The process of infection with bacteriophage phiX174, XXI. Replication and fate of the replicative form

Previous publications have described the formation of a double-stranded DNA intermediate(1) (replicative form, RF) during the replication of bacteriophage phiX174, the semiconservative replication at a bacterial "site"(2) of that RF containing the parental DNA strand,(3,4) and the persistence of the parental DNA strand at the site, accompanied by the release of the daughter RF.(5) The nascent RF molecules first appear as RFII,(6,7) and are rapidly converted to RFI. This paper considers the structure of the nascent RF molecules in more detail and inquires into the fate of the daughter RF molecules during the subsequent period of progeny single-strand DNA formation. A succeeding paper(8) will consider the process of formation of the progeny single-strands

The process of infection with bacteriophage phiX174, XXII. Synthesis of progeny single-stranded DNA

In a previous article,(1) it was demonstrated that of the labeled thymidine incorporated into the replicative form (RF) of phiX-DNA during the period of RF replication (3-12 or 15 min after infection), approximately half was transferred to virus during the subsequent period of single-strand DNA synthesis, while half remained in RF molecules. In this paper experiments are presented which permit a definition of the label remaining in the RF and which clarify the role of the RF in the synthesis of the progeny single-stranded DNA

Aureobasidin A, an antifungal cyclic depsipeptide antibiotic, is a substrate for both human MDR1 and MDR2/P-glycoproteins

AbstractThe human MDR1 gene encodes the multidrug transporter P-glycoprotein (Pgp). Although the MDR2/Pgp shares about 80% identity at the amino acid level with the MDRI/Pgp, the MDR2/Pgp cannot act as a multidrug transporter. We examined the drug sensitivity of Saccharomyces cerevisiae expressing either the human MDR1/Pgp or MDR2/Pgp. The human MDR1/Pgp conferred about 4-fold resistance to aureobasidin A, a cyclic depsipeptide antifungal antibiotic, on the drug-sensitive yeast strains. Interestingly the human MDR2/Pgp also conferred about 2.5-fold resistance to aureobasidin A. The resistance to aureobasidin A conferred by the MDR2/Pgp as well as by the MDR1/Pgp was overcome by vinblastine, verapamil, and cyclosporin A, depending on their concentrations, but not by colchicine. Aureobasidin A probably interacts directly with Pgps, because it overcame multidrug resistance of human cells and inhibited azidopine photoaffinity labeling of MDRI/Pgp in human cell membranes. These results suggest the possibility that the human MDR1 and MDR2/Pgps have conserved domain(s) for drug recognition

Anti-cancer drugs and glutathione stimulate vanadate-induced trapping of nucleotide in multidrug resistance-associated protein (MRP)

AbstractMultidrug resistance-associated protein (MRP), a member of the ABC superfamily transporters, functions as an ATP-dependent efflux pump that extrudes cytotoxic drugs from the cells. Although glutathione has been considered to play an important role in the function of MRP, there is no convincing evidence that glutathione directly interacts with MRP. Here we demonstrate that vanadate-induced trapping of 8-azido-ATP in MRP was stimulated in the presence of glutathione, oxidized glutathione and the anti-cancer drugs VP-16 and vincristine. MRP in membrane from a human MRP cDNA transformant was specifically photolabeled with 8-azido-[α-32P]ATP by the vanadate-trapping technique. Vanadate and Mg2+ were required for trapping of nucleotides, and vanadate trapping of nucleotides was inhibited by excess ADP as well as ATP. These results suggest that a stable inhibitory complex MRP·MgADP·Vi, an analog of the MRP·MgADP·Pi transition state complex, is formed in the presence of vanadate. Glutathione as well as anti-cancer drugs would directly interact with MRP, and stimulate the formation of the transition state of the ATPase reaction of MRP

Functional analysis of block 5, one of the highly conserved amino acid sequences in the 130-kDa CryIVA protein produced by Bacillus thuringiensis subsp. israelensis

AbstractThere are five amino acid sequences highly conserved among Bacillus thuringiensis δ-endotoxins. We have changed the amino acid residues in block 5, one of the conserved sequences, of CryIVA. When the amino acid residues with charged side chains were replaced by others, the amount of production of the altered CryIVA protein was markedly decreased. It is suggested that the decrease is caused by the unstable conformation of the altered CryIVA protein molecule, as judged by digestion with trypsin and thermolysin. On the other hand, the substitution of amino acid residues in block 5 did not affect the insecticidal activity of CryIVA. These results strongly suggest that block 5 of CryIVA is one of the stability-determining elements of the protoxin molecule

Clinical perspectives of Treponema pallidum subsp. Endemicum infection in adults, particularly men who have sex with men in the Kansai area, Japan: A case series

Bejel, caused by Treponema pallidum subsp. Endemicum (TEN), is a locally transmitted disease among children and juveniles in hot and dry regions. The number of adult cases of TEN infection outside of endemic areas has recently increased. We clinically examined five cases of TEN infection among adult cases previously reported in Japan. TEN infection mainly developed among young to middle-aged men who have sex with men (MSM). The clinical features of cases of TEN infection were similar to those of primary- and secondary-stage T. pallidum subsp. pallidum (TPA) infection. Genital lesions were common as the primary lesion. The clinical features and laboratory parameters of cases of TEN infection were similar to those of TPA infection. Most of the isolated strains had the A2058G mutation in 23S rDNA, which is responsible for resistance to macrolides. We also performed the systemic literature review of the TEN cases outside the endemic countries. The recent reported cases diagnosed with molecular methods shared the clinical features, occurred in young-to middle-aged sexually active persons in urban areas of developed countries and often accompanied with genital lesions, which were distinct from the classic description of bejel. This case series and the literature review provides important clinical insights and will contribute to the clinical detection of this rarely identified disease in developed countries. The surveillance of treponematoses, including TEN infection, using molecular diagnostic techniques is also warranted in developed countries, for the purpose of grasping the epidemic situation and control the local transmission

Molecular cloning and sequencing of the glycogen phosphorylase gene from Escherichia coli

AbstractThe glgP gene, which codes for glycogen phosphorylase, was cloned from a genomic library of Escherichia coli. The nucleotide sequence of the glgP gene contained a single open reading frame encoding a protein consisting of 790 amino acid residues. The glgP gene product, a polypeptide of Mr 87 000, was confirmed by SDS-polyacrylamide gel electrophoresis. The deduced amino acid sequence showed that homology between glgP of E. coli and rabbit glgP, human glgP, potato glgP, and E. coli malP was 48.6, 48.6, 42.3, and 46.1%, respectively. Within this homologous region, the active site, glycogen storage site, and pyridoxal-5′-phosphate binding site are well conserved. The enzyme activity of glycogen phosphorylase increased after introduction on a multicopy of the glgP gene