Downregulation of cell-to-cell communication by the viralsrc gene is blocked by TMB-8 and recovery of communication is blocked by vanadate

Summary The viralsrc gene downregulates junctional communication, closing cell-to-cell membrane channels presumably by way of the phosphoinositide signal route. We show that TMB-8 [8-N, N-(diethylamino) octyl-3,4,5-trimethoxybenzoate] counteracts this downregulation in cells transformed by temperature-sensitive mutant Rous sarcoma virus: TMB-8 (36–72 m) raises junctional permeability when applied during activity ofsrc protein kinase, i.e., at steady permissive temperature; and TMB-8 inhibits the fall of junctional permeability, when the activity ofsrc protein kinase gets turned on. TMB-8 also (reversibly) inhibits the growth of the cells at permissive temperature and reverses the morphological changes associated with transformation. The morphological reversal lags several hours behind the junctional-permeability reversal. Communication recovers within a few minutes when the activity of thesrc protein kinase is turned off (in absence of TMB-8). Sodium orthovanadate (20 m) prevents this recovery, but it has no major effect on junctional permeability on its own. We discuss possible modes of action of these agents on critical stages of the signal route, related to intracellular Ca²⁺ and protein kinase C.     

Enzyme Immunoassay for Cholecystokinin Octapeptide Sulfate and Its Application

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for cholecystokinin octapeptide sulfate (CCK-8S) has been developed using N-terminal specific antibody for CCK-8S. In this assay CCK-8S coupled with poly-L-Glu (CCK-poly-Glu), which is adsorbed on a solid phase, competes with CCK-8S for the binding sites of rabbit anti-CCK antibody, and the complex of the immobilized antibody and CCK-poly-Glu is measured using goat anti-rabbit immunoglobulin G conjugated with horseradish peroxidase. The total time for completion of the assay is less than 24 h. Near 50% bound levels, the intraassay coefficient of variation is 5.2-6.2% and the interassay coefficient of variation is 5.9-8.5%. This assay is sensitive enough to detect 9 pg of CCK-8S, and the data from rat brain regions using this ELISA are very similar to the data from those using radioimmunoassay (RIA). Therefore, this ELISA is simpler and more rapid in comparison with conventional RIA. In the preliminary experiments, we applied this method for determination of CCK content in the brain regions of adult rats treated with 6-hydroxy-dopamine or in newborn rats subjected to anoxia, and showed that this system is applicable to detection of changes of endogenous CCK content.     

Binding of the Novel Serotonin Agonist 8-Hydroxy-2-(Di-n-Propylamino) Tetralin in Normal and Alzheimer Brain

Binding of [3H]8-hydroxy-2-(di-n-propylamino) tetralin, a putative ligand for the 5-hydroxytryptamine (5-HT, serotonin) 1A recognition site, was measured in neocortex from postmortem human brain. The substance was found to bind to a saturable site with a KD value and pharmacological profile similar to that of rat. Binding to membranes from normal human temporal cortex was found to significantly correlate (inversely) with age. A significant reduction in binding, reflecting decreased density of recognition sites, was observed in the frontal cortex of patients with Alzheimer's disease (48% loss). This region in the dement brains showed unaltered presynaptic 5-HT function (5-HT and 5-hydroxyindoleacetic acid content) whereas 5-HT concentration was reduced in the temporal cortex.     

Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking

The small nuclear RNAs U4 and U6 display extensive sequence complementarity and co-exist in a single ribonucleoprotein particle. We have investigated intermolecular base-pairing between both RNAs by psoralen cross-linking, with emphasis on the native U4/U6 ribonucleoprotein complex. A mixture of small nuclear ribonucleoproteins U1 to U6 from HeLa cells, purified under non-denaturing conditions by immune affinity chromatography with antibodies specific for the trimethylguanosine cap of the small nuclear RNAs was treated with aminomethyltrioxsalen. A psoralen cross-linked U4/U6 RNA complex could be detected in denaturing polyacrylamide gels. Following digestion of the cross-linked U4/U6 RNA complex with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate cross-linked fragments. These fragments were analysed by chemical sequencing methods and their positions identified within RNAs U4 and U6. Two overlapping fragments of U4 RNA, spanning positions 52 to 65, were cross-linked to one fragment of U6 RNA (positions 51 to 59). These fragments show complementarity over a contiguous stretch of eight nucleotides. From these results, we conclude that in the native U4/U6 ribonucleoprotein particle, both RNAs are base-paired via these complementary regions. The small nuclear RNAs U4 and U6 became cross-linked in the deproteinized U4/U6 RNA complex also, provided that small nuclear ribonucleoproteins were phenolized at 0 degree C. When the phenolization was performed at 65 degrees C, no cross-linking could be detected upon reincubation of the dissociated RNAs at lower temperature. These results indicate that proteins are not required to stabilize the mutual interactions between both RNAs, once they exist. They further suggest, however, that proteins may well be needed for exposing the complementary RNA regions for proper intermolecular base-pairing in the course of the assembly of the U4/U6 RNP complex from isolated RNAs. Our results are discussed also in terms of the different secondary structures that the small nuclear RNAs U4 and U6 may adopt in the U4/U6 ribonucleoprotein particle as opposed to the isolated RNAs.     

Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

Stability enhancement and circular dichroism spectral anomaly as an indication of non-covalent interactions in histidine- and tyrosine-containing ternary copper(II)—amino acid systems

Visible absorption and CD spectral and potentiometric studies on the His- and Tyr-containing ternary copper(II) complexes Cu(A)(L-B), where A refers to L-His, D-His, or L-Tyr and B to Lys, Tyr, Trp, Phe, Ala, Val, Arg, Glu, Asn, Gln, Ser, or Thr, were made to study ligand-ligand interactions in the complexes. While the CD spectral magnitudes in the d—d region are additive in the absence of side chain interactions and can be estimated from the magnitudes for the ternary systems involving DL-A or DL-B, deviation from the additivity was observed for Cu(L-His)(L-B) (B = LysH, Tyr, Trp, or Phe) and Cu(L-Tyr)(L-Trp). From the stability constants determined at 25 °C and I = 0.1 M (KNO₃), the equilibrium constants, K, for the following hypothetical equilibria were calculated to be large (0.14–0.60) for formation of Cu(L-/D-His)(L-B)(B = Tyr or Trp) and Cu(D-His)(L-Phe) with Cu(en)(L-Ala) as standard: $Cu(A)(L?Ala)+Cu(en)(L?b)\stackrel{K}{?}Cu(A)(L?B)+Cu(en)(L?Ala)$ The positive values indicate the stabilization due to the stacking between the imidazole ring of His and the aromatic side chain of L-B. Solvent dependence of the CD spectra for Cu(L-His)(L-LysH) and Cu(L-His) L-Trp) further supported the existence of the intramolecular electrostatic and hydrophobic interactions.     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Central actions of calcitonin on body temperature and intestinal motility in rats: evidence for different mediations

The effects of intracerebroventricular (i.c.v.) administration of calcitonin and PGE2 on intestinal motility and body temperature were examined in conscious rats chronically fitted with intraparietal electrodes in the small intestine, a cannula in a cerebral lateral ventricle and a subcutaneous thermistor probe. Both calcitonin and PGE2 restored the fasted pattern of intestinal motility in fed rats and induced an increase in body temperature. Indomethacin, an inhibitor of the cyclooxygenase with calcium antagonistic properties, and TMB-8, an intracellular calcium antagonist, blocked the effects of calcitonin on intestinal motility and body temperature. Piroxicam, an inhibitor of the cyclooxygenase which does not affect calcium uptake blocked the thermic but not the intestinal effects of calcitonin. TMB-8 but not indomethacin or piroxicam partially blocked the effects of PGE2 on both intestinal motility and body temperature. It is concluded that the central hyperthermic effect of calcitonin is mediated through the formation and the release of prostaglandins whereas the central action of calcitonin on digestive motility results from intracerebral effects on calcium fluxes.     

Diversity in the immune system: “Preconceived ideas” or ideas preconceived?

Two concepts of the evolution and regulation of expression of the combining site repertoire of the immune system, are compared. One view is based on the Associative Recognition Theory as formulated by the author and the other is based on the Idiotype Network Idea as conceived by Jerne. The two concepts are analyzed from the point of view of their logic, internal consistency and factual support.     

Binding of 5-methoxypsoralen to human serum low density lipoproteins

5-methoxypsoralen (5-MOP) binds to human serum low density lipoproteins (LDL) according to a two-step process. Scatchard analysis of the first step yields K = 1.4 × 10⁵ M^?1 and 4 binding sites. It involves the LDL apoprotein. The second step corresponds to a solubilization, in the lipidic core, of ? 45 molecules of 5MOP per LDL molecule. It is accompanied by a large blue shift of the 5MOP fluorescence. The ability of LDL to bind 5MOP and to carry it into various cells may explain some biological effects sometimes encountered during PUVA therapy.