An essential role for the His-8 residue of the SDF-1alpha-chimeric, tropism-redirected Env protein of the Moloney murine leukemia virus in regulating postbinding fusion events

BACKGROUND: To use retroviral vectors for the cell-specific delivery of genes, it is necessary to redirect their receptor tropism to cell-specific receptors. Previously, we reported that a Moloney murine leukemia virus (MLV) retroviral vector containing a human stromal-derived factor-1alpha (SDF-1alpha)-chimeric envelope protein (Env) (S3) acquired the ability to transduce human cells via CXCR4, the cognate receptor for SDF-1alpha, while retaining the ability to transduce mouse cells via mCAT1. METHODS: We constructed expression plasmids for derivatives of the S3 Env protein; S3-D84K containing an Asp-84-to-Lys (D84K) substitution, S3-H8R-D84K containing D84K and an additional His-8-to-Arg substitution, and S3-D84K-RY containing D84K and additional Gln-227-to-Arg plus Asp-243-to-Tyr substitutions which have been suggested to suppress the loss of function of His-8. Cellular expression, virion incorporation, and entry functions of these derivatives were investigated. RESULTS: All three derivatives were incorporated into virions. The S3-D84K vector lost its ecotropism, but could transduce CXCR4-expressing human and mouse cells at titers of 10(3) to 10(4) colony-forming units (cfu)/ml. The S3-H8R-D84K vector did not show transduction, although its Env protein could bind to CXCR4. The transduction titer of the S3-D84K-RY vector via CXCR4 was slightly lower than that of the S3-D84K vector. These results indicate that the His-8 residue of the S3-D84K Env protein is indispensable and may be fully functional in postbinding membrane fusion. CONCLUSIONS: Insertion of a ligand at Pro-79 of the Moloney MLV Env protein has proved to be a valuable strategy for constructing direct targeting retroviral vectors, since it permits the formation of a redirected Env protein without ecotropism, and it does not disrupt the function of the essential His-8 residue.     

Synergistic effect of interleukin-15 and interleukin-12 on antitumor activity in a murine malignant pleurisy model

 Interleukin(IL)-15, which uses IL-2 receptor (R) β and γ chains for signal transduction, shares many of the biological activities of IL-2. We examined the effects of exogenous IL-15 on protection in a murine malignant pleurisy model using BALB/c mice and syngeneic MethA fibrosarcoma (MethA). Intrapleural administration of IL-15 significantly prolonged the survival time of mice after an intrapleural inoculation of MethA, whereas the same dose of IL-2 did not. The in vivo antitumor effect of IL-15 was synergistically enhanced by additive administration of IL-12. Combination therapy of IL-15 and IL-12 protected mice from death from bloody pleural fluid. Such treatment induced marked increases in the number of CD3^-IL-2Rβ⁺ cells corresponding to natural killer (NK) cells and the production of interferon γ (IFNγ) by T cells in the thoracic exudate cells (TEC). Administration of anti-IFNγ mAb partly inhibited the protective effect of a combination of IL-15 and IL-12. A tumor-neutralizing (Winn) assay revealed that the antitumor activity of effector cells in the TEC was abrogated by treatment with anti-CD8 mAb or anti-asialoGM1 Ab plus complement. Thus, treatment with IL-15 in combination with IL-12 may enhance the activities of NK and CD8⁺ T cells in the TEC, providing strong antitumor activity against the malignant pleurisy. These results suggest that IL-15 together with IL-12 may have potential for the immunotherapy of some types of malignant pleurisy. Received: 13 July 1999 / Accepted: 3 December 1999     

Structural and functional analysis of pTB6 from Bifidobacterium longum

The complete nucleotide sequence for pTB6 (3,624 bp) from Bifidobacterium longum was determined. This plasmid is 95% homologous in nucleotide (nuc) sequence, and also 92% in RepB aa sequence, to rolling circle replication (RCR) plasmids pKJ36 and pB44, suggesting that pTB6 replicates by the rolling circle mechanism. The putative MembB, MobA, and protein encoding from orf (Orf) I detected were nonessential for plasmid replication. We constructed an immobile shuttle vector from pTB6 and pUC18, which transformed B. longum with a high efficiency of 2.5 x 10(6) transformants/microg DNA.     

Overexpression of IL-15 in vivo enhances Tc1 response, which inhibits allergic inflammation in a murine model of asthma

IL-15, a pleiotropic cytokine, is involved in the inflammatory responses in various infectious and autoimmune diseases. We have recently constructed IL-15-transgenic (Tg) mice, which have an increased number of memory-type CD8+ T cells in the peripheral lymphoid tissues. In the present study, we found that eosinophilia and Th2-type cytokine production in the airway were severely attenuated in OVA-sensitized IL-15-Tg mice following OVA inhalation. IL-15-Tg mice preferentially developed Tc1 responses mediated by CD8+ T cells after OVA sensitization, and in vivo depletion of CD8+ T cells by anti-CD8 mAb aggravated the allergic airway inflammation in IL-15-Tg mice following OVA inhalation. Adoptive transfer of CD8+ T cells from OVA-sensitized IL-15-Tg mice into normal mice before OVA sensitization suppressed Th2 response to OVA in the normal mice. These results suggest that overexpression of IL-15 in vivo suppresses Th2-mediated-allergic airway response via induction of CD8+ T cell-mediated Tc1 response.     

Magnesium influx enhanced by nitric oxide in hypertensive rat proximal tubule cells

An abnormal handling of renal magnesium has been suggested to cause salt-sensitive hypertension. The filtered magnesium is first reabsorbed in the proximal tubule. Amiloride has been shown to enhance renal magnesium conservation, but the regulatory mechanisms are unknown yet. High-salt (8% NaCl) diet decreased serum magnesium concentration, while increased urinary magnesium in Dahl salt-sensitive (DS) rat. Furthermore, the expression of nitric oxide synthase type 3 and nitric oxide (NO) content were decreased in high-salt loaded DS rat. In isolated proximal tubule cells, amiloride (0.1 mM) increased intracellular free magnesium concentration ([Mg(2+)](i)). However, the net [Mg(2+)](i) increase in the high-salt loaded DS rat was smaller than other groups. NOR1 (0.1 mM), a NO donor, restored the increase of [Mg(2+)](i) to the same level of other groups. On the contrary, L-NMMA (0.1 mM), an inhibitor of NO production, inhibited the increase of [Mg(2+)](i) in all groups. These results suggest that intracellular NO has an important role to up-regulate amiloride-elicited magnesium influx.     

A novel mitogen-activated protein kinase phosphatase is an important negative regulator of lipopolysaccharide-mediated c-Jun N-terminal kinase activation in mouse macrophage cell lines

We have isolated a cDNA homologous to known dual-specificity phosphatases from a mouse macrophage cDNA library and termed it MKP-M (for mitogen-activated protein kinase phosphatase isolated from macrophages). Three other presumed splice variant isoforms have also been identified for MKP-M. The longest and most abundant mRNA contains an open reading frame corresponding to 677 amino acids and produces an 80-kDa protein. The deduced amino acid sequence of MKP-M is most similar to those of hVH-5 (or mouse M3/6) and VHP1, a Caenorhabditis elegans tyrosine phosphatase. It includes an N-terminal rhodanase homology domain, the extended active-site sequence motif (V/L)X(V/I)HCXAG(I/V)SRSXT(I/V)XXAY(L/I)M (where X is any amino acid), and a C-terminal PEST sequence. Northern blot analysis revealed a dominant MKP-M mRNA species of approximately 5.5 kb detected ubiquitously among all tissues examined. MKP-M was constitutively expressed in mouse macrophage cell lines, and its expression levels were rapidly increased by lipopolysaccharide (LPS) stimulation but not by tumor necrosis factor alpha (TNF-alpha), gamma interferon, interleukin-2 (IL-2), or IL-15 stimulation. Immunocytochemical analysis showed MKP-M to be present within cytosol. When expressed in COS7 cells, MKP-M blocks activation of mitogen-activated protein kinases with the selectivity c-Jun N-terminal kinase (JNK) > p38 = extracellular signal-regulated kinase. Furthermore, expression of a catalytically inactive form of MKP-M in a mouse macrophage cell line increased the intensity and duration of JNK activation and TNF-alpha secretion after LPS stimulation, suggesting that MKP-M is at least partially responsible for the desensitization of LPS-mediated JNK activation and cytokine secretion in macrophages.     

Potent anti-metastatic activity of dimeric sesquiterpene thioalkaloids from the rhizome of Nuphar pumilum

The methanolic extract and its alkaloid fraction from the rhizomes of Nuphar pumilum inhibited invasion of B16 melanoma cells across collagen-coated filters in vitro. Dimeric sesquiterpene thioalkaloids with the 6-hydroxyl group, 6-hydroxythiobinupharidine, 6,6′-dihydroxythiobinupharidine, and 6-hydroxythionuphlutine B, showed potent activity with IC₅₀ values of 0.029, 0.087, and 0.36 μM, respectively, but dimeric sesquiterpene thioalkaloids lacking the 6-hydroxyl group (thiobinupharidine, neothiobinupharidine, syn-thiobinupharidine sulfoxide, thionuphultine B β-sulfoxide, and neothiobinupharidine β-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, deoxynupharidine, 7-epideoxynupharidine, and nupharolutine) showed weak activity. The alkaloid fraction (20 mg/kg/d, po) and the principal dimeric sesquiterpene thioalkaloid 6-hydroxythiobinupharidine (5 mg/kg/d, po) significantly inhibited lung tumor formation by more than 90% 10 days after injection of B16 melanoma cells in mice.     

Tyk2-signaling plays an important role in host defense against Escherichia coli through IL-23-induced IL-17 production by gammadelta T cells

Tyrosine kinase 2 (Tyk2), a member of the JAK-signal transducer family, is involved in intracellular signaling triggered by various cytokines, including IL-23. We have recently reported that resident gammadelta T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23. In this study, we examined importance of Tyk2-mediated signaling in the IL-17 production by gammadelta T cells using Tyk2 deficient (-/-) mice. Gammadelta T cells in the peritoneal cavity of Tyk2(-/-) mice displayed effecter/memory phenotypes and TCR V repertoire similar to those in Tyk2(+/+) mice and produced comparable level of IL-17 to those in Tyk2(+/+) mice in response to PMA and ionomycin, indicating normal differentiation to IL-17-producing effectors in the absence of Tyk2-signaling. However, gammadelta T cells in Tyk2(-/-) mice produced less amount of IL-17 in response to IL-23 in vitro than those in Tyk2(+/+) mice. Similarly, gammadelta T cells in the peritoneal cavity of Tyk2(-/-) mice showed severely impaired IL-17 production after an i.p. infection with E. coli despite comparable level of IL-23 production to Tyk2(+/+) mice. As a consequence, Tyk2(-/-) mice showed a reduced infiltration of neutrophils and severely impaired bacterial clearance after Escherichia coli infection. These results indicate that Tyk2-signaling is critical for IL-23-induced IL-17 production by gammadelta T cells, which is involved in the first line of host defense by controlling neutrophil-mediated immune responses.     

Molecular determinants of sensitivity and conductivity of human TRPM7 to Mg2+ and Ca2+

It is known that extracellular Mg(2+) and Ca(2+) can permeate TRPM7 and at the same time block the permeation by monovalent cations. In the present study, we examined the molecular basis for the conductivity and sensitivity of human TRPM7 to these divalent cations. Extracellular acidification to pH 4.0 markedly reduced the blocking effects of Mg(2+) and Ca(2+) on the Cs(+) currents, decreasing their binding affinities: their IC(50) values increased 510- and 447-fold, respectively. We examined the effects of neutralizing each of four negatively charged amino acid residues, Glu-1047, Glu-1052, Asp-1054 and Asp-1059, within the putative pore-forming region of human TRPM7. Mutating Glu-1047 to alanine (E1047A) resulted in non-functional channels, whereas mutating any of the other residues resulted in functionally expressed channels. Cs(+) currents through D1054A and E1052A were less sensitive to block by divalent cations; the IC(50) values were increased 5.5- and 3.9-fold, respectively, for Mg(2+) and 10.5- and 6.7-fold, respectively, for Ca(2+). D1059A also had a significant reduction, though less marked compared to the reductions seen for D1054A and E1052A, in sensitivity to Mg(2+) (1.7-fold) and Ca(2+) (3.9-fold). The D1054A mutation largely abolished inward currents conveyed by Mg(2+) and Ca(2+). In the E1052A and D1059A mutants, inward Mg(2+) and Ca(2+) currents were sizable but significantly diminished. Thus, it is concluded that in human TRPM7, (1) both Asp-1054 and Glu-1052, which are located near the narrowest portion in the pore's selectivity filter, may provide the binding sites for Mg(2+) and Ca(2+), (2) Asp-1054 is an essential determinant of Mg(2+)and Ca(2+) conductivity, and (3) Glu-1052 and Asp-1059 facilitate the conduction of divalent cations.