Crotofolane-Type Diterpenoids: Crotocascarins R-V, Rearranged Trinorcrotofolane: Crotocascarin δ and a Phorbol Derivative from the Leaves of Croton cascarilloides.

Five new crotofolanes, named crotocascarins R-V (1-5), one rearranged trinorcrotofolane, crotocascarin δ, and one phorbol derivative were isolated from the EtOAc-soluble fraction of the MeOH extract of the leaves of Croton cascarilloides. Crotocascarins R (1), T (3), and U (4) possessed isobutyric acid as an acyl moiety and crotocascarin B (2) an acetyl group, whereas crotocascarin V (5) was elucidated to be a hydroxylated compound of crotocascarin K at the 9-position. Crotocascarin δ (6) was a trinor rearranged crotofolane with a tertiary hemiketal functional group at the 8-position. The absolute configuration of the 8-position was determined by the comparison of the experimental electronic circular dichroism (ECD) spectrum and calculated ECD spectra. Compound 7 was a phorbol ester derivative with a peroxide functional group. The fatty acid attached at the 12-position was found to be a single species-i.e., lauric acid (C-12)-from the evidence of the mass spectral data.

Omphalines A-E: ent-Rosane-Type Diterpenoids from the Madagascar Endemic Plant, Omphalea oppositifolia.

From the less polar fraction of the MeOH extract of the leaves and twigs of Omphalea oppositifolia, five new ent-rosane-type diterpenoids, named omphalines A-E (1-5), were isolated together with one known compound, 7-keto-ent-kaurane-16ß,17-diol (6), by a combination of various kinds of chromatography. The structure of omphaline A (1) was elucidated to be 19-nor-ent-rosane-4,15-diene-2ß,6α-diol-3-one. Omphalines B (2), C (3), D (4), and E (5) possessed two double bonds at 5- and 15-positions, and hydroxy functional groups at 3ß-, 2α,3α-, 2α,3ß-, and 2α,19-positions, respectively. The absolute configuration of 1 was determined by the comparison of the experimental electronic circular dichroism (ECD) spectrum and calculated ECD spectra.

[Surgical Strategy for Multiple Synchronous Primary Lung Cancer Showing Ground Glass Opacities:Percutaneous Preoperative Computed Tomography-guided Lipiodol Marking Followed by Video-assisted Thoracic Surgery].

From May 2006 to March 2018, a total of 30 patients with multiple ground glass opacities( GGO) lessor equal to 2.0 cm underwent percutaneous preoperative computed tomography (CT)-guided Lipiodolmarking and subsequent video-assisted thoracic surgery( VATS). There were 22 patients with 2 GGOs,6 patients with 3 GGOs, 1 patient with 4 GGOs, and 1 patient with 5 GGOs. Of the 71 CT-guided Lipiodolmarking procedures were successfully completed. All 71 Lipiodol spots were detected with C-armshapedfluoroscopy and completely resected. Pneumothorax was the most common complication andoccurred in 22 (73.3%) of 30 cases. There were severe complications in two (6.7%) cases of pneumothoraxrequiring chest tube placement and three( 10.0%) cases of bloody sputum. Of 30 cases multipleGGOs revealed 24 synchronous lung cancer (19 patients with double lung cancer, 3 patients with triplelung cancer, 1 patient with quadruple lung cancer, and 1 patient with quintuple lung cancer). PreoperativeCT-guided lipiodol marking and VATS resection is a safe and reliable method for synchronous multiplelung cancer showing GGO.

Ebenamariosides A-D: Triterpene Glucosides and Megastigmanes from the Leaves of Diospyros maritima.

The 1-BuOH-soluble fraction of the methanol (MeOH) extract of Diospyros maritima was separated by chromatographic techniques to give three new oleanane-type and one new ursane-type triterpene glucoside, named ebenamariosides A-D (1-4); two megastigmanes were also isolated. The structures of triterpene glucosides was elucidated with extensive investigation by one and two dimensional NMR spectroscopy and the structures were confirmed by partial enzymatic hydrolyses to give the corresponding mono-glucosides and aglycones. The structures of the megastigmanes, including their absolute stereochemistries, were elucidated by spectroscopic evidence and by the modified Mosher's method. Two megastigmanes were chemically correlated and their absolute structures were unambiguously determined. The cytotoxicity of the triterpene glucosides and their degradation products were assayed. They did not show any significant activity.

Molecular cloning and characterization of vanillin dehydrogenase from Streptomyces sp. NL15-2K.

BACKGROUND: Streptomyces sp. NL15-2K, previously isolated from the forest soil, features an extensive catabolic network for lignin-derived aromatic compounds, including pathways transforming ferulic acid to vanillin, vanillic acid, and protocatechuic acid. To successfully use Streptomyces sp. NL15-2K as a biocatalyst for vanillin production, it is necessary to characterize the vanillin dehydrogenase (VDH) that degrades the produced vanillin to vanillic acid, as well as the gene encoding this enzyme. Here, we cloned the VDH-encoding gene (vdh) from strain NL15-2K and comprehensively characterized its gene product. RESULTS: The vdh open reading frame contains 1488 bp and encodes a 496-amino-acid protein with a calculated molecular mass of 51,705 Da. Whereas the apparent native molecular mass of recombinant VDH was estimated to be 214 kDa by gel filtration analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a subunit molecular mass of ca. 56 kDa, indicating that VDH is a homotetramer. The recombinant enzyme showed optimal activity at 45 °C and pH 9.5. The VDH substrate specificity followed this order: vanillin (100%) > protocatechualdehyde (91%) > benzaldehyde (79%) > p-hydroxybenzaldehyde (56%) > isovanillin (49%) ≈ salicylaldehyde (48%) > anisaldehyde (15%) ≈ veratraldehyde (12%). Although peptide mass fingerprinting and BLAST searches indicated that this enzyme is a salicylaldehyde dehydrogenase (SALDH), the determined kinetic parameters clearly demonstrated that the enzyme is a vanillin dehydrogenase. Lastly, phylogenetic analysis revealed that VDH from Streptomyces sp. NL15-2K forms an independent branch in the phylogenetic tree and, hence, is evolutionarily distinct from other VDHs and SALDHs whose activities have been confirmed experimentally. CONCLUSIONS: Our findings not only enhance the understanding of the enzymatic properties of VDH and the characteristics of its amino acid sequence, but also contribute to the development of Streptomyces sp. NL15-2K into a biocatalyst for the biotransformation of ferulic acid to vanillin.

Eight ent-Kaurane Diterpenoid Glycosides Named Diosmariosides A-H from the Leaves of Diospyros maritima and Their Cytotoxic Activity.

From the leaves of Diospyros maritima, collected from Okinawa Island, eight new glycosides based on ent-kaurane-type diterpenoids, entitled diosmariosides A-H, were isolated. The absolute structure of diosmarioside E (5) was determined by X-ray crystallographic analysis. The structure of diosmarioside H was elucidated to be a dimeric compound between diosmarioside A and a sugeroside through a ketal bond. An assay of cytotoxicity towards the lung adenocarcinoma (A549) cell line was performed. Among the compounds isolated, only diosmarioside D (4) and sugeroside 9 showed strong activity. The anti-microbial activity toward multi-drug resistant strains was also determined, but no activity was observed.

Expression and substrate range of Streptomyces vanillate demethylase.

Vanillate is converted to protocatechuate by an O-demethylase consisting of VanA and VanB in Streptomyces sp. NL15-2K. In this study, vanillate demethylase from this strain was functionally expressed in Escherichia coli, and its substrate range for vanillate analogs was determined by an in vivo assay using recombinant whole cells. Among aromatic methyl ethers, vanillate, syringate, m-anisate, and veratrate were good substrates, whereas ferulate, vanillin, and guaiacol were not recognized by Streptomyces vanillate demethylase. After vanillate, 4-hydroxy-3-methylbenzoate was a better substrate than m-anisate and veratrate, and the 3-methyl group was efficiently oxidized to a hydroxymethyl group. These observations suggest that the combination of a carboxyl group on the benzene ring and a hydroxyl group in the para-position relative to the carboxyl group may be preferable for substrate recognition by the enzyme. (1)H-NMR analysis showed that the demethylation product of veratrate was isovanillate rather than vanillate. Therefore, it was concluded that O-demethylation of veratrate by Streptomyces vanillate demethylase occurred only at the meta-position relative to the carboxyl group.

Molecular cloning and expression of the Streptomyces coniferyl alcohol dehydrogenase gene in Escherichia coli.

Coniferyl alcohol dehydrogenase (CADH) is a key enzyme in catabolism of lignin-related aromatic compounds in bacteria. In Streptomyces sp. NL15-2K, CADH is a tetramer of identical subunits with an individual molecular mass of 39 kDa. This work describes the cloning and sequencing of the CADH gene from Streptomyces sp. NL15-2K, optimization of a protocol for high-level active CADH expression, and purification of recombinant CADH. A BLAST search and motif analyses of the predicted CADH amino acid sequence indicated the enzyme belongs to the medium-chain zinc-dependent alcohol dehydrogenase group. Cell density at heat-shock treatment, temperatures for heat shock and culture, duration of heat shock, concentration of isopropyl-ß-d-thiogalactopyranoside (IPTG) as an inducer, and culture time after induction were adjusted for optimal CADH expression. Expression of active CADH under optimized conditions was approximately 4-fold higher than in the absence of heat shock. CADH purified from recombinant Escherichia coli was in the tetrameric form, as was natural CADH from NL15-2K.

Clinical significance of spread of lipiodol marking for pulmonary nodules and its influence on surgery.

OBJECTIVE: Computed tomography (CT)-guided lipiodol marking is one of the targeting methods for resecting small pulmonary nodules or ground-glass nodules in video-assisted thoracic surgery (VATS). However, lipiodol spreading during marking has not been assessed, practically. In this study, we examined the clinical significance and the influence of lipiodol spreading on surgery. METHODS: From April 2010 to March 2021, 176 pulmonary nodules in 167 patients were marked with lipiodol under CT guided before VATS. The marking images after lipiodol injection were classified into "Spread" and "non-Spread." Lung resection was sequentially performed on the same day. RESULTS: All target nodules were successfully resected in VATS. In the classification of marking images, Spread was 32 (18%), non-Spread was 144 (82%). There was a significant difference in duration of surgery (mean; 138.7 min vs. 118.3 min, p = 0.0496) and amount of bleeding (mean; 32.7 g vs. 11.2 g, p = 0.0173). Provided that limited to the data of wedge resections without intrathoracic pleural adhesion (n = 117), there was no significant difference in duration of surgery (mean; 104.8 min vs. 99.6 min, p = 0.48), amount of bleeding (mean; 4.9 g vs. 5.3 g, p = 0.58). In the multivariate logistic regression analysis, the risk factor of lipiodol spreading was intrathoracic pleural adhesion (odds ratio: 3.16, 95% confidence interval: 1.12-8.97, p = 0.03). There was no relationship between marking image and complication rate. CONCLUSIONS: Lipiodol spreading did not directly influence surgery and complication. However, it was a sign of intrathoracic pleural adhesion, which could lead to increased duration of surgery and amount of bleeding.

Characterization of two isozymes of coniferyl alcohol dehydrogenase from Streptomyces sp. NL15-2K.

We purified two isozymes of coniferyl alcohol dehydrogenase (CADH I and II) to homogeneity from cell-free extracts of Streptomyces sp. NL15-2K. The apparent molecular masses of CADH I and II were determined to be 143 kDa and 151 kDa respectively by gel filtration, whereas their subunit molecular masses were determined to be 35,782.2 Da and 37,597.7 Da respectively by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Thus, it is probable that both isozymes are tetramers. The optimum pH and temperature for coniferyl alcohol dehydrogenase activity were pH 9.5 and 45 °C for CADH I and pH 8.5 and 40 °C for CADH II. CADH I oxidized various aromatic alcohols and allyl alcohol, and was most efficient on cinnamyl alcohol, whereas CADH II exhibited high substrate specificity for coniferyl alcohol, and showed no activity as to the other alcohols, except for cinnamyl alcohol and 3-(4-hydroxy-3-methoxyphenyl)-1-propanol. In the presence of NADH, CADH I and II reduced cinnamaldehyde and coniferyl aldehyde respectively to the corresponding alcohols.

Ardisiatetrons A and B: tetronic acid derivatives and triterpenes from the leaves of Ardisia quinquegona and their biological activity.

From the leaves of Ardisia quinquegona, two alkylated tetronic acid derivatives, named ardisiatetrons A and B (1, 2), and four triterpenoids (3-6) were isolated together with one known compound (7) by a combination of various kinds of chromatography. The structure of new methyl migrated triterpene (3) was confirmed by X-ray crystallographic analysis. Compounds 2, 3, and 7 showed moderate anti-Leishmania activity and cytotoxicity towards A549 cells.

Safety and reliability of computed tomography-guided lipiodol marking for undetectable pulmonary lesions.

OBJECTIVES: This study aimed to evaluate the safety and reliability of percutaneous computed tomography (CT)-guided lipiodol marking for undetectable pulmonary lesions before video-assisted thoracic surgery (VATS). METHODS: We retrospectively analysed the cases of CT-guided lipiodol marking followed by VATS in 9 institutes from May 2006 to March 2018. Lipiodol (0.2-0.5 ml) was percutaneously injected closely adjacent to undetectable pulmonary lesions with computed-tomography guidance. Lipiodol spots were identified using C-arm-shaped fluoroscopy during VATS. We grasped the lipiodol spots, including the target lesions, with ring-shaped forceps and resected them. RESULTS: Of 1182 lesions, 1181 (99.9%) were successfully marked. In 1 case, the injected lipiodol diffused, and no spot was created. Of the 1181 lesions, 1179 (99.8%) were successfully resected with intraoperative fluoroscopy. Two lipiodol spots were not detected because of the lipiodol distribution during the division of pleural adhesions. The mean lesion size was 9.1 mm (range 1-48 mm). The mean distance from the pleural surface was 10.2 mm (range 0-43 mm). Lipiodol marking-induced pneumothorax occurred in 495 (57.1%) of 867 cases. Of these, chest drainage was required in 59 patients (6.8%). The other complications were 19 (2.2%) cases of bloody sputum, 3 (0.35%) cases of intravascular air, 1 (0.12%) case of pneumonia and 1 (0.12%) case of cerebral infarction. There were no lipiodol marking-induced deaths or sequelae. CONCLUSIONS: Preoperative CT-guided lipiodol marking followed by VATS resection was shown to be a safe and reliable procedure with a high success rate and acceptably low severe complication rate.

Draft Genome Sequence of Streptomyces sp. Strain NL15-2K, a Degrader of Lignin-Derived Aromatic Compounds, Isolated from Forest Soil.

Streptomyces sp. strain NL15-2K is a degrader of lignin-derived aromatic compounds and was isolated from a forest soil sample. Here, we report the draft genome sequence of this strain and its annotation. This genome of 12,072,023 bp exhibits a GC content of 70.32% and encodes 10,874 predicted proteins and 75 RNAs.

Molecular cloning and characterization of gene encoding novel puromycin-inactivating enzyme from blasticidin S-producing Streptomyces morookaensis.

Puromycin (PM) is classified into a family of nucleoside antibiotics together with blasticidin S (BS). PM-producing Streptomyces alboniger is known to express a PM-inactivating enzyme as a self-resistance determinant, which catalyzes the acetylation of PM. We have shown that, although BS-producing Streptomyces morookaensis also produces a PM-inactivating enzyme, it catalyzes the hydrolysis of an amide linkage between the aminonucleoside and O-methyl-L-tyrosine moiety of PM. In the present study, we cloned and characterized a gene encoding PM hydrolase (PMH) from BS-producing S. morookaensis JCM4673. The nucleotide sequence analysis suggests that an open reading frame consisting of 1986 bp is a gene for PMH and encodes a protein consisting of 662 amino acids with a calculated molecular mass of 71,260 Da. The molecular mass of the recombinant PMH, which was produced using an Escherichia coli host-vector system, was the same as that of PMH purified from the JCM4673 strain. Our biochemical study of the recombinant PMH confirmed that the enzyme is an aminopeptidase with broad substrate specificity. The putative primary structure of PMH contains a Gly-X-Ser-X-Gly motif, which is commonly observed among serine proteases. In addition, the amino acid sequence of PMH displays a high similarity to that of the Streptomyces acyl-peptide hydrolase (ACPH), which is a member of the prolyl oligopeptidase (POP) family of serine proteases. Furthermore, the catalytic triad (Ser-Asp-His), which is observed in the POP family, is also present in the primary structure of PMH. These results suggest that PMH is an aminopeptidase classified into the POP family.

Isolation and characterization of Streptomyces sp. NL15-2K capable of degrading lignin-related aromatic compounds.

Strain NL15-2K was isolated from soil by screening for bacteria capable of catabolizing lignin-related aromatic acids. This isolate was identified as a Streptomyces sp. on the basis of morphology and an analysis of its 16S rRNA gene sequence. NL15-2K utilized caffeic acid, coniferyl alcohol, ferulic acid, isovanillic acid, protocatechuic acid, vanillic acid, vanillin, and veratric acid as sole carbon sources.

Malignant transformation of alveolar adenoma to papillary adenocarcinoma: a case report.

We present a case involving alveolar adenoma (AA) comprising a malignant component. An 83-year-old man was referred with a solitary pulmonary nodule, which was diagnosed as adenocarcinoma (AC) by transbronchial biopsy. Resected specimen after left S1+2 segmentectomy revealed that the tumor comprised two histologically distinct areas-a central multi-cystic area (AA) and a surrounding area (papillary AC). Careful examination clarified that the borderline lesion showed intermediate mindbomb homolog-1 index and cytological atypia with low papillary growth, suggesting the possibility of malignant transformation of the AA. The malignant potential and coexisting lung carcinoma should be considered in evaluating the tumor diagnosed as AA.

Frequent hemizygous deletion at 1p36 and hypermethylation downregulate RUNX3 expression in human lung cancer cell lines.

Runt-related transcription factor 3 (RUNX3) has been recognized as a tumor suppressor gene in gastric cancer because its expression level was reduced or disappeared due to epigenetic changes. To evaluate the usefulness of the RUNX3 gene as a biomarker of lung cancer, we have analyzed the expression of the RUNX3 gene in 15 lung cancer cell lines by real-time reverse transcription-polymerase chain reaction (RT-PCR), and demonstrated that RUNX3 gene expression was reduced or disappeared in all cell lines examined (100%). In addition, we have attempted to classify all the cell lines into three groups according to the expression level; less than 10% (group I), 10-30% (group II) and approximately 50% (group III). We further investigated methylation status of the CpG sites in the exon 1 region of RUNX3 by methylation specific PCR (MSP), and studied the correlation between the expression level and hemizygous deletion as revealed by bicolor fluorescence in situ hybridization (FISH). The CpG sites were hypermethylated in 8 cell lines (53%) and the RUNX3 loci were hemizygously deleted in another 8 cell lines (53%). Furthermore group I, II, and III corresponded well to methylation-positive cell lines, cell lines showing hemizygous deletion, and the rest of cell lines without methylation or hemizygous deletion, respectively. These results suggest that a comprehensive study on RUNX3 using real-time RT-PCR, MSP, and FISH could be beneficial in understanding the pathogenetic mechanisms of human lung cancer at the molecular level.

Improvements in thoracic surgery outcomes: a multi-institutional collaboration study.

BACKGROUND: Treatment protocols (including those for thoracic surgery) tend to be customized for individual hospitals. Procedural standardization is required to improve surgical tasks and patient outcomes. This study aimed to evaluate the effects of an initiative to standardize surgical tasks for efficient and safe performance. METHODS: Hospitals associated with the Division of Chest Surgery of the Kyoto Prefectural University of Medicine held joint meetings involving their thoracic surgeons and operating room nurses between February 2011 and November 2012 to standardize surgical tasks. Operation times and blood loss were compared before and after standardization. RESULTS: The implementation rate of standardized surgical tasks was 97%. The pre-operative (from entry to the operating room until commencement of surgery) and post-operative (from conclusion of surgery until departure from the operating room) times were significantly decreased after the standardization. When compared according to operative group (all thoracic surgery, lung lobectomy, and partial lung resection), operation times were shorter for all three groups; in addition, the amount of blood loss was lower in all three groups after standardization. A post-standardization survey showed improved morale among the meeting participants. CONCLUSIONS: Interdisciplinary standardization of surgical tasks across institutions improved thoracic surgery tasks and surgical outcomes.