The bovine papillomavirus E5 transforming protein can stimulate the transforming activity of EGF and CSF-1 receptors

The bovine papillomavirus E5 transforming gene encodes a 44 amino acid protein product that is localized to cytoplasmic membranes, including the plasma membrane. We now report that E5 can cooperate with human EGF receptors and with human CSF-1 receptors to induce cellular transformation of NIH 3T3 cells. Cooperation occurred in the absence of receptor stimulation by ligand, and it was further augmented by treatment with ligand. Cooperation was not seen between E5 and either c-fes or c-src. The cooperation between E5 and high levels of EGF receptors was associated with inhibition of receptor degradation and persistence of activated receptors on the cell surface. We conclude that E5 may enhance the receptor activity via inhibition of receptor down-modulation.     

Phosphorylation and down-regulation of CD4 and CD8 in human CTLs and mouse L cells

The CD4 and CD8 molecules are rapidly phosphorylated following exposure of CD4⁺ or CD8⁺ human cytotoxic T lymphocytes (CTL) clones to B-lymphoblastoid cell lines bearing the relevant target alloantigens. Treatment of CD4⁺ or CD8⁺ CTL clones with phorbol myristate acetate (PMA), phytohemagglutinin, or mitogenic combinations of CD2-specific antibodies also resulted in CD4 or CD8 phosphorylation. Down-regulation of the surface expression of these molecules could be demonstrated in both CD4⁺ and CD8⁺ clones following exposure to the relevant alloantigen or PMA. Parallel experiments were conducted using mouse L cells in which the human CD4 or CD8 antigens were stably expressed. Exposure of these transfectants to PMA induced rapid phosphorylation of the CD4 and CD8 molecules. As in CD4⁺ CTL clones, rapid modulation of the CD4 antigen could be demonstrated in L cells following PMA treatment. In contrast, there was no demonstrable down-regulation of the CD8 antigen in PMA-treated CD8⁺ L cell transfectants. These studies demonstrate a significant differential property of the CD4 and CD8 antigens and suggest that down-regulation of the CD8 antigen may require its expression in a T-cell environment and/or the association of CD8 with the T-cell receptor or other T cell-specific molecules.     

Low-dose apomorphine reduces serum homovanillic acid concentrations in schizophrenic patients

This study was carried out to evaluate the postulated dopaminergic autoreceptor regulatory effect in man of low-dose apomorphine. Behavior and serum homovanillic acid concentrations following low-dose apomorphine were investigated. Five medicated chronic schizophrenic patients had serum homovanillic acid concentrations measured by mass fragmentography before and after 0.005 mg/kg of apomorphine or saline placebo. Results demonstrate significant reductions in serum homovanillic acid concentrations in all five subjects following apomorphine as compared with placebo. These findings present direct evidence of a specific dopamine autoreceptor effect of low-dose apomorphine in schizophrenic patients.     

A thermodynamic description of the binding of iron to ferrioxamine B in aqueous solutions

The iron exchange reaction between nitrilotriacetate (NTA) and ferrioxamine B (DFO) has been investigated with titration calorimetry. Using characterization models for the initial and final states of the reaction solution based on the determination of the mole fractions of the individual species as a function of pH, the concentration of each participating species was calculated (B. L. Gould, 1980, Masters Thesis, Utah State University, Logan). The contribution of each component reaction to the overall exchange at various pH values was used to determine the enthalpy change for each of these individual reactions. The values determined for the enthalpy and entropy changes are shown below. An analysis of the thermodynamic parameters of the iron exchange system indicated the following interactions among the hydroxamate groups of the ferrioxamine B: (1) The ionization of the individual hydroxamate protons occurs independently with the same enthalpy change for each ionization, and (2) the heat of binding a hydroxamate group to the iron is dependent on the number of previously bound hydroxamates.     

Desmoplakins of Epithelial and Myocardial Desmosomes are Immunologically and Biochemically Related

Abstract. Guinea pig antibodies against desmoplakins from bovine muzzle epidermis showed specific reaction in several epithelial tissues with desmoplakin I (M_r 250,000) and desmoplakin II (M_r 215,000). By immunofluorescence microscopy, prominent punctate staining was observed in various lines of cultured epithelial cells, revealing desmosomal junctions at sites of established cell-to-cell contacts as well as hemidesmosomes and internalized desmosome-derived membrane domains. On frozen tissue sections punctate staining was observed along plasma membranes of epithelial cells, and electron microscopy using the immunoperoxidase technique revealed that the antibodies were specifically localized at the plaques associated with desmosomes and hemidesmosomes. Of a large number of non-epithelial cells examined positive staining was only observed on desmosome-like junctions of myocardial cells and Purkinje fiber cells. In both epithelial and myocardial tissues the antibodies showed a broad range of cross-reactivity between diverse vertebrate species such as man, cow, rodent, and chicken, indicating that desmoplakins contain determinants strongly conserved during evolution. When binding of these antibodies to cytoskeletal polypeptides separated by gel electrophoresis and blotted on nitrocellulose paper sheets was examined, specific reaction was noted with desmoplakin I and, to a variable degree, also desmoplakin II from various epithelial cells. Reaction was also observed with a myocardial polypeptide from bovine and human hearts which had a similar M_r value (250,000) and isoelectric pH range as desmoplakin I. We conclude that desmoplakins are the major proteins present in the desmosomal plaques of both epithelial and myocardial cells and that the desmoplakin polypeptides present in these two different cell types are very similar, if not identical.     

Restriction endonuclease analysis of plastid DNA from tomato, potato and some of their somatic hybrids

Summary The buoyant density and endonuclease restriction patterns of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) ptDNA were examined and compared with those of their somatic hybrids. The plastids from these plants, both of which belong to the family of Solanaceae, contain a single DNA species whose density of 1.697 gcm^-3 and size of approximately 156 kbp are similar to those of ptDNA from other higher plants. The Sal I restriction patterns were indistinguishable; however, those obtained with Kpn I, Pst I, and Eco RI disclosed that each species possesses a unique ptDNA. These observations suggest a relatively recent divergence of both species. Of the twelve hybrid lines screened, eight contained exclusively potato ptDNa and four contained only tomato ptDNA at a 0.1–3% level of detection. Rearrangements of modifications of DNA were not detected. The plastid identities of three hybrid lines that had previously been analyzed by isoelectric focusing of RuBPcase subunits (Melchers et al. 1978) agreed with those determined by restriction endonuclease analysis.Abbreviations used in the text ptDNA plastid DNA, chloroplast DNA - cDNA copy DNA - RuBPcase ribulose bisphosphate carboxylase/oxygenase - LSU large subunit of RuBPcase - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC standard saline citrate - IEF isoelectric focusing     

Electronic, steric, and hydrophobic factors influencing the action of enkephalin-like peptides on opiate receptors

The requirements of opiate receptors for electronic, steric, and hydrophobic properties of the amino acids in Pos. 4 and 5 of enkephalin-like peptides were studied. A series of [D-Ala2]-enkephalins containing carboranylalanine, adamantylalanine, t-butylglycine and p-nitrophenylalanine were synthesized and their pharmacological activities in the guinea pig ileum and their naloxone displacement in rat brain homogenates determined. An electronegative (-E) aromatic character of the amino acid in Pos. 4 strongly enhanced potency, overruling steric effects. The enhancement was not caused by exceptional enzyme resistance. Amino acid in Pos. 5 contributed to potency mainly through its effect on overall hydrophobicity. The two C-terminal amino acids seem to function as potentiator and address elements in the enkephalins.     

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: comparison of reconstituted vesicles with native membrane vesicles

A Na⁺-specific and Na⁺-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na⁺ could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K⁺, Li⁺, and choline⁺. The electrochemical effect of the lipophilic anion, SCN^?, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl^? or SO₄^2?. The Na⁺-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K_m of 2 mm. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na⁺-specific and Na⁺-stimulated concentrative α-aminoisobutyric acid uptake.     

Exploratory analysis of quantitative histopathology of cervical intraepithelial neoplasia: objectivity, reproducibility, malignancy-associated changes, and human papillomavirus.

BACKGROUND: As part of a project to evaluate emerging optical technologies for cervical neoplasia, our group is performing quantitative histopathological analyses of biopsy specimens from 1,190 patients. Objectives in the interim analysis are (a) quantitatively assessing progression of the neoplastic process of cervical intraepithelial neoplasia (CIN)/squamous intraepithelial lesions (SIL), (b) detecting malignancy-associated changes (MACs), and (c) phenotypically measuring human papillomavirus (HPV) detected by DNA testing. METHODS: The diagnostic region of interest (ROI) from immediately adjacent sections were imaged, and the basal lamina and surface of the superficial layer were delimited. Nonoverlapping quantitatively stained nuclei were selected from 1,190 samples with histopathological characteristics of normal (929), koilocytosis (130), CIN 1 (40), CIN 2 (23), and CIN 3/carcinoma in situ (CIS) (68). A fully automatic procedure located and recorded the center of every nucleus in the region of interest (ROI). We used linear discriminant analysis to assess the changes between normal and CIN 3/CIS. RESULTS: Scores computed from the cell-by cell features and the clinical grade of CIN/SIL were highly correlated, as were those of the architectural features and the clinical grade of CIN/SIL. We found even higher correlations between a combination of cell-by-cell and architectural scores, and clinical grade. Using these scores, we found MACs in the normal biopsy specimens from patients with high-grade CIN/SIL. Furthermore, the same scores correlated with the molecular detection of HPV. CONCLUSIONS: Quantitative histopathology can be used in large clinical trials as an objective and reproducible measure of CIN/SIL. Detectable phenotypic changes correlate well with CIN/SIL neoplastic progression. It can also be used to infer the presence of CIN/SIL (MACs) and molecular changes associated with increased risk of cancer development (high-risk HPV).     

Molecular origins of osmotic second virial coefficients of proteins.

The thermodynamic properties of protein solutions are determined by the molecular interactions involving both solvent and solute molecules. A quantitative understanding of the relationship would facilitate more systematic procedures for manipulating the properties in a process environment. In this work the molecular basis for the osmotic second virial coefficient, B22, is studied; osmotic effects are critical in membrane transport, and the value of B22 has also been shown to correlate with protein crystallization behavior. The calculations here account for steric, electrostatic, and short-range interactions, with the structural and functional anisotropy of the protein molecules explicitly accounted for. The orientational dependence of the protein interactions is seen to have a pronounced effect on the calculations; in particular, the relatively few protein-protein configurations in which the apposing surfaces display geometric complementarity contribute disproportionately strongly to B22. The importance of electrostatic interactions is also amplified in these high-complementarity configurations. The significance of molecular recognition in determining B22 can explain the correlation with crystallization behavior, and it suggests that alteration of local molecular geometry can help in manipulating protein solution behavior. The results also have implications for the role of protein interactions in biological self-organization.