Transformation, in vitro, of cerebral hamster cells by polyoma virus]

A cell line called HCxPy was obtained in vitro by transformation of dissociated hamster brain cell cultures by polyoma virus. The first foci of transformed cells became evident 90 to 120 days after viral infection. This cell line is now at the 46th passage. The cells appear tumorigenic for hamsters after subcutaneous and intracerebral injection. They carry the polyoma virus T and cell surface antigens. Good evidence for astrocytic differentiation can be found by morphological examination of the tumours and of the cultured cells.     

Immunologic blood-brain barrier in the polyoma virus--Syrian hamster system]

The authors report premilinary results of an experiment on permeability of the blood-brain barrier (BBB) to anti-tumor virus-induced immunological factors in the polyoma virus/Syrian Hamster system. The animals were protected by subcutaneous or intracranial injections with virus before challenge with polyoma virus transformed cells by both routes. BBB seemed to be permeable to the efferent part of the subcutaneously induced immune reaction. On the contrary, antigenic information introduced in the central nervous system was trapped inside the BBB. Thus the BBB might offer a "one-way" permeability in this system.     

The effect of temperature and protein synthesis on the renaturation of firefly luciferase in intact H9c2 cells

A mild increase in temperature that does not exert an effect on tolerance development or synthesis of heat shock proteins (Hsps) in control cells can stimulate these processes when applied to cells that have previously been heat shocked. To study the underlying mechanism of this effect, H9c2 cells were stably transfected with the gene encoding firefly luciferase (Luc). Heat-shock-induced inactivation of Luc and its subsequent reactivation is frequently used as a model for cellular protein denaturation and renaturation. Luc reactivation was determined following a damaging heat shock (43 or 44 degrees C for 30 min) in cells that were subsequently exposed to either control temperatures (37 degrees C) or various mild hyperthermic conditions (from 38.5 to 41.5 degrees C for 1 h). To prevent changes in Luc activity consequent to new synthesis of Luc, Luc reactivation was monitored in the presence of cycloheximide, an inhibitor of protein synthesis. The results showed that reactivation of Luc was inhibited when heat-treated cells were post-treated under mild hyperthermic conditions. The observed increase in Hsp synthesis under mild hyperthermic post-heat shock conditions therefore appears to be the result of an increase in the period during which denatured proteins are present. In addition, we studied Luc reactivation in the absence of protein synthesis inhibitors. This condition led to much higher Luc activity. By estimating half-life times of Luc, the contribution of new Luc synthesis in this recovery could be determined, and only partially explained the observed increase in Luc reactivation after heat shock. Thus the synthesis of other proteins must be important for the renaturation of heat-damaged proteins.     

Hemagglutination by simian adenovirus 7]

Simian adenovirus 7, either complete virus or its capsid subunits, agglutinates Rat (Sprague-Dowley) red blood cells in the presence of heterotypical antiserum. Haemagglutination takes place at 4 degrees C and room temperature. The antigen could not be eluted and its haemagglutinin properties are heat-stable. The reaction is specific. It is inhibited by homologous antiserum only. This property and its characteristics permit a camparison of this strongly oncogenic adenovirus to the human adenovirus of subgroup III of Rosen.     

Mid-anaphase arrest in S. cerevisiae cells eliminated for the function of Cin8 and dynein

S. cerevisiae anaphase spindle elongation is accomplished by the overlapping function of dynein and the kinesin-5 motor proteins, Cin8 and Kip1. Cin8 and dynein are synthetically lethal, yet the arrest phenotypes of cells eliminated for their function had not been identified. We found that at a non-permissive temperature, dyn1Δ cells that carry a temperature-sensitive cin8 – 3 mutation arrest at mid-anaphase with a unique phenotype, which we named TAN (two microtubule asters in one nucleus). These cells enter anaphase, but fail to proceed through the slow phase of anaphase B. At a permissive temperature, dyn1Δ, cin8 – 3 or dyn1Δcin8 – 3 cells exhibit perturbed spindle midzone morphologies, with dyn1Δcin8 – 3 anaphase spindles also being profoundly bent and nonrigid. Sorbitol, which has been suggested to stabilize microtubules, corrects these defects and suppresses the TAN phenotype. We conclude that dynein and Cin8 cooperate in anaphase midzone organization and influence microtubule dynamics, thus enabling progression through the slow phase of anaphase B. Received 10 August 2008; received after revision 22 October 2008; accepted 27 October 2008     

Functional mechanisms of the cellular prion protein (PrPC) associated anti-HIV-1 properties

The cellular prion protein PrP(C)/CD230 is a GPI-anchor protein highly expressed in cells from the nervous and immune systems and well conserved among vertebrates. In the last decade, several studies suggested that PrP(C) displays antiviral properties by restricting the replication of different viruses, and in particular retroviruses such as murine leukemia virus (MuLV) and the human immunodeficiency virus type 1 (HIV-1). In this context, we previously showed that PrP(C) displays important similarities with the HIV-1 nucleocapsid protein and found that PrP(C) expression in a human cell line strongly reduced HIV-1 expression and virus production. Using different PrP(C) mutants, we report here that the anti-HIV-1 properties are mostly associated with the amino-terminal 24-KRPKP-28 basic domain. In agreement with its reported RNA chaperone activity, we found that PrP(C) binds to the viral genomic RNA of HIV-1 and negatively affects its translation. Using a combination of biochemical and cell imaging strategies, we found that PrP(C) colocalizes with the virus assembly machinery at the plasma membrane and at the virological synapse in infected T cells. Depletion of PrP(C) in infected T cells and microglial cells favors HIV-1 replication, confirming its negative impact on the HIV-1 life cycle.     

Diffusion loading conditions determine recovery of protein synthesis in electroporated P3X63Ag8 cells

Using the suspension cell line P3X63Ag8 we have studied the impact of the composition of the diffusion medium on cellular protein synthesis under standard electroporation conditions in TBS-Na. This buffer contains the high saline concentration usually present in electroporation-mediated DNA transfection. Electroporation in the presence of TBS-Na resulted in an immediate shut-off of protein synthesis, even though both FITC-dextran (Mr 40 kD) and Semliki Forest virus core protein (Mr 33 kD) were incorporated efficiently into the cytoplasm across the electropores at 0 degrees C. Subsequent resealing of the pores was completed after a 5-min incubation at 37 degrees C. When compared with control cells, overall protein synthesis of electroporated cells recovered slowly to resume a 30% activity after 1 h of incubation at 37 degrees C. We have determined optimal conditions for diffusion loading (which necessitates the presence of ATP, GTP, amino acids, K+, Mg2+, and Ca2+) and resealing (in the presence of K+, Mg2+, and Ca2+), leading to a full and lasting recovery of protein synthesis within 5 min after pore closure.     

In vitro transmission of mouse leukemia virus to restrictive mouse cells: necessity for mixed infection by two different leukemia viruses]

The plaque formation of murine leukemia virus (MuLV) in non permissive mouse cells (N-tropic MuLV in B-type cells, or B-tropic MuLV in N-type cells) was increased by murine sarcoma virus whose plaque-forming activity was extremely low (MuSV XC-). The infection of N-tropic MuLV in B-type cells was increased by MuSV XC- propagated only in N-type cells but not in B-type cells, and the infection of B-tropic MuLV in N-type cells by MuSV XC- propagated only in B-type cells but not in N-type cells.     

Tumor specific surface antigen in the culture medium of a rat cell line transformed by Rous sarcoma virus]

TSSA is detected on transformed cells by a mixed hemadsorption reaction. The medium of cultures of Rat cells transformed by Rous sarcoma virus (Prague strain, sub-group C) contains a soluble factor which specifically inhibits this reaction. This factor thus possesses the antigenic activity of TSSA which is associated with the presence of a component of molecular weight 42,000 by polyacrylamide gel electrophoresis.     

Stimulation of the synthesis and non-competitive inhibition of alkaline phosphatase by theophylline in normal hamster fibroblasts and absence of response in transformed fibroblasts]

Theophylline increases the synthesis of alkaline phosphatase in normal Hamster fibroblasts (activity X6 after 24 hours with theophylline 10-3 M). The enzymatic activity of these cells is inhibited by theophylline in vitro (80% inhibition in the presence of theophylline 10-3 M). The inhibition seems to be non competitive, since the apparent Km of the enzyme is not modified. This stimulator and inhibitor effect of theophylline is absent in Hamster fibroblasts transformed by SV 40 virus.     

Different sensitivities to ultraviolet rays of various functions of the polyoma virus. Stimulation of the synthesis of cellular DNA]

Peritoneal Mouse macrophage were used to study the stimulation of cell DNA synthesis by polyoma virus. Using ultraviolet-irradiated polyoma virus, it was possible to show a difference between the inactivations of infectivity and of induction of DNA synthesis. By statistically analysis of these two phenomena it was found that 39% of the viral genome is necessary for the induction of cell DNA synthesis.     

Effects of photoperiod, temperature and testosterone-treatment on plasma T3 and T4 levels in the Djungarian hamster, Phodopus sungorus

The effects of photoperiod, temperature and testosterone treatment on plasma T3 and T4 levels were investigated in the Djungarian hamster. Plasma T3 level was affected by temperature (25 degrees C less than 7 degrees C) but not by photoperiod. Plasma T4 level was affected by photoperiod (short day less than long day) at 25 degrees C. Administration of testosterone increased plasma T4 level under short photoperiod at 25 degrees C. Thus, higher plasma T4 level under long photoperiod at 25 degrees C might be induced by testosterone.     

Hypothalamic histamine modulates adaptive behavior of rats at high environmental temperature

Histamine content in the rat hypothalamus was lower at 4 degrees C and higher at 31 degrees C compared to that at 21 degrees C. Pretreatment with alpha-fluoromethylhistidine, a 'suicide' inhibitor of histamine decarboxylase, attenuated both the increased level of hypothalamic histamine and rat adaptive behavior at 31 degrees C. Increase of histamine content in the hypothalamus appears to be an important factor contributing to rat adaptive behavior to high environmental temperature.     

Hepatic silver binding protein (Ag BP) from sparrow (Passer domesticus)

A silver binding protein (Ag BP) has been identified in the liver of sparrows administered a tracer dose of 110mAg. The protein as purified by gel-filtration shows a major absorption maximum at 260 nm and a minor one at 225 nm. It has a mol.wt of 9500 daltons and is stable when exposed to high temperature (64 degrees C for 15 min) as well as to acidic pH (2.2).     

Improved separation at low temperature of glycoproteins by Con A-Sepharose affinity chromatography in the presence of sodium dodecyl sulfate (SDS)

The Con A-Sepharose affinity chromatography of glycoproteins was even more effective at 4 degrees C than that at room temperature (26-28 degrees C) in the presence of sodium dodecyl sulfate (SDS). Application of this methodology to the separation of several glycoproteins from SDS-solubilized membrane proteins in rat cerebellum, including a glycoprotein characteristic of the Purkinje cells, was successful.     

Pimozide and p-chlorophenylalanine blockade in DL-amphetamine and pargyline-treated rats held at two environmental temperatures.

In rats, treated with DL-amphetamine + monoamine oxidase inhibitor, and held at an ambient temperature of 28.5 degrees C, hyperthermia was completely eliminated by treatment with pimozide + p-chlorophenyl-alanine. The same drugs markedly reduced the hypothermic effects in rats treated similarly at 4 degrees C. Results implied that serotoninergic and dopaminergic neurones were involved in the thermoregulatory effects of amphetamine.     

Antibody-dependent adherence of peritoneal cells of the rat to Trichinella spiralis larvae]

Washed peritoneal exudate cells from normal Rats firmly adhere to Trichinella spiralis larvae in the presence of serum containing anti-Trichinella antibodies. This effect is observed when muscle larvae, cells and dilutions of anti-sera are incubated for 1 hr. at 37 degrees C. No adherence takes place at 4 degrees C. Whole serum or its gammaglobulin fraction are active and effect is inhibited by the addition of Trichinella antigens. Complement is not essential since antiserum heated for 2 hrs. at 56 degrees C is active. Washed cells from infested animals do not adhere to the larvae in the absence of antiserum.     

Protein kinase-dependent overexpression of the nuclear protein pirin in c-JUN and RAS transformed fibroblasts

Signalling via the protein kinase Raf-MEK-ERK pathway is of major importance for transformation by oncogenes. To identify genes affected by inhibition of this pathway, c-JUN transformed rat fibroblasts were treated with a MEK1 inhibitor (PD98059) and subjected to two-dimensional gel electrophoresis after cell lysis. Gene products with expression influenced by MEK1 inhibition were determined by mass spectrometry of fragments from in-gel tryptic digestions. The expression of pirin, a nuclear factor I-interacting protein, was lowered after inhibition of MEK1. Western blot analysis revealed increased expression of pirin in RAS and c-JUN transformed cells in the absence of PD98059. Inhibition of MEK1 also led to reduced expression of α-enolase, phosphoglycerate kinase, elongation factor 2 and heterogeneous nuclear ribonucleoprotein A3, the latter two being detected as truncated proteins. In contrast, the level of ornithine aminotransferase was increased. We conclude that inhibition of MEK1 results in major alterations of protein expression in c-JUN transformed cells, suggesting that this pathway is important for oncogene-induced phenotypic changes. Received 30 December 1998; accepted 12 January 1999     

Cholecystokinin-octapeptide-induced hyperthermia in guinea-pigs

Intracerebroventricular administration of cholecystokinin-octapeptide (CCK-8) at room temperature (21 degrees C) induced dose-related hyperthermia in guinea-pigs and also produced hyperthermia at low (10 degrees C) and high (30 degrees C) ambient temperatures. CCK-8-induced hyperthermia was direct and not mediated by prostaglandins, norepinephrine, cyclic AMP or naloxone-sensitive receptors.