Intestinal active absorption of sugar-conjugated compounds by glucose transport system: implication of improvement of poorly absorbable drugs.

The intestinal absorption of glucose- and galactose-conjugated compounds was studied in the everted sac of the rat small intestine. The absorption clearance of p-nitrophenyl beta-D-glucopyranoside (p-NPglc) at 250 microM in the mucosal side (4.45 +/- 0.34 microL/min/cm, mean +/- SE, N = 4), calculated by dividing the absorption rate by the drug concentration, was significantly decreased (0.476 +/- 0.036 microL/min/cm) in the presence of 1 mM phloridzin, an inhibitor of glucose transport, and in the absence of Na+, a cosubstrate of the glucose transport carrier (0.424 +/- 0.018 microL/min/cm). The absorption clearance of p-NPglc was decreased as its concentration increased. In the same experiment, the absorption clearance of p-nitrophenyl beta-D-galactopyranoside (1.99 +/- 0.23 microL/min/cm) was also significantly decreased in the presence of phloridzin and in the absence of Na+. However, the absorption clearance of p-nitrophenyl beta-D-mannopyranoside (0.811 +/- 0.013 microL/min/cm) was low and not significantly decreased in the presence of phloridzin (P greater than 0.1). Furthermore, the absorption clearance of beta-naphthyl beta-D-glucopyranoside and beta-naphthyl beta-D-galactopyranoside was also significantly decreased in the presence of phloridzin (P less than 0.001). These results indicated that the glucose and galactose moieties provided these compounds with a new route by way of the glucose transport carrier for intestinal absorption.     

Reduction of osteoclasts in a critical embryonic period is essential for inhibition of mouse tooth eruption.

Alveolar bone resorption by osteoclasts is essential for tooth eruption. Osteoclast-deficient Csfm(op) homozygous (op/op) mice, which lack functional macrophage colony-stimulating factor (M-CSF), suffer from osteopetrosis and completely lack tooth eruption. Although osteoclasts appear, and osteopetrosis is cured with age in op/op mice, tooth eruption is never seen. This fact suggests that there is a critical period when osteoclasts are required for tooth eruption. In this study, to detect the critical period, we administered an antagonistic antibody directed against c-Fms, a receptor for M-CSF, to inbred C57BL/6 mice for various periods. Administration of this antibody decreased tartrate-resistant acid phosphatase-positive (TRAP) osteoclasts, and incisor eruption was completely inhibited by continual administration of this antibody from embryonic day 15.5 (E15.5) until postnatal day 12.5 (D12.5). A 1-day delay of this administration abolished the inhibition of incisor eruption. The number of TRAP-positive osteoclasts was significantly reduced between E16.5 and E18.5 in the mice treated with antibody from E15.5 compared with those treated from E16.5. These results indicate that this period, during which the number of osteoclasts decreases significantly, is critical for inhibiting incisor eruption in C57BL/6 mice.     

Vascularized rib strut technique for repair of pectus excavatum.

A vascularized rib strut based on the anterior intercostal branch of the internal mammary artery was applied to provide rigid internal fixation of the chest wall after correction of pectus excavatum. The procedure is simple and has substantial advantages when compared with techniques using metallic struts or nonvascularized free rib grafts.     

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.     

Blood pressure changes following extracorporeal shock wave lithotripsy for urolithiasis.

Hypertension after extracorporeal shock wave lithotripsy (ESWL) has been a controversial subject. Changes in blood pressure were studied in 262 patients (mean age 47.8 years) 18.6 months after ESWL. According to World Health Organization criteria the number of patients who showed a decrease exceeded those who showed an increase in blood pressure. The patients who have been on antihypertensive therapy showed a significantly greater decrease in blood pressure than those without medication. Of 192 normotensive patients diastolic pressure increased 1.2 mm. Hg and 2 (1.0%) had hypertension 18.4 months after ESWL. Annualized increase in diastolic pressure and new onset of hypertension were calculated to be 0.78 mm. Hg and 0.65%, respectively. Significant elevation of diastolic pressure was noted in the patients who received a larger number of shock waves. Blood pressure should be carefully followed after ESWL especially in patients who have been treated by a greater number of shock waves.     

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.     

Japanese guidelines for the management of herpes simplex encephalitis; comparison with those from the International Management Herpes Forum]

Herpes simplex encephalitis (HSE) is still recognized as a severe sporadic encephalitis, although the mortality and morbidity rates have been decreased to 10% and 30%, respectively. This disease is diagnosed using clinical symptoms, CSF, EEG, CT, MRI, and virologic tests such as polymerase chain reaction (PCR) or enzyme immunosorbent assay (EIA). Early diagnosis and treatment are essential for HSE. However, the early symptoms of this disease are various, and the laboratory diagnostic criteria are unclear to the non-specialist. In 2005, Japanese guidelines for the management of HSE have been issued via two sets of Workshops at the Japanese Neuroinfectious Disease Congress. The diagnostic and therapeutic criteria were discussed in comparison with those from the International Management Herpes Forum (IMHF) in 2004. For a definitive diagnosis, CSF PCR for herpes simplex virus (HSV) is recommended, and the detection rate has been reported to be 60 to 80% within the 7th day of the illness. In the IMHF, the PCR method has also been the primary method for early diagnosis and for monitoring the therapy. Further, quantitative real-time PCR has become available for measuring the effectiveness of aciclovir therapy. To measure HSV antibody levels, complement antibody (CF), neutralizing antibody (NT), or enzyme-linked immunosorbent assay (ELISA or EIA) are available. Significant elevation of EIA IgG or intrathecal HSV antibody production should be shown, although these antibody responses often appear two weeks after the onset of HSE. Regarding anti-herpesvirus drugs, in both Japanese and IMHF guidelines aciclovir is consistent with the first choice, and it is recommended that its administration would be started as soon as HSE is suspected on the basis of clinical pictures, CT * MRI, EEG, or CSF findings. However, antiviral therapy may be discontinued if a negative CSF HSV PCR is obtained at > 72 hours after onset. A recent Japanese study shows the efficacy of a combination therapy of aciclovir and corticosteroid for this disease. Further prospective investigation is expected.