mDia1/3-dependent actin polymerization spatiotemporally controls LAT phosphorylation by Zap70 at the immune synapse.

The mechanism by which the cytosolic protein Zap70 physically interacts with and phosphorylates its substrate, the transmembrane protein LAT, upon T cell receptor (TCR) stimulation remains largely obscure. In this study, we found that the pharmacological inhibition of formins, a major class of actin nucleators, suppressed LAT phosphorylation by Zap70, despite TCR stimulation-dependent phosphorylation of Zap70 remaining intact. High-resolution imaging and three-dimensional image reconstruction revealed that localization of phosphorylated Zap70 to the immune synapse (IS) and subsequent LAT phosphorylation are critically dependent on formin-mediated actin polymerization. Using knockout mice, we identify mDia1 and mDia3, which are highly expressed in T cells and which localize to the IS upon TCR activation, as the critical formins mediating this process. Our findings therefore describe previously unsuspected roles for mDia1 and mDia3 in the spatiotemporal control of Zap70-dependent LAT phosphorylation at the IS through regulation of filamentous actin, and underscore their physiological importance in TCR signaling.

Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

Strong coupling superconductivity at 8.4 K in an antiperovskite phosphide SrPt3P.

We report the discovery of a family of ternary platinum phosphides APt3P (A = Ca, Sr, and La), which crystallize in an antiperovskite-based structure closely related to that of the heavy fermion superconductor CePt3Si. All three phosphides showed superconductivity at low temperatures and the highest critical temperature T(c) = 8.4 K was observed for SrPt3P. The analysis of specific heat C(T) for SrPt3P shows clear evidence for very strong coupling s-wave superconductivity with a large ratio between superconducting gap Δ0 and T(c), 2Δ0/k(B)T(c) ∼ 5, and the presence of low-energy phonons. The presence of multiple Fermi surface pockets was inferred from the nonlinear magnetic field dependence of Hall resistivity, which we argue might play a role in realizing the strong coupling of charge carriers with the low-lying phonons.

Two distinct steps of immigration of hematopoietic progenitors into the early thymus anlage.

Thymic epithelial cells, which create a three-dimensionally organized meshwork structure peculiar to the thymus, develop from simple epithelia of the third pharyngeal pouch and cleft during organogenesis. We comparatively investigated the thymus anlages of normal and nude mice by immunohistochemical analysis with regard to epithelial organization and distribution of hematopoietic progenitor cells at early stages of organogenesis. Our results show that development of the mouse thymus anlage at early stages can be subdivided into at least two stages by the differences in epithelial organization, i.e. stratified epithelial stage on embryonic day (Ed) 11 and clustered epithelial stage on Ed12. At the former stage, hematopoietic progenitor cells are accumulated in the mesenchymal layer of the thymus anlage, and at the latter stage progenitor cells enter the epithelial cluster and proliferate. In nude mice, hematopoietic progenitor cells are found in the mesenchymal layer on Ed11.5, but they are not observed among epithelial cells on Ed12, even though epithelial cells form a cluster structure. The present results suggest that aberrant development of the nude mouse thymus anlage occurs at the clustered epithelial stage and that epithelial cells of the nude anlage lack the ability to induce the entrance of hematopoietic progenitor cells into the epithelial cluster.

The earliest stages of B cell development require a chemokine stromal cell-derived factor/pre-B cell growth-stimulating factor.

Environmental factors essential for the first stages of B lymphopoiesis remain elusive. Here, we report that immediately after commitment to B lineage, precursors become dependent on a chemokine SDF-1 and its receptor CXCR4 using mutant and radiation chimeric mice. In bone marrow, generation of the earliest identifiable B cell precursor populations requires CXCR4. In fetal liver, we identified Lin(-)CD19(-)c-kit(+)IL-7Ralpha(+)AA4.1(+), the earliest unipotent B cell precursor population, and found that its development was severely affected in SDF-1(-/-) embryos but not in IL-7(-/-) embryos. Lin(-) T cell progenitors appeared normal in SDF-1(-/-) embryos. Moreover, SDF-1 exhibited specific biologic activities on the earliest B cell precursors. SDF-1 provides the first example of a cytokine responsible for the earliest B lineage stages.

Expression of alpha(4)beta(7) integrin defines a distinct pathway of lymphoid progenitors committed to T cells, fetal intestinal lymphotoxin producer, NK, and dendritic cells.

During embryogenesis, the Peyer's patch anlagen are induced by a cell population that produces lymphotoxin (LT) alpha(1)beta(2) following stimulation of IL-7Ralpha. In this study, we show that the LT-producing cell is localized within the IL-7Ralpha(+) and integrin alpha(4)beta(7) (alpha(4)beta(7))(+) population in the embryonic intestine. Lineage commitment to the LT producer phenotype in the fetal liver coincides with expression of alpha(4)beta(7). Before expression of alpha(4)beta(7), the potential of IL-7Ralpha(+) population to generate B cells is lost. However, the progenitors for T cells and LT producer cells reside in the IL-7Ralpha(+)alpha(4)beta(7)(+) cells, but during subsequent differentiation, the potential to give rise to T cells is lost. This IL-7Ralpha(+)alpha(4)beta(7)(+) population migrates to the intestine, where it induces the Peyer's patch anlagen. When stimulated with IL-15 or IL-3 and TNF, the intestinal IL-7Ralpha(+)alpha(4)beta(7)(+) population can differentiate into fully competent NK1.1(+) NK cells or CD11c(+) APCs. Expression of alpha(4)beta(7) is lost during differentiation of both lineages; IL-7Ralpha expression is lost during NK1.1(+) cells differentiation. A newly discovered lineage(-)IL-7Ralpha(+)c-Kit(+)alpha(4)beta(7)(+) population in the fetal liver is committed to T, NK, dendritic, and fetal intestinal LT producer lineage, the latter being an intermediate stage during differentiation of NK and dendritic cells.

Effects of sensory feedback on variations on intertap interval and force in finger-tapping sequences.

This study was designed to examine effects of somatosensory feedback on variations of intertap interval and muscle force in finger-tapping sequences over 10 minutes. Although intertap intervals were decreased on the massed task as the time passed, the intervals were constant in the distributed task. In finger-tapping for a long time, impulses perhaps circulate within the loop circuits between the cerebral motor cortex and the peripheral nerve and subsequently increase further the excitability of the circuits. This increase in the excitability within the circuits may shorten the interval and increase variation of the interval. On the other hand, although peak force increased up to the 5-min. mark on the massed task, the force decreased after the 6-min. mark. This increase of force also may be produced by increasing activation of the corticoperipheral loop circuits. Although the decrease of force was perhaps produced by the fatigue of finger muscles for tapping during a few minutes, fatigue appeared more clearly in muscle force than in timing control. However, the force and the variation were constant in the distributed task.

Basic fibroblast growth factor up-regulates the surface expression of complement receptors on human monocytes.

OBJECTIVE AND DESIGN: To clarify the possible involvement of basic fibroblast growth factor (b-FGF) in inflammation, we examined the effect of b-FGF on the surface expression of complement receptors (CR) on human monocytes in vitro. MATERIALS AND METHODS: Heparinized venous blood was obtained from healthy adult donors. The surface expression of CR on blood monocytes was determined by two-color immunofluorescent staining using flow cytometry and monoclonal antibodies. A standard whole blood lysis technique was used to avoid any in vitro manipulation that would activate monocytes. RESULTS: b-FGF increased the expression of CR3 on monocytes in a dose- and time-dependent manner. The b-FGF concentrations used were up to 100 ng/ml. The values of mean fluorescence intensity (MFI) of CR3 expression on unstimulated monocytes were 12.6+/-1.3 (n = 3), whereas those on b-FGF-stimulated monocytes were 59.2+/-7.1 (n = 3). b-FGF also up-regulated the expression of CR1 on monocytes in a dose- and time-dependent manner. The MFI values of CR1 expression on unstimulated monocytes were 2.5+/-0.1 (n = 3), whereas those on b-FGF-stimulated monocytes were 11.1+/-0.6 (n=3). The magnitude of CR1 expression by monocytes was significantly smaller than that of CR3 expression. The maximal stimulatory effect of b-FGF on monocytes was observed using greater than 25 ng/ml of b-FGF and 90-120 min incubation period. CONCLUSION: b-FGF may participate in the inflammatory process by modulating the CR expression on blood monocytes.

Stepwise lineage restriction of progenitors in lympho-myelopoiesis.

It has long been controversial whether hematopoiesis progresses through ordered stages of determination as in embryonic development. This is due to the absence of a methodology capable of exactly determining the developmental potential of hematopoietic stem/progenitor cells. The multilineage progenitor (MLP) assay enabled us to discriminate among seven types of hematopoietic progenitors, which are multipotent progenitor p-MTB (capable of generating myeloid, T and B cells), bipotent progenitors p-MT, p-MB and p-TB, and unipotent progenitors p-M, p-T and p-B. Among these seven types, the p-TB type progenitor was found to be absent. These findings indicate that the process of lineage commitment proceeds through an ordered but not random process. By extending the area of investigation to include the erythroid lineage, more convincing evidence for the ordered process was obtained. Detailed and exact illustration of the process of hematopoiesis will provide an opportunity to revive hematopoiesis as one of the most fascinating targets of research in developmental biology.

Retention of relative force in the scaling of a serial force pattern with an attenuated-force tap.

The present study was designed to examine the retention of relative force in the scaling of a serial force pattern in a finger-tapping sequence using an attenuated tap. On practice trials, 12 undergraduate students tapped a force plate connected to strain gauges that gave them feedback about the force. On test trials, participants recalled the force pattern (200 gm-200 gm-200 gm-100 gm) and the intertap interval (400 msec.) practiced during the practice period without the feedback (recalled task). Then, they adaptively produced a halved (halved task) or doubled force profile (doubled task) at the fixed intertap interval. Analyses showed that mean peak forces at the first three tap positions of the tapping sequence undershot the expected forces across all tasks. Hence, the ratios of the forces in Serial Positions 1:4, 2:4, and 3:4 were considerably lower than 2.0. This is a contextual effect suggesting that the last attenuated tap affected the first three taps of the tapping sequence. Thus, because the relative force of movements appears to be a weaker invariant feature than sequencing and relative timing for generalized motor program theory of Schmidt and Lee, this finding does not support the relative force for a generalized motor program.

Commitment to natural killer cells requires the helix-loop-helix inhibitor Id2.

We have previously described how T and natural killer (NK) lineage commitment proceeds from common T/NK progenitors (p-T/NK) in the murine fetal thymus (FT), with the use of a clonal assay system capable of discriminating p-T/NK from unipotent T or NK lineage-committed progenitors (p-T and p-NK, respectively). The molecular mechanisms controlling the commitment processes, however, are yet to be defined. In this study, we investigated the progenitor activity of FT cells from Id2-/- mice that exhibit defective NK cell development. In the Id2-/- FT, NK cells were greatly reduced, and a cell population that exclusively contains p-NK in the wild-type thymus was completely missing. Id2-/- FT progenitors were unable to differentiate into NK cells in IL-2-supplemented-FT organ culture. Single progenitor analysis demonstrated that all Id2-/- fetal thymic progenitors are destined for the T cell lineage, whereas progenitors for T/NK, T, and NK cell lineages were found in the control. Interestingly, the total progenitor number was similar between Id2-/- and Id2+/+ embryos analyzed. Expression of Id2 was correlated with p-NK activity. Our results suggest that Id2 is indispensable in thymic NK cell development, where it most probably restricts bipotent T/NK progenitors to the NK cell lineage.

Immature multipotent hemopoietic progenitors lacking long-term bone marrow-reconstituting activity in the aorta-gonad-mesonephros region of murine day 10 fetuses.

Previous studies indicated that multipotent progenitors exist in early fetuses that do not contain long-term reconstituting (LTR) activity. However, it remained unclear whether these multipotent progenitors are committed to the hemopoietic lineage or are immature mesodermal cells or hemangioblasts. In this study, we have succeeded in enriching the multipotent progenitors that are capable of generating myeloid, T, and B cells in the LFA-1(-) subpopulation of TER-119(-)c-kit(+)CD45(+) cells from the aorta-gonad-mesonephros (AGM) region of day 10 fetuses. We found that these day 10 AGM LFA-1(-) cells do not show the LTR activity, whereas day 11 AGM LFA-1(-) cells do have such an activity. These results strongly suggest that multipotent progenitors lacking LTR activity emerge as CD45(+) hemopoietic progenitor cells in the AGM region on the 10th day of gestation, and such p-Multi mature into hemopoietic stem cells by acquiring LTR activity.

Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of lck proximal promoter activity.

The proximal promoter of lck directs gene expression exclusively in T cells. To investigate the developmental regulation of the lck proximal promoter activity and its relationship to T cell lineage commitment, a green fluorescence protein (GFP) transgenic (Tg) mouse in which the GFP expression is under the control of the proximal promoter of lck was created. In the adult GFP-Tg mice, >90% of CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes, and the majority of CD4(-)CD8(+) and CD4(-)CD8(-) [double-negative (DN)] thymocytes were highly positive for GFP. Slightly lower but substantial levels of expression of GFP was also observed in mature splenic T cells. No GFP(+) cells was detected in non-T lineage subsets, including mature and immature B cells, CD5(+) B cells, and NK cells, indicating a preserved tissue specificity of the promoter. The earliest GFP(+) cells detected were found in the CD44(+)CD25(-) DN thymocyte subpopulation. The developmental potential of GFP(-) and GFP(+) cells in the CD44(+)CD25(-) DN fraction was examined using in vitro culture systems. The generation of substantial numbers of alphabeta and gammadelta T cells as well as NK cells was demonstrated from both GFP(-) and GFP(+) cells. However, no development of B cells or dendritic cells was detected from GFP(+) CD44(+)CD25(-) DN thymocytes. These results suggest that the progenitors expressing lck proximal promoter activity in the CD44(+)CD25(-) DN thymocyte subset have lost most of the progenitor potential for the B and dendritic cell lineage. Thus, progression of T cell lineage restriction in the earliest thymic population can be visualized by lck proximal promoter activity, suggesting a potential role of Lck in the T cell lineage commitment.

Anti-Helicobacter pylori agents. 4. 2-(Substituted guanidino)-4-phenylthiazoles and some structurally rigid derivatives.

In order to find a new class of anti-Helicobacter pylori (H. pylori) agents, a series of 4-[(3-acetamido)phenyl]-2-(substituted guanidino)thiazoles and some structurally rigid analoges were synthesized and evaluated for antimicrobial activity against H. pylori. Among the compounds obtained, high anti-H. pyrori activities were observed in benzyl derivative 34 (MIC = 0.025 microg/mL) and phenethyl derivatives 35 and 36 (MIC = 0.037 microg/mL and 0.017 microg/mL). Though alkyl derivatives generally showed lower activity, the 2-methoxyethyl derivative 28 preserved significant activity (MIC = 0.32 microg/mL) and also exhibited more potent gastric antisecretory activity than ranitidine. Structural restriction by bridging between the thiazole and the phenyl rings with an alkyl chain did not improve the activity in this series.

Multivariate analysis of prognostic determinants after surgery for renal cell carcinoma at Himeji National Hospital.

A clinico-pathological study was performed retrospectively on 62 patients who underwent surgery for renal cell carcinoma between January 1992 and October 1998 at Himeji National Hospital to clarify the prognostic determinants for survival. The median follow-up period was 32 months and the cause-specific survival rates at 1, 3 and 5 years were 86.7, 81.3, 81.3%, respectively. Of the 62 patients, 11 (17.7%) patients died of renal cell carcinoma and 2 (3.2%) patients died of unrelated causes. Of the variables related to survival, presenting symptoms, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), alkaline phosphatase (ALP), tumor size, pathological tumor grade, infiltration pattern, pathological tumor stage, N classification and M classification were significant risk factors for survival by univariate analysis. However, ALP, N classification and M classification were significant for survival as determined by the step-wise procedure and M classification was the most significant factor according to Cox's proportional hazard model analysis.

Basic fibroblast growth factor modulates the surface expression of effector cell molecules and primes respiratory burst activity in human neutrophils.

Basic fibroblast growth factor (b-FGF) mediates a variety of biological responses such as angiogenesis and hematopoiesis. We examined the effect of b-FGF on human neutrophil functions in vitro. The surface expression of effector cell molecules on neutrophils was determined by flow cytometry and monoclonal antibodies. b-FGF increased the expression of CD11b leukocyte integrin and complement receptor type 1 on neutrophils and decreased the expression of L-selectin on neutrophils in a dose- and time-dependent manner. We also examined the effect of b-FGF on the respiratory burst activity in neutrophils. Although b-FGF alone did not induce intracellular oxidative product formation by neutrophils, it enhanced H(2)O(2) production in neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate. These findings suggest that b-FGF may participate in the inflammatory process via modulating the surface expression of effector cell molecules and enhancing respiratory burst activity in neutrophils.

The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage.

The antigen specificity of a rat monoclonal antibody TER-119 was investigated. In adult mice, TER-119 reacted with mature erythrocytes, 20-25% of bone marrow cells and 2-3% of spleen cells but not with thymocytes nor lymph node cells. In fetal haematopoietic tissues, 30-40% of d 10 yolk sac cells, 80-90% of d 14 fetal liver cells and 40-50% of newborn liver cells were reactive with TER-119. TER-119+ cells in adult bone marrow expressed significant levels of CD45 but not myeloid (Mac-1, Gr-1) or B-cell (B220) markers. Morphological examination and haematopoietic colony-forming assays for isolated TER-119+ cells revealed that TER-119 reacts with erythroid cells at differentiation stages from early proerythroblast to mature erythrocyte, but not with cells showing typical erythroid blast-forming unit (BFU-E) and erythroid colony-forming unit (CFU-E) activities. Erythroleukaemia cell lines do not express the TER-119 antigen even after stimulation with dimethylsulphoxide. TER-119 immunoprecipitated protein bands with molecular masses of 110 kDa, 60 kDa, 52 kDa and 32 kDa from erythrocyte membrane, whereas only a 52-kDa band was detected by TER-119 in Western blot analysis. Further molecular and cellular analyses indicated that the TER-119 antigen is a molecule associated with cell-surface glycophorin A but not with glycophorin A itself.

T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver.

The developmental potential of individual cells in the Lin-c-kit+CD45+IL-7R+ (IL-7R+) population from murine fetal liver was investigated using a clonal assay capable of determining the potential of a progenitor to give rise to myeloid, T, and B cells. Unipotent progenitors generating T cells (p-T) or B cells (p-B) but not other types of progenitors were found in the IL-7R+ population. A large proportion of progenitors at day 12 of gestation are p-T, whereas the frequency of p-T dramatically decreases with gestational age. In marked contrast, p-B are very rare by day 12, but they rapidly increase thereafter. These findings strongly suggest that the commitment of multipotent progenitors to T and B cell lineages occurs independently.

Type 1 diabetes mellitus caused by treatment with interferon-beta.

A 57-year-old man was referred to our outpatient clinic after interferon-beta (IFN-beta) treatment for 7 weeks. While IFN-beta therapy was continued in our outpatient clinic, his blood glucose level increased gradually, and he was admitted to our hospital for hyperglycemia. The patient was prescribed a 1,600-kcal diet and intensive insulin therapy was performed. GAD antibody became positive 15 months after the start of IFN therapy, and disappeared 27 months after the start of IFN therapy. Insulin secretion was depleted and the patient had HLA-DR4, B54, and DRB1*0405. This appears to be a case of type 1 diabetes mellitus induced by administration of IFN-beta alone.