Cellular gene expression in papillomas of the choroid plexus from transgenic mice that express the simian virus 40 large T antigen.

Transgenic mice that contain the simian virus 40 (SV40) enhancer-promoter and large tumor (T) antigen gene develop papillomas of the choroid plexus. The tumors remain well differentiated on histological examination and express normal levels of tissue-specific mRNAs for transthyretin (TTR) and the 5-HT1C serotonin receptor, two differentiated cell markers. Both Northern (RNA) blot analysis and in situ cytohybridization have been used to monitor the steady-state levels of the mRNAs from the viral oncogene (T antigen) and from several cellular oncogenes. In situ hybridization demonstrated, in serial sections, increased levels of both T antigen mRNA and p53 mRNA localized in the tumor tissue but not in the normal brain tissue. The ratios of the steady-state levels of mRNA for p53/TTR and p53/L32, a ribosomal protein gene, were 2- to 20-fold higher in the tumor tissue than in the normal choroid plexus tissue. Several other oncogenes did not show elevated levels of mRNA in these tumors. p53 protein levels were not detectable in normal brain tissue, but p53 levels were very high in tumor tissue in which all of the p53 was found in a complex with the SV40 large T antigen. These data continue to show a close relationship between SV40 T-antigen-mediated tumorigenesis and the role of p53 in these tumors.     

The Epstein-Barr virus (EBV) BZLF1 immediate-early gene product differentially affects latent versus productive EBV promoters.

The Epstein-Barr virus (EBV) BZLF1 gene product is thought to mediate the disruption of latent EBV infection. We have examined the regulatory effects of BZLF1 by studying its transactivating effects on seven different EBV promoters. We find that whereas the BZLF1 gene product increases the activity of the two early promoters, BMLF1 and BMRF1, it decreases the activity of three latent promoters (the BamHI-C and BamHI-W Epstein-Barr nuclear antigen promoters and the latent membrane protein promoter). The BZLF1-induced changes in promoter-directed chloramphenicol acetyltransferase activity occur in EBV-negative as well as EBV-positive cell lines and are accompanied by a similar change in chloramphenicol acetyltransferase mRNA. Deletion analysis of the BamHI Z fragment indicates that in a portion of the amino-terminal half of the BZLF1 gene product (amino acids 24 to 86) is not essential for positive transactivating effects but is required for down-regulating effects. Thus, different domains of the same EBV immediate-early gene product can either increase the function of EBV promoters active in productive infection or decrease the function of key promoters active in latent infection.     

On the sensitivity of intact cells to perturbation by ethanol

A comparison was made of ethanol's effects on the order of plasma membranes in intact cells and some isolated membrane preparations. Order was assessed by steady-state fluorescence polarization techniques using the non-permeant probe, TMA-DPH. The data show that two cultured cells, rat neonatal astroglial and N2A neuroblastoma, were sensitive to significant ethanol-induced disordering within the anesthetically relevant range (100 - 200 mM). Human erythrocytes, cultured fibroblasts and homogenized astroglial cells required higher ethanol concentrations (greater than 250 mM) to produce a similar effect. Intact erythrocytes were approximately twice as sensitive as erythrocyte ghost membranes to ethanol-induced perturbation. The neonatal glial and N2A cells were approximately five times more sensitive than synaptic membranes to ethanol effects. DMPC and DMPC + cholesterol liposomes and myelin membranes were insensitive to ethanol's effects. The incorporation of 10 mole % ganglioside GM1 sensitized the liposomes to ethanol-induced perturbation.     

Hypophysectomy decreases and growth hormone increases the turnover and mass of rat liver glutamine synthetase

Hypophysectomy diminishes rat liver glutamine synthetase (GS) activity and growth hormone (GH) administration restores this activity to normal levels; brain GS is unaffected. We have now investigated the effects of long-term hypophysectomy (45-day) and GH treatment on the GS mass (amount of enzyme) and turnover in rat liver and brain. Labeled GS was isolated by immunoprecipitation at intervals between one and six days after pulse administration of [U-14C] leucine and the GS half-life (t1/2) was determined. The GS mass was obtained by immunoassay and by calculation using the specific activity of purified GS. GS turnover was calculated by multiplying the GS mass by the first-order rate constant of degradation (kd). During the time course of each experiment, the GS mass did not change, indicating that in each of the three hormonal states studied, a steady state existed. Hypophysectomy increased the t1/2 of hepatic GS from 3.8 to 8.8 days and decreased GS turnover from 0.38 to 0.1 microgram/100 g body wt/day; the GH regimen used restored the turnover to above normal levels, 0.6 microgram/100 g body wt/day. The GS mass decreased from 2.0 to 1.2 micrograms/100 g body wt and GH restored the GS mass to normal levels. The brain enzyme was not affected by hypophysectomy or GH.     

Functional profile of the human fetal gamma-globin gene upstream promoter region.

We performed a systematic functional analysis of the human gamma-globin promoter to identify its activator domains. We used a panel of truncation and scanning mutants as well as transfection in human K562 fetal erythroid cells. The various mutations produced relatively small changes in promoter function in both transient and stable transfection assays. The CACCC region and the region containing the binding sites for protein GATA-1 behaved as activator domains. We also obtained evidence for a minor activator site in the - 200 to - 190 region. The results are consistent with the interpretation that gamma-globin gene regulation may occur in part through multiple small effects of promoter elements.     

Nucleotide sequence of cDNA for Acacia confusa trypsin inhibitor and implication of post-translation processing.

Synthetic oligonucleotides corresponding to all possible sequences of N-terminal and C-terminal region of Acacia confusa trypsin inhibitor were used to generate ACTI-related sequences using the polymerase chain reaction on the cDNAs encoding ACTI of the seeds of legume, A. confusa. The deduced amino acid sequence agreed with that determined by the peptide analysis except an extra amino acid residue, serine, was found at the junction of A and B chain, which was removed by post-translation processing with specific protease(s). The substrate specificity of the protease(s) was found to cleave at the C-terminal sites of asparagine and serine, which was also shown to be the same case for another plant protein, abrin, isolated from legume, Abrus precatorius.     

Deletion of C-terminal 12 amino acids of GLUT1 protein does not abolish the transport activity.

We engineered the GLUT1 cDNA to delete C-terminal 12 amino acids of encoded GLUT1 protein. This mutated GLUT1 protein expressed in CHO cells by transfection of its cDNA was demonstrated to reside on the plasma membrane by cell surface labeling technique, and retain the transport activity, similar to that of the wild-type GLUT1. In addition, metabolic labeling of the intact cells with 35S indicated that the half-life of the mutated GLUT1 was not significantly different from that of the wild-type GLUT1. These results suggest that C-terminal 12 amino acids of GLUT1 are not important for the transport activity and the stability of the protein. Taken together with our previous results on the mutant without C-terminal 37 amino acids, the amino acids between the 37th and the 13th from the C-terminus appear to be essential for the transport activity.     

Tissue factor and its extracellular soluble domain: the relationship between intermolecular association with factor VIIa and enzymatic activity of the complex.

We find that the isolated, extracellular domain of tissue factor (TF1-218; sTF) exhibits only 4% of the activity of wild-type transmembrane TF (TF1-263) in an assay that measures the conversion of factor X to Xa by the TF:VIIa complex. Further, the activity of sTF is manifest only when vesicles consisting of phosphatidylserine and phosphatidylcholine (30/70 w/w) are present. To determine whether the decreased activity results from weakened affinity of sTF for VIIa, we studied their interaction using equilibrium ultracentrifugation, fluorescence anisotropy, and an activity titration. Ultracentrifugation of the sTF:VIIa complex established a stoichiometry of 1:1 and an upper limit of 1 nM for the equilibrium dissociation constant (Kd). This value is in agreement with titrations of dansyl-D-Phe-L-Phe-Arg chloromethyl ketone active site labeled VIIa (DF-VIIa) with sTF using dansyl fluorescence anisotropy as the observable. Pressure dissociation experiments were used to obtain quantitative values for the binding interaction. These experiments indicate that the Kd for the interaction of sTF with DF-VIIa is 0.59 nM (25 degrees C). This value may be compared to a Kd of 7.3 pM obtained by the same method for the interaction of DF-VIIa with TF1-263 reconstituted into phosphatidylcholine vesicles. The molar volume change of association was found to be 63 and 117 mL mol-1 for the interaction of DF-VIIa with sTF and TF1-263, respectively. These binding data show that the sTF:VIIa complex is quantitatively and qualitatively different from the complex formed by TF1-263 and VIIa.     

A ferredoxin-type iron-sulfur protein gene,frx B,is expressed in the chloroplasts of tobacco and spinach

Previously, a ferredoxin-type iron-sulfur protein, frx B protein, was identified in a high-salt extract of the purified thylakoid membrane of Chlamydomonas reinhardtii, a unicellular green alga. Polyclonal antibody was raised against a synthetic pentadecameric peptide with an amino acid sequence corresponding to the highly conserved region of the putative frx B proteins of 3 land plants [21]. In this report, protein(s) reacting strongly and specifically with this antibody was detected in the equivalent high-salt extract prepared from purified chloroplast of spinach and tobacco. One strong reaction polypeptide band from tobacco chloroplast was purified from SDS-polyacrylamide gel and subjected to endoproteinase lys C digestion. The resulting polypeptides were separated by reversed-phase chromatography. N-terminal sequencing of 3 purified polypeptides revealed that the protein is encoded by the frxB gene identified from DNA sequence analysis.     

Oxidation and cleavage of 3-aminopyridine adenine dinucleotide phosphate with periodate

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate in the neutral pH resulted in oxidation of the ribose linked to 3-aminopyridine and cleavage of the dinucleotide into adenosine- and 3-aminopyridine-containing moieties. Separation of these moieties was afforded by thin-layer chromatography, high-performance liquid chromatography, and fast protein liquid chromatography. From fast atom bombardment mass spectra and nuclear magnetic resonance spectra, the adenosine-containing moiety was identified as 2'-phosphoadenosine 5'-phosphate while the aminopyridine moiety was present in a mixture of the hydrated 3-aminopyridine mononucleotide/nucleoside dialdehyde. Separation of the completely oxidized product by Pharmacia fast protein liquid chromatography gave three major peaks corresponding to 2'-phosphoadenosine 5'-phosphate, 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside, with minor amount of oxidized 3-aminopyridine mononucleotide. Thus the oxidized 3-aminopyridine adenine dinucleotide phosphate was shown to cleave by two pathways: it may either undergo beta-elimination to give 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside; or the phosphodiester linkage may be hydrolyzed to give 2'-phosphoadenosine 5'-phosphate and oxidized 3-aminopyridine mononucleotide. The latter compound may further undergo beta-elimination and eventually give oxidized 3-aminopyridine nucleoside. Hydrolysis could be prevented by storing the sample as lyophilized powder, while beta-elimination was diminished by lowering the storage temperature. We found that the lyophilized powder of oxidized 3-aminopyridine adenine dinucleotide phosphate can be stored at -50 degrees C for several months with minimum decomposition.