Mechanical regulation of bone homeostasis through p130Cas-mediated alleviation of NF-κB activity.

Mechanical loading plays an important role in bone homeostasis. However, molecular mechanisms behind the mechanical regulation of bone homeostasis are poorly understood. We previously reported p130Cas (Cas) as a key molecule in cellular mechanosensing at focal adhesions. Here, we demonstrate that Cas is distributed in the nucleus and supports mechanical loading-mediated bone homeostasis by alleviating NF-κB activity, which would otherwise prompt inflammatory processes. Mechanical unloading modulates Cas distribution and NF-κB activity in osteocytes, the mechanosensory cells in bones. Cas deficiency in osteocytes increases osteoclastic bone resorption associated with NF-κB-mediated RANKL expression, leading to osteopenia. Upon shear stress application on cultured osteocytes, Cas translocates into the nucleus and down-regulates NF-κB activity. Collectively, fluid shear stress-dependent Cas-mediated alleviation of NF-κB activity supports bone homeostasis. Given the ubiquitous expression of Cas and NF-κB together with systemic distribution of interstitial fluid, the Cas-NF-κB interplay may also underpin regulatory mechanisms in other tissues and organs.

IKK/NF-kappaB signaling pathway inhibits cell-cycle progression by a novel Rb-independent suppression system for E2F transcription factors.

E2Fs are key regulators of cell-cycle progression, and their transcriptional activities are regulated by histone acetyltransferases (HATs). Retinoblastoma (Rb) family proteins (pRb, p107 and p130) bind to E2Fs and inhibit their transcriptional activities by disrupting HAT binding and recruitment of histone deacetylases. In this study, we show that IkappaB kinases (IKKalpha or IKKbeta) activation inhibits cell growth and E2F-dependent transcription in normal human fibroblasts. The inhibition of E2F by IKKs was not observed in cells lacking nuclear factor (NF)-kappaB/p65; however, it was observed in cells lacking three Rb family genes. p65 disrupted the physical interaction between activator E2Fs (F2F1, E2F2 and E2F3) and the HAT cofactor transactivation/transformation-domain associated protein, resulting in a reduction in E2F-responsive gene expression. Furthermore, IKKalpha and IKKbeta directly phosphorylated E2F4, resulting in nuclear accumulation and enhanced DNA binding of the E2F4/p130 repressor complex. Our study describes a novel growth inhibitory system that functions by Rb-independent suppression of E2Fs by the IKK/NF-kappaB signaling pathway.

Human beta-defensin-3 induction in H. pylori-infected gastric mucosal tissues.

AIM: To examine human beta-defensin-3 (hBD-3) expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H. pylori infection for better understanding the innate immune response to H. pylori. METHODS: We used reverse transcription-polymerase chain reactions and immunohistochemistry to examine hBD-3 expression in inflamed gastric mucosal tissues or MKN45 gastric cancer cells with or without H. pylori. Effects of hBD-3 against H. pylori were also evaluated. RESULTS: The mean mRNA expression of hBD-3 in H. pylori-positive specimens was significantly higher than that in H pylori-negative specimens (P = 0.0002, Mann-Whitney). In addition, unlike uninfected samples, 8 of 15 (53.33%) infected mucosal samples expressed hBD-3 protein. H. pylori dose-dependently induced mRNA expression of hBD-3 in MKN45 cells, an effect inhibited by adding anti-toll-like receptor (TLR)-4 antibody. HBD-3 protein completely inhibited H. pylori growth. CONCLUSION: Our results suggest that like hBD-2, hBD-3 may be involved in the pathophysiology of H. pylori-induced gastritis.

[Testicular involvement in IgD multiple myeloma].

A 65-year-old man was admitted to our hospital in September 1997 because of back pain and renal dysfunction. He was diagnosed as having multiple myeloma due to the presence of IgD lambda monoclonal gammopathy and diffuse infiltration of plasma cells in the bone marrow. The patient achieved a partial response to DMVM-IFN-alpha combination therapy. His condition worsened in December 1998, but was ameliorated by VAD therapy. The patient's left testis became enlarged in December 1999, and an orchiectomy was performed. The normal testicular cells had been entirely displaced by myeloma cells comprising typical plasma cells and large lymphoid cells. Pleural, mediastinal, spinal, and right testicular involvement with myeloma subsequently developed. Despite attempts to treat the patient with more than one type of combination therapy, his condition worsened progressively, and he died in June 2000. Reports of IgD myeloma with testicular involvement are rare. The histopathology of our patient's resected testis, i.e. the two myeloma cell-plasmacytoid and lymphoid cell components showing differential immunostaining, was unique.

An Asian variant of intravascular large B-cell lymphoma: clinical, pathological and cytogenetic approaches to diffuse large B-cell lymphoma associated with haemophagocytic syndrome.

Diffuse large B-cell lymphoma with haemophagocytic syndrome (BCL-HS) has been reported mainly in Asia and is regarded as a distinct variant of intravascular lymphoma (IVL). However, it is unclear whether all cases of BCL-HS fall within the framework of IVL and available clinical information is limited. We analysed 25 cases with BCL-HS, including 11 autopsied cases (median, 66 years; male-female ratio, 1.1:1). The patients presented with fever, anaemia, thrombocytopenia, hepatosplenomegaly, haemophagocytosis, bone marrow invasion, respiratory disturbance and disseminated intravascular coagulopathy, but usually lacked lymphadenopathy, mass formation, neurological abnormalities and skin lesions. The clinical course was aggressive with a median survival of 7 months. The morphological findings were uniform: large lymphoid cells infiltrated vessels and/or sinusoids of the liver, marrow, lung, kidney and other organs. They were positive for CD19, CD20, CD79a and HLA-DR, but negative for CD10, CD23 and CD30. CD5 was positive in five out of 17 cases. Our critical review indicates that BCL-HS is the equivalent of the Asian variant of IVL.

Demonstration of Cl- requirement for inhibition of vacuolar acidification by cycloprodigiosin in situ.

Applying vacuole-perfusion and plasma membrane permeabilization techniques to internodal cells of Chara, we analyzed the requirement of Cl- for the action of cycloprodigiosin (cPrG) to inhibit vacuole acidification in situ. By combining the two techniques, the Cl- concentration on both sides of the tonoplast could be controlled. In permeabilized cell fragments lacking Cl- in the vacuole, the inhibitory effect of cPrG on vacuole acidification was cancelled. On the other hand, Cl- in the cytoplasm was not needed for the cPrG action. These results supported the function of cPrG as a H+/Cl- symporter. Requirement of Cl- for the cPrG action was also demonstrated in vacuole-perfused living cells. This is the first report on the mechanism of cPrG action in situ.

[Sustained long-term complete remission in an elderly aplastic anemia patient after cessation of combined therapy consisting of granulocyte-colony stimulating factor and erythropoietin].

An 83-year-old woman received a diagnosis of moderate aplastic anemia in November 1990. Immunosuppressive therapy consisting of anti-lymphocyte globulin combined with high-dose corticosteroids was effective until pancytopenia developed in August 1993. The patient was hospitalized again and recurrence of aplastic anemia was diagnosed on the basis of hematologic findings, including RBC 129 x 10(4)/microliter, Hb 5.5 g/dl, Ret 23,200/microliter, WBC 2,200/microliter with 27% neutrophils, platelets 2.2 x 10(4)/microliter, and hypoplastic bone marrow. Recombinant human granulocyte-colony stimulating factor (G-CSF) of 125 micrograms/day combined with recombinant human erythropoietin (EPO) of 6,000 U/day were started in November 1993. The doses of G-CSF and EPO were increased to 250 micrograms/day and 12,000 U/day, respectively. We stopped combination therapy in March 1995, after trilineage hematopoietic cell recovery had been achieved. Complete recovery in peripheral blood was sustained for more than 2 years despite the termination of G-CSF and EPO therapy. Combination therapy with G-CSF and EPO may be safe and effective for elderly patients with aplastic anemia when the choice of therapy is limited.

[Clinical characterization of B cell lymphoma-associated hemophagocytic syndrome].

Malignant lymphoma is a major cause of hemophagocytic syndrome (HPS), in which reactive macrophages, phagocytic red blood cells, white blood cells, and platelets proliferate in bone marrow, liver, and spleen. In contrast to T/NK-cell lymphoma-associated hemophagocytic syndrome (T/NK-LAHS), few cases of B-LAHS have been reported; thus, the clinical characterization of B-LAHS remains to be established. We describe here four cases of B-LAHS that include the following features: (1) HPS was the initial presentation; (2) bone marrow involvement with large-cell lymphomas was noted in all cases, despite lack of remarkable lymphadenopathy; (3) no active infection with Epstein-Barr virus as the etiological agent was confirmed; (4) except for the spleen in one case, primary site of lymphoma could not be determined; and (5) serum IL-6, soluble IL-2 receptor, and IFN-gamma- but not TNF-alpha and IL-1 beta-, were significantly elevated. Such characteristics are peculiar to and different from those usually seen in B-cell lymphoma, suggesting that B-LAHS is a unique clinical entity among B-cell lymphomas.

Histological evaluation of allograft bone after acetabular revision arthroplasty: report of two cases.

We recently had the opportunity to take histological sections from two patients who underwent acetabular reconstruction in which allograft and ME Müller acetabular roof reinforcement rings were used. In one patient (case 1), histological sections of the chipped allograft were taken on two separate occasions from the same area, at 7 months, and at 3 years and 11 months after the bone graft. The histology of the chipped allograft showed necrosis at 7 months, but almost normal morphology of trabecular bone formation at 3 years and 11 months after the bone graft. In the other patient (case 2) histological sections of the block allograft and chipped allograft were taken at 1 year and 8 months after the bone graft. The block allograft showed only a small amount of admixture of newly formed bone with the necrotic bone, while the chipped allograft showed a large amount of newly formed bone, with only a small amount of necrotic bone remaining. Therefore, we principally use chipped allograft for acetabular reconstruction, in order to achieve early and complete graft incorporation. If a block allograft is used in a weight-bearing area, it should be protected from excessive load by using an acetabular reinforcement device.

Cycloprodigiosin hydrochloride obtained from Pseudoalteromonas denitrificans is a potent antimalarial agent.

Cycloprodigiosin hydrochloride (cPrG*HCl) is a stable fluorescent red pigment obtained from the marine bacterium Pseudoalteromonas denitrificans. It was found that the compound was incorporated into Plasmodium falciparum cells upon incubation and exhibited a potent antimalarial activity with the concentration required for 50% of the activity being 11 nM, which is stronger than that of chloroquine, a well-known antimalarial agent. The compound did not affect growth rate of mammalian cells. Antimalarial activity of cPrG*HCl was also observed in vivo. These results indicate that cPrG*HCl is a potent antimalarial drug.

A possible immunosuppressant, cycloprodigiosin hydrochloride, obtained from Pseudoalteromonas denitrificans.

Cycloprodigiosin hydrochloride (cPrG.HCl), a member of the prodigiosin family, is a red pigment obtained from the marine bacterium Pseudoalteromonas denitrificans. cPrG.HCl markedly suppressed 3H-thymidine incorporation by concanavalin A stimulated murine splenocytes but had little effect on lipopolysaccharide dependent 3H-thymidine incorporation, indicating that cPrG.HCl acts as a selective inhibitor of T cell proliferation in the same way as other members of the prodigiosin family. cPrG.HCl inhibited the proliferation of the PMA stimulated Jurkat cells through an apoptotic process. Intriguingly, cPrG.HCl inhibited the H+ translocation by vacuolar type ATPase in chromaffin granule membranes without any effect on either its ATPase activity nor on the membrane conductance of phospholipid bilayers, suggesting that cPrG.HCl selectively uncouples H+ translocation from the ATPase reaction rather than acting as a non-specific ionophore. Since crystalline cPrG.HCl is highly stable, it raises the possibility of its therapeutic use as an immunosuppressant.

A novel technique for the study of Ras activity: electroporation of [alpha-32P]GTP.

An efficient, simple, and reproducible procedure for the assessment of Ras activity present in adherent mammalian cells is described. [alpha-32P]GTP was introduced by in situ electroporation into mouse C3H10T1/2 fibroblasts or their ras(val12)-transformed derivatives. After a 3-hr incubation at 37 degrees C, Ras was immunoprecipitated from cell extracts and the Ras-bound GTP/GTP + GDP ratio was determined by thin-layer chromatography. Contrary to Streptolysin-O permeabilization, the cells are not affected in any detectable way by the procedure, so that [alpha-32P]GTP binding and conversion to [alpha-32P]GDP can be studied over a period of time for the measurement of steady-state Ras activity. The results show that careful control of electric field intensity results in a great increase in the efficiency and specificity of labelling compared to the addition of [32P]orthophosphate to the culture medium, while the GTP/GTP + GDP ratios obtained were essentially the same as after in vivo labeling.

[The role of p21ras oncoprotein in signal transduction mediated through B cell antigen receptor (BCR)].

Ligation of B cell antigen receptor (BCR) with antigen or anti IgM leads to enter cells into the proliferation and differentiation to produce specific Ig. Protein tyrosine kinase (PTK) and CD 45 protein tyrosine phosphatase (PTP) both are involved in the early phase of BCR-mediated B cell signaling. We have investigated the role of p 21ras(ras) in B cell signal transduction using TNP-specific TA3 7.9 murine B cells. B cell stimulation with either TNP6-OVA(Ag) or anti-IgM resulted in rapid accumulation of GTP-bound(active) ras as well as induction of a number of tyrosine phosphorylated substrates. The accumulation of GTP-bound ras was blocked by the treatment with either PTK inhibitors(genistein) or PTP inhibitors(PAO), suggesting that BCR-mediated ras activation is regulated by both PTK and PTP including CD 45. As phosphorylation on tyrosine residues of Lyn, Fyn, and Blk protein tyrosine kinases occurred upon BCR stimulation, these PTKs may be candidates being involved in an induction of not only tyrosine phosphorylation of substrates but also p 21ras activation. Furthermore we found that rasGAP activity is suppressed following Ag stimulation, accompanied by the phosphorylation on tyrosine of rasGAP and its associated protein p 62. These data indicate that protein tyrosine kinases may alter rasGAP activity to induce p 21ras activation in B cells.

Regulation of BCR- and PKC/Ca(2+)-mediated activation of the Raf1/MEK/MAPK pathway by protein-tyrosine kinase and -tyrosine phosphatase activities.

Ligation of the B cell Ag receptor (BCR) activates a protein-tyrosine kinase (PTK) and CD45 protein-tyrosine phosphatase (PTPase)-dependent signaling cascade that results in the activation of Ras. This pathway of Ras activation can operate independently of protein kinase C (PKC) activity. Activation of Ras may lead to two distinct Ras-dependent pathways involving either a Raf1/MEK/MAPK module or a MEKK/SEK/SAPK module; however, it is unclear as to how Ras controls the independent activation of either of these pathways. We have used genistein and phenylarsine oxide (PAO) as inhibitors of PTK and PTPase, respectively, to investigate whether they regulate the BCR- and Ca2+/PKC-dependent activation of the Ras/Raf1/MEK/MAPK module. Assays of phosphotransferase activities conducted with Ag (TNP6-OVA)-specific 7.9 murine B lymphoma cells demonstrated that BCR-mediated stimulation of the Raf1/MEK/MAPK module is controlled by PTK and PTPase activities. An elevation in [Ca2+]i was required to optimally activate Raf1 and MEK through the BCR. However, when signaling through the BCR was bypassed by direct stimulation of the Raf1/MEK/MAPK module via a rise in [Ca2+]i and phorbol ester-induced PKC activation, the phosphotransferase activities of Raf1, MEK and MAPK were still regulated in a PTK-dependent manner that was also partially sensitive to the PTPase inhibitor PAO. Thus, at least two alternate routes, i.e. a BCR/PTK/Ras-dependent route and another PKC/Ca(2+)-dependent route, may converge at the level of Raf1 for activation of the Raf1/MEK/MAPK module in B cells.

[Effect of blood conservation in open-heart surgery: a comparison of 3 different methods].

Conventional blood conservation techniques have been insufficient to decrease blood transfusion requirement in open-heart surgery. Blood conservation and erythropoietin administration were performed to avoid homologous blood transfusion. Intraoperative autotransfusion has been routinely used in cardiac operations with cardiopulmonary bypass in our hospital. To evaluate the effect of conservation techniques, 286 patients were divided into four groups. In group I (23 patients), autologous whole blood was drawn and saved one to two weeks before operation. In group II (50 patients), erythropoietin preparation was given subcutaneously once a week and autologous blood conservation was also performed in the same manner as group I. In group III (48 patients), intra-operative hemodilutional autologous blood transfusion was performed. In group IV, as a control group (165 patients), only intra-operative autotransfusion was used. Homologous blood transfusion was avoided in 83% of group I patients, in 90% of group II, in 82% of group III, and 29% of group IV. In addition, in group II the hemoglobin value at the time of discharge was significantly higher than those of other groups (p < 0.05-0.01). Thus, conventional blood conservation techniques plus subcutaneous administration of erythropoietin was very effective to increase the rate of "non-blood" open-heart surgery.

Tyrosine kinase and CD45 tyrosine phosphatase activity mediate p21ras activation in B cells stimulated through the antigen receptor.

Cross-linking of the Ag receptor (AgR) induces intracellular signaling events in B cells, such as p21ras activation, that lead to their proliferation and differentiation. This event is accompanied by the tyrosine phosphorylation of the p21ras-associated GTPase-activating protein p120 ras.GAP, raising the possibility that AgR-stimulated p21ras activity is regulated by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPases) in B cells. To test this possibility, we examined the effects of PTK and PTPase inhibitors on protein tyrosine phosphorylation and p21ras activation induced by AgR cross-linking in TNP-specific TA3 7.9 murine B lymphoma cells. Although AgR-induced protein tyrosine phosphorylation was inhibited by the PTK inhibitors genistein and herbimycin A, it was enhanced by exposure to the PTPase inhibitor phenylarsine oxide (PAO). Cross-linking of the AgR by Ag or F(ab')2 anti-IgM induced a rapid (within 5 min) two- to threefold increase in p21ras activation in 7.9 B cells. Interestingly, a second peak of p21ras activation was evident at approximately 40 min after stimulation. Genistein and herbimycin A and PAO each blocked AgR-stimulated p21ras activation. Similarly, Ag-induced p21ras activation was inhibited by pretreatment of 7.9 B cells with an anti-CD45 mAb (detects the 220-kDa B cell isoform of CD45). Moreover, p21ras activation was induced by Ag and F(ab')2 anti-IgM in CD45+ but not CD45- J558L microns 3 B cells. These data indicate that p21ras activation induced by AgR cross-linking in B cells is regulated by both PTK and CD45 PTPase activities.

Near-infrared Fourier transform Raman spectroscopic study of cornea and sclera.

Near-infrared excited Fourier transform Raman spectroscopy was applied in situ on intact rabbit cornea and sclera. High quality Raman spectra were obtained noninvasively from these tissues. The characteristic bands assigned to proline and hydroxyproline appeared in the region of 800-1000 cm-1. Raman spectra of purified type I collagen and other components of the cornea and sclera were also determined. The main elements of the spectra of the cornea and sclera originated from the collagen proteins, and other components such as proteoglycan contributed little. Near-infrared excited Fourier transform Raman spectroscopy can be used for biophysical study of the cornea and sclera in situ or in vivo.

Antigen-induced B lymphocyte activation involves the p21ras and ras.GAP signaling pathway.

Ligation of a B lymphocyte surface immunoglobulin (sIg) antigen receptor (AgR) by its specific Ag ligand initiates a signaling pathway that culminates in B cell activation. However, many events of this pathway have not been elucidated. Here we present three novel findings that demonstrate directly that AgR-mediated signaling in B cells functions by the p21ras/ras.GAP-dependent pathway. First, stimulation of TA3 7.9 Ag-specific murine B lymphoma cells for 2 min with either Ag or F(ab')2 anti-IgM induces p21ras activation as measured by an increase in the GTP/GDP ratio of its bound nucleotides. This activation of p21ras does not occur via a change in its guanine nucleotide exchange rate. Second, Ag stimulation results in the inhibition of activity of p120 ras.GAP, a protein that regulates p21ras activation. Tyrosine phosphorylation of ras.GAP occurs within 1 min after Ag stimulation but is no longer detectable at 20 min after stimulation, at which time ras.GAP activity remains inhibited. Thus, tyrosine phosphorylation of ras.GAP is not required for the inhibition of its activity. Third, despite the role proposed for a ras.GAP-associated p190 protein in the control of ras.GAP activity in B cells, p190 was not detectable either in anti-ras.GAP immunoprecipitates of [35S]methionine labeled lysates of Ag-stimulated or -unstimulated 7.9 cells or as a tyrosine phosphoprotein in Western blots of anti-ras.GAP immunoprecipitates of Ag-stimulated 7.9 cell lysates. Inasmuch as the TA3 7.9 B lymphoma is representative of a mature, sIgM-bearing B cell, our observations raise the intriguing possibility that the capacity of p190 to associate with ras.GAP and regulate the activities of ras.GAP and p21ras in a B cell is dependent on the stage of differentiation of the B cell.

[Clinical evaluation of meropenem in the pediatric field].

A clinical study on a new carbapenem antibiotic, meropenem (MEPM), was carried out in acute pediatric infections. MEPM was administered to 8 patients including 3 patients with acute pneumonia, 2 with cervical lymphadenitis, 1 with acute tonsillitis, and 1 with cellulitis and 1 with sepsis. The overall efficacy rate was 100%. As an adverse reaction, diarrhea was observed in 1 patient. In clinical laboratory tests 1 patient was found to have S-GPT elevation which normalized after discontinuation of MEPM. MEPM appears to be effective and safe drug for pediatric acute infections.