Increased apoptosis and angiogenesis in gastric low-grade mucosa-associated lymphoid tissue-type lymphoma by Helicobacter heilmannii infection in C57/BL6 mice

Helicobacter heilmannii has been reported to cause gastric low-grade mucosa-associated lymphoid tissue-type (MALT) lymphoma, but its precise pathophysiological mechanism remains to be clarified. We recently established a model of gastric B-cell MALT lymphoma in C57BL/6 mice by means of peroral infection of H. heilmannii primarily obtained from cynomolgus monkeys. Using this model, macroscopic, immunohistochemical, and electron microscopic observations of MALT lymphomas were carried out in order to examine the development of apoptosis and angiogenesis. Enhancement of the microvascular network and an increase in vascular endothelial growth factor-A were detected in the central region of the MALT lymphoma tissue in the infected mouse stomach, while vascular endothelial growth factor-C was detected at the margins of the MALT lymphomas. In addition, many H. heilmannii-invaded parietal cells showed caspase-3 immunoreactivity in the fundic mucosal tissue surrounding the MALT lymphoma. In conclusion, in H. heilmannii-induced MALT lymphoma, enhanced immunoreactivity of vascular endothelial growth factor-A and factor-C was observed in areas encircled by increased parietal cell apoptosis, which indicates the pathophysiological relevance of both angiogenesis and apoptosis in MALT lymphoma formation.     

Cdk5 is required for multipolar-to-bipolar transition during radial neuronal migration and proper dendrite development of pyramidal neurons in the cerebral cortex

The mammalian cerebral cortex consists of six layers that are generated via coordinated neuronal migration during the embryonic period. Recent studies identified specific phases of radial migration of cortical neurons. After the final division, neurons transform from a multipolar to a bipolar shape within the subventricular zone-intermediate zone (SVZ-IZ) and then migrate along radial glial fibres. Mice lacking Cdk5 exhibit abnormal corticogenesis owing to neuronal migration defects. When we introduced GFP into migrating neurons at E14.5 by in utero electroporation, we observed migrating neurons in wild-type but not in Cdk5(-/-) embryos after 3-4 days. Introduction of the dominant-negative form of Cdk5 into the wild-type migrating neurons confirmed specific impairment of the multipolar-to-bipolar transition within the SVZ-IZ in a cell-autonomous manner. Cortex-specific Cdk5 conditional knockout mice showed inverted layering of the cerebral cortex and the layer V and callosal neurons, but not layer VI neurons, had severely impaired dendritic morphology. The amount of the dendritic protein Map2 was decreased in the cerebral cortex of Cdk5-deficient mice, and the axonal trajectory of cortical neurons within the cortex was also abnormal. These results indicate that Cdk5 is required for proper multipolar-to-bipolar transition, and a deficiency of Cdk5 results in abnormal morphology of pyramidal neurons. In addition, proper radial neuronal migration generates an inside-out pattern of cerebral cortex formation and normal axonal trajectories of cortical pyramidal neurons.     

Discovery and SAR study of novel dihydroquinoline-containing glucocorticoid receptor agonists

We have recently reported the discovery of a novel class of glucocorticoid receptor (GR) antagonists, exemplified by 3, containing a 1,2-dihydroquinoline molecular scaffold. Further SAR studies of these antagonists uncovered chemical modifications conveying agonist functional activity to this series. These agonists exhibit good GR binding affinity and are selective against other nuclear hormone receptors.     

S locus F-box brothers: multiple and pollen-specific F-box genes with S haplotype-specific polymorphisms in apple and Japanese pear

Although recent findings suggest that the F-box genes SFB/SLF control pollen-part S specificity in the S-RNase-based gametophytic self-incompatibility (GSI) system, how these genes operate in the system is unknown, and functional variation of pollen S genes in different species has been reported. Here, we analyzed the S locus of two species of Maloideae: apple (Malus domestica) and Japanese pear (Pyrus pyrifolia). The sequencing of a 317-kb region of the apple S9 haplotype revealed two similar F-box genes. Homologous sequences were isolated from different haplotypes of apple and Japanese pear, and they were found to be polymorphic genes derived from the S locus. Since each S haplotype contains two or three related genes, the genes were named SFBB for S locus F-box brothers. The SFBB genes are specifically expressed in pollen, and variable regions of the SFBB genes are under positive selection. In a style-specific mutant S haplotype of Japanese pear, the SFBB genes are retained. Apart from their multiplicity, SFBB genes meet the expected characteristics of pollen S. The unique multiplicity of SFBB genes as the pollen S candidate is discussed in the context of mechanistic variation in the S-RNase-based GSI system.     

Bacterial artificial chromosome library for genome‐wide analysis of Chinese hamster ovary cells

Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene‐amplified CHO DR1000L‐4N cell line for genome‐wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC‐FISH). Thirteen BAC‐FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR‐deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165‐kb DNA region containing exogenous Dhfr was cloned from the BAC library using high‐density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome. Biotechnol. Bioeng. 2009; 104: 986–994. © 2009 Wiley Periodicals, Inc.     

Synthesis of a new opioid ligand having the oxabicyclo[3.2.1]octane skeleton using a new rearrangement reaction

An attempt to prepare a trimer having the 1,3,5-trioxazatriquinane skeleton led to discovery of a novel rearrangement reaction that afforded a compound with an oxabicyclo[3.2.1]octane skeleton whose reaction mechanism was proposed. On the basis of this mechanism, we synthesized the rearranged product from a dimethyl acetal intermediate in excellent yield. The compound with an oxabicyclo[3.2.1]octane skeleton showed high affinity for μ and κ but not δ opioid receptor types. The compound expected to be a key intermediate for novel κ selective ligands.     

Aerobic oxidation of indolomorphinan without the 4,5-epoxy bridge and subsequent rearrangement of the oxidation product to spiroindolinonyl-C-normorphinan derivative

Aerobic oxidation of indolomorphinan 1 without a 4,5-epoxy bridge proceeded in the presence of platinum catalyst to give indoleninomorphinan 2 or quinolono-C-normorphinan 5. The 4-hydroxy group would play an important role in deciding the course of the reaction. Treatment of 2a with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave spiroindolinonyl-C-normorphinan 3a whose structure resembles that of δ opioid receptor agonist spiroindanyloxymorphone (SIOM). Boron trichloride was effective for the reverse reaction from 3a to 2a without side reaction. This practical interconversion method between hydroxyindolenine and spiroindolinone would be useful for the design and construction of drug-like compound libraries. Although the compound 3b was expected to show the selectivity for δ opioid receptor because of the structural resemblance to SIOM, it was rather selective for μ opioid receptor (μ: K_i = 0.75 nM; δ: K_i = 2.90 nM; κ: K_i = 13.4 nM). The result suggests that the slight difference of the spatial location of the benzene rings in these compounds may definitively affect the binding affinity for δ opioid receptor.     

Evolutional and Geographical Relationships of <Emphasis Type="Italic">Bartonella grahamii</Emphasis> Isolates from Wild Rodents by Multi-locus Sequencing Analysis

To clarify the relationship between Bartonella grahamii strains and both the rodent host species and the geographic location of the rodent habitat, we have investigated 31 B. grahamii strains from ten rodent host species from Asia (Japan and China), North America (Canada and the USA), and Europe (Russia and the UK). On the basis of multi-locus sequencing analysis of 16S rRNA, ftsZ, gltA, groEL, ribC, and rpoB, the strains were classified into two large groups, an Asian group and an American/European group. In addition, the strains examined were clearly clustered according to the geographic locations where the rodents had been captured. In the phylogenetic analysis based on gltA, the Japanese strains were divided into two subgroups: one close to strains from China, and the other related to strains from Far Eastern Russia. Thus, these observations suggest that the B. grahamii strains distributed in Japanese rodents originated from two different geographic regions. In the American/European group, B. grahamii from the North American continent showed an ancestral lineage and strict host specificity; by contrast, European strains showed low host specificity. The phylogenetic analysis and host specificity of B. grahamii raise the possibility that B. grahamii strains originating in the North American continent were distributed to European countries by adapting to various rodent hosts. An erratum to this article can be found at     

Multilocus patterns of nucleotide polymorphism and demographic change in Taxodium distichum (Cupressaceae) in the lower Mississippi River alluvial valley

? Premise of the Study: Studies of the geographic patterns of genetic variation can give important insights into the past population structure of species. Our study species, Taxodium distichum L. (bald-cypress), prefers riparian and wetland habitats and is widely distributed in southeastern North America and Mexico. We compared the genetic variation of T. distichum with that of its close relative, Cryptomeria japonica, which is endemic to Japan. ? Methods: Nucleotide polymorphisms of T. distichum in the lower Mississippi River alluvial valley, USA, were examined at 10 nuclear loci. ? Key Results: The average nucleotide diversity at silent sites, π(sil), across the 10 loci in T. distichum was higher than that of C. japonica (π(sil) = 0.00732 and 0.00322, respectively). In T. distichum, Tajima's D values were each negative at 9 out of 10 loci, which suggests a recent population expansion. Maximum-likelihood and Bayesian estimations of the exponential population growth rate (g) of T. distichum populations indicated that this species had expanded approximately at the rate of 1.7-1.0 × 10(-6) per year in the past. ? Conclusions: Taxodium distichum had significantly higher nucleotide variation than C. japonica, and its patterns of polymorphism contrasted strikingly with those of the latter, which previously has been inferred to have experienced a reduction in population size.     

Functional availability of γ-herpesvirus K-cyclin is regulated by cellular CDK6 and p16INK4a

Viral K-cyclin derived from Kaposi’s sarcoma-associated herpesvirus is homologous with mammalian D-type cyclins. Here, we demonstrated the regulatory mechanisms for K-cyclin function and degradation in human embryonic kidney HEK293 and primary effusion lymphoma JSC-1 cell lines. Proteasome inhibitor MG132 treatment induced an accumulation of ubiquitinated K-cyclin in these cells, and co-expression of CDK6 prevented K-cyclin ubiquitination. Also K-cyclin mutants incompetent for CDK6-binding were destabilized by proteasome pathway. Furthermore, silencing of p16INK4a promoted K-cyclin-CDK6 complex formation and hence induced K-cyclin-associated kinase activity in HEK293 cells. These observations indicate that CDK6-bound K-cyclin is functionally stable but monomeric K-cyclin is targeted to ubiquitin-dependent degradation pathway in these cells. Our data suggest that the balance between CDK6 and p16INK4a regulates the availability of functional K-cyclin in human cells.