Autonomous replicating sequences from intron of human ras gene in a simian virus 40 T antigen dependent system

Of the several DNA fragments present in the human lung cancer gene, 1.1 and 2.0 kilobase (kb) fragments corresponding to the intron of this gene were hybridized to a half part of the 27 nucleotides perfect palindrome present in the initiation part of replication in simian virus 40 (SV40) DNA. These two fragments cloned in pBR322 had good template activity, and the initiation of DNA replication started from the region of these fragments in an in vitro system, in which the initiation of DNA replication occurs on cloned DNA containing SV40 origin of DNA replication as described previously. Furthermore, these two clones could replicate autonomously in nuclei of SV40 transformed Cos cells, producing SV40 T antigen constitutively when the clones were transfected into Cos cells. These results show that functional SV40 origin-like sequences are present in human genomes, and they can replicate autonomously within the cells which are producing SV40 T antigen.     

Accumulation of unique globo-series glycolipids in PC 12h pheochromocytoma cells

In a previous paper, we reported the presence of a unique globo-series glycolipid as one of the major neutral glycolipid: Gal alpha 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1' Cer, in the subcloned PC 12h pheochromocytoma cells (Ariga, T., Yu, R. K., Scarsdale, J. N., Suzuki, M., Kuroda, Y., Kitagawa, H., and Miyatake, T. (1988) Biochemistry 27, 5335-5340). Recently we found that the subcloned PC 12h cells accumulated other unusual neutral glycolipids. In order to characterize these glycolipids, PC 12h cells were subcutaneously transplanted into rats. The induced tumor tissue accumulated four minor neutral glycolipids, which were purified by droplet counter-current, Iatrobeads column, and preparative thin-layer chromatographies. These glycolipid structures were determined by fast atom bombardment-mass spectrometry, proton nuclear magnetic resonance spectroscopy, permethylation study, and sequential degradation with various exoglycosidases to be as follows: A, Fuc alpha 1-2Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; B, GalNAc beta 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; C, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 4Gal beta 1-4Glc beta 1-1'Cer; and D, Gal alpha 1-3Gal alpha 1-3Gal alpha 1- 3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer. Glycolipids A and B were tentatively characterized in normal rat small intestine (Breimer, M. E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 557-568; Angstrom, J., Breimer, M. E., Falk, K.-E., Hansson, G. C., Karlsson, K.-A., and Leffler, H. (1982) J. Biol. Chem. 257, 682-688). Glycolipids C and D have not been reported in the literature.     

Study of fluorescence interaction and conformational changes of bovine serum albumin with histamine H1‐receptor–drug epinastine hydrochloride by spectroscopic and time‐resolved fluorescence methods

The fluorescence, ultraviolet (UV) absorption, time resolved techniques, circular dichroism (CD), and infrared spectral methods were explored as tools to investigate the interaction between histamine H₁ drug, epinastine hydrochloride (EPN), and bovine serum albumin (BSA) under simulated physiological conditions. The experimental results showed that the quenching of the BSA by EPN was static quenching mechanism and also confirmed by lifetime measurements. The value of n close to unity indicated that one molecule of EPN was bound to protein molecule. The binding constants (K) at three different temperatures were calculated (7.1 × 10⁴, 5.5 × 10⁴, and 3.9 × 10⁴M⁻¹). Based on the thermodynamic parameters (ΔH⁰, ΔG⁰, and ΔS⁰), the nature of binding forces operating between drug and protein was proposed. The site of binding of EPN in the protein was proposed to be Sudlow's site I based on displacement experiments using site markers viz, warfarin, ibuprofen, and digitoxin. Based on the Förster's theory of non‐radiation energy transfer, the binding average distance, r between the donor (BSA) and acceptor (EPN) was evaluated and found to be 4.48 nm. The UV–visible, synchronous fluorescence, CD, and three‐dimensional fluorescence spectral results revealed the changes in secondary structure of the protein upon its interaction with EPN. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 646–657, 2015.     

Incorporation of uracil into the growing strand of adenovirus 12 DNA.

The amount of rapidly labeled short DNA chains in adenovirus 12(Ad12)-infected cells was markedly increased in the presence of either uridine or deoxycytidine which could be converted to dUTP. When the infected cells were labeled with [³H]uridine or [³H] deoxycytidine and the labeled nucleotides in the short DNA chains from the Hirt supernatant were analysed by thin-layer chromatography, approximately 90 or 20% of the label was detected in dUTP. These results suggest that at least a portion of short DNA chains formed during Ad12 DNA replication is derived from an excision-repair mechanism of uracil containing nascent strands.     

Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain

Chronic mucocutaneous candidiasis (CMC) is a heterogeneous group of primary immunodeficiency diseases characterized by chronic and recurrent Candida infections of the skin, nails, and oropharynx. Gain-of-function mutations in STAT1 were very recently shown to be responsible for autosomal-dominant or sporadic cases of CMC. The reported mutations have been exclusively localized in the coiled-coil domain, resulting in impaired dephosphorylation of STAT1. However, recent crystallographic analysis and direct mutagenesis experiments indicate that mutations affecting the DNA-binding domain of STAT1 could also lead to persistent phosphorylation of STAT1. To our knowledge, this study shows for the first time that a DNA-binding domain mutation of c.1153C>T in exon 14 (p.T385M) is the genetic cause of sporadic CMC in two unrelated Japanese patients. The underlying mechanisms involve a gain of STAT1 function due to impaired dephosphorylation as observed in the coiled-coil domain mutations.