Recent progress on heterologous protein production in methylotrophic yeast systems.

Recombinant protein production technology is widely applied to the manufacture of biologics used as drug substances and industrial proteins such as recombinant enzymes and bioactive proteins. Various heterologous protein production systems have been developed using prokaryotic and eukaryotic hosts. Especially methylotrophic yeast in eukaryotic hosts is suggested to be particularly valuable because such systems have the following advantages: protein secretion into culture broth, eukaryotic quality control systems, a post-translational modification system, rapid growth, and established recombinant DNA tools and technologies such as strong promoters, effective selection markers, and gene knock-in and -out systems. Many methylotrophic yeasts such as the genera Candida, Ogataea, and Komagataella have been studied since methylotrophic yeast was first isolated in 1969. The methanol-consumption-related genes in methylotrophic yeast are strongly and strictly regulated under methanol-containing conditions. The well-regulated gene expression systems under the methanol-inducible gene promoter lead to the potential application of heterologous protein production in methylotrophic yeast. In this review, we describe the recent progress of heterologous protein production technology in methylotrophic yeast and introduce Ogataea minuta as an alternative production host as a substitute for K. phaffii and O. polymorpha.

Structure and Stereochemistry of Amphidinolide N Congeners from Marine Dinoflagellate Amphidinium Species.

Two highly potent cytotoxic 26-membered macrolides, isocaribenolide-I (1) and a chlorohydrin 2, together with known amphidinolide N (3), have been isolated from a free-swimming dinoflagellate Amphidinium species (KCA09053 and KCA09056 strains) collected off Iriomote Island, Japan. The structures of 1 and 2 were determined to be a congener of 3 with an isobutyl terminus and the chlorohydrin form of 3, respectively, by detailed analyses of spectroscopic data. The relative stereochemistries of 1 and 2 were elucidated by the conformational analyses based on NMR data.

Iriomoteolides-14a and 14b, New Cytotoxic 15-Membered Macrolides from Marine Dinoflagellate Amphidinium Species.

Two new macrolides, iriomoteolides-14a (1) and 14b (2) have been isolated from the marine dinoflagellate Amphidinium species (strain KCA09057). Compounds 1 and 2 are 15-membered macrolides, which are structural analogs of amphidinolides O (3) and P (4). The structures of 1 and 2 were assigned on the basis of detailed NMR analyses and chemical conversion studies. Compounds 1 and 2 showed moderate cytotoxic activity against human cervix adenocarcinoma HeLa cells.

Total Synthesis, Stereochemical Revision, and Biological Assessment of Iriomoteolide-2a.

Iriomoteolide-2a is a marine macrolide metabolite isolated from a cultured broth of the benthic dinoflagellate Amphidinium sp. HYA024 strain. This naturally occurring substance was reported to show remarkable cytotoxic activity against human cancer cell lines HeLa and DG-75 and in vivo antitumor activity against murine leukemia P388 cell line. Herein, the total synthesis, stereochemical revision, and biological assessment of iriomoteolide-2a are reported in detail. Total synthesis of the proposed structure 1 of iriomoteolide-2a featured a late-stage convergent assembly of three components by a Suzuki-Miyaura coupling, an esterification, and a ring-closing metathesis. However, the NMR data of synthetic 1 were not identical to those of the natural product. Careful analysis of the NMR data of the authentic material and synthesis/NMR analysis of appropriately designed model compounds led to consideration of four possible stereoisomers 2-5 as candidates for the correct structure. Accordingly, total syntheses of 2-5 were achieved by taking advantage of the convergent strategy, and comparison of the NMR spectra of synthetic 2-5 with those of the natural product led to the conclusion that 5 shows the correct relative configuration of iriomoteolide-2a. The absolute configuration of this natural product was finally established through chiral HPLC analysis of synthetic 5/ent-5 with the authentic sample. The antiproliferative activity of the synthetic compounds was assessed against HeLa and A549 cells to show that, in contrast to expectation, synthetic 5 and ent-5 were only marginally active in these cell lines. This work clearly underscores the vital role of total synthesis in the establishment of the structure and biological activity of natural products.

Total Synthesis and Stereochemical Revision of Iriomoteolide-2a.

Total syntheses of the proposed and correct structures of iriomoteolide-2a, a cytotoxic marine macrolide natural product with an unusual 23-membered macrolactone skeleton, have been accomplished for the first time. The synthesis of the correct structure involves an asymmetric epoxidation/diepoxide cyclization cascade for the construction of the bis(tetrahydrofuran) moiety, a Suzuki-Miyaura coupling for the fragment assembly, and a ring-closing metathesis for the closure of the macrocyclic backbone. In addition, the original stereochemical assignment of iriomoteolide-2a was revised.

Stereodivergent Synthesis and Configurational Assignment of the C1-C15 Segment of Amphirionin-5.

The relative configuration of the C3-C12 portion of amphirionin-5, a novel marine polyketide with potent cell proliferation-promoting activity, was established by the stereodivergent synthesis of six diastereomeric model compounds and comparison of their NMR spectroscopic data with those reported for the natural product. This study led to the elucidation of the relative configuration between C4/C5 and C9/C12 and to the reassignment of the proposed configuration of the C9 position of amphirionin-5.

Iriomoteolides-10a and 12a, Cytotoxic Macrolides from Marine Dinoflagellate Amphidinium Species.

Two new macrolides, iriomoteolides-10a (1) and -12a (2), have been isolated from a marine dinoflagellate Amphidinium sp. (KCA09053 strain), and their structures were elucidated on the basis of a detailed two dimensional (2D)-NMR analysis. Compound 1 is a novel 21-membered Amphidinium macrolide, which contains one tetrahydrofuran ring, two ketone carbonyls, two hydroxyl groups, and six one-carbon branches. Compound 2 is a new 12-membered macrolide related to amphidinolide Q. Compound 1 exhibited cytotoxic activity against human cervix adenocarcinoma HeLa and murine hepatocellular carcinoma MH134 cells.

Amphirionin-2, a novel linear polyketide with potent cytotoxic activity from a marine dinoflagellate Amphidinium species.

A novel linear polyketide, amphirionin-2 (1), with two unique hexahydrofuro[3,2-b]furan moieties has been isolated from the cultivated algal cells of a benthic dinoflagellate Amphidinium sp. (strain KCA09051). The structure was elucidated on the basis of detailed analyses of 2D NMR data, and the absolute configuration of C-5 was determined by using modified Mosher's method. Amphirionin-2 (1) exhibited potent cytotoxic activity against human colon carcinoma Caco-2 cells and human lung adenocarcinoma A549 cells.

Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum.

Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and ß-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

Direct and stereospecific interaction of amphidinol 3 with sterol in lipid bilayers.

Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. Although AM3 permeabilizes phospholipid membranes only in the presence of sterol, the detailed molecular basis by which AM3 recognizes sterols in membranes remains unknown. Here, we investigated the molecular interaction between sterols and AM3 in membranes from the viewpoint of stereospecific molecular recognition using ergosterol, cholesterol, and epicholesterol, which is the 3-OH epimer of cholesterol. Dye leakage assays, surface plasmon resonance experiments, (2)H and (31)P NMR measurements, and microscopic observations revealed that AM3 directly interacts with membrane sterols through the strict molecular recognition of the stereochemistry of the sterol 3-OH group. The direct interaction enhances the membrane binding efficiency of AM3, which subsequently permeabilizes membranes without altering membrane integrity.

Salinispora arenicola from temperate marine sediments: new intra-species variations and atypical distribution of secondary metabolic genes.

The obligate marine actinobacterium Salinispora arenicola was successfully cultured from temperate sediments of the Pacific Ocean (Tosa Bay, offshore Kochi Prefecture, Japan) with the highest latitude of 33°N ever reported for this genus. Based on 16S rRNA gene sequence analysis, the Tosa Bay strains are of the same phylotype as the type strain S. arenicola NBRC105043. However, sequence analysis of their 16S-23S rRNA intergenic spacer (ITS) revealed novel sequence variations. In total, five new ITS sequences were discovered and further phylogenetic analyses using gyrase B and rifamycin ketosynthase (KS) domain sequences supported the phylogenetic diversity of the novel Salinispora isolates. Screening of secondary metabolite genes in these strains revealed the presence of KS1 domain sequences previously reported in S. arenicola strains isolated from the Sea of Cortez, the Bahamas and the Red Sea. Moreover, salinosporamide biosynthetic genes, which are highly homologous to those of Bahamas-endemic S. tropica, were detected in several Tosa Bay isolates, making this report the first detection of salinosporamide genes in S. arenicola. The results of this study provide evidence of a much wider geographical distribution and secondary metabolism diversity in this genus than previously projected.

Observation of glycolytic metabolites in tumor cell lysate by using hyperpolarization of deuterated glucose.

Hyperpolarization of stable isotope-labeled substrates and subsequent NMR measurement of the metabolic reactions allow for direct tracking of cellular reactions in vitro and in vivo. Here, we report the hyperpolarization of (13)C6-glucose-d7 and evaluate its use as probes to observe glucose flux in cells. We measured the lifetime of the polarized signal governed by the spin-lattice relaxation time T1. (13)C6-Glucose-d7 exhibited a T1 that was over ten times as long as that of (13)C6-glucose, and metabolic NMR studies of hyperpolarized (13)C6-glucose-d7 using tumor cell lysate led to observation of the resonances due to phosphorylated fluctofuranoses generated through aerobic glycolysis.

Selection of the methylotrophic yeast Ogataea minuta as a high-producing host for heterologous protein expression.

Three Ogataea minuta var. minuta strains have been deposited as NBRC 0975, NBRC 10402, and NBRC 10746 in the National Institute of Technology and Evaluation (NITE) Biological Resource Center (NBRC) collection. We investigated the ability to produce secretory proteins and several genotypic and phenotypic characteristics in order to select the best strain for heterologous protein expression. NBRC 10746 showed the best performance as evaluated by Cypridina noctiluca luciferase expression. Subsequently, clone #5-30 named tat06213, which was obtained by single-colony isolation from NBRC 10746, was established as a promising host for heterologous protein expression. To deepen our understanding of the characteristics of O.minuta var. minuta strains, sequence analysis of the D1/D2 domain of large subunit rRNA was conducted and the resulting phylogenetic tree derived from the D1/D2 domain showed that NBRC 10402 and NBRC 10746 were grouped into a different cluster far from NBRC 0975. Furthermore, a chromosome structure topology with electrophoretic karyotype and AOX1 loci analyzed by pulsed-field gel electrophoresis with Southern blotting showed different chromosome patterns and AOX1-hybridization loci among the strains. Additionally, the sequences of the promoter regions of the cloned AOX1 genes were not identical among the three strains. These findings might explain the differences in heterologous protein expression among the tested O. minuta var. minuta strains.

Cardiac output obtained from test bolus injections as a factor in contrast injection rate revision of following coronary CT angiography.

BACKGROUND: Optimal contrast enhancement is crucial for the detection of coronary artery stenoses and atherosclerotic changes in coronary CT angiography (CTA). PURPOSE: To demonstrate the feasibility of using the cardiac output (CO) obtained from the test bolus injection data-set (COtest) as a factor in contrast injection rate revision of the following coronary CTA. MATERIAL AND METHODS: The test bolus injection data-sets of 52 consecutive coronary CTAs were examined. COtest was calculated from the test bolus data-set. Aortic peak enhancement (APE) was measured on the following coronary CTA. We simulated the APE at a fixed contrast injection rate of 4 mL/s (simAPE) in each patient. RESULTS: The ranges of COtest and simAPE were 2.82-7.56 L/min and 194-527 Hounsfield Units, respectively. There was a significant negative correlation (R = -0.802, P < 0.001) between simAPE and COtest. CONCLUSION: COtest can be used for injection rate revision on coronary CTA.

Intraductal papillary mucininous neoplasm of the bile ducts: multimodality assessment with pathologic correlation.

Mucin-producing intraductal papillary neoplasm (adenocarcinoma/adenoma) in the bile duct is becoming recognized as a specific type of neoplasm. Since, it bears a striking similarity to intraductal papillary mucinous neoplasms of the pancreas with regard to its histopathologic features, the term "intraductal papillary mucinous neoplasms of the bile duct" (IPMN-B) is frequently used, although no definite terminology or definition has been decided by World Health Organization. This neoplasm lacks ovarian-like stroma and communicates with the bile ducts, unlike biliary mucinous cystic neoplasm (MCN). On the other hand, malignant IPMN-B is categorized as an intraductal-growth type of intrahepatic cholangiocarcinoma (ICC). In comparison to other types of ICC, such as the mass-forming type and periductal-infiltrating type that have poor resectability and an unfavorable prognosis, malignant IPMN-B can be resected and demonstrates a more favorable prognosis. Meanwhile, unlike biliary MCN that is usually confined in a closed cyst, IPMN-B can spread along the mucosal surface of the bile ducts, and it should be widely resected. Therefore, multimodality assessment is needed to ensure the correct diagnosis of IPMN-B. We herein review the imaging findings of IPMN-B with pathologic correlation.

Total synthesis and complete configurational assignment of amphirionin-2.

Amphirionin-2 is a linear polyketide metabolite that exhibits potent and selective cytotoxic activity against certain human cancer cell lines. We disclose herein the first total synthesis of amphirionin-2 and determination of its absolute configuration. Our synthesis featured an extensive use of cobalt-catalyzed Mukaiyama-type cyclization of γ-hydroxy olefins for stereoselective formation of all the tetrahydrofuran rings found in the natural product, and a late-stage Stille-type coupling for convergent assembly of the entire carbon backbone. Four candidate diastereomers of amphirionin-2 were synthesized in a unified, convergent manner, and their spectroscopic/chromatographic properties were compared with those of the authentic material. The present study culminated in the reassignment of the C5/C7 relative configuration, assignment of the C12/C18 relative configuration, and determination of the absolute configuration of amphirionin-2.

Treatment of in-stent restenosis with beraprost sodium: an experimental study of short- and intermediate-term effects in dogs.

1. The aim of the present study was to evaluate the inhibitory effects of the short-term administration of beraprost sodium, a stable prostaglandin I(2) analogue, on neointimal thickening after stenting. 2. To examine the immediate and short-term effects, Z-stents were placed in the iliac veins of 12 dogs, which were randomly assigned to either a beraprost-treated or control (saline) group. Beraprost (0.35 microg/kg per min) or saline (1.5 mL/min) was administered 30 min before stenting and was continued for 5 h thereafter. Platelet aggregation was measured before and after drug administration. At 3, 7 and 14 days after stenting, dogs were killed and immunohistochemical staining for proliferating cell nuclear antigen was used to quantify the proliferation of vascular smooth muscle cells (SMC). To evaluate intermediate-term effects, a Z-stent was placed in the right iliac vein in 10 dogs, followed by beraprost treatment. Three days later, a second Z-stent was placed contralaterally with saline infusion as a control. After 4 weeks, dogs were killed and neointimal thickness was measured under a light microscope to calculate the intima : media area ratio. 3. Platelet aggregation was more significantly suppressed in the beraprost-treated than in the control group (P = 0.01). In addition, SMC proliferation was significantly lower in the beraprost-treated group 7 and 14 days after stenting (P < 0.05). Over the intermediate term, the intima : media area ratio was significantly lower in the beraprost-treated vein compared with control (P < 0.05). 4. In conclusion, short-term beraprost treatment during stenting suppresses in situ platelet aggregation and SMC proliferation, thus reducing neointimal thickening.

Correct diagnosis of vascular encasement and longitudinal extension of hilar cholangiocarcinoma by four-channel multidetector-row computed tomography.

Accurate diagnosis of local invasion of hilar cholangiocarcinomas is challenging due to their small size and the anatomic complexity of the hepatic hilar region. On the other hand, the correct diagnosis of local invasion is essential for assuring the possibility of curative surgery. The purpose of this study was to evaluate the feasibility of four-channel multidetector-row computed tomography (MDCT) in the assessment of vascular and bile duct involvement, by which we could obtain useful information for the surgical management of hilar cholangiocarcinoma. The subjects were 18 patients for whom the extent of tumor invasion was surgically and pathologically confirmed. All patients underwent preoperative multiphasic CT scanning by MDCT. Arterial and portal dominant phases were acquired using a detector configuration of 1.25 mm X 4 mm, and both axial and multiplanar reconstructed images were interpreted. Longitudinal extension was evaluated up to second-order branches. Vascular invasion is considered to be the degree of tumor contiguity to the hepatic arteries and portal vein and was graded by CT. The longitudinal extension was correctly diagnosed in 14 patients (77.8%). Hepatic artery invasion was correctly diagnosed in 17 patients with sensitivity of 100% and specificity of 90%, respectively. Portal vein invasion was correctly diagnosed in 47 of 51 branches with sensitivity and specificity of 92.3% and 90.2%, respectively. Multiplanar reconstructed images contributed to the correct diagnosis for both vascular encasement and longitudinal tumor extension. In conclusion, MDCT is useful in preoperative evaluation of hilar cholangiocarcinoma, especially when combined with multiplanar reconstructed images.

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies.

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.

Nagelamides K and L, dimeric bromopyrrole alkaloids from sponge Agelas species.

Two new dimeric bromopyrrole alkaloids, nagelamides K (1) and L (2), have been isolated from Okinawan marine sponges Agelas species, and the structures and stereochemistry were elucidated from spectroscopic data. Nagelamide K (1) is a bromopyrrole alkaloid possessing a rare piperidinoiminoimidazolone ring with an aminoimidazole ring and a taurine unit, while nagelamide L (2) is a unique dimeric bromopyrrole alkaloid containing an ester linkage. Nagelamides K (1) and L (2) exhibited antimicrobial activity.