Isolation and Characterization of an Escherichia coli ruv Mutant Which Forms Nonseptate Filaments After Low Doses of Ultraviolet Light Irradiation

Two ultraviolet light (UV)-sensitive mutants have been isolated from Escherichia coli K-12. These mutants, designated RuvA(-) and RuvB(-), were controlled by a gene located close to the his gene on the chromosome map. They were sensitive to UV (10- to 20-fold increase) and slightly sensitive to gamma rays (3-fold increase). Host cell reactivation, UV reactivation and genetic recombination were normal in these mutants. Irradiation of the mutants with UV resulted in the production of single-strand breaks in deoxyribonucleic acid, which was repaired upon incubation in a growth medium. After UV irradiation, these mutants resumed deoxyribonucleic acid synthesis at a normal rate, as did the parent wild-type bacteria, and formed nonseptate, multinucleate filaments. From these results we concluded that the mutants have some defect in cell division after low doses of UV irradiation, similar to the lon(-) or fil(+) mutant of E. coli. The ruv locus was divided further into ruvA and ruvB with respect to nalidixic acid sensitivity and the effect of minimal agar or pantoyl lactone on survival of the UV-irradiated cell. The ruvB(-)mutant was more sensitive to nalidixic acid than were ruvA(-) and the parent strain. There was a great increase in the surviving fraction of the UV-irradiated ruvB(-) mutant when it was plated on minimal agar or L agar containing pantoyl lactone. No such increase in survival was observed in the ruvA(-) mutant.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

Monitoring for dynamic biological processing by intramolecular bioluminescence resonance energy transfer system using secreted luciferase

Proteolytic processing plays crucial roles in physiological and pathophysiological cellular functions such as peptide generation, cell cycle, and apoptosis. We developed a novel biophysical bioluminescence resonance energy transfer (BRET) system between a secreted Vargula luciferase (Vluc) and an enhanced yellow fluorescent protein (EYFP) for visualization of cell biological processes. The bioluminescence spectrum of the fusion protein (Vluc-EYFP) is bimodal (lambdamax = 460 nm (Vluc) and 525nm (EYFP)), indicating that the excited-state energy of Vluc transfers to EYFP (in short, BRET). The BRET signal can be measured in the culture medium and pursue quantitative production of two neuropeptides, nocistatin (NST) and nociceptin/orphanin FQ (N/OFQ) in living cells. NST and N/OFQ are located in tandem on the same precursor, but NST exhibits antagonistic action against N/OFQ-induced central functions. Insertion of a portion of the NST-N/OFQ precursor (Glu-Gln-Lys-Gln-Leu-Gln-Lys-Arg-Phe-Gly-Gly-Phe-Tyr-Gly) in Vluc-EYFP makes the fusion protein cleavable at Lys-Arg in NG108-15 cells, and proprotein convertase 1 enhances this digestion. The change in BRET signals quantifies the processing of the fusion protein. Our novel intramolecular BRET system using a secreted luciferase is useful for investigating peptide processing in living cells.     

Monoclonal antibody A7 coupled to magnetic particles as a contrast enhancing agent for magnetic resonance imaging of human colorectal carcinoma

Background: Local recurrence, the most frequent pattern of recurrence of rectal carcinoma, is almost always fatal. The difficulty of diagnosing local recurrence contributes importantly to the poor prognosis. Methods: We coupled monoclonal antibody (Mab) A7, which reacts specifically with human colorectal carcinoma, to ferromagnetic lignosite (FML) particles to distinguish rectal carcinoma from other tissues by magnetic resonance (MR) imaging. We examined retention of immunoreactivity by the A7-FML complexes in vitro, and also their distribution in vivo according to radiolabeling and MR imaging when injected into nude mice bearing human colorectal carcinoma xenografts. Results: A7-FML retained binding activity nearly identical to that of Mab A7. Significantly more ¹²⁵I-labeled A7-FML accumulated in engrafted tumors than did ¹²⁵I-labeled normal mouse IgG-FML complexes (P<0.05). A7-FML disappeared rapidly from the blood. Normal tissues accumulated less ¹²⁵I-labeled A7-FML than tumors; this accumulation decreased linearly with time. In MR imaging, signal intensity was reduced in the tumor by the injection of A7-FML. Conclusions: A7-FML is potentially useful as a MR contrast enhancing agent for human colorectal carcinoma xenografts implanted subcutaneously.     

Impacts of icodextrin on integrin-mediated wound healing of peritoneal mesothelial cells

AimsExposure to glucose and its metabolites in peritoneal dialysis fluid (PDF) results in structural alterations of the peritoneal membrane. Icodextrin-containing PDF eliminates glucose and reduces deterioration of peritoneal membrane function, but direct effects of icodextrin molecules on peritoneal mesothelial cells have yet to be elucidated. We compared the impacts of icodextrin itself with those of glucose under PDF-free conditions on wound healing processes of injured mesothelial cell monolayers, focusing on integrin-mediated cell adhesion mechanisms.Main methodsRegeneration processes of the peritoneal mesothelial cell monolayer were investigated employing an in vitro wound healing assay of cultured rat peritoneal mesothelial cells treated with icodextrin powder- or glucose-dissolved culture medium without PDF, as well as icodextrin- or glucose-containing PDF. The effects of icodextrin on integrin-mediated cell adhesions were examined by immunocytochemistry and Western blotting against focal adhesion kinase (FAK).Key findingsCell migration over fibronectin was inhibited in conventional glucose-containing PDF, while icodextrin-containing PDF exerted no significant inhibitory effects. Culture medium containing 1.5% glucose without PDF also inhibited wound healing of mesothelial cells, while 7.5% icodextrin-dissolved culture medium without PDF had no inhibitory effects. Glucose suppressed cell motility by inhibiting tyrosine phosphorylation of FAK, formation of focal adhesions, and cell spreading, while icodextrin had no effects on any of these mesothelial cell functions.SignificanceOur results demonstrate icodextrin to have no adverse effects on wound healing processes of peritoneal mesothelial cells. Preservation of integrin-mediated cell adhesion might be one of the molecular mechanisms accounting for the superior biocompatibility of icodextrin-containing PDF.     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Rapid induction of large chromosomal deletions by a Cre/inverted loxP system in mouse ES Cell hybrids

Loss of heterozygosity by whole or partial loss of chromosomal regions is crucial to genetic disorders, cancers and diseases. It is difficult to analyze the mechanisms of pathogenesis caused by large-scale chromosomal abnormalities due to the extreme rarity of this mutagenesis. Using a Cre/inverted loxP system, we have generated a chromosome elimination cassette (CEC) that induces a selective loss of embryonic-stem-cell-derived chromosomes in undifferentiated embryonic stem cell-somatic cell hybrids. Here, due to the increased expression of Cre, rapid formation of Cre recombination products and immediate loss of CEC-tagged chromosomes were detected by fluorescence in situ hybridization. Cre also initiated intrachromosomal recombination between identical short sequences outside loxP, leading to large chromosomal deletions of CEC-tagged regions. The Cre-mediated antiparallel synapses likely act as a scaffold to bring the identical short sequences into close proximity for recombination. This CEC technology might allow better understanding of the modulator sequences responsible for the tangled structure formation and its solution mechanism, inducing mitotic recombination leading to chromosomal deletions.     

Attenuated response to liver injury in moesin-deficient mice: impaired stellate cell migration and decreased fibrosis

Hepatic stellate cells (HSCs) respond to injury with a coordinated set of events (termed activation), which includes migration and upregulation of matrix protein production. Cell migration requires an intact actin cytoskeleton that is linked to the plasma membrane by ezrin-radixin-moesin (ERM) proteins. We have previously found that the linker protein in HSCs is exclusively moesin. Here, we describe HSC migration and fibrogenesis in moesin-deficient mice. We developed an acute liver injury model that involved focal thermal denaturation and common bile duct ligation. HSC migration and collagen deposition were assessed by immunohistology and quantitative real-time PCR. Activated HSCs were isolated from wild-type or moesin-deficient mice for direct examination of migration. Activated HSCs from wild-type mice were positive for moesin. Migration of moesin-deficient HSCs was significantly reduced. In a culture assay, 22.1% of normal HSCs migrated across a filter in 36h. In contrast, only 1.3% of activated moesin-deficient HSCs migrated. Collagen deposition around the injury area similarly was reduced in moesin-deficient liver. The linker protein moesin is essential for HSC activation and migration in response to injury. Fibrogenesis is coupled to migration and reduced in moesin-deficient mice. Agents that target moesin may be beneficial for chronic progressive fibrosis.     

Hyperproduction of phosphate-binding protein, phoS, and pre-phoS proteins in Escherichia coli carrying a cloned phoS gene

A large amount of phosphate-binding protein, the phoS gene product, accumulated in the periplasmic space of the cells when an Escherichia coli strain carrying a multicopy plasmid containing a chromosomal fragment of the phoS-phoT region (pSN507) was grown in a low-phosphate medium. When the same strain carrying a plasmid containing only the phoS gene (pSN518 or pSN5182) was grown in low-phosphate medium, phosphate-binding protein accumulated in the periplasm, and in addition a larger protein accumulated in the non-periplasmic fraction. The apparent Mr of this protein and the phosphate-binding protein were 39000 and 35000 respectively, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. This larger protein showed immunological cross-reaction with the phosphate-binding protein. The 39000-Mr protein was also detected in cells carrying pSN507 when the proteins were pulse-labeled with radioactive amino acids. From these findings, together with the fact that this protein is recovered from the membrane fraction, we conclude that this protein is an unsecreted precursor protein of the phosphate-binding protein. Kinetics and regulation of accumulation of these proteins were studied. This system will be useful for preparation and purification of the precursor protein for biochemical studies in relation to the mechanism of protein secretion.