Bifidobacteria Abundance-Featured Gut Microbiota Compositional Change in Patients with Behcet’s Disease

Gut microbiota compositional alteration may have an association with immune dysfunction in patients with Behcet’s disease (BD). We conducted a fecal metagenomic analysis of BD patients. We analyzed fecal microbiota obtained from 12 patients with BD and 12 normal individuals by sequencing of 16S ribosomal RNA gene. We compared the relative abundance of bacterial taxa. Direct comparison of the relative abundance of bacterial taxa demonstrated that the genera Bifidobacterium and Eggerthella increased significantly and the genera Megamonas and Prevotella decreased significantly in BD patients compared with normal individuals. A linear discriminant analysis of bacterial taxa showed that the phylum Actinobacteria, including Bifidobacterium, and the family Lactobacillaceae exhibited larger positive effect sizes than other bacteria in patients with BD. The phylum Firmicutes and the class Clostridia had large effect sizes in normal individuals. There was no significant difference in annotated species numbers (as numbers of operational taxonomic unit; OTU) and bacterial diversity of each sample (alpha diversity) between BD patients and normal individuals. We next assigned each sample to a position using three axes by principal coordinates analysis of the OTU table. The two groups had a significant distance as beta diversity in the 3-axis space. Fecal sIgA concentrations increased significantly in BD patients but did not correlate with any bacterial taxonomic abundance. These data suggest that the compositional changes of gut microbes may be one type of dysbiosis (unfavorable microbiota alteration) in patients with BD. The dysbiosis may have an association with the pathophysiology of BD.     

Ultrahigh resolution structure of a class A beta-lactamase: on the mechanism and specificity of the extended-spectrum SHV-2 enzyme

Bacterial beta-lactamases hydrolyze beta-lactam antibiotics such as penicillins and cephalosporins. The TEM-type class A beta-lactamase SHV-2 is a natural variant that exhibits activity against third-generation cephalosporins normally resistant to hydrolysis by class A enzymes. SHV-2 contains a single Gly238Ser change relative to the wild-type enzyme SHV-1. Crystallographic refinement of a model including hydrogen atoms gave R and R(free) of 12.4% and 15.0% for data to 0.91 A resolution. The hydrogen atom on the O(gamma) atom of the reactive Ser70 is clearly seen for the first time, bridging to the water molecule activated by Glu166. Though hydrogen atoms on the nearby Lys73 are not seen, this observation of the Ser70 hydrogen atom and the hydrogen bonding pattern around Lys73 indicate that Lys73 is protonated. These findings support a role for the Glu166-water couple, rather than Lys73, as the general base in the deprotonation of Ser70 in the acylation process of class A beta-lactamases. Overlay of SHV-2 with SHV-1 shows a significant 1-3 A displacement in the 238-242 beta-strand-turn segment, making the beta-lactam binding site more open to newer cephalosporins with large C7 substituents and thereby expanding the substrate spectrum of the variant enzyme. The OH group of the buried Ser238 side-chain hydrogen bonds to the main-chain CO of Asn170 on the Omega loop, that is unaltered in position relative to SHV-1. This structural role for Ser238 in protein-protein binding makes less likely its hydrogen bonding to oximino cephalosporins such as cefotaxime or ceftazidime.     

Efficient release of overproduced gene products from Escherichia coli BL21(DE3) by lytic infection with newly isolated bacteriophages

Overproduced proteins from Escherichia coli BL21(DE3) were efficiently released with virulent bacteriophages. Leviviridae-like bacteriophages were isolated from soil and used to lyse BL21(DE3) cells transformed with beta-glucosidase, chitinase, or chitosanase genes. This method caused lysis of bacterial cells similar to that by conventional sonication and enabled us to effectively recover and purify the enzymes.     

Two new taxa of Achnanthidium and Encyonema (Bacillariophyceae) from the Yom River,Thailand, with special reference to the areolae occlusions implying ontogenetic relationship

We discovered two new taxa of Achnanthidium and Encyonema occurring with high abundance in the upstream of the Yom River, which is one of four major rivers in Northern Thailand. Achnanthidium pseudoconspicuum var. yomensis var. nov. closely resembles Achnanthidium pseudoconspicuum var. pseudoconspicuum in scanning electron microscopy (SEM), but has clearly different valve morphometry under the light microscopy (LM). Achnanthidium pseudoconspicuum var. yomensis is also similar to A. japonicum in valve morphology under LM, but differs to this species based on SEM observation. Encyonema yuwadeeanum sp. nov. shows similarities to Encyonema leei and Encyonema subkukenanum, but differs in valve shape and/or polar fissure shape. We discuss the possible valve ontogenetic relationship between Achnanthidium and Cymbellales based on the detailed areolae structures of these two new taxa.     

The effect of C3 transferase on human adipose-derived stem cells

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, which have the ability to differentiate into fat, cartilage, bone, or nerves that can be applied in tissue engineering. On the other hand, the exoenzyme C3 transferase (C3) is a Rho inhibitor. Once in the cytosol, the cell-penetrating moiety is released, thereby allowing C3 transferase to freely diffuse intracellularly and inactivate RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1. In this study, we investigated ASC cytoskeletal changes induced by the addition of C3 employing immunofluorescence staining, changes in alpha-smooth muscle actin (a-SMA) gene expression employing real-time RT-PCR, and the Rho-inhibitory effect employing the pull-down assay. C3 significantly reduced stress fiber disruption and a-SMA expression 24 h after its addition at a concentration of 1 μg/ml, and it also reduced the Rho activity level. While the correlation of the occurrence can be assumed, it requires further examination to verify it. C3 may be an effective inhibitor of intracellular signal transmission in ASC cytoskeletal control involving Rho.     

Victorin triggers programmed cell death and the defense response via interaction with a cell surface mediator

The host-selective toxin victorin is produced by Cochliobolus victoriae, the causal agent of victoria blight of oats. Victorin has been shown to bind to the P protein of the glycine decarboxylase complex (GDC) in mitochondria, and induce defense-related responses such as phytoalexin synthesis, extracellular alkalization and programmed cell death. However, evidence demonstrating that the GDC plays a critical role in the onset of cell death is still lacking, and the role of defense-like responses in the pathogenicity has yet to be elucidated. Here, cytofluorimetric analyses, using the fluorescein (VicFluor) or bovine serum albumin-fluorescein derivative of victorin (VicBSA), demonstrated that victorin-induced cell death occurs before these conjugates traverse the plasma membrane. As with native victorin, VicBSA clearly elicits apoptosis-like cell death, production of phytoalexin, extracellular alkalization, and generation of nitric oxide and reactive oxygen intermediates. These results suggest that the initial recognition of victorin takes place on the cell surface, not in mitochondria, and leads to the activation of a battery of victorin-induced responses. Pharmacological studies showed that extracellular alkalization is the essential regulator for both victorin- and VicBSA-induced cellular responses. We propose a model where victorin may kill the host cell by activating an HR-like response, independent of the binding to the GDC, through ion fluxes across the plasma membrane.     

Coordinate involvement of cysteine protease and nuclease in the executive phase of plant apoptosis

We have developed an oat cell-free apoptosis system to investigate the execution mechanisms of plant apoptosis. Cell extracts derived from oat tissues undergoing toxin (victorin)-induced apoptosis caused nuclear collapse and internucleosomal DNA fragmentation in isolated nuclei. Pharmacological studies revealed that cysteine protease, which is E-64-sensitive but insensitive to caspase-specific inhibitors, is a crucial component in the morphological change of isolated nuclei, and that nuclease and the cysteine protease act cooperatively to induce the apoptotic DNA laddering. Interestingly, this finding is contrasted with those in well-studied animal cell-free systems in which an apoptotic endonuclease is solely responsible for the DNA fragmentation.