Cells transformed with a ts viral src mutant are temperature sensitive for in vivo growth.

Studies on ts mutants of avian sarcoma viruses have previously implicated the src gene product (pp60src) kinase function in in vitro transformation. The role of src in vivo, however, has not been clearly defined. Using a sensitive and quantitative assay that was developed in chicken embryos (Chambers et al., Cancer Res. 42:4018-4025, 1982), we tested the in vivo tumorigenic properties of cells transformed with LA23, an avian sarcoma virus that is temperature sensitive for in vitro transformation. We found that the in vivo growth ability of these cells was temperature sensitive and that this in vivo behavior correlated with the in vitro transformation behavior (growth in soft agar and saturation density).     

Glucocorticoid receptors are associated with particles containing DNA and RNA in vivo

Chemical crosslinking of glucocorticoid-receptor complexes to associated components in living cells was performed by the use of formaldehyde. Glucocorticoid binding sites were predominantly located in nuclei, and could not be efficiently extracted by 0.3 M NaCl. Sonication was found to cause the release of about 40% of nuclear receptor complexes. By sucrose density gradient centrifugation of soluble extracts from nuclear sonicates, crosslinked receptor complexes were found in oligomeric forms under high salt conditions. Treatment of these extracts with hydrolytic enzymes showed that DNA and RNA were associated with crosslinked receptor complexes.     

Brain somatostatin receptors are up-regulated in somatostatin-deficient mice

The peptide somatostatin (SST) is widely synthesized in the brain and periphery and acts through a family of five receptors (SSTR1-5) to exert numerous effects. A gene product related to SST, cortistatin (CST), also interacts with SSTR1-5. Here we have investigated the regulation of SSTR1-5 and of CST in SST knockout (SSTKO) mice. The five SSTRs were quantitated individually by subtype-selective binding analysis, by immunocytochemistry, and by mRNA measurement and showed, in the brain of SSTKO mice, up-regulation of subtypes 1, 2, 4, and 5, and down-regulation of SSTR3. Peripheral tissues displayed both subtype- and tissue-specific changes in SSTR1-5 mRNA levels of expression. Lack of SST did not up-regulate normal CST expression in brain nor did it induce its expression in the periphery. SST-like immunoreactivity, however, was induced in the proximal midgut in SSTKO animals, suggesting intestinal expression of a novel SST-like gene.     

Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells. methyl-beta-cyclodextrin does not affect cell surface transport of a GPI-anchored protein

Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergent-resistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-beta-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-beta-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties.     

Translation initiation factors are not required for Dicistroviridae IRES function in vivo

The cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry site (IRES) uses an unusual mechanism of initiating translation, whereby the IRES occupies the P-site of the ribosome and the initiating tRNA enters the A-site. In vitro experiments have demonstrated that the CrPV IGR IRES is able to bind purified ribosomes and form 80S complexes capable of synthesizing small peptides in the absence of any translation initiation factors. These results suggest that initiation by this IRES is factor-independent. To determine whether the IGR IRES functions in the absence of initiation factors in vivo, we assayed IGR IRES activity in various yeast strains harboring mutations in canonical translation initiation factors. We used a dicistronic reporter assay in yeast to determine whether the CrPV IGR IRES is able to promote translation sufficient to support growth in the presence of various deletions or mutations in translation initiation factors. Using this assay, we have previously shown that the CrPV IGR IRES functions efficiently in yeast when ternary complexes (eIF2•GTP•initiator tRNA^met) are reduced. Here, we demonstrate that the CrPV IGR IRES activity does not require the eukaryotic initiation factors eIF4G1 or eIF5B, and it is enhanced when eIF2B, the eIF3b subunit of eIF3, or eIF4E are impaired. Taken together, these data support a model in which the CrPV IGR IRES is capable of initiating protein synthesis in the absence of any initiation factors in vivo, and suggests that the CrPV IGR IRES initiates translation by directly recruiting the ribosomal subunits in vivo.     

In vitro tracheal responses from mice chosen for in vivo lung cholinergic sensitivity

We selected two inbred strains of mice based on their different in vivo lung responses to intravenous acetylcholine for studies on the in vitro tracheal responses to contractile and relaxing agents. In addition, we studied the role of cyclooxygenase products on the in vitro responses. Tracheal rings were contracted with increasing concentrations of carbachol and KCl and relaxed with increasing concentrations of isoproterenol after contraction with carbachol at the concentration that produced 30, 50, and 70% of the maximal contraction (EC30, EC50, and EC70, respectively) and KCl at the EC50. Half the tracheae simultaneously underwent the same protocols after pretreatment with indomethacin (3 X 10(-6) M). Despite a severalfold difference in the maximal response to cholinergic agents in vivo, there were no significant differences between the strains in the tracheal responses to carbachol (P = 0.78) or KCl (P = 0.13) in vitro. Both strains showed inhibition of the isoproterenol relaxation by carbachol (P less than 0.0001). Multiple linear regression analysis showed that the strain that was more sensitive to carbachol in vivo was also more sensitive to isoproterenol in vitro after carbachol contraction (P = 0.014). The greater isoproterenol sensitivity of the tracheae from this strain was not present after contraction with KCl, nor were these tracheae more sensitive to relaxation with sodium nitroprusside. Indomethacin pretreatment of the tissues in vitro augmented the maximal response and the sensitivity to carbachol (P less than 0.001) and KCl (P = 0.0006), and this effect was similar in both strains. Evaluation of isoproterenol relaxation after indomethacin pretreatment was confounded by the lower concentrations of carbachol needed for contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

PRINS-labeled knobs are not associated with increased chromosomal stickiness in the maize st1 mutant

In maize, the st1 mutant has been observed to result in chromosomes that stick together during both mitotic and meiotic anaphase. These sticky chromosomes result in abnormal chromosome separation at anaphase. Although the mechanism producing the st1 mutant phenotype is unknown, delayed replication of knob heterochromatin has been implicated in similar phenomena that result in sticky chromosomes. Primed in situ labeling (PRINS) was used to locate the 180-bp knob DNA sequences on mitotic metaphase chromosomes of several maize lines. The chromosomal regions labeled by PRINS corresponded to the reported C bands found in these lines. Additionally, PRINS was used to identify knob-bearing regions in anaphase spreads of a line carrying the st1 mutant and a nonmutant line having a similar number of chromosome knobs. The increase in abnormal anaphase figures in the st1 mutant was not accompanied by an increase in association of knob DNA with abnormal anaphases. Thus, the increase in chromosomal stickiness appears to be due to an increase in stickiness of knob and nonknob chromosomal regions. The mechanism responsible for the st1 mutant, therefore, is hypothesized to be different from that implicated in the other previously described sticky chromosomes situations.     

Exercise training-induced adaptations of immune response are mediated by beta-adrenergic receptors in aged but not young mice.

Beta-adrenergic blockade was used to determine whether the exercise training-induced adaptations of immune response to viral infection were mediated by catecholamines in young and old mice. Young (2 mo) and older (16 mo) male BALB/c mice were randomly assigned to an exercise or control group, and half of the mice in each group received the beta-adrenergic receptor antagonist nadolol. After 8 wk of moderate exercise training, mice were challenged with herpes simplex virus (HSV) 24 h postexercise. The results showed that exercise treatment increased anti-HSV IgM antibody, enhanced IL-10, and altered the kinetics of IFN-gamma and IL-2 production in young and old mice. Unique to older mice, exercise decreased mitogen-induced proliferation, increased splenocytes, and tended to decrease memory cells (CD44(hi+)). In contrast, exercise increased mitogen-induced proliferation but decreased the number of splenic lymphocyte and CD4+ cells in young mice. beta-Adrenergic blockade blunted the exercise-induced changes in anti-HSV IgM, IL-2, IFNgamma, and mitogen-induced proliferation in old but not young mice. The findings suggest that some of the immunomodulatory effects of chronic exercise are mediated via beta-adrenergic receptors and that the role of beta-adrenergic receptors is age dependent.     

Evidence that benzodiazepine receptors reside on cerebellar purkinje cells: Studies with “nervous” mutant mice

The age-related, regionally-specific loss of ³H-diazepam binding sites in nervous mutant mice paralleled the loss of cerebellar Purkinje cells. These results suggest that benzodiazepine receptors reside on cerebellar Purkinje cells.     

Novel object exploration in mice: not all objects are created equal

Object exploration is an increasingly popular experimental paradigm in behavioral sciences. We have begun a series of studies with mice specifically looking at the parameters that influence behaviors in this test. The aim of the present study was to examine the effect of object type on performance in the object exploration test. More specifically, adult male C57BL/6J mice were trained and tested using objects that could be climbed (CLIMB) or with those that could only be touched (TOUCH). The results show that activity is affected by the presentation of objects, with object type interacting with some of these changes. C57 mice explored objects that can be climbed over significantly longer than objects that can only be touched and a more rapid habituation was observed using objects that could only be touched. Robust recognition memory was observed in both groups of mice, however mice in the CLIMB group exhibited a significantly greater discrimination index compared to mice in the TOUCH group. Taken together, these findings demonstrate that the selection of objects is of critical importance and it is recommended that special attention be given to the functional (affordant) properties of the objects to-be-used in future studies.     

NK cells are not required for spontaneous autoimmune diabetes in NOD mice

NK cells have been shown to either promote or protect from autoimmune diseases. Several studies have examined the role of receptors preferentially expressed by NK cells in the spontaneous disease of NOD mice or the direct role of NK cells in acute induced disease models of diabetes. Yet, the role of NK cells in spontaneous diabetes has not been directly addressed. Here, we used the NOD.NK1.1 congenic mouse model to examine the role of NK cells in spontaneous diabetes. Significant numbers of NK cells were only seen in the pancreas of mice with disease. Pancreatic NK cells displayed an activated surface phenotype and proliferated more than NK cells from other tissues in the diseased mice. Nonetheless, depletion of NK cells had no effect on dendritic cell maturation or T cell proliferation. In spontaneous disease, the deletion of NK cells had no significant impact on disease onset. NK cells were also not required to promote disease induced by adoptively transferred pathogenic CD4(+) T cells. Thus, NK cells are not required for spontaneous autoimmune diabetes in NOD mice.     

S-(+)-aporphines are not selective for human D3 dopamine receptors

Summary 1. Our aim was to test the hypothesis that selectivity for D₃ dopamine (DA) receptors may contribute to limbic anti-DA selectivity ofS-(+)-aporphine DA partial agonists.2. Affinity was tested with³H-emonapride, using human D₃ receptors in mouse fibroblasts and D₂ receptors in rat striatal tissue.3. D₃ receptors showed a picomolar affinity for³H-emonapride, Na⁺ dependence, and reversible saturability, as well as stereoselectivity. Confirmatory or novel D₃/D₂ pharmacologic selectivity was found with several benzamides, thioxanthenes, buspirone, GBR-12909, and DA agonists including hydroxyaminotetralins [ADTN, (+)-7-OH-DPAT, (–)-PPHT and its fluorescein derivative], (–)-N-propylnorapomorphine, (–)-3-PPP, (–)-quinpirole, and SDZ-205-502, but neither aminoergoline nor (+)-aporphine partial agonists.4. The results extend pharmacologic characterization of D₃-transfected cell membranes but fail to account for the high limbic anti-DA selectivity ofS-(+)-aporphines.     

To phosphorylate or not to phosphorylate: Selective alterations in tyrosine kinase-inhibited EphB mutant mice

EphB tyrosine kinase receptors have been implicated in multiple developmental processes; however, the signaling mechanism underlying these events remains unclear. Through a triple knock-in mouse line for three neurally expressed EphBs, Sokis et al. demonstrated that EphB tyrosine kinase activity is required for axon guidance but does not influence synapse formation. This short communication highlights their study and appealing molecular approach that elucidated the functions of EphB tyrosine kinase during developmental events.     

Enhanced in vivo sensitivity to interferon with in vitro resistant B16 tumor cells in mice

Mouse B16 melanoma cells rapidly develop resistance to the antiproliferative effects of interferon (IFN) and interferon (IFN) when they are exposed to the interferons in vitro. This resistance was characterized to be non-genetic and dose-dependent, and does not alter other IFN-induced effects such as antiviral effects and elevation of 2,5-oligoadenylate synthetase activity in IFN-treated cells. The study of these IFN-resistant cells has been extended to an in vivo tumor model. Resistance, if it occurred in vivo, did not adversely affect the survival of IFN-treated mice. Further, IFN-treated mice inoculated with B16 cells that were resistant in vitro (B16^res cells) survived significantly longer than IFN-treated mice inoculated with B16 cells that were sensitive in vitro. The IFN-treated B16^res-inoculated mice had a significantly higher cure rate as well. The prolonged survival of the mice bearing B16^res cell tumors did not seem to be caused by the slower growth rate of the B16^res cells, since experiments performed with a tenfold higher B16^res cell inoculum and a tenfold lower B16 cell inoculum did not show any change in the survival pattern. It is clear that in vitro resistant B16^res cells are more sensitive to antitumor effects induced by IFN in vivo than in vitro sensitive B16 cells.Supported by U. S. Public Health Service grant no. CA50752 awarded by the National Cancer Institute, Department of Health and Human Services (W. R. F.) and by a James W. McLaughlin Fellowship (C. M. F.)     

Infection by and protective immune responses against Plasmodium berghei ANKA are not affected in macrophage scavenger receptors A deficient mice

Background  

Scavenger receptors (SRs) recognize endogenous molecules modified by pathological processes as well as components of diverse microorganisms. Mice deficient for both SR-AI and II are more susceptible to infections by a variety of bacterial and viral pathogens.     

Foot-and-mouth disease virus mutant with decreased sensitivity to ribavirin: implications for error catastrophe

The nucleoside analogue ribavirin (R) is mutagenic for foot-and-mouth disease virus (FMDV). Passage of FMDV in the presence of increasing concentrations of R resulted in the selection of FMDV with the amino acid substitution M296I in the viral polymerase (3D). Measurements of progeny production and viral fitness with chimeric viruses in the presence and absence of R documented that the 3D substitution M296I conferred on FMDV a selective replicative advantage in the presence of R but not in the absence of R. In polymerization assays, a purified mutant polymerase with I296 showed a decreased capacity to use ribavirin triphosphate as a substrate in the place of GTP and ATP, compared with the wild-type enzyme. The results suggest that M296I has been selected because it attenuates the mutagenic activity of R with FMDV. Replacement M296I is located within a highly conserved stretch in picornaviral polymerases which includes residues that interact with the template-primer complex and probably also with the incoming nucleotide, according to the three-dimensional structure of FMDV 3D. Given that a 3D substitution, distant from M296I, was associated with resistance to R in poliovirus, the results indicate that picornaviral polymerases include different domains that can alter the interaction of the enzyme with mutagenic nucleoside analogues. Implications for lethal mutagenesis are discussed.