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Dimethyl fumarate (DMF), a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, has been proven effective for the systemic treatment of multiple sclerosis. The aim of this study is to evaluate the anti-inflammatory effects of Nrf2 activators on human renal mesangial cells (HRMCs) and the development of lupus nephritis (LN) in mice. To assess Nrf2 activation in vitro, HRMCs were treated with safe doses of Nrf2 activators and prednisolone. The expression levels of Nrf2 and its target genes were measured using quantitative reverse transcription PCR and enzyme-linked immunosorbent assay. The anti-inflammatory effects of these compounds were assessed by measuring tumor necrosis factor alpha-induced cytokine secretion. Experimental LN was induced in female BALB/c mice by a single intraperitoneal injection of pristane. The urine albumin-to-creatinine ratio was measured at 20 weeks after injection. Pathological changes as well as protein and mRNA expression levels were assessed in the kidney obtained at the experimental end point. Oral administration of DMF or prednisolone to these mice was initiated after pristane injection. Nrf2 activators such as sulforaphane and DMF showed anti-inflammatory effects in HRMCs, whereas glucocorticoid (prednisolone) showed partial effects. Moreover, DMF ameliorated the development of kidney diseases in pristane-induced LN mice, whereas glucocorticoid had no effect. Nrf2 activators showed stronger anti-inflammatory and organ-protective effects than glucocorticoid in the kidney. Thus, Nrf2 activators are potential therapeutic targets in glucocorticoid-resistant LN in humans.

Introduction:Diagnosis of cardiogenic thrombosis is important because anticoagulant therapy is recommended for its secondary prevention. However, it is difficult to make a clear distinction in the pathogenesis between atherothrombosis and cardiogenic thrombosis.Hypothesis:Role of macrophage in thrombus formation is different between atherothrombosis and cardiogenic thrombosis.Methods:The Macrophage in Thrombus (MITO) study is a prospective observational study examining pathological and biological differences of arterial thrombus between atherothrombosis and cardiogenic thrombosis. Patients with ST elevation myocardial infarction (STEMI) or acute ischemic stroke (AIS) whose solid thrombus could be retrieved from the infarct-related artery were enrolled. Patients were divided into 2 groups as follows; STEMI with sinus rhythm: Group A (presumed atherothombosis) and AIS with atrial fibrillation (presumed cardiogenic thrombosis): Group C, and compared macrophage score as follows; 1:almost no macrophages detected byх40; 2:difficult to detect the macrophages in х20 but detected by х40; and 3:easy to detect macrophages in х20 in the thrombus. Serum levels of soluble CD163 were measured all the patients.Results:The score for CD163 positive macrophage of Group C (n=33) was significantly higher than that of Group A (n=32) (p<0.01): Figure. Serum level of soluble CD163 in Group C (median 603 ng/ml, IQR 405, 788) tended to be higher than that in Group A (median 520 ng/ml, IQR: 314, 634); p=0.08.Conclusions:CD163 positive macrophages are more involved in the thrombus formation of cardiogenic thrombus than that of atherothrombosis. Detection of CD163 positive macrophages are likely to help discriminate between atherothrombosis thrombus versus cardiogenic thrombus.

Homogeneous Bi-Pb alloy samples in the ε-phase are treated under mega-gravity centrifugal acceleration field. Atomic sedimentation in solid and associated partial crystallization occurs during the treatment. Small differences of densities in the ε-phase are enhanced under the mega-gravity, and they cause the separation of the ε-phase between fully packed hcp structure of the Bi3Pb7 and partially defected hcp structure of the Bi3Pb7-δ. The compositional graded superconducting alloy is obtained in the latter. Partially melt-growth occurs in the latter and microstructure of Pb is formed along the gravity field associating with the formation of highly oriented the (211) phase of Bi3Pb7-δ. The superconductivity parallel to gravity is Pb, and perpendicular to gravity is Bi3Pb7-δ as the result. Superconducting properties differs between the separated two phases.

Ultracentrifuge experiments were performed on the twinned Y1Ba2Cu3O7-x (Y123) single crystal at much lower temperatures than the melting point. Two layers structure with slightly different compositions was observed in the sample ultracentrifuged at 250℃(380,000 G), which might be due to the sedimentation of atoms. In the strong gravity layer, it was found that the Y123 phase disappeared, and unknown XRD peaks appeared. Decomposition occurred in the sample ultracentrifuged at 400℃.

A visible four-layers structure with anomalous nano-sturucture was formed from a homogeneous -phase Bi3Pb7 intermetallic compound under a strong gravitational field (1.02x106 G, 130°C, 100 hours). In the 4th layer (lowest-gravity region), pure Bi particles precipitate. In the 2nd 3rd layers, composition graded structures, where Pb content increased along the gravity direction, were formed. It was found that the very broad XRD peak appeared in the 2nd layer, which indicated that an amorphous structure was contained.

An atomic-scale graded structure has been formed by sedimentation of substitutional atoms under an ultra-strong gravitational field of 1 million G level in alloys and compounds. In this study, we investigate the sedimentation of impurity atoms in semiconductor materials under a strong gravitational field. High-temperature ultracentrifuge experiments (0.59×106 G, 400°C, 60 hours) have been performed on an InSb single crystal wafer which surface was coated with Ge by means of Physical Vapor Deposition (PVD). It was observed that the penetration depth of diffused Ge atoms under the gravitational field was several times larger than under terrestrial field at the same temperatures.

The atomic-scale graded structure of In-Pb alloy was formed by an ultracentrifuge under a gravitational field of 0.81 x 106 g for 100 hours at 150 ℃ in solid state. The isotope ratio measurements were performed on the centrifuged sample with secondary ion mass spectrometer (SIMS, CAMECA IMS-6f). 206Pb/208Pb and 207Pb/208Pb isotope ratio changed with negative gradient in the direction of centrifugal force approximately 1.5% and 0.8%, respectively. There was a tendency that the heavy 208Pb isotope abundance increased and the light 206Pb isotope abundance decreased in the direction of centrifugal force. Three-isotope diagram of 206Pb/208Pb versus 207Pb/208Pb proved that the isotope fractionation depends on the isotopic mass difference. These results showed that a strong gravitational field not only affected the inter-diffusion but also the self-diffusion in this alloy by causing isotope fractionation effect, which was dependent on the mass-difference.

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrsgene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrsmice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrsflox/flox;SynI-cremice. Notably, the hrsflox/flox;SynI-cremice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrsflox/flox;SynI-cremice. These data suggest that Hrs plays an important role in neural cell survival in vivoand provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.

In this study, we investigate the crystalline states and conditions for the grain-refinement of Bi70Sb30 (at.%) alloy. It was considered under an ultra strong gravity field, the crystals were fine-grained from the primary grain sizes of several mm to several tens of mm, and the crystal growth followed with formation of graded-composition structure due to sedimentation of atoms along the direction of gravity. It was found that for the crystal-grain refinement in Bi70Sb30 alloy the minimum gravitational field and the minimum time duration were at least 160,000 G and 10 hours, respectively at about 200 ºC.

Ultra-strong gravitational field can induce sedimentation of even atoms in condensed matter. We had realized sedimentation of substitutional solute atoms in some miscible alloys. In this study, the ultra-centrifuge experiments were performed on an intermetallic compound of Bi-Pb system (Bi3Pb7) by changing time duration of experiment time (experimental conditions; maximum centrifugal force: 1.0x106g level, temperature: 130-150 ℃, duration: 30-150h, state: solid). Composition changes were observed in the centrifuged samples. And, it was found that the Bi phase appeared from starting state of Bi3Pb7 around the weak gravitational field region of the sample. These results showed that sedimentation of substitutional solute atoms occurred, and induced the structure change in intermetallic compounds.

Estrogen has been postulated to be involved in inhibition of vascular smooth muscle cell (VSMC) proliferation mainly via estrogen receptor (ER), but the detailed mechanism has remained primarily unknown. Therefore, in this study, microarray analysis was used in two types of cultured human VSMCs: one positive for ERα, and the other for ERβ, which were treated by estrogens to detect the estrogen-responsive genes. We also used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate mRNA levels of selective target gene (TG) in these cells. We further studied whether the TG product was involved in inhibition of proliferation using small interfering RNA (siRNA) of the TG transfection. We subsequently used quantitative RT-PCR and in situhybridization analysis to evaluate the expression of these gene products in human aorta. E4F1, a possible inducer of cell growth arrest, was markedly increased only in ERα-positive VSMCs by estrogens in both microarray and RT-PCR analyses. Blocking of E4F1 using siRNA suppressed estrogenic inhibition of ERα-positive VSMC proliferation. E4F1 mRNA was abundant in premenopausal female aorta with mild atherosclerotic changes. E4F1 is therefore considered one of the estrogen-responsive genes involving ERα-mediated inhibition of VSMC proliferation and may play an important role in estrogen-related atheroprotection of human aorta.

The Src family kinase Lyn initiates intracellular signal transduction by associating with a variety of immune receptors such as antigen receptor on B cells and high-affinity Fc receptor (FcR) for immunoglobulin Ig(E) (FcεRI) on mast cells. Involvement of Lyn in the IgE-mediated immediate-type hypersensitivity is well documented, but the physiological significance of Lyn in IgG-dependent, type III low-affinity FcR for IgG (FcγRIII)-mediated responses is largely unknown. In this study, we generated a double-mutant mouse strain deficient in both type II FcR for IgG (FcγRIIB) and Lyn to exclude any involvement of inhibitory signaling by FcγRIIB, which otherwise downregulates FcγRIII-mediated cellular responses. FcγRIIB-deficient but Lyn-sufficient mice served as controls. The Lyn deficiency attenuated IgG-mediated systemic anaphylaxis in vivo, and significantly reduced calcium mobilization and degranulation responses of bone marrow–derived mast cells (BMMCs) in vitro. However, we found that either interleukin 4 or tumor necrosis factor α release by BMMCs was comparable to that from Lyn-deficient and control mice, and the reverse-passive Arthus reaction was equally induced in both mutant mice, indicating that Lyn is not involved in the onset of the IgG-mediated, FcγRIII-dependent late phase responses of mast cells. These findings provide us with insight into distinct signaling mechanisms in mast cells underlying the development of diverse pathologies as well as a therapeutic potential for selective treatment of allergic disorders.

ABSTRACTA possible role for the γ subunit of immunoglobulin Fc receptors (FcR) in mucosal defenses against intestinal nematode parasites was studied using age-matched FcRγ-knockout (FcRγ−/−) and wild-type (FcRγ+/+) C57BL/6 mice. Mice were infected subcutaneously with 3,000 infective larvae of Strongyloides venezuelensis, and the degree of infection was monitored by daily fecal egg counts and adult worm recovery on days 8 and 13 postinfection. Mucosal mast cell (MMC) responses were assayed by in situ intestinal mast cell counts in stained histological sections of the jejunum and by measuring mouse mast cell protease 1 (MMCP-1) release in serum using sandwich enzyme-linked immunosorbent assay. FcRγ−/−mice had significantly higher egg counts (P< 0.01) and numbers of adult worms (P< 0.05) than FcRγ+/+mice, but mastocytosis and serum MMCP-1 release were comparable. It was concluded that MMCP-1 release may be spontaneous, does not depend on mast cell degranulation via the FcRγ signaling system, and appears to play no role in the expulsion of S. venezuelensis. The delay in worm expulsion in the FcRγ−/−mice might be related to inability of the MMC to degranulate and release effector molecules other than MMCP-1, since FcRγ deletion abrogates mast cell degranulative responses.

The combination of hemorrhagic pneumonitis and rapidly progressive glomerulonephritis is a characteristic feature of Goodpasture's syndrome (GPS), an autoimmune disease resulting from the interaction of pathogenic anti–collagen type IV (C-IV) antibodies with alveolar and glomerular basement membranes. Lack of a suitable animal model for this fatal disease has hampered both a basic understanding of its etiology and the development of therapeutic strategies. We now report a novel model for GPS using mice deficient in a central regulatory receptor for immunoglobulin (Ig)G antibody expression and function, the type IIB Fc receptor for IgG (FcγRIIB). Mutant mice immunized with bovine C-IV reproducibly develop massive pulmonary hemorrhage with neutrophil and macrophage infiltration and crescentic glomerulonephritis. The distinctive linear, ribbon-like deposition of IgG immune complex seen in GPS was observed along the glomerular and tubulointerstitial membranes of diseased animals. These results highlight the role of FcγRIIB in maintaining tolerance and suggest that it may play a role in the pathogenesis of human GPS.

Paired Ig-like receptors (PIR) are polymorphic type I transmembrane proteins belonging to an Ig superfamily encoded by multiple isotypic genes. They are expressed on immune cells such as mast cells, macrophages, and B lymphocytes. Two subtypes of PIR have been classified according to the difference in the primary structure of the PIR transmembrane and cytoplasmic regions. These subtypes are designated as PIR-A and PIR-B. In this study, the transmembrane and cytoplasmic regions of the PIR-A subtype were shown to mediate activation signal events such as cytoplasmic calcium mobilization, protein tyrosine phosphorylations, and degranulation in rat mast cell line RBL-2H3. The association of the Fc receptor γ and β subunits with PIR-A was shown to be responsible for PIR-A function but not required for membrane expression of PIR-A on COS-7 cells. We further revealed the role of two charged amino acid residues in the transmembrane region, namely arginine and glutamic acid, in PIR-A function and its association with the above subunits. In contrast to the inhibitory nature of the PIR-B subtype, present findings reveal that PIR-A potentially acts as a stimulatory receptor in mast cells, suggesting a mechanism for regulation of mast cell functions by the PIR family.

The Kit and PDGFRa receptor tyrosine kinases are encoded in close proximity at the murine white spotting (W) and patch (Ph) loci. Whereas W mutations affect hematopoiesis, melanogenesis, and gametogenesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. TheWsh, W57, and Phmutations diminish Kit expression in certain cell types such as mast cells and enhance it in others. The Wsh,W57, and Ph mutations arose from deletions and inversions affecting sequences in between the Kit andPDGFRa genes. We have determined the precise location of the breakpoint of the Wshinversion and the endpoints of the W57deletion upstream of the Kittranscription start site and examined the effect of these mutations on Kit expression in mast cells and hematopoietic stem cells and lineage progenitors. Our results indicate that positive elements controlling Kit expression in mast cells mapping in between −23 and −154 kb from the transcription start site can be dissociated from negative elements controlling Kit misexpression during embryonic development in the vicinity of the PDGFRa gene. In addition, we have identified two clusters of hypersensitive sites in mast cells at −23 −28 kb and −147 −154 kb from the Kit gene transcription start site. Analysis of these hypersensitive sites in mutant mast cells indicates a role for HS4-6 in Kit expression in mast cells. These findings provide a molecular basis for the phenotype of these Kit expression mutations and they provide insight into the complex mechanisms governing the regulation ofKit expression.

Autoimmune diseases, like rheumatoid arthritis, result from a dysregulation of the immune response culminating in hyperactivation of effector cells leading to immune-mediated injury. To maintain an appropriate immune response and prevent the emergence of autoimmune disease, activation signals must be regulated by inhibitory pathways. Biochemical and genetic studies indicate that the type IIB low-affinity receptor for immunoglobulin (Ig)G (FcγRIIB) inhibits cellular activation triggered through antibody or immune complexes and may be an important component in preventing the emergence of autoimmunity. To investigate the role of FcγRIIB in the development of type II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans, we have examined its contribution in determining the susceptibility to CIA in the nonpermissive H-2b haplotype. H-2b mice immunized with bovine CII do not develop appreciable disease. In contrast, immunization of the FcγRIIB-deficient, H-2b mice with bovine CII induced CIA at an incidence of 42.2%. The maximal arthritis index of the FcγRIIB-deficient mice developing CIA (6.9 ± 3.6) was comparable to that of DBA/1 mice (8.6 ± 1.9), an H-2q strain susceptible for CIA induction. IgG1, IgG2a, and IgG2b antibody responses against CII were elevated in the FcγRIIB-deficient animals, especially in those mice showing arthritis, but less pronounced than DBA/1 mice. Histological examinations of the arthritic paws from FcγRIIB-deficient mice revealed that cartilage was destroyed and bone was focally eroded in association with marked lymphocyte and monocyte/macrophage infiltration, very similar to the pathologic findings observed in DBA/1 mice. These results indicate that a nonpermissive H-2b haplotype can be rendered permissive to CIA induction through deletion of FcγRIIB, suggesting that FcγRIIB plays a critical role in suppressing the induction of CIA.

It is widely accepted that immunoglobulin (Ig)E triggers immediate hypersensitivity responses by activating a cognate high-affinity receptor, FcεRI, leading to mast cell degranulation with release of vasoactive and proinflammatory mediators. This apparent specificity, however, is complicated by the ability of IgE to bind with low affinity to Fc receptors for IgG, FcγRII and III. We have addressed the in vivo significance of this interaction by studying IgE-mediated passive systemic anaphylaxis in FcγR-deficient mice. Mice deficient in the inhibitory receptor for IgG, FcγRIIB, display enhanced IgE-mediated anaphylactic responses, whereas mice deficient in an IgG activation receptor, FcγRIII, display a corresponding attenuation of IgE-mediated responses. Thus, in addition to modulating IgG-triggered hypersensitivity responses, FcγRII and III on mast cells are potent regulators of IgE-mediated responses and reveal the existence of a regulatory pathway for IgE triggering of effector cells through IgG Fc receptors that could contribute to the etiology of the atopic response.

Autoantibodies and immune complexes are major pathogenic factors in autoimmune injury, responsible for initiation of the inflammatory cascade and its resulting tissue damage. This activation results from the interaction of immunoglobulin (Ig)G Fc receptors containing an activation motif (ITAM) with immune complexes (ICs) and cytotoxic autoantibodies which initiates and propagates an inflammatory response. In vitro, this pathway can be interrupted by coligation to FcγRIIB, an IgG Fc receptor containing an inhibitory motif (ITIM). In this report, we describe the in vivo consequences of FcγRII deficiency in the inflammatory response using a mouse model of IC alveolitis. At subthreshold concentrations of ICs that fail to elicit inflammatory responses in wild-type mice, FcγRII-deficient mice developed robust inflammatory responses characterized by increased hemorrhage, edema, and neutrophil infiltration. Bronchoalveolar fluids from FcγRII−/− stimulated mice contain higher levels of tumor necrosis factor and chemotactic activity, suggesting that FcγRII deficiency lowers the threshold of IC stimulation of resident cells such as the alveolar macrophage. In contrast, complement- and complement receptor–deficient mice develop normal inflammatory responses to suprathreshold levels of ICs, while FcRγ−/− mice are completely protected from inflammatory injury. An inhibitory role for FcγRII on macrophages is demonstrated by analysis of FcγRII−/− macrophages which show greater phagocytic and calcium flux responses upon FcγRIII engagement. These data reveal contrasting roles for the cellular receptors for IgG on inflammatory cells, providing a regulatory mechanism for setting thresholds for IC sensitivity based on the ratio of ITIM to ITAM FcγR expression. Exploiting the FcγRII inhibitory pathway could thus provide a new therapeutic approach for modulating antibody-triggered inflammation.

In the region between the polyadenylation site of the rat skeletal muscle (SkM) Na-channel gene and the 5′ end of the growth hormone (GH) gene, a gene coding for B-cell-specific membrane protein B29/Ig-β was found and noted to have the same orientation as the Na-channel and GH genes. Rat B29/Ig-β gene was 3.1 kb in length with six exons and was separated by 3.3 and 9.3 kb from Na-channel and GH genes, respectively. Rat B29/Ig-β protein comprised 228 amino acids, and its amino acid sequence was 85 and 69% identical with the mouse and human counterparts, respectively. With the long-area PCR method, genomic DNA connecting human SkM Na-channel (SCN4A) and B29/Ig-β (CD79B) genes and CD79B and GH (GH1) genes was amplified, and the physical linkage of SCN4A/CD79B/GH1 genes in the human genome was established. The human CD79B gene was separated by 6.3 and 10.5 kb from the SCN4A and GH1 genes, respectively.

Summary The expression of T-cell receptors (TCR) in malignant lymphomas was examined immunohistochemically by monoclonal antibodies which react with the TCR? or TCRd chain. TCR? was expressed in 16 out of 47 non-Hodgkin's lymphomas. These included 15 T-cell lymphomas and 1 Ki-1 lymphoma. The anti-TCR? chain antibody,?F1, did not react with 26 B-cell lymphomas, 1 Ki-1 lymphoma or 6 Hodgkin's disease. The anti-TCRd chain antibody, TCRd1, did not react with any type of malignant lymphoma. Although TCR? and CD3 were co-expressed in normal lymphoid tissues and most T-cell lymphomas, 3 cases of CD3+CD4+ CD8-T-cell lymphoma failed to express TCR0. TCR? and Ig JH gene configurations in malignant lymphomas were examined by Southern hybridization. Although each of 9 T-cell lymphomas had a rearranged TCR? locus, TCR? gene rearrangement in the 3 cases of?F1-CD3+T-cell lymphomas was demonstrated by Southern blot. No transcripts of the TCR? gene could be found in 2 out of the 3?F1-CD3+T-cell lymphomas by Northern blot, indicating the lack of TCR? protein expression to be due to non-transcription of the TCR gene. Loss of TCR? proteins in these T-cell lymphomas is thus quite likely to be associated with T-cell tumour activation and progression, since 3?F1-CD3+T-cell lymphomas expressed CD25 (interleukin-2 receptor) to a high degree.

The nucleotide sequences of cloned cDNAs were used to determine the primary structures of the precursors of vasotocin (sVT) and isotocin (sIT) from the hypothalamus of the chum salmon,Oncorhynchus keta. Two different cDNAs were obtained for each of sVT and sIT precursors (sVT-I and sVT-II; sIT-I and sIT-II). Both sVT and sIT precursors were found to contain a signal peptide and hormone that is connected to a neurophysin by a Gly-Lys-Arg sequence. Northern and Southern blot analyses showed that the sVT and sIT genes are expressed by the same chum salmon hypothalamus, but not by the liver and kidney. Microheterogeneity was found in the nucleotide and amino acid sequences of sVT precursors between our results and the previously reported data (Heierhorst et al. 1990). The conspicuous difference is the occurrence of a stop codon in the middle of sVT-II cDNA. The carboxyl termini of both sVT and sIT neurophysins are about 30 amino acids longer than neurophysins of toad and mammalian neurohypophysial hormone precursors. Although these extended regions do not contain a glycosylation site, they show striking similarity with the glycopeptide moiety (copeptin) of toad vasotocin and mammalian vasopressin precursors. The central portion of the neurophysins shows highest homology among corresponding regions of sVT and sIT precursors. Moreover, calculation of nucleotide substitution rates suggests that a recent gene conversion may have occurred which encompasses the exon that encodes the central segment of the sVT and sIT precursors. A possible pathway for the evolution of precursor molecules of neurohypophysial hormones is discussed.

We have analyzed the molecules participating in the inhibitory function of gp49B1, a murine type I transmembrane glycoprotein expressed on mast cells and natural killer cells, as well as the chromosomal location of its gene. As assessed by SDS-polyacrylamide gel electrophoresis and immunoblot analysis, tyrosine-phosphorylated, but not nonphosphorylated, synthetic peptides matching each of the two immunoreceptor tyrosine-based inhibitory motif (ITIM)-like sequences found in the cytoplasmic portion of gp49B1 associated with the ∼65-kDa tyrosine phosphatase SHP-1 and ∼70-kDa SHP-2 derived from RBL-2H3 cells. In addition, the phosphotyrosyl peptide matching the second ITIM-like sequence also bound the ∼145-kDa inositol polyphosphate 5-phosphatase SHIP. Thus, it has been strongly suggested that the inhibitory nature of gp49B involves the recruitment of SHP-1, SHP-2, and SHIP for the delivery of inhibitory signal to the cell interior upon phosphorylation of tyrosine residues in their ITIMs. The gp49B gene has been found to be in the juxtaposition of its cognate gene, gp49A. The gene pair was shown to locate in the B4 band of mouse chromosome 10. In this region, no conserved linkage homology to human chromosome 19, where the genes for killer cell inhibitory receptors are found, has been identified.

Humoral and cellular immune responses communicate with each other via Fc receptors (FcR) expressed on various hematopoietic cells. Recent studies on several FcR knockout mice demonstrated pivotal roles of an IgG/FcγR system in the regulation of immune responses and the onset of hypersensitivity. The γ subunit of FcR is an essential component of the complex and is required for both receptor assembly and signal transduction. FcR γ chain-deficient mice have lost the functional expression of FcεRI, FcγRI, and FcγRIII and are unable to mount several types of hypersensitive reactions, including the skin Arthus reaction. In contrast, FcγRII-deficient mice exhibit augmented humoral immune responses and IgG-mediated anaphylaxis reactions. Thus, the regulatory system of murine hypersensitive responses involves both positive and negative signaling through FcR. In B cells, FcγRIIb modulates membrane Ig-induced Ca2+mobilization by inhibiting Ca2+influx through phosphorylation of its immunoreceptor tyrosine-based inhibition motif and recruitment of cytoplasmic phosphatases. Elucidation of the detailed mechanisms of negative regulatory signaling in the inflammatory effector cells by FcγRIIb as well as several groups of potent inhibitory molecules expressed on such cells should be valuable in the development of novel therapeutic procedures for allergic disorders.

The lpr and gld genes are thought to result in an incapacity for Fas-mediated apoptosis of T and B cells and the development of subsequent autoimmune disease. A newly established gld-congenic strain of mice, MRL/MpTn-gld/gld (MRL/gld), was found to develop vascular lesions involving arteritis and glomerulonephritis (GN), which were similar to those observed in the MRL/Mp-lpr/lpr (MRU/lpr) strain. However, comparative studies with a C3H/HeJ strain bearing Ipr or gld revealed that these lesions developed only in mice with an MRL background. We were successful in transferring GN to normal MHC-compatible gld/gld and irradiated +/+ mice by bone marrow cells of MRUgld mice, but were unsuccessful using those of C3H/gld mice. Transfer of arteritis, however, was only successful in mice with an MRL background. Nephritogenic monoclonal antibodies obtained from an MRL/lpr and an MRL/gld mouse were shown to be bone marrow-derived and rich in clonal diversity, and at least two of these were capable of causing glomerular injury by different mechanisms. Development of GN and systemic arteritis in MRL/lpr and MRL/gld mice will be dependent not only on their incapacity for Fas-mediated apoptosis but also on bone marrow cells and peripheral cells with intrinsic defects.

Calcitonin (CT) and the calcitonin generelated peptide (CGRP) are generated by alternative RNA processing from a single CT/CGRP gene. Recently, we reported the existence of CGRP-immunoreactivity and CGRP mRNA in endocrine cells or Kulchitsky (K) cells of human and rat lung [Wada et al. 1987b]. In this report, an examination was made of developmental changes in the expression of the CGRP gene in rat lungs by immunohistochemistry, radioimmunoassay (RIA) and Northern hybridization. CGRP-positive K-cells in lung tissue appeared on the 18th day of gestation. Their number was greatest on the 20th day of gestation and then decreased postnatally. The level of CGRP in rat lung was found to be highest in a 1-day-old neonate by RIA. In the Northern hybridization of rat lung using the CGRP 3′ non-coding region (exon 6) of the first human CT/CGRP gene as the probe, 1.0 kilobase (kb) CGRP mRNA was found to be abundant on the 20th day of gestation and in a 1 day-old neonate. It thus appears that CGRP in rat lung is essential for pulmonary adaptation at birth and/or from the last intrauterine stage to the early neonatal period.

By Southern hybridization using a genomic DNA fragment carrying a human IgE heavy chain constant region gene (Ce) as a probe, we analyzed the organization of human Ce genes and their flanking regions in 23 atopic dermatitis and 6 senile erythroderma patients with elevated serum IgE levels, and 6 atopic dermatitis patients with normal IgE levels. On Barn HI, Hind III, and Eco RI digestions, we detected three hybridizable fragments containing three human Ce genes, Ce1, Ce2, and Ce3, respectively, in all leukocyte DNAs. These fragments were almost identical in size among patients and healthy donors. Pst I digestion generated a genetic polymorphism. We, however, could find no correlation between this polymorphism and the disorders. It was concluded that among the patients and healthy donors, there was no marked difference in the organization of the functional Ce gene and its flanking region containing a class switch region. Our conclusion cannot rule out the presence of genetic abnormalities of this region in some atopic dermatitis patients which are not resolvable by our method. In the course of this study, we found a novel Ce-like gene in placenta DNA which differs from the three Ce genes commonly present in normal human DNA.

The Kit ligand (KL)/Kit receptor pair functions in hematopoiesis, gametogenesis, and melanogenesis. KL is encoded at the murine steel (Sl) locus and encodes a membrane growth factor which may be proteolytically processed to produce soluble KL. The membrane-associated form of KL is critical in mediating Kit function in vivo. Evidence for a role of cytoplasmic domain sequences of KL comes from the Sl17H mutation, a splice site mutation that replaces the cytoplasmic domain with extraneous amino acids. Using deletion mutants and the Sl17H allele, we have investigated the role of the cytoplasmic domain sequences of KL in biosynthetic processing and cell surface presentation. The normal KL protein products are processed for cell surface expression, where they form dimers. Both Sl17H and the cytoplasmic deletion mutants of KL were processed to the cell surface; however, the rate of transport and protein stability were affected by the mutations. Deletion of cytoplasmic domain sequences of KL did not affect dimerization of KL. In contrast, dimerization of the Sl17H protein was reduced substantially. In addition, we have characterized the hematopoietic cell compartment in Sl17H mutant mice. The Sl17H mutation has only minor effects on hematopoiesis. Tissue and peritoneal mast cell numbers were reduced in mutant mice as well as in myeloid progenitors. Interestingly, long-term bone marrow cultures from Sl17H mice did not sustain the long-term production of hematopoietic cells. In addition, homing of normal hematopoietic progenitors to the spleen of irradiated Sl17H/Sl17H recipient mice was diminished in transplantation experiments, providing evidence for a role of Kit in homing or lodging. These results demonstrate that the membrane forms of KL exist as homodimers on the cell surface and that dimerization may play an important role in KL/Kit-mediated juxtacrine signaling.

Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B–mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5′-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B–mediated inhibition requires redundant functions of SHP-1 and SHP-2.

The ζ family includes ζ, η, and FcεRIγ (Fcγ). Dimers of the ζ family proteins function as signal transducing subunits of the T cell antigen receptor (TCR), the pre-TCR, and a subset of Fc receptors. In mice lacking ζ/η chains, T cell development is impaired, yet low numbers of CD4+ and CD8+ T cells develop. This finding suggests either that pre-TCR and TCR complexes lacking a ζ family dimer can promote T cell maturation, or that in the absence of ζ/η, Fcγ serves as a subunit in TCR complexes. To elucidate the role of ζ family dimers in T cell development, we generated mice lacking expression of all of these proteins and compared their phenotype to mice lacking only ζ/η or Fcγ. The data reveal that surface complexes that are expressed in the absence of ζ family dimers are capable of transducing signals required for α/β–T cell development. Strikingly, T cells generated in both ζ/η−/− and ζ/η−/−–Fcγ−/− mice exhibit a memory phenotype and elaborate interferon γ. Finally, examination of different T cell populations reveals that ζ/η and Fcγ have distinct expression patterns that correlate with their thymus dependency. A possible function for the differential expression of ζ family proteins may be to impart distinctive signaling properties to TCR complexes expressed on specific T cell populations.

The sedimentation of isotope atoms has been realized in monoatomic liquid and solid Se. It was observed that the concentration ratio 82Se/76Se increased by % level in the specimen ultracentrifuged at 0.7–0.9 million G under liquid and solid states. The present result is evidence of sedimentation of substitutional atoms in condensed matter via self-diffusion, and suggests its possible application to isotope separation by condensed matter centrifugation.

The atomic-scale graded structure of In-Pb alloy was formed by an ultracentrifuge experiment under conditions that a gravitational field of 0.81 × 106G for 100 hours at 150°C (solid state) in our previous study. The isotope fluctuation on this sample was measured using secondary ion mass spectrometer (SIMS). The 115In/113In isotope ratio changed with positive gradient in the direction of centrifugal force approximately 1.2%. The isotope ratio fluctuation of centrifuged sample was 4 times larger than that of starting sample (<0.3%). This showed that the isotope fractionation effect was induced by solid centrifugation in this alloy, although achieved concentration gradients were small.

Radical addition of bromotrichloromethane to ethyl cinnamate was carried out under an ultrastrong gravitational field (>2.5 × 105G), and ethyl 3-chloro-3-phenyl-2-(trichloromethyl)propionate (A), ethyl 3-bromo-3-phenyl-2-(trichloromethyl)propionate (B), and ethyl 2-bromo-3-phenyl-3-(trichloromethyl)propionate (C) were isolated by reversed-phase HPLC. To date, there has been no report of the synthesis of Aand Cat 1 G, and this would be the first example of a selectivity change induced by ultrastrong gravity.

The molecular-graded structures have been realized through direct copolymerization of halogenated styrene and 2-ethylhexyl methacrylate under ultra-strong gravitational field. The graded structures were clearly confirmed by EPMA and NMR spectroscopy.

Immunoglobulin-binding factors are known to regulate the synthesis of B-cell-derived immunoglobulin heavy-chain isotypes1. Cloning and nucleotide sequence determination of complementary DNA encoding rodent IgE-binding factors (IgE-BF) revealed that messenger RNA encodes a glycoprotein of 557 amino acids which is expressed as a precursor of relative molecular mass (Mr) 60,000 (60K) in COS7 monkey cells2. We report here that the 3′ two-thirds of the IgE-BF coding sequence shows a surprising homology (72%) at the DNA level with coding sequences of the gag and pol (DNA endonuclease) genes of the Syrian hamster intracisternal A particle (IAP H18)3, an endogenous retrovirus. This marked homology demonstrates that the rodent gene encoding IgE-BF is a hybrid gene which evolved very recently by integrating genes of viral origin, and that the encoded polypeptide comprises three separate domains: an IgE-BF domain and retrovirus-derived gag and DNA endonuclease-like domains. This may represent the first report of a cellular gene containing a virus-derived coding sequence.

A magnetic field is predicted to emerge on a particle in a rotating material body even if the body is electrically neutral. This emergent field is called a Barnett field. We show that nuclear magnetic resonance (NMR) enables direct measurement of the Barnett field in solids. We rotated both a sample and an NMR coil synchronously at high speed and found an NMR shift whose sign reflects that of the nuclear magnetic moments. This result provides direct evidence of the Barnett field. The use of NMR for Barnett field measurement enables the unknown signs of nuclear magnetic moments in solids to be determined.

Anti-endothelial cell antibodies (AECAs) are thought to be critical for vasculitides in collagen diseases, but most were directed against molecules localized within the cell and not expressed on the cell surface. To clarify the pathogenic roles of AECAs, we constructed a retroviral vector system for identification of autoantigens expressed on the endothelial cell surface.

In a human genome, we found dispersed repetitive sequences homologous to part of a human endogenous retrovirus termed HERV-K which resembled mouse mammary tumor virus. For elucidation of their structure and organization, we cloned some of these sequences from a human gene library. The sequence common to the cloned DNA was ca. 630 base-pairs (bp) in length with an A-rich tail at the 3′ end and was found to be a SINE (short interspersed repeated sequence) type nonviral retroposon. In this retroposon, the 5′end had multiple copies of a 40 bp direct repeat very rich in GC content and about the next 510 nucleotides were homologous to the 3′ long terminal repeat and its upstream flanking region of the HERV-K genome. This retroposon was thus given the name, SINE-R element since most of it derived from a retrovirus. SINE-R elements were present at 4,000 to 5,000 copies per haploid human genome. The nucleotide sequence was ca. 90% homologous among the cloned elements.

We have determined the nucleotide sequences of long terminal repeat (LTR) regions of Syrian hamster intracisternal A particle (IAP) genes . The size of the LTRs was 350 base-pairs (bp) and 376 bp in two clones, H10 and H18, respectively. Two LTRs at both ends of the IAP gene were linked to directly repeating 6 bp hamster sequences. Many structural features common to the integrated retroviral LTRs such as “CAT” box, “TATAA” box, polyadenylation signal, and terminal inverted repeat (3 bp), were present on each LTR. The estimated length of R region (about 60 bp) was similar to that of the murine leukemia-sarcoma virus. In contrast, the calculated U5 region of 54 bp was the shortest among those of the retrovirusea so far studied. Furthermore, from the analysis of primer binding sites, phenylalanine tRNA was for the first time identified as a presumed primer tRNA for reverse transcription. These results clearly distinguish Syrian hamster IAP LTRa from other retroviral ones. Based on the comparison of the sequences between Syrian hamster and laboratory mouse LTRs, the structural features peculiar to the IAP LTRa and the origin of the IAP genes are discussed.

In this study, a determination was made of the chromatin structure of the rat growth hormone (GH) gene locus by DNase I sensitivity analysis using GC [GH+, prolactln (PRL)−], 235 (GH−, PRL+), GH3 (GH+, PRL+) and liver (GH−, PRL−) cells. From 7 kb upstream from the transcription start site to 19 kb downstream from the polyadenylation site, two major DNase I-hypersensrtlve sites (M-DHS; UIA, UNA) and three M-DHS (DIA, DM, Dill) were found within 2 kb upstream and 7 kb downstream regions, respectively. Two minor DHS (m-DHS; UIB, 1MB) in the upstream region and one m-DHS (DIB) downstream were shown to be associated with M-DHS. Thus, a total of five M-DHS and three m-DHS were mapped on the rat GH gene locus. Among these, five (UIIB, UIA, UIB, DIB, DIA) Including two (UIA, DIA) M-DHS were specific for GH-producing cells. UNA and Dill were M-DHS only in PRL-producing 235 cells while the major hypersensrtivtty of Dll was detected in GH-producing cells and liver cells. Assessment of the enhancing activity of the DHS regions indicated novel enhancers in one upstream and two downstream regions that function well with the GH promoter In GC cells. These enhancers, each appearing different, coincided with m-DHS but not M-DHS in GC cells, and were not activated by Pit-1. Based on these observations, the following functions of five M-DHS and three m-DHS regions were defined: enhancer; locus control region (LCR); switch region serving for conversion from GH/PRL-producing cells to PRL-producing cells; and a region having a structural function in chromatin.

Endogenous retrovirus-like sequences homologous to intracisternal type-A particle (IAP) genes, which are present in the inbred mouse (Mus musculus) genome, were cloned from a Syrian hamster gene library. A typical hamster IAP gene was 7 kb long and segments homologous to long terminal repeat (IAP) sequences present in Mus musculus IAP genes were located at both ends of the gene. Contrary to the pattern found in the Mus musculus IAP genes, the organization of the cloned hamster IAP genes was not markedly polymorphic and deletion was not observed among these cloned genes. A sequence about 0.8 kb long and located close to the 3′ end of the hamster IAP gene was well conserved in both IAP gene families, although they showed less overall homology with one another. The reiteration frequency of the hamster IAP genes was calculated to be 950 copies per haploid genome. Since such IAP genes with the above properties were not found in the genome of the Chinese hamster, whose progenitors diverged from those of the Syrian hamster about 7.5 Myr ago, the integration of a huge number of Syrian hamster IAP genes must have occurred subsequent to such divergence.