Construction of Streptomyces coelicolor A3(2) mutants that exclusively produce NA4/NA6 intermediates of agarose metabolism through mutation induction

Abstract NA4/NA6, an intermediate degradation product of β-agarase, is a high value-added product with anticancer, anti-obesity, and anti-diabetic effects. Therefore, a method that enables the efficient production of NA4/NA6 would be useful from economic and medical perspectives. In this study, we aimed to generate a Streptomyces coelicolor A3(2) mutant M22-2C43 that produces NA4/NA6 as a final product; this method serves as a more efficient alternative to the enzymatic conversion of β-agarase for the generation of these products. The M22-2C43 strain was generated through two rounds of mutagenesis and screening for increased β-agarase activity and effective production of NA4/NA6. We assembled the complete genomes of two mutants, M22 and M22-2C43, which were identified following a two-round screening. Large and small genetic changes were found in these two mutants, including the loss of two plasmids present in wild-type S. coelicolor A3(2) and chromosome circularization of mutant M22-2C43. These findings suggest that mutant M22-2C43 can produce NA4/NA6 as a degradation product due to functional inactivation of the dagB gene through a point mutation (G474A), ultimately preventing further degradation of NA4/NA6 to NA2. To our knowledge, this is the first report of a microbial strain that can effectively produce NA4/NA6 as the main degradation product of β-agarase, opening the door for the use of this species for the large-scale production of this valuable product

A case of pigmentary orthochromatic leukodystrophy with findings of proton MR spectroscopy and serial brain MRIs

Despite a few case reports over the last 60 years, little progress has been made in defining the phenotype, genotype and pathophysiological mechanisms involved in pigmentary orthochromatic leukodystrophy (POLO). Furthermore, there is currently no data available regarding MRI in patients in the relatively early stages of POLD. Here, we present a 37 year old male patient with brain biopsy-proven POLD who had brain MRIs three times during the first year of his clinical course and proton MR spectroscopy (MRS) throughout his diagnostic evaluation. This patient with POLD was clinically characterized by seizures, rapidly progressive frontally predominant dementia and gait disturbance. The brain MRIs taken serially over the first year revealed progressive development of frontal-predominant white matter changes in the periventricular areas during the earlier periods, which later spread into the deep white matter. His MRS was helpful in the diagnostic approach because the results enabled demyelinating changes to be distinguished from other disease processes such as ischemia, gliosis or tumors. The MRS findings also reflected the disease dynamics because metabolic derangement was observed, even in the white matter that appeared normal. The findings presented here provide insight into the dynamics of POLD. (C) 2010 Elsevier B.V. All rights reserved.Wider C, 2009, NEUROLOGY, V72, P1953, DOI 10.1212/WNL.0b013e3181a826c0Freeman SH, 2009, BRAIN PATHOL, V19, P39, DOI 10.1111/j.1750-3639.2008.00163.xVan Gerpen JA, 2008, NEUROLOGY, V71, P925Keegan BM, 2008, NEUROLOGY, V70, P1128Shannon P, 1997, CAN J NEUROL SCI, V24, P146Knopman D, 1996, NEUROLOGY, V46, P429CONSTANTINIDIS J, 1991, ACTA NEUROPATHOL, V82, P483OKEDA R, 1989, ACTA NEUROPATHOL, V78, P533BELEC L, 1988, REV NEUROL, V144, P347YAMADERA H, 1985, SEISHIN SHINKEIGAKU, V87, P93OEPEN H, 1964, ARCH PSYCHIAT NERVEN, V206, P115PEIFFER J, 1959, ARCH PSYCHIAT NERVEN, V199, P417van Bogaert L, 1936, REV NEUROL-FRANCE, V65, P21

Complementary Regulation of BfmRS Two-Component and AbaIR Quorum Sensing Systems to Express Virulence-Associated Genes in Acinetobacter baumannii

Acinetobacter baumannii expresses various virulence factors to adapt to hostile environments and infect susceptible hosts. This study investigated the regulatory network of the BfmRS two-component and AbaIR quorum sensing (QS) systems in the expression of virulence-associated genes in A. baumannii ATCC 17978. The ΔbfmS mutant exhibited a significant decrease in surface motility, which presumably resulted from the low expression of pilT and A1S_0112-A1S_0119 gene cluster. The ΔbfmR mutant displayed a significant reduction in biofilm and pellicle formation due to the low expression of csu operon. The deletion of abaR did not affect the expression of bfmR or bfmS. However, the expression of abaR and abaI was upregulated in the ΔbfmR mutant. The ΔbfmR mutant also produced more autoinducers than did the wild-type strain, suggesting that BfmR negatively regulates the AbaIR QS system. The ΔbfmS mutant exhibited no autoinducer production in the bioassay system. The expression of the A1S_0112-A1S_0119 gene cluster was downregulated in the ΔabaR mutant, whereas the expression of csu operon was upregulated in this mutant with a high cell density. In conclusion, for the first time, we demonstrated that the BfmRS-AbaIR QS system axis regulated the expression of virulence-associated genes in A. baumannii. This study provides new insights into the complex network system involved in the regulation of virulence-associated genes underlying the pathogenicity of A. baumannii

Nitrergic Pathway Is the Main Contributing Mechanism in the Human Gastric Fundus Relaxation: An In Vitro Study

<div><p>Background</p><p>Human gastric fundus relaxation is mediated by intrinsic inhibitory pathway. We investigated the roles of nitrergic and purinergic pathways, two known inhibitory factors in gastric motility, on spontaneous and nerve-evoked contractions in human gastric fundus muscles.</p><p>Methods</p><p>Gastric fundus muscle strips (12 circular and 13 longitudinal) were obtained from patients without previous gastrointestinal motility disorder who underwent gastrectomy for stomach cancer. Using these specimens, we examined basal tone, peak, amplitude, and frequency of spontaneous contractions, and peak and nadir values under electrical field stimulation (EFS, 150 V, 0.3 ms, 10 Hz, 20 s). To examine responses to purinergic and nitrergic inhibition without cholinergic innervation, atropine (muscarinic antagonist, 1 μM), MRS2500 (a purinergic P2Y1 receptor antagonist, 1 μM), and N-nitro-L-arginine (L-NNA, a nitric oxide synthase inhibitor, 100 μM) were added sequentially for spontaneous and electrically-stimulated contractions. Tetrodotoxin was used to confirm any neuronal involvement.</p><p>Results</p><p>In spontaneous contraction, L-NNA increased basal tone and peak in both muscle layers, while amplitude and frequency were unaffected. EFS (up to 10 Hz) uniformly induced initial contraction and subsequent relaxation in a frequency-dependent manner. Atropine abolished initial on-contraction and induced only relaxation during EFS. While MRS2500 showed no additional influence, L-NNA reversed relaxation (<i>p</i> = 0.012 in circular muscle, and <i>p</i> = 0.006 in longitudinal muscle). Tetrodotoxin abolished any EFS-induced motor response.</p><p>Conclusions</p><p>The relaxation of human gastric fundus muscle is reduced by nitrergic inhibition. Hence, nitrergic pathway appears to be the main mechanism for the human gastric fundus relaxation.</p></div

Differential Expression of -Associated Genes in Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of multiple fluid-filled cysts that expand over time and destroy renal architecture. The proteins encoded by the PKD1 and PKD2 genes, mutations in which account for nearly all cases of ADPKD, may help guard against cystogenesis. Previously developed mouse models of PKD1 and PKD2 demonstrated an embryonic lethal phenotype and massive cyst formation in the kidney, indicating that PKD1 and PKD2 probably play important roles during normal renal tubular development. However, their precise role in development and the cellular mechanisms of cyst formation induced by PKD1 and PKD2 mutations are not fully understood. To address this question, we presently created Pkd2 knockout and PKD2 transgenic mouse embryo fibroblasts. We used a mouse oligonucleotide microarray to identify messenger RNAs whose expression was altered by the overexpression of the PKD2 or knockout of the Pkd2. The majority of identified mutations was involved in critical biological processes, such as metabolism, transcription, cell adhesion, cell cycle, and signal transduction. Herein, we confirmed differential expressions of several genes including aquaporin-1, according to different PKD2 expression levels in ADPKD mouse models, through microarray analysis. These data may be helpful in PKD2-related mechanisms of ADPKD pathogenesis

Mxi1 influences cyst formation in three-dimensional cell culture

Cyst formation is a major characteristic of ADPKD and iscaused by the abnormal proliferation of epithelial cells. Renalcyst formation disrupts renal function and induces diversecomplications. The mechanism of cyst formation is unclear.mIMCD-3 cells were established to develop simple epithelialcell cysts in 3-D culture. We confirmed previously that Mxi1plays a role in cyst formation in Mxi1-deficient mice. Cysts inMxi1 transfectanted cells were showed by collagen or mebiolgels in 3-D cell culture system. Causative genes of ADPKDwere measured by q RT-PCR. Herein, Mxi1 transfectants rarelyformed a simple epithelial cyst and induced cell death.Overexpression of Mxi1 resulted in a decrease in the PKD1,PKD2 and c-myc mRNA relating to the pathway of cystformation. These data indicate that Mxi1 influences cystformation of mIMCD-3 cells in 3-D culture and that Mxi1 maycontrol the mechanism of renal cyst formation. [BMB reports2012; 45(3): 189-193

Serial administration of atropine, MRS2500, L-NNA, and TTX under EFS on circular and longitudinal human gastric fundus muscles (10 Hz, 150 V, 0.3 ms and 20 s).

<p>(A) A representative tracing of EFS-evoked motor responses of circular muscle. NOS inhibitor reverses the EFS-induced relaxation. (B)A typical tracing of EFS-evoked motor responses of longitudinal muscle. EFS-induced relaxation is also reversed by L-NNA.</p

The four parameters of spontaneous contraction in the circular smooth muscle of human gastric fundus.

<p>Basal tone and peak amplitude increase to a significant degree by inhibition of NOS. Mean amplitude and frequency, on the other hand, are not affected by inhibition of purinergic and nitrergic pathways.</p