CrkL is recruited through its SH2 domain to the erythropoietin receptor and plays a role in Lyn-mediated receptor signaling.

The erythropoietin (Epo) receptor transduces its signals by activating physically associated tyrosine kinases, mainly Jak2 and Lyn, and thereby inducing tyrosine phosphorylation of various substrates including the Epo receptor (EpoR) itself. We previously demonstrated that, in Epo-stimulated cells, an adapter protein, CrkL, becomes tyrosine-phosphorylated, physically associates with Shc, SHP-2, and Cbl, and plays a role in activation of the Ras/Erk signaling pathway. Here, we demonstrate that Epo induces binding of CrkL to the tyrosine-phosphorylated EpoR and SHIP1 in 32D/EpoR-Wt cells overexpressing CrkL. In vitro binding studies showed that the CrkL SH2 domain directly mediates the EpoR binding, which was specifically inhibited by a synthetic phosphopeptide corresponding to the amino acid sequences at Tyr(460) in the cytoplasmic domain of EpoR. The CrkL SH2 domain was also required for tyrosine phosphorylation of CrkL in Epo-stimulated cells. Overexpression of Lyn induced constitutive phosphorylation of CrkL and activation of Erk, whereas that of a Lyn mutant lacking the tyrosine kinase domain attenuated the Epo-induced phosphorylation of CrkL and activation of Erk. Furthermore, Lyn, but not Jak2, phosphorylated CrkL on tyrosine in in vitro kinase assays. Together, the present study suggests that, upon Epo stimulation, CrkL is recruited to the EpoR through interaction between the CrkL SH2 domain and phosphorylated Tyr(460) in the EpoR cytoplasmic domain and undergoes tyrosine phosphorylation by receptor-associated Lyn to activate the downstream signaling pathway leading to the activation of Erk and Elk-1.

Rac is activated by tumor necrosis factor alpha and is involved in activation of Erk.

Tumor necrosis factor alpha (TNFalpha) activates various signal transduction pathways including those involving phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinases (Erk), c-Jun N-terminal protein kinases (JNK), and p38 kinases. Using the Rac binding domain of PAK (PAK-RBD) as an activation-specific probe, here we demonstrate that TNFalpha very rapidly and transiently activates the Rho family GTPase Rac in L929 cells. The PI3K inhibitor LY294002 significantly inhibited TNFalpha activation of Rac as well as Erk and abolished that of the PI3K target Akt, without showing any inhibitory effects on JNK and p38 activation. Furthermore, TNFalpha activation of Erk was abolished by a dominant negative Rac mutant, Rac17N, or by an activated Rac mutant, Rac12V. These findings suggest that Rac is activated by a mechanism that is at least partly dependent on PI3K in TNFalpha stimulated cells and plays a critical role in activation of the Erk signaling pathway.

Rap1 is activated by erythropoietin or interleukin-3 and is involved in regulation of beta1 integrin-mediated hematopoietic cell adhesion.

The CrkL adaptor protein is involved in signaling from the receptor for erythropoietin (Epo) as well as interleukin (IL)-3 and activates beta(1) integrin-mediated hematopoietic cell adhesion through its interaction with C3G, a guanine nucleotide exchange factor for Rap1. We demonstrate here that Epo as well as IL-3 activates Rap1 in an IL-3-dependent hematopoietic cell line, 32D, expressing the Epo receptor. The cytokine-induced activation of Rap1 was augmented in cells that inducibly overexpress CrkL or C3G. The CrkL-mediated enhancement of cell adhesion was inhibited by expression of a dominant negative mutant of Rap1, Rap1A-17N, whereas an activated mutant of Rap1, Rap1A-63E, activated beta(1) integrin-dependent adhesion of hematopoietic cells. In 32D cells, Rap1 was also activated by phorbol 12-myristate 13-acetate and ionomycin, which also enhanced cell adhesion to fibronectin, whereas, an inhibitor of phospholipase C, inhibited both cytokine-induced activation of Rap1 and cell adhesion. It was also demonstrated that Rap1 as well as CrkL is involved in signaling from the EpoR endogenously expressed in a human leukemic cell line, UT-7. These results suggest that Epo and IL-3 activate Rap1 at least partly through the CrkL-C3G complex as well as through additional pathways most likely involving phospholipase Cgamma and strongly implicate Rap1 in regulation of beta(1) integrin-mediated hematopoietic cell adhesion.

Association of vitamin D receptor gene polymorphism with periodontal diseases in Japanese and Chinese.

We examined whether polymorphisms in the vitamin D receptor (VDR) gene are associated with the incidence of adult periodontitis (AP) and early-onset periodontitis (EOP) in case-controlled studies of Japanese and Chinese subjects. Restriction fragment length polymorphisms in the VDR gene detected by digestion with Taq I were found to be significantly associated with the occurrence of AP or EOP, suggesting that the VDR genotype a risk factor for periodontitis.

Placement of implants in distraction osteogenesis: a pilot study in dogs.

This study investigated the possibility of achieving osseointegration of implants placed in a distracted site during the consolidation period. Four healthy male mongrel dogs were used in this experiment. A subperiosteal corticotomy around the mandible was performed between the left mandibular premolar and first molar. After a 7-day latency period for soft tissue healing, the distraction was performed at the rate of 1 mm per day for 14 consecutive days to allow for 14 mm of elongation, using an extraoral distraction device. Three weeks after the completion of distraction, screw-type implants were placed in the distracted site. Twenty-four weeks after placement of the implants, they were stable, and osseointegration had been achieved physically, radiographically, and histologically. These results suggest the possibility of shortening the period of implant treatment by using the distraction osteogenesis technique.

CrkL mediates Ras-dependent activation of the Raf/ERK pathway through the guanine nucleotide exchange factor C3G in hematopoietic cells stimulated with erythropoietin or interleukin-3.

CrkL is an SH2 and SH3 domain-containing adaptor protein implicated in pathogenesis of chronic myelogenous leukemia. Here, we demonstrate that overexpression of CrkL enhances the erythropoietin (Epo)- or interleukin (IL)-3-induced activation of Elk-1 and the c-fos gene promoter activity in 32D/EpoR-Wt cells. Moreover, the Epo-induced activation of ERK1 and ERK2 was augmented and prolonged in cells inducibly overexpressing CrkL. A moderate increase in Epo-induced activation of JNK was also observed in cells overexpressing CrkL. Overexpression of C3G enhanced the Elk-1 activation synergistically with CrkL, while a C3G mutant lacking the guanine nucleotide exchange domain showed an inhibitory effect. Studies using a dominant negative Ha-Ras mutant demonstrated that the Elk-1 and ERK2 activation enhanced by CrkL and C3G was dependent on Ras. Consistent with this, the Epo-induced activation of Ras was augmented in cells inducibly overexpressing CrkL. Most importantly, a CrkL mutant defective in the SH2 or N-terminal SH3 domain showed an inhibitory effect on the Epo-induced activation of ERK2. These data indicate that the CrkL-C3G complex plays a role in Epo- or IL-3-induced, Ras-dependent activation of the Raf/ERK pathway leading to the activation of Elk-1 and the c-fos gene transcription.

Posttraumatic intestinal stenosis presenting as a perforation: report of a case.

A 78-year-old woman was admitted to the hospital after falling into a ditch approximately 1 m deep and sustaining a blunt abdominal trauma with a left femur fracture. On the tenth day after admission, symptoms of a small bowel obstruction occurred. A nasogastric tube was inserted, and the symptoms thus improved. She sometimes complained of abdominal pain during the 12 months after the fall, but recovered with conservative management. The next year, she was readmitted to the hospital for a pin extraction of the left femur bone. During this admission, 15 months since her admission after her fall, she again complained of abdominal pain. Abdominal pain increased with a muscular defense, and abdominal X-rays revealed free air. She was referred to our hospital with a diagnosis of perforative peritonitis, and emergency surgery was performed. Upon laparotomy, circumferential stenoses of the small bowel were recognized in the proximal segments about 40cm, 80cm, and 100cm from the ileocecal region. In addition, a perforation and prominent dilatation of the bowel segment was observed just proximal to the stenosis about 100cm from the ileocecal region. She underwent a small intestinal resection at two sites. There were no findings of an intestinal specific ulcer, such as Crohn's disease, intestinal tuberculosis, or malignancy, based on the results of a histopathological examination.

CrkL activates integrin-mediated hematopoietic cell adhesion through the guanine nucleotide exchange factor C3G.

CrkL is a member of the Crk family of adapter proteins consisting mostly of SH2 and SH3 domains. CrkL is most abundantly expressed in hematopoietic cells and has been implicated in pathogenesis of chronic myelogenous leukemia. However, its function has not been precisely defined. Here, we show that overexpression of CrkL enhances the adhesion of hematopoietic 32D cells to fibronectin. The CrkL-induced increase in cell adhesion was blocked by antibodies against VLA-4 (alpha4beta1) and VLA-5 (alpha5beta1) but was observed without changes in surface expression levels of these integrins. Studies using CrkL mutants demonstrated that the SH2 domain is partially required for enhancing cell adhesion, whereas the C-terminal SH3 domain as well as the tyrosine phosphorylation site (Y207) is dispensable. In contrast, the N-terminal SH3 domain, involved in binding C3G and other signaling molecules, was showed to play a crucial role, because a mutant defective of this domain showed an inhibitory effect on the cell adhesion to fibronectin. Furthermore, overexpression of C3G also increased the adhesion of hematopoietic cells to fibronectin, whereas a C3G mutant lacking the guanine nucleotide exchange domain abrogated the CrkL-induced increase in cell adhesion. On the other hand, a dominant negative mutant of H-Ras or that of Raf-1 enhanced the basal and CrkL-induced cell adhesion and that of R-Ras modestly decreased the adhesion. Taken together, these results indicate that the CrkL-C3G complex activates VLA-4 and VLA-5 in hematopoietic cells, possibly by activating the small GTP binding proteins, including R-Ras, through the guanine nucleotide exchange activity of C3G.

A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors.

Synthetic benzamide derivatives were investigated for their ability to inhibit histone deacetylase (HDA). In this study, one of the most active benzamide derivatives, MS-27-275, was examined with regard to its biological properties and antitumor efficacy. MS-27-275 inhibited partially purified human HDA and caused hyperacetylation of nuclear histones in various tumor cell lines. It behaved in a manner similar to other HDA inhibitors, such as sodium butyrate and trichostatin A; MS-27-275 induced p21(WAF1/CIP1) and gelsolin and changed the cell cycle distribution, decrease of S-phase cells, and increase of G1-phase cells. The in vitro sensitivity spectrum of MS-27-275 against various human tumor cell lines showed a pattern different than that of a commonly used antitumor agent, 5-fluorouracil, and, of interest, the accumulation of p21(WAF1/CIP1) tended to be faster and greater in the cell lines sensitive to MS-27-275. MS-27-275 administered orally strongly inhibited the growth in seven of eight tumor lines implanted into nude mice, although most of these did not respond to 5-fluorouracil. A structurally analogous compound to MS-27-275 without HDA-inhibiting activity showed neither the biological effects in cell culture nor the in vivo therapeutic efficacy. These results suggest that MS-27-275 acts as an antitumor agent through HDA inhibition and may provide a novel chemotherapeutic strategy for cancers insensitive to traditional antitumor agents.

[Thoraco-abdominal impalement injury with diaphragmatic injury: a case report].

A 60-year-old man was admitted to our hospital because of impalement injury due to traffic accident. Chest X-ray on admission revealed normal lung field. CT scans of the chest and abdomen revealed slight pneumothorax and intra-abdominal organ protruding from abdominal cavity. An emergency operation was performed. Diaphragmatic injury was not detected during the abdominal procedure. On exploring the back wound, we found a laceration of 8 cm in diameter in the diaphragm and repaired it. Impalement injuries which have aspects of both blunt and penetrating trauma are uncommon. Accordingly, wound exploration and debridement of fistulous tract are necessary. In the case of thoraco-abdominal injuries by impalement, one should bear in mind the existence of diaphragmatic injury even with normal diaphragmatic shadow on chest X-ray.

[A refractory case of relapsing polychondritis].

We report here a case of relapsing polychondritis (RPC), who responded to salazosulfapyridine (SASP) after 1.5 years of active disease. A 15-year-old girl having chondritis of bilateral auricles, nose and respiratory tract, ocular inflammation, cochlear and vestibular dysfunction and seronegative polyarthritis was diagnosed as RPC, and oral corticosteroid was initially effective. She was admitted to our hospital with obstructive tract chondritis and was refractory to any of the treatment such as intravenous (i.v.) pulse steroid therapy, i.v. pulse cyclophosphamide (CPA), oral intermittent methotrexate (MTX) and high dose i.v. gamma globulin. Although she was partly responsive to oral cyclosporine A (CsA), but clinical symptoms did not completely subside. SASP, initiated in combination with oral corticostreroid and CsA, however, was successful not only to show steroid sparing effect but also to induce complete remission. SASP might be useful to refractory case of RPC.

A Japanese multicenter study of osseointegrated implants placed in irradiated tissues: a preliminary report.

A survey was undertaken to analyze implants placed in irradiated tissues. It was found that nine centers had placed 118 implants in 24 patients in Japan. Of 118 implants, 39 were in the maxilla, 71 were in the mandible, and 8 were in the orbital region. Seven patients underwent adjunctive hyperbaric oxygen treatment. The treatment decreased implant loss only in the maxilla. (The success rate without hyperbaric oxygen treatment was 62.5%, and that with hyperbaric oxygen treatment was 80.0% for the maxilla.) Implants 7 and 10 mm in length were at a greater risk of being lost than longer implants in the maxilla.

Distribution of IL-15 receptor alpha-chains on human peripheral blood mononuclear cells and effect of immunosuppressive drugs on receptor expression.

IL-15, a newly described cytokine exerting IL-2-like in vitro activities, binds to and induces proliferation of cells co-expressing IL-15R alpha, IL-2R beta, and IL-2R gamma chains. To study the expression of human IL-15R alpha chains, we have utilized tagged human IL-15 protein and FACS analysis. In contrast to resting cells, mitogen-activated macrophages, NK cells, and CD4+ and CD8+ T cells express IL-15R alpha chains. Neither IL-2R alpha nor IL-2R beta chains are required for IL-15 binding. Dexamethasone, but not cyclosporine or rapamycin, blocks mitogen-induced IL-15R alpha expression. Dexamethasone-pretreated cells respond to IL-15 poorly, while the response to IL-2 is not affected. Thus, despite structural and functional similarities between IL-2R alpha and IL-15R alpha chains, the activation-triggered mechanisms of induction are different. Since IL-15R alpha chain is necessary and sufficient for IL-15 binding, regulation of IL-15R alpha expression may represent a new target for T cell-directed pharmacologic intervention.

Minute hepatocellular carcinoma originating in the caudate lobe with a long-term surviving of 9 years 6 months after surgical resection: a case report.

An extremely rare case of minute hepatocellular carcinoma originating in the caudate lobe who was resected and surviving for 9 years 6 months is presented. A 52 year-old male patient was diagnosed as having a minute hepatocellular carcinoma originating in the caudate lobe by lipiodol computed tomography and celiac angiography. Although the caudate lobectomy was successfully performed, refractory ascites developed for 4 months after surgical procedure. After remission of ascites, the patient is well and surviving for 9 years 6 months without recurrence of hepatocellular carcinoma. This case suggests that early detection and treatment of hepatocellular carcinoma can lead to a long-term survival even when the caudate lobe of the liver is involved.

The effect of minocycline in rat models of inflammatory arthritis: correlation of arthritis suppression with enhanced T cell calcium flux.

Adjuvant and collagen arthritis in the rat are widely accepted T-cell-dependent counterparts of rheumatoid arthritis and were used to examine the antiinflammatory properties of minocycline. Administration of oral minocycline, a semisynthetic tetracycline, significantly decreased (P < 0.01) the incidence of arthritis in both models. In vivo exposure to minocycline also significantly increased the percentage of splenocytes exhibiting a rise in free intracellular calcium concentration ([Ca2+]i) following concanavalin A stimulation (P < 0.05 in adjuvant and P < 0.01 in collagen). This enhancement was mitogen dose-dependent and supported exclusively by extracellular Ca2+. Resting [Ca2+]i levels were unaffected by minocycline and predominantly the CD4+ subset was involved. No changes were observed in weight, IgG antibodies to collagen, synoviocyte release of collagenase and prostaglandin E2, acute inflammation in an air-pouch system, or cell surface expression of activation markers (interleukin-2 and transferrin receptors) by splenocytes or lymph node cells. As a controlled [Ca2+]i rise is a critical event in normal T cell activation, minocycline's antiarthritic profile in vivo may relate to perturbed Ca2+ influx during T cell activation, an alteration that could promote the development of clinical tolerance to otherwise arthritogenic stimuli.

Effect of renal prostaglandins on survival in patients with liver cirrhosis.

To assess the effect of renal prostaglandins on the prognosis of liver cirrhosis, we studied the relationship between the effect of prostaglandin synthetase inhibition by indomethacin on glomerular filtration rate (GFR) and effective renal plasma flow (RPF) and survival in 30 patients with liver cirrhosis. After indomethacin administration, GFR dropped significantly from 86 +/- 2 ml/min to 73 +/- 3 ml/min (p less than 0.01) and RPF from 421 +/- 10 to 349 +/- 14 ml/min (p less than 0.01). Survival was not correlated with baseline GFR or RPF, but was correlated with changes in both GFR and RPF after indomethacin administration (p less than 0.01). The cumulative survival rate from 1 to 7 yr was significantly higher (p less than 0.01) in patients with a decrease in GFR of less than or equal to 15% after indomethacin administration than in patients with a decrease in GFR of more than 15%. Findings for changes in RPF after indomethacin administration were similar. These results suggest that the augmentation of prostaglandin inhibition by indomethacin is associated with a poor prognosis of liver cirrhosis. It may be involved in the depletion of renal prostaglandins to maintain renal hemodynamics in patients with liver cirrhosis.