Multipurpose robot for automated cycle sequencing

Paramagnetic beads have the superior advantages of easy separation and resuspension by controlling the magnetic filed. Previously, we have developed Magtration technology to automate paramagnetic bead handling and have built several automated instruments that handle 1-12 samples simultaneously. To achieve more high-throughput sample processing, two types of a 96-arrayed Integrated Magtration Unit (IMU) were developed, one installed with electromagnets and the other with thin rod-shaped magnets made of neodymium. A multipurpose robot (SX-96GC) equipped with the IMU was also developed for fully automatic processing of 96 samples in parallel. The cleanup of dye-terminator sequencing products was performed using the robot installed with the permanent magnet version of IMU. The results had quality comparable to those by the same protocol in manual handling or to those by the conventional protocols. The robot processed 96 samples in a microplate within 30 min. The protocol that can purify 384 samples within 1 h by processing two microplates concurrently was successfully designed.     

Reproductive success of two male morphs in a free-ranging population of Bornean orangutans

The reproductive success of male primates is not always associated with dominance status. For example, even though male orangutans exhibit intra-sexual dimorphism and clear dominance relationships exist among males, previous studies have reported that both morphs are able to sire offspring. The present study aimed to compare the reproductive success of two male morphs, and to determine whether unflanged males sired offspring in a free-ranging population of Bornean orangutans, using 12 microsatellite loci to determine the paternity of eight infants. A single flanged male sired most of the offspring from parous females, and an unflanged male sired a firstborn. This is consistent with our observation that the dominant flanged male showed little interest in nulliparous females, whereas the unflanged males frequently mated with them. This suggests that the dominant flanged male monopolizes the fertilization of parous females and that unflanged males take advantage of any mating opportunities that arise in the absence of the flanged male, even though the conception probability of nulliparous females is relatively low.     

A point mutation of low-density-lipoprotein receptor causing rapid degradation of the receptor.

The exons of the low-density-lipoprotein-(LDL)-receptor gene from a Japanese patient with homozygous familial hypercholesterolemia were amplified by the polymerase chain reaction (PCR), and their nucleotide sequences were determined. A point mutation from G to C was found in exon 9, which was expected to change Asp at position 412 to His. This amino acid change occurred within the epidermal-growth-factor-precursor homology domain of the LDL receptor, slightly impairing the processing from the precursor to the mature form and causing rapid degradation of the mature form in the fibroblasts of the patient. The mutant LDL-receptor gene transfected into COS-1 cells expressed a LDL-receptor protein with the same properties as the protein expressed in the fibroblasts of the patient; impaired processing and rapid degradation of the synthesized receptor protein. The mutation was identified in family members of the patient by dot-blot hybridization of PCR-amplified DNA with the mutant oligonucleotide. The family members carrying the mutant gene showed higher serum cholesterol levels than the others. However, their cholesterol levels were also greatly influenced by the apolipoprotein-E phenotype.     

Single-Molecule Motions of MHC Class II Rely on Bound Peptides

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.     

Desensitization to gonadotropic hormones: a model system for the regulation of a G-protein-coupled receptor with 7-transmembrane spanning regions

Gonadotropic hormone, luteinizing hormone, and follicle-stimulating hormone exert their effect via activation of G-coupled receptors, which activate the hormone sensitive adenylyl cyclase, protein kinase A, and cyclic AMP responsive elements. This activation leads to specific de novo synthesis of steroidogenic factors and steroidogenic enzymes. In normal cells and following activation of this signaling pathway, desensitization period will be followed. This down-regulation, which was studied in detail for the last three decays, was found to take place at various steps of these signal transduction pathways as well as at different kinetics. A common and diverse feature of the mechanism of desensitization in other G-coupled-7-transmembrane receptor system is also discussed.     

Production of completely flavinylated histamine dehydrogenase, unique covalently bound flavin, and iron-sulfur cluster-containing enzyme of nocardioides simplex in Escherichia coli, and its properties

The hmd gene of histamine dehydrogenase from Nocardioides simplex was overexpressed in Escherichia coli, and the resulting enzyme was purified to homogeneity. The purified recombinant enzyme is almost identical with the native enzyme in view of molecular weight and specific activity, and is stoichiometrically assembled with the three cofactors 6-S-cysteinyl FMN, 4Fe-4S cluster, and ADP.     

Reduction of plastocyanin by tyrosine-containing oligopeptides

Oxidized plastocyanin (PC) was reduced with TyrTyrTyr and LysLysLysLysTyrTyrTyr (KKKKYYY) oligopeptides at neutral pH. The TyrTyrTyr site of the peptides provided an electron to the copper active site of PC, whereas the tetralysine site of KKKKYYY functioned as the recognition site for the negative patch of PC. The reciprocal initial rate constant (1/k(int)) increased linearly with the reciprocal TyrTyrTyr concentration and proton concentration, although the electron transfer rate decreased gradually with time. The results showed that PC was reduced by the deprotonated species of TyrTyrTyr. A linear increase of log k(int) with increase in the ionic strength was observed due to decrease in the electrostatic repulsion between negatively charged PC and deprotonated (TyrTyrTyr)(-). PC was reduced faster by an addition of KKKKYYY to the PC-TyrTyrTyr solution, although KKKKYYY could not reduce PC without TyrTyrTyr. The ESI-LCMS spectrum of the products from the reaction between PC and TyrTyrTyr showed molecular ion peaks at m/z 1015.7 and 1037.7, which suggested formation of a dimerized peptide that may be produced from the reaction of a tyrosyl radical. The results indicate that PC and the tyrosine-containing oligopeptides form an equilibrium, PC(ox)/(oligopeptide)(-)-->/<--PC(red)/(oligopeptide)(*). The equilibrium is usually shifted to the left, but could shift to the right when the produced oligopeptide radical reacts with unreacted peptides. For the reaction of PC with KKKKYYY in the absence of TyrTyrTyr, the produced KKKK(YYY)(*) radical peptide could not react with other KKKKYYY peptides, since they were positively charged. In the presence of both KKKKYYY and TyrTyrTyr, PC may interact effectively with KKKKYYY through its tetralysine site and receive an electron from its TyrTyrTyr site, where the produced KKKK(YYY)(*) may interact with TyrTyrTyr peptides.     

Abnormally differentiated subsets of intestinal macrophage play a key role in Th1-dominant chronic colitis through excess production of IL-12 and IL-23 in response to bacteria

Disorders in enteric bacteria recognition by intestinal macrophages (Mphi) are strongly correlated with the pathogenesis of chronic colitis; however the precise mechanisms remain unclear. The aim of the current study was to elucidate the roles of Mphi in intestinal inflammation by using an IL-10-deficient (IL-10-/-) mouse colitis model. GM-CSF-induced bone marrow-derived Mphi (GM-Mphi) and M-CSF-induced bone marrow-derived Mphi (M-Mphi) were generated from bone marrow CD11b+ cells. M-Mphi from IL-10-/- mice produced abnormally large amounts of IL-12 and IL-23 upon stimulation with heat-killed whole bacteria Ags, whereas M-Mphi from wild-type (WT) mice produced large amounts of IL-10 but not IL-12 or IL-23. In contrast, IL-12 production by GM-Mphi was not significantly different between WT and IL-10-/- mice. In ex vivo experiments, cytokine production ability of colonic lamina propria Mphi (CLPMphi) but not splenic Mphi from WT mice was similar to that of M-Mphi, and CLPMphi but not splenic Mphi from IL-10-/- mice also showed abnormal IL-12p70 hyperproduction upon stimulation with bacteria. Surprisingly, the abnormal IL-12p70 hyperproduction from M-Mphi from IL-10-/- mice was improved by IL-10 supplementation during the differentiation process. These results suggest that CLPMphi and M-Mphi act as anti-inflammatory Mphi and suppress excess inflammation induced by bacteria in WT mice. In IL-10-/- mice, however, such Mphi subsets differentiated into an abnormal phenotype under an IL-10-deficient environment, and bacteria recognition by abnormally differentiated subsets of intestinal Mphi may lead to Th1-dominant colitis via IL-12 and IL-23 hyperproduction. Our data provide new insights into the intestinal Mphi to gut flora relationship in the development of colitis in IL-10-/- mice.     

Isolation of Ashbya gossypii mutant for an improved riboflavin production targeting for biorefinery technology

AIMS: To isolate a strain overproducing riboflavin and to improve riboflavin production for practical use in a biorefinery technology. METHODS AND RESULTS: Ashbya gossypii spores were mutagenized by exposure to UV light and mutant ZP4 strain, producing riboflavin threefold the riboflavin that of the wild-type strain, was isolated by the first and second screenings. Proteomic analysis of ZP4 strain showed the expression patterns of eight types of genes related to riboflavin biosynthesis different from those of the wild-type strain and those enzyme activities were investigated. When activated bleaching earth (ABE) containing 75 g l(-1) rapeseed oil was added in the culture of the ZP4 strain with oxygen-enriched air supplied, riboflavin concentration increased to 8.7 g l(-1) at 5 days of culture. Riboflavin production yield was 0.17 g g(-1) of consumed oil, which was eightfold higher than that of the wild-type strain. CONCLUSIONS: The results show that the mutant ZP4 strain shows potential for improving riboflavin production for practical utilization using vegetable oil as the sole carbon source. SIGNIFICANCE AND IMPACT OF STUDY: Our results indicate that the mutant ZP4 strain shows potential for producing riboflavin from vegetable oil, and therefore will be contributed to biorefinery technology.