Binding sites of HeLa cell nuclear proteins on the upstream region of adenovirus type 5 E1A gene.

Twenty one binding sites of HeLa cell nuclear proteins were identified on the upstream region of adenovirus type 5 E1A gene using DNase I footprint assay. The proximal promoter region contained five binding sites that overlapped the cap site, TATA box, TATA-like sequence, CCAAT box, and -100 region relative to the E1A cap site(+1). The -190 region was a potential site for octamer-motif binding proteins, such as NFIII and OBP100. An upstream copy of the E1A enhancer element 1 was the site for a factor (E1A-F) with the binding specificity of XGGAYGT (X = A, C; Y = A, T). E1A-F factor also bound to three other sites, one of which coincided with the distal E1A enhancer element. The distal element also contained a potential site for ATF factor. The adenovirus minimal origin of DNA replication competed for DNA-protein complex formation on the CCAAT and TATA box region and the -190 region, suggesting that these regions interacted with a common or related factor.     

The membrane domain of 3-hydroxy-3-methylglutaryl-coenzyme A reductase confers endoplasmic reticulum localization and sterol-regulated degradation onto beta-galactosidase

A hybrid gene has been constructed consisting of coding sequence for the membrane domain of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase linked to the coding sequence for the soluble enzyme Escherichia coli beta-galactosidase. Expression of the hybrid gene in transfected Chinese hamster ovary cells results in the production of a fusion protein (HMGal) which is localized in the endoplasmic reticulum. The fusion protein contains the high-mannose oligosaccharides characteristic of HMG-CoA reductase. Importantly the beta-galactosidase activity of HMGal decreases when low density lipoprotein is added to the culture media. Therefore, the membrane domain of HMG-CoA reductase is sufficient to determine both correct intracellular localization and sterol-regulation of degradation. Mutant fusion proteins which lack 64, 85, or 98 amino acid residues from within the membrane domain of HMG-CoA reductase are found to be localized in the endoplasmic reticulum and to retain beta-galactosidase activity. However, sterol-regulation of degradation is abolished.     

Cytoplasmic Alkalization and Cytoplasmic Streaming Induced by Light and Histidine in Leaf Cells of Egeria densa: in vivo 31P-NMR study

Rotational streaming of the cytoplasm including chloroplastswas induced by L-histidine, as well as by light, on the anticlinalface of leaf cells of Egeria densa. In the case of treatmentwith L-histidine some of the chloroplasts remained stationaryon the periclinal face of cells after rotational cytoplasmicstreaming was initiated. However, these chloroplasts were easilydislodged and translocated to the centrifugal end of the histidine-treatedcells by application of a centrifugal force that barely affectedthe location of chloroplasts in cells incubated in the darkwithout L-histidine. This result indicates that the anchoringof chloroplasts was weakened by L-histidine. Thus only the releaseof chloroplasts from anchoring was not enough for initiationof their streaming. The cytoplasmic pH (pH_c) and vacuolar pH(pH_v) were noninvasively monitored by in vivo ³¹P-nuclear magneticresonance (NMR) spectroscopy. Compared with the dark controlvalue, both illumination and treatment with L-histidine increasedthe pH_c by 0.3 units. In contrast, pH_v changed only a littlewith both illumination and treatment with L-histidine. Releaseof chloroplasts from anchoring and initiation of cytoplasmicstreaming are discussed in relation to the increase in pH_c inducedby both light and L-histidine. ⁴ Present address: Department of Cell Biology, National Instituteof Agrobiological Resources, Kannondai, Tsukuba, Ibaraki, 305Japan ⁵ Present address: Marine Biotechnology Institute Co., Ltd.,Head Office, 2-35-10 Hongo, Bunkyo-ku, Tokyo, 113 Japan (Received July 16, 1990; Accepted December 20, 1990)     

Establishment of a new perchloric acid treatment method to allow determination of the total endotoxin content in human plasma by the limulus test and clinical application.

We established a new method of plasma treatment for the removal of interfering factors in the plasma to allow detection of endotoxin by limulus test. The limulus test used was an endotoxin-specific chromogenic test, the Endospecy test. Perchloric acid (PCA) treatment and centrifugation (PCA method) is usually used to remove interfering factors from plasma, with the precipitate being discarded and the supernatant used to detect endotoxin. As the solubilized precipitates of endotoxin-spiked plasma and some patient plasma were found to contain the Endospecy activity, we have devised a new method assaying endotoxin in both the supernatant and precipitate. This study confirmed that the solubilized precipitate of endotoxin-spiked plasma had Endospecy activity and found that the precipitate had other endotoxin activities, such as lethality in galactosamine-sensitized mice and pyrogenicity in rabbits. We also confirmed that interfering factors were completely removed from plasma samples by this new method. The endotoxin level after the new PCA method was found to be about 8 times higher than that determined after PCA treatment and the new PCA method surpasses the conventional PCA method with regard to the positive rate of endotoxin contents in clinical samples. These results indicate that the new PCA method is superior to the PCA method as a plasma pretreatment method for limulus test.     

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Cytoplasmic Ca2+ has No Effect on the Excitability of Chara Plasmalemma

Internodal cells of Chara australis were subjected to two consecutiveintracellular perfusions with a Ca²⁺-free EGTA medium whichdisintegrated the tonoplast within about 10 minutes and thenwith a Ca²⁺-buffered medium. All perfusion media usually contained1 mM ATP. To stop the electrogenic pump, the internode was depletedof intracellular ATP. The excitability of the plasmalemma wasnot significantly influenced by intracellular free Ca²⁺ concentrationsup to 10^–4 M. To trigger action potentials, minimum currentdensities of 1 to 2 µA cm^–2 had to be applied atall tested Ca²⁺ concentrations. In the absence of cytoplasmicATP, excitability was completely lost at all Ca²⁺ concentrations. ¹ Present address: Botanisches Institut der Universit?t Bonn,Venusbergweg 22, D-5300 Bonn, FRG. (Received September 22, 1984; Accepted March 6, 1985)     

Ionic Activity Gradients across the Surface Membrane of Cytoplasmic Droplets Prepared from Chara australis

The membrane potential and the ionic activity gradients of K⁺and Cl^– across the surface membrane of cytoplasmic dropletsprepared from Chara australis internodal cells, were measuredin high and low ionic strength bathing solutions using liquidion exchange microelectrodes selective for K⁺ and Cl^–.Our results indicate that K⁺ is close to electrochemical equilibriumwhereas Cl^– is not. ¹ Present address: ICI Japan, Palace Hotel Annex Building, Marunouchi,Tokyo, Japan.     

A sensitive method for quantitative analysis of phospholipid molecular species by high-performance liquid chromatography

A simple and sensitive method was developed for quantitative analysis of phospholipid molecular species. Diacylglycerols were prepared from phospholipids by phospholipase C treatment and converted to the corresponding dinitrobenzoyl derivatives, which could be sensitively detected at 254 nm. The derivatives of 21 molecular species were resolved by high-performance liquid chromatography with an octadecylsilyl reversed-phase column. All the derivatives had the same peak area per mol, and peak areas were proportional to the amounts of the derivatives. Quantification was carried out at the picomole level.