Apoptotic Effect of Ganciclovir on Primary Effusion Lymphoma Cells Infected with Kaposi's Sarcoma-Associated Herpesvirus

We evaluated the cytotoxic and apoptotic effects of two purine nucleoside analogues, acyclovir (ACV) and ganciclovir (GCV), on lymphoma cells stably harboring Kaposi's sarcoma-associated herpesvirus (KSHV). Colorimetric caspase assay, flow cytometry, and immunoblotting with antibodies against apoptosis-related molecules revealed that GCV has cytotoxic activity toward KSHV-infected primary effusion lymphoma cells, while ACV has weak or little activity. In addition to the GCV-induced cytotoxicity, apoptosis via caspase-7/8, cleavage of poly(ADP-ribose) polymerase, and accumulation of p53 and p21 were induced by GCV treatment. In contrast, neither ACV nor GCV have cytotoxicity- or apoptosis-inducing activities toward uninfected cells.     

A physical model for the simultaneous membrane transport and metabolism of drugs.

A simple model for the simultaneous passive membrane transport and bioconversion of a drug, which may be a weak electrolyte or a neutral molecule, is mathematically described. It includes an aqueous diffusion layer and an operational aqueous pore pathway. The applicability of the model is shown for the in situ rat intestinal transport of prostaglandin F_2a.     

Identification of differentially expressed genes in senescence-accelerated mouse testes by suppression subtractive hybridization analysis

Senescence-accelerated mouse (SAM) strains constitute a model of accelerated senescence coupled with a short lifespan and the early development of various age-related disorders. To identify differential gene expression in testes between senescence-accelerated SAMP1 and control SAMR1 mice, we performed suppression subtractive hybridization. We observed that the expression of three genes related to cell proliferation (myosin regulatory light chain B, aldolase 1A isoform, and cytochrome c oxidase subunit VIc) were upregulated and four genes implicated in spermatogenesis were downregulated in SAMP1 mice. Asb-8, a member of ankyrin repeat-containing proteins, was abundantly expressed in the testes and downregulated in SAMP1. The other three downregulated genes (germ cell-specific gene 1, T-complex polypeptide 1b, and activator of cAMP responsive element modulator in testis) have been reported to regulate late-stage spermatogenesis. These gene expression profiles might explain the findings of early testicular maturation and rapid decline in the ability to produce spermatozoa with advancing age in SAMP1 mice.     

Cooperative DNA‐binding and sequence‐recognition mechanism of aristaless and clawless

To achieve accurate gene regulation, some homeodomain proteins bind cooperatively to DNA to increase those site specificities. We report a ternary complex structure containing two homeodomain proteins, aristaless (Al) and clawless (Cll), bound to DNA. Our results show that the extended conserved sequences of the Cll homeodomain are indispensable to cooperative DNA binding. In the Al–Cll–DNA complex structure, the residues in the extended regions are used not only for the intermolecular contacts between the two homeodomain proteins but also for the sequence‐recognition mechanism of DNA by direct interactions. The residues in the extended N‐terminal arm lie within the minor groove of DNA to form direct interactions with bases, whereas the extended conserved region of the C‐terminus of the homeodomain interacts with Al to stabilize and localize the third α helix of the Cll homeodomain. This structure suggests a novel mode for the cooperativity of homeodomain proteins.     

Enhancement of display efficiency in yeast display system by vector engineering and gene disruption

Vector engineering and gene disruption in host cells were attempted for the enhancement of α-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2-d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology.     

Crim1C140S mutant mice reveal the importance of cysteine 140 in the internal region 1 of CRIM1 for its physiological functions

Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (CRIM1) is a type I transmembrane protein involved in the organogenesis of many tissues via its interactions with growth factors including BMP, TGF-β, and VEGF. In this study, we used whole-exome sequencing and linkage analysis to identify a novel Crim1 mutant allele generated by ENU mutagenesis in mice. This allele is a missense mutation that causes a cysteine-to-serine substitution at position 140, and is referred to as Crim1^C140S. In addition to the previously reported phenotypes in Crim1 mutants, Crim1^C140S homozygous mice exhibited several novel phenotypes, including dwarfism, enlarged seminal vesicles, and rectal prolapse. In vitro analyses showed that Crim1^C140S mutation affected the formation of CRIM1 complexes and decreased the amount of the overexpressed CRIM1 proteins in the cell culture supernatants. Cys140 is located in the internal region 1 (IR1) of the N-terminal extracellular region of CRIM1 and resides outside any identified functional domains. Inference of the domain architecture suggested that the Crim1^C140S mutation disturbs an intramolecular disulfide bond in IR1, leading to the protein instability and the functional defects of CRIM1. Crim1^C140S highlights the functional importance of the IR1, and Crim1^C140S mice should serve as a valuable model for investigating the functions of CRIM1 that are unidentified as yet.

     

Brain Endothelial Cells Produce Amyloid β from Amyloid Precursor Protein 770 and Preferentially Secrete the O-Glycosylated Form

Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia.     

Myosin-X Induces Filopodia by Multiple Elongation Mechanism

Filopodia are actin-rich finger-like cytoplasmic projections extending from the leading edge of cells. Unconventional myosin-X is involved in the protrusion of filopodia. However, the underlying mechanism of myosin-X-induced filopodia formation is obscure. Here, we studied the movements of myosin-X during filopodia protrusion using a total internal reflection microscope to clarify the mechanism of myosin-X-induced filopodia formation. Myosin-X was recruited to the discrete site at the leading edge where it assembles with exponential kinetics before the filopodia extension. The myosin-X-induced filopodia showed repeated extension-retraction cycles with each extension of 2.4 μm, which was critical to produce long filopodia. Myosin-X, lacking the FERM domain, could move to the tip as does the wild type. However, it was transported toward the cell body during filopodia retraction, did not undergo multiple extension-retraction cycles, and failed to produce long filopodia. During the filopodia protrusion, the single molecules of full-length myosin-X moved within filopodia. The majority of the fluorescence spots showed two-step photobleaching, suggesting that the moving myosin-X is a dimer. Deletion of the FERM domain did not change the movement at the single molecule level with the same velocity of ∼600 nm/s as wild-type, suggesting that the myosin-X in filopodia moves without interaction with the attached membrane via the FERM domain. Based upon these results, we have proposed a model of myosin-X-induced filopodia protrusion.     

Structural and Dynamic Features of the MutT Protein in the Recognition of Nucleotides with the Mutagenic 8-Oxoguanine Base

Escherichia coli MutT hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, an event that can prevent the misincorporation of 8-oxoguanine opposite adenine in DNA. Of the several enzymes that recognize 8-oxoguanine, MutT exhibits high substrate specificity for 8-oxoguanine nucleotides; however, the structural basis for this specificity is unknown. The crystal structures of MutT in the apo and holo forms and in the binary and ternary forms complexed with the product 8-oxo-dGMP and 8-oxo-dGMP plus Mn²⁺, respectively, were determined. MutT strictly recognizes the overall conformation of 8-oxo-dGMP through a number of hydrogen bonds. This recognition mode revealed that 8-oxoguanine nucleotides are discriminated from guanine nucleotides by not only the hydrogen bond between the N7-H and Oδ (N119) atoms but also by the syn glycosidic conformation that 8-oxoguanine nucleotides prefer. Nevertheless, these discrimination factors cannot by themselves explain the roughly 34,000-fold difference between the affinity of MutT for 8-oxo-dGMP and dGMP. When the binary complex of MutT with 8-oxo-dGMP is compared with the ligand-free form, ordering and considerable movement of the flexible loops surrounding 8-oxo-dGMP in the binary complex are observed. These results indicate that MutT specifically recognizes 8-oxoguanine nucleotides by the ligand-induced conformational change.