The Dissection of Meiotic Chromosome Movement in Mice Using an In Vivo Electroporation Technique

During meiosis, the rapid movement of telomeres along the nuclear envelope (NE) facilitates pairing/synapsis of homologous chromosomes. In mammals, the mechanical properties of chromosome movement and the cytoskeletal structures responsible for it remain poorly understood. Here, applying an in vivo electroporation (EP) technique in live mouse testis, we achieved the quick visualization of telomere, chromosome axis and microtubule organizing center (MTOC) movements. For the first time, we defined prophase sub-stages of live spermatocytes morphologically according to ^GFP-TRF1 and ^GFP-SCP3 signals. We show that rapid telomere movement and subsequent nuclear rotation persist from leptotene/zygotene to pachytene, and then decline in diplotene stage concomitant with the liberation of SUN1 from telomeres. Further, during bouquet stage, telomeres are constrained near the MTOC, resulting in the transient suppression of telomere mobility and nuclear rotation. MTs are responsible for these movements by forming cable-like structures on the NE, and, probably, by facilitating the rail-tacking movements of telomeres on the MT cables. In contrast, actin regulates the oscillatory changes in nuclear shape. Our data provide the mechanical scheme for meiotic chromosome movement throughout prophase I in mammals.     

Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose,as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome

The cellulosome is a supramolecular multienzyme complex formed by species-specific interactions between the cohesin modules of scaffoldin proteins and the dockerin modules of a wide variety of polysaccharide-degrading enzymes. Cellulosomal enzymes bound to the scaffoldin protein act synergistically to degrade crystalline cellulose. However, there have been few attempts to reconstitute intact cellulosomes due to the difficulty of heterologously expressing full-length scaffoldin proteins. We describe the synthesis of a full-length scaffoldin protein containing nine cohesin modules, CipA; its deletion derivative containing two cohesin modules, ΔCipA; and three major cellulosomal cellulases, Cel48S, Cel8A, and Cel9K, of the Clostridium thermocellum cellulosome. The proteins were synthesized using a wheat germ cell-free protein synthesis system, and the purified proteins were used to reconstitute cellulosomes. Analysis of the cellulosome assembly using size exclusion chromatography suggested that the dockerin module of the enzymes stoichiometrically bound to the cohesin modules of the scaffoldin protein. The activity profile of the reconstituted cellulosomes indicated that cellulosomes assembled at a CipA/enzyme molar ratio of 1/9 (cohesin/dockerin = 1/1) and showed maximum synergy (4-fold synergy) for the degradation of crystalline substrate and ∼2.4-fold-higher synergy for its degradation than minicellulosomes assembled at a ΔCipA/enzyme molar ratio of 1/2 (cohesin/dockerin = 1/1). These results suggest that the binding of more enzyme molecules on a single scaffoldin protein results in higher synergy for the degradation of crystalline cellulose and that the stoichiometric assembly of the cellulosome, without excess or insufficient enzyme, is crucial for generating maximum synergy for the degradation of crystalline cellulose.     

The Structure of an Arabinogalactan Isolated from Bamboo Shoot

It was found that azide bound to alcohol oxidase non-covalently and caused a color change from yellow to red. Alcohol oxidase was purified with a high yield from methanol-limited chemostat-grown cells of Candida boidinii S2 as an enzyme-azide complex by a simple procedure. That is, a cell-free extract was prepared from cells treated with a cationic detergent, Cation M2, and alcohol oxidase amounted to more than 80% of the soluble protein. Inactivation of the enzyme by H₂O₂ and aldehydes was decreased in the complex. The results were discussed as to the production of various aldehydes with the yeast cells.     

Regulated genes in mesenchymal stem cells and gastric cancer

AIM: To investigate the genes regulated in mesenchymal stem cells(MSCs) and diffuse-type gastric cancer(GC),gene expression was analyzed. METHODS: Gene expression of MSCs and diffuse-type GC cells were analyzed by microarray. Genes related to stem cells, cancer and the epithelial-mesenchymal transition(EMT) were extracted from human gene lists using Gene Ontology and reference information. Gene panels were generated, and messenger RNA gene expression in MSCs and diffuse-type GC cells was analyzed. Cluster analysis was performed using the NCSS software.RESULTS: The gene expression of regulator of G-protein signaling 1(RGS1) was up-regulated in diffuse-type GC cells compared with MSCs. A panel of stem-cell related genes and genes involved in cancer or the EMT were examined. Stem-cell related genes, such as growth arrest-specific 6, musashi RNA-binding protein 2 and hairy and enhancer of split 1(Drosophila), NOTCH family genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated, and genes related to stem cells and NOTCH signaling are altered in diffuse-type GC compared with MSCs.     

Real-Time Dynamic Adsorption Processes of Cytochrome c on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy

An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (R_RMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.