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.     

Measurement of free and total hydroxyproline by automated flow injection of serum or urine samples from maintenance hemodialysis patients with renal osteodystrophy

An automated measurement of total and free hydroxyproline in serum or urine is presented that uses flow injection analysis. After exclusion of nonspecific substances, hydroxyproline was oxidized by chloramine- T and L-cysteine with Ehrlich's reagent. The linearity obtained was from 3.8μmole/ L to 1.22 mmole/L with good precision (CV <3%). Comparison of the proposed method with HPLC yielded r = 0.939 as the correlation coefficient. Reference intervals of free and total hydroxyproline are 1.4–9.7 μmole/L, 3.8–27.2 μmole/L for serum, and 10.0–72.5 μmole/L, 25.2–303.6 μmole/L for urine, respectively. Serum free and total hydroxyproline levels in renal osteodystrophy patients on maintenance hemodialysis (N = 71) were significantly higher than in controls (P<0.0001). This method is superior to the use of HPLC with regard to stability of the color reaction. The measurement of serum free and total hydroxyproline is a useful marker for therapeutic observation of renal osteodystrophy patients. © 1994 Wiley-Liss, Inc.     

Epidemiology of limb-body wall complex in Japan

Limb-body wall complex is a malformation of body and limbs with craniofacial defects. We describe here the epidemiology of this complex using the population-based registry data in the Kanagawa Birth Defects Monitoring Program during the period 1982–1991. Eleven infants (11/428,599 births) with the complex were ascertained in the study. The incidence and spectrum of the defects observed in our cases were similar to those of other studies. The parental ages in the study group were not significantly different from those in the general population. No teratogenic agents and factors were identified in the present study. Most cases were diagnosed prenatally. © 1994 Wiley-Liss, Inc.     

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)     

Effect of FK-506 on xenografted human Graves' thyroid tissue in severe combined immunodeficient mice

OBJECTIVE We studied the macrolide antibiotic FK-506, an immunosuppressive agent, in an attempt to ameliorate the lesion of autoimmune thyroid disease in human thyroid tissue xenografted into severe combined immunodeficient (SCID) mice. It was not felt appropriate to employ this agent directly in patients with autoimmune thyroid disease because adequate therapeutic modalities are available and the introduction of new, experimental agents could not be justified. Moreover, the study of the tissue before and after treatment could not have been undertaken directly in patients. DESIGN Human thyroid xenografts from four patients with Graves' disease and two normal persons were xenografted into SCID mice. Two weeks after xenograft-ing, human immunoglobulin G (IgG) was detectable in all SCID mice xenografted with Graves' thyroid tissue. Mice were divided into two groups with human IgG levels similar to each other. Mice in the first group were treated with FK-506 daily for 6 weeks; mice in the second (similar) group were given phosphate-buffered saline (PBS) only (control group). MEASUREMENTS Blood samples were taken every 2 weeks from the tail veins for human IgG, thyroid stimulating antibody, thyroperoxidase antibodies, thyroglobulin antibodies, and interferon-gamma (IFN-7). After 8 weeks treatment, animals were sacrificed; thyroid tissue was examined histologically and for thyrocyte HLA-DR expression. FK-506 was also added to thyrocytes in in-vitro tissue culture conditions. RESULTS After 4–6 weeks of FK-506 therapy, human IgG, all thyroid antibodies and IFN-7 were suppressed, while the levels remained elevated in the control group. Lymphocytic infiltration virtually disappeared in the human thyroid tissue of the FK-506-treated mice and thyrocyte HLA-DR expression markedly declined; in the control mice, lymphocytic infiltration remained heavy and HLA-DR expression remained high. On the other hand, FK-506 added directly to thyrocytes in vitro (without lymphocytes) did not reduce thyrocyte HLA-DR expression. CONCLUSIONS FK-506 appears to suppress the activation of intrathyroidal lymphocytes, but not thyrocytes. From these observations, it is concluded that this agent, by its action on intrathyroidal lymphocytes, is able to ameliorate the immunologically mediated histological and serological disturbance in human autoimmune thyroid disease, at least under these circumstances.     

Curative resection of gastric cancer: Limitation of peritoneal lavage cytology in predicting the outcome

Patients with stage T3N0～2M0 gastric carcinoma (n = 108) were studied for relevant prognostic factors. Peritoneal lavage cytology (PLC) was performed in all. In univariate analysis, 5-year survival rates were better with smaller serosal invasion (diameter <3.0 cm vs. ≥3.0 cm, 61% vs. 37%, P < 0.05) and fewer metastatic nodes (≤5 vs. ≥6, 57% vs. 29%, P < 0.05). In multivariate analyses, only these two factors were significant. The predictive value of PLC was not shown in both univariate and multivariate analyses. Peritoneal recurrence occured in 14 (22%) of 77 patients with negative PLC, and in 3 (18%) of 17 with positive PLC, the difference being not significant. Our results indicate that PLC is insensitive in predicting the development of peritoneal recurrence. Its role in the estimation of survival is limited, as many will die of visceral or locoregional recurrence if not of peritoneal dissemination.     

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.