Flow cytometric micronucleus test with mouse peripheral erythrocytes.

Flow cytometric (FC) analysis was applied to micronucleus test with mouse peripheral blood erythrocytes. The method is based on the measurement of peak fluorescence (PFL) of sphered glutaraldehyde-fixed erythrocytes before and after staining with 4',6-diamidino-2-phenylindole (DAPI), in an EPICS V flow cytometer. The frequency of micronucleated erythrocytes (MNEs) is calculated by a computer program comparing PFL data obtained with and without DAPI. To evaluate the method, male ddY mice were treated with 6-mercaptopurine and benzene and blood was collected from tail vein at intervals of 4-7 days. Both microscopic and FC analysis showed a steady increase in the incidence of MNEs, reaching a plateau when about a month had passed from the start of the treatments. The effects of benzo[a]pyrene, mitomycin C,N-ethyl-N-nitrosourea, bromodichloromethane and potassium chromate (VI) were also studied with both the manual and FC assay in samples collected a week after five weekly treatments. The percentages of MNEs obtained manually and by the FC measurements showed good correlation, the former three chemicals being positive and the latter two negative or, in the FC analysis, difficult to classify. Because of the high number of cells examined (50,000/animal), the FC analysis was probably more sensitive than the manual method where only 2000 cells were scored per animal. This was further suggested by (i) steady time responses, also for individual animals, in the FC results on 6-mercaptopurine and benzene, (ii) overall reduced inter-individual variation in the FC measurements, and (iii) detection of MNE induction by mitomycin C at a lower dose level with the FC than the manual analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arginine vasotocin (AVT) and AVT-related peptide are major aldosterone-releasing factors in the bullfrog neurointermediate lobe.

Two major components which stimulate aldosterone release from Xenopus adrenocortical tissue were isolated from an acid-acetone extract of the neurointermediate lobes of the bullfrog (Rana catesbeiana) using C18 Sep-Pak cartridges, Sephadex G-50, and reverse-phase HPLC columns. One of the components was identified as arginine vasotocin (AVT) from its HPLC profile and amino acid sequence analysis. The other was an AVT-like decapeptide with an extra glycine residue at the C-terminus of nonamidated AVT, which was recently termed hydrin 2. The yields of these two peptides were almost the same. They also showed equipotent activity in stimulating water flux from the isolated urinary bladder of the toad (Bufo japonicus).     

Enhancement of hepatitis-B surface-antigen expression by 5-azacytidine in a hepatitis-B-virus-transfected cell line.

The human hepatoblastoma-derived cell line HB611 secretes hepatitis-B surface antigen (HBsAg) and hepatitis-B e antigen (HBeAg) into the medium. Hepatitis-B-virus (HBV) DNA integrated into the cellular genome was found to be hypermethylated. When the cells were treated with 5-azacytidine for 3 days, the level of HBsAg in the medium increased, while the level of HBeAg remained constant. The level of alpha-fetoprotein (AFP) decreased with the 5-azacytidine treatment. Southern blot analysis of DNA digested with HpaII or MspI showed that 5-azacytidine treatment resulted in hypomethylation of the integrated HBV DNA, suggesting that 5-azacytidine increased HBsAg production in the cells through hypomethylation of the HBV genomic DNA.     

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.     

Eosinophilic fasciitis associated with Borrelia afzelii infection]

A 58-year-old woman suffered from stiffness, swelling, rubor and muscle pain in the extremities one month after she climbed a mountain in Kyushu, an island in southern Japan. On admission, mild proximal weakness was present in the extremities. Her range of motion in the extremities was limited due to firm skin and subcutaneous stiffness which was similar to scleroderma. She showed peripheral blood eosinophilia and hypergammaglobulinemia as well as a high erythrocyte sedimentation rate. An IgM antibody against Borrelia afzelii was positive. MRI of the skeletal muscles demonstrated enhancing fascia around the biceps brachii muscle, and a biopsy specimen revealed perivascular infiltration of mononuclear cells within the hypertrophic fascia. Eosinophilic infiltration was absent. We treated the patient with prednisolone, doxycycline and amoxicillin, which alleviated the symptoms. This is the first report to show that Borrelia afzelii was involved in eosinophilic fasciitis.     

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.