“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Contrasting effects of the protein kinase C inhibitor staurosporine on the interleukin-1 and phorbol ester activation pathways in the EL4-6.1 thymoma cell line.

EL 4-6.1 cells, variants of the murine EL4 thymoma cell line, can be activated by interleukin 1 (IL-1) or phorbol 12-myristate-13-acetate (PMA), or PMA+IL-1 to secrete interleukin 2 (IL-2) and interleukin 4 (IL-4) and to express the IL-2 receptor (IL-2R). To compare the different activation pathways, we examined the effects of staurosporine (STAR) and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), two protein kinase C (PKC) inhibitors, on the induction of interleukin secretion and IL-2R expression in these cells. We report here that nanomolar concentrations of STAR strongly potentiated (20- to 30-fold) the production of IL-2 or IL-4, when EL 4-6.1 cells were induced by IL-1 alpha (or IL-1 beta) alone. By contrast, at identical concentrations, STAR dose-dependently inhibited the production of IL-2 and IL-4 resulting from PMA or PMA+IL-1 cell treatment. STAR also negatively affected the expression of IL-2R, which was dependent on PMA-sensitive PKC with either IL-1, PMA, or PMA+IL-1 stimulation. The changes in interleukin production and IL-2R expression in EL 4-6.1 activated cells were correlated with changes at the mRNA level measured by quantitative polymerase chain reaction (PCR). This finding suggests a pretranslational effect of the drug. At micromolar concentrations, H7 showed the same effects as STAR, but only increased IL-1-triggered interleukin secretions twofold. We observed that the action of PKC inhibitors did not result from modification of IL-1 receptor (IL-1R) expression in EL 4-6.1 cells. Thus, our data show that PKC inhibitors clearly distinguish between IL-1 and PMA stimulatory pathways. In addition, they suggest that the IL-1 stimulatory pathway involves PKC(s) [or other undefined kinase(s)] which regulate this pathway and differ from PKC(s) activated by PMA.     

Using the longest significance run to estimate region-specific p-values in genetic association mapping studies

Background  

Association testing is a powerful tool for identifying disease susceptibility genes underlying complex diseases. Technological advances have yielded a dramatic increase in the density of available genetic markers, necessitating an increase in the number of association tests required for the analysis of disease susceptibility genes. As such, multiple-tests corrections have become a critical issue. However the conventional statistical corrections on locus-specific multiple tests usually result in lower power as the number of markers increases. Alternatively, we propose here the application of the longest significant run (LSR) method to estimate a region-specific p-value to provide an index for the most likely candidate region.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Preparation and properties of recombinant DNA derived tobacco mosaic virus coat protein

Recombinant DNA derived tobacco mosaic virus (vulgare strain) coat protein (r-TMVP) was obtained by cloning and expression in Escherichia coli and was purified by column chromatography, self-assembly polymerization, and precipitation. SDS-PAGE, amino terminal sequencing, and immunoblotting with polyclonal antibodies raised against TMVP confirmed the identify and purity of the recombinant protein. Isoelectric focusing in 8 M urea and fast atom bombardment mass spectrometry demonstrated that the r-TMVP is not acetylated at the amino terminus, unlike the wild-type protein isolated from the tobacco plant derived virus. The characterization of r-TMVP with regard to its self-assembly properties revealed reversible endothermic polymerization as studied by analytical ultracentrifugation, circular dichroism, and electron microscopy. However, the details of the assembly process differed from those of the wild-type protein. At neutral pH, low ionic strength, and 20 degrees C, TMVP forms a 20S two-turn helical rod that acts as a nucleus for further assembly with RNA and additional TMVP to form TMV. Under more acidic conditions, this 20S structure also acts as a nucleus for protein self-assembly to form viruslike RNA-free rods. The r-TMVP that is not acetylated carries an extra positive charge at the amino terminus and does not appear to form the 20S nucleus. Instead, it forms a 28S four-layer structure, which resembles in size and structure the dimer of the bilayer disk formed by the wild-type protein at pH 8.0, high ionic strength, and 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance

The replication and stable maintenance of latent Epstein-Barr virus (EBV) DNA episomes in human cells requires only one viral protein, Epstein-Barr nuclear antigen 1 (EBNA1). To gain insight into the mechanisms by which EBNA1 functions, we used a yeast two-hybrid screen to detect human proteins that interact with EBNA1. We describe here the isolation of a protein, EBP2 (EBNA1 binding protein 2), that specifically interacts with EBNA1. EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system, and the EBP2-EBNA1 interaction was confirmed by coimmunoprecipitation from insect cells expressing these two proteins. EBP2 is a 35-kDa protein that is conserved in a variety of organisms and is predicted to form coiled-coil interactions. We have mapped the region of EBNA1 that binds EBP2 and generated internal deletion mutants of EBNA1 that are deficient in EBP2 interactions. Functional analyses of these EBNA1 mutants show that the ability to bind EBP2 correlates with the ability of EBNA1 to support the long-term maintenance in human cells of a plasmid containing the EBV origin, oriP. An EBNA1 mutant lacking amino acids 325 to 376 was defective for EBP2 binding and long-term oriP plasmid maintenance but supported the transient replication of oriP plasmids at wild-type levels. Thus, our results suggest that the EBNA1-EBP2 interaction is important for the stable segregation of EBV episomes during cell division but not for the replication of the episomes.     

A therapeutic antibody and its antigen form different complexes in serum than in phosphate-buffered saline: A study by analytical ultracentrifugation

During the development of protein therapeutics, characterization of the active pharmaceutical ingredient is performed extensively to ensure the stability, safety, and efficacy of the drug. Little is known, however, about the characteristics of protein drugs circulating in the blood. The recent availability of a fluorescence detection system (FDS) in analytical ultracentrifugation (AUC) instruments enables the characterization of fluorescently labeled proteins in biological fluids. AUC provides information about protein size, shape, self-association, and binding while avoiding many limitations associated with size exclusion chromatography. Furthermore, with the specificity and sensitivity of FDS, measurements can be performed at physiological concentrations directly in serum. In the current study, we used omalizumab, an anti-immunoglobulin E (IgE) monoclonal antibody, to demonstrate the potential of using AUC-FDS for the study of a monoclonal antibody and its complexes directly in human serum. Omalizumab properties were essentially unaltered after labeling with the fluorescent dye Alexa Fluor 488. In addition, omalizumab and IgE formed different complexes in serum than in phosphate-buffered saline in terms of both size and affinity.     

Evidence for the involvement of histidine A(12) in the aggregation and precipitation of human relaxin induced by metal-catalyzed oxidation

The metal-catalyzed oxidation (ascorbate/cupric chloride/oxygen) of recombinant human relaxin (rhRlx, type II) was shown by Li et al. [Li, S., Nguyen, T. H., Sch?neich, C., and Borchardt, R. T. (1995) Biochemistry 34, 5762-5772] to result in the chemical modification of His A(12), Met B(4), and Met B(25). Considering the fact that His A(12) exists in an extended loop that joins two alpha-helices in this protein, we hypothesized that oxidation of this specific amino acid leads to alterations in the secondary and tertiary structures of the protein, resulting in the pH-dependent aggregation/precipitation phenomena observed in our earlier studies (i.e., at pH >6.0 most of the degradants of rhRlx are insoluble). Evidence obtained in the current study that supports this hypothesis includes the following: (i) oxidation of rhRlx with hydrogen peroxide (H(2)O(2)), which leads only to modification of Met B(4) and Met B(25), does not result in the pH-dependent aggregation/precipitation of the protein; and (ii) metal-catalyzed oxidation of porcine relaxin (pRlx), which does not contain His at position A(12), leads to chemical degradation of the protein [e.g., Met A(2) is oxidized] but produces only slight pH-dependent aggregation/precipitation of the protein. In addition, experimental evidence is provided to show that the physical instability of rhRlx observed at pH >6.0 does not appear to be related to the pH-dependent solubility of a common protein degradant. Instead, it appears that several oxidation products of His A(12) are produced in a pH-dependent manner and that these oxidation products produce different effects on the physical stability of the protein. Evidence in support of this conclusion includes the observation that the soluble degradants of rhRlx showed reduced levels of His, reduced levels of the T(2)-T(7) tryptic fragment that contained His A(12), and the presence of 2-oxo-His. Similarly, the precipitated degradants of rhRlx showed reduced levels of His but no 2-oxo-His. In addition, the soluble degradants, which contain 2-oxo-His, appear to exist as monomers having an average molecular weight similar to that of rhRlx. These results suggest that the metal-catalyzed oxidation of His A(12) leads to other, as yet unidentified oxidation products of His A(12) that affect the secondary/tertiary structure of the protein more significantly than does 2-oxo-His and ultimately lead to the physical instability of the protein observed at higher pH values.     

Studies on the mechanism of assembly of tobacco mosaic virus.

Sedimentation and proton binding studies on the endothermic self-association of tobacco mosaic virus (TMV) protein indicate that the so-called "20S" sedimenting protein is an interaction system involving at least the 34-subunit two-turn yield cylindrical disk aggregate and the 49-subunit three-turn helical rod. The pH dependence of this overall equilibrium suggests that disk formation is proton-linked through the binding of protons to the two-turn helix which is not present as significant concentrations near pH 7. There is a temperature-induced intramolecular conformation change in the protein leading to a difference spectrum which is complete in 5 x 10(-6) s at pH 7 and 20 degrees C and is dominated at 300 nm by tryptophan residues. Kinetics measurements of protein polymerization, from 10(-6) to 10(3) s, reveal three relaxation processes at pH 7.0, 20 degrees C, 0.10 M ionic strength K (H) PO4. The fastest relaxation time is a few milliseconds and represents reactions within the 4S protein distribution. The second fastest relaxation is 50-100 x 10(-3) s and represents elementary polymerization steps involved in the formation of the approximately 20 S protein. Analysis of the slowest relaxation, approximately 5 x 10(4) s, suggests that this very slow formation of approximately 20 S protein may be dominated by some first order process in the overall dissociation of approximately 20S protein. Sedimentation measurements of the rate of TMV reconstitution, under the same conditions, show by direct measurements of 4S and approximately 20S incorporation at various 4S to approximately 20S weight ratios that the relative rate of approximately 20S incorporation decreases almost linearly, from 0 to 50% 4S. There appears to be one or more regions of TMV-RNA, approximately 1-1.5 kilobases long, which incorporates approximately 20S protein exclusively. Solutions of approximately 95-100% approximately 20S protein have been prepared for the first time and used for reconstitution with RNA. Such protein solutions yield full size TMV, but at a slower rate than if 4S protein is added. Thus the elongation reaction in TMV assembly, following nucleation with approximately 20S protein, is not exclusively dependent upon the presence of either 4S or approximately 20S protein aggregates. The initial, maximum, rate of reconstitution increases about threefold when the protein composition is changed from 5% to 30% 4S protein, at constant total protein concentration at pH 7.0, 20 degrees C in 0.10 M ionic strength K (H)PO4. The probable binding frame at the internal assembly nucleation site of TMV-RNA has been determined by measuring the association constants for the binding of various trinucleoside diphosphates to helical TMV protein rods. The -CAG-AAG-AAG-sequence at the nucleation site is capable of providing at least 10-14 kcal/mol of sites of binding free energy for the nucleation event in TMV self-assembly.