Conversion of sucrose to isomaltulose by Klebsiella planticola CCRC 19112

An isomaltulose-producing bacterium was isolated and taxonomically characterized. Its morphological and biochemical properties conform best to those described for Klebsiella planticola. When cultured under optimal conditions, the organism simultaneously converted sucrose into both isomaltulose (α-D-glucopyranosyl-1,6-fructose) and trehalulose (α-D-glucopyranosyl-1,1-fructose) with substrate conversion rates of 80% and 15%, respectively. Sucrose and Bacto-tryptone were the most effective carbon and supplemental nitrogen sources, respectively, for producing cells of high isomaltulose-forming ability. None of several inorganic salts tested had any significant effect. The major product formed in the reaction mixture was verified to be isomaltulose by co-chromatography and IR spectroscopy. Received 21 April 1998/ Accepted in revised form 7 July 1998     

Temperature dependence of human ether-a-go-go-related gene K+ currents

The function of voltage-gated human ether-à-go-gorelated gene (hERG) K⁺ channels is critical for both normal cardiac repolarization and suppression of arrhythmias initiated by premature excitation. These important functions are facilitated by their unusual kinetics that combine relatively slow activation and deactivation with rapid and voltage-dependent inactivation and recovery from inactivation. The thermodynamics of these unusual features were examined by exploring the effect of temperature on the activation and inactivation processes of hERG channels expressed in Chinese hamster ovary cells. Increased temperature shifted the voltage dependence of activation in the hyperpolarizing direction but that of inactivation in the depolarizing direction. This increases the relative occupancy of the open state and contributes to the marked temperature sensitivity of hERG current magnitude observed during action potential voltage clamps. The rates of activation and deactivation also increase with higher temperatures, but less markedly than do the rates of inactivation and recovery from inactivation. Our results also emphasize that one cannot extrapolate results obtained at room temperature to 37°C by using a single temperature scale factor. potassium channel; kinetics; voltage-dependent gating     

A genome-wide study of preferential amplification/hybridization in microarray-based pooled DNA experiments

Microarray-based pooled DNA methods overcome the cost bottleneck of simultaneously genotyping more than 100000 markers for numerous study individuals. The success of such methods relies on the proper adjustment of preferential amplification/hybridization to ensure accurate and reliable allele frequency estimation. We performed a hybridization-based genome-wide single nucleotide polymorphisms (SNPs) genotyping analysis to dissect preferential amplification/hybridization. The majority of SNPs had less than 2-fold signal amplification or suppression, and the lognormal distributions adequately modeled preferential amplification/hybridization across the human genome. Comparative analyses suggested that the distributions of preferential amplification/hybridization differed among genotypes and the GC content. Patterns among different ethnic populations were similar; nevertheless, there were striking differences for a small proportion of SNPs, and a slight ethnic heterogeneity was observed. To fulfill appropriate and gratuitous adjustments, databases of preferential amplification/hybridization for African Americans, Caucasians and Asians were constructed based on the Affymetrix GeneChip Human Mapping 100 K Set. The robustness of allele frequency estimation using this database was validated by a pooled DNA experiment. This study provides a genome-wide investigation of preferential amplification/hybridization and suggests guidance for the reliable use of the database. Our results constitute an objective foundation for theoretical development of preferential amplification/hybridization and provide important information for future pooled DNA analyses.     

Nonlinearity between action potential alternans and restitution, which both predict ventricular arrhythmic properties in Scn5a+/- and wild-type murine hearts

Electrocardiographic QT- and T-wave alternans, presaging ventricular arrhythmia, reflects compromised adaptation of action potential (AP) duration (APD) to altered heart rate, classically attributed to incomplete Na(v)1.5 channel recovery prior to subsequent stimulation. The restitution hypothesis suggests a function whose slope directly relates to APD alternans magnitude, predicting a critical instability condition, potentially generating arrhythmia. The present experiments directly test for such correlations among arrhythmia, APD alternans and restitution. Mice haploinsufficient in the Scn5a, cardiac Na(+) channel gene (Scn5a(+/-)), previously used to replicate Brugada syndrome, were used, owing to their established arrhythmic properties increased by flecainide and decreased by quinidine, particularly in right ventricular (RV) epicardium. Monophasic APs, obtained during pacing with progressively decrementing cycle lengths, were systematically compared at RV and left ventricular epicardial and endocardial recording sites in Langendorff-perfused Scn5a(+/-) and wild-type hearts before and following flecainide (10 μM) or quinidine (5 μM) application. The extent of alternans was assessed using a novel algorithm. Scn5a(+/-) hearts showed greater frequencies of arrhythmic endpoints with increased incidences of ventricular tachycardia, diminished by quinidine, and earlier onsets of ventricular fibrillation, particularly following flecainide challenge. These features correlated directly with increased refractory periods, specifically in the RV, and abnormal restitution and alternans properties in the RV epicardium. The latter variables were related by a unique, continuous higher-order function, rather than a linear relationship with an unstable threshold. These findings demonstrate a specific relationship between alternans and restitution, as well as confirming their capacity to predict arrhythmia, but implicate mechanisms additional to the voltage feedback suggested in the restitution hypothesis.     

The potential of mesenchymal stem cells in the management of radiation enteropathy

Although radiotherapy is effective in managing abdominal and pelvic malignant tumors, radiation enteropathy is still unavoidable. This disease severely affects the quality of life of cancer patients due to some refractory lesions, such as intestinal ischemia, mucositis, ulcer, necrosis or even perforation. Current drugs or prevailing therapies are committed to alleviating the symptoms induced by above lesions. But the efficacies achieved by these interventions are still not satisfactory, because the milieus for tissue regeneration are not distinctly improved. In recent years, regenerative therapy for radiation enteropathy by using mesenchymal stem cells is of public interests. Relevant results of preclinical and clinical studies suggest that this regenerative therapy will become an attractive tool in managing radiation enteropathy, because mesenchymal stem cells exhibit their pro-regenerative potentials for healing the injuries in both epithelium and endothelium, minimizing inflammation and protecting irradiated intestine against fibrogenesis through activating intrinsic repair actions. In spite of these encouraging results, whether mesenchymal stem cells promote tumor growth is still an issue of debate. On this basis, we will discuss the advances in anticancer therapy by using mesenchymal stem cells in this review after analyzing the pathogenesis of radiation enteropathy, introducing the advances in managing radiation enteropathy using regenerative therapy and exploring the putative actions by which mesenchymal stem cells repair intestinal injuries. At last, insights gained from the potential risks of mesenchymal stem cell-based therapy for radiation enteropathy patients may provide clinicians with an improved awareness in carrying out their studies.     

Spatial and temporal heterogeneities are localized to the right ventricular outflow tract in a heterozygotic Scn5a mouse model

Ventricular tachycardia (VT) in Brugada Syndrome patients often originates in the right ventricular outflow tract (RVOT). We explore the physiological basis for this observation using murine whole heart preparations. Ventricular bipolar electrograms and monophasic action potentials were recorded from seven epicardial positions in Langendorff-perfused wild-type and Scn5a+/- hearts. VT first appeared in the RVOT, implicating it as an arrhythmogenic focus in Scn5a+/- hearts. RVOTs showed the greatest heterogeneity in refractory periods, response latencies, and action potential durations, and the most fractionated electrograms. However, incidences of concordant alternans in dynamic pacing protocol recordings were unaffected by the Scn5a+/- mutation or pharmacological intervention. Conversely, particularly at the RVOT, Scn5a+/- hearts showed earlier and more frequent transitions into discordant alternans. This was accentuated by flecainide, but reduced by quinidine, in parallel with their respective pro- and anti-arrhythmic effects. Discordant alternans preceded all episodes of VT. The RVOT of Scn5a+/- hearts also showed steeper restitution curves, with the diastolic interval at which the gradient equaled one strongly correlating with the diastolic interval at which discordant alternans commenced. We attribute the arrhythmic tendency within the RVOT to the greater spatial heterogeneities in baseline electrophysiological properties. These, in turn, give rise to a tendency to drive concordant alternans phenomena into an arrhythmogenic discordant alternans. Our findings may contribute to future work investigating possible pharmacological treatments for a disease in which the current mainstay of treatment is implantable cardioverter defibrillator implantation.     

Suppression of tumor angiogenesis by targeting the protein neddylation pathway

Inhibition of protein neddylation, particularly cullin neddylation, has emerged as a promising anticancer strategy, as evidenced by the antitumor activity in preclinical studies of the Nedd8-activating enzyme (NAE) inhibitor MLN4924. This small molecule can block the protein neddylation pathway and is now in clinical trials. We and others have previously shown that the antitumor activity of MLN4924 is mediated by its ability to induce apoptosis, autophagy and senescence in a cell context-dependent manner. However, whether MLN4924 has any effect on tumor angiogenesis remains unexplored. Here we report that MLN4924 inhibits angiogenesis in various in vitro and in vivo models, leading to the suppression of tumor growth and metastasis in highly malignant pancreatic cancer, indicating that blockage of angiogenesis is yet another mechanism contributing to its antitumor activity. At the molecular level, MLN4924 inhibits Cullin–RING E3 ligases (CRLs) by cullin deneddylation, causing accumulation of RhoA at an early stage to impair angiogenic activity of vascular endothelial cells and subsequently DNA damage response, cell cycle arrest and apoptosis due to accumulation of other tumor-suppressive substrates of CRLs. Furthermore, we showed that inactivation of CRLs, via small interfering RNA (siRNA) silencing of its essential subunit ROC1/RBX1, recapitulates the antiangiogenic effect of MLN4924. Taken together, our study demonstrates a previously unrecognized role of neddylation in the regulation of tumor angiogenesis using both pharmaceutical and genetic approaches, and provides proof of concept evidence for future development of neddylation inhibitors (such as MLN4924) as a novel class of antiangiogenic agents.     

Analysis of Antarctic proteobacteria by PCR fingerprinting and screening for antimicrobial secondary metabolites

Fifty-seven proteobacterium species were successfully isolated from soils of Barrientos Island of the Antarctic using 11 different isolation media. Analysis of 16S rDNA sequencing of these isolates showed that they belonged to eight different genera, namely Bradyrhizobium, Sphingomonas, Methylobacterium, Caulobacter, Paracoccus, Ralstonia, Rhizobium, and Staphylococcus. All isolates were studied for capability of producing antimicrobial and antifungal secondary metabolites using high-throughput screening models. Approximately 23 (13/57) and 2% (1/57) of isolates inhibited growth of Candida albicans ATCC 10231(T) and Staphylococcus aureus ATCC 51650(T), respectively. These results indicated that proteobacterium species isolates from Antarctic could serve as potential source of useful bioactive metabolites. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting produced nine clusters and 13 single isolates, with a high D value of 0.9248. RAPD fingerprinting produced six clusters and 13 single isolates, with a relatively low D value of 0.7776. ERIC-PCR analysis proved to have better discrimination capability than RAPD analysis and generated better clustering for all proteobacterium species isolates. We conclude that ERIC-PCR is a robust, reliable and rapid molecular typing method for discriminating different genera of proteobacteria.