Toshio Hattori

Inhibition of infection of incoming HIV‐1 virus by RNA‐cleaving DNA enzyme

Nine different DNA enzymes (DzV3‐n, n=1–9) targeting the V3 loop region of HIV‐1 HXB2 were synthesized. One of those, DzV3‐9, efficiently cleaved the target in the conserved sequence in the RNA transcript in vitro. DzV3‐9 was stable in the cells and inhibited replication of both NL432 and SF162 strains in U87 cells expressing CD4 and co‐receptors. The inhibitory effect of DNAzyme on incoming HIV‐1 was also demonstrated with pseudotype virions generated by NL432‐based luciferase reporter genes. Thus, an efficient, stable DNAzyme against a functionally important region of HIV‐1 was identified, and it may be useful for prevention of HIV‐1 infection.

Xanthine oxidase inhibition reduces reactive nitrogen species production in COPD airways

Reactive nitrogen species (RNS) have been reported to be involved in the inflammatory process in chronic obstructive pulmonary disease (COPD). However, there are no studies on the modulation of RNS in COPD. It was hypothesised that inhibition of xanthine oxidase (XO) might decrease RNS production in COPD airways through the suppression of superoxide anion production. Ten COPD and six healthy subjects participated in the study. The XO inhibitor allopurinol (300u2005mg·day−1 p.o. for 4 weeks) was administered to COPD patients. RNS production in the airway was assessed by 3‐nitrotyrosine immunoreactivity and enzymic activity of XO in induced sputum as well as by exhaled nitric oxide (eNO) concentration. XO activity in the airway was significantly elevated in COPD compared with healthy subjects. Allopurinol administration to COPD subjects significantly decreased XO activity and nitrotyrosine formation. In contrast, eNO concentration was significantly increased by allopurinol administration. These results suggest that oral administration of the xanthine oxidase inhibitor allopurinol reduces airway reactive nitrogen species production in chronic obstructive pulmonary disease subjects. This intervention may be useful in the future management of chronic obstructive pulmonary disease.

Oligodeoxynucleotides Without CpG Motifs Work as Adjuvant for the Induction of Th2 Differentiation in a Sequence-Independent Manner

The outcomes of immune responses are regulated by various parameters including how Ags are handled by APCs. In this study, we describe the intrinsic immunomodulatory characteristics of oligodeoxynucleotides (ODNs) that improve the Ag presentation by APCs. ODNs (20-mer) containing CpG motifs induced strong Th1-skewed responses. In contrast, those without CpG motifs enhanced cytokine production by effector Th cells without particular skewing toward Th1 responses or induced the differentiation of unprimed CD4+ T cells toward Th2 cells. These functional features were prominently envisaged when ODNs were conjugated to the Ag, and were underlain by the facilitated binding of ODN-conjugated Ag to Ia+ cells. Despite the functional differences between ODNs with CpG motifs and those without CpG motifs, both ODNs bound to Ia+ cells with similar affinity and kinetics. Immunoenhancing activities of the ODNs were not sequence-dependent; the characteristics, including the facilitation of Ag capture, enhancement of effector Th cell responses, and induction of Th2 cells, were shared by randomly synthesized ODNs conjugated to Ag. This is the first study suggesting that ODNs, independent of the sequences, enhance immune responses through the promoted capture of ODN-conjugated Ag by APCs.

Regiospecific oxygenations during ring cleavage of a secondary metabolite, 3,4-dimethoxybenzyl alcohol catalysed by lignin peroxidase

Enzymatic oxidation of veratryl alcohol yielded a new ring cleavage product (δ‐lactone) in addition to the two known γ‐lactone products. The experiment with 18O‐enriched water and dioxygen clearly showed that one oxygen atom each from water and dioxygen is specifically incorporated into the cleavage product at the original C3 or C4 position of 3,4‐dimethoxybenzyl alcohol. A new type of reaction mechanism proposed for the ring cleavage of this compound is rationally explained in good accord with the one‐electron transfer mechanism.

Modification of virus infectivity by cytoplasmic tail of HIV-1 TM protein.

Envelope glycoprotein incorporation is an essential process in formation of infectious particles of human immunodeficiency virus. Accumulated data have indicated that the cytoplasmic tail of Env gp41 is required for efficient incorporation. By analyzing mutant viruses with truncated cytoplasmic tails, we found that the domain was required in a cell-type-dependent manner for maintaining virus infectivity. Although the viruses with truncated cytoplasmic tails produced from HeLa, A3.01 and SupT1 cells showed a greatly reduced infectivity, those from SW480 and MT-4 cells retained a significant infectivity. To clarify the different effect of the cytoplasmic tail mutation on virus infectivity, we performed biochemical studies on the virions produced from HeLa and SW480 cells. Although the truncation of cytoplasmic tail appeared to reduce the Env incorporation in both cell lines, it caused a significant incorporation of Env precursor with HeLa cells. The results suggested that the cytoplasmic tail regulated selective incorporation of processed Env into virions in a cell-type-dependent manner.

Adult T-cell Leukemia Derived from S100β Positive Double-Negative (CD4− CD8−) T Cells

Adult T-cell leukemia (ATL) is a mature T-cell malignancy which is caused by human T lymphotropic virus type-I (HTLV-I). Most of the ATL cells are CD3+, CD4+, CD8−, and T-cell receptor (TCR) aβ+ and also express activated antigens such as HLA-DR and inter-leukin-2 receptor (IL2R) α chain (CD25). Diminished surface expression of the TCRαβ/CD3 complex is a specific feature of ATL cells. Since the gene transcript for each chain of this complex has been detected and surface expression of this complex is further decreased, accompanied by the induction of IL2Ra chain, after stimulation with anti-CD3 monoclonal antibody (mAb), the TCRαβ/CD3 complex is considered to be down-modulated in vivo. We recently reported four ATL patients whose leukemic cells were CD4−, CD8− (double-negative; DN), TCRαβ+. These DN-ATL cells expressed S100αβ protein which was not detected in CD4+ ATL cells. Similar to CD4+ ATL cells, surface expression of the TCRαβ/CD3 complex on DN-ATL cells was decreased in vivo despite the lack of CD4 ...

The possible involvement of CXCR4 in the inhibition of HIV-1 infection mediated by DP178/gp41.

The N‐ (N36/DP107) and C‐terminal peptides (C34/DP178) from two α‐helical domains of human immunodeficiency virus type 1 (HIV‐1) gp41 inhibited HIV infection. A single‐round infection using pseudotyped virus clarified that a greater amount of gp41‐derived peptides was necessary for the inhibition of R5 virus (ADA) infection than for that of X4 virus (LAI) infection. Furthermore, R5X4 virus (89.6) infection via CCR5 needs more peptides for inhibition than its infection via CXCR4 does. A high sensitivity of X4 virus was partially ascribed to the inhibition of the 12G5 binding to CXCR4 by DP178LAI.

Presence of human B-lymphocyte antigens on adult T-cell leukemia cells.

Abstract Leukemic cells from nine patients with adult T-cell leukemia were examined for Fcγ and Fcμ receptors, human B-lymphocyte antigens (HBLA), and effect on PWM-induced B-cell differentiation. Fcγ receptors were detected in one and Fcμ receptors in another. Leukemic cells in both cases bore HBLA and expressed a suppressive effect on PWM-induced B-cell differentiation. However, leukemic cells from the remaining seven cases did not bear Fc receptors. In four, leukemic cells possessed HBLA and all expressed a suppressive effect on the PWM system. Some leukemic cells which did not express HBLA had the same effect on the PWM system. The origin of adult T-cell leukemia was discussed.

Triazine dyes inhibit HIV-1 entry by binding to envelope glycoproteins.

We have attempted to purify envelope (Env) glycoproteins of human immunodeficiency virus (HIV) from the culture supernatants of CHO‐Sec cells that secreted truncated 140‐kDa precursor and mature 120‐kDa Env glycoproteins. The concentrated culture supernatants were applied to a column coupled with cibacron blue 3GA (CB3GA) to separate albumin from the Env proteins because CB3GA, a triazine dye, has been known to have a high affinity to albumin. Unexpectedly, Env proteins as well as albumin bound to the column, and the bound Env proteins were eluted by increasing the ionic strength using KC1. Gp120 was eluted at 0.5–0.9 m of KC1, while a higher concentration (0.9–1.5 m) was necessary for the elution of gp140. The agarose gel coupled with reactive red 120 (RR120), another triazine dye with similar characteristics, also retained both Env proteins, and the bound Env proteins could be eluted in a similar manner. In addition, these agents inhibited syncytium formation caused by HTLV‐IIIB and HTLV‐IIMN. Inhibition was also seen when a virus‐free fusion assay between Env protein expressed in CHO cells and fluorescent labeled SupT1 cells were used. These findings indicate that triazine dyes bind to the functional regions of Env proteins of HIV‐1 that play important role(s) for HIV infection.

A VSV-G Pseudotyped HIV Vector Mediates Efficient Transduction of Human Pulmonary Artery Smooth Muscle Cells

Attempts were made to infect human vascular smooth muscle cells derived from the pulmonary artery (hPASMC) with two different human immunodeficiency virus (HIV) vector systems. ADA/Luc or HXB2/Luc were generated by cotransfection of luciferase reporter gene vector, pNL4‐3‐Lue‐E–R–, and one of two envelope expressing vectors, pSMADA (R5) or pSMHXB2 (X4). The VSV‐G/Luc or VSV‐G/GFP were produced by a three‐plasmid expression system which consisted of vesicular stomatitis virus G protein (VSV‐G) expressing vector, packaging plasmid, and one of two reporter genes (pHR‐CMV‐Luc or pHR‐CMV‐GFP). We used hPASMC, U87.CD4.CCR5 and U87.CD4.CXCR4 for infection. Neither ADA/Luc nor HXB2/Luc could infect hPASMC, though they could infect U87.CD4 with corresponding coreceptors. On the other hand, the transduction of both VSV‐G/Luc and VSV‐G/GFP to hPASMC was remarkable. At day 3, the relative proportion of positive cells of hPASMC infected with VSV‐G/GFP was 15%. The above finding indicates a direct role of HIV‐1 infection in pulmonary hypertension ‘a rare complication of HIV‐1 infection’ and HIV‐based vectors could introduce foreign genes into hPASMC for gene therapy of pulmonary hypertension.