Simple and straightforward construction of a mouse gene targeting vector using in vitro transposition reactions

In a gene targeting experiment, the generation of a targeting construct often requires complex DNA manipulations. We developed a set of cassettes and plasmids useful for creating targeting vectors to modify the mammalian genome. A positive selection marker cassette (PGK/EM7p-npt), which included dual prokaryotic and eukaryotic promoters to permit consecutive selection for recombination in Escherichia coli and then in mouse embryonic stem cells, was flanked by two FRT-loxP sequences. The PGK/EM7p-npt cassette was placed between the minimum regions of a Tn7 transposable element for insertion into another DNA by means of Tn7 transposase in vitro. We also constructed a plasmid having a loxP-Zeo-loxP cassette between the modified Tn5 outer elements. These cassettes can be integrated randomly into a given genomic DNA through the in vitro transposition reaction, thus producing a collection of genomic segments flanked by loxP sites (floxed) at various positions without the use of restriction enzymes and DNA ligase. We confirmed that this system remarkably reduced the time and labor for the construction of complex gene targeting vectors.     

Purification and characterization of a novel class IIa bacteriocin, piscicocin CS526, from surimi-associated Carnobacterium piscicola CS526

The bacteriocin piscicocin CS526 was inactivated by proteolytic enzymes, was stable at 100 degrees C for 30 min, had a pH range of 2 to 8, and was active against Enterococcus, Listeria, Pediococcus, and Leuconostoc. The N-terminal sequence was YGNGL, not the YGNGV consensus motif common in class IIa bacteriocins (alternate residues underlined). The molecular mass of piscicocin CS526, which had a bactericidal mode of action, was approximately 4,430 Da.     

A novel mutagen, 2-(5-hydroxy-4,6-dinitroindolyl) ethanol, formed in the reaction between 5-hydroxytryptamine and nitrite under acid conditions, especially in the presence of L-cysteine

We examined the mutagenic activity of each of 29 amino acids mixed under acidic conditions with 5-hydroxytryptamine (5-HT) and nitrite using Salmonella typhimurium strain TA 100 with or without a metabolic activation system (S9 mix). The reaction mixture containing L-cysteine was strongly mutagenic without S9 mix. We subjected an ethyl acetate extract of the reaction mixture to HPLC, isolated a mutagenic component, and investigated its chemical structure by LC-mass spectrometry (MS), high-resolution fast atom bombardment (HRFAB)-MS, and 1H and 13C NMR. We identified the mutagen as 2-(5-hydroxy-4,6-dinitro-3-indolyl) ethanol (2HDIE). We injected 8 mg/kg 2HDIE i.p. into male ICR mice and found that the compound increased the frequency of micronuclei in peripheral reticulocytes. Our results suggest that 2HDIE might be formed in vivo by consumption of 5-HT, nitrite and L-cysteine in foods, and might act as a mutagen.     

Serinc, an activity-regulated protein family, incorporates serine into membrane lipid synthesis

Cell membranes contain various transporter proteins, some of which are responsible for transferring amino acids across membrane. In this study, we report another class of carrier proteins, termed Serinc1-5, that incorporates a polar amino acid serine into membranes and facilitates the synthesis of two serine-derived lipids, phosphatidylserine and sphingolipids. Serinc is a unique protein family that shows no amino acid homology to other proteins but is highly conserved among eukaryotes. The members contain 11 transmembrane domains, and rat Serinc1 protein co-localizes with lipid biosynthetic enzymes in endoplasmic reticulum membranes. A Serinc protein forms an intracellular complex with key enzymes involved in serine and sphingolipid biosyntheses, and both functions, serine synthesis and membrane incorporation, are linked to each other. In the rat brain, expression of Serinc1 and Serinc2 mRNA was rapidly up-regulated by kainate-induced seizures in neuronal cell layers of the hippocampus. In contrast, myelin throughout the brain is enriched with Serinc5, which was down-regulated in the hippocampus by seizures. These results indicate a novel mechanism linking neural activity and lipid biosynthesis.     

Matrix metalloproteinase-9 in macrophages induces thymic neovascularization following thymocyte apoptosis

Matrix metalloproteinase-9 (MMP-9) has been implicated in the degradation of the extracellular matrix in a variety of physiological and pathological processes. We found that MMP-9 expression in thymuses of BALB/c mice that had been injected with anti-CD3 Ab to induce thymocyte apoptosis was increased both at mRNA and protein levels. Macrophages are shown to be the principal stromal cells responsible for phagocytosis of dying thymocytes, and macrophages were found to constitutively express MMP-9. The activity of plasmin, which is known as one of the activators for MMP-9, was increased in the thymuses with MMP-9 activation. Binding of Ab HUIV26, which recognizes a cryptic epitope on collagen type IV following proteolytic cleavage, was found to be reduced in MMP-9 knockout mice, suggesting that collagen type IV is a substrate of MMP-9. Although the formation of thymic neovessels was found following thymocyte apoptosis, it was diminished in anti-CD3 Ab-injected MMP-9 knockout mice. In vivo administration of Ab HUIV26 resulted in a reduction of thymic neovascularization. After clearance of apoptotic thymocytes, the number of macrophages in the thymuses was decreased, and this decrease was delayed by blocking of HUIV26 epitope. Taken together, our results suggest that MMP-9 expression in macrophages mediates degradation of collagen type IV and facilitates their migration from the thymus after clearance of apoptotic thymocytes. These studies demonstrate a potential role of macrophage MMP-9 in the remodeling of thymic extracellular matrix following thymocyte apoptosis.     

ACE1 polymorphism and progression of SARS

We have hypothesized that genetic predisposition influences the progression of SARS. Angiotensin converting enzyme (ACE1) insertion/deletion (I/D) polymorphism was previously reported to show association with the adult respiratory distress syndrome, which is also thought to play a key role in damaging the lung tissues in SARS cases. This time, the polymorphism was genotyped in 44 Vietnamese SARS cases, with 103 healthy controls who had had a contact with the SARS patients and 50 controls without any contact history. SARS cases were divided into either non-hypoxemic or hypoxemic groups. Despite the small sample size, the frequency of the D allele was significantly higher in the hypoxemic group than in the non-hypoxemic group (p=0.013), whereas there was no significant difference between the SARS cases and controls, irrespective of a contact history. ACE1 might be one of the candidate genes that influence the progression of pneumonia in SARS.     

Human parvovirus B19 transgenic mice become susceptible to polyarthritis

Human parvovirus B19 (B19) often causes acute polyarthritis in adults. In this paper, we analyzed nucleotide sequences of the B19 genome of patients with rheumatoid arthritis (RA), and then introduced the nonstructural protein 1 (NS1) gene of B19 into C57BL/6 mice that had a genetic origin not susceptible to arthritis. The transgenic mice developed no lesions spontaneously, but were susceptible to type II collagen (CII)-induced arthritis. B19 NS1 was expressed in synovial cells on the articular lesions that were histologically characteristic of granulomatous synovitis and pannus formation in cartilage and bone. Serum levels of anti-CII Abs and TNF-alpha increased in NS1 transgenic mice to the same levels as those of DBA/1 mice, which were susceptible to polyarthritis. Stimulation with CII increased secretion of Th1-type- and Th2-type cytokines in NS1 transgenic mice, indicating that a nonpermissive H-2(b) haplotype in the wild type of C57BL/6 mice can be made susceptible to polyarthritis through the expression of NS1. This study is the first to show that a viral agent from the joints in humans can cause CII-induced arthritis resembling RA.     

Higher plant RecA-like protein is homologous to RadA

A novel RecA-like protein, differing from Dmc1 and Rad51, was characterized in Oryza sativa L. cv. Nipponbare. Because the protein is homologous to bacterial RadA, the gene was designated OsRadA. The open reading frame was predicted to encode a 66kDa protein of 619 amino acid residues and was found in plants but not animals or yeast. OsRadA showed D-loop and single-stranded DNA-dependent ATPase activities. Gene expression was found to be high in meristematic tissues, and was localized in the nucleus. An RNAi mutant of Arabidopsis thaliana RadA (AtRadA) was sensitive to mutagenic agents such as UV and MMC, suggesting that RadA functions in DNA repair.     

Generation of a mouse for conditional excision of progesterone receptor

The progesterone receptor (PR) is required for several aspects of mammalian female reproduction. PR null mice have overlapping defects that preclude an understanding of its multiple functions in ovulation, pregnancy, mammary gland biology, and sexual behavior. We have generated a PR conditional excision (PRCE) allele in which loxP sites flank exon 1. Homozygous PRCE females are fertile and appear to be functionally normal. Global cre mediated excision of the floxed exon 1 using EIIa-cre mice resulted in systemic loss of exon 1 and PR protein. Female mice homozygous for this null allele were sterile, as expected for PR knockout (PRKO) females. Conditional loss of PR will facilitate investigation of the spatial and temporal roles of PR in both normal development and disease.