Origin of cortical microtubules organized at M/G1 interface: recruitment of tubulin from phragmoplast to nascent microtubules

The origin of cortical microtubules (CMTs) was investigated in transgenic BY-2 cells stably expressing a GFP (green fluorescent protein) -tubulin fusion protein (BY-GT16). In a previous study, we found that CMTs were initially organized in the perinuclear regions but then elongated to reach the cell cortex where they formed bright spots, and that the appearance of parallel MTs from the bright spots was followed by the appearance of transverse MTs (Kumagai et al., Plant Cell Physiol. 42, 723-732, 2001). In this study, we investigated the migration of tubulin to the reorganization sites of CMTs at the M/G1 interface. After synchronization of the BY-GT16 cells by aphidicolin, the localization of GFP-tubulin was monitored and analyzed by deconvolution microscopy. GFP-tubulin was found to accumulate on the nuclear surface near the cell plate at the final stage of phragmoplast collapse. Subsequently, GFP-tubulin accumulated again on the nuclear surface opposite the cell plate, where nascent MTs elongated to the cell cortex. The significance of these observations on the mode of CMT organization is discussed.     

1H and 13C NMR assignments for the glycans in glycoproteins by using 2H/13C-labeled glucose as a metabolic precursor

In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-¹³C₆,²H₇]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with ¹³C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-¹³C₆,²H₇]glucose.     

Pairing of oligosaccharides in the Fc region of immunoglobulin G

The Fc portion of immunoglobulin G (IgG) expresses paired oligosaccharides with microheterogeneities, which are associated with efficiencies of effector functions and with pathological states. A comparison of electrospray ionization mass spectrometry data obtained using a variety of Fc fragments derived from human and mouse IgG that do and do not retain the inter-chain disulfide bridge(s) revealed that (1) the Fc portion can be asymmetric as well as symmetric with respect to glycosylation and (2) the ratios of the individual glycoforms are different from what is expected from the random pairing.     

A novel NMR method for determining the interfaces of large protein-protein complexes

Identification of the interfaces of large (Mr > 50,000) protein-protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein-protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system.     

Recommendations for the presentation of NMR structures of proteins and nucleic acids – IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.     

Conversion of cholic acid and chenodeoxycholic acid into their 7-oxo derivatives by Bacteroides intestinalis AM-1 isolated from human feces

Secondary bile acid-producing bacteria were isolated from human feces to improve our appreciation of the functional diversity and redundancy of the intestinal microbiota. In total, 619 bacterial colonies were isolated using a nutrient-poor agar medium and the level of secondary bile acid formation was examined in each by a liquid culture, followed by thin-layer chromatography. Of five strains analyzed by 16S rRNA gene sequencing and biochemical testing, one was identified as Bacteroides intestinalis AM-1, which was not previously recognized as a secondary bile-acid producer. GC-MS revealed that B. intestinalis AM-1 converts cholic acid (CA) and chenodeoxycholic acid into their 7-oxo derivatives, 7-oxo-deoxycholic acid (7-oxo-DCA) and 7-oxo-lithocholic acid, respectively. Thus, B. intestinalis AM-1 possesses 7α-hydroxysteroid dehydrogenase (7α-HSDH) activity. In liquid culture, B. intestinalis AM-1 showed a relatively higher productivity of 7-oxo-DCA than Escherichia coli HB101 and Bacteroides fragilis JCM11019^T, which are known to possess 7α-HSDH activity. The level of 7α-HSDH activity was higher in B. intestinalis AM-1 than in the other two strains under the conditions tested. The 7α-HSDH activity in each of the three strains is not induced by CA; instead, it is regulated in a growth phase-dependent manner.     

An approach to canine behavioural genetics employing guide dogs for the blind

The purpose of this study was to attempt to find related variables of the canine genome with behavioural traits of dogs maintained and tested in a guide dog facility which provided a relatively uniform environment. The study involved 81 Labrador Retrievers that were being trained as guide dogs. Each dog was taken on walk-out sessions in which the trainer weekly recorded observations that were related to behavioural traits. The records were subjected to key-word analysis of 14 behaviour-related words. A factor analysis on the appearance rate of the 14 key words or phrases resulted in the extraction of six factors that accounted for 67.4% of the variance. Factor 1, referred to as aggressiveness, was significantly related to the success or failure of the dog in qualifying as a guide dog, and was also related to the variable of litter identification. Factor 2, referred to as distraction, was related to the variable of trainer. Factor 3, activity level, was related to the variable of sex, and was significantly related to the polymorphisms of c.471T>C in the solute carrier family 1 ( neuronal/epithelial high affinity glutamate transporter ) member 2 gene and c.216G>A in the catechol-O-methyltransferase gene. The involvement of polymorphisms c.471T>C and c.216G>A in behavioural patterns related to activity level is similar to comparable genetic studies in other mammalian species. These results contribute to a greater understanding of the role of these genes in behaviour.     

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.     

Microarray analysis of tonsils in immunoglobulin A nephropathy patients

Background

Recently, combination of tonsillectomy and steroid pulse therapy was reported to be effective as the treatment of the immunoglobulin A nephropathy (IgAN). However, the gene expression difference between the tonsils in patients with IgAN and those in control patients is not established.

Methods

We performed tonsillectomy combined with steroid pulse as a treatment to IgAN, analyzed the gene expression in the tonsils (N = 23) using microarray, compared with those with patients suffering from chronic tonsillitis (N = 22). From some candidate genes related with IgAN, we confirmed the apolipoprotein B messenger RNA-editing enzyme catalytic polypeptides 2 (APOBEC2) gene expression in the tonsil and we also analyzed its expression levels and clinical features.

Results

Up-regulated genes seem to be categorized into two groups. One group belongs to the muscle related genes which might be caused by structural differences. The other group includes the immune system-related genes, such as APOBEC2, CALB2, DUSP27, and CXCL11. APOBEC2 was positively stained in the epithelium and the peripheral region of the germinal center in both tonsils. APOBEC2 expression level was negatively related with serum igg level, but did not correlate with clinical course after tonsillectomy.

Conclusion

We confirmed gene expression differences related with immune system and muscle structure. The APOBEC2 was confirmed to be elevated in the tonsils with IgAN patients, and the gene expression level was negatively related with serum igg level in overall patients. These results might be helpful to reveal the mechanism of IgAN.