CCR1 acts downstream of NFAT2 in osteoclastogenesis and enhances cell migration.

We found that a chemokine receptor gene, CCR1, acts downstream of NFAT2 in RANKL-stimulated RAW264 and bone marrow cells. The upstream regulatory region of CCR1 showed RANKL-dependent and CsA-suppressible promoter activity. Downregulation of the expression and function of CCR1 suppressed cell migration. INTRODUCTION: We previously reported that the expression of NFAT2 induced by RANKL is a key process for progression to multinucleated cells in an in vitro osteoclastogenesis system. Identifying the target genes of NFAT2 would thus be informative about the differentiation process. We focused here on chemokine and chemokine receptor genes that act downstream of NFAT2 in RAW264 cells as well as osteoclast precursors prepared from bone marrow cells. MATERIALS AND METHODS: RAW264 mouse monocyte/macrophage line cells were cultured with or without cyclosporin A (CsA) in the presence of RANKL or glutathione S-transferase (GST). Osteoclast precursors were prepared from bone marrow cells. RANKL-inducible and CsA-suppressible genes were searched for by microarray analysis, and expression was confirmed by quantitative RT-PCR. Promoter activity was measured by luciferase gene reporter assay. Short interfering (si)RNA for CCR1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay. RESULTS AND CONCLUSIONS: We identified the chemokine receptor gene CCR1 as a gene showing significant differential expression profiles in osteoclastogenesis in the presence versus the absence of CsA, an inhibitor of NFAT. This property was unique to CCR1 among the chemokine and chemokine receptor genes examined in both RAW264 and bone marrow cells. The upstream regulatory region was isolated from CCR1, and its RANKL-dependent and CsA-suppressible promoter activity was confirmed. The functional significance of CCR1 was assessed by monitoring the migration of cells in a transwell migration assay, and this activity was abolished when either CsA- or CCR1 siRNA-treated cells were used. Moreover, treatment with a Galpha inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (Met-RANTES), an antagonist of CCR1, suppressed multinucleated cell formation in the bone marrow cell system. Together, these results suggest that the CCR1 signaling cascade is under the control of NFAT2 and seems to enhance the migration of differentiating osteoclasts.     

Pre- and post-synaptic effects of spiradoline and U-50488H, selective kappa opioid receptor agonists, in isolated ileum.

In guinea pig ileum, spiradoline (2 x 10(-6) M or greater) and U-50488H (3 x 10(-6) M or greater) suppressed contractile responses to acetylcholine (ACh), histamine, and BaCl2. Inhibition by spiradoline (2 x 10(-5) M) of ACh-induced contractions was not antagonized by pretreatment with naloxone (3 x 10(-4) M). Spiradoline (2 x 10(-8) M or greater) and U-50488H (3 x 10(-8) M or greater) caused dose-dependent inhibition of the contractile response of guinea pig ileum to transmural electric stimulation. The inhibitory effect of spiradoline or of U-50488H at low concentrations was reduced by a high concentration of naloxone (3 x 10(-4) M). Spiradoline at low concentrations ranging from 2 x 10(-9) to 2 x 10(-7) M reduced spontaneous contractions in rabbit ileum. Naloxone (3 x 10(-4) g/ml) antagonized the spiradoline-induced inhibition, but marked inhibition by spiradoline at 10(-4) g/ml was not restored by naloxone. These results suggest that both kappa agonists exert presynaptic inhibitory action on cholinergic nerve endings in the myenteric plexus at a low concentration range of 10(-9) to 10(-7) M and directly inhibit the smooth-muscle motility of the gut at greater concentrations.     

Analysis of the determinant factors for open heart surgery without blood transfusion by the quantification theory--possibility of application of maximum surgical blood order schedule for open heart surgery]

In order to complete operations without blood transfusion we have chosen means of preoperative autologous blood saving and intraoperative autotransfusion, but we have not always achieved our purpose. We examined 29 patients (13 patients without blood transfusion and 16 with blood transfusion) to analyze the determinant factors as to whether open heart surgery without blood transfusion may be indicated or not, according to the quantification theory (type II) and to examine the possibility to apply the maximum surgical blood order schedule (MSBOS) for the open heart surgery by the quantification theory (type I). The analysis of determinant factors revealed hematocrit (Ht) value before saving of blood (more than 40%) as the best contributor of possibility of non-blood transfusion surgery, followed by the amount of blood loss during operation (less than 600 ml), the amount of saving blood (more than 800 ml), body weight (less than 70 kg), calculated Ht value on the beginning of cardiopulmonary bypass (CPB) (more than 24%), CPB time (less than 120 minutes) and the amount of postoperative blood loss (less than 600 ml). The prospective using blood volume at the operation was precisely calculated by the values of 4 preoperative factors, that is, the amount of saving blood, calculated Ht value on the beginning of CPB, CPB time and body weight. Therefore it is important to increase the amount of preoperative saving blood and decrease the amount of surgical bleeding in order to perform operations without blood transfusion, and is possible to apply the MSBOS for the open heart surgery.     

Differential induction responses of delta-aminolevulinate synthase mRNAs during erythroid differentiation: use of nonradioactive in situ hybridization.

Expression of mRNAs encoding the erythroid-specific delta-aminolevulinate synthase (ALAS-E) and the nonspecific delta-aminolevulinate synthase (ALAS-N) were examined in murine Friend virus-transformed erythroleukemia (MEL) cells using nonradioactive in situ hybridization. Following dimethyl sulfoxide (DMSO) treatment, ALAS-E mRNA increased markedly, while ALAS-N mRNA did not increase in wild-type MEL cells. In contrast, in a DMSO-resistant clone of MEL cells, ALAS-E was not detectable before and after DMSO treatment. These findings suggest that ALAS-E and ALAS-N mRNAs are under separate controls and that the expression of ALAS-E mRNA is a critical event in erythroid differentiation.     

Yucca leaf protein (YLP) stops the protein synthesis in HSV-infected cells and inhibits virus replication.

Yucca leaf protein (YLP), an inhibitor of tobacco mosaic virus isolated from the leaves of Yucca recurvifolia Salisb., exhibited potent activity against herpes simplex virus type 1 (HSV-1) with no cytotoxicity below 300 micrograms/ml. The inhibitory dose was varied with the time of addition; 50% effective concentrations (ED50) of YLP were 3, 19 and 95 micrograms/ml when YLP exposure was begun 3 h before virus infection, 0 h and 3 h after infection, respectively. This protein also inhibited the multiplication of herpes simplex virus type 2 and human cytomegalovirus. YLP has been shown to have a weak virucidal activity at higher concentrations. Analysis of early events following infection showed that YLP affected viral penetration in HeLa cells but did not interfere with adsorption to the cells. YLP was found to exert strong inhibition of protein synthesis in virus-infected cells but not in uninfected cells. This selective effect can be considered to attribute mainly to the antiviral activity of YLP.     

Mapping corpus callosum deficits in autism: an index of aberrant cortical connectivity.

BACKGROUND: Volumetric studies have reported reductions in the size of the corpus callosum (CC) in autism, but the callosal regions contributing to this deficit have differed among studies. In this study, a computational method was used to detect and map the spatial pattern of CC abnormalities in male patients with autism. METHODS: Twenty-four boys with autism (aged 10.0 +/- 3.3 years) and 26 control boys (aged 11.0 +/- 2.5 years) underwent a magnetic resonance imaging (MRI) scan at 3 Tesla. Total and regional areas of the CC were determined using traditional morphometric methods. Three-dimensional (3D) surface models of the CC were also created from the MRI scans. Statistical maps were created to visualize morphologic variability of the CC and to localize regions of callosal thinning in autism. RESULTS: Traditional morphometric methods detected a significant reduction in the total callosal area and in the anterior third of the CC in patients with autism; however, 3D maps revealed significant reductions in both the splenium and genu of the CC in patients. CONCLUSIONS: Statistical maps of the CC revealed callosal deficits in autism with greater precision than traditional morphometric methods. These abnormalities suggest aberrant connections between cortical regions, which is consistent with the hypothesis of abnormal cortical connectivity in autism.     

Ulnar recurrent adipofascial flap for reconstruction of massive defects around the elbow and forearm.

An ulnar recurrent adipofascial flap was raised and turned over to reconstruct massive defects around the elbow and forearm after wide resection of malignant tumours. Compared to the fasciocutaneous flap, the ulnar recurrent adipofascial flap has the following advantages: (1) a larger flap can be harvested without the problem of primary closure of the donor site, (2) the contour and scar of the donor site is reasonably acceptable since no tension is presented, (3) the flap can be turned over to cover a wider area, and (4) debulking of the flap can be performed during the operation if needed. The rotation arc of the ulnar recurrent adipofascial flap reaches a wide region, including the distal one-half of the upper arm, the elbow, and the proximal two-thirds of the forearm. Sensory deficit in the forearm was avoided in our patients because meticulous separation and preservation of the medial cutaneous nerve of the forearm was achieved without jeopardising the blood supply to the flap. The ulnar recurrent adipofascial flap is an easy and reliable option for one-stage reconstruction of massive defects around the elbow.     

The long-term results of meniscus transplantation for articular cartilage defects in the knee joint

The purpose of this study was to examine the long-term clinical results of meniscus transplantation for articular cartilage defects in the knee joint. The type of study was case series. From October 1990 to June 1995, eight cases underwent allogenic or autogenic meniscus transplantations for articular cartilage defects, and seven cases were available for follow-up evaluations. The age at surgery ranged from 14 to 42 years of age (average 22.5). In one case, transplantation of tissue-engineered cartilage was performed due to pain 5 years after surgery. The other six cases were followed up for 8–13 years (average 10.1). The size of the cartilage defect ranged from 1.0 to 6.3 cm² (average 2.8 cm²). Patients were evaluated with the Lysholm score and MR images. We also performed arthroscopic examinations in three cases at the final evaluation. This study leads to the conclusion that meniscus transplantation for articular cartilage damage is not comparable to autologous chondrocyte transplantation. Two cases showed a good clinical outcome but the tissue remained as fibrocartilage tissue in the long-term.     

A Tissue-Engineered Artificial Bile Duct Grown to Resemble The Native Bile Duct

The aim of this study was to fabricate an artificial bile duct for the development of a new treatment for biliary diseases. Eighteen hybrid pigs were implanted with a bile duct organoid unit (BDOU) made of a bioabsorbable polymer. Twelve of the transplanted BDOUs had been seeded with autologous bone marrow cells (BMCs) in advance. Six animals, the controls, were grafted with the scaffold alone with no BMCs seeded. The common bile duct was cut, the hepatic cut end of the native common bile duct was anastomosed to the BDOU and the other end was anastomosed to the duodenum. The controls underwent a similar operation. The neo-bile duct was removed at pre-determined time points and investigated histologically. All 18 recipient pigs survived until their sacrifice at 6 weeks, 10 weeks or 6 months. Histological examination revealed incomplete epithelialization of the neo-bile duct at 6 weeks and 10 weeks after transplantation. At 6 months, the organoid exhibited a morphology almost identical to that of the native common bile duct. No differences were found between the controls and BMC-seeded pigs. These results show that the artificial bile duct thus fabricated can serve as a substitute for the native bile duct.