Yoichi Fujii

HIV-1 nef suppression by virally encoded microRNA

BackgroundMicroRNAs (miRNAs) are 21~25-nucleotides (nt) long and interact with mRNAs to trigger either translational repression or RNA cleavage through RNA interference (RNAi), depending on the degree of complementarity with the target mRNAs. Our recent study has shown that HIV-1 nef dsRNA from AIDS patients who are long-term non-progressors (LTNPs) inhibited the transcription of HIV-1.ResultsHere, we show the possibility that nef-derived miRNAs are produced in HIV-1 persistently infected cells. Furthermore, nef short hairpin RNA (shRNA) that corresponded to a predicted nef miRNA (~25 nt, miR-N367) can block HIV-1 Nef expression in vitro and the suppression by shRNA/miR-N367 would be related with low viremia in an LTNP (15-2-2). In the 15-2-2 model mice, the weight loss, which may be rendered by nef was also inhibited by shRNA/miR-N367 corresponding to suppression of nef expression in vivo.ConclusionsThese data suggest that nef/U3 miRNAs produced in HIV-1-infected cells may suppress both Nef function and HIV-1 virulence through the RNAi pathway.

Dietary n-3 fatty acid deficiency decreases nerve growth factor content in rat hippocampus

Dietary deprivation of alpha-linolenic acid (n-3) through two generations has been shown to lower performance in an operant-type brightness-discrimination learning test in rats. Here, we examined a possible correlation between nerve growth factor (NGF) content and n-3 fatty acid status in the brain. Female rats were fed a semipurified diet supplemented with safflower oil (n-3 fatty acid-deficient) and their offsprings were fed a diet supplemented with either 3% safflower oil (Saf group) or a mixture of 2.4% safflower oil plus 0.6% ethyl eicosapentaenoate (Saf+EPA group) after weaning. The brain docosahexaenoic acid (22:6n-3, DHA) content in the Saf group was less than half of that in the Per group fed a diet supplemented with 3% perilla oil (n-3 fatty acid-sufficient) throughout the duration of the experiment. The DHA level of the Saf+EPA group was restored to the level of the Per group. However, the NGF contents in the hippocampus of the Saf and Saf+EPA groups were half that of the Per group. In the piriform cortex, the NGF content tended to be higher in the Saf and Saf+EPA groups than in the Per group. These results indicate that dietary n-3 fatty acid deficiency and restoration affect NGF levels differently among different brain regions.

Soluble Nef antigen of HIV-1 is cytotoxic for human CD4+ T cells

We have previously shown that Nef‐gene 10 fusion protein induces marked growth arrest of human primary CD4+ T cells. Here, in vitro cytostatic and cytotoxic activities of human immunodeficiency virus type 1 (HIV‐1) Nef against CD4+ T cells were extensively investigated. Growth of human CD4+ cells was inhibited significantly just by the addition of purified full‐length Nef to cultures. When Nef was cross‐linked by anti‐Nef antibodies, it became very cytocidal for CD4+ T cells. A high percentage of sera from HIV‐1‐infected individuals contained soluble Nef. Thus, soluble Nef in vivo may play an important role in immunodysfunction of CD4+ T lymphocytes in HIV‐1 infection.

DNA methylation analysis at distal and proximal promoter regions of the oestrogen receptor gene in breast cancers

SummaryOestrogen receptor α (ER-α) gene has two specific promoters, distal (P0) and proximal (P1), which induce almost identical transcripts in size due to different splicing. We examined the methylation at both promoter regions of the ER-α gene using HpaII, a methylation-sensitive restriction enzyme, prior to polymerase chain reaction (PCR) amplification. To confirm the results of PCR-based methylation analysis, Southern hybridization was also performed. Twenty of 29 patients with ER-α-positive tumours and five of 27 with ER-α-negative tumours were unmethylated at the P1 promoter region of the ER-α gene. The incidence of methylation was highly negatively correlated with ER-α expression (P = 0.0002). A similarly negative correlation was observed at the P0 promoter region of the ER-α gene (P = 0.0154). Additionally, the tumours with the ER-α gene hypermethylated at both promoter regions had definitely negative ER-α values. It was suggested that this epigenetic change might control ER-α expression, and might play an important role in the loss of hormone-dependence in breast cancer.

Double‐Stranded nef RNA Interferes with Human Immunodeficiency Virus Type 1 Replication

RNA interference (RNAi) has been reported to be post‐transcriptional gene silencing (PTGS) by approximately 500 nucleotide‐(nt)‐long double‐stranded (ds) RNA that specifically targets homologous sequences of messenger RNA. In this report, we describe inhibition of HIV‐1 transcription by synthetic dsRNAs constructed with mutated nef genes (nef dsRNAs) derived from long‐term non‐progressors (LTNPs) using cotransfection of the target gene‐expressing plasmid and dsRNA. The effects of nef dsRNAs were examined with luciferase (Luc) reporter which is combined with the HIV‐1 (SF2) LTR in persistently HIV‐1‐infected T cell and macrophage cell lines. At 48 hr, a defective nef dsRNA (556 nt) suppressed Luc activity more potently than did SF2 full‐length nef dsRNA (744 nt), suggesting that approximately 500 nt‐long nef dsRNA could interfere with the HIV‐1 transcription.

HIV-1 Nef protein in the nucleus influences adipogenesis as well as viral transcription through the peroxisome proliferator-activated receptors.

Background: Although the HIV-1 Nef protein (27 kDa) localizes primarily in cytoplasm, there is considerable evidence suggesting its occasional localization in the nucleus. Nef is known to play an important role in transcriptional events and viral replication, but the actual target of Nef in the nucleus remains to be identified. Objective: To examine the functional roles of Nef in the nucleus and its possible interactions with other unknown factors in the nucleus. Methods: High-density microarray analysis was used to screen directly the unique functions of Nef on host gene transcription. The nuclear localization of Nef and its effects on the expression of peroxisome proliferator-activated receptors (PPAR) was examined using PPAR promoter/reporter assay and immunoblotting. A long terminal repeat/reporter assay was used to investigated the effects of Nef and PPAR on viral transcription. Results: Nef in the nucleus suppressed PPARγ expression and reduced fatty acid levels in human T and macrophage cell lines. Expression of Nef or PPAR suppressed viral replication; the effect of PPARγ or retinoid X receptor-α on viral replication were reduced by coexpression of Nef in MT(−)4 T cells. Conclusion: Nef may be involved in both viral replication and the wasting syndrome associated with AIDS.

Estrogen receptor-positive breast cancer in Japanese women: trends in incidence, characteristics, and prognosis

BACKGROUND The incidence of breast cancer in Japanese women has doubled in all age groups over the past two decades. PATIENTS AND METHODS We examined the characteristics of the tumors treated in three time periods between 1982 and 2010. Estrogen receptor (ER), progesterone receptor (PgR) and HER2 status were assessed by immunohistochemistry. Correlation of hormone receptor levels with clinicopathological factors and prognosis was analyzed in ER-positive, HER2-negative breast cancer in two age groups (≤50 years versus >50 years). RESULTS The frequency of ER-positive breast cancer in women aged 50 years or younger increased greatly over the interval studied (1982-1991: 52.5%, 1992-2001: 72.6%, 2002-2010: 87.1%, P < 0.0001). The frequency of ER-positive tumors also significantly increased in women over 50 years of age (1982-1991: 69.4%, 1992-2001: 73.3%, 2002-2010: 78.6%, P = 0.029). In ER-positive, HER2-negative breast cancer, tumor grade was negatively correlated with expression levels of ER and PgR. Prognosis for patients with ER-positive, HER2-negative disease significantly improved over time, due to advances in adjuvant therapies. CONCLUSION It is necessary to establish risk factors, both genetic and environmental, capable of predicting the risk of ER-positive breast cancer and thus enable the efficient selection of candidates for hormone receptor-targeted chemoprevention.

Human immunodeficiency virus type 1 Nef protein on the cell surface is cytocidal for human CD4+ T cells

We have previously shown that the carboxyl‐terminal region of human immunodeficiency virus type 1 (HIV‐1) Nef antigen present on the outer surface of virus‐infected cells has affinity for uninfected T cells. Here, the in vitro cytotoxic potential of HIV‐1 Nef on the T cell surface against CD4+ T cells was investigated in detail. Human T cells expressing Nef on the cell surface by transfection with non‐infectious mutant HIV‐1 proviruses were demonstrated to kill CD4+ T cells effeciently. Furthermore, it was shown that the carboxyl‐terminal portion of Nef was cytotoxic for CD4+ T cells and that monoclonal antibody against the carboxyl‐terminal region of Nef inhibited Nef induced‐cytolysis. Thus, we concluded that Nef protein on CD4+ T cells may play an important role in the specific loss of CD4+ T lymphocytes during HIV‐1 infection.

Expression of human immunodeficiency virus type 1 Nef antigen on the surface of acutely and persistently infected human T cells.

The authors have previously shown the role of human immunodeficiency virus type 1 (HIV-1) Nef protein as a growth inhibitor to CD4+ T lymphocytes. We now report that the Nef antigen is partly expressed on both acutely and persistently infected human T cells. To investigate the cell surface expression of the Nef antigen, murine monoclonal antibodies (mAbs) were prepared by immunization with a recombinant Nef protein. The recombinant Nef was expressed by a baculovirus expression system as a truncated Nef protein with 22 kDa containing the middle to the C-terminus. Three clones were found to produce mAbs with IgM isotype against Nef protein by ELISA with the same truncated Nef. All these mAbs reacted on immunoblotting with two forms of Nef, 25 kDa and 27 kDa, in an HIV-1-infected human T-cell line and with the 27 kDa Nef in retroviral vector-infected mouse fibroblasts expressing a full-length Nef protein. Membrane immunofluorescence and flow cytometry with these mAbs revealed the Nef antigen to be expressed, at least in part, on the surface of these cells. Thus, the cell-surface form of Nef might play an important role in the selective depletion of CD4+ cells in HIV-1 infection.

Dietary, but not Topical, Alpha-linolenic Acid Suppresses UVB-induced Skin Injury in Hairless Mice when Compared with Linoleic Acid¶

Abstract Peroxidizability of fatty acids in the air is roughly proportional to the number of double bonds, but in vivo peroxidation proceeds in a more complex manner. Here, we compared the effects of dietary and topically applied oils enriched with linoleic acid (LA, 18:2n-6) or alpha-linolenic acid (ALA, 18:3n-3) on UV-induced skin injury in a strain of hairless mice. The UVB-induced erythema score was significantly lower in mice with topically applied creams containing LA and ALA than in mice with the basal cream; no significant increase in the score was detected in the ALA group compared with the LA group. However, dietary ALA inhibited the increase in erythema score after UVB irradiation compared with LA. The peroxidizability index of the skin total lipids was significantly higher, but UVB-induced prostaglandin E2 (PGE2) production was significantly lower in the group fed an ALA-rich diet compared with the group fed an LA-rich diet. The levels of thiobarbituric acid–reactive substances, estimated in the presence of butylated hydroxytoluene in the assay mixture, were not affected by UVB treatment or by the dietary fatty acids, but the severity of the skin lesion was associated with PGE2 levels. These results indicate that the type of fatty acids, n-6 or n-3, is critical for the suppression of UVB-induced skin lesion when the skin fatty acids are modified by dietary manipulation. Anti-inflammatory activity of dietary flaxseed oil with relatively high ALA and low LA contents was demonstrated in UVB-irradiated hairless mice.