Genetic organization and conjugal plasmid DNA transfer of pHP69, a plasmid from a Korean isolate of Helicobacter pylori

We isolated pHP69, a 9,153 bp plasmid from Helicobacter pylori with a 33.98% (G+C) content. We identified 11 open reading frames (ORFs), including replication initiation protein A (repA), fic (cAMP-induced filamentation protein), mccC, mccB, mobA, mobD, mobB, and mobC, as well as four 22 bp tandem repeat sequences. The nucleic acid and predicted amino acid sequences of these ORFs exhibited significant homology to those of other H. pylori plasmids. pHP69 repA encodes a replication initiation protein and its amino acid sequence is similar to those of replicase proteins from theta-type plasmids. pHP69 contains two types of repeat sequences (R1 and R2), a MOBHEN family mobilization region comprising mobC, mobA, mobB, and mobD, and genes encoding microcin B and C. Among the 36 H. pylori strains containing plasmids, mobA or mccBC are present in 12 or 6, respectively and 3 contain both genes. To examine intrinsic capability of H. pylori for conjugative plasmid transfer, a shuttle vector pBHP69KH containing pHP69 and replication origin of pBR322 was constructed. It was shown that this vector could stably replicate and be mobilized among clinical H. pylori strains and demonstrated to gene transfer by natural plasmid.     

Type I interferon signaling regulates Ly6C(hi) monocytes and neutrophils during acute viral pneumonia in mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1 ^−/−) mice develop significant defects in the infiltration of Ly6C^hi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6C^hi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6C^int monocytes of Ifnar1 ^−/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1 ^−/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6C^hi monocytes. By using BM chimeric mice (WT BM into Ifnar1 ^−/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6C^hi monocytes. Of note, WT BM reconstituted Ifnar1 ^−/− chimeric mice with increased numbers of Ly6C^hi monocytes survived longer than influenza-infected Ifnar1 ^−/− mice. In contrast, WT mice that received Ifnar1 ^−/− BM cells with alternative Ly6C^int monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.     

The establishment and characterization of immortal hepatocyte cell lines from a mouse liver injury model

Hepatocytes are an important research tool used for numerous applications. However, a short life span and a limited capacity to replicate in vitro limit the usefulness of primary hepatocyte cultures. We have hypothesized that in vivo priming of hepatocyte could make them more susceptible to growth factors in the medium for continuous proliferation in vitro. Here, a novel approach used to establish hepatocyte cell lines that included hepatocyte priming in vivo prior to culture with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet was attempted. The cell line grew in a monolayer while maintaining a granular cytoplasm and a round nucleus. Electron microscopy displayed hepatocyte-like features including mitochondria, glycogen granules, and the presence of bile canaliculi. This cell line expressed many mature hepatocyte-specific genes including albumin, alpha1-antitrypsin, glucose 6-phosphatase, and tyrosine aminotransferase. Functional characteristic of hepatocytes like the ability to store glycogen, lipid, and synthesis of urea is well demonstrated by this cell line. These cells demonstrated anchorage dependent growth properties in soft agar and did not form tumors after transplantation into nude mice. This cell line can be sustained in culture for more than 100 passages (>1.5 years) without undergoing noticeable morphological changes or transformation. This novel method resulted in the establishment of an immortal, non-transformed hepatocyte cell line with functional characteristics that may aid research of cell metabolism, toxicology, and hepatocyte transplantation.     

Cloning and expression of bovine imc-415 cDNA from mammary gland

Bovine imc-415 cDNA was cloned from mammary gland using RACE PCR; it coded for 245 amino acids. The deduced amino acid sequence of mouse and human showed about 94% identity. Expression of bovine imc-415 increased about 40% in involuted mammary tissues compared with lactating tissues.     

Structure and function of the potent cyclic and linear melanocortin analogues

The MC3R and MC4R proteins comprise two melanocortin receptor subtypes that are involved in obesity, with each protein displaying a unique mechanism of action. To enable the design of a selective drug candidate, the solution structures of four peptidyl analogues of the melanocyte stimulating hormones, NDP-MSH, NDP-MSH(4-10) and two cyclic forms ([C5,C10]NDP-MSH(5-10), [C5,C10]NDP-MSH(5-11)), were characterized by two-dimensional nuclear magnetic resonance (NMR) spectroscopy and simulated annealing calculations. Using data from c-AMP assays in combination with structural analysis of melanocortin receptor/ligand models, we conclude that a lysine residue at the C-terminus of the His-Phe-Arg-Trp core sequence of melanocortin hormone is an important determinant for receptor selectivity in the both cyclic and linear MSH analogues. Our results suggest that side-chain orientation and charge-charge interactions with the ligand molecule play critical roles in receptor selectivity, whereas the overall backbone conformation or turn type contributes mainly to receptor binding.     

Identification of High-Risk Plaques by MRI and Fluorescence Imaging in a Rabbit Model of Atherothrombosis

IntroductionThe detection of atherosclerotic plaques at risk for disruption will be greatly enhanced by molecular probes that target vessel wall biomarkers. Here, we test if fluorescently-labeled Activatable Cell Penetrating Peptides (ACPPs) could differentiate stable plaques from vulnerable plaques that disrupt, forming a luminal thrombus. Additionally, we test the efficacy of a combined ACPP and MRI technique for identifying plaques at high risk of rupture.ConclusionsOur targeted fluorescence ACPP probes distinguished disrupted plaques from stable plaques with high sensitivity and specificity. The combination of anatomic, MRI-derived predictors for disruption and ACPP uptake can further improve the power for identification of high-risk plaques and suggests future development of ACPPs with molecular MRI as a readout.