Improvement of short straws for sperm cryopreservation: installing an air-permeable filter facilitates handling.

Saving space for sperm cryopreservation would aid mouse genetics research. We previously developed the ST (sperm freezing in ShorT STraw to reduce STorage space) method for cryopreserving mouse sperm in a smaller storage space than conventional methods. However, our ST method has two drawbacks: difficulties during freeze-thaw procedures and the potential risk of sperm loss during storage. Here, we refine ST, terming the new method improved ST (iST). In iST, the straw has an air-permeable filter and the straw container (2-ml cryotube) is endowed with air vents. As in our ST method, iST frozen-thawed sperm showed good performance upon in vitro fertilization. Moreover, up to nine straws can be stored in one cryotube, occupying less storage space than conventional methods. This method provides an easy and space-saving cryopreservation method for mouse sperm, and thus will be valuable for mouse genetics researchers.

Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylation of FLT1 gene in choriocarcinoma cells.

BACKGROUND: Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells. METHODS: qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulfite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model. RESULTS: Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells. CONCLUSIONS: Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo. We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.

Freezing sperm in short straws reduces storage space and allows transport in dry ice.

Efficient cryopreservation and transportation of mouse sperm are among the most desirable strategies for current and future research on mouse genetics. However, the current method for sperm cryopreservation uses an 11-cm plastic straw, which is a bulky and fragile container. Developing an alternative to overcome the limitations associated with this method would accelerate biomedical research. Here, we developed the ST (sperm-freezing in ShorT STraw to reduce STorage space) method for cryopreserving mouse sperm in short 3.8-cm plastic straws. Up to nine short straws can be stored in a cryotube, reducing storage space. We further show that sperm frozen by the ST method can be transported in liquid nitrogen or dry ice without any detrimental effects on subsequent fertilization and the birth rate. Our findings suggest that this sperm-freezing method is beneficial not only for individual laboratories but also for large-scale mutagenesis/knockout and phenotyping programs.

CKR-L3, a deletion version CCR6-isoform shows coreceptor-activity for limited human and simian immunodeficiency viruses.

BACKGROUND: The chemokine receptors (CKRs), mainly CCR5 and CXCR4 function as major coreceptors in infections caused by human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Approximately 20 G protein-coupled receptors (GPCRs) have been identified as minor coreceptors, alike CCR6 that we reported recently. Since CKR-L3 is indentified as a natural isoform of CCR6, we attempted in this study to explore the coreceptor function of CKR-L3. METHODS: NP-2 cells transduced with CD4-receptor (NP-2/CD4) normally remain resistant to HIV or SIV infection. However, the introduction of functional coreceptors can make these cells susceptible to these viruses. NP-2/CD4/CKR-L3 cells were produced to examine the coreceptor activity of CKR-L3. Likely, CCR6-isoform and the major coreceptors, CCR5 and CXCR4 were also examined in parallel. Presence of viral antigen in infected NP-2/CD4/coreceptor cells was detected by indirect immunofluorescence assay (IFA). The results were validated by detection of syncytia, proviral DNA and by measuring reverse transcriptase (RT) activities. RESULTS: HIV-2MIR and SIVsmE660 were found to infect NP-2/CD4/CKR-L3 cells, indicative of the coreceptor function of CKR-L3. Viral antigens appeared faster in NP-2/CD4/CKR-L3 cells than in NP-2/CD4/CCR6, indicating that CKR-L3 is a more efficient coreceptor. Moreover, syncytia formation was more rapid and RT release evidenced earlier and at higher levels with CKR-L3 than with CCR6. Sequence analysis in the C2-V3 envelope region of HIV-2MIR replicated through CKR-L3 and CCR6 coreceptor showed two and three amino acid substitutions respectively, in the C2 region compared to the CCR5-variant. The SIVsmE660-CKRL3 variant showed three amino acid substitutions in the V1 region, one change in the V2 and two changes in the C2 region. The SIVsmE660-CCR6 variant produced two changes in the V1 region, and three in the C2 region. CONCLUSIONS: Isoform CKR-L3 exhibited coreceptor activity for limited primary HIV and SIV isolates with better efficiency than the parent CCR6-isoform. Amino acid substitutions in the envelope region of these viruses may confer selective pressure towards CKR-L3-use. CKR-L3 with other minor coreceptors may contribute to HIV and SIV pathogenesis including dissemination, trafficking and latency especially when major coreceptors become compromised. However, further works will be required to determine its clinical significance in HIV and SIV infection.

Entry of human T-cell leukemia virus type 1 is augmented by heparin sulfate proteoglycans bearing short heparin-like structures.

Three molecules have been identified as the main cellular factors required for binding and entry of human T-cell leukemia virus type 1 (HTLV-1): glucose transporter 1 (GLUT1), heparan sulfate (HS), and neuropilin 1 (NRP-1). However, the precise mechanism of HTLV-1 cell tropism has yet to be elucidated. Here, we examined the susceptibilities of various human cell lines to HTLV-1 by using vesicular stomatitis virus pseudotypes bearing HTLV-1 envelope proteins. We found that the cellular susceptibility to HTLV-1 infection did not correlate with the expression of GLUT1, HS, or NRP-1 alone. To investigate whether other cellular factors were responsible for HTLV-1 susceptibility, we conducted expression cloning. We identified two HS proteoglycan core proteins, syndecan 1 and syndecan 2, as molecules responsible for susceptibility to HTLV-1. We found that treatment of syndecan 1-transduced cells (expressing increased HS) with heparinase, a heparin-degradative enzyme, reduced HTLV-1 susceptibility without affecting the expression levels of HS chains. To further elucidate these results, we characterized the expression of HS chains in terms of the mass, number, and length of HS in several syndecan 1-transduced cell clones as well as human cell lines. We found a significant correlation between HTLV-1 susceptibility and the number of HS chains with short chain lengths. Our findings suggest that a combination of the number and the length of HS chains containing heparin-like regions is a critical factor which affects the cell tropism of HTLV-1.

X4-tropic human immunodeficiency virus IIIB utilizes CXCR4 as coreceptor, as distinct from R5X4-tropic viruses.

Human immunodeficiency viruses initiate infections via CCR5 coreceptors and then change their tropism to C-X-C chemokine receptor type 4 (CXCR4), this change being associated with rapid disease progression. HIV-1IIIB, a widely described pure X4-tropic strain, is distinct from R5X4-tropic viruses. In this study, the requirement for amino terminal regions (NTRs) of CXCR4 for entry of HIV-1IIIB virus into host cells was examined and compared to that of R5X4-tropic viruses. CXCR4 and its deletion mutant (CXCR4ΔNTR23; first 23 amino acids removed from NTR) were amplified to examine their coreceptor activities. NP-2/CD4/CXCR4 and NP-2/CD4/CXCR4ΔNTR23 cell lines were prepared accordingly. Indirect immune fluorescence assay (IFA), PCR, and reverse transcriptase (RT) activity were used to compare the process of infection of host cells by HIV-1IIIB virus, one R5-tropic and five other R5X4-tropic viruses. All the R5X4-tropic HIVs were found to utilize both CCR5 and CXCR4 but unable to use CXCR4ΔNTR23 as coreceptors. In contrast, X4-tropic HIV-1IIIB was found to preferentially infect through CXCR4ΔNTR23. Viral antigens in infected NP-2/CD4/CXCR4ΔNTR23 cells were detected by IFA and confirmed by detection of proviral DNA and by performing RT assays on the spent cell-supernatants. In dual tropic viruses, deletion of 23 amino acids from NTR abrogates the coreceptor activity of CXCR4. This observation demonstrates that NTR of CXCR4 have an obligatory coreceptor role for dual tropic viruses. However, HIV-1IIIB may have different requirements for NTR than R5X4 viruses or may infect host cells independent of NTR of CXCR4.

Tax1-expressing feline 8C cells are useful to monitor the life cycle of human T-cell leukemia virus type I.

Extremely low infectivity has hampered direct (cell-free) infection studies of human T-cell leukemia virus type I (HTLV-I). In order to break through this barrier, we examined the susceptibility of many kinds of cells to HTLV-I and found a feline kidney cell line, 8C, that is highly susceptible to HTLV-I and produced remarkable amounts of infectious progeny viruses. Tax1 protein encoded by HTLV-I is known as a transcription activator for viral and cellular genes. We found that the 8C cells expressing the Tax1 protein (8C/TaxWT cells) can produce more progeny viruses than 8C cells when the cells were exposed to cell-free HTLV-I. A large number of syncytia were also induced in these cells. Here, we propose 8C/TaxWT cells as a useful tool to study the cell-free HTLV-I infection.

Human T-cell leukemia viruses are highly unstable over a wide range of temperatures.

The biological properties of human T-cell leukemia virus type I (HTLV-I) and HTLV type II (HTLV-II) are not well elucidated as cell-free viruses. We established new assay systems to detect the infectivity of cell-free HTLVs and examined the stability of cell-free HTLVs at different temperatures. HTLVs lost infectivity more rapidly than did bovine leukemia virus (BLV), which is genetically related to HTLVs. The half-lives of three HTLV-I strains (two cosmopolitan strains and one Melanesian strain) at 37 °C were approximately 0.6 h, whereas the half-life of a BLV strain was 8.5 h. HTLV-I rapidly lost infectivity unexpectedly at 0 and 4 °C. We examined the stability of vesicular stomatitis virus pseudotypes with HTLV-I, HTLV-II or BLV Env proteins, and the Env proteins of HTLVs were found to be more unstable at 4 and 25 °C than the Env proteins of the BLV. Over the course of the viral life cycle, heat treatment inhibited HTLV-I infection at the phase of attachment to the host cells, and inhibition was more marked upon entry into the cells. The HTLV-I Env surface (SU) protein (gp46) was easily released from virions during incubation at 37 °C. However, this release was inhibited by pre-treatment of the virions with N-ethylmaleimide, suggesting that the inter-subunit bond between gp46 SU and gp21 transmembrane (TM) proteins is rearranged by disulfide bond isomerization. HTLVs are highly unstable over a wide range of temperatures because the disulfide bonds between the SU and TM proteins are labile.

Ionising irradiation alters the dynamics of human long interspersed nuclear elements 1 (LINE1) retrotransposon.

It is important to identify the mechanism by which ionising irradiation induces various genomic alterations in the progeny of surviving cells. Ionising irradiation activates mobile elements like retrotransposons, although the mechanism of its phenomena consisting of transcriptions and insertions of the products into new sites of the genome remains unclear. In this study, we analysed the effects of sparsely ionising X-rays and densely ionising carbon-ion beams on the activities of a family of active retrotransposons, long interspersed nuclear elements 1 (L1). We used the L1/reporter knock-in human glioma cell line, NP-2/L1RP-enhanced GFP (EGFP), that harbours full-length L1 tagged with EGFP retrotransposition detection cassette (L1RP-EGFP) in the chromosomal DNA. X-rays and carbon-ion beams similarly increased frequencies the transcription from L1RP-EGFP and its retrotransposition. Short-sized de novo L1RP-EGFP insertions with 5'-truncation were induced by X-rays, while full-length or long-sized insertions (>5 kb, containing ORF1 and ORF2) were found only in cell clones irradiated by the carbon-ion beams. These data suggest that X-rays and carbon-ion beams induce different length of de novo L1 insertions, respectively. Our findings thus highlight the necessity to investigate the mechanisms of mutations caused by transposable elements by ionising irradiation.

A formylpeptide receptor, FPRL1, acts as an efficient coreceptor for primary isolates of human immunodeficiency virus.

BACKGROUND: More than 10 members of seven-transmembrane G protein-coupled receptors (GPCRs) have been shown to work as coreceptors for human immunodeficiency virus type 1 (HIV-1), HIV type 2 (HIV-2), and simian immunodeficiency viruses (SIVs). As a common feature of HIV/SIV coreceptors, tyrosine residues are present with asparagines, aspartic acids or glutamic acids in the amino-terminal extracellular regions (NTRs). We noticed that a receptor for N-formylpeptides, FPRL1, also contains two tyrosine residues accompanied by glutamic acids in its NTR. It was reported that monocytes expressing CCR5 and FPRL1 in addition to CD4 are activated by treatment with ligands or agonists of FPRL1. Activated monocytes down-modulate CCR5 and become resistant to infection by HIV-1 strains. Thus, FPRL1 plays important roles in protection of monocyptes against HIV-1 infection. However, its own coreceptor activity has not been elucidated yet. In this study, we examined coreceptor activities of FPRL1 for HIV/SIV strains including primary HIV-1 isolates. RESULTS: A CD4-transduced human glioma cell line, NP-2/CD4, is strictly resistant to HIV/SIV infection. We have reported that when NP-2/CD4 cells are transduced with a GPCR having coreceptor activity, the cells become susceptible to HIV/SIV strains. When NP-2/CD4 cells were transduced with FPRL1, the resultant NP-2/CD4/FPRL1 cells became markedly susceptible to some laboratory-adapted HIV/SIV strains. We found that FPRL1 is also efficiently used as a coreceptor by primary HIV-1 isolates as well as CCR5 or CXCR4. Amino acid sequences linked to the FPRL1 use could not be detected in the V3 loop of the HIV-1 Env protein. Coreceptor activities of FPRL1 were partially blocked by the forymyl-Met-Leu-Phe (fMLF) peptide. CONCLUSION: We conclude that FPRL1 is a novel and efficient coreceptor for HIV/SIV strains. FPRL1 works as a bifunctional factor in HIV-1 infection. Namely, the role of FPRL1 in HIV-1 infection is protective and/or promotive in different conditions. FPRL1 has been reported to be abundantly expressed in the lung, spleen, testis, and neutrophils. We detected mRNA expression of FPRL1 in 293T (embryonal kidney cell line), C8166 (T cell line), HOS (osteosarcoma cell line), Molt4#8 (T cell line), U251MG (astrocytoma cell line), U87/CD4 (CD4-transduced glioma cell line), and peripheral blood lymphocytes. Roles of FPRL1 in HIV-1 infection in vivo should be further investigated.

Suppression of HIV-1 Infectivity by Human Glioma Cells.

HIV-1 infection to the central nervous system (CNS) is very common in AIDS patients. The predominant cell types infected in the brain are monocytes and macrophages, which are surrounded by several HIV-1-resistant cell types, such as astrocytes, oligodendrocytes, neurons, and microvascular cells. The effect of these HIV-1-resistant cells on HIV-1 infection is largely unknown. In this study, we examined the stability of HIV-1 cultured with several human glioblastoma cell lines, for example, NP-2, U87MG, T98G, and A172, to determine whether these HIV-1-resistant brain cells could enhance or suppress HIV-1 infection and thus modulate HIV-1 infection in the CNS. The HIV-1 titer was determined using the MAGIC-5A indicator cell line as well as naturally occurring CD4(+) T cells. We found that the stability of HIV-1 incubated with NP-2 or U87MG cells at 37°C was significantly shorter (half-life, 2.5-4 h) compared to that of HIV-1 incubated with T98G or A172 cells or in culture medium without cells (half-life, 8-18 h). The spent culture media (SCM) of NP-2 and U87MG cells had the ability to suppress both R5- and X4-HIV-1 infection by inhibiting HIV-1 attachment to target cells. This inhibitory effect was eliminated by the treatment of the SCM with chondroitinase ABC but not heparinase, suggesting that the inhibitory factor(s) secreted by NP-2 and U87MG cells was chiefly mediated by chondroitin sulfate (CS) or CS-like moiety. Thus, this study reveals that some but not all glioma cells secrete inhibitory molecules to HIV-1 infection that may contribute in lowering HIV-1 infection in the CNS in vivo.

Littermate influence on infant growth in mice: comparison of SJL/J and ICR as cotransferred carrier embryos.

In mice, a minimum number of healthy embryos is required to trigger and maintain pregnancy. Therefore, when recovering mouse embryos from a limited litter, one useful technique is to transfer carrier ICR embryos along with the embryos of interest, a technique referred to as cotransfer. In this study, we examined suitable mouse strains for cotransfer with C57BL/6J (B6) embryos in regards to the maintenance of pregnancy, number of pups born, intrauterine growth, and postnatal growth. Because the coat color of B6 is black, we compared two white coat-colored strains, SJL/J and ICR. Cotransfer of SJL/J and ICR embryos had similar effects on maintenance of pregnancy, number of pups born, and intrauterine growth. However, the postnatal growth of B6 mouse pups cotransferred and grown with SJL/J pups was better than for B6 mouse pups cotransferred and grown with ICR pups, suggesting competition among littermates. These results demonstrate that cotransfer of SJL/J embryos will be useful not only as carrier embryos with B6-background embryos but also as a model system to examine littermate competition.

Characterisation of cytoplasmic DNA complementary to non-retroviral RNA viruses in human cells.

The synthesis and subsequent genomic integration of DNA that is complementary to the genomes of non-retroviral RNA viruses are rarely observed. However, upon infection of various human cell lines and primary fibroblasts with the vesicular stomatitis virus (VSV), we detected DNA complementary to the VSV RNA. The VSV DNA was detected in the cytoplasm as single-stranded DNA fully complementary to the viral mRNA from the poly(A) region to the 7-methyl guanosine cap. The formation of this DNA was cell-dependent. Experimentally, we found that the transduction of cells that do not produce VSV DNA with the long interspersed nuclear element 1 and their infection with VSV could lead to the formation of VSV DNA. Viral DNA complementary to other RNA viruses was also detected in the respective infected human cells. Thus, the genetic information of the non-retroviral RNA virus genome can flow into the DNA of mammalian cells expressing LINE-1-like elements.

CCR6 functions as a new coreceptor for limited primary human and simian immunodeficiency viruses.

More than 12 chemokine receptors (CKRs) have been identified as coreceptors for the entry of human immunodeficiency virus type 1 (HIV-1), type 2 (HIV-2), and simian immunodeficiency viruses (SIVs) into target cells. The expression of CC chemokine receptor 6 (CCR6) on Th17 cells and regulatory T cells make the host cells vulnerable to HIV/SIV infection preferentially. However, only limited information is available concerning the specific role of CCR6 in HIV/SIV infection. We examined CCR6 as a coreceptor candidate in this study using NP-2 cell line-based in-vitro studies. Normally, CD4-transduced cell line, NP-2/CD4, is strictly resistant to all HIV/SIV infection. When CCR6 was transduced there, the resultant NP-2/CD4/CCR6 cells became susceptible to HIV-1HAN2, HIV-2MIR and SIVsmE660, indicating coreceptor roles of CCR6. Viral antigens in infected cells were detected by IFA and confirmed by detection of proviral DNA. Infection-induced syncytia in NP-2/CD4/CCR6 cells were detected by Giemsa staining. Amount of virus release through CCR6 has been detected by RT assay in spent culture medium. Sequence analysis of proviral DNA showed two common amino acid substitutions in the C2 envelope region of HIV-2MIR clones propagated through NP-2/CD4/CCR6 cells. Conversely, CCR6-origin SIVsmE660 clones resulted two amino acid changes in the V1 region and one change in the C2 region. The substitutions in the C2 region for HIV-2MIR and the V1 region of SIVsmE660 may confer selection advantage for CCR6-use. Together, the results describe CCR6 as an independent coreceptor for HIV and SIV in strain-specific manner. The alteration of CCR6 uses by viruses may influence the susceptibility of CD4+ CCR6+ T-cells and dendritic cell subsets in vivo and therefore, is important for viral pathogenesis in establishing latent infections, trafficking, and transmission. However, clinical relevance of CCR6 as coreceptor in HIV/SIV infections should be investigated further.

Inhibitory effect of chondroitin sulfate type E on the binding step of human T-cell leukemia virus type 1.

Cell surface heparan sulfate proteoglycans (HSPGs) are involved in the binding and entry of human T-cell leukemia virus type 1 (HTLV-1) into host cells, while sulfated polysaccharides such as heparin inhibit HTLV-1 infection. Chondroitin sulfate proteoglycans (CSPGs) are classified as another major type of proteoglycans. Here, we examined the effect of four types of chondroitin sulfate (CS) on HTLV-1 infection. Accordingly, a human T cell line, MOLT-4, was inoculated with cell-free HTLV-1 in the presence or absence of soluble CS, and the synthesis of reverse-transcribed HTLV-1 DNA within cells 20 h after inoculation was detected using polymerase chain reaction (PCR). Among the four types of CS (A, C, D, and E), the E type (CSE), which was derived from the squid cartilage, exhibited anti-HTLV-1 activity. Furthermore, we observed that CSE directly interacted with recombinant HTLV-1 envelope (Env) proteins and inhibited the binding of HTLV-1 virions to MOLT-4 cells, indicating that the interaction between Env and CSE plays a significant role in its anti-HTLV-1 activity. In addition, CSE inhibited syncytium formation that was induced by HTLV-1-producing cells. When CSE was mixed with the synthetic fusion inhibitor peptide corresponding to the ectodomain of the Env transmembrane subunit (TM) gp21, the HTLV-1 infection was further inhibited when compared with the inhibitory effect of each compound alone. Thus, further elucidation of the in vitro antiviral mechanism of CSE shown in this study will lead to the development of CSE-like molecules for the entry inhibition of HTLV-1.

Lack of the trans-receptor mechanism of HIV-1 infection: CD4- and coreceptor-independent incorporation of HIV-1-resistant cells into syncytia induced by HIV-1.

Human immunodeficiency virus type 1 (HIV-1) infects cells through an interaction of HIV-1 envelope protein with CD4 and an appropriate coreceptor on target cells. This interaction often leads to cell fusion, and formation of syncytia. HIV-1-resistant cells expressing either CD4 or a coreceptor are often surrounding HIV-1-susceptible cells, expressing both CD4 and a compatible coreceptor, in vivo. It is therefore worthwhile to investigate whether these HIV-1-resistant cells could cooperate in HIV-1 infection or cell fusion leading to their incorporation into syncytia. When CD4-positive, coreceptor-negative cells were co-cultured with CD4-negative, coreceptor-positive cells and exposed to HIV-1, HIV-1 infection was not established, indicating that CD4 and the coreceptor expressed on different cell surfaces could not cooperate in HIV-1 entry. However, when HIV-1-resistant cells expressing CD4 or a coreceptor or lacking both were mixed with HIV-1-susceptible cells and inoculated with HIV-1, all these HIV-1-resistant cells were similarly incorporated into syncytia induced by HIV-1, indicating a CD4- and coreceptor-independent incorporation of HIV-1-resistant cells into syncytia. This incorporation was impaired by the transfection of these cells with siRNAs for adhesion molecules. Our study demonstrates that HIV-1-resistant cells can be incorporated into syncytia induced by HIV-1 and this incorporation may partially be mediated through adhesion molecules.

Short communication: identification of the conformational requirement for the specificities of coreceptors for human and simian immunodeficiency viruses.

More than 10 G protein-coupled receptors (GPCRs) work as coreceptors for human and simian immunodeficiency viruses (HIVs/SIVs); however, structural features critical for coreceptor activity have not been identified. Our objective was to elucidate the structural requirement of coreceptor activities. Amino-terminal regions (NTRs), extracellular loops (ECLs), and the undecapeptidyl arch (UPA) in the second ECL have been shown to be important for coreceptor function. We made chimeric coreceptors for these regions between CCR5 and GPR1, which is genetically distant from CCR5, and analyzed their activities. The coreceptor activity and specificity of CCR5 were maintained when its NTR or UPA was replaced with GPR1. In contrast, the GPR1 chimera with CCR5 NTR was used by HIV-1 strains that can use only CCR5, but not both CCR5 and CXCR4, or GPR1. GPR1 chimera with CCR5 UPA almost lost activity. All ECL chimeras could hardly maintain activity. Thus, CCR5 is more flexibly acceptable to heterologous NTR and UPA than GPR1, suggesting the existence of conformational differences made by the integration of multiple extracellular regions. This conformation may specifically interact with HIV-1 in a strain-dependent manner. Identification of a factor that is critical to make this conformation will contribute to understanding the mechanism of coreceptor function of GPCRs. For this, the coreceptor activity of GPR1, which is genetically distant from CCR5, will be a useful tool.

Irradiation with carbon ion beams induces apoptosis, autophagy, and cellular senescence in a human glioma-derived cell line.

PURPOSE: We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). METHODS AND MATERIALS: A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated beta-galactosidase (SA-beta-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependent kinase inhibitor p21(WAF1/CIP1) in the irradiated cells was analyzed by Western blotting. RESULTS: When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells (<1% of initially irradiated cells) could not form a colony: however, they showed a morphological phenotype consistent with cellular senescence, that is, enlarged and flattened appearance. The senescent nature of these attached cells was further indicated by staining for SA-beta-gal. The mean telomere length was not changed after irradiation with C-ions. Phosphorylation of p53 at serine 15 as well as the expression of p21(WAF1/CIP1) was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. CONCLUSIONS: Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.

Isolation of the feline alpha1,3-galactosyltransferase gene, expression in transfected human cells and its phylogenetic analysis.

The enzyme alpha 1,3-galactosyltransferase (alpha1,3-GT), which catalyzes synthesis of terminal alpha-galactosyl epitopes (Gal alpha1,3Gal beta1-4GlcNAc-R), is produced in non-primate mammals, prosimians and new-world monkeys, but not in old-world monkeys, apes and humans. We cloned and sequenced a cDNA that contains the coding sequence of the feline alpha1,3-GT gene. Flow cytometric analysis demonstrated that the alpha-galactosyl epitope was expressed on the surface of a human cell line transduced with an expression vector containing this cDNA, and this alpha-galactosyl epitope expression subsided by alpha-galactosidase treatment. The open reading frame of the feline alpha1,3-GT cDNA is 1,113 base pairs in length and encodes 371 amino acids. The nucleotide sequence and its deduced amino acid sequence of the feline alpha1,3-GT gene are 88-90% and 85-87%, respectively, similar to the reported sequences of the bovine, porcine, marmoset and cebus monkey alpha1,3-GT genes, while they are 88% and 82-83%, respectively, similar to those of the orangutan and human alpha1,3-GT pseudogenes, and 81% and 77%, respectively, similar to the murine alpha1,3-GT gene. Thus, the alpha1,3-GT genes and pseudogenes of mammals are highly similar. Ratios of non-synonymous nucleotide changes among the primate pseudogenes as well as the primate genes are still higher than the ratios of non-primates, suggesting that the primate alpha1,3-GT genes tend to be divergent.

Formation of vesicular stomatitis virus pseudotypes bearing surface proteins of hepatitis B virus.

It has been difficult to propagate and titrate hepatitis B virus (HBV) in tissue culture. We examined whether vesicular stomatitis virus (VSV) pseudotypes bearing HBV surface (HBs) proteins infectious for human cell lines could be prepared. For this, expression plasmids for three surface proteins, L, M, and S, of HBV were made. 293T cells were then transfected with these plasmids either individually or in different combinations. 293T cells expressing HBs proteins were infected with VSVdeltaG*-G, a recombinant VSV expressing green fluorescent protein (GFP), to make VSV pseudotypes. Culture supernatants together with cells were harvested and sonicated for a short time. The infectivities of freshly harvested supernatants were determined by quantifying the number of cells expressing GFP after neutralization with anti-VSV serum and mouse monoclonal antibodies (MAbs) against HBs protein. Among 14 cell lines tested for susceptibility to HBV pseudotype samples, HepG2, JHH-7, and 293T cells were judged to be the most susceptible. Namely, the infectious units (IU) of the culture supernatant samples neutralized with anti-VSV in the absence and presence of anti-HBs S MAbs and titrated on HepG2 cells ranged from 1,000 to 4,000 IU/ml and 200 to 400 IU/ml, respectively, suggesting the presence of VSVdeltaG*(HBV) pseudotypes. This infectivity was inhibited by treatment with lactoferrin or dextran sulfate. Pretreatment of the cells with trypsin or tunicamycin inhibited plating of the pseudotype samples. The HBV pseudotypes can be used to analyze early steps of HBV infection, including the entry mechanism of HBV.