Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer.

BACKGROUND: Functional microRNAs (miRNAs) in exosomes have been recognised as potential stable biomarkers in cancers. The aim of this study is to identify specific miRNAs in exosome as serum biomarkers for the early detection of recurrence in human colorectal cancer (CRC). METHODS: Serum samples were sequentially obtained from six patients with and without recurrent CRC. The miRNAs were purified from exosomes, and miRNA microarray analysis was performed. The miRNA expression profiles and copy number aberrations were explored using microarray and array CGH analyses in 124 CRC tissues. Then, we validated exosomal miRNAs in 2 serum sample sets (90 and 209 CRC patients) by quantitative real-time RT-PCR. RESULTS: Exosomal miR-17-92a cluster expression level in serum was correlated with the recurrence of CRC. Exosomal miR-19a expression levels in serum were significantly increased in patients with CRC as compared with healthy individuals with gene amplification. The CRC patients with high exosomal miR-19a expression showed poorer prognoses than the low expression group (P<0.001). CONCLUSIONS: Abundant expression of exosomal miR-19a in serum was identified as a prognostic biomarker for recurrence in CRC patients.

Accurate and rapid novel genetic diagnosis for detection of sentinel lymph node metastasis in breast cancer patients.

BACKGROUND: The transcription-reverse transcription concerted reaction (TRC) test is a novel molecular-based procedure, which can assess nodal metastasis accurately and quickly. We examined the usefulness of the TRC test with a double marker, cytokeratin 19 (CK19) and carcinoembryonic antigen (CEA) mRNA, to detect sentinel lymph nodes (SLN) metastasis in breast cancer patients. METHODS: A total of 264 SLNs from 131 breast cancer patients were assigned to a training set (109 SLNs from 50 patients) and validation set (155 SLNs from 81 patients). Cytokeratin 19 and CEA mRNA were detected by TRC tests, and the sensitivity and specificity of the SLN metastasis between the TRC and histology cohorts were compared. RESULTS: Mean copy numbers of CK19 and CEA by TRC tests were increased according to the metastatic size. In the training set, TRC test showed 100% sensitivity, specificity and concordance rates against the permanent histopathology test. In the validation set, sensitivity was 97.1%, specificity was 99.2% and the concordance rate was 99.4%. CONCLUSION: Our results showed that the detection of CK19 and CEA mRNA using the TRC test is, an accurate and rapid method for detection of SLN metastasis and can be applied as an intraoperative molecular diagnosis in breast cancer patients.

Two cases of diverticulitis in patients with Williams syndrome.

Williams syndrome is rare and associated with physical anomalies and mental retardation. It is a disease resulting from a gene deletion of chromosome 7. The main concurrent medical conditions typically associated with Williams syndrome are heart defects such as supravalvular aortic stenosis, mental retardation, and unusual physical characteristics. It is also associated with colon diverticulosis and diverticulitis. In the present article, we report on 2 cases of diverticulitis in patients with Williams syndrome, in whom surgery was performed. In many cases of diverticulitis in patients with Williams syndrome, surgical treatment is indicated. It is important to take diverticulitis into consideration when examining a patient with Williams syndrome presenting with abdominal pain and consider surgical treatment if necessary.

Effect of immunotherapy and spleen preservation on immunological function in patients with gastric cancer.

Splenectomy is often performed in patients who undergo total gastrectomy for cancer of the upper stomach. Although splenectomy facilitates lymph node dissection of the splenic hilum and recent reports advocate spleen preservation, the role of the spleen is not fully elucidated in gastric cancer treatment. This prospective randomized study was performed to evaluate the role of the spleen in immunological function in gastric cancer patients who underwent total gastrectomy and received postoperative immunochemotherapy. Forty-five patients with gastric cancer were randomly allocated to four groups: 1. splenectomy without immunotherapy (OK-432 administration), 2. splenectomy with immunotherapy, 3. spleen preservation without immunotherapy, 4. spleen preservation with immunotherapy. Postoperative immunological function of these patients was compared among the four groups. NK cell activity of the peripheral blood lymphocytes (PBL) in spleen-preserved patients who received immunotherapy was significantly higher for 24 weeks after surgery than that of the splenectomized patients with and without OK-432 administration. IL-2 production of PBL in spleen-preserved patients with immunotherapy was significantly higher between 4 and 24 weeks after surgery compared with that of the splenectomized patients without immunotherapy. The results suggest that spleen preservation might be beneficial in patients with less advanced gastric cancer who receive postoperative immunochemotherapy after total gastrectomy.

Purification of a low molecular weight factor that induces differentiation and inhibits growth in myeloid leukemia cells.

A procedure is described for purifying a low molecular weight peptide that induces differentiation in mouse myeloid leukemia M1 cells. The factor comes from the conditioned medium of macrophage-like cells differentiated from mouse myeloid leukemia M1 cells. The procedure for purification includes gel filtration on Sephadex G-15, anionic exchange chromatography, thin-layer chromatography, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography gel filtration. The molecular weight of the factor estimated from the amino acid composition was approximately 1280, which agrees well with that obtained by high-performance liquid chromatography gel filtration. The half-maximal concentration of the purified factor for inducing differentiation of M1 cells was approximately 3.2 x 10(-7) M as judged by nitroblue tetrazolium staining ability. The purified factor also inhibits the growth of human leukemia HL-60 cells.

Inhibition of experimental metastasis by an alpha-glucosidase inhibitor, 1,6-epi-cyclophellitol.

Isolated from a culture filtrate of Phellinus sp., cyclophellitol is a specific inhibitor of beta-glucosidase, but unlike castanospermine, it does not inhibit experimental metastasis. However, its structural analogue, 1,6-epi-cyclophellitol, inhibited alpha-glucosidase as well as beta-glucosidase, and inhibited experimental metastasis. 1,6-Epi-cyclophellitol depressed alpha-glucosidase activity in cultured B16/F10 cells after 48 h of incubation. Preincubation of B16/F10 cells for 48 h with 1,6-epi-cyclophellitol inhibited invasion of the cells in a Boyden chamber assay at the doses effective in inhibiting alpha-glucosidase in situ. Pulmonary metastasis of B16/F10 cells in mice was inhibited by pretreatment of the cells with 1,6-epi-cyclophellitol in culture. The inhibitor reduced the collagen type I- and IV-mediated attachment of the cells, whereas it had no effect on laminin-mediated attachment. These results suggest that alpha-glucosidase in tumor cells is essential for the metastatic process through the cellular interaction with collagen type I and IV.

Inhibition of brain prostaglandin D synthetase and prostaglandin D2 dehydrogenase by some saturated and unsaturated fatty acids.

The activities of rat brain prostaglandin D synthetase and swine brain prostaglandin D2 dehydrogenase were inhibited by some saturated and unsaturated fatty acids. Myristic acid was most potent among saturated straight-chain fatty acids so far tested. The IC50 values of this acid were 80 microM for prostaglandin D synthetase and 7 microM for prostaglandin D2 dehydrogenase, respectively. Little inhibition was found with methyl myristate and myristyl alcohol. The IC50 values of these derivatives were more than 200 microM for both enzymes, suggesting that the free carboxyl group was essential for the inhibition. The effects of cis double bond structure of fatty acids on the inhibition potency were examined by the use of the carbon 18 and 20 fatty acids. The inhibition potencies for both enzymes increased with the number of cis double bonds; the IC50 values of stearic, oleic, linoleic and linolenic acid were, respectively, more than 200, 60, 30 and 30 microM for prostaglandin D synthetase, and 20, 10, 8.5 and 7 microM for prostaglandin D2 dehydrogenase. Arachidonic acid also inhibited the activities of both enzymes with respective IC50 values of 40 microM for prostaglandin D synthetase and 3.9 microM for prostaglandin D2 dehydrogenase, while arachidic acid showed little inhibition. The kinetic studies with myristic acid and arachidonic acid demonstrated that the inhibition by these fatty acids was competitive and reversible for both enzymes. Myristic acid and other fatty acids also inhibited the activities of several enzymes in prostaglandin metabolism, although to a lesser extent. The IC50 values of myristic acid for prostaglandin E isomerase, thromboxane synthetase and NAD-linked prostaglandin dehydrogenase (type I) were 200, 700 and 100 microM, respectively. However, this fatty acid showed little inhibition on fatty acid cyclooxygenase (20% at 800 microM), glutathione-requiring prostaglandin D synthetase from rat spleen (20% at 800 microM), and NADP-linked prostaglandin dehydrogenase (type II) (no inhibition at 200 microM).

3,4-dihydroxystyrene, a novel microbial inhibitor for phenylalanine hydroxylase and other pteridine-dependent monooxygenases.

A new microbial inhibitor for rat-liver phenylalanine hydroxylase (L-phenylalanine, tetrahydropteridine: oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1) was isolated from a culture medium of Fomes tasmanicus, and its structure was determined as 3,4-dihydroxystyrene. This compound inhibited the enzyme by 50% at a concentration of 5 X 10(-6) M and 5 X 10(-7) M, respectively, without or with preincubation at 25 degrees C for 15 min. Without preincubation, dihydroxystyrene inhibited phenylalanine hydroxylase noncompetitively with phenylalanine and a pteridine cofactor, 6,7-dimethyltetrahydropterin, and uncompetitively with oxygen. A change in kinetic properties of the inhibition was observed when the enzyme was preincubated with dihydroxystyrene; the degree of inhibition was increased, and the purely noncompetitive-type inhibition with phenylalanine changed to a mixed-type inhibition. A study concerning the structure-inhibitory activity relationship using several compounds similar to dihydroxystyrene, indicated that the catechol structure is essential and that the structure of the aliphatic side-chain affects the inhibitory potency. A similar degree of inhibition was observed using 6,7-dimethyl- or 6-methyltetrahydropterin or tetrahydrobiopterin as a cofactor. Dihydroxystyrene also inhibited other pteridine-dependent monooxygenases, tyrosine hydroxylase (EC 1.14.16.2) and tryptophan hydroxylase (EC 1.14.16.4), indicating that dihydroxystyrene is a general inhibitor for pteridine-dependent monooxygenases.

Influence of the site of stimulation on atrioventricular nodal refractory periods and the effect of verapamil.

The refractory periods of the atrioventricular (AV) node appear dependent on the pattern of AV nodal input. In 21 superfused AV rabbit heart preparations stimulated from each of the 2 principal input regions, crista terminalis or atrial septum, the effect of changing the site of stimulation of the AV nodal refractoriness and the relative effect of verapamil on AV nodal refractoriness was determined. In 6 of 21 preparations the functional AV refractory curve became discontinuous only when stimulation was applied at the atrial septum and suggested dual AV nodal pathways (dual pathways group). In the 15 other preparations no interruption of the curve occurred with either crista terminalis or atrial septal stimulation (normal conduction group). In the normal conduction group, the difference in the effective refractory period of the AV node obtained by crista terminalis vs atrial septal stimulation was not significant (154 +/- 25 vs 150 +/- 28 ms). However, the functional refractory period was significantly longer with crista terminalis vs atrial septal stimulation (232 +/- 19 vs 239 +/- 19 ms, p less than 0.001). After verapamil administration, the effective and functional refractory periods during crista terminalis vs atrial septal stimulation were prolonged to 270 +/- 49 vs 285 +/- 55 ms (p less than 0.01) and 335 +/- 43 vs 351 +/- 41 ms (p less than 0.001), respectively. Thus, the difference in refractory periods associated with changing the stimulation site was exaggerated with verapamil.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison between Japan and North America in the post-hospital course after recovery from an acute coronary event.

We compared the post-hospital prognosis after an acute coronary event (acute myocardial infarction and unstable angina) in 106 patients in Japan vs. 789 patients in North America who were prospectively enrolled in the Multicenter Study of Myocardial Ischemia and were followed-up for an average of 26 months per patients. Risk factors more frequent in Japan were older age, males and smoking at enrollment, but the rest of many risk factors were similar. After adjusting for differences in clinical and medication variables, Cox analyses indicated patients in North America had a significantly greater risk of experiencing a primary end-point (cardiac death, non-fatal myocardial infarction or unstable angina) than patients in Japan (hazard ratio [North America:Japan] = 3.1, P = 0.003). There was a non-significant trend in the restricted end-points (cardiac death or non-fatal myocardial infarction) with North America having more frequent events than Japan (hazard ratio = 2.2, P = 0.12). The long-term outcome after recovery from an acute coronary event is more favorable in Japan than in North America, mostly due to a reduction in subsequent hospitalization for unstable angina. The reason for these findings cannot be explained by differences in the measured risk factors or medications.

2-Amino-5-methyl-5-hexenoic acid, a methionine analog produced by Streptomyces sp. MF374-C4.

2-Amino-5-methyl-5-hexenoic acid (AMHA), a new methionine analog, was isolated from a fermentation broth of Streptomyces sp. MF374-C4 based on its reversal of the effect of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in a test system that determines the size of growth zones of revertants (His+) of Salmonella typhimurium TA1535. AMHA also inhibited growth of the tester strain in a synthetic medium. These AMHA activities were abolished by methionine. The incidence of spontaneous streptomycin-resistant mutations of Escherichia coli K12 was not decreased by AMHA at concentrations where cell growth was partially inhibited. AMHA inhibited protein synthesis but not DNA or RNA synthesis in S. typhimurium TA1535 and E. coli K-12. The analog inhibited formation of methionyl-tRNA but not of valyl-tRNA in a cell-free system of E. coli, and supported ATP-PPi exchange in the cell-free system. At concentrations where it inhibited cell growth, AMHA decreased the number of foci, induced by ROUS sarcoma virus, on cultured sheets of chick-embryo fibroblasts. The effects of AMHA on focus formation and on the cell growth were overcome by methionine.

Biological activities of cyclophellitol.

Cyclophellitol [1S,2R,3S,4R,5R,6R)-5-hydroxymethyl-7-oxabicyclo[4,1,0] heptane-2,3,4-triol) was tested against 9 glycosidases and found to be a specific inhibitor of almond beta-glucosidase. Cyclophellitol inhibited almond beta-glucosidase activity by 50% at 0.8 micrograms/ml and was a competitive inhibitor of almond beta-glucosidase as revealed by Lineweaver-Burk plot. Cyclophellitol was inactive against yeast alpha-glucosidase, beta-galactosidase, beta-glucuronidase, alpha-L-fucosidase, end-beta-N-acetyl glucosaminidase, alpha-mannosidase, and cellulase. It was weakly active toward fungal beta-xylosidase. Cyclophellitol-treated almond beta-glucosidase was equally suppressed after dialysis; thus cyclophellitol is likely to bind to almond beta-glucosidase irreversibly. The inhibitor was found by fluorimetric assay to be active against beta-glucosidase but inactive toward alpha-glucosidase in Molt-4 microsomal fraction. It also inhibited Molt-4 beta-glucocerebrosidase completely at 2 micrograms/ml when the enzyme was assayed with a synthetic labeled substrate, and the inhibitory activity was more than one hundred times higher than that of nojirimycin, castanospermine, or of deoxynojirimycin. Mice administered 1 mg of cyclophellitol daily for 5 days began to exhibit severe abnormalities of nervous system similar to those found in Gaucher's mouse.

Novel antibiotics, amythiamicins. I. Taxonomy, fermentation, isolation, physico-chemical properties, and antimicrobial activity.

Novel antibacterial antibiotics, amythiamicins A, B, C and D, have been isolated from the fermentation broth of Amycolatopsis sp. MI481-42F4. In this paper, the taxonomy of the producing strain, fermentation, isolation, physico-chemical properties and biological activities of amythiamicins are reported. Amythiamicins inhibit the growth of Gram-positive bacteria including multi-drug resistant strains.

Novel antibiotics, amythiamicins. II. Structure elucidation of amythiamicin D.

The structure of a unique polythiazole-containing cyclic peptide antibiotic, amythiamicin D, was elucidated by chemical degradations and NMR spectral analyses. Acid hydrolysis of amythiamicin D gave one mole of glycine and three new amino acids. Structures of N-acetyl-O-methyl derivatives of these new amino acids were determined by NMR and UV spectral analyses. Connectivities of these amino acids were determined by HMBC experiments.

Novel antibiotics, amythiamicins. III. Structure elucidations of amythiamicins A, B and C.

The structures of novel antimicrobial antibiotics, amythiamicins A, B and C, were elucidated by chemical degradations and NMR spectral analyses. The main frame from C-1 to C-41 of these antibiotics was the same as that of amythiamicin D. Amino acid autoanalyses of amythiamicins A, B and C showed that these have another one mole of serine and proline in comparison with amythiamicin D. Stereochemistries of both amino acids were determined to be L by chiral HPLC. These seryl-prolyl residues in amythiamicins A, B and C are attached at C-41 through an oxazoline ring, amide and ester bond, respectively.

The structure of histargin.

The structure of histargin, an inhibitor of carboxypeptidase B, produced by Streptomyces roseoviridis MF118-A5 strain, has been defined as N-[(S)-1-carboxy-4-guanidinobutyl]-N'-[(S)-1-carboxy-2-(imidazol-4 -yl) ethyl]ethylenediamine, by spectral analysis and chemical synthesis.

Dehydrodicaffeic acid dilactone, an inhibitor of catechol-O-methyl transferase.

In the screening of catechol-O-methyltransferase inhibitors, three compounds were isolated from the culture filtrate of a mushroom, Inonotus sp. One was 3,4-dihydroxycinnamic acid (caffeic acid) which had been reported as an inhibitor of this enzyme. The others were the dextrorotatory 2,6-bis-(3',4'-dihydroxyphenyl)-3,7-dioxabicyclo-[3,3,0]-octane 4,8-dione (dehydrodicaffeic acid dilactone) andits antipode. These new compounds inhibited both dopamine beta-hydroxylase and dopa decarboxylase and showed hypotensive activity in the SH rat.

Cytotoxicity of spergualin and amine oxidase activity in medium.

The cytotoxicity of spergualin on cultured L5178Y cells is dependent on the kind of serum contained in culture media. Spergualin has stronger cytotoxicity to L5178Y cells in calf serum (IC50 = 2 micrograms/ml) than in horse serum (IC50 = 60 micrograms/ml). This was thought to be caused by amine oxidase in sera. Because calf serum was rich in amine oxidase and horse serum was very poor. Spergualin was found to be oxidized by either calf serum or amine oxidase purified from beef plasma. Aminoguanidine, an amine oxidase inhibitor suppressed the spergualin effect to inhibit the growth of L5178Y cells in calf serum to the level in horse serum. On the other hand, in horse serum the spergualin cytotoxicity was enhanced by addition of amine oxidase. These results suggested to us that spergualin might be inactive in itself and that the amine oxidase-oxidized product might play an essential role in inhibiting the growth of cells.

New triene-ansamycins, thiazinotrienomycins F and G and a diene-ansamycin, benzoxazomycin.

New triene-ansamycins designated thiazinotrienomycins F (TT-F) and G (TT-G) and a new diene-ansamycin, benzoxazomycin, were isolated from a culture broth of Streptomyces sp. MJ672-m3 and their structures were elucidated by spectroscopic analyses. The Mean Graphs of TT-G suggests that the tumor growth inhibitory activities are almost as strong as TT-B, in respect of GI50 and TGI against several human cancer cell lines.

Delaminomycins, novel extracellular matrix receptor antagonists. V. Biosynthesis.

The biosynthesis of delaminomycin A, produced by Streptomyces albulus MJ202-72F3, was investigated by feeding 13C-labeled compounds followed by 13C NMR analyses. The results indicate that delaminomycin A is derived from six acetate units, five propionate units and one glycine unit.