Synthesis and biological evaluation of a 6-aminofuro[3,2-c]pyridin-3(2H)-one series of GPR 119 agonists.

G protein-coupled receptor 119 (GPCR 119 (GPR119)) agonists have received considerable attention as a promising therapeutic option for treatment of type 2 diabetes mellitus. GPR119 is one of the GPCRs expressed in pancreatic islet ß-cells and its activation enhances stimulation of insulin secretion in a glucose-dependent manner. We have recently described a series of 6-amino-1H-indan-1-ones as potent, selective, and orally bioavailable GPR119 agonists with an amino group that plays important roles not only in their drug-like properties, such as high aqueous solubility, but also in their potent agonistic activity. However, many of these compounds displayed strong to moderate inhibition of human ether-à-go-go related gene channel. Attenuation of the basicity of the amino group by replacing the adjacent benzene ring with electron-deficient heteroaromatic rings provided several heterocyclic cores among which 6-aminofuro[3,2-c]pyridin-3(2H)-one was selected as a promising scaffold. Further optimization around the side chain moiety led to the discovery of 17i, which showed not only strong human GPR119 agonistic activity (EC50=14 nM), but also beneficial effects on gastric emptying and plasma total glucagon-like peptide-1 levels in mice.

Oxygen requirement for denitrification by the fungus Fusarium oxysporum.

The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrate (NO3-) and about 250 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrite (NO2-). The reduction of NO3- required more O2 than that of NO2- . With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3- and NO2-, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3- assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3- reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3-.

Multi-atmospheric pressure ionisation interface for liquid chromatography-mass spectrometry.

We describe the structure of a multi-atmospheric pressure ionisation (multi-API) interface for liquid chromatography-mass spectrometry (LC-MS). This interface includes five modes of atmospheric pressure spray with electron impact ionisation (APEI), atmospheric pressure chemical ionisation (APCI), atmospheric pressure spray ionisation (APSI), electrospray ionisation (ESI) and sonic spray ionisation (SSI). This LC-MS system was realised by developing an APEI interface which resembles other API interfaces and a highly sensitive API interface using advanced collision induced dissociation in a differential pumping region and an electrostatic ion guide. This system can deal with a wide variety of organic compounds from hydrocarbons with low polarity to proteins with high polarity by using the five modes.

A sonic spray interface for the mass analysis of highly charged ions from protein solutions at high flow rates.

We have improved the sonic spray interface to enable the analysis of multiply charged ions of protein from a solution at a flow rate of 1 mL/min using a conventional liquid chromatograph/mass spectrometer. In this interface, we added a multihole plate in front of the sampling orifice of a mass spectrometer. This plate does not have a hole coaxial to the sampling orifice but has small holes around the central region of the plate. The plate reduces the density of the solvent molecules in the sprayed gas introduced into the vacuum region through the sampling orifice from the atmosphere and prevents the ions from being solvated and becoming charged droplets due to the cooling that follows adiabatic expansion of the sprayed gas. With this improvement, multiply charged ions whose charge distribution ranged from 11+ to 16+ were analyzed from a 1 µM cytochrome c solution at a high flow rate of 1 mL/min without using a splitter.

Sonic spray mass spectrometry.

We have developed a sonic spray ionization method, in which a methanol and water solution is sprayed from a fused-silica capillary with gas flow coaxial to the capillary. Ions as well as charged droplets are produced under atmospheric pressure, and their intensities depend on the gas flow rate (gas velocity). Positive ions produced from dilute solutions of molecules regarded as neurotransmitters, such as catecholamine, by this ionization method have been analyzed with a quadrupole mass spectrometer. The protonated dopamine molecule is detected in the spray of the 10 nM solution, and the mass spectrum is compared with that obtained by the ion spray ionization method. A comparison between the mass-analyzed ion intensity and the ion current, which represents the sum of ions and charged droplets, shows that most ions are produced from the charged droplets after spraying. Furthermore, we found that the charged droplet formation cannot be ascribed to the traditional models of friction electrification, electrical double layer, or statistical charging. An explanation is proposed based on the ion concentration distribution in a small droplet.

Detection of gamma-aminobutyric acid in a living rat brain using in vivo microdialysis-capillary electrophoresis/mass spectrometry.

Coupling of in vivo microdialysis with capillary electrophoresis/mass spectrometry (MD-CE/MS) was demonstrated. A microdialysis probe was inserted in the striatum of a rat brain and gamma-aminobutyric acid (GABA) in the brain was detected by CE/MS without any chemical treatment of the sample. MD-CE/MS is expected to be a useful technique for analyzing brain substances, because of the relatively small damage it causes to a living structure and the high selectivity of the analytical technique.

Comparison of peptide analysis results for horse, bovine and dog cytochrome c using capillary electrophoresis/mass spectrometry with untreated capillary column and basic buffer condition.

Peptide analysis of cytochrome c using capillary electrophoresis/mass spectrometry was demonstrated to be a rapid, sensitive and useful technique for providing information concerning the location of variation. We analyzed tryptic digests of horse, bovine and dog cytochrome c, and compared the peptide analysis results for information concerning amino acid sequences in proteins. The differences in amino acid sequence were confirmed by taking into consideration that all the other fragments are identical in each map. The use of an untreated capillary column with basic buffer condition (water+acetonitrile+(20 g/L) ammonium carbonate (70/20/10), pH 9.2) had a reproducibility of migration times to within about 2%.

Direct peptide mapping of sickle-cell hemoglobin using electrospray mass spectrometry.

Direct peptide mapping of sickle-cell hemoglobin (Hb S) using electrospray mass spectrometry is demonstrated to be a rapid, sensitive and useful technique for providing information concerning the location of variation. By fully comparing electrospray mass spectra of digests of sickle-cell and normal hemoglobins, using L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin, the point mutation of B1 fragment of beta-chain in Hb S can be confirmed, taking into consideration that all the other fragments are completely identical in the two proteins.

Four interfaces for liquid chromatography/atmospheric-pressure-ionization mass spectrometry.

Mixture analyses are demonstrated using the liquid chromatograph/atmospheric-pressure-ionization mass spectrometric system with four modes. These modes are atmospheric-pressure spray with electron ionization, atmospheric-pressure chemical ionization, atmospheric-pressure spray ionization, and electrospray ionization modes. This system can deal with a wide variety of compounds from hydrocarbons with low polarity to proteins with high polarity.

Comparison of intensities between doubly charged ions [M + 2H]2+ and singly charged ions [M + H]+ of gramicidin S by electrospray mass spectrometry.

The reason why the intensity of doubly charged ions [M + 2H]2+ of gramicidin S is higher than that of singly charged ions [M + H]+ in electrospray is investigated by ion evaporation theory. As a result of comparison between the total free energies of extracting [M + 2H]2+ and [M + H]+ from a charged droplet to infinity, it is found that the total free energy of [M + 2H]2+ is estimated to be lower than that of [M + H]+. This clearly supports the experimental result. In addition, the importance of the electrostatic contribution in electrospray is demonstrated by showing the result that the total free energy of [M + 2H]2+ without electrostatic contribution is higher than that of [M + H]+.

Electrospray mass spectrometry: application of ion evaporation theory to amino acids.

We describe the result of applying the ion evaporation theory to a series of amino acids. The very good correlation (r = 0.98) of the natural logarithms of protonated molecule intensities observed by electrospray with the difference between the hydration free energies of molecules and the gas-phase binding free energies of molecules and protons in amino acids is consistent with the ion evaporation model. It seems that the difference in the protonated molecule intensities of amino acids obtained by electrospray can be explained by a scheme in which protonated molecules in the liquid phase are extracted into the gas phase after a charged droplet is formed.

[Glial fibrillary acidic protein and neurofilament protein in medulloblastoma].

Medulloblastoma is the most common primitive neuroectodermal tumor (PNET) with the potential to differentiate along glial or neuronal lines. Thirty cases of medulloblastoma were tested by the peroxidase-antiperoxidase (PAP) method with anti-GFAP serum (DAKO) and by the avidin-biotin peroxidase complex (ABC) method with 68kd subunit of anti-NF antibody. All the cases were classified into three subtypes based on these immunohistochemical findings and were analyzed in relation to clinico-pathological features. Fifteen of thirty medulloblastomas contained GFAP positive cells, seventeen showed cells reacting to NF. The reactions for both proteins were present in eight medulloblastomas (PNET-BD, bipotential differentiation). Seventeen medulloblastomas reacted to only one protein (PNET-MD, monopotential differentiation). No reaction for either was found in five cases (PNET-NOS, not otherwise specified). The two year survival rate was 12.5% for PNET-BD compared to 49.2% for PNET-MD and 53.3% for PNET-NOS. Nine variables, i.e. age, tumor stage, metastatic stage, operation, radiotherapy, chemotherapy, histology, GFAP and NF, were analyzed using Cox's proportional hazard model. This revealed that the significant factors were tumor stage (p = 0.0002), GFAP (p = 0.0008) and operation (p less than 0.05). In conclusion, GFAP is the most important histological factor for prognosis and medulloblastoma without glial differentiation has a much better prognosis than one with glial differentiation.

[Clinicopathological study of oligodendroglioma with special reference to immunohistochemical investigation].

The present study was undertaken to evaluate the utility of pathologic features and specific immunohistochemical studies in estimating the prognosis of oligodendroglioma. The pathological diagnosis of an oligodendroglioma was made on HE stained-sections according to WHO classification. Sixteen oligodendrogliomas, twelve mixed oligoastrocytomas and ten anaplastic oligodendrogliomas were immunotested by the peroxidase-antiperoxidase (PAP) method with anti-GFAP serum, anti-S-100 serum and anti-MBP (Myelin basic protein) serum and by the avidin biotin peroxidase-complex (ABC) method with anti-vimentin serum and ant-Leu 7 monoclonal antibody. GFAP positive cells were interpreted as reactive astrocytes, neoplastic astrocytes and neoplastic oligodendrocytes, S-100 positive cells were interpreted as reactive astrocytes and neoplastic astrocytes. Leu 7 positive cells were found in only one case of anaplastic oligodendroglioma. Anti-Leu 7 could not be considered as a specific marker for oligodendroglioma. Of the anaplastic oligodendroglioma 60% displayed MBP positively and 70% displayed vimentin positively. NSE positive cells were found in a few anaplastic oligodendrogliomas. The present study has not so far uncovered any marker that is restricted to oligodendrogliomas. However GFAP may be useful to assess the extent of reactive astrocytes and neoplastic astrocytes in the oligodendroglioma or mixed oligoastrocytoma. MBP and vimentin will help to determine the malignancy of oligodendroglioma.

[Treatment of malignant brain tumors with slowly releasing anticancer drug-polymer composites].

The purpose of this study is to present the methodology and results of a clinical trial of local chemotherapy of malignant brain tumors based on slowly-releasing anticancer drug-polymer composites. The slowly releasing drugs were prepared by combining and mutually dispersing anticancer agents with glassified monomers containing 10% polymetacrylic methyl acid and then this compound was frozen at -78 degrees C and exposed to 1 X 10(6) rad of gamma rays from cobalt 60. Thus we prepared a compound of polymers and anticancer agents. We used needle-shaped capsules of this compound. These capsules release the drug very slowly over 40 days. We administered locally to the malignant brain tumors with either slowly releasing mitomycin, slowly releasing adriamycin, slowly releasing ACNU or slowly releasing 5 Fu drugs. The following techniques were employed in implantation these capsules. Implantation into the remaining tumor wall at the time of excision. Implantation into the tumor by CT-guided stereotactic method. We implanted these drugs into tumor of 55 cases, thereafter we conducted both radiation and chemotherapy with ACNU in most patients. This method has the following advantages: It is possible to be employed to different types of anticancer agents. Both dosage and releasing time can be adjusted. It is possible to administer these capsules postoperatively by the stereotactic method. The clinical study consists of 55 patients, 20 cases of anaplastic astrocytoma, 23 cases of glioblastoma multiforme, 5 cases of oligodendroglioma, 3 cases of medulloblastoma and 4 cases of others. Survival rate estimated by Kaplan-Meier method was 47% in glioblastoma at 12 months and 91% in anaplastic astrocytoma at 18 months.(ABSTRACT TRUNCATED AT 250 WORDS)

[Clinicopathological study on malignant glioma--with special reference to GFAP].

In surgical specimens of 91 cases of malignant glial tumor, the correlation between the clinical malignancy of the tumors and the presence of GFA protein in the surgical specimen was examined. In anaplastic astrocytomas, 20 of 27 cases revealed positive staining of GFAP. In the cases of glioblastoma multiforme 27 of 35 cases showed GFAP positive staining. On the other hand, in medulloblastomas, all 29 cases were negative. In anaplastic astrocytomas and glioblastoma multiforms, the difference between the survival curves of GFAP positive groups and negative groups were examined. Between two groups, there was no statistical differences. In this study, the presence of GFAP was not proportional to the degree of tumor differentiation, and the GFAP staining of the tumor tissue was not a valuable examination showing the prognosis of the patient.

[Clinicopathological study of supratentorial tumors with multipotential differentiation in childhood. Primitive neuroectodermal tumor].

Three cases of supratentorial tumor in childhood were studied clinico-pathologically in an attempt to clarify its histological character. Case 1: A 3-year-old boy. Carotid angiogram revealed avascular lesion in the left parietal lobe. Twice operations and radiotherapy were performed. Ten months after the second operation, he died. Surgical specimen at the first operation was composed mainly of round tumor cells. The tumor tissue contained many collagen fibers. At the periphery of this tissue, medulloblastomatous areas consisting of closely aggregated hyperchromatic small round cells were found. There were perivascular rosettes and Homer Wright rosettes. In part, tubular and papillary arrangement of cells was also present. Astrocytomatous and oligodendrogliomatous structures were also present. Surgical specimens at the second operation showed the predominance of sarcomatous areas consisting of spindle-shaped cells with abundant argyrophilic fibers. Case 2: A 10-year-old girl. CT scan revealed a heterogeneous enhanced mass with a cyst and calcification in the right parietal lobe. Operation and radiotherapy were performed. Twelve months after operation, she is still alive. Most of the surgical specimens showed sarcomatous structure with abundant argyrophilic fibers. In these tissues, there were medulloblastomatous and ependymomatous features. Papillary arrangement of cells was also present. In part, there was oligodendrogliomatous structure. Case 3: A 2-year-old girl. CT scan revealed a heterogeneous enhanced mass in the right frontal lobe. The tumor tissue was composed of an aggregation of undifferentiated small round cells with Homer Wright rosettes.(ABSTRACT TRUNCATED AT 250 WORDS)