Domain structure and function of α-1,3-glucanase Agl-EK14 from the gram-negative bacterium Flavobacterium sp. EK-14.

The α-1,3-glucanase Agl-EK14 from Flavobacterium sp. EK-14 comprises a signal peptide (SP), a catalytic domain (CAT), a first immunoglobulin-like domain (Ig1), a second immunoglobulin-like domain (Ig2), a ricin B-like lectin domain (RicinB), and a carboxy-terminal domain (CTD). SP and CTD are predicted to be involved in extracellular secretion, while the roles of Ig1, Ig2, and RicinB are unclear. To clarify their roles, domain deletion enzymes Agl-EK14ΔRicinB, Agl-EK14ΔIg2RicinB, and Agl-EK14ΔIg1Ig2RicinB were constructed. The insoluble α-1,3-glucan hydrolytic, α-1,3-glucan binding, and fungal cell wall hydrolytic activities of the deletion enzymes were almost the same and lower than those of Agl-EK14. Kinetic analysis revealed that the Km values of the deletion enzymes were similar and uniformly higher than those of Agl-EK14. These results suggest that the deletion of RicinB causes a decline in binding and hydrolytic activity and increases the Km value. To confirm the role of RicinB, Ig1, Ig2, and RicinB were fused with green fluorescent protein (GFP). As a result, RicinB-fused GFP (GFP-RicinB) showed binding to insoluble α-1,3-glucan and Aspergillus oryzae cell walls, whereas Ig1- and Ig2-fused GFP did not. These results indicated that RicinB is involved in α-1,3-glucan binding. The fusion protein GFP-Ig1Ig2RicinB was also constructed and GFP-Ig1Ig2RicinB showed strong binding to the cell wall of A. oryzae compared to GFP-RicinB. Gel filtration column chromatography suggested that the strong binding was due to GFP-Ig1Ig2RicinB loosely associated with itself.

Synthesis and evaluation of catecholamine derivatives as amyloid-beta aggregation inhibitors.

Effectively inhibition of amyloid ß (Aß) aggregation is considered an important method for treatment of the Alzheimer's disease. Herein, inspired by the ability of trans-clovamide to effectively inhibit Aß aggregation, we synthesized a series of structurally related catecholamine derivatives and tested them as Aß aggregation inhibitors using the Thioflavin T assay. The results show that they demonstrated a higher inhibitory rate against Aß aggregation. Furthermore, these compounds exhibited high water solubilities and low cytotoxicities. Additionally, transmission electron microscopy images and dynamic light scattering of their Aß aggregations were observed. Docking simulations revealed that the catechol moiety of the synthesized compounds can form hydrogen bonds with the key regions of Aß and thereby inhibit Aß aggregation.

Redesign of a thioflavin-T-binding protein with a flat ß-sheet to evaluate a thioflavin-T-derived photocatalyst with enhanced affinity.

Amyloids, proteinous aggregates with ß-sheet-rich fibrils, are involved in several neurodegenerative diseases such as Alzheimer's disease; thus, their detection is critically important. The most common fluorescent dye for amyloid detection is thioflavin-T (ThT), which shows on/off fluorescence upon amyloid binding. We previously reported that an engineered globular protein with a flat ß-sheet, peptide self-assembly mimic (PSAM), can be used as an amyloid binding model. In this study, we further explored the residue-specific properties of ThT-binding to the flat ß-sheet by introducing systematic mutations. We found that site-specific mutations at the ThT-binding channel enhanced affinity. We also evaluated the binding of a ThT-based photocatalyst, which showed the photooxygenation activity on the amyloid fibril upon light radiation. Upon binding of the photocatalyst to the PSAM variant, singlet oxygen-generating activity was observed. The results of this study expand our understanding of the detailed binding mechanism of amyloid-specific molecules.

Synthesis and Structural Revision of the Cyclic Hexapeptide Dimers Antatollamides A and B.

The synthesis and structural revision of the dimerized cyclic hexapeptides antatollamides A (1) and B (2) are reported. These are unique peptides with two proline residues and bicyclic peptides combined by a disulfide bond. Cyclization and disulfide bond formation of the linear peptide led to antatollamide A (1). However, the 1H and 13C NMR spectra of synthetic antatollamide A (1) were not consistent with those of isolated antatollamide A (1). Meanwhile, the NMR spectra of the monomeric cyclic hexapeptide cyclo(Pro-Pro-Phe-dCys-Ile-Val) (3) and the isolated antatollamide A (1) were identified completely. In addition, we found that isolated antatollamide B (2) is cyclo(Pro-Pro-dPhe-dCys-Ile-Val) (4).

SpyTag Peptide with Alkoxyl Aspartic Acids for pH-Dependent Activation of the SpyCatcher/Tag System.

The SpyCatcher/SpyTag system is a protein pair that forms a covalent isopeptide bond without an additional energy supply. The ability to connect fused proteins makes this system an attractive tool for several protein engineering applications. Conditional activation of the SpyCatcher/SpyTag complex formation further expands the use of this system. Here, we evaluated the pH activation of SpyTag using alkoxyaspartic acids in the isopeptide-forming residue. We found that a peptide with an ethoxy group can be activated by hydrolysis under high pH conditions. However, the hydrolysis induces isoaspartate (isoAsp) formation, which is confirmed by an isoAsp-inserted short peptide. We overcame this problem by changing the C-terminal side of the aspartic acid position to Pro, which does not form isoAsp under high pH conditions. The findings of this study provide fundamental knowledge of the synthetic construction of the modified SpyTag peptide.

Addition of α-1,3-glucan-binding domains to α-1,3-glucanase Agn1p from　 Schizosaccharomyces pombe enhances hydrolytic activity of insoluble α-1,3-glucan.

The glycoside hydrolase (GH) 71 α-1,3-glucanase (Agn1p) from Schizosaccharomyces pombe consists of an N-terminal signal sequence and a catalytic domain. Meanwhile, the GH87 α-1,3-glucanase (Agl-KA) from Bacillus circulans KA-304 consists of an N-terminal signal sequence, a first discoidin domain (DS1), a carbohydrate-binding module family 6 (CBM6), a threonine and proline repeat linker (TP), a second discoidin domain (DS2), an uncharacterized domain, and a catalytic domain. DS1, CBM6, and DS2 exhibit α-1,3-glucan binding activity. This study involved genetically fusing TP, DS1, CBM6, TP, and DS2 to the C-terminus of Agn1p, generating the fusion enzyme Agn1p-DCD. The fusion enzyme was then expressed in Escherichia coli and purified from the cell-free extract. Agn1p-DCD and Agn1p exhibited similar characteristics, such as optimal pH, optimal temperature, pH stability, and thermostability. Insoluble α-1,3-glucan (1%) hydrolyzing assay showed that Agn1p-DCD and Agn1p released approximately 7.6 and 5.0 mM of reducing sugars, respectively, after 48 h of reaction. Kinetic analysis and an α-1,3-glucan binding assay indicated that the addition of DS1, CBM6, and DS2 enhanced the affinity of Agn1p for α-1,3-glucan. Moreover, Agn1p-DCD contributed to enhancing the fungal growth inhibition activity when combined with a mixture of GH19 chitinase and GH16 ß-1,3-glucanase.

Development of 1,5-diarylpyrazoles as EGFR/JNK-2 dual inhibitors: design, synthesis, moleecular docking, and bioactivity evaluation.

The eradication of multifactorial diseases, such as cancer, requires the design of drug candidates that attack multiple targets that contribute to the progression and proliferation of such diseases. Here, 1,5-diarylpyrazole derivatives bearing vanillin or sulfanilamide are developed as potential dual inhibitors of epidermal growth factor receptor (EGFR)/c-Jun N-terminal kinase 2 (JNK-2) for possible anticancer activity. These derivatives inhibited the growths of DLD-1, HeLa, K-562, SUIT-2 and HepG2 cancer cell lines, with minimum concentration required to inhibit half of the cellular growth (IC50) values of 2.7-63 µM. The tests confirmed that 5b and 5d were potent JNK-2 inhibitors, with IC50 of 2.0 and 0.9 µM, respectively, whereas 6 h selectively inhibited EGFR protein kinase (EGFR-PK) (IC50 = 1.7 µM). Notably, 6c inhibited both kinases, with IC50 values of 2.7 and 3.0 µM against EGFR-PK and JNK-2, respectively, offering a reference for designing mutual inhibitors of EGFR/JNK-2. The docking studies revealed the ability of the pyrazole ring to bind to the hinge region of the ATP binding site, thereby supporting the experimental inhibitory results. Furthermore, the developed compounds could induce apoptosis and induce cell cycle arrest at different cell phases.

α-1,3-Glucanase from the gram-negative bacterium Flavobacterium sp. EK-14 hydrolyzes fungal cell wall α-1,3-glucan.

The glycoside hydrolase (GH) 87 α-1,3-glucanase (Agl-EK14) gene was cloned from the genomic DNA of the gram-negative bacterium Flavobacterium sp. EK14. The gene consisted of 2940 nucleotides and encoded 980 amino acid residues. The deduced amino acid sequence of Agl-EK14 included a signal peptide, a catalytic domain, a first immunoglobulin-like domain, a second immunoglobulin-like domain, a ricin B-like lectin domain, and a carboxyl-terminal domain (CTD) involved in extracellular secretion. Phylogenetic analysis of the catalytic domain of GH87 enzymes suggested that Agl-EK14 is distinct from known clusters, such as clusters composed of α-1,3-glucanases from bacilli and mycodextranases from actinomycetes. Agl-EK14 without the signal peptide and CTD hydrolyzed α-1,3-glucan, and the reaction residues from 1 and 2% substrates were almost negligible after 1440 min reaction. Agl-EK14 hydrolyzed the cell wall preparation of Aspergillus oryzae and released glucose, nigerose, and nigero-triose from the cell wall preparation. After treatment of A. oryzae live mycelia with Agl-EK14 (at least 0.5 nmol/ml), mycelia were no longer stained by red fluorescent protein-fused α-1,3-glucan binding domains of α-1,3-glucanase Agl-KA from Bacillus circulans KA-304. Results suggested that Agl-EK14 can be applied to a fungal cell wall lytic enzyme.

Early enteral nutrition with arginine compensates for negative nitrogen balance in patients undergoing curative total gastrectomy.

The effects of early enteral arginine-rich nutrition (EAN) were analyzed among patients undergoing curative-intent total gastrectomy for gastric cancer. There were 19 patients in this prospective study, all randomly assigned to either a parenteral nutrition (PN) group or an EAN group for the first seven days after surgery. The EAN group received 1.8-fold greater arginine (10.1 g/day) compared with the PN group, which was administered through an enteral tube inserted into the jejunal loop. Both groups were provided almost identical amounts of total amino acids (54 g/day), and the total energy was set at 65% of the total requirement (25 kcal/kg/day). No significant differences were observed between the two groups in postoperative complications, length of hospital stay, oral intake, nutritional status, or body weight. The serum arginine profile was similar in the two groups, as it decreased significantly on postoperative day (POD) 1, and gradually returned to preoperative levels by POD 7. The nitrogen balance remained negative until POD 7 in the PN group, but turned neutral at POD 7 in the EAN group. While we could not confirm body weight loss improvement, these results suggested that early arginine-rich enteral nutrition could improve the nitrogen balance after total gastrectomy. J. Med. Invest. 70 : 325-333, August, 2023.

Development and Assessment of 1,5-Diarylpyrazole/Oxime Hybrids Targeting EGFR and JNK-2 as Antiproliferative Agents: A Comprehensive Study through Synthesis, Molecular Docking, and Evaluation.

New 1,5-diarylpyrazole oxime hybrid derivatives (scaffolds A and B) were designed, synthesized, and then their purity was verified using a variety of spectroscopic methods. A panel of five cancer cell lines known to express EGFR and JNK-2, including human colorectal adenocarcinoma cell line DLD-1, human cervical cancer cell line Hela, human leukemia cell line K562, human pancreatic cell line SUIT-2, and human hepatocellular carcinoma cell line HepG2, were used to biologically evaluate for their in vitro cytotoxicity for all the synthesized compounds 7a-j, 8a-j, 9a-c, and 10a-c. The oxime containing compounds 8a-j and 10a-c were more active as antiproliferative agents than their non-oxime congeners 7a-j and 9a-c. Compounds 8d, 8g, 8i, and 10c inhibited EGFR with IC50 values ranging from 8 to 21 µM when compared with sorafenib. Compound 8i inhibited JNK-2 as effectively as sorafenib, with an IC50 of 1.0 µM. Furthermore, compound 8g showed cell cycle arrest at the G2/M phase in the cell cycle analysis of the Hela cell line, whereas compound 8i showed combined S phase and G2 phase arrest. According to docking studies, oxime hybrid compounds 8d, 8g, 8i, and 10c exhibited binding free energies ranging from -12.98 to 32.30 kcal/mol at the EGFR binding site whereas compounds 8d and 8i had binding free energies ranging from -9.16 to -12.00 kcal/mol at the JNK-2 binding site.

Heterologous expression of α-1,3-glucanase Agn1p from Schizosaccharomyces pombe, and efficient production of nigero-oligosaccharides by enzymatic hydrolysis from solubilized α-1,3;1,6-glucan.

The glycoside hydrolase family 71 α-1,3-glucanase (Agn1p) of Schizosaccharomyces pombe was expressed in Escherichia coli Rosetta-gami B (DE3). Agn1p (0.5 nmol/mL) hydrolyzed insoluble α-1,3-glucan (1%), and about 3.3 mm reducing sugars were released after 1440 min of reaction. The analysis of reaction products by high-performance liquid chromatography revealed that pentasaccharides accumulated in the reaction mixture as the main products, along with a small amount of mono-, di-, tri-, tetra-, and hexasaccharides. Soluble glucan was prepared from insoluble α-1,3;1,6-glucan by alkaline and sonication treatment to improve the hydrolytic efficiency. As a result, this solubilized α-1,3;1,6-glucan maintained a solubilized state for at least 6 h. Agn1p (0.5 nmol/mL) hydrolyzed the solubilized α-1,3;1,6-glucan (1%), and about 8.2 mm reducing sugars were released after 240 min of reaction. Moreover, Agn1p released about 12.3 mm reducing sugars from 2% of the solubilized α-1,3;1,6-glucan.

Annual report on National Clinical Database 2020 for gastroenterological surgery in Japan.

Aim: The National Clinical Database (NCD) of Japan is a nationwide data entry system for surgery, and it marked its 10th anniversary in 2020. The aim was to present the 2020 annual report of gastroenterological surgery of the NCD. Methods: The data of the surgical procedures stipulated by the training curriculum for board-certified surgeons of the Japanese Society of Gastroenterological Surgery in the NCD from 2011 to 2020 were summarized. Results: In total, 5 622 845 cases, including 593 088 cases in 2020, were extracted from the NCD. The total number of gastroenterological surgeries increased gradually in these 10 years, except for the year 2020 due to the COVID-19 pandemic. The annual number of surgeries of each organ, except the pancreas and liver, decreased by 0.4%-13.1% in 2020 compared to 2019. The surgical patients were consistently aging, with more than 20% of all gastroenterological surgeries in 2020 involving patients aged 80 years or older. The participation of board-certified surgeons increased for each organ (75.9%-95.7% in 2020). The rates of endoscopic surgery also increased constantly. Although the incidences of postoperative complications of each organ increased by 0.7%-7.9% in these 10 years, postoperative mortality rates decreased by 0.2%-1.5%. Conclusions: We present here the short-term outcomes of each gastroenterological operative procedure in 2020. This review of the 10-years of NCD data of gastroenterological surgery revealed a consistent increase of the number of surgeries (except for in 2020), especially endoscopic procedures, and aging of the Japanese population. The good safety of Japanese gastroenterological surgeries was also indicated.

Structure-Activity Relationship Study of N-Hydroxyphtalimide Derivatives for the Detection of Amines during Fmoc-Solid-Phase Peptide Synthesis.

Previously, we developed a method for the detection of unprotected amino groups based on their reversible reaction with N-hydroxyphthalimide (NHPI) to form intensely colored products, which can be useful when conducting solid-phase peptide synthesis. Here, we describe a structure-activity relationship study of NHPI derivatives to identify the derivative best suited for this method using a spectrophotometer toward the estimation of chemical yields. We found that the products resulting from the reaction of the derivative with an unprotected amino group were only intensely colored if the structure of the derivative incorporated an NHPI framework. We also prepared five peptides, including those containing N-methyl and D-amino acid, and Pro residues, using our reversible detection method to detect unprotected amino groups. The mechanism of the detection reaction was also studied by the structural analysis of the NHPI (1) and diisopropylamine complex and concluded to entail salt formation between the N-hydroxy group and amine.

Nigero-oligosaccharide production by enzymatic hydrolysis from alkaline-pretreated α-1,3-glucan.

Nigero-oligosaccharides are α-1,3-linked oligomers of glucose. Glycoside hydrolase 87 type α-1,3-glucanase Agl-KA from Bacillus circulans KA304 is an endo-lytic enzyme that releases nigero-oligosaccharides (tetra-, tri-, and di-saccharide) from α-1,3-glucan. α-1,3-Glucan is insoluble under natural conditions, thus the efficiency of enzymatic hydrolysis is low and only 5 mM of reducing sugars were released from 1% glucan by Agl-KA. To improve hydrolytic efficiency, α-1,3-glucan was solubilized by 1 M NaOH and alkaline-solubilized glucan was adjusted to approximately pH 8. As a result, glucan maintained a solubilized state. This alkaline-pretreated α-1,3-glucan (1%) was hydrolyzed by Agl-KA (0.64 nmol/mL) and approximately 11.6 mM of reducing sugars were released at 240 min of reaction. When 0.016, 0.032, and 0.13 nmol/mL enzyme were added, reducing sugar reached approximately 5.1, 7.5, and 9.8 mM, respectively, and reaction mixtures containing 0.016 and 0.032 nmol/mL enzyme gradually became cloudy. Our findings suggest α-1,3-glucan cannot maintain its solubilized state and gradually becomes insoluble. For deletion enzyme of α-1,3-glucan binding domains from Agl-KA (AglΔDCD-UCD) on glucan hydrolysis (2%), reducing sugar concentrations released by AglΔDCD-UCD were almost the same as Agl-KA. These findings suggest that alkaline-pretreated α-1,3-glucan maintains a soluble state during a short time period and that glucan is efficiently hydrolyzed even by α-1,3-glucanase without α-1,3-glucan binding domains.

Comprehensive evaluation of three-dimensional anatomy of perigastric vessels using enhanced multidetector-row computed tomography.

BACKGROUND: To perform laparoscopic gastrectomy safely, we aimed to comprehensively re-evaluate perigastric vessel anatomies using a three-dimensional angiography reconstructed from enhanced multidetector-row computed tomography data. METHODS: Perigastric vessel anatomy was preoperatively analyzed using a multidetector-row computed tomography-based three-dimensional angiography reconstructed in 127 patients undergoing gastric surgery. RESULTS: Of the 67 left gastric veins that ran along the dorsal side of the arteries, 59 (88.1%) ran along the dorsal side of the common hepatic artery and flowed into the portal vein. In 18 cases, a common trunk of one to three left gastric arteries and the replaced left hepatic artery was observed. The left inferior phrenic artery ramified from the left gastric artery in 5.5% of the cases. The right gastric artery was classified into distal (73.2%), caudal (18.1%), and proximal (8.7%) types. The infra-pyloric artery was also classified into distal (64.6%), caudal (26.0%), and proximal (9.4%) types. The posterior gastric artery branched as a common trunk with the superior polar artery in the proximal (37.9%) and distal (18.4%) regions of the splenic artery. The left gastroepiploic artery ramified from the splenic (18.1%) and inferior terminal arteries (81.9%). No, one, and two gastric branches of the left gastroepiploic artery, which ramified between the roots of the left gastroepiploic artery and its omental branch, were found in 36.5%, 49.2%, and 14.3% of the cases, respectively. CONCLUSIONS: Preoperative 3D angiography is useful for the precise evaluation of perigastric vessel anatomies, and may help us to perform laparoscopic gastrectomy and robotic surgery safely.

Antimicrobial Activity of Polysorbate 80-iodine Complex: Polysorbate 80 Firmly Retains Iodine.

Considering that iodine is highly volatile and has low solubility in water, it is utilized as an antiseptic in its complex form (iodophor) with a carrier material. Herein, we prepared the polysorbate 80-iodine complex and investigated its properties. In the presence of 0%, 0.01%, 0.1%, and 1% polysorbate, Pseudomonas putida NBRC 100650 growth was inhibited at 75, 75, 50, and 25 ppm iodine, respectively, indicating that high concentrations of polysorbate 80 enhanced the antibacterial activity of iodine. Absorption spectra of the mixtures of polysorbate 80 and iodine were analyzed; we observed that two peaks at 287 and 350 nm, derived from triiodide ions, shifted to the longer wavelength side in the presence of 0.1% and 1% polysorbate 80. Further, when 1% polysorbate 80 was added to the mixture of soluble starch and iodine, the peak around 580 nm arising from the amylose-iodine complex disappeared, indicating that polysorbate 80 captured iodine from the starch-iodine complex. We also found that polysorbate 80 retained iodine for approximately 4 months and prevented its volatilization; moreover, the mixture did not lose its growth inhibitory activity upon storage for approximately 4 months. Collectively, our data indicated that polysorbate 80 firmly retains low concentrations of iodine and that the polysorbate 80-iodine complex can serve as an antiseptic that can be stably stored for a long time.

GH-16 Type ß-1,3-Glucanase from Lysobacter sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase, and Its Glucan-binding Domain Binds to Fungal Cell-wall.

The GH-16 type ß-1,3-glucanase (BgluC16MK) gene of Lysobacter sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from L. enzymogenes strain N4-7. BgluC16MK includes a signal sequence, a catalytic domain and carbohydrate-binding module family 6-type ß-glucan binding domain (B-GBD). The expression of the BgluC16MK gene in Escherichia coli without the signal sequence resulted in antifungal activity at a dose of 0.6-0.8 nmol/disk. However, BgluC16MK displayed antifungal activity at a dose of 0.025 nmol/disk in combination with Chi19MK. Substrate-specific assay revealed that purified BgluC16MK hydrolyzed insoluble curdlan more readily than the soluble substrate. Furthermore, to explore the binding selectivity of B-GBD of BgluC16MK, we constructed a fusion protein (B-GBD-GFP) using the B-GBD and green fluorescent protein. The activity of the fusion protein against various substrates indicates that B-GBD was selective for glucans with ß-1,3-linkages. An additional study demonstrated the binding ability of B-GBD-GFP to the cell-wall of living fungi, such as T. reesei and Aspergillus oryzae. These findings suggest that BgluC16MK can be utilized to generate antifungal enzyme preparations and that the fusion protein B-GBD-GFP can be used to identify the fungal cell surface structure using ß-glucans.

Artificial intelligence-based classification of peripheral blood nucleated cells using label-free imaging flow cytometry.

Label-free image identification of circulating rare cells, such as circulating tumor cells within peripheral blood nucleated cells (PBNCs), the vast majority of which are white blood cells (WBCs), remains challenging. We previously described developing label-free image cytometry for classifying live cells using computer vision technology for pattern recognition, based on the subcellular structure of the quantitative phase microscopy images. We applied our image recognition methods to cells flowing in a flow cytometer microfluidic channel, and differentiated WBCs from cancer cell lines (area under receiver operating characteristic curve = 0.957). We then applied this method to healthy volunteers' and advanced cancer patients' blood samples and found that the non-WBC fraction rates (NWBC-FRs), defined as the percentage of cells classified as non-WBCs of the total PBNCs, were significantly higher in cancer patients than in healthy volunteers. Furthermore, we monitored NWBC-FRs over the therapeutic courses in cancer patients, which revealed the potential ability in monitoring the clinical status during therapy. Our image recognition system has the potential to provide a morphological diagnostic tool for circulating rare cells as non-WBC fractions.

Saturated Fatty Acids in Cell Membrane Lipids Induce Resistance to 5-Fluorouracil in Colorectal Cancer Cells.

BACKGROUND/AIM: Resistance to chemotherapy is a major obstacle for patients with unresectable colorectal cancer (CRC); however, the factors that induce chemoresistance have not been elucidated. Lipid composition influences neoplastic behaviour. Therefore, this study examined whether lipid composition affects sensitivity to chemotherapeutic agents in CRC. MATERIALS AND METHODS: We performed a lipidomic analysis of a CRC xenograft-derived spheroid model to identify potential relationships between the lipid profile and chemoresistance to 5-fluorouracil (5-FU). Genetic and pharmacological modulation of lipid synthesis were also used in the HCT-116 and DLD-1 CRC cell lines to further characterize resistance to 5-FU. RESULTS: Our lipidomic profiling revealed that phospholipids with saturated fatty acids (SFAs) were more abundant in 5-FU-resistant spheroids. The importance of phospholipids containing SFA in chemoresistance was confirmed by showing that in HCT-116 and DLD-1 cells, genetic or pharmacological inactivation of stearoyl-CoA desaturase-1, a key enzyme that converts SFAs to monounsaturated fatty acids, increased the proportion of SFAs in membranous phospholipids and reduced cell membrane fluidity, and this ultimately resulted in resistance to 5-FU. CONCLUSION: These data suggest that the saturated to monounsaturated fatty acid ratio in cellular membranous phospholipids affects sensitivity to chemotherapeutic agents.

Synthesis and Configuration Confirmation of the ATHOD Fatty Amino Acid Residue in the Burkholdines.

Eight possible diastereomers of the 3-amino-5,6,7-trihydroxy octadecanoic acid (ATHOD) moiety of the burkholdines (Bks) have been synthesized and their configurations assigned. Though the relative configuration of the triol in the ATHOD residue of the Bks was proposed to be anti-anti-anti in the literature, 1H NMR spectra of our synthesized anti-anti-anti ATHOD derivative was inconsistent with that of the isolated ATHOD residue, suggesting that the assignment of the relative configuration of that residue in the literature was incorrect. However, by comparison of the NMR data of our ATHOD derivatives with that of configurationally defined samples of 2-amino-4-pentanol using Kishi's NMR database method, we conclude that the absolute configuration of the ATHOD moiety is (3R,5S,6R,7S). In addition, we revealed that the ATHOD residue present in the occidiofungins A-D has the same configuration as that in all the Bks.