Non-Heme Iron Enzymes Catalyze Heterobicyclic and Spirocyclic Isoquinolone Core Formation in Piperazine Alkaloid Biosynthesis.

We report the discovery and biosynthesis of new piperazine alkaloids-arizonamides, and their derived compounds-arizolidines, featuring heterobicyclic and spirocyclic isoquinolone skeletons, respectively. Their biosynthetic pathway involves two crucial non-heme iron enzymes, ParF and ParG, for core skeleton construction. ParF has a dual function facilitating 2,3-alkene formation of helvamide, as a substrate for ParG, and oxidative cleavage of piperazine. Notably, ParG exhibits catalytic versatility in multiple oxidative reactions, including cyclization and ring reconstruction. A key amino acid residue Phe67 was characterized to control the formation of the constrained arizonamide B backbone by ParG.

A lectin from Crenomytilus grayanus as a probe for the detection of widespread cancerous and metastatic cells.

A novel Gal-binding lectin from mussels (Crenomytilus grayanus, CGL) with 6 binding sites in the dimeric structure has been previously shown to have antifungal, anticancer, and antibacterial activities. In this study, a glycan array was used to confirm that CGL recognizes a range of non-reducing end α- or ß-linked Gal glycans on normal cells but not sialic acid-capped glycans. This finding suggests that CGL has potential in the tumor detection due to the hyper-sialylation present in cell surface glycans from cancer cells. To evaluate the feasibility of this possibility, we labeled CGL with biotin and then mixed it with streptavidin-horseradish peroxidase (HRP) to create a CGL-biotin-SP complex as a probe for use in enzyme-linked lectin assays. CGL-biotin-SP successfully distinguished not only HeLa cells and de-sialylated HeLa cells that mimic normal cell surface glycans but also lung and breast cancer cells with different metastatic abilities. This work provides the insights into a new Gal-binding lectin by establishing its specificity and also demonstrates practical applications in cancer diagnosis greater than other reported lectins.

Structural and biological insights into Klebsiella pneumoniae surface polysaccharide degradation by a bacteriophage K1 lyase: implications for clinical use.

BACKGROUND: K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge. METHODS: We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes. RESULTS: We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1-3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric ß-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose. CONCLUSIONS: Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.

An Immunological Polysaccharide from Tremella fuciformis: Essential Role of Acetylation in Immunomodulation.

The edible fungus Tremella fuciformis was shown to have a high molecular weight (1.87 × 103 kDa) bioactive polysaccharide, denoted as TFP-F1. Monosaccharide composition and NMR analysis of the polysaccharide and its derivatives indicated it contained fucose (Fucp), xylose (Xylp), mannose (Manp), and glucuronic acid (GlcAp) in a ratio of 0.9:1.0:3.2:1.2. Using IR, NMR, and GC-MS spectroscopic data, the structure of TFP-F1 was elucidated as {→3)-[ß-D-GlcAp-(1→2)]-α-D-Manp-(1→3)-α-D-Manp-(1→3)-[α-L-Fucp-(1→2)-ß-D-Xylp-(1→2)]-α-D-Manp-(1→}n, with partial acetylation of C6-OH in mannoses. Furthermore, at a concentration of 1 µg/mL, TFP-F1 was found to stimulate the secretion of TNF-α and IL-6 in J774A.1 macrophage cells in vitro via interaction with toll-like receptor 4 (TLR4). The removal of O-acetyl groups led to the loss of immunomodulatory activities, demonstrating that O-acetyl groups play an essential role in enhancing the production of pro-inflammatory cytokines.

Bacteriophage Tail-Spike Proteins Enable Detection of Pseudaminic-Acid-Coated Pathogenic Bacteria and Guide the Development of Antiglycan Antibodies with Cross-Species Antibacterial Activity.

Pseudaminic acid (Pse), a unique carbohydrate in surface-associated glycans of pathogenic bacteria, has pivotal roles in virulence. Owing to its significant antigenicity and absence in mammals, Pse is considered an attractive target for vaccination or antibody-based therapies against bacterial infections. However, a specific and universal probe for Pse, which could also be used in immunotherapy, has not been reported. In a prior study, we used a tail spike protein from a bacteriophage (ΦAB6TSP) that digests Pse-containing exopolysaccharide (EPS) from Acinetobacter baumannii strain 54149 (Ab-54149) to form a glycoconjugate for preparing anti-Ab-54149 EPS serum. We report here that a catalytically inactive ΦAB6TSP (I-ΦAB6TSP) retains binding ability toward Pse. In addition, an I-ΦAB6TSP-DyLight-650 conjugate (Dy-I-ΦAB6TSP) was more sensitive in detecting Ab-54149 than an antibody purified from anti- Ab-54149 EPS serum. Dy-I-ΦAB6TSP also cross-reacted with other pathogenic bacteria containing Pse on their surface polysaccharides (e.g., Helicobacter pylori and Enterobacter cloacae), revealing it to be a promising probe for detecting Pse across bacterial species. We also developed a detection method that employs I-ΦAB6TSP immobilized on microtiter plate. These results suggested that the anti-Ab-54149 EPS serum would exhibit cross-reactivity to Pse on other organisms. When this was tested, this serum facilitated complement-mediated killing of H. pylori and E. cloacae, indicating its potential as a cross-species antibacterial agent. This work opens new avenues for diagnosis and treatment of multidrug resistant (MDR) bacterial infections.

Pseudaminic Acid on Exopolysaccharide of Acinetobacter baumannii Plays a Critical Role in Phage-Assisted Preparation of Glycoconjugate Vaccine with High Antigenicity.

Pseudaminic acid (Pse) has been known for participating in crucial bacterial virulence and thus is an attractive target in the development of glycoconjugate vaccine. Particularly, this therapeutic alternative was suggested to be a potential solution against antibiotic resistant Acinetobacter baumannii that poses a serious global health threat. Also, Pse was found to be involved in the exopolysaccharide (EPS) of mild antibiotic resistant A. baumannii strain 54149 ( Ab-54149) of which specific glycosyl linkage can be depolymerized by phage ΦAB6 tailspike protein (ΦAB6TSP). In this study, we found that the antibodies induced by Ab-54149 EPS was capable of recognizing a range of EPS of other clinical A. baumannii strains, and deemed as a great potential material for vaccination. To efficiently acquire homogeneous EPS-derived oligosaccharide with significant immunogenic activity for the production of glycoconjugate, we used the ΦAB6TSP for the fragmentation of Ab-54149 EPS instead of chemical methods. Moreover, insight into the ligand binding characterization of ΦAB6TSP suggested the branched Pse on the Ab-54149 EPS served as a recognition site of ΦAB6TSP. The serum boosted by ΦAB6TSP-digested product and carrier protein CRM197 conjugate complex displayed specific sensitivity toward Ab-54149 EPS with bacterial killing activity. Strikingly, Pse is an ideal epitope with strong antigenicity, profiting the application of the probe for pathogen detection and glyco-based vaccine.

Capsular Polysaccharide Is Involved in NLRP3 Inflammasome Activation by Klebsiella pneumoniae Serotype K1.

Klebsiella pneumoniae (strain 43816, K2 serotype) induces interleukin-1ß (IL-1ß) secretion, but neither the bacterial factor triggering the activation of these inflammasome-dependent responses nor whether they are mediated by NLRP3 or NLRC4 is known. In this study, we identified a capsular polysaccharide (K1-CPS) in K. pneumoniae (NTUH-K2044, K1 serotype), isolated from a primary pyogenic liver abscess (PLA K. pneumoniae), as the Klebsiella factor that induces IL-1ß secretion in an NLRP3-, ASC-, and caspase-1-dependent manner in macrophages. K1-CPS induced NLRP3 inflammasome activation through reactive oxygen species (ROS) generation, mitogen-activated protein kinase phosphorylation, and NF-κB activation. Inhibition of both the mitochondrial membrane permeability transition and mitochondrial ROS generation inhibited K1-CPS-mediated NLRP3 inflammasome activation. Furthermore, IL-1ß secretion in macrophages infected with PLA K. pneumoniae was shown to depend on NLRP3 but also on NLRC4 and TLR4. In macrophages infected with a K1-CPS deficiency mutant, an lipopolysaccharide (LPS) deficiency mutant, or K1-CPS and LPS double mutants, IL-1ß secretion levels were lower than those in cells infected with wild-type PLA K. pneumoniae. Our findings indicate that K1-CPS is one of the Klebsiella factors of PLA K. pneumoniae that induce IL-1ß secretion through the NLRP3 inflammasome.

Phosphoproteomic analysis reveals the effects of PilF phosphorylation on type IV pilus and biofilm formation in Thermus thermophilus HB27.

Thermus thermophilus HB27 is an extremely thermophilic eubacteria with a high frequency of natural competence. This organism is therefore often used as a thermophilic model to investigate the molecular basis of type IV pili-mediated functions, such as the uptake of free DNA, adhesion, twitching motility, and biofilm formation, in hot environments. In this study, the phosphoproteome of T. thermophilus HB27 was analyzed via a shotgun approach and high-accuracy mass spectrometry. Ninety-three unique phosphopeptides, including 67 in vivo phosphorylated sites on 53 phosphoproteins, were identified. The distribution of Ser/Thr/Tyr phosphorylation sites was 57%/36%/7%. The phosphoproteins were mostly involved in central metabolic pathways and protein/cell envelope biosynthesis. According to this analysis, the ATPase motor PilF, a type IV pili-related component, was first found to be phosphorylated on Thr-368 and Ser-372. Through the point mutation of PilF, mimic phosphorylated mutants T368D and S372E resulted in nonpiliated and nontwitching phenotypes, whereas nonphosphorylated mutants T368V and S372A displayed piliation and twitching motility. In addition, mimic phosphorylated mutants showed elevated biofilm-forming abilities with a higher initial attachment rate, caused by increasing exopolysaccharide production. In summary, the phosphorylation of PilF might regulate the pili and biofilm formation associated with exopolysaccharide production.

Lon protease affects the RdxA nitroreductase activity and metronidazole susceptibility in Helicobacter pylori.

BACKGROUND: The lon gene of Helicobacter pylori strains is constitutively expressed during growth. However, virtually nothing is understood concerning the role of Lon in H. pylori. This study examined the function and physiological role of Lon in H. pylori (HpLon) using a trapping approach to identify putative Lon binding partners in the bacterium. MATERIALS AND METHODS: Protease-deficient Lon was expressed and served as the bait in trapping approach to capture the interacting partners in H. pylori. The antibiotic susceptibility of wild-type and lon derivative mutants was determined by the E test trips and the disc diffusion assay. The effect of HpLon on RdxA activity was detected the change in NADPH oxidation and metronidazole reduction by spectrophotometer. RESULTS: Lon in Helicobacter pylori (HpLon) interacting partners are mostly associated with metronidazole activation. lon mutant presents more susceptible to metronidazole than that of the wild type, and this phenotype is recovered by complementation of the wild-type Lon. We found that the ATPases associated with a variety of cellular activities (AAA(+) ) module of HpLon causes a decrease in both NADPH oxidase and Mtz reductase activity in RdxA, a major Mtz-activating enzyme in H. pylori. CONCLUSION: Metronidazole resistance of H. pylori causes the serious medical problem worldwide. In this study, HpLon is involved in metronidazole susceptibility among H. pylori strains. We provide the evidence that HpLon alters RdxA activity in vitro. The decrease in metronidazole activation caused by HpLon is possibly prior to accumulate mutation in rdxA gene before the metronidazole-resistant strains to be occurred.

Klebsiella pneumoniae K2 capsular polysaccharide degradation by a bacteriophage depolymerase does not require trimer formation.

K2-capsular Klebsiella pneumoniae is a hypervirulent pathogen that causes fatal infections. Here, we describe a phage tailspike protein, named K2-2, that specifically depolymerizes the K2 capsular polysaccharide (CPS) of K. pneumoniae into tetrasaccharide repeating units. Nearly half of the products contained O-acetylation, which was thought crucial to the immunogenicity of CPS. The product-bound structures of this trimeric enzyme revealed intersubunit carbohydrate-binding grooves, each accommodating three tetrasaccharide units of K2 CPS. The catalytic residues and the key interactions responsible for K2 CPS recognition were identified and verified by site-directed mutagenesis. Further biophysical and functional characterization, along with the structure of a tetrameric form of K2-2, demonstrated that the formation of intersubunit catalytic center does not require trimerization, which could be nearly completely disrupted by a single-residue mutation in the C-terminal domain. Our findings regarding the assembly and catalysis of K2-2 provide cues for the development of glycoconjugate vaccines against K. pneumoniae infection. IMPORTANCE: Generating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of K. pneumoniae into tetrasaccharide fragments while retaining the vital O-acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2 K. pneumoniae, we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center.

3,4-Bis[1-(prop-2-yn-yl)-1H-indol-3-yl]-1H-pyrrole-2,5-dione.

In the title mol-ecule, C26H17N3O2, both indole ring systems are essentially planar, with maximum deviations of 0.019 (2) and 0.033 (1) Šfor the N atoms, and form dihedral angles of 34.40 (9) and 45.06 (8)° with the essentially planar pyrrole ring [maximum deviation = 0.020 (2) Å]. The dihedral angle between the two indole ring systems is 58.78 (6)°. In the crystal, mol-ecules are connected by pairs of N-H⋯O hydrogen bonds, forming inversion dimers and generating R 2 (2)(8) rings. Weak π-π stacking inter-actions, with a centroid-centroid distance of 3.983 (2) Å, are also observed.

Structure and immunological characterization of the capsular polysaccharide of a pyrogenic liver abscess caused by Klebsiella pneumoniae: activation of macrophages through Toll-like receptor 4.

The active components of a primary pyrogenic liver abscess (PLA) Klebsiella pneumoniae in stimulating cytokine expression in macrophages are still unclear. The capsular polysaccharide (CPS) of PLA K. pneumoniae is important in determining clinical manifestations, and we have shown that it consists of repeating units of the trisaccharide (→3)-ß-D-Glc-(1→4)-[2,3-(S)-pyruvate]-ß-D-GlcA-(1→4)-α-L-Fuc-(1→) and has the unusual feature of extensive pyruvation of glucuronic acid and acetylation of C(2)-OH or C(3)-OH of fucose. We demonstrated that PLA K. pneumoniae CPS induces secretion of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by macrophages through Toll-like receptor 4 (TLR4) and that this effect was lost when pyruvation and O-acetylation were chemically destroyed. Furthermore, expression of TNF-α and IL-6 in PLA K. pneumoniae CPS-stimulated macrophages was shown to be regulated by the TLR4/ROS/PKC-δ/NF-κB, TLR4/PI3-kinase/AKT/NF-κB, and TLR4/MAPK signaling pathways.

6-Benz-yloxy-2-phenyl-pyridazin-3(2H)-one.

In the title compound, C(17)H(14)N(2)O(2), the central pyridazine ring forms dihedral angles of 47.29 (5) and 88.54 (5)° with the benzene rings, while the dihedral angle between the benzene rings is 62.68 (6)°. In the crystal, molecules are linked by two weak C-H⋯O hydrogen bonds and three weak C-H⋯π inter-actions.

Development of Klebsiella pneumoniae Capsule Polysaccharide-Conjugated Vaccine Candidates Using Phage Depolymerases.

Klebsiella pneumoniae is an important pathogen associated with nosocomial infection and has developed increasing resistance to antibiotics such as extended-spectrum ß-lactams and carbapenem. In recent years, K. pneumoniae isolates have emerged as a major cause of global community-acquired infections such as pneumonia and pyogenic liver abscess. Although serotypes K1 and K2 have been identified as the predominant capsular types associated with invasive infections, no K. pneumoniae vaccine is commercially available, probably due to immunogenicity loss in the traditional depolymerization method to obtain capsule polysaccharide (CPS) for the preparation of conjugated vaccine. In this study, we successfully retained immunogenicity by using K1 (K1-ORF34) and K2 (K2-ORF16) CPS depolymerases that were identified from phages to cleave K1 and K2 CPSs into intact structural units of oligosaccharides with intact modifications. The obtained K1 and K2 oligosaccharides were separately conjugated with CRM197 carrier protein to generate CPS-conjugated vaccines. Immunization experiments of mice showed both K1 and K2 CPS-conjugated vaccines induced anti-CPS antibodies with 128-fold and 64-fold increases of bactericidal activities, respectively, compare to mice without vaccinations. Challenge tests indicated that K1 or K2 CPS-conjugated vaccine and divalent vaccine (a mixture of K1 and K2 CPS-conjugated vaccines) protected mice from subsequent infection of K. pneumoniae by the respective capsular type. Thus, we demonstrated K1 and K2 CPS-conjugated vaccines prepared by CPS depolymerases is a promising candidate for developing vaccines against human K. pneumoniae infections.

A hexasaccharide from capsular polysaccharide of carbapenem-resistant Klebsiella pneumoniae KN2 is a ligand of Toll-like receptor 4.

Klebsiella pneumoniae serotype KN2 is a carbapenem-resistant strain and leads to the health care-associated infections, such as bloodstream infections. Its capsular polysaccharide (CPS) was isolated and cleaved by a specific enzyme from a bacteriophage into a hexasaccharide-repeating unit. With GC-MS, NMR, and Mass analyses, the structure of KN2 CPS was determined to be {→3)-ß-D-Glcp-(1→3)-[α-D-GlcpA-(1→4)-ß-D-Glcp-(1→6)]-α-D-Galp-(1→6)-ß-D-Galp-(1→3)-ß-D-Galp-(1→}n. We demonstrated that 1 µg/mL CPS could stimulate J774A.1 murine macrophages to release tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in vitro. Also, we proved that KN2 CPS induced the immune response through Toll-like receptor 4 (TLR4) in the human embryonic kidney (HEK)-293 cells. Strikingly, the hexasaccharide alone shows the same immune response as the CPS, suggesting that the hexasaccharide can shape the adaptive immunity to be a potential vaccine adjuvant. The glucuronic acid (GlcA) on other polysaccharides can affect the immune response, but the GlcA-reduced KN2 CPS and hexasaccharide still maintain their immunomodulatory activities.

From nanoplates to microtubes and microrods: a surfactant-free rolling mechanism for facile fabrication and morphology evolution of Ag2S films.

By a simple and facile wet-chemistry technique without any surfactant, various shapes of Ag(2)S crystals--including leaflike pentagonal nanoplates, crinkly nanoscrolls, hexagonal prismlike microtubes, and microrods--were fabricated in situ on a large-area silver-foil surface separately. Detailed experiments revealed that the Ag(2)S nanoplates were formed just by immersing the silver foil in a sulfur/ethanol solution at room temperature and atmospheric pressure, and they subsequently rolled into nanoscrolls and further grew into microtubes and microrods under solvothermal conditions. Inspired by the natural curling of a piece of foliage, we proposed a surfactant-free rolling mechanism to interpret the observed morphological evolution from lamellar to tubular structures. Based on these simple, practical, and green chemical synthetic routes, we can easily synthesize lamellar, scrolled, tubular, and clubbed Ag(2)S crystals by simply adjusting the reaction temperature, pressure, and time. It is very interesting to note that the current rolling process is quite different from the previous reported rolling mechanism that highly depends on the surfactants; we revealed that the lamellar Ag(2)S could be rolled into tubular structures without using any surfactant or other chemical additives, just like the natural rolling process of a piece of foliage. Therefore, this morphology-controlled synthetic route of Ag(2)S crystals may provide new insight into the synthesis of metal sulfide semiconducting micro-/nanocrystals with desired morphologies for further industrial applications. The optical properties of the pentagonal Ag(2)S nanoplates/film were also investigated by UV/Vis and photoluminescence (PL) techniques, which showed large blue-shift of the corresponding UV/Vis and PL spectra.

Proteomannans in biofilm of Helicobacter pylori ATCC 43504.

BACKGROUND: The human bacterial pathogen Helicobacter pylori forms biofilms. However, the constituents of the biofilm have not been extensively investigated. In this study, we analyzed the carbohydrate and protein components of biofilm formed by H. pylori strain ATCC 43504 (NCTC 11637). MATERIALS AND METHODS: Development of H. pylori biofilm was analyzed using scanning electron microscopy (SEM) and quantified using crystal violet staining. The extracted extracellular polysaccharide (EPS) matrix was analyzed using GC-MS and nuclear magnetic resonance (NMR) analyses. Proteomic profiles of biofilms were examined by SDS-PAGE while deletion mutants of upregulated biofilm proteins were constructed and characterized. RESULTS: Formation of H. pylori biofilm is time dependent as shown by crystal violet staining assay and SEM. NMR reveals the prevalence of 1,4-mannosyl linkages in both developing and mature biofilms. Proteomic analysis of the biofilm indicates the upregulation of neutrophil-activating protein A (NapA) and several stress-induced proteins. Interestingly, the isogenic mutant napA revealed a different biofilm phenotype that showed reduced aggregated colonial structure when compared to the wild type. CONCLUSIONS: This in vitro study shows that mannose-related proteoglycans (proteomannans) are involved in the process of H. pylori biofilm formation while the presence of upregulated NapA in the biofilm implies the potency to increase adhesiveness of H. pylori biofilm. Being a complex matrix of proteins and carbohydrates, which are probably interdependent, the H. pylori biofilm could possibly offer a protective haven for the survival of this gastric bacterial pathogen in the extragastric environments.

Dioscorea phytocompounds enhance murine splenocyte proliferation ex vivo and improve regeneration of bone marrow cells in vivo.

Specific cytokines have been tested clinically for immunotherapy of cancers; however, cytotoxicity has often impaired their usefulness. Consequently, alternative approaches are increasingly desirable. Dioscorea spp. tuber is a widely used traditional Chinese medicinal herb claimed to confer immunostimulatory activity. In this study, we evaluated Dioscorea as an adjuvant therapy for use alongside chemotherapy for cancer. Phytocompounds from Dioscorea tubers were ethanol fractioned and used for ex vivo splenocyte proliferation assay or in vivo force-feeding of mice pre-treated with the chemotherapy agent 5-fluorouracil. Co-treatment with a 50-75% ethanol-partitioned fraction of the tuber extract of D. batatas (DsCE-II) and interleukin (IL)-2 resulted in a significantly higher rate of murine splenocyte cell proliferation ex vivo than treatment with DsCE-II or IL-2 alone. This DsCE-II fraction, which contains a polysaccharide with a high proportion of ß-1,4-linkage mannose (≥64%), also promoted the regeneration of specific progenitor cell populations in damaged bone marrow tissues of 5-fluorouracil-treated mice. Colony-forming unit (CFU) analyses demonstrated that the population of CFU-GM cells, but not CFU-GEMM or BFU-E cells, preferentially recovered to ~67% in the bone marrow of immune-suppressed mice fed with DsCE-II. DsCE-II efficacy level was ~85% of that obtained by subcutaneous administration of recombinant G-CSF proteins (5 µg kg(-1)) in mice tested in parallel. This study suggests that the DsCE-II fraction of D. batatas extract may be considered for further development as a dietary supplement for use alongside chemotherapy during cancer treatment.

Ethyl 4-hy-droxy-6-(4-hy-droxy-phen-yl)-4-trifluoro-methyl-2-sulfanyl-idene-1,3-diazinane-5-carboxyl-ate ethanol monosolvate.

The title compound, C(14)H(15)F(3)N(2)O(4)S·C(2)H(5)OH, was prepared by reaction of 4-hy-droxy-benzaldehyde, ethyl 4,4,4-trifluoro-3-oxobutano-ate and thio-urea. The hexa-hydro-pyrimidine ring adopts a half-chair conformation, the mean plane formed by the ring atoms excluding the C atom bonded to the eth-oxy-carbonyl group has an r.m.s. deviation of 0.0333 Å, and the dihedral angle between this plane and the benzene ring is 56.76 (5)°. The mol-ecular conformation is stabilized by an intra-molecular O-H⋯O hydrogen bond, generating an S(6) ring. The crystal structure is stabilized by inter-molecular O-H⋯O, O-H⋯S, N-H⋯O and N-H⋯S hydrogen bonds. The ethyl group of the ester unit is disordered over two positions, with an occupancy ratio of 0.757 (10):0.243 (10).

2-Benzyl-6-benz-yloxypyridazin-3(2H)-one.

In the title compound, C(18)H(16)N(2)O(2), the central pyridazine ring forms dihedral angles of 77.08 (5)° and 84.62 (5)° with the two benzene rings. The dihedral angle between the two benzene rings is 68.18 (4)°. A very weak intra-molecular C-H⋯N hydrogen bond and an intra-molecular C-H⋯π inter-action occur. The crystal structure is stabilized by weak inter-molecular C-H⋯O hydrogen bonds and weak C-H⋯π and π-π stacking inter-actions [centroid-centroid distance = 3.6867 (10) Å].