c-Abl phosphorylates Dok1 to promote filopodia during cell spreading

Filopodia are dynamic F-actin structures that cells use to explore their environment. c-Abl tyrosine kinase promotes filopodia during cell spreading through an unknown mechanism that does not require Cdc42 activity. Using an unbiased approach, we identified Dok1 as a specific c-Abl substrate in spreading fibroblasts. When activated by cell adhesion, c-Abl phosphorylates Y361 of Dok1, promoting its association with the Src homology 2 domain (SH2)/SH3 adaptor protein Nck. Each signaling component was critical for filopodia formation during cell spreading, as evidenced by the finding that mouse fibroblasts lacking c-Abl, Dok1, or Nck had fewer filopodia than cells reexpressing the product of the disrupted gene. Dok1 and c-Abl stimulated filopodia in a mutually interdependent manner, indicating that they function in the same signaling pathway. Dok1 and c-Abl were both detected in filopodia of spreading cells, and therefore may act locally to modulate actin. Our data suggest a novel pathway by which c-Abl transduces signals to the actin cytoskeleton through phosphorylating Dok1 Y361 and recruiting Nck.     

Membrane-associated IL-1 contributes to chronic synovitis and cartilage destruction in human IL-1 alpha transgenic mice

IL-1 molecules are encoded by two distinct genes, IL-1alpha and IL-1beta. Both isoforms possess essentially identical activities and potencies, whereas IL-1alpha, in contrast to IL-1beta, is known to act as a membrane-associated IL-1 (MA-IL-1) and plays an important role in a variety of inflammatory situations. The transgenic (Tg) mouse line (Tg1706), which was generated in our laboratory, overexpresses human IL-1alpha (hIL-1alpha) and exhibits a severe arthritic phenotype characterized by autonomous synovial proliferation with subsequent cartilage destruction. Because the transgene encoded Lys(64) to Ala(271) of the hIL-1alpha amino acid sequence, Tg mice may overproduce MA-IL-1 as well as soluble IL-1alpha. The present study investigated whether MA-IL-1 contributes to synovial proliferation and cartilage destruction in the development of arthritis. Flow cytometric analysis revealed that both macrophage-like and fibroblast-like synoviocytes constitutively produce MA-IL-1. D10 cell proliferation assay revealed MA-IL-1 bioactivity of paraformaldehyde-fixed synoviocytes and the further induction of endogenous mouse MA-IL-1 via autocrine mechanisms. MA-IL-1 expressed on synoviocytes triggered synoviocyte self-proliferation through cell-to-cell (i.e., juxtacrine) interactions and also promoted proteoglycan release from the cartilage matrix in chondrocyte monolayer culture. Interestingly, the severity of arthritis was significantly correlated with MA-IL-1 activity rather than with soluble IL-1alpha activity or concentration of serum hIL-1alpha. Moreover, when the Tg1706 line was compared with the Tg101 line, which selectively overexpresses the 17-kDa mature hIL-1alpha, the severity of arthritis was significantly higher in the Tg1706 line than in the Tg101 line. These results suggest that MA-IL-1 contributes to synoviocyte self-proliferation and subsequent cartilage destruction in inflammatory joint disease such as rheumatoid arthritis.     

migS, a cis-acting site that affects bipolar positioning of oriC on the Escherichia coli chromosome

During replication of the Escherichia coli chromosome, the replicated Ori domains migrate towards opposite cell poles, suggesting that a cis-acting site for bipolar migration is located in this region. To identify this cis-acting site, a series of mutants was constructed by splitting subchromosomes from the original chromosome. One mutant, containing a 720 kb subchromosome, was found to be defective in the bipolar positioning of oriC. The creation of deletion mutants allowed the identification of migS, a 25 bp sequence, as the cis-acting site for the bipolar positioning of oriC. When migS was located at the replication terminus, the chromosomal segment showed bipolar positioning. migS was able to rescue bipolar migration of plasmid DNA containing a mutation in the SopABC partitioning system. Interestingly, multiple copies of the migS sequence on a plasmid in trans inhibited the bipolar positioning of oriC. Taken together, these findings indicate that migS plays a crucial role in the bipolar positioning of oriC. In addition, real-time analysis of the dynamic morphological changes of nucleoids in wild-type and migS mutants suggests that bipolar positioning of the replicated oriC contributes to nucleoid organization.     

Development of agmatine sensor using the combination of putrescine oxidase and agmatinase for squid freshness

Agmatine (Agm) is an indicator of squid freshness. The Agm sensor was developed using flow injection analysis (FIA) that consisted of the putrescine oxidase (PuOx) reactor, the agmatinase (AUH)-PuOx reactor and two oxygen electrodes. In the proposed sensor, the first step is that coexisting cadaverine (Cad) and putrescine (Put) are removed by passing through the PuOx reactor and the initial decomposition is determined by the amount of oxygen consumed, simultaneously. The second step is that the amount of Agm is determined by the amount of oxygen consumed in the AUH-PuOx reactor. The optimum conditions for the use of the Agm sensor were as follows: 50 mM HEPES containing MnSO4 at a final concentration of 5 mM, pH 8.0, flow rate of 0.6 mL min(-1) and injection volume of 50 microL. A single assay could be completed in approximately 3 min. A linear relationship was obtained between the output and the Agm concentration in the range of 0.01-1 mM Agm with a correlation coefficient of 0.999. The detection limit was 0.005 mM. The relative standard deviations (RSDs) were 3.14 and 1.19% (n = 20) for 0.1 and 0.3 mM Agm, respectively. The extracts of squid were injected into the proposed sensor and the results were compared with those obtained using the conventional high-performance liquid chromatography (HPLC) method. A correlation was observed between the results obtained by the proposed sensor and those obtained by the conventional method. The determination of squid freshness is one of the good uses of the proposed Agm sensor.     

Amperometric determination of laminarin using immobilized beta-1,3-glucanase

A novel sensor system equipped with a reactor packed with beads containing immobilized beta-1,3-glucanase and glucose oxidase was developed for the amperometric determination of laminarin concentration. The proposed sensor system consisted of a reactor, an oxygen electrode, a flow cell, a pump, a buffer tank, and a recorder. The measurement was performed with a flow injection system. The optimum conditions for the sensor system were as follows: transfer solution, pH 7.0; 0.1 M phosphate buffer solution; flow rate, 0.15 ml/min; and sample volume, 50 microl. The response was correlated to the laminarin concentration. The calibration curve was obtained between 50 and 0.5 mg/ml laminarin (R2 = 0.994). The detection limit was 50 microg/ml laminarin (the ratio of signal/noise = 3). The relative standard deviations were 2.0% (n = 15) and 2.5% (n = 15) for 0.4 and 1.0 mg/ml laminarin solutions, respectively. One assay was completed within 5 min. Results suggest that the sensor can be used not only for the analysis of seaweed and health-enhancing foods but also for monitoring the initial pollution of the marine environment.     

Null mutation of the dam or seqA gene suppresses temperature-sensitive lethality but not hypersensitivity to novobiocin of muk null mutants

Escherichia coli mukF, mukE, and mukB null mutants have common phenotypes such as temperature-dependent colony formation, anucleate cell production, chromosome cutting by septum closure, and abnormal localization of SeqA-DNA clusters. We show here that the associated muk null mutations cause hypersensitivity to novobiocin. Null mutation of either dam or seqA suppressed partially the temperature-sensitive lethality but failed to suppress the anucleate cell production and the hypersensitivity to novobiocin caused by muk null mutations.     

Retinoic acid induction of sialyltransferase activity in neuroblastoma cells of differing sialylation potentials

In order to determine how glycosylation changes associated with cellular differentiation may be influenced by the basal cellular sialylation potential, the effect of retinoic acid (RA)-induced differentiation was investigated in neuroblastoma cells expressing differing levels (and activities) of the 2,6(N) sialyltransferase (ST6N) enzyme. The increase in ST activity was proportional to the basal cellular sialylation potentials with the high activity clones showing the greatest increase. This was paralleled by an up-regulation of the level of overall sialoglycoprotein glycosylation level. An increase in the levels of the polysialic acid (PSA) epitope was associated with a parallel increase in the levels of the neural cell adhesion molecule (NCAM) protein backbone although there was no overall change in the PSA:NCAM ratio following RA treatment.     

Effects of acupuncture on vasopressin-induced emesis in conscious dogs

Although acupuncture has a significant clinical benefit, the mechanism of acupuncture remains unclear. Vasopressin, a posterior pituitary hormone, is involved in nausea and vomiting in humans and dogs. To investigate the antiemetic effects of acupuncture on vasopressin-induced emesis, gastroduodenal motor activity and the frequency of retching and vomiting were simultaneously recorded in conscious dogs. In seven dogs, four force transducers were implanted on the serosal surfaces of the gastric body, antrum, pylorus, and duodenum. Gastroduodenal motility was continuously monitored throughout the experiment. Vasopressin was intravenously infused at a dose of 0.1 U x kg(-1) x min(-1) for 20 min. Electroacupuncture (EA, 1-30 Hz) at pericardium-6 (PC6), bladder-21 (BL21), or stomach-36 (ST36) was performed before, during, and after the vasopressin infusion. To investigate whether the opioid pathway is involved in EA-induced antiemetic effects, naloxone (a central and peripheral opioid receptor antagonist) or naloxone methiodide (a peripheral opioid receptor antagonist) was administered before, during, and after EA and vasopressin infusion. Intravenous infusion of vasopressin induced retching and vomiting in all dogs tested. Retrograde peristaltic contractions occurred before the onset of retching and vomiting. EA (10 Hz) at PC6 significantly reduced the number of episodes of retching and vomiting. EA at PC6 also suppressed retrograde peristaltic contractions. In contrast, EA at BL21 or ST36 had no antiemetic effects. The antiemetic effect of EA was abolished by pretreatment with naloxone but not naloxone methiodide. It is suggested that the antiemetic effect of acupuncture is mediated via the central opioid pathway.     

Degradation of Ferric Chelate of Ethylenediaminetetraacetic Acid by Bacterium Isolated from Deep-Sea Stalked Barnacle

Twenty strains of marine bacteria that degrade ferric chelate of ethylenediaminetetraacetic acid (Fe-EDTA) were isolated from among 117 strains collected from a marine environment. Among them strain 02-N-2, which was isolated from stalked barnacle collected from the deep sea in the Indian Ocean, had the highest Fe-EDTA degradation ability and was selected for further study. The strain showed high Fe-EDTA degradation ability at different seawater concentrations. In addition, the intact cells of this strain had the ability to degrade such metal-EDTAs as Ca, Cu, and Mg. The strain was an aerobic, gram-variable, rod-shaped organism. The results of various taxonomic studies revealed that the strain had significant similarity to Bacillus jeotgali JCM 10885^T, which was isolated from a Korean traditional fermented seafood, Jeotgal.