A high throughput colorimetric cell proliferation assay for the identification of human cytomegalovirus inhibitors.

A colorimetric assay based on the cleavage of the tetrazolium salt WST-1 has been developed for human cytomegalovirus (HCMV) antiviral susceptibility testing and adapted to a microtiter plate format. Optimal conditions were determined and the standard routine assay was calibrated with a viral input of 0.05-0.10 plaque forming unit (p.f.u.)/cell with a density of 2000 cells/well in a 96-well microtiter plate for an incubation period of 7 days. Ganciclovir (9-(2-hydroxy-1(hydroxymethyl) ethyoxymethyl) guanine; DHPG), and cidofovir ((S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine; HPMPC) were used as positive control test compounds to validate the assay. The effective EC50 concentration values obtained with the two antiviral compounds in the present assay were in good agreement with plaque reduction assay results performed in parallel experiments. This method presents the advantage of being easy and rapid to perform, reliable, reproducible, and convenient for use in a high throughput screening capacity.

Lewis and Fischer rats: a comparison of dopamine transporter and receptors levels.

Previous reports have shown that the inbred strains of rat, Lewis (LEW) and Fischer 344 (F344), differ in several behavioural and biochemical indices of mesolimbic dopamine (DA) function. Specifically, these two strains differ in their behavioural and neurochemical response to novel environments, and acute amphetamine or cocaine challenge as well as in their susceptibility to addiction. To investigate if differences in DA D1-like, D2-like, D3 receptors and DA transporter could be correlated with these behavioural differences between strains, a comparative autoradiographic study of DA receptors and transporter within the striatal and accumbal regions was undertaken. We observed strain and region specific differences in binding levels for DA D2-like and D3 receptors and for the DA transporter. Namely, DA transporter levels in the striatum, nucleus accumbens and olfactory tubercle of LEW rats were significantly lower than in F344 rats. DA D3 densities in the shell of the nucleus accumbens and olfactory tubercle of LEW rats were lower than the levels found in the F344 rats. Finally, LEW rats have a lower levels of D2-like receptors in the striatum and the core of the nucleus accumbens compared to F344 rats. These data suggest that differences in DA transporter and DA receptors may in part contribute to differences in DA related behaviour seen between these two strains.

Molecular weight determination of the hepatic vasopressin receptor with a high-affinity photoprobe.

We report here a study of photoaffinity labeling of the V1a-vasopressin receptor with high-affinity, V1-specific radioiodinated antagonist ligands: one containing an azidophenylalanine residue ([beta,beta-dimethyl-beta-mercaptopropionyl(1), p-azido-Phe2,Val4,Lys8,D-Tyr9] vasopressin), two others containing nitrophenylalanine, and one, highly similar but without a photosensitive function, as control. All analogues competed in the dark for the same binding site with vasopressin. Long-wavelength UV irradiation of rat liver membranes incubated in presence of the radio-iodinated azido photolabel produced a specifically labeled protein band at 53 kDa in SDS-PAGE. Identical experiments with the nitrophenylalanyl peptides produced only non-specific labeling and control experiments with the non-photosensitive analogue produced no labeling at all. Chemical crosslinking of 3H-VP to the same membrane preparation produced a result identical to that of the azido photolabel, confirming the receptor nature of the labeled protein. Deglycosylation of the labeled receptor with endoglycosidase F reduced the observed molecular weight of 53 kDa to 43 kDa. The molecular parameters reported herein of the presumed hepatic vasopressin receptor confirm the values deduced from the molecular cloning of the rat V1a receptor.

Importance of the Ser-132 phosphorylation site in cell transformation and apoptosis induced by the adenovirus type 5 E1A protein.

The 289-residue (289R) and 243R early region 1A (E1A) proteins of human adenovirus type 5 induce cell transformation in cooperation with either E1B or activated ras. Here we report that Ser-132 in both E1A products is a site of phosphorylation in vivo and is the only site phosphorylated in vitro by purified casein kinase II. Ser-132 is located in conserved region 2 near the primary binding site for the pRB tumor suppressor and, in 289R, just upstream of the conserved region 3 transactivation domain involved in regulation of early viral gene expression. Mutants containing alanine or glycine in place of Ser-132 interacted with pRB-related proteins at somewhat reduced efficiency; however, all Ser-132 mutants transformed primary rat cells in cooperation with E1B as well as or better than the wild type when both major E1A proteins were expressed. Such was not the case with mutants expressing only 289R. In cooperation with E1B, the Asp-132 and Gly-132 mutants yielded reduced numbers of smaller transformed foci. With activated ras, all Ser-132 mutants were significantly defective for transformation and the rare foci produced were small and contained extensive areas populated by low densities of flat cells. In the absence of E1B, all Ser-132 mutants induced p53-independent cell death more readily than virus expressing wild-type 289R. These results suggested that phosphorylation at Ser-132 may enhance the binding of pRB and related proteins and also reduce the toxicity of E1A 289R, thus increasing transforming activity.

Decreased binding of dopamine D3 receptors in limbic subregions after neonatal bilateral lesion of rat hippocampus.

Neonatal, bilateral lesion of the ventral hippocampus (VH) in rats recently has been proposed as a model of schizophrenia because these animals show postpubertal hypersensitivity to stress and to dopamine (DA) agonists that can be reversed by neuroleptic treatment. In search of the mechanisms of postpubertal emergence of hyperdopaminergic behavior in this model, we investigated developmental expressions of DA D1, D2, and D3 receptors in various striatal and limbic subregions of rats that had received bilateral ibotenic acid lesion of the VH at postnatal day 7 (PD7). D-Amphetamine-, apomorphine-, and stress-induced changes in locomotor activity were measured and, in accordance with previous reports, we observed an increased locomotor activity at PD56 in the hippocampal-lesioned group. The expression of DA D1, D2, and D3 receptors was then estimated in these rats by ligand autoradiography at PD41 and PD62. We observed that the levels of DA D3 receptors, as measured by tritiated 7-hydroxy-N,N-di-n-propyl-2-amino-tetralin ([3H]7-OH-DPAT) binding, are markedly reduced at PD62 in the limbic areas of lesioned rats compared with sham controls particularly in the nucleus accumbens, olfactory tubercles, and islands of Calleja. A small but significant increase in D1 receptors was also seen in the caudate-putamen of the lesioned animals at PD62, whereas no significant change in the overall expression of D2 receptors ([3H]spiperone binding) was noted. In view of the inhibitory role of D3 receptors on locomotion and, presumably, other DA-mediated behaviors, it is suggested that behavioral changes in the neonatally hippocampal-lesioned rats may be mediated by altered D3 receptor levels.

Decreased expression of the embryonic form of the neural cell adhesion molecule in schizophrenic brains.

The regulated expression of neural cell adhesion molecule (NCAM) isoforms in the brain is critical for many neurodevelopmental processes including neurulation, axonal outgrowth, and the establishment of neuronal connectivity. We have investigated the expression of the major adult isoforms of NCAM (NCAM-180, NCAM-140, and NCAM-120) and its embryonic highly polysialylated isoform (PSA-NCAM) in the hippocampal region of postmortem brains from 10 schizophrenic and 11 control individuals. Immunohistochemical analysis with a monoclonal antibody recognizing the PSA-NCAM revealed immunoreactivity primarily in the dentate gyrus and in a subset of cells in the hilus region. We have observed a 20-95% reduction in the number of hilar PSA-NCAM-immunoreactive cells in the great majority of schizophrenic brains. The change in PSA-NCAM immunoreactivity is not obvious in other hippocampal subfields. Western blots of tissues from the hippocampal region (as well as from the frontal cortex) probed with a polyclonal antibody recognizing all NCAM isoforms did not reveal significant changes in the overall expression of NCAM, suggesting that the decrease in PSA-NCAM-immunoreactive cells may be related to post-translational processing of the molecule. The expression of this embryonic form of NCAM has been proposed to be related to synaptic rearrangement and plasticity. Therefore, the decrease in PSA-NCAM immunoreactivity in schizophrenic hippocampi may suggest an altered plasticity of this structure in a large proportion of schizophrenic brains. These findings may bear significance to the "neurodevelopmental hypothesis" of schizophrenia.

Immunomodulatory and antiviral activities of 2',3'-dideoxy-beta-L-cytidine and 2',3'-dideoxy-beta-L-5-fluorocytidine.

Two dideoxynucleosides, 2',3'-dideoxy-beta-L-cytidine and 2',3'-dideoxy-beta-L-5-flurocytidine, containing unnatural L-configuration in their sugar moieties, were synthesized and assayed for antiviral activities. Both compounds were shown to possess potent anti-human immunodeficiency virus type 1 and antihepatitis B virus activities, while demonstrating no anti-herpes simplex viruses 1 and 2 activity. These two compounds exhibited in vitro cellular toxicities for several leukocytic cell lines and were shown to inhibit phytohemagglutinin-stimulated human peripheral blood mononuclear leukocyte proliferations. At inhibitory concentrations, both compounds caused accumulations of cells in the S phase. While demonstrating no obvious morphological toxicity in vivo in mice at concentrations of 75 and 150 mg/kg, 2',3'-dideoxy-beta-L-5-fluorocytidine- treated animals were shown to have considerable increases in CD4/CD8 double positive T lymphocyte population in their blood circulation.

Functional interactions within adenovirus E1A protein complexes.

The transforming potential of adenovirus E1A oncogene products derives largely from the formation of complexes with cellular proteins, including the p105Rb tumor suppressor and a related p107 species, p130 and p300 proteins, and cyclin A (p60cycA). Extensive quantitative analyses using E1A deletion mutants identified unique binding patterns for each of these polypeptides within the amino terminus and conserved regions 1 and 2 (CR1 and CR2) of E1A proteins. A novel protein, termed p400, was found by peptide mapping to be related to p300, and, like p300, to require the E1A amino terminus and a portion of CR1 for binding. p130 was shown to be related to p107, and like p107, to associate with p60cycA. p107, p130 and p105Rb all interacted primarily with CR2, however, sequences within CR1 and the amino terminus were capable of weak interactions and appeared to function cooperatively with CR2 to bind these proteins. Protein kinase activity present in E1A complexes probably derives at least in part from p60cycA-linked p33cdk2 associated with p107 and p130. In vitro phosphorylation of complexes purified by immunoprecipitation resulted in labeling of several proteins. p60cycA was phosphorylated to about the same extent in cyclin A complexes prepared from either AD5- or mock-infected KB cells, however, that of p130 and p107 was dramatically higher in p60cycA complexes from infected cells. p300 was also phosphorylated in complexes prepared using E1A-specific antibodies. Thus one role of E1A proteins in signal transduction and regulation of the cell cycle may be to control the biological activity of p107, p130 and p300 by enhancing their phosphorylation through complex formation.

Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins.

Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36-69 within conserved region 1 (CR1), and residues 121-138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably.

Fluorescent peptide hormones: development of high affinity vasopressin analogues.

Highly potent and specific peptide hormone analogues with fluorescent reporter groups are current research goals. Until now, however, only moderately potent analogues have been described. We report here several types of vasopressin (VP) analogues with different fluorophores attached to the peptide. In a first series, fluorophores were attached to the free epsilon amino function of [des-amino1-lysine8]VP (dLVP), producing agonistic analogues. In a second series, reporter groups were added to the N-terminal of open-chain antagonist structures. The biological activities of these analogues were assessed by two different sets of experiments: 1) The measurement of their binding affinities towards the V1a-vasopressin receptor subtype from WRK1 cells or rat liver membrane preparations; 2) Their ability to stimulate the phospholipase C activity in WRK1 cells. As expected, a simple acylation of fluorophores to dLVP resulted in a considerable loss of affinity. If however, the Lys8 side chain was extended through double Schiff-base formation with glutaraldehyde-ethylenediamine followed by reduction to an aminoalkyl aminoalkylamine, single fluorophores could be added without loss of affinity compared to VP. The open-chain analogues, on the other hand, while displaying weak affinity, nevertheless exhibited pure antagonistic behavior.

Preparation and biological activities of potential vasopressin photoaffinity labels.

Several potential photoaffinity analogues of the peptide hormone vasopressin (VP) were prepared by classical solid-phase peptide synthesis using two different pathways. Peptide sequences were built by introduction of (a) Nar-protected aminophenylalanine or (b) nitrophenylalanine in the photolabeling position. Conversion to the azido peptide was completed in pathway a after cleavage and before purification and in pathway b from small quantities of purified nitrophenylalanine-containing precursor peptides. V1 receptor binding properties were measured using membranes prepared from rat liver cells. The binding potential of agonistic VP structures was abolished by the introduction of an azido or a nitro group into the aromatic side chain at position 3. Cyclo desamino-beta,beta-dialkyl-Cys1-type VP antagonist structures were prepared with the photoactivable moiety in position 2 and an iodination residue in position 9. One particular compound, [Dmpa1, Phe(N3)2, Val4, Lys8,D-Tyr9]VP (8), containing beta,beta-dimethyl-beta-mercaptopropionic acid in position 1, had excellent binding properties, both in the radioiodinated (Kd = 4.8 +/- 1.9 x 10(-10) M) and noniodinated form (Kd = 6.4 +/- 0.98 x 10(-10) M). The analogues with long-chain beta-alkylation (diethyl and pentamethylene) and the linear antagonist photolabel showed significantly less affinity. Optimal binding properties were obtained within a very narrow range of hydrophobicity; greater or lesser hydrophobicity was correlated to less potent binding. The precursor analogues, containing nitrophenylalanine, displayed a structure-activity relationship similar to that of the azido peptides. The most potent analogues will be used for receptor labeling studies. A linear antagonist structure having a photosensitive group in position 1, has also been prepared, but this compound displayed much less affinity than the cyclic antagonists. The most potent compounds were also highly selective for the V1 receptor and did not recognize the V2 receptor from other preparations.

Sedimentation behavior of native and reduced apolipoprotein A-II from human high density lipoproteins.

The solution properties of human serum apolipoprotein A-II, both in the native and in the reduced forms, were investigated by the technique of sedimentation equilibrium in the analytical ultracentrifuge. For both proteins, the apparent weight average molecular weights determined in neutral buffer systems were found to be dependent on protein concentration and invariant with the rotor speeds used (16,000 to 44,000 rpm) indicating a reversible self-association. These results were also found to be independent of temperature between 5 and 30 degrees C. The pattern of self-association of native apolipoprotein A-II could best be described by a monomer-dimer-trimer equilibrium, in agreement with previously reported data (Vitello, L B., and Scanu, A. M. (1975), Biochemistry 15, 1161). The self-association pattern of apolipoprotein A-II reduced in the presence of 50 mM dithiothreitol conformed with a monomer-dimer-tetramer equilibrium similar to that reported for the native single chain apolipoprotein A-II of the rhesus monkey (Barbeau, D. L., et al. (1977), J. Biol. Chem. 252, 6745), but differing significantly from that reported for the reduced and carboxymethylated human product (Osborne, J. C. , et al. (1975), Biochemistry 14, 3741).

An ultracentrifugal study of the self-association of canine apolipoprotein A-I in solution.

The sedimentation behavior of canine apolipoprotein (apo) A-I in 0.02 M EDTA, pH 8.6, was studied as a function of protein concentration by the techniques of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge. At concentrations of less than 1 g/liter, apo-A-I exhibited a monomodal sedimentation pattern, with apparent sedimentation coefficients which varied from 2.3 to 3.5 S with increasing protein concentrations. Above 1.5 g/liter, apo-A-I had two well resolved peaks with s20,w values of 4.15 S and 5.75 S. The proportion of the 5.75 S component increased with increasing apo-A-I concentrations, with a concomitant decrease of the 4.15 S component. By sedimentation equilibrium ultracentrifugation with both the conventional and meniscus-depletion methods, the apparent weight-average molecular weight of apo-A-I was found to be concentration-dependent. At a protein concentration of 5.25 g/liter, an apparent weight average molecular weight of 138,000 was determined, indicating that molecular species larger than a tetramer (monomer molecular weight = 28,000) were present in solution. When analyzed in terms of a reversible self-associating system, the experimental data could best be described according to a monomer-dimer-tetramer-octamer model, as previously reported from human apo-A-I (Vitello, L. B., and Scanu, A. M. (1975) J. Biol. Chem. 251, 1131-1136). The equilibrium constants were: K2 = 4.5 liters/g, K4 = 470 liters3/g3, and K8 = 41,600 liters7/g7, respectively.

Ultracentrifugal behavior of apolipoprotein A-I of rhesus monkey (Macaca mulatta).

The sedimentation equilibrium and sedimentation velocity of apolipoprotein AI (apo-A-I) from high density lipoproteins of rhesus monkey plasma were studied in aqueous solutions of 0.02 M EDTA, pH 8.6, as a function of protein concentration. The sedimentation equilibrium results showed that, in the concentration range between 9 and 36 muM (0.25 to 1.0 g/liter), apo-A-I behaved as a single species of molecular weight 28,200. This molecular weight corresponds to that of the monomeric form as previously estimated from electrophoretic and chemical data. As the apo-A-I concentration was increased above 36 muM, the log c versus r2 plots from sedimentation equilibrium experiments became curvilinear, suggesting self-association. An analysis of these data generated nonlinear, nonoverlapping molecular weight versus concentration plots pointing at the existence of a heterogeneous population of apo-A-I. By sedimentation velocity studies apo-A-I at concentrations between 68 and 330 muM exhibited two distinct components, with S0/20,w = 1.9 and S0/20,w = 3.9. When separated by gel permeation chromatography, these two components had an identical amino acid composition and retained similar S20,w values as before column fractionation. The slow component had a diffusion coefficient of 8.27 X 10(-7) cm2/s and, assessed by the criteria of sedimentation equilibrium ultracentrifugation, remained monomeric even at concentrations of 125 muM. The fast component, when analyzed by low speed sedimentation equilibrium, behaved as a self-associating system which could best be fitted into a monomer-hexamer model in a rapid equilibrium. The equilibrium constant for this association was found to be 9.5 X 10(23) M(-5). Thus, rhesus apo-A-I, dissolved in 0.02 M EDTA, pH 8.6, consisted of two distinct species, one monomeric over a relatively wide range of concentrations, the other readily self-associating. Their structural relationship remains to be established. This ultracentrifugal behavior of rhesus apo-A-I differs markedly from that reported for human apo-AI (Vitello, L. B., and Scanu, A. M. (1976) J. Biol. Chem. 251, 1131-1136).