transfected cells (J774A.1 C.1) containing antisense tumor necrosis factor α (TNF-α) plasmid to determine the role of endogenous TNF-α on nitric oxide production as well as on the growth ofMycobacterium microti in interferon γ (IFN-γ)- and lipopolysaccharide (LPS)-treated cells. On stimulation with IFN-γ and LPS a higher level of NO was observed in J774A.1 cells compared to J774A.1 C.1 which indicated that endogenous TNF-α is required for the production of NO. Comparing the effect of IFN-γ and LPS on the intracellular growth ofM. microti, the growth-reducing activity was higher in J774A.1 cells than in J774A.1 C.1 cells and was not completely abrogated in the presence of the nitric oxide inhibitorN ^G-methyl-l-arginine (l-NMA). J774A.1 C.1 cells infected withM. microti produced a significant amount of NO when exogenous TNF-α was added along with IFN-γ and LPS and the concentration of intracellular bacteria decreased almost to that in IFN-γ and LPS treated parental J774A.1 cells. Addition of exogenous TNF-α even in the presence ofl-NMA in J774.1 C.1 cells could also partially restore intracellular growth inhibition ofM. microti caused by IFN-γ and LPS. TNF-α is probably required for the production of NO in J774A.1 cells by IFN-γ and LPS but TNF-α and NO are independently involved in the killing of intracellularM. microti with IFN-γ and LPS.     

Bradykinin-induced oscillations of cell membrane potential in cells expressing the Ha-ras oncogene

Products of ras genes are putative elements of growth factor signal transduction. However, the mechanism of action of these proteins in normal and malignant growth is as yet obscure. To test for functional consequences of ras oncogene expression, electrophysiological experiments were performed on NIH-3T3 fibroblasts transfected with a transforming Ha-ras MMTV-LTR construct expressing the oncogene on treatment with dexamethasone (+ras). Transfected cells in the absence of dexamethasone (-ras) and nontransfected cells in the presence of dexamethasone (oras) served as controls. In -ras and oras, bradykinin induces a single, transient hyperpolarization. In +ras, bradykinin elicits oscillations of cell membrane potential throughout the presence of the hormone by activation of calcium-sensitive K+ channels. The oscillations of cell membrane potential are abolished in the absence of extracellular calcium. As evident from fura 2 fluorescence, bradykinin leads to a transient increase of intracellular calcium both in the presence and absence of extracellular calcium. Oscillations of intracellular calcium could be observed in +ras cells, if bradykinin was applied at reduced extracellular sodium concentration possibly to impair calcium extrusion via the sodium/calcium exchange. Bradykinin induces oscillations of cell membrane potential similarly in -ras cells loaded with GTP[S], a nonhydrolyzable analogue of GTP. Thus, the altered response of ras oncogene expressing cells to bradykinin relates to the GTP binding property of the ras protein. It is concluded that in cells expressing ras oncogene but not in other fibroblasts bradykinin mimicks the effect of growth factors on the cell membrane.     

Interferon-γ Promotes Differentiation of Neural Progenitor Cells via the JNK Pathway

It has been reported that interferon-γ (IFN-γ) facilitates differentiation of PC-12 cells and murine adult neural stem cells. Here we show that IFN-γ promotes the differentiation of C17.2 neural progenitor cells (NPC) into a neuronal phenotype characterized by neurite outgrowth and the expression of the neuronal marker protein β-III tubulin. IFN-γ induced an increase in the activity c-jun N-terminal kinase (JNK) without affecting activities of extracellular signal-regulated kinases (ERKs 1 and 2). An inhibitor of JNK blocked the ability of IFN-γ to promote differentiation of NPC into neurons, whereas an inhibitor of ERKs 1 and 2 did not. Our findings show that the pro-inflammatory cytokine, IFN-γ has the potential to stimulate neurogenesis, suggesting roles for this cytokine in development and repair of the nervous system.     

Up-regulation of E-cadherin and β-catenin in human hepatocellular carcinoma cell lines by sodium butyrate and interferon-α

Summary Human E-cadherin is a homophilic cell adhesion molecule and its expression is well preserved in normal human hepatocytes; a decrease in its expression has been observed in poorly differentiated hepatocellular carcinoma cells. We examined the alteration of E-cadherin and catenin expressions caused by differentiation inducers in human hepatocellular carcinoma cells. Hepatocellular carcinoma cell lines, HCC-T and HCC-M, were cultured with all-trans retinoic acid (ATRA), dexamethasone (DEX), sodium butyrate, and interferon-α. E-cadherin expression was only up-regulated by butyrate and interferon-α (IFN-α) in both cell lines, studied by means of fluorescence immunostaining and flow cytometry. The localization of E-cadherin staining was shown at their cell membrane. According to the increase in E-cadherin expression, β-catenin expression appeared at the cell membrane of both cell lines when treated with butyrate and IFN-α. Such an appearance was not observed when cells were treated with ATRA and DEX. Western blotting showed that α-and γ-catenin expression was not changed, while only the expression of β-catenin increased. β-Catenin oncogenic activation as a result of amino acid substitutions or interstitial deletions within or including parts of exon 3, which has been demonstrated recently, was not detected in these cell lines by direct deoxyribonucleic acid sequencing. These results suggest that the expression and interaction between E-cadherin and wild-type β-catenin are potentially modulated by butyrate and IFN-α, and that these two agents are potent inhibitors of hepatocellular carcinoma cell invasion and metastasis.     

Interferon-α/β upregulate IL-15 expression in vitro and in vivo: analysis in human hepatocellular carcinoma cell lines and in chronic hepatitis C patients during interferon-α/β treatment

Type I interferon (IFN) possesses antiviral and antitumor activities and also having an immune regulatory effect, activating cellular immune response and upregulating several cytokines. Recent study has shown that type I IFN upregurates the dendritic cell production of IL-15 capable of activating natural killer cells and CD8⁺ memory T lymphocytes. However, it is still unknown if type I IFN induces IL-15 production in non-immune cells and if type I IFN affects IL-15 production in vivo. The present study investigated the effect of type I IFNs on IL-15 expression in hepatocellular carcinoma (HCC) cell lines in vitro and in patients with chronic hepatitis C in vivo. When three HCC cell lines, Huh7, HepG2, and JHH4 were cultured in vitro, IFN upregulation of IL-15 expression was observed at both the mRNA and protein levels. In experiments using Huh7 cells, upregulation of IL-15 expression occurred within 24 h of the start of IFN stimulation, and both IFN-α and -β dose-dependently increased IL-15 production in the range from 100 U/ml to 10,000 U/ml of concentration. IFN-β showed stronger activity in IL-15 production induction in vitro than IFN-α. For in vivo examination, sera were obtained from 21 chronic hepatitis C patients treated with IFN and 29 healthy individuals, and the serum IL-15 level was quantified by ELISA. The serum IL-15 level of chronic hepatitis C patients before IFN treatment was similar to that of the healthy controls and significantly increased only during the IFN administration period. These results confirm that IFN-α/β induce IL-15 production and also suggest that IL-15 may be associated with type I IFN-induced immune response.     

Effects of tumor necrosis factor and interferon-β on proliferation and epidermal growth factor binding in young and senescent WI-38 cells

Summary Tumor necrosis factor-α (TNF) and various interferons (IFN) have potent cytostatic or cytotoxic effects on a variety of human tumor-derived cell lines. Their effects on normal cells are more controversial. We have examined the effects of TNF and IFN-β on the proliferation of WI-38 cells in a serum-free, growth factor-supplemented medium and in serum-containing medium. These cells respond to the combination of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and dexamethasone by DNA synthesis at a rate and extent equivalent to serum-stimulated cells. TNF has no effect on this growth factor-stimulated proliferation. However, it is stimulatory in serum-containing medium. IFN-β inhibits DNA synthesis 60 to 70% in both young and senescent cells. TNF and IFN-β together have a synergistic effect and completely inhibit growth factor-stimulated DNA synthesis in young cells. No synergism was observed with senescent cells. TNF stimulated an increase in the number of EGF specific binding sites two- to threefold in 24 h in both young and senescent cells. This seems to result from a proportional increase in a very high affinity binding site. IFN-β has little or no effect on EGF binding either alone or in combination with TNF.     

Treatment with TNF-α and IFN-γ alters the activation of SER/THR protein kinases and the metabolic response to IGF-I in mouse c2c12 myogenic cells

The aim of this study was to compare the effects of TNF-α, IL-1β and IFN-γ on the activation of protein kinase B (PKB), p70^S6k, mitogen-activated protein kinase (MAPK) and p90 ^rsk , and on IGF-I-stimulated glucose uptake and protein synthesis in mouse C2C12 myotubes. 100 nmol/l IGF-I stimulated glucose uptake in C2C12 myotubes by 198.1% and 10 ng/ml TNF-α abolished this effect. Glucose uptake in cells differentiated in the presence of 10 ng/ml IFN-γ increased by 167.2% but did not undergo significant further modification upon the addition of IGF-I. IGF-I increased the rate of protein synthesis by 249.8%. Neither TNF-α nor IFN-γ influenced basal protein synthesis, but both cytokines prevented the IGF-I effect. 10 ng/ml IL-1β did not modify either the basal or IGF-I-dependent glucose uptake and protein synthesis. With the exception of TNF-α causing an 18% decrease in the level of PKB protein, the cellular levels of PKB, p70^S6k, p42^MAPK, p44^MAPK and p90 ^rsk were not affected by the cytokines. IGF-I caused the phosphorylation of PKB (an approximate 8-fold increase above the basal value after 40 min of IGF-I treatment), p42^MAPK (a 2.81-fold increase after 50 min), and the activation of p70^S6k and p90 ^rsk , manifesting as gel mobility retardation. In cells differentiated in the presence of TNF-α or IFN-γ, this IGF-I-mediated PKB and p70^S6k phosphorylation was significantly diminished, and the increase in p42^MAPK and p90 ^rsk phosphorylation was prevented. The basal p42^MAPK phosphorylation in C2C12 cells treated with IFN-γ was high and comparable with the activation of this kinase by IGF-I. Pretreatment of myogenic cells with IL-1β did not modify the IGF-I-stimulated phosphorylation of PKB, p70^S6k, p42^MAPK and p90 ^rsk . In conclusion: i) TNF-α and IFN-γ, but not IL-1β, if present in the extracellular environment during C2C12 myoblast differentiation, prevent the stimulatory action of IGF-I on protein synthesis. ii) TNF-α- and IFN-γ-induced IGF-I resistance of protein synthesis could be associated with the decreased phosphorylation of PKB and p70^S6k. iii) The activation of glucose uptake in C2C12 myogenic cells treated with IFN-γ is PKB independent. iv) The similar effects of TNF-α and IFN-γ on the signalling and action of IGF-I on protein synthesis in myogenic cells could suggest the involvement of both of these cytokines in protein loss in skeletal muscle.     

Ultrastructure before freezing,while frozen,and after thawing in assessing cryoinjury of mouse epididymal spermatozoa

Tails of mouse epididymides were treated as follows: control, unfrozen with and without cryoprotective agents (CPA); frozen (to below ?80 °C), slowly (8 °C/min), and rapidly (18 °C/sec), with and without CPA. Intracellular and/or extracellular location of CPA, at least glycerol, was influenced, respectively, by high (22 °C) or low (0 °C) exposure temperature. Standard procedures in electron microscopy were employed and the frozen state preserved by freeze-substitution. Motility before freezing and after thawing was the criterion of cryosurvival.Results showed no evidence of deleterious ultrastructural effects of freezing at rates compared, or of benefits of CPA, regardless of their cellular location. Differences were noted, however, in the appearance of spermatozoa in the frozen state, as a function of the rate of freezing but not as a function of the presence, absence, or location of either glycerol of DMSO. Rapidly frozen cells showed intracellular ice formation in the acrosome, neck, midpiece, and tail regions; there was no intranuclear ice, and extracellular ice artifacts were small. Slowly frozen cells showed large extracellular ice artifacts with evidence of shrinkage distortion due to the dehydration induced by extracellular ice. No spermatozoa survived any of the freezing treatments, showing the lethal effect of both extracellular ice during slow freezing and of intracellular and/or extracellular ice during rapid freezing.     

Conversion of human interferon-β from a secreted to a phosphatidylinositol anchored protein by fusion of a 17 amino acid sequence to its carboxyl terminus

A number of cell-surface proteins are anchored in plasma membranes by a glycosylated phosphatidylinositol (PI) moiety that is covalently attached to the carboxyl-terminal amino acid of the mature protein. We have previously reported the construction of a cDNA clone of a truncated Platelet-derived growth factor (PDGF) receptor that consists of the extracellular domain without the transmembrane and cytoplasmic domains. In the construction of the vector, a sequence of 51 base pairs (bp) from the 3′-untranslated region of the receptor cDNA was linked in frame with the external domain coding sequence. The truncated receptor protein with the peptide VTSGHCHEERVDRHDGE fused to its carboxyl terminus was covalently attached to the membrane by a PI linkage and it was released by phosphatidylinositol specific-phospholipase C (PI-PLC). When the 51 bp sequence was deleted, the external domain receptor protein was secreted into the media. To determine whether the PI linkage of the protein was due to the 17 amino acids added, the peptide was fused to the carboxyl terminus of the secreted protein human Interferon-β (hu-IFN-β). Chinese hamster ovary (CHO) cells transfected with the hu-IFN-β cDNA secreted the protein to theconditioned media, whereas CHO cells transfected with the carboxyl terminus modified-hu-IFN-β cDNA did not secrete detectable levels of protein. CHO cells expressing the carboxyl terminus modified-hu-IFN-β were treated with PI-PLC, the media and cell lysates were analyzed by SDS-PAGE after immunoprecipitation with antibodies against hu-IFN-β. The modified protein is anchored to the plasma membrane by a PI linkage and it is specifically released by PI-PLC, whereas a control preparation of CHO cells expressing wild type hu-IFN-β does not show the same pattern. The 17 amino acid peptide fused to the carboxyl terminus of IFN-β directs attachment of a PI anchor and targets the fusion protein to the plasma membrane.     

19F NMR studies of changes in membrane potential and intracellular volume during dexamethasone-induced apoptosis in human leukemic cell lines

The induction of apoptosis in leukemic cells by dexamethasone is well known, but the mechanism of this type of cell death and of dexamethasone resistance by some variants is still poorly understood. Apoptotic cell death is preceded by many changes in cellular properties, such as glucose metabolism, cell size, cell density, and others. In this study, ¹⁹F-NMR has been used to characterize changes in cell membrane potential and intracellular accessible volume during dexamethasone induced apoptosis. One dex-sensitive (CEM-C7) and three dex-resistant variants (CEM-C1, CEM-ICR27, and CEM-4R4) were examined. We have observed separate intracellular and extracellular resonances for trifluoroacetate and trifluoroacetamide added to suspended leukemic cells. From the equilibrium distribution of these fluoro-compounds between intra and extracellular spaces, the changes in membrane potential and intracellular accessible volume were calculated. The membrane potential for CEM-C7 cells was found to significantly decrease in the presence of dexamethasone (9-mV decrease within 18 h of dexamethasone treatment), while that of CEM-ICR27 was found in some samples to increase on dexamethasone incubation. The membrane potential for CEM-C1 decreased slightly, while that of CEM-4R4 was not appreciably affected by dexamethasone. The reduction of membrane potential seems to be an early step in the mechanism of dexamethasone induced apoptosis. Although the intracellular volume varied with cell type and dexamethasone incubation (for CEM-C7), the fractional intracellular volume (α = V_in/V_cell was found to be the same (0.82 ± 0.06) for all the cell lines in the presence and absence of dexamethasone. © 1993 Wiley-Liss, Inc.     

Effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii (Cyanophyta)

The effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii were determined. Of the three temperatures tested, 10, 25 and 35°C, the maximal geosmin concentration and geosmin productivity were yielded at 10°C, while the highest chl a production was observed at 25°C. In the studies on light intensity, the maximal geosmin concentration and geosmin productivity were observed at 10 μmol m⁻² s⁻¹, while the highest chl a production was at 20 μmol m⁻² s⁻¹. It was suggested that more geosmin was synthesized with lower chl a demand. Meanwhile, the relative amounts of extra- and intracellular geosmin were investigated. Under optimum growth conditions (20 μmol m⁻² s⁻¹, 25°C; BG-11 medium), the amounts of extracellular geosmin increased as the growth progressed and reached the maximum in the stationary phase, while the intracellular geosmin reached its maximum value in the late exponential phase, and then began to decline. However, under the low temperature (10°C) or light (10 μmol m⁻² s⁻¹) conditions, more intracellular geosmin was synthesized and mainly accumulated in the cells. The proportions of extracellular geosmin were high, to 33.33 and 32.27%, respectively, during the stationary phase at 35°C and 20 μmol m⁻² s⁻¹. It was indicated that low temperature or light could stimulate geosmin production and favor the accumulation of geosmin in cells, while more intracellular geosmin may be released into the medium at higher temperatures or optimum light intensity.     

β-Galactosidase activity of Escherichia coli under long-term starvation, alterations in temperature, and different nutrient conditions in lake water

β-Galactosidase activity of Escherichia coli was investigated in response to long-term starvation, changes in temperature and the presence of certain nutrient sources in lake water. β-Galactosidase activity decreased markedly in filtered-autoclaved lake water at 25 °C and 37 °C, whereas it remained almost constant at 4 °C and 15 °C for 60 days. Increases in β-galactosidase activity were observed in response to the following nutrient sources: glycine, serine, methionine and ammonium sulfate at 4 °C; glycine and ammonium sulfate at 15 °C; glycine, serine, methionine and ammonium sulfate at 30 °C. Glycine addition led to an increase in β-galactosidase activity of almost five and seven orders of magnitude at 15 °C and 30 °C, respectively. In addition, L-methionine had the strongest influence on β-galactosidase activity, which was detected as an increase of seven and eleven orders of magnitude at 4 °C and 30 °C, respectively. The effect of several amino acids and other nitrogen sources depended on the concentration of the nutrient source and the temperature. The results showed that, in lake water, long-term starvation, temperature change, and variations in nitrogen sources alter β-galactosidase activity. Those effects should be taken into account when monitoring coliforms from the environment. Electronic Publication     

Treatment with recombinant interferon-β reduces inflammation and slows cartilage destruction in the collagen-induced arthritis model of rheumatoid arthritis

We investigated the therapeutic potential and mechanism of action of IFN-β protein for the treatment of rheumatoid arthritis (RA). Collagen-induced arthritis was induced in DBA/1 mice. At the first clinical sign of disease, mice were given daily injections of recombinant mouse IFN-β or saline for 7 days. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were assessed histologically 8 days after the onset of arthritis. Proteoglycan depletion was determined by safranin O staining. Expression of cytokines, receptor activator of NF-κB ligand, and c-Fos was evaluated immunohistochemically. The IL-1-induced expression of IL-6, IL-8, and granulocyte/macrophage-colony-stimulating factor (GM-CSF) was studied by ELISA in supernatant of RA and osteoarthritis fibroblast-like synoviocytes incubated with IFN-β. We also examined the effect of IFN-β on NF-κB activity. IFN-β, at 0.25 μg/injection and higher, significantly reduced disease severity in two experiments, each using 8–10 mice per treatment group. IFN-β-treated animals displayed significantly less cartilage and bone destruction than controls, paralleled by a decreased number of positive cells of two gene products required for osteoclastogenesis, receptor activator of NF-κB ligand and c-Fos. Tumor necrosis factor α and IL-6 expression were significantly reduced, while IL-10 production was increased after IFN-β treatment. IFN-β reduced expression of IL-6, IL-8, and GM-CSF in RA and osteoarthritis fibroblast-like synoviocytes, correlating with reduced NF-κB activity. The data support the view that IFN-β is a potential therapy for RA that might help to diminish both joint inflammation and destruction by cytokine modulation.     

Comparative study of extracellular and intracellular β-glucosidases of a new strain of Zygosaccharomyces bailii isolated from fermenting agave juice

A yeast strain isolated in the laboratory was studied and classified as a Zygosaccharomyces bailii. Both intracellular and extracellular β-glucosidases of this yeast were purified by ion-exchange chromatography, gel filtration and hydroxylapatite (only for the intracellular enzyme). The tetrameric structure of the two β-glucosidases was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum activity at 65°C and pH 5.5. The extracellular enzyme exhibited optimum catalytic activity at 55°C and pH 5. The molecular mass of purified intracellular and extracellular β-glucosidases, estimated by gel filtration, was 440 and 360 kDa, respectively. Both enzymes are active against glycosides with (1 → 4)-β, (1 → 6)-β and (1 → 4)-α linkage configuration. The intracellular enzyme possesses (1 → 6)-α-arabinofuranosidase activity and extracellular enzyme (1 → 6)-α-rhamno-pyranosidase activity. The two β-glucosidases are competitively inhibited by glucose and by D-gluconic-acid-lactone and a slight glucosyl transferase activity is observed in the presence of ethanol. Since the glycosides present in wine and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast β-glucosidases for the liberation of the bound aroma is discussed.     

The mechanism of cryohemolysis: by direct observation with the cryomicroscope and the electron microscope.

Blood films (3–8 μm thick) supported between two glass coverslips were frozen to ?20 °C. In the extracellular areas, ice cavities of the order of 0.2 μm separated by bands of dense plasma were evident when examined with the electron microscope; intracellular ice was not observed with the light microscope. Electron microscopy also showed the presence of intracellular ice particles of the order of 0.2–0.7 μm, these appeared as fine reticulations when observed with the light microscope. Upon gradual rewarming the following changes were observed: recrystallization in the extracellular matrix (?18 to ?8 °C), intracellular recrystallization (?13 to ?10 °C), transfer of water from erythrocytes to extracellular areas (?9 to ?7 °C), and melting and hemolysis (?6 to ?2 °C).Freezing of blood at ?3 °C and subsequent thawing did not cause hemolysis of the red cells. In blood frozen at ?3 °C and cooled to ?20 °C or frozen by abrupt exposure to 20 °C the erythrocytes hemolyzed in 7/16–11/16 of a second, whereas in blood frozen at ?3 °C and cooled to ?10 °C the cells hemolyzed in 5–15 sec even though the mode if lysis (i.e., uniform seepage of hemoglobin from the surface of the cell) was similar in all cases. This indicates that the presence of intracellular ice does not seem to play a major role in the injury to the erythrocytes. The mechanism of cryoinjury demonstrated by hemolysis has been discussed.     

Production of halostable β-mannanase and β-mannosidase by strain NN, a new extremely halotolerant bacterium

An extremely halotolerant mannan-degrading bacterium (strain NN) was isolated from the Great Salt Lake, Utah, USA. Strain NN grew at salinities from 0 to 20% NaCl with optimal growth at 0% NaCl. When grown on 0.2% (w/v) locust bean gum as the carbon source at 10% NaCl, both β-mannanase and β-mannosidase activities were produced. β-Mannosidase activity was shown to be cell-associated, while at least 23% of the total β-mannanase activity was extracellular. The optimum temperature and pH for β-mannanase activity were 70 °C and 7.6, and for β-mannosidase 25 °C and 7.0. The β-mannanase system retained full activity after 24 h of incubation at 60 °C and 10% NaCl. β-Mannanase activity was maximal at 1% NaCl and β-mannosidase activity at 0.5% NaCl. Despite these low salinity optima, 50% and 100% respectively of the initial β-mannanase and β-mannosidase activities remained after 48 h of incubation at 20% NaCl, indicating a high degree of halostability. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis revealed the presence of at least eight different mannan-degrading proteins in the cell-free culture supernatant of cultures grown on locust bean gum. Received: 19 March 1998 /  Received revision: 8 June 1998 / Accepted: 14 June 1998     

Ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

One phase of a study on cryosurvival and cryoprotection of mammalian cells, in terms of ultrastructural alteration of rough endoplasmic reticulum (RER) within rat pancreatic acinar cells, is presented. Small (2–3 mm) squares of tissue, 0.7–0.9 mm in thickness, were compared as unfrozen controls, with (w) and without (wo) glycerol pretreatment (15% $\text{v}\text{v}$ in mammalian Ringer's solution) at 0 °C and 22 °C (to regulate glycerol permeability); as well as parallel frozen-thawed samples, after combinations of slow (3.8 °C/min) freezing (SF) and rapid (38 °C/sec) freezing (RF) with either slow (1.5 °C/min) thawing (ST) or rapid (8 °C/sec) thawing (RT). Regimens compared were SFRT, SFST, RFRT, and RFST, all w and wo glycerol pretreatment at 0 °C and 22 °C. Tissue from each treatment was prepared for electron microscopic observations. The results on rates of freezing and thawing and relative cryoprotection of intracellular and extracellular glycerol under conditions described are intended to serve as a correlative basis for subsequent parallel studies on function (protein synthesis) and ultrastructure of the frozen state. They now indicate the following: (1) Cryoinjury of RER, which occurred during all treatments compared, was manifested in irregularity, dilatation, vesiculation, and altered matrix density of cisternae, and ribosomal derangement or disjunction. Least injury was shown by some disorientation and dilatation with increasing degrees of damage involving accentuation of these and other alterations. Such ultrastructural alterations to RER are not unique to cryoinjury, since they have been induced by treatments and agents other than freeze-thawing in experimental pathology. (2) Cryoinjury is unique, however, in that it can be regulated to demonstrate a spectrum of degrees of injury to cells and their organelles, immediately after cryoexposure. Controlled cryoinjury is suggested as a research tool for studies on injury, in general, on an ultrastructural-functional level. (3) Glycerol is injurious or toxic during pretreatment. Toxicity, which resembles cryoinjury, is greater during 22 ° C (intracellular) than 0 °C (extracellular) glycerol pretreatment, especially with respect to dilatation of cisternae. (4) Extra-cellular glycerol is cryoprotective during both slow and rapid freezing followed by either slow or rapid thawing, while little or no cryoprotection is afforded when glycerol is located simultaneously in the intracellular and extracellular location. (5) Rate of freezing is more important than rate of thawing as a factor in cryosurvival. Rapid freezing is more injurious than slow freezing, in the absence of glycerol or in the presence of extracellular glycerol, with slight or no differences seen as a function of thawing rate. Neither rate of freezing nor rate of thawing is of serious consequence when glycerol is intracellular. (6) Rate of thawing has importance after slow freezing, when slow thawing is more injurious than rapid, but not after rapid freezing, either in the presence or absence of extracellular glyeerol.     

Involvement of dopamine receptors and beta-arrestin in metamphetamine-induced inclusions formation in PC12 cells

Exposure of PC12 cells to metamphetamine (MA) induces the formation of multilamellar structures (whorls) resembling autophagic granules that subsequently develop as intracellular inclusions. These inclusions stain for a variety of antigens belonging to the ubiquitin proteasome pathway. Since MA-induced intracellular bodies require the presence of dopamine in the present study we analyzed the role of dopamine (DA) receptors in producing neuronal inclusions. Moreover, we investigated potential signaling pathways which could lead to ubiquitination in the presence of MA. Based on recent reports that ubiquitination of β-adrenergic receptors is promoted by β-arrestin which shuttles proteins from the plasma membrane to the ubiquitin proteasome system we investigated whether β-arrestin is involved in MA-induced inclusion formation. Our experiments document that (i) β-arrestin was associated with MA-induced intracellular bodies; (ii) MA induced a rapid and reversible ubiquitination of β-arrestin; (iii) dopamine antagonists reduced both MA-induced β-arrestin ubiquitination and intracellular whorls formation; (iv) the number of MA-induced intracellular bodies was reduced in cells transfected with the β-arrestin dominant negative mutant, βarrV53D and was increased by the persistently ubiquitinated β-arrestin-ubiquitin fusion protein. In conclusion, the present study demonstrates the involvement of β-arrestin in MA-induced intracellular bodies and the participation of dopamine receptors in this process.     

Thermosensitization of tumor cells with inhibitors of chaperone activity and expression

Effects of inhibitors of the heat shock protein 90 (HSP90) chaperone activity and inhibitors of the heat shock protein (HSP) expression on sensitivity of HeLa tumor cells to hyperthermia were studied. It was found that nanomolar concentrations of inhibitors of the HSP90 activity (17AAG or radicicol) slowed down the chaperone-dependent reactivation of a thermolabile reporter (luciferase) in heat-stressed HeLa cells and slightly enhanced their death following the incubation for 60 min at 43°C. The inhibitors of HSP90 activity stimulated de novo induction of additional chaperones (HSP70 and HSP27) that significantly increased intracellular HSP levels. Treatment of the cells with 17AAG or radicicol along with an inhibitor of the HSP induction (e.g. quercetin or triptolide, or NZ28) completely prevented the increase in the intracellular chaperone levels resulting from the inhibition of HSP90 activity and subsequent heating. Combination of all three treatments (inhibition of the HSP90 activity + inhibition of the HSP induction + heating at 43°C for 60 min) resulted in more potent inhibition of the reporter reactivation and a sharp (2–3-fold) increase in cell death. Such enhancement of the cytotoxicity may be attributed to the “chaperone deficiency” when prior to heat stress both the functional activity of constitutive HSP90 and the expression of additional (inducible) chaperones are blocked in the cells.