Sensitive detection of EGFR mutations using a competitive probe to suppress background in the SMart Amplification Process.

In a previous study, a single nucleotide polymorphism (SNP) diagnostic system named the SMart Amplification Process version 2 (SMAP 2) was reported, which enabled rapid gene diagnostics from crude samples such as whole blood. The asymmetric primer design and use of Taq MutS were reported as innovative background suppression technologies employed by SMAP 2, but Taq MutS is known to display differential affinities for various mismatch combinations, and hence may not be entirely effective for all possible applications. To address this issue we developed another approach using a competitive probe (CP) to enhance background suppression technology instead of Taq MutS. CP is a 3'-end aminated oligonucleotide that competes with 3'-end of a discrimination primer or the self-priming elongation site on intermediate product 2 (IM2) for non-target sequences, such as the alternative allele. The preferred hybridization kinetics for the full-match CP on the non-target sequence results in effective background suppression in SMAP 2 assays. By using a CP, we demonstrated the sensitive detection of EGFR gene mutations in purified genomic DNA from mixed cell populations. The CP approach is another tool enhancing the effectiveness and versatility of SMAP 2 assays, expanding its potential applications, and reinforcing its position as a highly effective technology for molecular diagnostics.     

In vivo fascicle behavior of synergistic muscles in concentric and eccentric plantar flexions in humans.

Ultrasonography was used to directly measure in vivo fascicle behavior of the medial gastrocnemius (MG) and soleus (SOL) muscles while the subjects (n=6 men) performed maximal voluntary concentric and eccentric plantar flexions at 60, 120, 180 and 240 deg/s. Fascicle shortening and lengthening velocities of MG, obtained from fascicle length changes over time, were significantly higher than those of SOL at +/-120, +/-180 and +240 deg/s, possibly reflecting physiological and mechanical differences between these muscles. On the other hand, the effective fascicle shortening and lengthening velocities, defined as the velocities in the longitudinal direction of muscle belly, were not significantly different between MG and SOL. This could be due to difference in fascicle architecture and/or the existence of mechanical linkages between these muscles. Moreover, when the contribution of tendinous tissues to muscle-tendon complex length change was determined from fascicle length, pennation angle, moment arm and joint angle, it accounted for approximately 50% in both concentric and eccentric trials, but showed considerable intra-subject variations. This result quantifiably demonstrates the importance of tendinous tissues in isokinetically controlled joint movements.     

Isolation and Characterization of the N-linked Oligosaccharides in Nacrein from <Emphasis Type="Italic">Pinctada fucata</Emphasis>

We analyzed the structure of the N-linked oligosaccharides enzymatically liberated from the organic matrix (OM) component in the nacreous layer of Japanese pearl oyster: Pinctada fucata. The lectin-blot analysis of the soluble OM after separation by SDS-PAGE, four components, with sizes of approximately 55 kDa, 35 kDa, 25 kDa, and 21 kDa were detected with GNA lectin, which recognized terminal mannose of high mannose and hybrid types of N-glycan. The 55-kDa component of the soluble OM detected by lectin blotting was identified as nacrein by using liquid chromatography/mass spectrometry (LC/MS). LC/MS analysis of the N-glycan liberated from nacrein detected a hybrid-type N-glycan, which contained sulfite and sialic acid at its terminus. The data strongly imply that nacrein, a sulfated OM glycoprotein, participates in molluscan biomineralization by creating a favorable environment for calcium ion uptake through sulfonic acid and sialic acid.     

Autophagy Negatively Regulates Cell Death by Controlling NPR1-Dependent Salicylic Acid Signaling during Senescence and the Innate Immune Response in Arabidopsis

Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. In higher plants, autophagy defects result in early senescence and excessive immunity-related programmed cell death (PCD) irrespective of nutrient conditions; however, the mechanisms by which cells die in the absence of autophagy have been unclear. Here, we demonstrate a conserved requirement for salicylic acid (SA) signaling for these phenomena in autophagy-defective mutants (atg mutants). The atg mutant phenotypes of accelerated PCD in senescence and immunity are SA signaling dependent but do not require intact jasmonic acid or ethylene signaling pathways. Application of an SA agonist induces the senescence/cell death phenotype in SA-deficient atg mutants but not in atg npr1 plants, suggesting that the cell death phenotypes in the atg mutants are dependent on the SA signal transducer NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. We also show that autophagy is induced by the SA agonist. These findings imply that plant autophagy operates a novel negative feedback loop modulating SA signaling to negatively regulate senescence and immunity-related PCD.     

Reproductive interference and salinity tolerance differentiate habitat use between two alien cockleburs: <Emphasis Type="Italic">Xanthium occidentale</Emphasis> and <Emphasis Type="Italic">X. italicum</Emphasis> (Compositae)

In recent years, reproductive interference (RI), the fitness cost of reproductive activities among species, has received much attention as a factor in competitive exclusion by alien species. In this study, we aimed to explain the distribution of two annual alien Xanthium species (X. occidentale and X. italicum) found in the northern Kinki Distinct of Japan from the viewpoint of RI. First, specimen records demonstrated that Xanthium occidentale was more dominant in all habitats except seaside habitats. Subsequently, using artificial patches of potted plants, we demonstrated that X. italicum suffered intense RI from X. occidentale. Finally, X. italicum was superior to X. occidentale in tolerating salinity stress. Combining these results, we concluded that the asymmetrical RI caused by X. occidentale displaced X. italicum except in seaside habitats, where X. occidentale could not establish colonies. Furthermore, we discuss the possibility that a similar RI effect caused the extinction of native species.     

Alien pollen grains interfere with the reproductive success of native congener

The effects of invasive species on native species comprise important conservation issues. Determining the mechanisms by which invasives exclude natives is indispensable to efficiently control their impact, but most invasives remain poorly studied. The purpose of this study was to elucidate potentially important but neglected mechanisms, reproductive interference, in wild Taraxacum systems, in which invasive Taraxacum officinale has displaced its native congener T. japonicum in Japan. Hand-pollination of mixed pollen grains significantly reduced the native seed-set compared to conspecific-only pollination. Moreover, natives with a high ratio of invasive pollen on their stigmas suffered severe seed-set reduction, and the proportion of invasive pollen on native stigmas increased as frequencies of the alien neighbor increased. These results, combined with those of previous studies, revealed that depositing invasive pollen on native stigmas contributes to the observed alien-frequency-dependent reduction of native seed-set, and strongly suggest that reproductive interference was the primary cause of displacement in the Taraxacum systems.     

The effects of refreezing on the viscoelastic and tensile properties of ligaments

Biomechanical testing protocols for ligaments can be extensive and span two or more days. During this time, a specimen may have to undergo more than one cycle of freezing and thawing. Thus, the objective of this study was to evaluate the effects of refreezing on the viscoelastic and tensile properties of ligaments. The femur-medial collateral ligament-tibia complexes (FMTC) from six pairs of rabbit knees were used for this study. Following sacrifice, one leg in each pair was assigned to the fresh group and the FMTC was immediately dissected and prepared for testing. The contralateral knees were fresh-frozen at -20 degrees C for 3 weeks, thawed, dissected and then refrozen for one additional week before being tested as the refrozen group. The cross-sectional area and shape of the medial collateral ligament (MCL) was measured using a laser micrometer system. Stress relaxation and cyclic stress-relaxation tests in uniaxial tension were performed followed by a load to failure test. When the viscoelastic behavior of the MCL was described by the quasi-linear viscoelastic (QLV) theory, no statistically significant differences could be detected for the five constants (A, B, C, tau1, and tau2) between the fresh and refrozen groups (p > or = 0.07) based on our sample size. In addition, the structural properties of the FMTCs and the mechanical properties of the MCLs were also found to be similar between the two groups (p > or = 0.68). These results suggest that careful refreezing of the specimens had little or no effect on the biomechanical properties measured.     

Cytokeratin 18 is a specific marker of bovine intestinal M cell

Microfold (M) cells in the follicle-associated epithelium (FAE) of Peyer's patches have an important role in mucosal immune responses. A primary difficulty for investigations of bovine M cells is the lack of a specific molecular marker. To identify such a marker, we investigated the expression of several kinds of intermediate filament proteins using calf Peyer's patches. The expression patterns of cytokeratin (CK) 18 in jejunal and ileal FAE were very similar to the localization pattern of M cells recognized by scanning electron microscopy. Mirror sections revealed that jejunal CK18-positive cells had irregular and sparse microvilli, as well as pocket-like structures containing lymphocytes, typical morphological characteristic of M cells. However, CK18-negative cells had regular and dense microvilli on their surface, typical of the morphology of enterocytes. In contrast, CK20 immunoreactivity was detected in almost all villous epithelial cells and CK18-negative cells in the FAE. CK18-positive proliferating transit-amplifying cells in the crypt exchanged CK18 for CK20 above the mouth of the crypt and after moving to the villi; however, CK18-positive M cells in the crypt continued their expression of CK18 during movement to the FAE region. Terminal deoxynucleotidyl-transferase-mediated deoxyuridine-triphosphate-biotin nick-end labeling-positive apoptotic cells were specifically detected at the apical region of villi and FAE in the jejunum and ileum, and all were also stained for CK20. These data indicate that CK18 may be a molecular marker for bovine M cells in FAE and that M cells may transdifferentiate to CK20-positive enterocytes and die by apoptosis in the apex of the FAE.