A shift in sphingolipid composition from C24 to C16 increases susceptibility to apoptosis in HeLa cells

Sphingolipids, major lipid components of the eukaryotic plasma membrane, have a variety of physiological functions and have been associated with many diseases. They have also been implicated in apoptosis. Sphingolipids are heterogeneous in their acyl chain length, with long-chain (C16) and very long-chain (C24) sphingolipids being predominant in most mammalian tissues. We demonstrate that knockdown of ELOVL1 or CERS2, which catalyze synthesis of C24 acyl-CoAs and C24 ceramide, respectively, drastically reduced C24 sphingolipid levels with a complementary increase in C16 sphingolipids. Under ELOVL1 or CERS2 knockdown conditions, cisplatin-induced apoptosis significantly increased. Enhanced sensitivity to cisplatin-induced apoptosis exhibited close correlation with increases in caspase-3/7 activity. No significant alterations in sphingolipid metabolism such as ceramide generation were apparent with the cisplatin-induced apoptosis, and inhibitors of ceramide generation had no effect on the apoptosis. Apoptosis induced by UV radiation or C6 ceramides also increased in ELOVL1 or CERS2 knockdown cells. Changes in the composition of sphingolipid chain length may affect susceptibility to stimuli-induced apoptosis by affecting the properties of cell membranes, such as lipid microdomain/raft formation.     

Fluorescence in situ hybridization using bacterial artificial chromosome (BAC) clones for the analysis of chromosome rearrangement in Chinese hamster ovary cells

Chromosome identification using Chinese hamster ovary (CHO) genomic bacterial artificial chromosome (BAC) clones has the potential to contribute to the analysis and understanding of chromosomal instability of CHO cell lines and to improve our understanding of chromosome organization during the establishment of recombinant CHO cells. Fluorescence in situ hybridization imaging using BAC clones as probes (BAC-FISH) can provide valuable information for the identification of chromosomes. In this study, we identified chromosomes and analyzed the chromosome rearrangement in CHO cells using BAC-FISH methods.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm^?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm^?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm^?1(amide II band) and 1,240?cm^?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm^?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.     

Reevaluation of host ranges of feline leukemia virus subgroups

We reevaluated the host ranges of feline leukemia virus (FeLV) subgroups A, B and C using pseudotype assays based on recombinant NB-tropic murine leukemia virus, which is not usually blocked after viral entry in mammalian cells. Pseudotype viruses of FeLV-B and -C infected a variety of cell lines from many mammalian species. Unexpectedly, FeLV-A pseudotype viruses of two independent isolates from the UK and US also infected a variety of non-feline cell lines including cells from humans, rabbits, pigs and minks. Moreover, both isolates of FeLV-A productively infected human embryonic kidney 293 and mink Mv-1-Lu cells. We conclude that FeLV-A is not strictly ecotropic.     

A functional variant of Fcγ receptor IIIA is associated with rheumatoid arthritis in individuals who are positive for anti-glucose-6-phosphate isomerase antibodies

Anti-glucose-6-phosphate isomerase (GPI) antibodies are known to be arthritogenic autoantibodies in K/B×N mice, although some groups have reported that few healthy humans retain these antibodies. The expression of Fcγ receptors (FcγRs) is genetically regulated and has strong implications for the development of experimental arthritis. The interaction between immune complexes and FcγRs might therefore be involved in the pathogenesis of some arthritic conditions. To explore the relationship between functional polymorphisms in FcγRs (FCGR3A-158V/F and FCGR2A-131H/R) and arthritis in individuals positive for anti-GPI antibodies, we evaluated these individuals with respect to FCGR genotype. Genotyping for FCGR3A-158V/F and FCGR2A-131H/R was performed by PCR amplification of the polymorphic site, followed by site specific restriction digestion using the genome of 187 Japanese patients with rheumatoid arthritis (including 23 who were anti-GPI antibody positive) and 158 Japanese healthy individuals (including nine who were anti-GPI antibody positive). We report here on the association of FCGR3A-158V/F functional polymorphism with anti-GPI antibody positive status. Eight out of nine healthy individuals who were positive for anti-GPI antibodies possessed the homozygous, low affinity genotype FCGR3A-158F (odds ratio = 0.09, 95% confidence interval 0.01–0.89; P = 0.0199), and probably were 'protected' from arthritogenic antibodies. Moreover, among those who were homozygous for the high affinity genotype FCGR3A-158V/V, there were clear differences in anti-human and anti-rabbit GPI titres between patients with rheumatoid arthritis and healthy subjects (P = 0.0027 and P = 0.0015, respectively). Our findings provide a molecular model of the genetic regulation of autoantibody-induced arthritis by allele-specific affinity of the FcγRs.     

Congenic method in the chick limb buds by electroporation

Electroporation is a powerful tool with which to study limb development. Limb development, however, remains an intricate series of events, requiring the precise dissection of developmental processes using relevant transgenes. In this review, we describe the anatomy of the limb field as the basis of targeted electroporation, and specific expression vectors are discussed. We share a useful protocol for electroporation of chick limb buds, and the expression pattern of enhanced green fluorescent protein in the limb buds is used to demonstrate relevant embryonic patterning. Finally, useful trouble-shooting techniques are described.     

Methyl-beta-cyclodextrin improves fertilizing ability of C57BL/6 mouse sperm after freezing and thawing by facilitating cholesterol efflux from the cells

Sperm cryopreservation provides an economical means of storing genetically engineered mouse strains in resource facilities. In general, relatively high fertilization rates are obtained for frozen/thawed sperm of the CBA/JN, DBA/2N, and C3H inbred strains and some F1 hybrid strains. However, the fertilization rate for frozen/thawed sperm of C57BL/6, which is the main strain of genetically engineered mice, remains very low. Therefore, it is necessary to establish an in vitro fertilization (IVF) method for cryopreserved C57BL/6 sperm that can obtain a high rate of fertilization after thawing. In the present study, we focused on the effects of methyl-beta-cyclodextrin (MBCD) on the fertilizing ability of frozen/thawed C57BL/6 sperm. Our results have shown that the highest fertilization rate for frozen/thawed sperm was obtained with MBCD at 1.0 mM for 30 min (63.7% +/- 11.0%), but the effects were attenuated by long-term incubation for 120 min at 1.0 or 2.0 mM. The embryos with frozen/thawed sperm showed good developmental potential, and the offspring had normal fertility. The efflux of cholesterol from frozen/thawed sperm was increased by MBCD in a dose-dependent manner and occurred much earlier and to a greater extent than bovine serum albumin. The localization of cholesterol labeled by filipin in the sperm plasma membrane was drastically decreased by MBCD. In summary, we suggest that MBCD is useful for developing an IVF method for frozen/thawed C57BL/6 mouse sperm achieving a high fertilization rate, being involved in the capacity to sequester cholesterol from sperm membrane.     

Biochemical characterization of phospholipids, sulfatide and heparin as potent stimulators for autophosphorylation of GSK-3beta and the GSK-3beta-mediated phosphorylation of myelin basic protein in vitro

The stimulatory effects of SH (sulfatide and heparin) and two phospholipids (PI and PS) on autophosphorylation of GSK-3beta and the GSK-3beta-mediated phosphorylation of myelin basic protein (MBP) and two synthetic MBP peptides (M86 and M156) were comparatively examined in vitro. It was found that (i) both PI and SH highly stimulated the GSK-3beta-mediated phosphorylation of MBP, but not glycogen synthase, and two MBP peptides through their direct binding to these substrates and (ii) both PI and heparin, as compared with sulfatide, highly stimulated autophosphorylation of GSK-3beta. The K(m) value of MBP for GSK-3beta was highly reduced and the V(max) value was significantly increased in the presence of these acidic modulators, which augmented further phosphorylation of MBP by the kinase. Under our experimental condition, similar stimulatory effects of PI and heparin were observed with the GSK-3beta-mediated phosphorylation of tau protein (TP) in vitro. These results presented here suggest that these two phospholipids and SH may function as effective stimulators for autophosphorylation of GSK-3beta and for the GSK-3beta-mediated high phosphorylation of SH-binding proteins, including MBP and TP, in the highly accumulated levels of these acidic and sulfated modulators in the brain.