CD27 and CD40 inhibit p53-independent mitochondrial pathways in apoptosis of B cells induced by B cell receptor ligation

B cells in the germinal center are known to undergo apoptosis after B cell receptor (BCR) ligation, a process relevant to immunological tolerance. Human CD27 is a B cell co-stimulatory molecule. The aim of this study was to compare the effects of CD27 and CD40 signals on BCR-mediated apoptosis of B cells. BCR ligation activated mitochondrial apoptotic pathways including down-regulation of Bcl-X(L), dissipation of mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-9. Each of these effects was significantly inhibited by CD27 and CD40. Bik expression was weakly but significantly down-regulated by CD27 but up-regulated by CD40. BCR ligation resulted in p53 activation including its phosphorylation at Ser(15), nuclear translocation, and target gene p53AIP1 induction. CD27 and CD40 clearly suppressed these processes. Analyses that used dominant-negative p53 variants revealed a low but still substantial level of BCR-mediated apoptosis and intact mitochondria-mediated apoptotic pathway. These pathways were further inhibited by CD27 and CD40, although the cells showed no p53 phosphorylation or p53AIP1 expression. Our results suggested that, at the mitochondrial level, CD27 and CD40 co-stimulatory signals regulated the p53-amplified apoptotic pathway in B cells through the inhibition of p53-independent apoptotic pathway primarily induced by BCR ligation.     

Positional effect of cell inactivation on root gravitropism using heavy-ion microbeams

When primary root apical tissues of Arabidopsis thaliana were irradiated by heavy-ion microbeams with 120 microm diameter, strong inhibition of root elongation and curvature were observed at the root tip. Irradiation of the cells that become the lower part of the root cap after gravistimulation showed strong inhibition of root curvature, whereas irradiation of the cells that become the upper part of the root cap after gravistimulation did not show severe damage in either root curvature or root growth. Further analysis using smaller area microbeams with 40 microm diameter indicated that the greatest inhibition of curvature occurred at the root tip and the next greatest inhibition occurred in the cells in the lower part of the root cap. These results indicate not only that the root tip and columella cells are the most sensitive sites for root gravity, but also that signalling of root gravity would go through the lower part of the cap cells after perception.     

Cranial integration in Homo: singular warps analysis of the midsagittal plane in ontogeny and evolution

This study addresses some enduring issues of ontogenetic and evolutionary integration in the form of the hominid cranium. Our sample consists of 38 crania: 20 modern adult Homo sapiens, 14 sub-adult H. sapiens, and four archaic Homo. All specimens were CT-scanned except for two infant H. sapiens, who were imaged by MR instead. For each specimen 84 landmarks and semi-landmarks were located on the midsagittal plane and converted to Procrustes shape coordinates. Integration was quantified by the method of singular warps, a new geometric-statistical approach to visualizing correlations among regions. The two classic patterns of integration, evolutionary and ontogenetic, were jointly explored by comparing analyses of overlapping subsamples that span ranges of different hypothetical factors. Evolutionary integration is expressed in the subsample of 24 adult Homo, and ontogenetic integration in the subsample of 34 H. sapiens. In this data set, vault, cranial base, and face show striking and localized patterns of covariation over ontogeny, similar but not identical to the patterns seen over evolution. The principal differences between ontogeny and phylogeny pertain to the cranial base. There is also a component of cranial length to height ratio not reducible to either process. Our methodology allows a separation of these independent processes (and their impact on cranial shape) that conventional methods have not found.     

The apparent uptake of fluorescently labeled siRNAs by electroporated cells depends on the fluorochrome

Transfection of mammalian cells with preformed small interfering RNAs (siRNAs) permits a transient and often specific reduction of gene expression. It is possible to rapidly examine the uptake of siRNAs by transfection with fluorescently labeled siRNAs. We examined the apparent uptake of such siRNAs by several leukemic cell lines after electroporation. We show that Cy3 and Cy5-labeled siRNAs cause a significant amount of cell fluorescence, as judged by flow cytometry. In contrast, several fluorescein-labeled siRNAs could not be detected. Nevertheless, such fluoresceinated siRNAs efficiently suppressed a leukemic target gene, demonstrating that siRNA uptake must have taken place. Therefore, for cell electroporation, fluorescein-labeled siRNAs may lead to false negative results and should not be used to examine electroporation-mediated siRNA uptake.     

Isolation of lectins by affinity chromatography with porcine plasma proteins immobilized on agarose

To develop a convenient method to isolate lectins, we prepared an affinity gel by coupling plasma proteins with agarose beads under conditions where the pH did not exceed 7.5. The validity of the use of this affinity gel in combination with elution using a hapten saccharide was confirmed by isolation of concanavalin A from Jack bean meal. Successful application of the method was demonstrated by isolation of two novel vegetable lectins from udo (Aralia cordate) and wasabi (Wasabia japonica). The method would be useful to isolate new lectins from various sources including plant and animal tissues.     

Development of catheter-type optical oxygen sensor and applications to bioinstrumentation

A catheter-type optical oxygen sensor based on phosphorescence lifetime was developed for medical and animal experimental use. Since the sensor probe should have biocompatibility and high oxygen permeability in vivo, we focused attention on acceptable polymer materials for contact lenses as the substrates of probes. Pd-porphyrin was doped in silicone-based polymer, and was fixed at the edge of an optical fiber inserted in a catheter tube. The shape of the probe was 600 μm in diameter and 100 μm in thickness, and the probe had high oxygen permeability of Dk value 455. In accuracy evaluation, there found an excellent correlation between the pO₂ values measured through phosphorescence lifetime using the oxygen sensors and those measured as the calibrating data using oxygen electrodes. The response time required to achieve 90% from reversible default value to be from 150 to 0 mmHg, and from 0 to 150 mmHg was 15.43 and 7.52 s, respectively. In addition, other properties such as temperature and pH dependency, response, and durability of our optical oxygen sensor were investigated. In animal experiments, the catheter-type oxygen sensor was inserted via the femoral artery of a rat, and arterial oxygen pressure was monitored under asphyxiation. The sensor was valid in the range of oxygen concentration sufficient for biometry, and expected to be integrated with an indwelling needle.     

Nortropinyl-arylsulfonylureas as novel, reversible inhibitors of human steroid sulfatase

Steroid sulfatase (STS) has emerged as an attractive target for a range of estrogen- and androgen-dependent diseases. Searching for novel chemotypes as STS inhibitors, we identified nortropinyl-arylsulfonylurea 3 as a hit from high-throughput screening. A series of analogues was prepared in order to explore the essential structural elements for STS inhibition, and first structure-activity relationships were established. Mechanistic investigations revealed that the compounds are reversible, competitive inhibitors of STS.