Anorexigenic effects of miglitol in concert with the alterations of gut hormone secretion and gastric emptying in healthy subjects.

Although the α-glucosidase inhibitor miglitol (MG) has been reported to have anorexigenic effects, the mechanism remains to be elucidated. The objective of this study was to explore the effects of MG on appetite in relation to concomitant changes in postprandial gut hormone levels. This randomized open-label crossover study included 20 healthy volunteers. The effects of 50 mg MG on glucagon-like peptide-1 (GLP-1), peptide YY (PYY), and ghrelin levels were assessed in conjunction with a simultaneous determination of appetite scores using visual analogue scales (VAS) over 3 h after the ingestion of a 592 kcal test cookie. Additionally, the gastric emptying rate (GER) was measured using breath ¹³CO2 appearance in 10 subjects. 12 subjects were administered 50 mg MG thrice a day for 1 week, and alterations of the gut hormone levels and the VAS scores for appetite were evaluated. MG pre-administration resulted in a significant enhancement of GLP-1 and PYY responses induced by the cookie ingestion. Following MG administration, ghrelin level declined at 1 h, with a persistent suppression during the postprandial phase in contrast to the restoration to the basal level without MG. Furthermore, MG pre-administration suppressed appetite and maintained satiety evaluated using a VAS rating with concomitant inhibition of GER after cookie ingestion. One-week administration of MG did not influence either gut hormone levels before a meal or VAS rating during a whole day. These observations suggest that MG exerts an anorexigenic effects with concomitant alterations of gut hormone secretions and gastric emptying after meal ingestion.

Octamer binding protein 2 (Oct2) regulates PD-L2 gene expression in B-1 cells through lineage-specific activity of a unique, intronic promoter.

Programmed death-1 ligand 2 (PD-L2) expression extends beyond macrophages/dendritic cells to B-1 B cells, a distinct B-cell lineage that is responsible for natural immunoglobulin and which is repertoire skewed toward autoreactive specificities. PD-L2 expression is constitutive in B-1 cells, whereas it is inducible in other cell types, suggesting that PD-L2 is regulated differently in the former versus the latter, and this proved to be the case, both in transcription and promotion. B-1 cells express a PD-L2 transcript that lacks exon 1, in contrast to macrophages/dendritic cells for which exon1 is included, reflecting a unique start site upstream of exon 2. PD-L2 transcription in B-1 cells is regulated by a novel intronic promoter located between exons 1 and 2. This intronic promoter binds Octamer binding protein 1 (Oct1) and Oct2, and although these transcription factors are present in all B cells, Oct2 binding is found in vivo only in B-1 cells and not PD-L2-negative B-2 cells. Moreover, the proximal promoter upstream of exon 1 that is active in macrophages is inactive in B-1 cells. Thus, PD-L2 expression is regulated by two different promoters that function in a lineage-specific manner, with the B-1-specific promoter being constitutively active as a result of Oct1 and Oct2 binding.

The risk of post-molar gestational trophoblastic neoplasia is higher in heterozygous than in homozygous complete hydatidiform moles.

BACKGROUND: Complete hydatidiform mole (CHM) is a high-risk pregnancy for gestational trophoblastic neoplasia (GTN). Patients with CHM have a 10-30% chance of trophoblastic sequelae. CHM includes androgenic homozygous (monospermic) and androgenic heterozygous (dispermic) moles. It is controversial whether the risk of GTN is higher with heterozygous than with homozygous CHM. A prospective cohort study was conducted to assess risk of GTN in homozygous and heterozygous CHM using short tandem repeat (STR) polymorphisms, and a meta-analysis of previous reports. METHODS: Twenty-eight consecutive molar pregnancies were evacuated and followed by regular hCG measurements to detect GTN. Persistent GTN was diagnosed according to the International Federation of Gynecology and Obstetrics 2000 system. Cytogenesis of the mole was determined by STR polymorphisms of molar tissue and parental blood. A meta-analysis of the GTN rate from previous reports was conducted using Mantel-Haenszel methods. RESULTS: Of 28 molar pregnancies, 24 were homozygous and three were heterozygous CHM. The remaining mole was diandric triploidy (a partial hydatidiform mole). Of the 24 homozygous CHMs, six (25%) cases developed GTN and received chemotherapy. Meanwhile, all three cases (100%) of heterozygous mole developed GTN and needed chemotherapy. The GTN risk was higher in heterozygous (P = 0.029, Fisher's exact test) than homozygous moles. A systematic review revealed only five previous reports (with more than 15 cytogenetically diagnosed cases), and the pooled relative risk of persistent GTN for heterozygous mole was not significant (odds ratio, 2.0; 95% confidence interval, 0.98-4.07). CONCLUSIONS: Heterozygous CHM had a higher risk for GTN than homozygous CHM.

[Successful resection of left ventricular myxoma in a toddler; report of a case].

A 4-year-old girl was found to have large left ventricular myxoma without any tumor-related symptoms. She underwent an urgent surgery and the myxoma was successfully removed through a left ventriculectomy. Great care was taken to prevent tumor-embolization during surgery, and to resect the endocardium attaching directly to the tumor. Future surveillance of this case warrants our operative technique described in this report.

Enhanced resistance to seed-transmitted bacterial diseases in transgenic rice plants overproducing an oat cell-wall-bound thionin.

Bacterial attack is a serious agricultural problem for growth of rice seedlings in the nursery and field. The thionins purified from seed and etiolated seedlings of barley are known to have antimicrobial activity against necrotrophic pathogens; however, we found that no endogenous rice thionin genes alone are enough for resistance to two major seed-transmitted phytopathogenic bacteria, Burkholderia plantarii and B. glumae, although rice thionin genes constitutively expressed in coleoptile, the target organ of the bacteria. Thus, we isolated thionin genes from oat, one of which was overexpressed in rice. When wild-type rice seed were germinated with these bacteria, all seedlings were wilted with severe blight. In the seedling infected with B. plantarii, bacterial staining was intensively marked around stomata and intercellular spaces. However, transgenic rice seedlings accumulating a high level of oat thionin in cell walls grew almost normally with bacterial staining only on the surface of stomata. These results indicate that the oat thionin effectively works in rice plants against bacterial attack.

Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco.

We demonstrate here that induced expression of sarcotoxin IA, a bactericidal peptide from Sarcophaga peregrina, enhanced the resistance of transgenic tobacco plants to both bacterial and fungal pathogens. The peptide was produced with a modified PR1a promoter, which is further activated by salicylic acid treatment and necrotic lesion formation by pathogen infection. Host resistance to infection of bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci was shown to be dependent on the amounts of sarcotoxin IA expressed. Since we found antifungal activity of the peptide in vitro, transgenic seedlings were also inoculated with fungal pathogens Rhizoctonia solani and Pythium aphanidermatum. Transgenic plants expressing higher levels of sarcotoxin were able to withstand fungal infection and remained healthy even after 4 weeks, while control plants were dead by fungal infection after 2 weeks.

Preparation of a stable subunit of Japanese elderberry (Sambucus sieboldiana) bark lectin and its application for the study of cell surface carbohydrates by flow cytometry.

A stable subunit of Sambucus sieboldiana bark lectin (MSSA) was prepared by selective reduction of disulfide bridges between the subunits and alkylation with 4-vinylpyridine. Amino acid analysis of MSSA revealed that 1.4 cysteine residues per subunit were selectively modified. MSSA failed to agglutinate rabbit erythrocytes and precipitate fetuin. However, MSSA retained the ability to bind to fetuin, as detected by ELISA. Neu5Ac alpha 2-6lactose inhibited the binding to fetuin of both SSA and MSSA. Flow cytometric analysis showed that human histocytic lymphoma U937 cells were clearly stained with FITC-labeled MSSA (FITC-MSSA) without any detectable agglutination and that this staining was almost completely inhibited by the addition of Neu5Ac alpha 2-6lactose (2 mM). Treatment of U937 cells with native FITC-SSA at the sub-agglutinating concentration (0.3 microgram/ml) showed much poorer fluorescence intensity than that of MSSA, suggesting that MSSA is an invaluable tool for the detection of cell surface glycoconjugates containing NeuAc alpha 2-6Gal/GalNAc sequences by flow cytometry.

Identification of a novel high-affinity binding site for N-acetylchitooligosaccharide elicitor in the membrane fraction from suspension-cultured rice cells.

Binding experiments using a 125I-labeled tyramine conjugate of N-acetylchitooctaose, a highly potent elicitor for the induction of phytoalexin production in rice cells, and a microsomal membrane preparation from suspension-cultured rice cells showed the presence of a novel high-affinity binding site for this oligosaccharide. The binding of the ligand was saturable and the Scatchard plot analysis of the results indicated the presence of a single class of binding site with a Kd of 5.4 nM which is comparable with that reported for the binding of the hepta-beta-glucoside elicitor in soybean membrane. The ligand binding was inhibited by unlabeled N-acetylchitoheptaose but not by its deacetylated form. These characteristics of this binding site coincide well with the specificity and sensitivity for the elicitor in several assay systems, suggesting the possible involvement of this binding site in the recognition of the elicitor in vivo.

Characterization of hemopoietic precursor cells in juvenile-type chronic myelocytic leukemia.

In order to study the pathogenesis of juvenile-type chronic myelocytic leukemia (CML), we examined the colony-forming capacity and colony composition in the bone marrow (BM) and peripheral blood (PB) of three children with juvenile-type CML. Large numbers of granulocytes and macrophage colonies were formed by BM and PB cells. Whole agar culture staining revealed that especially macrophage colonies increased in comparison with normal controls. After removal of carbonyl iron-laden cells with a magnet or deprivation of cells adherent to glass from BM cells, the number of macrophage colonies markedly reduced in comparison with the number of colonies formed by untreated BM cells, suggesting that some of the macrophage colony-forming cells (M-CFC) may have phagocytic and/or adherent activity. Radiation sensitivity and thymidine suicide rate of these M-CFC were not different from those of granulocyte colony-forming cells (G-CFC). The predominance of M-CFC in juvenile-type CML may be one of the reflections of fetal-type myelopoiesis since M-CFC are predominant in cord blood and PB in the neonatal period. Moreover, considerable numbers of erythroid-colony-forming units (CFU-E) were present in PB of all patients. It may be concluded that juvenile-type CML is a panmyelopathy with the predominance of M-CFC.

Effect of continuous intravenous administration of high-dose G-CSF on hematologic recovery following autologous purged bone marrow transplantation.

Delay in peripheral blood recovery is a common complication of autologous purged bone marrow transplantation. To overcome this problem, we examined the effect of continuous intravenous administration of high-dose G-CSF on hematologic recovery following autologous bone marrow transplantation (auto-BMT) with purged bone marrow. Continuous intravenous administration of high dose G-CSF significantly facilitated the recovery of platelet counts and reticulocyte counts compared to one-hour bolus intravenous injection of the usual-dose G-CSF, although both ways of administration facilitated the recovery of leukocyte counts. The results showed continuous intravenous administration of high-dose G-CSF was useful to facilitate the recovery of not only leukocytes but also reticulocytes and platelets following auto-BMT with purged bone marrow in certain situations. Continuous i.v. administration of high-dose G-CSF may be one of the safest and most useful modes facilitating the hematopoietic recovery following auto-BMT with purged bone marrow.

Predictive markers for hepatic veno-occlusive disease after hematopoietic stem cell transplantation in adults: a prospective single center study.

Hepatic veno-occlusive disease (VOD) is a major complication after hematopoietic stem cell transplantation (HSCT). Aetiological determinants, diagnosis and treatment remain unclear. Changes in coagulation-fibrinolysis parameters and N-terminal propeptide for type III procollagen (P-III-P) have been studied in patients with or without VOD after HSCT. We prospectively measured protein C activity, tissue plasminogen activator (t-PA), antithrombin III (AT-III), plasminogen activity (PLG), thrombin-antithrombin III (TAT), alpha2-plasmin inhibitor (alpha2-PI),fibrinogen (Fbg) and P-III-P in 44 consecutive adult patients undergoing allogeneic HSCT. Each parameter was determined before conditioning, on day 0 of HSCT and weekly for 5 weeks. Five of the 44 patients developed VOD at a median post HSCT of day 3 (range, day 3 to 12). On repeated analysis of variance (ANOVA), there were significant differences between patients with and without VOD in P-III-P (P < 0.0001), protein C (P < 0.0001), t-PA (P < 0.0001), PLG (P < 0.0001), AT-III(P < 0.0001), Fbg (P < 0.0001), alpha2-PI (P = 0.0002). Levels of P-III-P were significantly higher in patients with VOD than without VOD, before preparative chemotherapy (P < 0.005) and on days 0 and 7 (P < 0.001). On day 0, levels of t-PA were significantly higher in patients with VOD than without VOD (P < 0.05). On day 7, levels of protein C were significantly lower in patients with VOD than without VOD (P < 0.01). On day 0, there were trends of differences (P = 0.0515) between patients with and without VOD in the levels of protein C. These results suggest P-III-P, t-PA and protein C are predictive markers for VOD after HSCT in adults. Moreover, the serum P-III-P level before start of conditioning might indicate patients at risk for developing VOD.

Comparison of cytomegalovirus (CMV) antigenemia and CMV in bronchoalveolar lavage fluid for diagnosis of CMV pulmonary infection after bone marrow transplantation.

A comparative cytomegalovirus (CMV) diagnostic study was carried out on 30 bone marrow transplant patients. Forty-three bronchoalveolar lavage fluid (BALF) samples from these patients were examined for CMV by viral culture, polymerase chain reaction (PCR), shell vial and cytology. In parallel, peripheral blood samples were subjected to CMV antigenemia assay. CMV was detected in 12 (27.9%) of the 43 BALF samples (10 samples in viral culture, 10 samples in PCR, eight samples in shell vial and three samples in cytology). The CMV antigenemia assay yielded a positive result for six samples. The rates of agreement between results of the CMV antigenemia assay and results of each of the BALF tests were as follows: 81.4% with viral culture, 76.7% with PCR, 86.0% with shell vial, and 88.4% with cytology. Although the sensitivity of the CMV antigenemia assay was inferior to the sensitive tests of BALF samples, statistically significant correlations were demonstrated between the CMV antigenemia assay, viral culture, shell vial and cytology. Although the CMV antigenemia assay was shown to be useful for detection of CMV, it may be necessary to confirm not only the sensitivity but also the specificity of this method for prevention of CMV disease after BMT.

Risk factors for hepatic veno-occlusive disease after bone marrow transplantation: retrospective analysis of 137 cases at a single institution.

One hundred and thirty-seven consecutive patients who received bone marrow or peripheral blood stem cell transplantation were studied retrospectively to identify the risk factors for hepatic veno-occlusive disease (VOD). Of the 137 recipients, twenty (14.6%) patients were diagnosed with VOD using the McDonald's criteria. In these 20 patients with VOD, we analyzed various clinical parameters, including age, sex, HLA status, conditioning regimen, irradiation, immunosuppressive agents, mode of transplantation, history of hepatic dysfunction, pre-transplant hepatic and renal function, infectious episodes, antibiotics use, and serum viral titers. A history of hepatic dysfunction and low levels of pseudocholinesterase before transplantation were found to be statistically significant (P = 0.04 and 0.04). Low levels of pseudocholinesterase were significant by multivariate analysis using the logistic regression model (P = 0.02). These results suggest that pseudocholinesterase levels before transplant are important markers of VOD in patients receiving BMT.

Enhanced resistance to bacterial diseases of transgenic tobacco plants overexpressing sarcotoxin IA, a bactericidal peptide of insect.

Sarcotoxin IA is a bactericidal peptide of 39 amino acids found in the common flesh fly, Sarcophaga peregrina. Many agronomically important bacteria in Japan are killed by this peptide at sub-micro molar levels, and the growth of tobacco and rice suspension cultured cells is not inhibited with less than 25 microM. Transgenic tobacco plants which overexpress the peptide, i.e. over 250 pmol per gram of fresh leaf, under the control of a high expression constitutive promoter showed enhanced resistance to the pathogens for wild fire disease (Pseudomonas syringae pv. tabaci) and bacterial soft rot disease (Erwinia carotovora subsp. carotovora).

Construction and characterization of a Xanthomonas oryzae pv. oryzae bacterial artificial chromosome library.

Xanthomonas oryzae pv. oryzae is an important plant pathogen which causes bacterial blight of rice. To facilitate genome studies of this bacterium, we have constructed a bacterial artificial chromosome (BAC) library of strain MAFF 311018. It consisted of 750 clones representing 16 genome equivalents, and had an insert size ranging from 20 to 220 kb with an average size of 107 kb. This library is the first to be constructed from a X. oryzae pv. oryzae strain. The usefulness of this library was demonstrated through polymerase chain reaction screening of 11 genes and the 16S--23S rDNA spacer region in a 192-clone subset, representing five genome equivalents. The results obtained showed an average of 5.9 BAC clones per screening. This result is in good agreement with the estimated size of the test library, indicating that the constructed BAC library can be used to facilitate genome analysis of X. oryzae pv. oryzae.

Second allogeneic peripheral blood stem cell transplantation with fludarabine-based low-intensity conditioning regimen for relapsed myelodysplastic syndrome after allogeneic bone marrow transplantation.

We describe the case of a 51-year-old patient with relapsed myelodysplastic syndrome after allogeneic bone marrow transplantation (BMT), who underwent allogeneic peripheral blood stem cell transplantation (PBSCT) after conditioning with a novel regimen consisting of fludarabine, busulfan, and antithymocyte globulin. The second PBSCT was performed early, at 3 months after the initial allogeneic BMT, but it was well tolerated and complete hematologic remission was documented. The patient did not experience any early transplantation-related organ toxicity but died from opportunistic infection 6 months after the second transplantation. Our experience suggests that this novel regimen may induce remission and could be offered to patients relapsing after the first transplantation; however, the fludarabine-containing regimen might be accompanied by profound immunosuppression.

A cell wall-bound beta-glucosidase from germinated rice: purification and properties.

A large portion of beta-glucosidase (EC 3.2.1.21) in germinating rice seeds, which appears to be ionically bound to cell walls, can be solubilized with 1 M NaCl. Its activity increased more than eight-fold within five days of germination. It was purified to electrophoretic homogeneity from the extracts of germinated rice seeds by fractionation with (NH4)2SO4 followed by CM-Sepharose, Polybuffer exchanger 118, Concanavalin A-Sepharose and Bio-Gel P-100. The Mr of the purified enzyme, estimated by SDS-PAGE, was 56,000 and the isoelectric point was > 10.0. Its N-terminal amino acid sequence (44 residues) exhibited high homology to those of beta-glucosidases from other plants, such as barley and white clover. Its activity was optimal at pH 4.5 and 50 degrees, and it was strongly inhibited by glucono-1,5-lactone. The enzyme showed hydrolytic as well as transglycosylation activity towards (1-->3)-beta- and (1-->4)-beta-linked oligosaccharides with degree of polymerization of 2-4. The results suggest that the beta-glucosidase is probably involved not only in hydrolysis but also in modification of oligosaccharides in cell walls of germinating rice seeds.

Gene therapy for prostate cancer: toxicological profile of four HSV-tk transducing adenoviral vectors regulated by different promoters.

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1beta was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.

Urinary trace amine excretion and platelet monoamine oxidase activity in schizophrenia.

The relationship between subtypes of schizophrenia classified by ICD-9 and 24-hour urinary beta-phenylethylamine (PEA) and phenylacetic acid (PAA) excretion has been studied. Schizophrenia was divided into two types: paranoid and nonparanoid. Increased urinary PEA excretion was found in paranoid schizophrenics, but urinary PAA excretion did not show any significant difference between schizophrenics and normal subjects. A relationship between platelet monoamine oxidase (MAO) activity and urinary PEA excretion was found. These findings offer some indication that PEA may play a role in the pathogenesis of schizophrenia.

Genetic Diversity of Xanthomonas oryzae pv. oryzae Strains from Sri Lanka.

ABSTRACT Sixty strains of Xanthomonas oryzae pv. oryzae, collected from 29 locations in Sri Lanka in 1995, were analyzed by restriction fragment length polymorphism using either polymerase chain reaction-amplified 16S and 23S rDNA or the repetitive DNA element IS1112 from X. oryzae pv. oryzae as hybridization probes. Two different ribogroups were observed in the Sri Lankan strains using rDNA probes, whereas five clusters were identified by the IS1112 probe. Bootstrap analysis revealed that the five clusters defined by IS1112 were relatively robust. Our results suggest that the Sri Lankan strains are phylogenetically composed of five different groups. Each cluster was partially associated with climatic conditions (intermediate zone and wet zone) and was related to groups based on ribotyping. Based on virulence analysis using 12 rice cultivars, each containing a single resistance gene, 14 pathotypes were identified among the Sri Lankan strains. All strains were virulent to resistance genes Xa1, Xa2, Xa4, Xa10, Xa11, and Xa14. Only one strain (pathotype 1) was virulent to all major resistance genes including Xa21, while strains of the other pathotypes were all avirulent to Xa21. A partial relationship was found between the determined phylogenetic groups using the IS1112 probe and pathotypes for all but two clusters. The results of this study will facilitate the further understanding of the population structure of X. oryzae pv. oryzae in Sri Lanka.