Two new adenopeptins B and C inhibit sphere formation of pancreatic cancer cells.

Cancer remains one of the leading causes of death worldwide, particularly pancreatic cancer being lethal because of its aggressiveness and lack of early detection methods. A factor that contributes to malignancy are cancer stem cell-like characteristics promoted by the tumor-stromal interaction. Given that fibroblast conditioned medium (CM) promotes sphere formation of cancer cells, a cancer stem cell-like characteristic, its inhibitor could be a new anticancer agent. By exploring microbial cultures as a source, we found new compounds, namely, adenopeptins B (1) and C (2), from Acremonium sp. ESF00140. 1 and 2 selectively and potently inhibited the sphere formation of pancreatic cancer cells cultured in the fibroblast CM compared with the control medium. Oxygen consumption rate (OCR) assays showed that 1 and 2 inhibit OCR in pancreatic cancer cells. Studies of similar compounds suggested mitochondrial complex V inhibition. Therefore, results of measuring the activity of human mitochondrial complex V revealed that 1 and 2 inhibited its activity. Oligomycin A, an inhibitor of mitochondrial complex V, as well as 1 and 2, strongly inhibited the sphere formation of pancreatic cancer cells cultured in fibroblast CM. The addition of 1 and 2 to pancreatic cancer cells cultured in fibroblast CM increased reactive oxygen species (ROS) production compared with that in the control medium. In pancreatic cancer cells cultured in fibroblast CM, mitochondria significantly influence sphere formation, and targeting their function with 1 and 2 might provide a new therapeutic approach for pancreatic cancer.

Catenulopyrizomicins, new anti-Hepatitis B virus compounds, from the rare actinomycete Catenuloplanes sp. MM782L-181F7.

Hepatitis B virus (HBV) causes chronic hepatitis in humans, and current antiviral therapies rarely treat viral infections. To improve the treatment efficacy, novel therapeutic agents, especially those with different mechanisms of action, need to be developed for use in combination with the current antivirals. Here, we isolated new anti-HBV compounds, named catenulopyrizomicins A-C, from the fermentation broth of rare actinomycete Catenuloplanes sp. MM782L-181F7. Structural analysis revealed that these compounds contained a structure that is composed of thiazolyl pyridine moiety. The catenulopyrizomicins reduced the amount of intracellular viral DNA in HepG2.2.15 cells with EC50 values ranging from 1.94 to 2.63 µM with small but notable selectivity. Mechanistic studies indicated that catenulopyrizomicin promotes the release of immature virion particles that fail to be enveloped through alterations in membrane permeability.

Isolation of new derivatives of the 20-membered macrodiolide bispolide from Kitasatospora sp. MG372-hF19.

New three macrocyclic diolides, named bispolides C-E (1-3), were isolated from a fermentation broth of the actinomycete strain MG372-hF19, which produces an indole glycoside and leptomycins as we reported previously. The absolute structures of compounds 1-3 were elucidated by NMR and X-ray crystallography. Compounds 1-3 diverge from the known nine bispolides in their different alkylation patterns on the 20-membered macrocyclic diolide skeleton and the side chain in their planar structures. Furthermore, compounds 1-3 exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci and cytotoxic activity against human cancer cell lines. Among them, compound 3 has the most potent biological activities against bacteria and tumor cells. Additionally, using a membrane-potential-sensitive fluorescence probe, we found that compounds 1-3 and elaiophylin have a similar effect on membrane potential in A549 human lung cancer cells.

Micromonosporamide A with Glutamine-Dependent Cytotoxicity from Micromonospora sp. MM609M-173N6: Isolation, Stereochemical Determination, and Synthesis.

An acyldipeptide, micromonosporamide A, was isolated from the fermentation broth of Micromonospora sp. MM609M-173N6 by bioassay-guided fractionation using a glutamine compensation assay. The planar structure was elucidated on the basis of comprehensive one- and two-dimensional nuclear magnetic resonance and high-resolution mass spectrometry. The relative and absolute configuration of the entire molecule were determined using a combined approach, involving chromatographic analysis by liquid chromatography-mass spectrometry, advanced Marfey's method, and total synthesis. Micromonosporamide A exhibited glutamine-dependent antiproliferative activity.

Proteolytic Processing, Maturation, and Unique Synteny of the Streptomyces Hemagglutinin SHA.

SHA is an l-rhamnose- and d-galactose-binding lectin that agglutinates human group B erythrocytes and was first purified almost 50 years ago. Although the original SHA-producing Streptomyces strain was lost, the primary structure of SHA was more recently solved by mass spectrometry of the archived protein, which matched it to a similar sequence in the Streptomyces lavendulae genome. Using genomic and protein biochemical analyses, this study aimed to identify SHA-secreting Streptomyces strains to further investigate the expression and binding activities of these putative proteins. Of 67 strains genetically related to S. lavendulae, 17 secreted pro-SHAs in culture. Seven SHA homologues were purified to homogeneity and then subjected to liquid chromatography-high-resolution multistage mass spectrometry (LC-MS/MS) and hemagglutination (HA) assays. Processing of pro-SHAs occurred during and after purification, indicating that associated proteases converted pro-SHAs into mature SHAs with molecular masses and HA activities similar to that of the archived SHA. Previously, the SHA monomer was shown to have two carbohydrate binding sites. The present study, however, found no HA activity in pro-SHAs, suggesting that pro-SHAs have only one binding site. Genetically, the SHA gene resides in conserved syntenic regions. The published genomes of 1,234 Streptomyces strains were analyzed, revealing 18 strains with SHA genes, 16 of which localized to a unique syntenic region. The SHA syntenic region consists of ∼17 open reading frames (ORFs) and is specific to S. lavendulae-related strains. Notably, a lipoprotein gene excludes SHA from the synteny in some strains, suggesting that horizontal gene transfer events during the course of evolution shaped the distribution of SHA genes. IMPORTANCE Lectins are extremely useful molecules for the study of glycans and carbohydrates. Here, we show that homologous genes encoding the l-rhamnose- and d-galactose-binding lectins, SHAs, are present in multiple bacterial strains, genetically related to Streptomyces lavendulae. SHA genes are expressed as precursor pro-SHA proteins that are truncated and mature into fully active lectins with two carbohydrate binding sites, which exhibit hemagglutination activity for type B red blood cells. The SHA gene is located within a conserved syntenic region, hinting at specific but yet-to-be-discovered biological roles of this carbohydrate-binding protein for its soil-dwelling microbial producer.

The potent protein phosphatase 2A inhibitors aminocytostatins: new derivatives of cytostatin.

Specific inhibitors of protein phosphatase 2A (PP2A) mediate anticancer effects by augmenting the tumor-killing activity of natural killer (NK) cells. In this study, new PP2A inhibitors, aminocytostatins A-E, were isolated from Kitasatospora sp. MJ654-NF4 and structurally characterized. Aminocytostatins are derivatives of cytostatin, which is a specific PP2A inhibitor isolated from the same organism, and aminocytostatins have a characteristic amino group within the lactone moiety. Compared to cytostatin, aminocytostatin A showed a stronger inhibitory activity against PP2A in vitro and augmented the tumor-killing activity of NK cells in vivo. Furthermore, a docking model was generated to demonstrate the favorable activities of aminocytostatin A.

Saccharobipyrimicin, a new antibiotic from the leaf-litter actinomycete Saccharothrix sp. MM696L-181F4.

In the course of screening for new antimicrobial compounds, a new antibiotic substance named saccharobipyrimicin was isolated from the leaf-litter actinomycete Saccharothrix sp. MM696L-181F4. The structure of saccharobipyrimicin was elucidated by various spectral methods, mainly single-crystal X-ray analysis and chemical degradation. It revealed that saccharobipyrimicin contained a 2,2'-bipyridine skeletal structure. Saccharobipyrimicin showed moderate and broad-spectrum antimicrobial activity. Two chemical derivatives of saccharobipyrimicin showed weaker antimicrobial activities than that of saccharobipyrimicin against most test microorganisms except two tolC mutants of Escherichia coli and Neisseria gonorrhoeae.

A new indole glycoside from Kitasatospora sp. MG372-hF19 carrying a 6-deoxy-α-L-talopyranose moiety.

Small cell lung cancer (SCLC) is a severe malignancy with early and widespread metastasis, and novel therapeutic drugs are needed. To identify cytotoxic natural compounds against SCLC, we screened libraries of microbial fermentation broths using several lung cancer cell lines. We found that the actinomycete strain MG372-hF19 produces a compound that has not been isolated from natural sources but previously chemically synthesized, 6-chloro-1H-indole-3-carboxaldehyde (1), and an entirely new compound, named 6-deoxy-α-L-talopyranose 1-(6-chloro-1H-indole-3-carboxylate) (2), together with leptomycins. The molecular formulas of the compounds were established as C9H6ClNO and C15H16ClNO6, respectively, via high-resolution electrospray ionization mass spectrometry, and their structures were determined using detailed NMR. Absolute configurational analysis of the sugar unit of compound 2 revealed that the compound incorporates the rare deoxyhexose 6-deoxy-α-L-talopyranose. Both compounds exhibited weak growth-inhibiting activities against human lung cancer cell lines.

Changes in the Dermal Structure during Cultured Epidermal Autograft Engraftment Process.

The use of cultured epithelial autografts for the treatment of extensive burn wounds has become popular in recent years. We examined extensive burn wounds in 14 patients by using a combination of autograft and cultured epithelial autografts developed in Japan (JACE). METHODS: We undertook a skin biopsy at 2, 4, and 6 weeks after transplantation with JACE. By using electron microscopy we observed the engraftment process. RESULTS: In transmission electron microscope findings, we recognized the engraftment process of JACE. Keratinocytes matured gradually. Collagen fibers formed thick bundles in the dermis layer. In scanning electron microscope findings, we observed papillary dermis development on the artificial dermis. CONCLUSIONS: After managing wound bed preparation by using artificial dermis, we were able to recognize the good result of grafting JACE on meshed 6:1 split thickness autografts. This is because the auto dermis from autograft extended under the JACE, binding between JACE, and the dermis became strong.

Antitragicus muscle resection: A key to correction of prominent lobules.

The antitragicus muscle arises from the outer part of the antitragicus cartilage, and inserts into the helical tail and antihelix. Overdevelopment or malpositioning of the antitragicus muscle exerts an anterior pull on the helical tail, and it can cause prominent lobules. We attempted prominent lobule correction using antitragicus muscle resection and helical tail setback in combination with a Mustarde or Furnas suture technique. Seventeen children with prominent lobules underwent this technique, and all had satisfactory outcomes. Resection of the antitragicus muscle is minimally invasive and easy to perform. This procedure is a key to successful lobular setback.

Combination Treatment of Artificial Dermis and Basic Fibroblast Growth Factor for Skin Defects: A Histopathological Examination.

UNLABELLED: This study examined a combination of artificial dermis and basic fibroblast growth factor (bFGF) to treat skin defects in clinical cases, and it histopathologically examined the effects on the conditions of recipient beds. MATERIALS AND METHODS: The subjects were 11 patients with skin defects from burn ulcers or traumatic ulcers. In each subject, debridement was performed and subsequently artificial dermis was applied to the defect. The bFGF was used on 1 side (combination therapy) of the artificial dermis and not used on the other side (artificial dermis monotherapy). A histopathological examination was performed on the granulation tissue collected from the recipient bed. The authors also measured skin hardness 6 months after the skin graft. RESULTS: Histologically, the combination therapy site had more extensive capillary angiogenesis than the monotherapy site. The combination therapy site also had capillary walls consisting of thick, large endothelial cells; fibroblast proliferation and activation; and more severe infiltration of inflammatory cells. Skin hardness after the graft was also much softer in the combination therapy. CONCLUSION: The results suggest the usefulness of this combination therapy in the preparation of skin graft beds to improve skin hardness after skin grafts in clinical cases.

Difference in nasolabial features between awake and asleep infants with unilateral cleft lip: Anthropometric measurements using three-dimensional stereophotogrammetry.

PURPOSE: Cleft lip repair is performed in the supine position, tilting the head back under general anesthesia. However, postoperative results are evaluated in the upright position while patients are awake. The purpose of this study was to anthropometrically assess whether nasolabial features of infants with unilateral cleft lip are influenced by posture and anesthesia. MATERIAL AND METHODS: Three-dimensional facial images in a preoperative upright position and operating supine position under general anesthesia were captured from 51 consecutive infants with unilateral cleft lip. Twenty-four indirect anthropometric measurements (11 for the nose and 13 for the lip elements) were considered on each infant. RESULTS: In the supine position under general anesthesia, alar surface distance was significantly shorter (p < 0.001). Regarding lip measurements, medial lip height of the cleft side and philtrum height were significantly smaller (p < 0.05 and p < 0.05, respectively), whereas vermilion height was greater (p < 0.01). In addition, the cleft width and lip width were significantly broader (p < 0.001 and p < 0.001, respectively) after general anesthesia. CONCLUSIONS: Several nasolabial alteration patterns are found after general anesthesia that are presumably attributable to cessation of nasal breathing and the action of muscle relaxation. Surgeons should take these nasolabial changes into account during preoperative planning and postoperative assessment.

Bioconversion of FR901459, a novel derivative of cyclosporin A, by Lentzea sp. 7887.

FR901459, a product of the fungus Stachybotrys chartarum No. 19392, is a derivative of cyclosporin A (CsA) and a powerful immunosuppressant that binds cyclophilin. Recently, it was reported that CsA was effective against hepatitis C virus (HCV). However, FR901459 lacks active moieties, which are essential for synthesizing more potent and safer derivatives of this anti-HCV agent. Here we identified an actinomycete strain (designated 7887) that was capable of efficient bioconversion of FR901459. Structural elucidation of the isolated bioconversion products (1-7) revealed that compounds 1-4 were mono-hydroxylated at the position of 1-MeBmt or 9-MeLeu, whereas compounds 5-7 were bis-hydroxylated at both positions. The results of morphological and chemical characterization, as well as phylogenetic analysis of 16S ribosomal DNA (rDNA), suggested that strain 7887 belonged to the genus Lentzea. Comparison of the FR901459 conversion activity of strain 7887 with several other Lentzea strains revealed that although all examined strains metabolized FR901459, strain 7887 had a characteristic profile with respect to bioconversion products. Taken together, these findings suggest that strain 7887 can be used to derivative FR901459 to produce a chemical template for further chemical modifications that may provide more effective and safer anti-HCV drugs.

Vulgatibacter incomptus gen. nov., sp. nov. and Labilithrix luteola gen. nov., sp. nov., two myxobacteria isolated from soil in Yakushima Island, and the description of Vulgatibacteraceae fam. nov., Labilitrichaceae fam. nov. and Anaeromyxobacteraceae fam. nov.

Two myxobacterial strains (designated B00001(T) and B00002(T)) were isolated from forest soil samples collected from Yakushima Island, Kagoshima, Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains B00001(T) and B00002(T) respectively formed independent branches within the suborders Cystobacterineae and Sorangiineae and were most closely related to Cystobacter armeniaca DSM 14710(T) (90.4% similarity) and Byssovorax cruenta DSM 14553(T) (91.3%). Neither strain showed typical features of myxobacteria such as bacteriolytic action or fruiting body formation, but both had high DNA G+C contents (66.3-68.3 mol%). Swarming motility was observed in strain B00002(T) only. Cells of both strains were vegetative, chemoheterotrophic, mesophilic, strictly aerobic, Gram-negative, motile rods, and both strains exhibited esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and ß-galactosidase activities. Strain B00001(T) contained MK-7 as the predominant respiratory quinone and the major fatty acid was iso-C15:0. In contrast, strain B00002(T) contained MK-8 as the major cellular quinone and the major fatty acids were C16 : 1ω5c and iso-C17 : 0. Based on the phenotypic and genotypic data presented, strains B00001(T) and B00002(T) represent novel genera and species, for which we propose the names Vulgatibacter incomptus gen. nov., sp. nov. and Labilithrix luteola gen. nov., sp. nov., respectively. The type strains of Vulgatibacter incomptus and Labilithrix luteola are B00001(T) ( = NBRC 109945(T) = DSM 27710(T)) and B00002(T) ( = NBRC 109946(T) = DSM 27648(T)), respectively. The new genera are assigned to the new families Vulgatibacteraceae fam. nov. and Labilitrichaceae fam. nov., respectively. In addition, Anaeromyxobacteraceae fam. nov., is proposed to accommodate the genus Anaeromyxobacter, which is related to the genus Vulgatibacter.

Experience of using cultured epithelial autografts for the extensive burn wounds in eight patients.

UNLABELLED: In Japan, the cultured epithelial autografts "JACE" was accepted as a health insurance adaptation from January 1, 2009. We examined the extensive burn wounds in 8 patients by using a combination of autograft and JACE. After debridement, we managed the wound bed preparation by using artificial dermis. The wound bed was covered with fine tissue 2 weeks after we implanted artificial dermis and trafermin was used every day. Meshed 6:1 split-thickness autografts were placed onto the recipient wound bed under the JACE. The epidermalization was nearly complete within 3 to 4 weeks. RESULTS: A total of 39 patients underwent medical treatment of burns. All patients burned more than 30% total body surface area (TBSA). We divided them into 2 groups. The control group consisted of 31 patient, 23 men and 8 women. They underwent operation not using JACE but only autograft. The average age of the patients was 59.61 (3.85) years. The TBSA burned in this control group was 58.94% (3.89%). Operation times were 2.16 (0.24) hours. The overall survival rate was 35.5%. The study group consisted of 8 patients, 5 men and 3 women. The average age of the patients was 56.38 (7.04) years. The TBSA burned in this study group was 51.63% (4.17%). Operation times were 4.25 (0.59) hours, and the overall survival rate in this study group was 87.5%. The average take rate of JACE was 80.0% (3.09%) 4 weeks postoperatively. CONCLUSIONS: JACE is one of the cultured epithelial autografts. Although we managed the wound bed preparation by using artificial dermis instead of cryopreserved cadaver allograft, we were able to recognize a good result from grafting JACE on meshed 6:1 split-thickness autografts. The study group observed a significant difference in operation times compared with the control group. However, this treatment contributed to reducing the area of the donor site.

Identification of ten KB425796-A congeners from Paenibacillus sp. 530603 using an antifungal assay against Aspergillus fumigatus in combination with micafungin.

The discovery and characterization of natural congeners is one approach for understanding the relationship between chemical structure and biological function. We recently isolated the novel antifungal metabolite KB425796-A produced by the recently isolated bacterium Paenibacillus sp. 530603. On the basis of morphological changes of Aspergillus fumigatus induced by KB425796-A in combination with micafungin, we developed a highly sensitive screening method for the specific detection of KB425796-A congeners. Using this method, we isolated ten congeners of KB425796-A, named KB425796-B, -C, -D, -E, -F, -G, -H, -I, -J and -K, which exhibited diverse antifungal potencies against A. fumigatus. One of the most potent congeners, KB425796-C, had antifungal activities against several micafungin-resistant infectious fungi. KB425796-C can be a potential drug candidate for treating micafungin-resistant fungal infections.

KB425796-A, a novel antifungal antibiotic produced by Paenibacillus sp. 530603.

The novel antifungal macrocyclic lipopeptidolactone, KB425796-A (1), was isolated from the fermentation broth of bacterial strain 530603, which was identified as a new Paenibacillus species based on morphological and physiological characteristics, and 16S rRNA sequences. KB425796-A (1) was isolated as white powder by solvent extraction, HP-20 and ODS-B column chromatography, and lyophilization, and was determined to have the molecular formula C79H115N19O18. KB425796-A (1) showed antifungal activities against Aspergillus fumigatus and the micafungin-resistant infectious fungi Trichosporon asahii, Rhizopus oryzae, Pseudallescheria boydii and Cryptococcus neoformans.

Surgical treatment of facial fracture by using unsintered hydroxyapatite particles/poly l-lactide composite device (OSTEOTRANS MX(®)): a clinical study on 17 cases.

BACKGROUND: In Japan we currently use absorption properties for facial fractures. OSTEOTRANS MX(®) (Takiron co., ltd, Japan) is an absorption device, which is called Super FIXSORB MX(®) in Japan. This absorbable osteosynthetic device constitutes unsintered hydroxyapatite particles/poly l-lactide (u-HA/PLLA) composites. This study focuses on reporting clinical cases of using OSTEOTRANS MX(®). MATERIALS AND METHODS: Seventeen patients (16 men and 1 woman) aged 10-80 years (mean: 39.9 years, SD: ±20.7) with 86 fracture sites were treated. In all cases we used 1.0 mm plates and 5 mm or 7 mm screws. The postoperative observation period was 6-60 months (mean: 21.8 months, SD: ±14.5). RESULTS: The fracture site recovered in all cases. Complications included one bone excess on the forehead and one foreign-body reaction on the frontozygomatic suture, but the fracture sites were recovered and had no problems. In the case with the longest observation time 60 months, the plate was almost fully absorbed. However, in other cases the plate was not fully absorbed because of a shorter observation time. CONCLUSION: OSTEOTRANS MX(®) is a useful device because of its suitable intensity, thinness, radiopaque, and few complications. A longer observation time is required for a plate to be absorbed completely.