Structure and physical map of 64 variable segments in the 3'0.8-megabase region of the human immunoglobulin heavy-chain locus.

We have constructed the physical map of the 0.8 megabase DNA fragment which contains the 3' 64 variable region (V) gene segments of the human immunoglobulin heavy chain (H) locus. The organization of the VH locus showed several features that indicate dynamic reshuffling of this locus. The sequenced 64 VH segments include 31 pseudogenes, of which 24 are highly conserved except for a few point mutations. Comparison of the 64 germline VH sequences shows that each VH family has conserved sequences, suggesting that there might be some genetic or selection mechanisms involved in maintenance of each family. The total number of the human VH segments was estimated to be about 120, including at least 7 orphons.

Role of glycosaminoglycans of biglycan in BMP-2 signaling.

Recently we have reported that biglycan (BGN) promotes osteoblast differentiation and that this function is due in part to its ability to positively modulate bone morphogenetic protein (BMP) functions. In this study we investigated the role of glycosaminoglycans (GAGs) of BGN in this function using in vitro and in vivo models. C2C12 myogenic cells were treated or untreated with BMP-2 alone or in combination with glycanated, partially glycanated or de-glycanated BGN, and the effects on BMP signaling and function were assessed by Smad1/5/8 phosphorylation and alkaline phosphatase (ALP) activity. Furthermore, the effect of de-glycanation of BGN on BMP-2 induced osteogenesis was investigated employing a rat mandible defect model. The defects were filled with collagen scaffolds loaded with glycanated or de-glycanated BGN alone or in combination with a sub-optimal dose of BMP-2 (subBMP). In in vitro experiments, BMP signaling and function were the greatest when BMP-2 was combined with de-glycanated BGN among the groups tested. In the rat mandible experiments, µCT analyses revealed that the newly formed bone was significantly increased only when subBMP was combined with de-glycanated BGN. The data indicate that the GAG component of BGN functions as a suppressor for the BGN-assisted BMP function.

Increased matrix metalloproteinase-2 and bone sialoprotein response to human coronal caries.

BACKGROUND: It has been suggested that host matrix metalloproteinase-2 (MMP-2) present in dentin may be involved in caries progression, however, its response to caries is not known. Bone sialoprotein (BSP) has been implicated in dentin mineralization and MMP-2 modulation. OBJECTIVE: To identify and compare the distribution of MMP-2 and BSP in healthy human coronal dentin and those with early caries. METHODS: Freshly extracted 3rd molars and premolars with and without early caries were fixed, demineralized and subjected to immunohistochemistry using a monoclonal anti-MMP-2 antibody and monoclonal anti-BSP antibody with an avidin-biotin complex method. Immunoreactivity was visualized with 3,3'-diaminobenzidine substrate and observed under light microscopy. RESULTS: Immunohistochemical analysis revealed that MMP-2 and BSP are not detected in the tubule lumens of healthy dentin. However, intense immunoreactivity for MMP-2 and BSP was detected in association with the full length of the caries-affected dentinal tubules. The MMP-2 and BSP at the dentino-enamel junction appeared unaltered. CONCLUSION: The results indicate that MMP-2 and BSP may be actively secreted by odontoblasts in response to carious insult. MMP-2 and BSP accumulation in the caries-affected dentinal tubules may indicate their potential involvement in the host defense mechanism which results in calcification of regions affected by the carious process.

Simulation of membrane fouling considering mixed liquor viscosity and variation of shear stress on membrane surface.

Simulation of membrane fouling in MBR was conducted considering accumulation, detachment and consolidation of extracellular polymeric substances accumulated on membrane surface. The fluctuation of shear stress working on membrane surface and the influence of the viscosity of mixed liquor were considered for the evaluation of shear stress. A flat-sheet-type membrane module was used and the change of trans-membrane pressure was measured in a laboratory-scale MBR reactor. Shear stress working on membrane surface caused by aeration was measured by a shear force sensor changing viscosity of bulk liquid. Effective shear stress on membrane surface was defined in the model as the sum of time-averaged shear stress and three times of standard deviation. The increase in the trans-membrane pressure was accurately simulated by the developed model suggesting validity of the developed fouling model and the idea of the effective shear stress on membrane surface.

Immunization and infection change the number of recombination activating gene (RAG)-expressing B cells in the periphery by altering immature lymphocyte production.

Recombination activating gene (RAG) expression in peripheral B cells increases after immunization with (4-hydroxy-3-nitrophenyl) acetyl coupled to chicken gamma globulin (NP-CGG) in alum. This increase could result from reinduction of RAG expression or, alternatively, from accumulation of RAG-expressing immature B cells in the periphery. We have used mice that carry a green fluorescent protein (GFP) RAG indicator transgene (RAG2-GFP) to characterize the RAG-expressing B cells in immunized spleens. Most of the RAG2-GFP-expressing B cells in unimmunized spleen are immature B cells. Injection with NP-CGG in alum initially suppresses lymphopoiesis in the bone marrow and decreases the number of immature RAG2-GFP-expressing B cells in the spleen. Recovery of lymphopoiesis in the bone marrow coincides with accumulation of RAG-expressing immature B cells in the spleen. Most of the RAG-expressing cells that accumulate in the spleen after immunization do not proliferate and they are not germinal center cells. Neither the initial suppression of lymphopoiesis nor the subsequent accumulation of RAG-expressing cells in the spleen is antigen dependent, since similar changes are seen with alum alone. Furthermore, such changes in the numbers of developing and circulating immature lymphoid cells are seen after injection with complete Freund's adjuvant or malaria infection. Our experiments suggest that adjuvants and infectious agents cause previously unappreciated alterations in lymphopoiesis resulting in the accumulation of RAG-expressing immature B cells in the spleen.

B cell development is arrested at the immature B cell stage in mice carrying a mutation in the cytoplasmic domain of immunoglobulin beta.

The B cell receptor (BCR) regulates B cell development and function through immunoglobulin (Ig)alpha and Ig beta, a pair of membrane-bound Ig superfamily proteins, each of which contains a single cytoplasmic immunoreceptor tyrosine activation motif (ITAM). To determine the function of Ig beta, we produced mice that carry a deletion of the cytoplasmic domain of Ig beta (Ig beta Delta C mice) and compared them to mice that carry a similar mutation in Ig alpha (MB1 Delta C, herein referred to as Ig alpha Delta C mice). Ig beta Delta C mice differ from Ig alpha Delta C mice in that they show little impairment in early B cell development and they produce immature B cells that respond normally to BCR cross-linking as determined by Ca(2+) flux. However, Ig beta Delta C B cells are arrested at the immature stage of B cell development in the bone marrow and die by apoptosis. We conclude that the cytoplasmic domain Ig beta is required for B cell development beyond the immature B cell stage and that Ig alpha and Ig beta have distinct biologic activities in vivo.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) stimulates extracellular signal-regulated kinase (ERK) activity in CD40 signaling along a ras-independent pathway.

CD40 activates nuclear factor kappa B (NF kappa B) and the mitogen-activated protein kinase (MAPK) subfamily, including extracellular signal-regulated kinase (ERK). The CD40 cytoplasmic tail interacts with tumor necrosis factor receptor-associated factor (TRAF)2, TRAF3, TRAF5, and TRAF6. These TRAF proteins, with the exception of TRAF3, are required for NF kappa B activation. Here we report that transient expression of TRAF6 stimulated both ERK and NF kappa B activity in the 293 cell line. Coexpression of the dominant-negative H-Ras did not affect TRAF6-mediated ERK activity, suggesting that TRAF6 may activate ERK along a Ras-independent pathway. The deletion mutant of TRAF6 lacking the NH2-terminal domain acted as a dominant-negative mutant to suppress ERK activation by full-length CD40 and suppress prominently ERK activation by a deletion mutant of CD40 only containing the binding site for TRAF6 in the cytoplasmic tail (CD40 delta 246). Transient expression of the dominant-negative H-Ras significantly suppressed ERK activation by full-length CD40, but marginally suppressed ERK activation by CD40 delta 246, compatible with the possibility that TRAF6 is a major transducer of ERK activation by CD40 delta 246, whose activity is mediated by a Ras-independent pathway. These results suggest that CD40 activates ERK by both a Ras-dependent pathway and a Ras-independent pathway in which TRAF6 could be involved.

Ras mediates effector pathways responsible for pre-B cell survival, which is essential for the developmental progression to the late pre-B cell stage.

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21(ras) in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21(Asn-17) (Ha-ras) was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21(ras) activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21(ras) activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21(ras) mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.

Regulation of RAG expression in developing lymphocytes.

Proper expression of products of the recombination-activating genes (RAGs) is essential for the development of the adaptive immune system. A major advance in the past year toward understanding RAG regulation is the establishment of green fluorescent protein (GFP)-RAG indicator mouse strains. In vivo visualization of RAG expression in single cells has helped to define the cells that express RAGs in secondary lymphoid organs and revealed differential cis requirements for stage- and lineage-specific RAG expression.

[Mitral valve repair with concomitant coronary bypass for partial rupture of an anterior papillary muscle].

We report a case of severe mitral regurgitation due to partial rupture of an anterior papillary muscle. A 63-year-old man was admitted to a hospital with heart failure. He was treated with diuretic agents effectively. Echocardiography demonstrated severe mitral regurgitation with prolapse of posterior leaflet and small mass-like structure on the prolapsed segment that was diagnosed the thickened leaflet. Coronary angiography revealed total occlusion of left anterior descending artery (LAD) filled with good collateral from right posterior descending artery and severe diffuse stenosis of circumflex artery (Cx). The patient underwent surgery on the 33rd day after admission with heart failure. At surgery, we recognized rupture of one of the heads of anterior papillary muscle that was entangled in chordae of the prolapsed segment. Mitral valve repair and coronary revascularization to LAD and Cx was successfully performed. His postoperative course was uneventful, and he was discharged on the 28th postoperative day.

Effect of bubble flow velocity on drag-force and shear stress working on submerged hollow fibre membrane.

This study is aimed at elucidating the mechanism by which rising air bubbles induce shear stress on hollow fibre membrane surfaces. Shear stress on hollow fibre membrane surfaces (laterally-set and vertically-set) caused by aeration was measured directly using a two-direction load sensor. In the laterally-set hollow fibre module, time-averaged upward-direction shear stress on the membrane surface was compared to theoretical shear stress values considering the effect of water flow on membrane surface. Measured time-average shear stress values were almost 200 times larger than theoretical values implying strong interactions between bubbles and solid surface. In the vertically-set membrane module, velocity measurement of bubble flow using laser Doppler velocimeter revealed that drag force working on membrane surface was closely related to upward-direction water velocity. Also fluctuation of drag force and shear force on membrane surface was found to be related to velocity fluctuation (turbulence).

Influence of extracellular polymeric substances on nitrogen removal in an intermittently-aerated membrane bioreactor.

Influence of EPS on fouling of intermittent aeration MBR reactor (denitrification MBR) was investigated changing intermittent aeration cycle (10 minute-cycle and 120 minute-cycle) in laboratory-scale reactors using synthetic wastewater. EPS were extracted from bacterial cells using cation resin method and molecular weight fractioning of EPS was conducted using gel chromatography. In both of the reactors, nitrogen removal rate was almost 100% after 50th day although DO concentration was not very high during the aerated phase because of accumulation of nitrifying bacteria in the reactors. In the 120 minutes-cycle reactor, trans-membrane pressure increased more rapidly than in the 10 minutes-cycle reactor. The reason might be that EPS of more than 1000 kDa, which are the main fouling substances, are produced more rapidly in the 120 minute-cycle condition. It was also found that three peaks at around 100 kDa, 500 kDa and 2000 kDa are prominent in EPS in intermittent-aeration MBR irrespective of cycle and higher molecular weight EPS are decomposed to smaller molecular weight EPS on membrane surface.

Package plant of extended aeration membrane bioreactors: a study on aeration intensity and biofouling control.

Biofouling control is important for effective process of membrane bioreactor (MBR). In this study, phenomena of biofouling for immersed type extended aeration MBR with two different anti-fouling aeration intensities were studied through a laboratory set up. The objectives of this study were (a) to observe biofouling phenomena of MBR that operates under different anti-fouling bubbling intensity, and simultaneously monitors performance of the MBR in organic carbon and nutrients removal; (b) to compare effectiveness of detergent and detergent-enzyme cleaning solutions in recovering biofouled membranes that operated in the extended aeration MBR. For MBR, which operated under continuous anti-fouling aeration, deposition and accumulation of suspended biomass on membrane surface were prohibited. However, flux loss was inescapable that biofilm layer was the main problem. Membrane cleaning was successfully carried out with detergent-enzyme mixture solutions and its effectiveness was compared with result from cleaning with just detergent solution.

Highest microvessel count as a long-term prognostic factor in Japanese breast cancer patients.

The aim of this study was to determine whether microvessel density (MVD) could add useful information in predicting the prognosis of breast cancer patients. In our study, MVD was calculated by counting microvessels per x200 field in the highest neovascularized area of the tumor (highest microvessel count, HMC). HMC significantly increased according to the increased number of positive nodes. Higher HMC significantly correlated with worse relapse-free survival (RFS) of patients with negative node, one to three positive nodes in the axilla or with stage I and II tumors. HMC, however, was not predictive for RFS of patients with four or more positive nodes or with stage III tumors. Multivariate analysis revealed that HMC was second only to nodal status and tumor size as being predictive for RFS. These results suggest that HMC could be used in selection of patients with early-stage breast cancer who are at high risk for having occult metastasis.

Platelet-derived endothelial cell growth factor/thymidine phosphorylase expression in macrophages correlates with tumor angiogenesis and prognosis in invasive breast cancer.

Expression of platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), an angiogenic factor, was immunohistochemically analyzed in 117 specimens of invasive breast carcinoma (IBC). PD-ECGF/TP expression was observed in cancer cells and/or stromal cells; most of these stromal cells were activated macrophages. Therefore, we assessed the PD-ECGF/TP expression separately in cancer cells and stromal cells. Sixty-one (52.1%) cases were classified as PD-ECGF/TP-positive in cancer cells and 44 (37.6%) were classified as positive in stromal cells. The PD-ECGF/TP expression in cancer cells did not correlate with any prognostic factors. However, its expression in stromal cells positively correlated with both tumor size and microvessel count, and inversely correlated with estrogen receptor status. Relapse-free survival and overall survival (OS) were significantly worse in patients with PD-ECGF/TP-positive stromal cells than in patients with negative cells. A multivariate analysis using the Cox proportional hazards model showed that the PD-ECGF/TP expression in stromal cells independently predicted OS as well as nodal status and tumor size. In conclusion, PD-ECGF/TP expression in stromal cells correlates with tumor angiogenesis and can be used to predict the prognosis of patients with IBC.

Angiogenesis and stromal fibronectin expression in invasive breast carcinoma.

Angiogenesis and stromal fibronectin (SFN) expression were immunohistochemically analyzed in 83 breast cancer specimens. Microvessel count (MVC), which correlated with lymph node metastasis, TNM stage, recurrence, and mortality, was relatively low in SFN-positive tumors. SFN expression did not correlate with lymph node metastases or tumor size. However, SFN positivity was less likely in patients with recurrent disease than in those without recurrence, and relapse-free survival was significantly better in patients with SFN-positive tumors than in those with SFN-negative tumors. MVC and SFN positivity were significant independent predictors of relapse-free survival as well as tumor size and axillary nodal status (Cox's proportional hazards regression analysis). Angiogenesis and SFN expression, both of which are inversely related, can be used prognostically in patients with breast cancer.

Left ventricular aneurysm, normal coronary arteries and embolization in a patient with systemic lupus erythematosus.

We describe a 30-year-old woman with a normal appearing coronary artery associated with SLE who suffered myocardial infarction with left ventricular aneurysm and systemic embolization including cerebral infarctions and saddle embolism. The patient was surgically treated with good results. To our knowledge this is the first reported case of systemic embolism due to myocardial infarction occurring in SLE with a normal coronary artery.

Relation between depressed cardiac response to exercise and autonomic nervous activity in mildly symptomatic patients with idiopathic dilated cardiomyopathy.

We investigated whether the depressed cardiac response to adrenergic stimulation is accompanied with impaired autonomic function in mildly symptomatic patients with idiopathic dilated cardiomyopathy (DCM). Twenty-seven patients with DCM (New York Heart Association class I or II) and 7 normal control subjects underwent exercise radionuclide ventriculography and 24-h ambulatory ECG. The following frequency components of heart rate variability were calculated: the areas under the low (low frequency component [LF], 0.04 to 0.15 Hz), high (high frequency component [HF], 0.15 to 0.40 Hz), and total frequency portions of the spectrum. HF and HF% (the ratio of HF to total power) were calculated as indexes of specific vagal influences, and LF% (the ratio of LF to total power) and the ratio of LF to HF were of sympathetic tone. The left ventricular ejection fraction (LVEF) increased by more than 5% in all normal control subjects during exercise, whereas 17 (63%) of patients failed to show more than a 5% increase in LVEF. The profile of the mean hourly HF% and LF/HF showed circadian variations in normal control subjects but not in patients. The HF and HF% during sleep were significantly lower and the LF/HF during sleep was higher in patients than in normal control subjects. In patients, the LVEF during exercise minus LVEF at rest was significantly correlated with HF, LF%, and LF/HF during sleep, and with the ratios of the mean values during early morning to the mean daytime values for those spectral indexes. Our results demonstrated that mildly symptomatic patients with DCM showed an attenuated cardiac response to exercise and altered autonomic function, and their close relationship, suggesting that autonomic nervous activity contributes to cardiac desensitization in DCM.

Myocardial contractile reserve as prognostic determinant in patients with idiopathic dilated cardiomyopathy without overt heart failure.

To assess the prognostic significance of myocardial contractile reserve in patients with idiopathic dilated cardiomyopathy (DCM) without overt heart failure (New York Heart Association functional class I or II), seventy-one patients underwent exercise radionuclide angiography in addition to clinical, radiographic, hemodynamic, and echocardiographic evaluations. Myocardial contractile reserve was assessed as left ventricular ejection fraction (LVEF) during peak exercise minus LVEF at rest (delta LVEF). During an average of 49 months, 18 patients died of the disease. Cox's proportional-hazards regression analysis showed that the delta LVEF was the most powerful and independent discriminator for survival (p = 0.0002). Ejection time (p = 0.0029) and cardiothoracic ratio (p = 0.017) were the second and third most predictive variables, respectively. Evaluation of the delta LVEF, which reflects residual myocardial contractile reserve, can provide important information about the prognosis of patients with DCM and mild symptoms.

Trichosporon beigelii pneumonia in patients with hematologic malignancies.

Trichosporon beigelii is a causative agent of hypersensitivity pneumonia in immunocompetent individuals and of invasive pneumonia in immunocompromised patients. The actual incidence and clinical manifestations of T beigelii pneumonia are obscure because the diagnosis is sometimes difficult. We studied eight patients with T beigelii pneumonia diagnosed by immunohistochemical investigation of lung tissue sections and/or isolation of the organism from the lung, sputum, or blood. All patients had underlying hematologic malignancies for which they had received cytotoxic chemotherapy, resulting in profound neutropenia. The clinical manifestations were persistent fever unresponsive to broad-spectrum antibiotic therapy, cough, bloody sputum, and rapidly progressive dyspnea. The chest radiographs showed diffuse alveolar infiltrates in four patients, diffuse interstitial infiltrates in one, patchy reticulonodular infiltrates in one, and focal alveolar infiltrates in two. Histopathologic examination demonstrated numerous centrally necrotic foci with minimal cellular inflammatory reaction, intra-alveolar hemorrhage, and edema. Trichosporon beigelii consisting of both yeast and hyphal forms was located predominantly in the alveolar vessels. In neutropenic patients with hematologic malignancies, this fungus appears to enter the lung not only through the airways but also via the hematogenous route. In vitro susceptibility testing indicated borderline susceptibility to amphotericin B and showed that some azoles were active against T beigelii at safely achievable serum concentrations.