Characterization of inhibition of M2 ion channel activity by BL-1743, an inhibitor of influenza A virus.

The influenza A virus M2 integral membrane protein has ion channel activity that can be inhibited by the antiviral drug amantadine. Recently, a spirene-containing compound, BL-1743 (2-[3-azaspiro (5,5)undecanol]-2-imidazoline), that inhibits influenza virus growth was identified (S. Kurtz, G. Lao, K. M. Hahnenberger, C. Brooks, O. Gecha, K. Ingalls, K.-I. Numata, and M. Krystal, Antimicrob. Agents Chemother. 39:2204-2209, 1995). We have examined the ability of BL-1743 to inhibit the M2 ion channel when expressed in oocytes of Xenopus laevis. BL-1743 inhibition is complete as far as can be measured by electrophysiological methods and is reversible, with a reverse reaction rate constant of 4.0 x 10(-3) s(-1). In contrast, amantadine inhibition is irreversible within the time frame of the experiment. However, BL-1743 inhibition and amantadine inhibition have similar properties. The majority of isolated influenza viruses resistant to BL-1743 are also amantadine resistant. In addition, all known amino acid changes which result in amantadine resistance also confer BL-1743 resistance. However, one BL-1743-resistant virus isolated, designated M2-I35T, contained the change Ile-35-->Thr. This virus is >70-fold more resistant to BL-1743 and only 10-fold more resistant to amantadine than the wild-type virus. When the ion channel activity of M2-I35T was examined in oocytes, it was found that M2-I35T is BL-1743 resistant but is reversibly inhibited by amantadine. These findings suggest that these two drugs interact differently with the M2 protein transmembrane pore region.     

A t(3;8) chromosomal translocation associated with hepatitis B virus intergration involves the carboxypeptidase N locus.

Integrated hepatitis B virus (HBV) DNA is found in the great majority of human hepatocellular carcinomas, suggesting that these viral integrations may be implicated in liver oncogenesis. Besides the insertional mutagenesis characterized in a few selected cases and the contribution of viral transactivators to cell transformation to malignancy, HBV has been shown to generate gross chromosomal rearrangements potentially involved in carcinogenesis. Here, we report a t(3;8) chromosomal translocation present in a hepatocellular carcinoma developed in noncirrhotic liver tissue. One side of the translocation, in 8p23, is shown to be in the vicinity of the carboxypeptidase N gene, a locus that is heavily transcribed in liver tissue and frequently deleted in hepatocellular carcinomas and other epithelial tumors. The other side of the translocation, in 3q27-29, is widely implicated in several types of translocations occurring in different malignancies, such as large-cell lymphomas. The present data strongly support a model in which HBV-induced chromosomal rearrangements play a key role during multistep liver oncogenesis.     

Subcellular compartmentation of different lipophilic fluorescein derivatives in maize root epidermal cells

Summary Fluorescence microscopy offers some distinct advantages over other techniques for studying ion transport processes in situ with plant cells. However, the use of this technology in plant cells has been limited by our lack of understanding the mechanisms that influence the subcellular distribution of dyes after loading with the lipophilic precursors. In this study, the subcellular distribution of 5-(and 6-)carboxydichlorofluorescein (CDCF), carboxy-SNAFL-1, and carboxy-SNARF-1 was compared to that of 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (BCECF) after incubation of maize roots with their respective lipophilic precursors. Previously, we reported that incubation of roots with BCECF-acetomethyl ester (BCECF-AM) led to vacuolar accumulation of this dye. Similar results were found when roots were incubated with CDCF-diacetate. In contrast, carboxy-SNAFL-1 appeared to be confined to the cytoplasm based on the distribution of fluorescence and the excitation spectra of the dye in situ. On the other hand, incubation of roots with carboxy-SNARF-1-acetoxymethyl acetate yielded fluorescence throughout the cell. When the cytoplasm of epidermal cells was loaded with the BCECF acid by incubation at pH 4 in the absence of external Ca, the dye was retained in the cytoplasm at least 3 h after the loading period. This result indicated that vacuolar accumulation of BCECF during loading of BCECF-AM was not due to transport of BCECF from cytoplasm to vacuole. The esterase activities responsible for the production of either carboxy-SNAFL-1 or BCECF from their respective lipophilic precursor by extracts of roots were compared. The characterization of esterase activities was consistent with the subcellular distribution of these dyes in root cells. The results of these experiments suggest that in maize root epidermal cells the subcellular distribution of these fluorescein dyes may be determined by the characteristics of the esterase activities responsible for hydrolysis of the lipophilic precursor.Abbreviations BCECF (BCECF-AM) 2,7-bis-(2-carboxyethyl)-5-(and 6-)carboxyfluorescein (its acetoxymethyl ester) - BTB bis-trispropane - CDCF (CDCF-DA) 5-(and 6-)carboxy-2,7-dichlorofluorescein (its diacetate derivative) - DAPI 4,6-diamidino-2 phenylindole dihydrochloride - DMSO dimethylsulfoxide - HEPES N-[2-hydroxyethyl] piperazine-N-[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethane-sulfonic acid - SNAFL-1 (SNAFL-1-DA) carboxyl SNAFL-1 (its diacetate) - SNARF-1 (SNARF-1-AM) carboxyl SNARF-1 (its acetoxymethyl acetate)     

Dielectric properties of human red blood cells in suspension at radio frequencies

Dielectric properties of human red blood cells (RBCs) in suspension (hematocrit 50%) from 243 healthy persons (120 males, 123 females) were measured at 25 °C in a frequency range of 1–500 MHz, with a coaxial transmission line reflection method (one-side measurement). The measuring system, controlled by an IBM-PC computer, was composed of a network analyzer (HP4195A), an impedance test adapter (HP41951-61001), a coaxial line sensor, and a temperature-controlling set. The data measured revealed a statistically significant age dependence, with a critical age of about 49 years, above which permittivity and conductivity of human RBCs in suspension decreased significantly. © 1994 Wiley-Liss, Inc.     

The GLC7 type 1 protein phosphatase is required for glucose repression in Saccharomyces cerevisiae.

We cloned the GLC7/DIS2S1 gene by complementation of the cid1-226 mutation, which relieves glucose repression in Saccharomyces cerevisiae. GLC7 encodes the catalytic subunit of type 1 protein phosphatase (PP1). Genetic analysis and sequencing showed that cid1-226 is an allele of GLC7, now designated glc7-T152K, which alters threonine 152 to lysine. We also show that the glc7-1 and glc7-T152K alleles cause distinct phenotypes: glc7-1 causes a severe defect in glycogen accumulation but does not relieve glucose repression, whereas glc7-T152K does not prevent glycogen accumulation. These findings are discussed in light of evidence that interaction with different regulatory or targeting subunits directs the participation of PP1 in diverse cellular regulatory mechanisms. Finally, genetic studies suggest that PP1 functions antagonistically to the SNF1 protein kinase in the regulatory response to glucose.     

Artificial mosaic protein containing antigenic epitopes of hepatitis E virus.

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.     

A surface mutant (G82R) of a human α-glutathione S-transferase shows decreased thermal stability and a new mode of molecular association in the crystal

A chimeric enzyme (GST121) of the human α-glutathione S-transferases GST1-1 and GST2-2, which has improved catalytic efficiency and thermostability from its wild-type parent proteins, has been crystallized in a space group that is isomorphous with that reported for crystals of GST1-1. However, a single-site (G82R) mutant of GST121, which exhibits a significant reduction both in vitro and in vivo in protein thermostability, forms crystals that are not isomorphous with GST1-1. The mutant protein crystallizes in space group P2₁2₁2₁, with cell dimensions a = 49.5, b = 92.9, c = 115.9 Å, and one dimer per asymmetric unit. Preliminary crystallographic results show that a mutation of the surface residue Gly 82 from a neutral to a charged residue causes new salt bridges to be formed among the GST dimers, suggesting that the G82R mutant might aggregate more readily than does GST121 in solution resulting in a change of its solution properties. © 1994 Wiley-Liss, Inc.     

NADH-Linked Ferricyanide and Cytochrome c Reduction Activities in Corn Root Plasma Membrane

Corn root plasma membrane catalyzed NADH reduction of ferricyanideand cytochrome c over a wide pH range. At pH 7.5, apparent K_msof NADH-cytochrome c pair were significantly lower than thoseof NADH-ferricyanide pair. FMN and polylysine respectively enhancedthe reduction of ferricyanide and cytochrome c. Yet, polyaspartatedecreased the ferricyanide reduction. NADH oxidation observedin the presence of both ferricyanide and cytochrome c was significantlyslower than the sum of rates obtained with individual acceptors.The results suggest that the membrane may contain differentbut not totally independent reduction sites for cytochrome cand ferricyanide. (Received April 13, 1993; Accepted August 23, 1993)     

Divalent cation and lipid-protein interactions of biomembranes

Divalent cations play an important role in the functions of biomembranes. This review deals with three topics: (1) Mg²⁺-mediated change in physical state of phospholipid induces conformation and activity change of reconstituted mitochondrial H⁺-ATPase, (2) a proper transmembrane Ca²⁺ gradient is essential for the higher enzymatic activity of adenylate cyclase, and (3) role of transmembrane Ca²⁺ gradient in the modulation of reconstituted sarcoplasmic reticulm Ca²⁺-ATPase activity.