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AmyI-1-18, an antimicrobial peptide, is a cationic α-helical octadecapeptide derived from α-amylase of rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antifungal activity of AmyI-1-18 against Candida albicans, 11 analogs bearing substitutions with alanine, leucine, and/or arginine, which were designed on the basis of the helical wheel projection of AmyI-1-18, were synthesized, and their antifungal activity was investigated. The antifungal activities of four analogs obtained by replacing arginine or lysine with alanine were significantly reduced. The results suggested that the cationic arginine and lysine residues in AmyI-1-18 are important for its antifungal activity. The antifungal activities of two single leucine-substituted analogs were not improved, but among three single arginine-substituted analogs, AmyI-1-18(D15R) had approximately a twofold higher antifungal activity [50% growth-inhibitory concentration (IC50 ): 31 μM] than AmyI-1-18 (IC50 : 64 μM) and exhibited low hemolytic activity (4% at 100 μM). Flow cytometric analysis using propidium iodide revealed that the antifungal activity of AmyI-1-18(D15R) was dependent on its membrane-disrupting activity in a manner different from that of AmyI-1-18. Further enhancement of the cationicity and hydrophobicity of AmyI-1-18(D15R) resulted in no improvement in antifungal activity and a significant increase in hemolytic activity. In this study, the results demonstrated that the antifungal activity of AmyI-1-18 against C. albicans was enhanced through increasing its membrane-disrupting activity by replacing aspartic acid at position 15 with arginine without a significant increase in hemolytic activity. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 219-229, 2016.

Responses to future changes in climatic and socio-economic conditions can be expected to vary between sectors and regions, reflecting differential sensitivity to these highly uncertain factors. A sensitivity analysis was conducted using a suite of impact models (for health, agriculture, biodiversity, land use, floods and forestry) across Europe with respect to changes in key climate and socio-economic variables. Depending on the indicators, aggregated grid or indicative site results are reported for eight rectangular sub-regions that together span Europe from northern Finland to southern Spain and from western Ireland to the Baltic States and eastern Mediterranean, each plotted as scenario-neutral impact response surfaces (IRSs). These depict the modelled behaviour of an impact variable in response to changes in two key explanatory variables. To our knowledge, this is the first time the IRS approach has been applied to changes in socio-economic drivers and over such large regions. The British Isles region showed the smallest sensitivity to both temperature and precipitation, whereas Central Europe showed the strongest responses to temperature and Eastern Europe to precipitation. Across the regions, sensitivity to temperature was lowest for the two indicators of river discharge and highest for Norway spruce productivity. Sensitivity to precipitation was lowest for intensive agricultural land use, maize and potato yields and Scots pine productivity, and highest for Norway spruce productivity. Under future climate projections, North-eastern Europe showed increases in yields of all crops and productivity of all tree species, whereas Central and East Europe showed declines. River discharge indicators and forest productivity (except Holm oak) were projected to decline over southern European regions. Responses were more sensitive to socio-economic than to climate drivers for some impact indicators, as demonstrated for heat-related mortality, coastal flooding and land use. ISSN:1436-3798 ISSN:1436-378X

AmyI-1-18, an octadecapeptide derived from α-amylase (AmyI-1) of rice (Oryza sativa L. japonica), is a novel cationic α-helical antimicrobial peptide (AMP) that contains two lysine and two arginine residues. The antimicrobial activity of AmyI-1-18 against human pathogens was quantitatively evaluated using a chemiluminescence method that measures ATP derived from viable cells. Of the ten kinds of human pathogens, AmyI-1-18 exhibited antimicrobial activity against nine. Its 50% growth-inhibitory concentrations (ICs50 ) against Porphyromonas gingivalis, Propionibacterium acnes, Pseudomonas aeruginosa, Candida albicans, and Streptococcus mutans were 13, 19, 50, 64, and 77 μM, respectively. AmyI-1-18 had little or no hemolytic activity even at 500 μM, and showed negligible cytotoxicity up to 1200 μM. The degree of 3,3'-dipropylthiadicarbocyanine iodide release from P. gingivalis cells induced by the addition of AmyI-1-18 was significantly lower than that induced by the addition of melittin. Flow cytometric analysis showed that the percentages of P. aeruginosa, S. mutans, and C. albicans cells stained with propidium iodide (PI), a DNA-intercalating dye, were 89%, 43%, and 3%, respectively, when AmyI-1-18 was added at a concentration equal to its 4×IC50 . Therefore, the antimicrobial activity of AmyI-1-18 against P. aeruginosa and S. mutans appears to be mainly attributable to its membrane-disrupting activity. In contrast, its antimicrobial activity against P. gingivalis and C. albicans most likely depends upon interactions with intracellular targets other than their cell membranes. Collectively, these results indicate that AmyI-1-18 is useful as a safe and potent AMP against the pathogens described above in many fields of healthcare. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 73-83, 2015.

The antimicrobial peptide AmyI-1-18 is a cationic α-helical octadecapeptide derived from α-amylase in rice (Oryza sativa L. japonica) that contains four cationic amino acid residues (two arginines and two lysines). To enhance the antibacterial activity of AmyI-1-18 against Porphyromonas gingivalis (a bacterium associated with periodontal disease), we synthesized 12 analogs bearing substitutions with alanine, leucine, and/or arginine that were designed based on helical wheel projections and investigated their antibacterial properties. The antibacterial properties of four analogs bearing substitution of a single arginine or lysine with alanine were almost similar to those of AmyI-1-18, suggesting that the antibacterial properties depend on the presence of three cationic amino acid residues. Of three single arginine-substituted analogs, AmyI-1-18(G12R) exhibited an antibacterial activity 2.8-fold higher [50% growth-inhibitory concentration (IC50): 4.6 μM] than that of AmyI-1-18 (IC50: 13 μM). Likewise, the antibacterial properties of two single leucine-substituted analogs were significantly enhanced; in particular, AmyI-1-18(N3L) exhibited an antibacterial activity (IC50: 2.5 μM) 5.2-fold higher than that of AmyI-1-18. The hemolytic activity of AmyI-1-18(N3L) against mammalian red blood cells was low (2% at 50 μM). A membrane-depolarization assay using a membrane potential-sensitive fluorescent dye revealed that, similar to AmyI-1-18, the antibacterial activity of AmyI-1-18(N3L) was not dependent on its membrane-disrupting activity. Our results demonstrate that the antibacterial properties of AmyI-1-18 against P. gingivalis are significantly improved, without a significant increase in hemolytic activity, by replacing asparagine with leucine at position 3.

Cell cycle arrest is associated with differentiation, senescence and apoptosis. We investigated alterations in the cell cycle during the development of hypertrophy induced by hydrogen peroxide (H(2)O(2)) in the H9c2 clonal myoblastic cell line. H(2)O(2) induced hypertrophy in H9c2 cells that was indicated by an increase in atrial natriuretic peptide (ANP) gene expression, a marker of cardiomyocyte hypertrophy, and a larger cell size. On induction of hypertrophy by H(2)O(2) in H9c2 cells, cell proliferation was arrested, indicated by the number of cells remaining constant during a 72-h incubation period. The cell cycle was arrested at the G1 and G2/M phases with an increase in p21 expression, a negative cell cycle regulator. Cell cycle arrest and increase in p21 expression were significantly inhibited by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), an intracellular calcium chelator. Although ANP gene expression was induced significantly, H(2)O(2) failed to induce hypertrophy in the presence of BAPTA-AM, and the cell cycle progressed. We concluded that H(2)O(2) induced cell cycle arrest in H9c2 cells, which was related to cellular hypertrophy.

An early event in the induction of apoptosis is cytochrome c (Cyt c) release from mitochondria. We investigated the involvement of mitochonrial permeability transition (MPT) and mitochondrial swelling in Cyt c release from mitochondria treated with alloxan and/or calcium (Ca2+). When mitochondria were treated with a high concentration of Ca2+. alone or Ca2+. with alloxan (alloxan-Ca2+), the MPT was accompanied by mitochondrial swelling and the release of Cyt c. Cyclosporin A prevented the induction of MPT but only slightly decreased the release of Cyt c. High molecular weight polyethylene glycol almost completely inhibited MPT-dependent osmotic mitochondrial swelling and Cyt c release. However, MPT-independent mitochondrial swelling and Cyt c release induced by exogenous K+. were inhibited by the high molecular weight polyethylene glycol. Ruthenium red strongly decreased the amount of Cyt c released. These results suggest that mitochondrial swelling but not MPT is necessary for Cyt c release induced by Ca2+ alone or alloxan and Ca2+.

Tri a Bd 27K, a major wheat allergen, is a glycoprotein. Tri a Bd 27K was found to occur in multiple forms by two-dimensional polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against the allergen. Furthermore, it was found that only Tri a Bd 27K components, which have N-linked glycan moieties with fucose residues, bound to IgE antibodies in the sera of wheat-sensitive patients.

Aberrant CpG island methylation contributes to the pathogenesis of various malignancies. However, little is known about the association of epigenetic abnormalities with multistep tumorigenic events in adult T cell leukemia/lymphoma (ATLL). To determine whether epigenetic abnormalities induce the progression of ATLL, we analyzed the methylation profiles of the SHP1, p15, p16, p73, HCAD, DAPK, hMLH-1, and MGMT genes by methylation specific PCR assay in 65 cases with ATLL patients. The number of CpG island methylated genes increased with disease progression and aberrant hypermethylation in specific genes was detected even in HTLV-1 carriers and correlated with progression to ATLL. The CpG island methylator phenotype (CIMP) was observed most frequently in lymphoma type ATLL and was also closely associated with the progression and crisis of ATLL. The high number of methylated genes and increase of CIMP incidence were shown to be unfavorable prognostic factors and correlated with a shorter overall survival by Kaplan-Meyer analysis. The present findings strongly suggest that the multistep accumulation of aberrant CpG methylation in specific target genes and the presence of CIMP are deeply involved in the crisis, progression, and prognosis of ATLL, as well as indicate the value of CpG methylation and CIMP for new diagnostic and prognostic biomarkers.

Hydrogen peroxide (H2O2) induces the hypertrophy in cultured H9c2 cardiomyocytes and cell death in glutathione (GSH)-depleted H9c2 cells. In the present study, we observed that pretreatment with a serine protease inhibitor, N-a-tosyl-L-lysine chloromethyl ketone (TLCK), significantly prevented the H2O2-induced cell damages in GSH-depleted H9c2 cells in a concentration-dependent manner. The phase contrast microscopy revealed that although the exposure of the GSH-depleted H9c2 cells to H2O2 resulted in a globular shape of the cells, TLCK prevented the occurrence of H2O2-induced morphological changes. TLCK also inhibited the generation of reactive oxygen species in the cells after addition of H2O2, suggesting that the antioxidant action of TLCK is involved in the protection against the cell damages by H2O2. Application of TLCK after ~30 min of exposure to H2O2 could significantly protect the cells from cell damages. The other serine protease inhibitors that were tested could not prevent the cell damages in GSH- depleted H9c2 cells. Pretreatment with an inhibitor of nuclear factor-kB translocation into the nucleus and a proteasome inhibitor did not prevent the cell damages in GSH-depleted H9c2 cells. An inhibitor of p53 significantly prevented the cell damages in GSH-depleted H9c2 cells. These results suggest that antioxidative action of TLCK prevents the death of GSH-depleted H9c2 cardiomyocytes induced by H2O2.

Questions: Are there any sustainable or vulnerable habitats in which beech (Fagus crenata) forests could survive in Japan under 110 hypothetical climate change scenarios? Location: Six islands of Japan on which beech grows naturally. Methods: An ecological habitat model was used to simulate the potential habitat shifts of beech forests under 110 climate change scenarios. The amount of suitable habitat loss and gain was calculated with three migration options and risk surfaces. Vulnerable and sustainable habitats were identified to evaluate the potential risks and survival of beech forests. Results: The total areas of potential suitable habitats differed considerably depending on the future temperature and precipitation changes. Some areas on the Sea of Japan (SOJ) side showed higher probability of maintaining suitable habitats, whereas there were wider areas in which suitable habitats could not persist under any of the 110 climate change scenarios. Conclusions: The risk surfaces of the suitable habitats showed that decreases in precipitation along with increases in temperature reduced the total areas of suitable habitats. Increases in precipitation with increases in temperature of more than or equal to 2°C always reduce the areas of suitable habitats. Under increased precipitation with a temperature increase of

Many researchers have hypothesized that differences in reactive oxygen species levels can trigger the cellular decision between hypertrophy and cell death in cardiomyocytes. In the present study, we examined the relationship between reactive oxygen species levels and hypertrophy or cell death in H9c2 cardiomyocytes after the addition of hydrogen peroxide. Following addition of hydrogen peroxide, we observed a slight increase in fluorescence intensity of 2',7'-dichlorofluorescein, a probe of intracellular reactive oxygen species, and cell hypertrophy in H9c2 cells (normal cells). In contrast, a dramatic increase in fluorescence intensity was followed by cell death in glutathione-depleted H9c2 cells. In the presence of the antioxidant Trolox or the iron chelator deferoxamine, both normal and glutathione-depleted cells developed hypertrophy without a concomitant increase in levels of reactive oxygen species. An inhibitor of p53, pifithrin-alpha, prevented cell death after the addition of hydrogen peroxide; instead a substantial increase in levels of reactive oxygen species and hypertrophy were observed. These results suggest that H9c2 cells exhibit differential sensitivity to intracellular reactive oxygen species generation with regard to their hypertrophic versus death responses to exogenously added hydrogen peroxide.

Until now, no method has been available to discriminate mature plasmacytoid DC (pDC) from myeloid DC (mDC) immunohistochemically. In this study, we report that these DC-subsets can be distinguished in routine pathological sections. Immature and mature monocyte-derived DCs (MoDCs) were S100 calcium binding protein B (S100B)+, while pDCs generated from pDC-precursors were S100B-. In contrast, both mature MoDC and pDC were fascin+. Epidermal Langerhans cells (LCs) were S100B+/fascin-. Although the majority of DCs were S100B+/fascin+ in the dermis with nonspecific inflammation, dermal DCs were mostly S100B-/fascin+ in psoriasis vulgaris, in which type I interferon secreted by pDC-precursors is thought to play a major role. S100B+/fascin+ DCs were accumulated in the superficial lymph node (LN), while they were scarce in the deep LN. In the superficial LN with dermatopathic lymphadenitis, a large number of S100B+/fascin+ DCs were accumulated in the T-zones, where numerous LC-derived DCs are accumulated. In contrast, almost all DCs were S100B-/fascin+ in the superficial LN with Kikuchi's lymphadenitis, in which numerous pDC-precursors are known to be present. In contrast to the superficial LN, the deep LN contained numerous S100B-/fascin+ DCs and a few S100B+ DCs. Thus, the distributions of S100B+ DC or S100B-/fascin+ DC correspond to the putative distribution of mDC or mature pDC, respectively.

One case of small-cell neuroendocrine carcinoma in the ampullary region of the duodenum is reported. The histological appearance of the tumor was identical to pulmonary small-cell carcinoma. Neuroendocrine differentiation was demonstrated immunohistochemically by positive immunoreaction for neuron specific enolase, Leu-7 and chromogranin, and ultrastructurally by the presence of scanty dense-core neurosecretory type granules. Small-cell neuroendocrine carcinoma in the ampulla of Vater is extremely rare. To our knowledge, this is the sixth reported case.

We identify and characterize a special type of macrophage in the human thymic cortex that may act as professional scavengers of apoptotic thymocytes. These are large cells with clear cytoplasm, evenly distributed exclusively in the thymic cortex, and usually contain degraded nuclei in their cytoplasm. They are distinct from ordinary macrophages (OM) in the thymic cortex in expressing fascin, an actin-bundling protein specific for dendritic cells (DC), and in lacking lysozyme (LZM) and CD68. They are also different from DC in lacking major histocompatibility complex (MHC)-class II molecules. To distinguish them from OM and DC, we called them thymic cortical dendritic macrophages (TCDM). Both TCDM and OM are positive for DC-SIGN (CD209) and HAM56, whereas fascin(hi) MHC-class II(hi) medullary DC (mDC) are negative for these antigens. TCDM exhibit either dendritic or plump feature depending on cases examined. Plump TCDM usually contain several degraded nuclei, while dendritic TCDM contain one or two. These degraded nuclei are positive for active caspase-3 (aCasp-3), indicating that they are apoptotic thymocytes. In contrast to TCDM, LZM(hi) CD68(hi) OM are smaller round cells, distributed unevenly throughout the thymus, and do not contain apoptotic thymocytes at all. TCDM tend to adhere to capillaries with their dendrites or they make extensive contacts covering a large portion of the capillaries. Electron microscopic analysis confirmed the extensive contact between TCDM and capillaries and indicated that TCDM possess extremely electron-lucent, abundant cytoplasm with numerous tubulovesicular structures and secondary lysosomes. The finding of numerous condensed nuclei in most of the TCDM indicates that these cells represent a special type of fixed macrophages in the human thymic cortex, and that they play a central role in the clearance of apoptotic thymocytes.

The IgE-binding proteins in beer were examined by immunoblotting analysis with sera of patients sensitive to beer. Several proteins were immunoblotted with the sera, and among these, 18-kDa proteins were identified as new IgE-binding proteins in beer. Perhaps they originated from barley as a raw material.

The authors developed an impact function with two explanatory variables (change in annual mean temperature and changc in annual precipitation) for each prefecture of Japan in order to give a simple estimation of the impact of climate change on the potential distribution of Buna (Fagus crenata) forests. The impact function predicts that areas suitable for Buna forests will not shrink in response to a temperature increase of less than 2°C in Hokkaido, Gunma, and Nagano prefectures. However, if the temperature increases more than 2°Cand there is no increase in the amount of precipitation, the areas suitable for such forests will decrease in those prefectures. In Tohoku, Hokuriku, and San-in districts, the areas suitable for Buna forests cover a comparatively large area. In these districts, though climate change will cause a decrease in the areas suitable for Buna forests, a relatively large amount of suitable land will still remain. In contrast to this situation, the suitable areas in coastal prefectures along the Pacific Ocean will be drastically reduced with even a slight increase in temperature.

We investigated the relationship of gastric mucosa-associated lymphoid tissue (MALT) lymphoma tumorigenesis to Helicobacter pylori infection, the t (11;18) translocation, and alterations in cell cycle regulators. We sought to assess the implications of altered expression of p27(Kip1), a cyclin-dependent kinase inhibitor, on high-grade transformation and responsiveness to eradication therapy. We used immunohistochemistry to examine p27(Kip1), p53, and Ki-67 expression in 23 MALT lymphomas, five diffuse large B-cell lymphomas (DLBCLs), and four DLBCLs with associated MALT lymphoma. All of the MALT lymphomas were positive for p27(Kip1) expression and negative for p53 with a low Ki-67 index, regardless of the sensitivity of these cells to eradication. All DLBCLs were negative for p27(Kip1) and positive for p53, exhibiting a high Ki-67 index. In DLBCLs with MALT lymphoma, p27(Kip1) expression was absent from both the MALT and large cells components. In all of these lymphomas, the MALT components were negative for p53 and displayed a low Ki-67 index, while the large cell components were positive for p53 with a high Ki-67 index. The expression patterns of the DLBCLs differed significantly from those of the MALT lymphomas. p27(Kip1) was not detected in either component of DLBCL with MALT lymphoma, suggesting that decreased expression of p27(Kip1) in the MALT component may be related to high-grade transformation. Thus, p27(Kip1) expression in morphological MALT lymphomas could be useful tool to predict high-grade transformation to DLBCL.

Summary Background A number of studies support the belief that human basophils play an important role in allergic inflammation. The exact mechanism of basophil activation at the site of allergic inflammation, however, has not been well understood, mainly due to their low number in blood and difficulty in obtaining a sufficient number of highly purified basophils for investigation. Objective The purpose of this study is to expand human basophils in vitro with high yield and purity by culturing peripheral blood stem cells (PBSCs). Methods We collected PBSC-rich mononuclear cells containing CD34+ cells (0.15–4.9%) by leukapheresis from patients with malignant lymphoma and lung cancer during haematopoietic recovery after chemotherapy plus granulocyte colony-stimulating factor-induced mobilization. PBSC-rich mononuclear cells were cultured in the presence of IL-3. Results When PBSC-rich mononuclear cells containing more than 1% of CD34+ cells were cultured, 20.0–83.3% of the cells, mostly with a yield of >10%, were metachromatic cells after 3 weeks of culture. These cells resembled mature peripheral blood basophils morphologically when examined by light and electron microscopy. Flow cytometric analysis showed that they expressed both FcɛRI and FcγRII. FcɛRI cross-linking resulted in intracellular calcium mobilization, histamine release and synthesis of cysteinyl leukotrienes. The intracellular histamine content and the release of these chemical mediators triggered by anti-IgE antibodies were comparable to those of peripheral blood basophils. Conclusion These findings suggest that PBSC-derived basophils expanded in vitro are morphologically and functionally mature and will be a useful tool for the analysis of basophil functions.

Epstein-Barr virus-associated hemophagocytic syndrome (EBV-AHS) is often associated with fatal infectious mononucleosis or T-cell lymphoproliferative diseases (LPD). To elucidate the true nature of fatal LPD observed in Herpesvirus papio (HVP)-induced rabbit hemophagocytosis, reactive or neoplastic, we analyzed sequential development of HVP-induced rabbit LPD and their cell lines. All of the seven Japanese White rabbits inoculated intravenously with HVP died of fatal LPD 18 to 27 days after inoculation. LPD was also accompanied by hemophagocytic syndrome (HPS) in five of these seven rabbits. Sequential autopsy revealed splenomegaly and swollen lymph nodes, often accompanied by bleeding, which developed in the last week. Atypical lymphoid cells infiltrated many organs with a “starry sky” pattern, frequently involving the spleen, lymph nodes, and liver. HVP-small RNA-1 expression in these lymphoid cells was clearly demonstrated by a newly developed in situ hybridization (ISH) system. HVP-ISH of immunomagnetically purified lymphoid cells from spleen or lymph nodes revealed HVP-EBER1+ cells in each CD4+, CD8+, or CD79a+ fraction. Hemophagocytic histiocytosis was observed in the lymph nodes, spleen, bone marrow, and thymus. HVP-DNA was detected in the tissues and peripheral blood from the infected rabbits by PCR or Southern blot analysis. Clonality analysis of HVP-induced LPD by Southern blotting with TCR gene probe revealed polyclonal bands, suggesting polyclonal proliferation. Six IL-2-dependent rabbit T-cell lines were established from transplanted scid mouse tumors from LPD. These showed latency type I/II HVP infection and had normal karyotypes except for one line, and three of them showed tumorigenicity in nude mice. These data suggest that HVP-induced fatal LPD in rabbits is reactive polyclonally in nature.

Background: We previously reported that crosslinking of IgG Fc receptor II (FcγRII) induces intracellular calcium mobilization, but not histamine release in human basophils. To clarify functional activities of FcγRII on human basophils, we analyzed the FcγRII-mediated signaling events in the human basophilic leukemia cell line KU812F. Methods: Flow cytometric methods were used to investigate the effect on intracellular calcium mobilization of cross-linking of FcγRII. KU812F cells were pre-incubated with anti-FcγRII monoclonal antibody (IV.3). After the addition of various concentrations of the tyrosine kinase inhibitor genistein or buffer alone, cells were stimulated with goat antimouse IgG F(ab') 2 (GAM) and analyzed with the flow cytometer. Next, in order to test the signaling events after cross-linking of FcγRII, we examined tyrosine kinase activity. The time-course of tyrosine phosphorylation after cross-linking of FcγRII and the effect of genistein on this tyrosine phosphorylation were tested by immunoblotting. Immunoprecipitation was also performed to identify the type of tyrosine kinase associated with signal transduction of FcγRII. Results: The tyrosine kinase inhibitor genistein inhibited intracellular calcium mobilization caused by cross-linking of FcγRII in a dose-dependent manner. Rapid tyrosine phosphorylation after FcγRII cross-linking was shown by immunoblot analysis and this phosphorylation was inhibited by genistein. Furthermore, tyrosine phosphorylation of Lyn and Syk was observed upon cross-linking of FcγRII. Conclusions: Tyrosine phosphorylation is necessary for the signaling pathway through FcγRII and tyrosine phosphorylation of Lyn and Syk, at least, is actively involved in this signal transduction.

Recent immunohistochemical demonstration of advanced glycation end products (AGE) in several tissues suggests potential involvement of AGEs in aging and age-related disorders. However, little is known about the generation of AGEs during development, and systematic experimental studies of the production of AGEs in aging are few. The aim of the present immunohistochemical study was to examine localization of AGE in fetal, young, and adult rats by using four monoclonal antibodies against AGE-structures—6D12 for Ne-(carboxymethyl)lysine (CML), KNH-30 for Ne-(carboxyethyl)lysine (CEL), 1F6 for fluorolink, and 2A2 for which the epitope is as yet unknown. CML was also determined by RP-HPLC. The results revealed that in rats at birth, all four AGE structures were found in most tissues and cells. The AGE accumulation and distribution increased gradually with aging. In the fetuses at 10 fetal days, all AGE-structures except for fluorolink were found in all ecto-, meso-, and endodermal tissues. AGE-deposition tended to increase with progression of fetal development. HPLC analysis revealed that 724.8±87.3 mmol CML/mol lysine was detected in fetuses at 13 fetal days. These results strongly suggest that AGE accumulation in tissues and cells occurs not only during aging but also from an early stage of ontogeny.

Class A macrophage scavenger receptor (SR-A) is one of the major receptors of macrophages and plays important roles in atherogenesis and host defense mechanisms. To assess the role of SR-A, monoclonal antibodies were generated by immunizing SR-A-deficient mice with a recombinant protein of human type I SR-A as immunogen. Four antibodies (SRA-C6, SRA-D10, SRA-E5, and SRA-F8) were confirmed to be specific for SR-A by Western blot analysis. In early atherosclerotic lesions, these antibodies recognized scattered macrophages in intima and foamy macrophages in the periphery of atheromatous cores. Interestingly, foamy macrophages in the core lesion were only weakly stained. In other organs, the antibodies recognized tissue macrophages such as alveolar macrophages, Kupffer cells in the liver, red pulp macrophages in the spleen, sinus macrophages in lymph nodes, and interstitial macrophages in various organs. Perivascular macrophages in the brain (Mato cells) were also positive for these antibodies. Freshly isolated blood monocytes were negative; however, they became positive for these antibodies after 1 day in culture. At 3–5 days in culture, the reaction intensity became stronger along their differentiation towards macrophages. Dendritic cells such as interdigitating cells of lymphoid tissues and epidermal Langerhans cells were invariably negative. In the reaction with animal tissues, each antibody showed a unique reaction pattern. Among four antibodies, SRA-E5 recognized SR-A molecules in all animal species examined, including rats and mice. These antibodies will be useful tools for the study of SR-A in atherogenesis and various other pathological conditions in humans and animal species.

Background Methods for determining the origin of BK virus (BKV)-infected cells (decoy cells) in clinical urine samples have not been established although they could enhance the diagnosis of BKV infection in immunocompromised patients. Methods We performed simultaneous immunostaining with anti-S100P (a urothelial marker) and anti-SV40 antibodies in 66 clinical urine samples exhibiting SV40 positivity and a decoy-cell appearance on Papanicolaou staining. The clinical voided urine samples included seven cases of renal transplantation, 47 cases of cancer therapy and 12 cases of non-neoplastic disease. SurePath™ liquid-based cytology was used for the urine samples. Results BKV-infected cells were categorized as SV40(+)/S100P(+) and SV40 (+)/S100p(−). SV40(+)/S100P(−) cells were found in 55 cases (83.4%); nine cases (13.6%) carried both SV40(+)/S100P(−) and SV40(+)/S100P(+) cells. The former were identified as BKV infection in renal tubules and the latter in both the renal tubules and urothelial epithelia. The remaining two cases (3.0%) had only SV40(+)/S100P(+) cells of urothelial origin. Conclusion Simultaneous immunostaining with anti-S100P and anti-SV40 is a useful method for determining the origin of BKV-infected cells in clinical urine samples from immunocompromised patients such as renal transplantation recipients.

ASPERGILLOSIS IN CLINICAL CONTEXT Aspergillosis is a fungal infection that can assume a broad spectrum of clinical forms depending on immune status and the presence of underlying lung diseases, ranging from allergic bronchopulmonary aspergillosis to aspergilloma and also to the invasive aspergillosis forms that are a major cause of mortality in severely immunocompromised patients. Pulmonary aspergillosis follows the inhalation of airborne conidia of Aspergillus fumigatus, and pre-existing conditions such as pulmonary functional abnormalities such as cystic fibrosis or chronic obstructive pulmonary disease provides a favorable environment for Aspergillus colonization and biofilm formation in the lungs (Zmeili and Soubani, 2007; Ramage et al., 2011). Due to its great importance in clinical context, a better understanding of the process in filamentous growth and biofilm formation can help to manage Aspergillus infection, especially when dealing with resistant strains to current antifungal drugs (Arendrup, 2013).

Background In the course of graft-versus-host disease (GVHD) or diseases that histologically mimic GVHD (e.g. toxic epidermal necrolysis, Stevens–Johnson syndrome), it is known that epidermal Langerhans cells (LCs) are depleted from the epidermis. However, the mechanism and significance of LC depletion is not well known. Objectives To investigate the numerical, morphological and phenotypic changes in LCs and apoptosis of LCs in the course of GVHD using a non-irradiated mouse GVHD model. Methods BALB/c nu/nu mice and C57BL/6 mice were used as recipients and donors, respectively. Recipient mice were injected with T-cell-enriched donor spleen cells. Skin samples were harvested at various times after the inoculation. The numerical and morphological changes were examined by an immunofluorescence study of epidermal sheets. Apoptosis was studied by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling method and flow cytometric analysis using annexin V. Phenotypic change was studied by flow cytometric analysis of epidermal cell suspensions. The mixed epidermal cell lymphocyte reaction (MELR) was performed to examine functional changes in the epidermal cells. Results Five days after inoculation, a graft-versus-host reaction occurred. Epidermal LCs began to decrease from the sixth day. On the fifth day, the LCs became larger and had prominent dendrites. Immediately before the LCs began to decrease, many LCs became round in shape, with scanty dendrites. LC apoptosis was not observed in the epidermis either on the fifth or seventh day. Phenotypically, the expression of CD40, CD80, CD86 and major histocompatibility complex class II antigen on the LCs was upregulated on the fifth and seventh day. Epidermal cells from GVHD mice showed an increased allostimulatory capacity in the secondary MELR. Conclusions These results suggest that at early GVHD onset, most LCs may not undergo apoptosis in the epidermis but are phenotypically activated, resulting in further activation of alloreactive T cells and aggravation of the disease.

To investigate the lymphomagenesis of NK/T lymphoma, we comprehensively and systematically analyzed the expression pattern of the human NK/T cell line (NK-YS) genome by cDNA expression array and tissue microarray. We detected significant changes in the gene expression of NK-YS cell line: an increase in 18 and a decrease in 20 genes compared to normal NK cells or peripheral blood mononuclear cells. Among these genes, we found a strong decrease in hematopoietic cell specific protein-tyrosine-phosphatase SH-PTP1 (SHP1) mRNA by cDNA expression array and reverse transcriptase-polymerase chain reaction. Further analysis with standard immunohistochemistry and tissue microarray, which used 207 paraffin-embedded specimens of various kinds of malignant lymphomas, showed that 100% of NK/T lymphoma specimens and more than 95% of various types of malignant lymphoma were negative for SHP1 protein expression. On the other hand, SHP1 protein was strongly expressed in the mantle zone and interfollicular zone lymphocytes in reactive lymphoid hyperplasia specimens. In addition, various kinds of hematopoietic cell lines, particularly the highly aggressive lymphoma/leukemia lines, lacked SHP1 expression in vitro, suggesting that loss of SHP1 expression may be related to not only malignant transformation, but also tumor cell aggressiveness. SHP1 expression could not be induced in either of two NK/T cell lines by phorbol ester, suggesting that genetic impairment or modification with methylation of SHP1 DNA could be one of the critical events in the pathogenesis of NK/T lymphoma. This evidence strongly suggests that loss of SHP1 gene expression plays an important role in multistep tumorigenesis, possibly as an anti-oncogene in the wide range of lymphomas/leukemias as well as NK/T lymphomas.

Interdigitating dendritic cells (IDC) of the human mesenteric lymph nodes (LN) were examined by two-color immunofluorescent microscopy and flow cytometry to clarify their exact localization, immunophenotype, and relationships with T and B cells. IDC were identified as HLA-DR(bright) large dendriform cells of the T cell areas co-expressing CD40, CD54 (ICAM-1), CD80 (B7/B7-1), CD83, and CD86 (B70/B7-2). The majority of IDC directly attached to a few IgD+ naive B cells as well as to numerous CD4+ T cells. When LN cells were singly suspended and briefly incubated in vitro, IDC formed clusters with IgD+ IgM+ naive B cells, but not with IgA+ or IgG+ B cells. When suspended LN cells were cultured, clustered B cells disappeared within 7 days, and on prolonged culture, some IDC developed into extensively dendriform cells forming stable complexes with several or sometimes numerous CD4+ IL-2R+ CD40L+ activated T cells. These findings indicate that resting naive B cells actually interact with IDC directly in T cell areas of human secondary lymphoid tissues. IDC have a non-antigen (Ag)-specific, strong affinity for resting naive B cells, but this affinity is transient and IDC cannot form stable complexes with B cells, although they can form stable complexes with activated T cells. It is suggested that the stable IDC/Ag-activated T cell complexes make it possible to capture and to stimulate rare Ag-specific resting naive B cells with high efficiency.

We used immunohistochemical methods and four monoclonal antibodies for specific molecular structures of advanced glycation end products (AGE)-6D12, KNH-30, 1F6, and 2A2-to examine localization of AGE in fetal, young, and adult rats, and rats with streptozotocin-induced diabetes. 6D12 recognized N(epsilon)-(carboxymethyl)lysine (CML); KNH-30, N(epsilon)-(carboxyethyl)lysine (CEL); and 1F6, fluorolink. The epitope of 2A2 is as yet unknown. Immunoreactivities for these monoclonal antibodies were found in various organs and tissues in postnatal and adult rats, and accumulation increased with aging. In the fetuses, AGE structures were detected at 10 fetal days, and their accumulation increased during ontogeny. Reversed-phase high-performance liquid chromatography revealed CML in fetuses at 13 fetal days and in lungs of 28-week-old rats. In various organs and tissues of fetal, young, and adult rats, CML, CEL, 2A2-positive AGE, and fluorolink accumulated, in that order, which suggests that the accumulation of CML, a nonfluorescent/noncross-linked AGE, occurs earlier than accumulation of fluorolink, a fluorescent/cross-linked AGE. In diabetic rats, hepatocytes, splenic macrophages, renal glomerular endothelial and mesangial cells, testicular Leydig cells, and erythrocytes showed excessive accumulation of AGE, leading to the pathologic changes characteristic of diabetes mellitus. For the induction of these changes, persistent hyperglycemia and hyperketonemia might be important for acceleration of intracellular AGE accumulation in diabetic rats. Thus, AGE accumulation in tissues and cells occurs not only during aging and in diabetes mellitus but also from an early stage of ontogeny.

Epstein-Barr virus-associated hemophagocytic syndrome (EBV-AHS) is often associated with fatal infectious mononucleosis. However, the animal model for EBV-AHS has not been developed. We reported the first animal model for EBV-AHS using rabbits infected with EBV-related herpesvirus of baboon (HVP). Eleven of 13 (85%) rabbits inoculated intravenously with HVP-producing cells developed fatal lymphoproliferative disorders (LPD) between 22 and 105 days after inoculation. LPD was also accompanied by hemophagocytic syndrome (HPS) in nine of these 11 rabbits. The peroral spray of cell-free HVP induced the virus infection with increased anti-EBV-viral capsid antigen-IgG titers in three of five rabbits, and two of these three infected rabbits died of LPD with HPS. Autopsy revealed hepatosplenomegaly and swollen lymph nodes. Atypical lymphoid T cells expressing EBV-encoded small RNA-1 infiltrated diffusely in many organs, frequently involving the lymph nodes, spleen, and liver. Hemophagocytic histiocytosis was observed in the lymph nodes, spleen, bone marrow, and thymus. HVP-DNA was detected in the tissues and peripheral blood from the infected rabbits by polymerase chain reaction or Southern blot analysis. Reverse transcriptase-polymerase chain reaction revealed both HVP-EBNA1 and HVP-EBNA2 transcripts, suggesting latency type III infection. These data indicate that the high rate of rabbit LPD with HPS induction is caused by HVP. This system is useful for studying the pathogenesis, prevention, and treatment of human EBV-AHS.

It is well known that the number of peritoneal macrophages is increased in patients with pelvic endometriosis. We measured the concentration of monocyte chemoattractant protein-1 (MCP-1) using an enzyme-linked immunosorbent assay (ELISA) in the peritoneal fluid of women with and without endometriosis. The expression of MCP-1 in pelvic endometriotic lesions obtained from the peritoneum was also examined using immunohistochemistry and nonradioactive in situ hybridization. The mean concentration of MCP-1 in the peritoneal fluid was significantly higher in the patients with endometriosis (P

Two major theories concerning the development and differentiation of macrophages-the reticuloendothelial system proposed by Aschoff (1924) and the mononuclear phagocyte system developed by van Furth (1972)-are critically reviewed. Phylogenetically, mononuclear phagocytic cells (macrophages) develop in all animals; monocytes are not detected in invertebrates, and both macrophages and monocytes appear in vertebrates. The phylogenetic principle that the development and differentiation of macrophages precede those of monocytes during the evolutionary processes of animals applies to human and murine ontogeny of macrophages. In early ontogeny, macrophages develop from hematopoietic stem cells during yolk sac hematopoiesis, and the stage of monocytic cells is bypassed. Monocytic cells develop during hepatic hematopoiesis, and their development proceeds from the middle stage of ontogeny.In postnatal and adult life, macrophages are differentiated from macrophage precursor cells at different stages or through different pathways of differentiation. In addition to developing via the differentiation pathway of monocytic cells into macrophages, tissue macrophages develop from macrophage precursor cells at or before the stage of granulocyte/macrophage colony-forming cells, and some macrophage populations are derived from B lymphoid precursor cells. Dendritic cells are also derived from different precursor cells and are classified into myeloid dendritic cells, monocyte-derived dendritic cells, and lymphoid dendritic cells according to their precursor cell origin. Thus, macrophages and their related cells are believed to be differentiated from hematopoietic stem cells through multiple pathways. Finally, the roles of two major macrophage populations, Kupffer cells and monocyte-derived macrophages, in hepatic granuloma formation are analyzed by considering various mouse models.

A basic chitinase was secreted into culture medium of pumpkin cell suspension cultures. The chitinase was purified from the culture medium. A cDNA encoding the pumpkin chitinase was cloned by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE) methods. The chitinase gene was strongly expressed in pumpkin callus cells, but little or not at all in mature leaf, young leaf, cotyledon, stem, hypocotyl and root of pumpkin. No chitinase mRNA was detected in intact pumpkin fruit tissues. However, chitinase was induced during callus formation from sliced pumpkin fruit tissues. Induction also occurred in the absence of 2,4-D, a chemical causing callus formation, suggesting that it may be independent of the presence of 2,4-D. Perhaps, induction is caused by osmotic or wounding stress. Levels of chitinase mRNA markedly increased at 4 h after transfer of pumpkin callus cells into fresh culture liquid medium. They were also high at later stages of cell suspension culture. In transgenic tobacco BY-2 cells, into which the pumpkin chitinase cDNA was introduced, the recombinant pumpkin chitinase was expressed and secreted into the culture medium, suggesting that the signal peptide of pumpkin chitinase also functions for secretion from tobacco BY-2 cells.

We report an autopsy case of a pedigree of familial amyloidotic polyneuropathy (FAP) with a mutation of isoleucine-50 transthyretin (ATTR Ser50Ile). A 47-year-old man started developing severe diarrhea and weight loss at age 41 years, followed by urinary incontinence, autonomic-nervous-system abnormalities and serious heart failure; the diagnosis of FAP (ATTR Ser50Ile) was made on the basis of genetic, histochemical and immunohistochemical analysis. Six years after the initial symptoms, he died of septic shock. Autopsy revealed suppurative peritonitis, perforation of the sigmoid colon and marked systemic amyloid deposition. The total amount of amyloid deposited in the heart was greatly increased and was much lower in the thyroid gland and kidneys compared with amyloid deposits in ordinary FAP (ATTR Val30Met). Amyloid deposition in peripheral vessel walls was prominent, particularly in lymphatics and veins. His elder sister, 54 years old, started to develop orthostatic hypotension at age 49 years, followed by dysesthesia, diarrhea and severe congestive heart failure. Endomyocardial biopsy revealed severe TTR–amyloid deposition; ultrastructural examination demonstrated that amyloid fibrils were deposited disproportionately and extended radially around microvessels. These characteristic patterns of systemic amyloid deposition and distinct clinical manifestations, especially in the cardiovascular system, are considered to be a characteristic feature of ATTR Ser50Ile amyloidosis.

Almost all nasopharyngeal carcinomas (NPCs) are infected by Epstein-Barr virus (EBV), but most ex vivo NPC cells lose EBV genomes during passages. In this study, an EBV-negative NPC cell line, TW03, established from EBV-carrying NPC was reinfected with EBV by cocultivation with irradiated Akata cells carrying recombinant EBV containing a neomycin-resistant gene. The reinfected EBV (+) TW03 cells expressed EBERs and EBNA1, but not EBNA2, lytic proteins (ZEBRA and EA-D), or LMP1. They had an epithelial appearance similar to that of EBV (-) TW03 cells. The doubling times of EBV (+) and EBV (-) TW03 cells were almost identical. However, the EBV (+) TW03 cells formed larger colonies with ragged contours in anchorage-independent cultures. An in vitro invasion assay showed that EBV (+) TW03 cells had a higher invasive activity than EBV (-) TW03 cells (p < 0.01). Both EBV (-) and EBV (+) TW03 cells formed poorly differentiated squamous cell carcinomas in SCID and nude mice. EBV (+) TW03 cells showed a higher tumorigenicity to nude mice (12 of 13) than EBV (-) TW03 cells (1 of 9) (p < 0.001). In the severe combined immunodeficiency (SCID) tumors of EBV (+) TW03 cells, not all of the tumor cells were EBER-1 positive. EBER-1 was more frequently detected in the peripheral regions and daughter nodules of the tumors than in the central areas. The microdissection polymerase chain reaction showed that the EBER-1-negative TW03 cells in the EBV (+) TW03 SCID tumors lost EBV genomes. EBER-1-negative cells showed as high a rate of Ki-67 positivity as EBER-1-positive cells, indicating that the former were proliferating rather than dead or dying. In horny pearls, keratinizing cells were ZEBRA-positive and EBER-negative. Loss of EBV genomes was not associated with squamous differentiation. These data indicated that reinfection of EBV promotes the tumorigenicity of EBV (-) TW03 cells by enhancing the invading activity.

Acyl coenzyme A:cholesterol acyltransferase(ACAT) is a critical enzyme participating in both intracellular cholesterol homeostasis and systemic sterol metabolism and are classified into two ACAT−isozymes termed ACAT−1 and ACAT−2. In humans, ACAT−1 is expressed ubiquitously in a variety of cells such as macrophages, enterocytes/hepatocytes, and steroid hormone−producing cells. In macrophages, immunoelectron microscopic examination revealed that ACAT−1 is localized in the endoplasmic reticulum(ER). When the cells are exposed to modified low density lipoprotein to induce foam cell transformation, a portion of ACAT−1 is shifted from ER to ER−derived small vesicles. The histological distribution of ACAT−2 in humans is still unclear, but recent data demonstrated that this isozyme is expressed in enterocytes.

We investigated the relationship between the forms of emergence and the seedling growth of rice(Oryza sativa L.)and the effects of plant growth regulators on the forms of emergence and seedling growth. In the first experiment, two cultivars of M type(Tchampa, Hei Chiao Chui Li Hsiang Keng)and two of S type(Haginomae Mochi, Shan Kiu Ju)were used as materials according to the results of the previous experiments. All cultivars were sown at a depth of 5.5cm. The mesocotyl growth was promoted in the case of M type, whereas the elongation growth of coleptile and the first leaf were inhibited when compared with S type. The growth of mesocotylar toots was also stimulated. The emergence rate of cultivars of S type was smaller than that of M type, but the growth after emergence was faster. The leaf number of seedlings of S type was greater than that of M type at 15 days after seeding. In the second experiment, we studied regulation of the forms of emergence and seedling growth by using five plant growth regulators, including three plant hormones, ABA, GA3 and ET, and two inhibitors of gibberellin biosynthesis, BX-112 and PP-333. The sowing depth was 5.5cm. As a result, in S type or F type cultivar, the form of emergence was changed to M type by an application of ABA. In F type, the form of emergence changed to S type by BX-112 or PP-333 application. These results suggest that the form of emergence is determined by plant hormones.

Luminol (3-aminophthaloylhydrazine) is well known as a chemiluminescent reagent that can be used to measure peroxidase (POD) activity. We have developed a more sensitive assay for POD using a new chemiluminescent reagent based on 10,10'-dimethyl-9, 9'-biacridinium nitrate (lucigenin). This new reagent was obtained from lucigenin by light irradiation in an organic polar solvent such as N,N-dimethylacetamide (DMAc). The detection limit for POD activity using this reagent is 10(-19) mol/assay. In an application for enzyme immunoassay based on a POD label, we were able to detect human alpha-fetoprotein (AFP) at 1pg/mL. The characterization of this chemiluminescent reagent was obtained by optical measurements, and the formation of lucigenin charge transfer complex (CT-complex) was confirmed. We also investigated the stability of lucigenin CT-complex as a chemiluminescent reagent for the measurement of POD activity, and confirmed that the reagent was stable for more than a year.

In mice homozygous for the gene mutation for type I and type II macrophage scavenger receptors (MSR-A), MSR-A-/-, the formation of hepatic granulomas caused by a single intravenous injection of heat-killed Corynebacterium parvum was delayed significantly for 10 days after injection, compared with granuloma formation in wild-type (MSR-A+/+) mice. In the early stage of granuloma formation, numbers of macrophages and their precursor cells were significantly reduced in MSR-A-/- mice compared with MSR-A+/+ mice. In contrast to MSR-A+/+ mice, no expression of monocyte chemoattractant protein-1, tumor necrosis factor-alpha, and interferon-gamma mRNA was observed in MSR-A-/- mice by 3 days after injection. Also in MSR-A-/- mice, uptake of C. parvum by Kupffer cells and monocyte-derived macrophages in the early stage of granuloma formation was lower and elimination of C. parvum from the liver was slower than in MSR-A+/+ mice. In the livers of MSR-A+/+ mice, macrophages and sinusoidal endothelial cells possessed MSR-A, but this was not seen in the livers of MSR-A-/- mice. In both MSR-A-/- and MSR-A+/+ mice, expression of other scavenger receptors was demonstrated. These data suggest that MSR-A deficiency impairs the uptake and elimination of C. parvum by macrophages and delays hepatic granuloma formation, particularly in the early stage.

Although young mice homozygous for the osteopetrosis ( op ) mutation usually developed prominent osteopetrosis, its severity was markedly reduced in aged op / op mice. This age-associated reversal of osteopetrosis was accompanied by the expansion of bone marrow cavities and increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive cells and of macrophages in the bone marrow. The TRAP-positive cells were mononuclear and developed ruffled borders and numerous vesicles, vacuoles, and granules. Enzyme-linked immunosorbent assay demonstrated a significant elevation of serum granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 levels in the aged op / op mice. To examine whether GM-CSF and/or IL-3 could correct osteopetrosis in young op / op mice, 5 ng of recombinant murine (rm)GM-CSF and/or 100 ng of rmIL-3 were injected daily into young op / op mice. In these treated young op / op mice, the bone marrow cavities were expanded significantly at 2 weeks after administration, associated with significantly increased numbers of TRAP-positive cells and bone marrow macrophages. TRAP-positive cells increased in number with days after injection. These results suggest that GM-CSF and IL-3 induce the development of osteoclasts to correct osteopetrosis in the op / op mice with aging.

To examine the factors affecting the emergence and establishment of rice seedlings, we tested FAO-designated germ plasm 207 cultivars (indica rice 137 cultivars, japonica rice 56 cultivars, and unexplained ecospecies 14 cultivars) collected from throughout the world. In the first experiment, the seeds were sown in nursery soil at a seeding depth of 3.0cm. Seeded pots were kept in a greenhouse at 30/25°C for 21 days. The ratio of the established plants (REP) to total plants was measured at 21 days after seeding. As a result, 13 cultivars, 6.3% of the total tested showed the best REP, i.e., 100-80%. These include 7 indica types and 6 japonica types of rice. In the second experiment, 75 cultivars were selected as the materials. The plants were grown under the same conditions as the first experiment, but the seeding depths were 1.0 and 3.0 cm. The chlorophyll contents of the second leaf blades and REP were measured at 2 and 3 weeks after seeding, respectively. The REP showed no correlation to seed weight, whereas correlation existed between REP and the values of chlorophyll contents of the second leaf blades at 1 cm and 3 cm seeding depths 2 weeks after seeding. In conclusion, seedling establishment seemed to be affected by the rate of increase in the chlorophyll contents of the leaves.

Rice seedlings with the mesocotyl and coleoptile (the undeveloped leaves enclosed in the coleoptile) are here referred to as ‘MC’ type seedlings and are considered to be suitable for deep sowing. We investigated the effects of abscisic acid (ABA) and several of its related compounds on the occurrence of MC type seedlings and on rice mesocotyl growth. Rice (Oryza sativa L. cv. JC 91) seedlings were grown on 0.8% agar medium in the presence or absence of various kinds of ABAs under aseptic conditions at 30 °C in the dark for 14 days. The activity of the R isomer of ABA (R-ABA) was slightly less than that of the naturally occurring S form (S-ABA) concerning the occurrence of MC type rice seedlings and the growth of the rice mesocotyl. In addition, the racemate of R-and S-ABA (RS-ABA) is less effective than R-ABA and S-ABA alone.Trans-ABA had no activity in relation to both percent occurrence of MC type seedlings and mesocotyl growth. The results of the present study suggest that the occurrence of MC type rice seedlings and the growth of rice mesocotyls were closely related to structure-activity relationships with analogs of ABA.

To examine the localization of von Hippel–Lindau (VHL) protein in human tissues, we produced four novel monoclonal antibodies against human VHL protein. Western blot analysis revealed that two of these antibodies recognized the epitope in amino acid sequence 60–89 of the VHL protein and the others recognized sequences 54–60 and 189–213. In a wild-type VHL gene-transfected cell line, immunocytochemistry and immunoelectron microscopy demonstrated the intracytoplasmic localization of VHL protein, particularly in mitotic cells. In normal human tissues, VHL protein was detected immunohistochemically in epithelial cells covering the body surface and the alimentary, respiratory, and genitourinary tracts; in secretory epithelial cells of exocrine and endocrine organs; in parenchymal cells of visceral organs; in cardiomyocytes; in neurons in nervous tissue; in lymphocytes in lymphoid tissue; and in macrophages. In pathological specimens, VHL protein was expressed in VHL-related tumor, as well as in endothelial cells, fibroblasts, and pericytes, all of which are involved in active angiogenesis. These findings suggest that these monoclonal antibodies can be useful for various immunological assays and that the VHL protein plays fundamental roles in physiological and pathological situations, especially in neovascularization.

We report an autopsy case of familial amyloidotic polyneuropathy (FAP) Type I with mutations in both transthyretin (TTR) and extracellular superoxide dismutase (EC-SOD). This patient started to develop peripheral neuropathy at age 25, followed by cardiac, renal, and autonomic nervous system failure due to massive amyloid deposition. Thirteen years after the initial symptoms, he died of septic shock. Autopsy revealed suppurative peritonitis, multiple abscesses in the bile ducts and urinary tract, and more marked amyloid deposition than commonly seen in FAP Amyloid deposition occurred in various organs and tissues, especially prominently around blood vessels and in interstitial tissues, and was demonstrated immunohistochemically to be composed of TTR but not amyloid A (AA) and not amyloid L (AL) proteins. The serum EC-SOD content of the patient was 10 fold higher than those seen often in other FAP patients and in healthy controls. Genetic analysis demonstrated the single amino acid substitutions in Val30Met TTR and Arg213G1y EC-SOD. Since these data suggest the dissociation of EC-SOD from the vascular wall, massive amyloid deposition in the present case may be related to increased oxidative stress in loco.

A two-color immunofluorescent analysis indicated that dendritic cells (DCs) in the human axillar lymph nodes (ie, lymph nodal DCs (LnDCs)) can be classified into three subsets. The first subset consists of CD1a + /CD86 − or dim /CD83 − or dim nondendriform DCs found mainly in lymph sinuses, the second is of CD1a − /CD86 + /CD83 + dendriform DCs scattered in normal T zones, and the third is of large CD1a bright /CD86 + /CD83 + dendriform DCs occasionally found in hyperplastic T zones. A three-color flow cytometric analysis, immunoperoxidase staining, and electron microscopic observation indicated that the majority of LnDCs corresponded to the first subset, which showed distinctive characteristics of DCs but did not fulfill the ultrastructural criteria for interdigitating reticulum cells (IDCs) and did not contain Birbeck granules. When LnDCs were cultured for 7 days, they became large CD1a dim /CD86 + /CD83 + dendriform cells, which formed large complexes with many T cells and exhibited distinctive ultrastructural features of interdigitating reticulum cells. LnDCs cultured in the presence of granulocyte/macrophage colony-stimulating factor became markedly larger CD1a bright /CD86 + /CD83 + dendriform cells forming large complexes with numerous T cells. These findings suggest that cells of the first subset represent immature LnDCs just migrating from epidermis, those of the second subset represent interdigitating reticulum cells, and those of the third subset represent interdigitating reticulum cells probably stimulated with certain immunostimulatory cytokines such as granulocyte/macrophage colony-stimulating factor. It is also suggested that either the second or the third subsets of LnDCs are derived from the first subset.

Background: Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant for blood monocytes in vitro. Recent studies in MCP-1-transgenic mice revealed that the local production of MCP-1 caused monocyte infiltration. However, the kinetics of monocyte infiltration after the production of MCP-1 or the amount of MCP-1 necessary for monocyte recruitment are not known. Methods: We purified recombinant rat MCP-1 expressed in COS-7 cells, and injected it into rat skin. The infiltrating cells were examined by immunohistochemistry and ultrastructural peroxidase cytochemistry. Results: Rat recombinant MCP-1 had a molecular mass of approximately 30 kD and exhibited the peak monocyte chemotactic activity at 10–9 M. One microgram of MCP-1 caused intra- and extravascular accumulation of mononuclear cells 3 h after injection. The cells were ED1+, indicating they were blood monocytes. The infiltration of mononuclear cells peaked at 12–24 h, and most of them were TRPM-3+ and ED3+, characteristic to exudate macrophages. None of the cells expressed ED2 or Ki-M2R antigens, markers for resident macrophages, until 3 days after injection. There was no uptake of [3H]thymidine by the infiltrating cells. Ultrastructural peroxidase cytochemistry confirmed that the infiltrating cells were monocytes and exudate macrophages. The number of OX8+ lymphocytes also peaked at 12 h, consisting of approximately 9% of the total infiltrating cells. Conclusion: These results indicate that MCP-1 attracts blood monocytes as early as 3 h and the infiltrating monocytes differentiate into exudate macrophages in loco. However, this effect was transient and the infiltration of monocytes did not result in tissue damage.

In mice homozygous for the 'viable motheaten' (mev) mutation, numbers of macrophage progenitor cells, particularly monocytes, were markedly increased in the bone marrow and spleen. Increased mobilization of these precursor cells to peripheral tissues and their differentiation to macrophages were evidenced by striking increases in macrophage numbers. Immunohistochemical double staining of tissue sections and flow cytometry analyses of single cell suspensions from these mice demonstrated CD5 (Ly-1)-positive macrophages in the peritoneal cavity, spleen and other tissues. Ly-1-positive macrophage precursor cells were demonstrated in the peritoneal cavity of the mev mice and developed in the omental milky spots. The development of marginal metallophilic and marginal zone macrophages was poor in the splenic white pulp and related macrophage populations were absent in the other lymphoid tissues. The numbers of epidermal Langerhans cells in the skin and T cell-associated dendritic cells in the thymic medulla, lymph nodes, and the other peripheral lymphoid tissues were decreased. However, increased numbers of dendritic cells accumulated in the lungs, liver, and kidneys. These abnormalities in development and differentiation of macrophages and dendritic cells may be ascribed to the deficiency in haematopoietic cell SHP-1 tyrosine phosphatase or may be a secondary consequence of abnormal microenvironments, (either constitutive or in response to inflammatory stimuli) in the haematopoietic and lymphopoietic organs and tissues of these mice.

Interleukin-2 (IL-2) receptor gamma chain-deficient mice with a truncated mutation showed the absence or severe reduction of natural killer cells, decreased numbers of T- and B-cells, marked hypoplasia of the thymus and peripheral lymphoid tissues, defective formation of lymphoid follicles and germinal centre in the peripheral lymphoid tissues, and the absence of Peyer's patches in the intestinal mucosa. In addition, marked splenomegaly with extramedullary haematopoiesis, increased level of IgM and decreased levels of IgG and IgE in serum, severe reduction of conventional B cells (B-2) in the peripheral lymphoid tissues, the presence of IgM-producing CD5+ B cells (B-1) and their differentiation into plasma cells and Motto cells in the spleen, and increased production and differentiation of macrophages in various tissues were found in the mutant mice. However, the development of both marginal metallophilic macrophage populations in the spleen and of their related macrophages in the other tissues of the mutant mice was severely impaired. All these abnormalities seem to be induced by the loss-of-function of the IL-2 receptor gamma chain. From 8 weeks of age on, inflammatory changes occurred in the intestines, mesenteric lymph nodes, lungs, liver, and kidneys of the mutant mice. Besides the absence of Hassall's corpuscles, thymic cysts were frequently observed in the mutant mice. These pathological abnormalities suggest that the gamma chain is implicated not only in lymphoid and haematopoietic development but also in thymic epithelial cell ontogeny.

Clinicopathological features of three autopsy cases of extremely rare late onset type I familial amyloidotic polyneuropathy were presented and compared with 19 autopsy cases of the ordinary type. In the late onset cases, the ages at onset and at death were 27.5 and 24.5 years older, respectively, compared with the ordinary type. Also, duration of the total clinical course from onset to death was 3.7 years less than in the late onset cases. The degree of amyloid deposition was more marked in the heart of the late onset cases, causing prominent cardiac hypertrophy. It was also marked in the kidneys or thyroid of two cases, but slight to moderate in the peripheral or autonomic nervous tissues in all cases. Immunohistochemical investigation demonstrated the presence of transthyretin (TTR) as an amyloid precursor protein and of serum amyloid P-component in amyloid deposits in various organs and tissues of the late onset type. These findings, as well as serum levels of variant TTR, were similar to those of the ordinary type. These results suggest that there are some factors other than the amyloid precursor protein that effect the degree of amyloid deposition.

To elucidate the differentiation mechanisms of macrophages in the murine omentum, we studied the repopulation of these cells and the expression of macrophage colony-stimulating factor (M-CSF) in the milky spots and omental tissues in mice depleted of macrophages following administration of liposome-encapsulated dichloromethylene diphosphonate (clodronate). The macrophages in the omentum were spindle or dendritic in shape, expressed several macrophage-specific antigens and Ia antigen, and phagocytized intraperitoneally injected carbon particles. In the milky spots, macrophages and macrophage precursors were detected, and the number of precursors increased alter elimination of macrophages by intraperitoneal injection of liposome-encapsulated clodronate. Macrophage precursors in the milky spots proliferated, moved to the omentum, and transformed into dendritic-shaped macrophages. Expression of M-CSF mRNA extracted from the milky spots was markedly enhanced at 2 and 3 days after macrophage depletion. Localization of M-CSF protein and mRNA was observed in the stromal cells of the milky spots. In osteopetrosis (op/op) mutant mice that are defective in the production of functional M-CSF omental macrophages were absent. These results indicate that M-CSF locally produced in the milky spots plays an important role in providing a microenvironment for development and differentiation of omental macrophages.