The Application of Sulfur Influences Microbiome of Soybean Rhizosphere and Nutrient-Mobilizing Bacteria in Andosol.

This study aimed to determine the effect of sulfur (S) application on a root-associated microbial community resulting in a rhizosphere microbiome with better nutrient mobilizing capacity. Soybean plants were cultivated with or without S application, the organic acids secreted from the roots were compared. High-throughput sequencing of 16S rRNA was used to analyze the effect of S on microbial community structure of the soybean rhizosphere. Several plant growth-promoting bacteria (PGPB) isolated from the rhizosphere were identified that can be harnessed for crop productivity. The amount of malic acid secreted from the soybean roots was significantly induced by S application. According to the microbiota analysis, the relative abundance of Polaromonas, identified to have positive association with malic acid, and arylsulfatase-producing Pseudomonas, were increased in S-applied soil. Burkholderia sp. JSA5, obtained from S-applied soil, showed multiple nutrient-mobilizing traits among the isolates. In this study, S application affected the soybean rhizosphere bacterial community structure, suggesting the contribution of changing plant conditions such as in the increase in organic acid secretion. Not only the shift of the microbiota but also isolated strains from S-fertilized soil showed PGPB activity, as well as isolated bacteria that have the potential to be harnessed for crop productivity.

Forest understories controlled the soil organic carbon stock during the fallow period in African tropical forest: a 13C analysis.

Soil organic carbon (SOC) dynamics after slash-burn agriculture are poorly understood in African tropical forest, though recent studies have revealed C4 grass invasion as a forest understory influences SOC dynamics after deforestation. This study aimed to quantify the relative SOC contribution of C4 and C3 plants separately through the sequential fallow periods of forest (cropland, or 4-7, 20-30, or >50 years of fallow forest) in the tropical forest of eastern Cameroon. We evaluated the SOC stock and natural 13C abundance for each layer. The SOC stock was largest in 4-7 years fallow forest (136.6 ± 8.8 Mg C ha-1; 100 cm depth, and C4:C3 = 58:42), and decreased with increasing fallow period. SOC from C4 plants was larger in the 4-7 and 20-30 years fallow forests (57.2-60.4 ± 5.8 Mg C ha-1; 100 cm depth), while it clearly decreased in >50 years fallow forest (35.0 ± 4.1 Mg C ha-1; 100 cm depth), resulting in the smallest SOC in this mature forest (106.4 ± 12.9 Mg C ha-1; 100 cm depth). These findings indicate that C4 grass understories contributed to the SOC restoration during early fallow succession in the tropical forest of eastern Cameroon.

[Evaluation of Patient Adherence to Anastrozole Therapy for Breast Cancer].

PURPOSE: There are many reports that describe the effectiveness of aromatase inhibitors as endocrine therapy after breast cancer surgery. However, there are few detailed evaluations of patient adherence to anastrozole(ANA)therapy. Here, we evaluated the adherence to ANA therapy in postoperative patients with primary breast cancer. METHODS: We investigated 102 postoperative patients with primary breast cancer without distant metastasis, who received ANA at JCHO Kyusyu Hospital. We calculated the medication continuation rate and disease-free survival at 5 years from the initiation of medication. The reasons for medication discontinuation and alternative drug therapy after ANA therapy discontinuation were also investigated. Re- sults: The 5-year continuation rate of ANA treatment alone was 79%(81/102). The rate of all patients who continued ANA treatment, including 9 who changed to other drugs, was 88%(90/102). The most frequent ANA discontinuation reasons were progressive disease(8 cases), arthritis(5 cases), and nausea(3 cases). The disease-free survival rate was 92%(94/102), and the overall survival rate was 97%(99/102). CONCLUSION: ANA showed a high continuation rate. Adherence of ANA in postoperative patients with breast cancer was well maintained, even when the treatment was changed to other drugs due to adverse events.

[Mediastinal Hemangioma with Literature Review;Report of a Case].

A 58-year-old woman visited a local physician for evaluation of collagen disease and screening computed tomography (CT) showed a posterior mediastinal tumor. After referral to our hospital, CT and magnetic resonance imaging (MRI) showed a mass, approximately 2 cm in diameter, located on the right of the 1st thoracic vertebra. Since a neurogenic tumor was suspected, thoracoscopic excision was performed. Surgical findings revealed a tumor between the 1st and 2nd ribs in the close vicinity of the right brachiocephalic vein. We severed blood vessels flowing into the tumor and removed it. The tumor, 25 mm in maximal diameter, was diagnosed as hemangioma by histological examinations. The prevalence of a hemangioma is less than 0.5% of all mediastinal tumors. Since it lacks specific imaging findings, its preoperative diagnosis is quite difficult to establish. Although a hemangioma in the mediastinum is quite rare, it should be included in the differential diagnoses of mediastinal tumors.

The characteristic structure of anti-HIV actinohivin in complex with three HMTG D1 chains of HIV-gp120.

The anti-HIV lectin actinohivin (AH) specifically interacts with HMTG (high-mannose-type glycan), which is attached to the glycoprotein gp120 of HIV-1 in a process in which the three branched mannotriose chains (D1, D2, and D3) of HMTG exhibit different binding affinities, it being estimated that that of D1 is the strongest, that of D3 is weaker, and that of D2 is undetectable. These properties have been ascribed to the stereochemical differences in linkages between the second and the third mannose residues of the three chains. In order to clarify the interaction geometry between AH and the major target D1, an X-ray determination of the crystal structure of AH in complex with D1-which is α(1,2)mannotriose composed of three mannose (Man) residues linked together only by α(1,2) bonding-has been performed. In each of the three D1-binding pockets of AH, two Man residues of D1 are accommodated at zones 1 and 2 in the pocket, in the same way as those found in the α(1,2)mannobiose-bound AH crystals. However, an OMIT map shows poor densities at both ends of the two residues. This suggests the existence of positional disorder of D1 in the pocket: the two zones are each occupied by two Man residues in two different modes, with mode A involving the Man1 and Man2 residues and mode B the Man2 and Man3 residues. In each mode, D1 is stabilized by adopting a double-bracket-shaped conformation through CH⋅⋅⋅O interactions. In mode B, however, the Man1 residue, which is the most sensitive residue to AH binding, protrudes wholly into the solvent region without contacts with AH. In mode A, in contrast, the Man3 residue interacts with the essential hydrophobic amino acid residues (Tyr and Leu conserved between the three pockets) of AH. Therefore, mode A is likely to be the one that occurs when whole HMTG is bound. In this mode, the two hydroxy groups (O3 and O4) of the Man2 residue are anchored in zone 2 by four hydrogen bonds with Asp, Asn, and Tyr residues of AH. In addition, it has been found that an isolated water molecule buried in the hydrophobic long loop bridges between Asp of AH and the hydroxy group of Man2 through hydrogen bonds. The most interesting feature is found in the interaction of the Man1 and Man3 residues with AH. All eight hydroxy groups of the two residues are completely exposed in the solvent region, whereas their hydrophobic parts make contacts with a Leu residue and two Tyr residues so that the shape of D1 and the surface of AH fit well over a wide area. These structural characteristics are potentially useful for development of AH to produce more effective antiretroviral drugs to suppress the infectious expansion of HIV/AIDS and to help expedite an end to the HIV/AIDS pandemic in the near future.

Peculiarity in crystal packing of anti-HIV lectin actinohivin in complex with α(1-2)mannobiose.

Previously, the anti-HIV lectin actinohivin (AH) was cocrystallized with the target α(1-2)mannobiose (MB) in the apparent space group P213. However, three MB-bound AH rotamers generated by ±120° rotations around the molecular pseudo-threefold rotation axis are packed randomly in the unit cell according to P212121 symmetry [Hoque et al. (2012). Acta Cryst. D68, 1671-1679]. It was found that the AH used for crystallization contains short peptides attached to the N-terminus [Suzuki et al. (2012). Acta Cryst. F68, 1060-1063], which cause packing disorder. In the present study, the fully mature homogeneous AH has been cocrystallized with MB into two new crystal forms at different pH. X-ray analyses of the two forms reveal that they have peculiar character in that the space groups are the same, P22121, and the unit-cell parameters are almost the same with the exception of the length of the a axis, which is doubled in one form. The use of homogeneous AH resulted in the absence of disorder in both crystals and an improvement in the resolution, thereby establishing the basis for AH binding to the target MB. In addition, the two crystal structures clarify the interaction modes between AH molecules, which is important knowledge for understanding the multiple binding effect generated when two AH molecules are linked together with a short peptide [Takahashi et al. (2011). J. Antibiot. 64, 551-557].

Minimum of the order parameter fluctuations of seismicity before major earthquakes in Japan.

It has been shown that some dynamic features hidden in the time series of complex systems can be uncovered if we analyze them in a time domain called natural time χ. The order parameter of seismicity introduced in this time domain is the variance of χ weighted for normalized energy of each earthquake. Here, we analyze the Japan seismic catalog in natural time from January 1, 1984 to March 11, 2011, the day of the M9 Tohoku earthquake, by considering a sliding natural time window of fixed length comprised of the number of events that would occur in a few months. We find that the fluctuations of the order parameter of seismicity exhibit distinct minima a few months before all of the shallow earthquakes of magnitude 7.6 or larger that occurred during this 27-y period in the Japanese area. Among the minima, the minimum before the M9 Tohoku earthquake was the deepest. It appears that there are two kinds of minima, namely precursory and nonprecursory, to large earthquakes.

Inhibition of infection and transmission of HIV-1 and lack of significant impact on the vaginal commensal lactobacilli by carbohydrate-binding agents.

OBJECTIVES: A selection of carbohydrate-binding agents (CBAs) with different glycan specificities were evaluated for their inhibitory effect against HIV infection and transmission, and their interaction with vaginal commensal bacteria. METHODS: Several assays were used for the antiviral evaluation: (i) cell-free virus infection of human CD4+ T lymphocyte C8166 cells; (ii) syncytium formation in co-cultures of persistently HIV-1-infected HUT-78/HIV-1 and non-infected CD4+ SupT1 cells; (iii) DC-SIGN-directed capture of HIV-1 particles; and (iv) transmission of DC-SIGN-captured HIV-1 particles to uninfected CD4+ C8166 cells. CBAs were also examined for their interaction with vaginal commensal lactobacilli using several viability, proliferation and adhesion assays. RESULTS: The CBAs showed efficient inhibitory activity in the nanomolar to low-micromolar range against four events that play a crucial role in HIV-1 infection and transmission: cell-free virus infection, fusion between HIV-1-infected and non-infected cells, HIV-1 capture by DC-SIGN and transmission of DC-SIGN-captured virus to T cells. As candidate microbicides should not interfere with the normal human microbiota, we examined the effect of CBAs against Lactobacillus strains, including a variety of vaginal strains, a gastrointestinal strain and several non-human isolates. None of the CBAs included in our studies inhibited the growth of these bacteria in several media, affected their viability or had any significant impact on their adhesion to HeLa cell monolayers. CONCLUSIONS: The CBAs in this study were inhibitory to HIV-1 in several in vitro infection and transmission models, and may therefore qualify as potential microbicide candidates. The lack of significant impact on commensal vaginal lactobacilli is an important property of these CBAs in view of their potential microbicidal use.

HIV-1 envelope trimer has similar binding characteristics for carbohydrate-binding agents as monomeric gp120.

The native HIV-1 Env complex consists of a gp120/gp41 trimer, but surface plasmon resonance (SPR)-directed binding studies for gp120-binding agents were almost exclusively performed on monomeric gp120. SPR-directed binding kinetics of monomeric gp120 and trimeric gp140 were investigated for a broad variety of envelope (Env)-binding agents. Similar kinetics for carbohydrate-binding agents (CBAs), the antibody 2G12 and sCD4 were observed, irrespective of the oligomeric state of gp120 that either contain the native mixture of complex and high-mannose N-glycans or that contain exclusively oligomannose N-glycans. The generally comparable kinetic properties of CBA, 2G12 and sCD4 binding to monomeric gp120 and trimeric gp140 indicate that monomeric gp120 is a good surrogate molecule for native HIV-1 Env trimer to investigate the binding affinities of Env-binding compounds.

Anti-inflammatory effect of berkeleyacetal C through the inhibition of interleukin-1 receptor-associated kinase-4 activity.

Berkeleyacetal C (BAC) isolated from Penicillium sp. which had isolated from a soil sample collected in Fukushima, inhibited NO production and induction of iNOS protein in RAW264.7 cells stimulated by the Toll-like receptor (TLR) 2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS). The other inflammatory mediator production by these stimulators was also suppressed by BAC in a concentration-dependent manner. BAC inhibited LPS- or PGN-activated nuclear translocation of nuclear factor (NF)-κB and MyD88-dependent signaling molecules. However, it showed no effect on LPS-induced nuclear translocation of interferon regulatory factor (IRF)-3, a MyD88-independent signaling molecule. To clarify the mechanistic basis for BAC ability to inhibit translocation of NF-κB and activated MyD88-dependent signaling molecules, we examined interleukin-1 receptor-associated kinase (IRAK)-4, existing to the most upstream on MyD88-dependent signaling molecules, in vitro kinase assay. BAC suppressed IRAK-4 kinase activity in a concentration-dependent manner. These findings suggest that BAC inhibits LPS- and PGN- induced NO production and iNOS expression by decreasing the level of the translocating of NF-κB in nuclear through inhibiting the kinase activity of IRAK-4 in inflammatory cells.

Structural insights into the specific anti-HIV property of actinohivin: structure of its complex with the α(1-2)mannobiose moiety of gp120.

Actinohivin (AH) is an actinomycete lectin with a potent specific anti-HIV activity. In order to clarify the structural evidence for its specific binding to the α(1-2)mannobiose (MB) moiety of the D1 chains of high-mannose-type glycans (HMTGs) attached to HIV-1 gp120, the crystal structure of AH in complex with MB has been determined. The AH molecule is composed of three identical structural modules, each of which has a pocket in which an MB molecule is bound adopting a bracket-shaped conformation. This conformation is stabilized through two weak C-H...O hydrogen bonds facilitated by the α(1-2) linkage. The binding features in the three pockets are quite similar to each other, in accordance with the molecular pseudo-threefold symmetry generated from the three tandem repeats in the amino-acid sequence. The shape of the pocket can accept two neighbouring hydroxyl groups of the O(3) and O(4) atoms of the equatorial configuration of the second mannose residue. To recognize these atoms through hydrogen bonds, an Asp residue is located at the bottom of each pocket. Tyr and Leu residues seem to block the movement of the MB molecules. Furthermore, the O(1) atom of the axial configuration of the second mannose residue protrudes from each pocket into an open space surrounded by the conserved hydrophobic residues, suggesting an additional binding site for the third mannose residue of the branched D1 chain of HMTGs. These structural features provide strong evidence indicating that AH is only highly specific for MB and would facilitate the highly specific affinity of AH for any glycoprotein carrying many HMTGs, such as HIV-1 gp120.

Crystallographic study of the interaction of the anti-HIV lectin actinohivin with the α(1-2)mannobiose moiety of gp120 HMTG.

Actinohivin (AH) is a new potent anti-HIV lectin of microbial origin. In order to modify it to produce a more efficient drug, its three-dimensional structure has previously been determined with and without the target α(1-2)mannobiose moiety of the high-mannose-type glycan (HMTG) attached to HIV-1 gp120. However, ambiguity remained in the structures owing to packing disorder that was possibly associated with peptide fragments attached at the N-terminus. To resolve these problems, the duration of cultivation of the AH-producing strain was examined and it was found that in a sample obtained from a 20 d culture the heterogeneous fragments were completely removed to produce mature AH with high homogeneity. In addition, the purification procedures were simplified in order to increase the yield of AH and the addition of solvents was also examined in order to increase the solubility of AH. AH thus obtained was successfully crystallized with high reproducibility in a different form to the previously obtained crystals. The crystal diffracted well to beyond 1.90 Å resolution and the crystallographic data suggested that it contained no packing disorder.

Targeting the hedgehog signaling pathway with interacting peptides to Patched-1.

BACKGROUND: The hedgehog (Hh) signaling pathway is aberrantly activated in many cancers. Overproduction of sonic hedgehog (Shh), a ligand in the Hh pathway, increases Hh signaling activity by inhibiting Patched-1 (Ptch1), a suppressive receptor in the Hh pathway. The purpose of this study was to establish a novel strategy for treating pancreatic cancer and other Hh-dependent cancers through control of the tumor-suppressive function of Ptch1. METHODS: We synthesized seven interacting peptides to the amino-acid sequence of the Ptch1 docking site for Shh. Human pancreatic cancer cell lines (AsPC-1, SUIT2) were cultured in the presence or absence of the peptides. Cell proliferation was assessed by cell counting and by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The activity of the Hh pathway was estimated by real-time polymerase chain reaction of the target gene product Gli1. To confirm their anti-tumor activity in vivo, the effect of the peptides in a mouse model of pancreatic cancer was determined. Finally, the Hh signaling activity of the xenograft was examined. RESULTS: Three of the interacting peptides to Ptch1 suppressed the proliferation of the two pancreatic cancer cell lines and decreased the expression of Gli1, both in vitro and in vivo. CONCLUSIONS: This study suggests that interacting peptides to Ptch1 may be a new tool for controlling the Hh-dependent growth of pancreatic cancer.

Semi-quantitative evaluation of CD44(+) /CD24(-) tumor cell distribution in breast cancer tissue using a newly developed fluorescence immunohistochemical staining method.

CD44(+) /CD24(-) tumor cells are reported to contain cancer stem cells in breast cancer. The main purpose of the present study is to develop an immunohistofluorescence method that can quantitatively analyze CD44(+) /CD24(-) tumor cell distribution in breast cancer tissue and help better define the role of CD44(+) /CD24(-) tumor cells in breast cancer. The samples used were from 21 primary breast cancer patients who underwent neoadjuvant chemotherapy and 17 cases with sentinel lymph nodes that had lymph node micrometastasis. CD44(+) /CD24(-) tumor cells were distinguished at a single cell level using improved triple-staining immunohistofluorescence and a simulated laser capture microdissection method. The percentage of CD44(+) /CD24(-) cells significantly increased following neoadjuvant chemotherapy treatment (0.93% and 2.78%, before and after, respectively, P = 0.0043). The percentage of CD44(+) /CD24(-) cells was also significantly high in micrometastatic sentinel lymph nodes (0.49% and 1.91%, primary tumors and lymph nodes, respectively, P = 0.0246). The CD44(+) /CD24(-) tumor cell distribution was heterogeneous in both breast cancer tissue and lymph node metastasis. In a xenograft model using immunodeficient mice, the hedgehog signaling inhibitor cyclopamine repressed the tumorigenicity of CD44(+) /CD24(-) cells. Our results suggest that this semi-quantitative immunohistochemical analysis is valuable for detecting a small population of cells in cancer tissues and that the hedgehog signaling pathway inhibitor cyclopamine is useful for regulating the CD44(+) /CD24(-) tumor cells in breast cancer.

The high mannose-type glycan binding lectin actinohivin: dimerization greatly improves anti-HIV activity.

The actinomycete-derived lectin actinohivin (AH) inhibits entry of HIV-1 to susceptible cells at low nM concentrations. The cooperative binding of three segments of AH to three high mannose-type glycans (HMTGs) of HIV-1 gp120 generates specific and strong anti-HIV activity. Dimerization of AH effectively improves anti-HIV activity by increasing the number of HMTG-binding pockets. AH dimers were prepared using an Escherichia coli expression system and their anti-syncytium formation and anti-HIV activities were evaluated. Each dimer was constructed by a head-to-tail fusion of two AH molecules, with or without a spacer. As a result, His-TEV-AH/RTB(132-143)/AH, which has the residues 132-143 of ricin toxin B-chain (RTB) as a spacer, had 20-fold higher anti-syncytium formation activity and also exhibited 2-30-fold higher anti-HIV activity than AH against various clinically isolated HIV-1 strains, including drug-resistant ones. Mutation analysis implies that all six HMTG-binding pockets of the dimer participated in HMTG binding. Several AH dimers with different spacer sequences showed diverse activities, suggesting that the spacer sequence is an important factor to create higher anti-HIV activity. A dimer with improved anti-HIV activity would be a good candidate for investigation as a potential microbicide to prevent HIV transmission.

Hedgehog signaling pathway mediates the progression of non-invasive breast cancer to invasive breast cancer.

The purpose of this study is to clarify the contribution of the Hedgehog signaling pathway (Hh pathway) to the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). A total of 149 surgically resected mammary disease specimens and 12 sentinel lymph nodes with micro-metastasis (Ly-met) were studied. The degree of Hh pathway activation was estimated from the Gli1 nuclear staining ratio (%Gli1 nuclear translocation) in cancer cells. The invasiveness of breast cancer cells was determined using Matrigel assays. A serial increase of %Gli1 nuclear translocation to IDC from non-neoplastic diseases was confirmed. In tumor specimens, %Gli1 nuclear translocation correlated with the invasiveness of each type of mammary disease and also correlated with invasion-related histopathological parameters. The %Gli1 nuclear translocation in lymph nodes with micro-metastasis was similar to that in primary sites and higher than that in DCIS with microinvasion and DCIS. Blockade of the Hh pathway decreased the invasiveness of breast cancer cells. In IDC, %Gli1 nuclear translocation correlated with the expression of estrogen receptor-α. Estrogen increased %Gli1 nuclear translocation and the invasiveness of estrogen receptor-α-positive cells. The Hh pathway mediates progression from a non-invasive phenotype to an invasive phenotype and %Gli1 nuclear translocation may be useful as a predictive marker for evaluating the ability of invasiveness.

Enantioselective total synthesis of (-)- and (+)-petrosin.

The enantioselective total synthesis of (-)- and (+)-petrosin is described. The union of two key segments was executed by Suzuki-Miyaura coupling. The quinolizidine rings were stereoselectively constructed via a diastereoselective Mannich reaction and an aza-Michael reaction. The 16-membered ring was constructed by ring-closing metathesis with the second-generation Grubbs catalyst.

Actinohivin: specific amino acid residues essential for anti-HIV activity.

Actinohivin (AH) is a microbial lectin containing 114 amino acids, which inhibits human immunodeficiency virus (HIV) infection. This effect is brought about by its specific binding to Man-α(1-2)-Man unit(s) of high-mannose type glycan (HMTG) bound to HIV gp120. The recently determined crystal structure of AH suggests that three repeated segments (the residue numbers 1-38, 39-76 and 77-114 for segments 1, 2 and 3, respectively) form three sugar-binding pockets to accommodate Man-α(1-2)-Man units. The strong specific binding of AH to gp120 is considered to be due to multivalent interaction of the three sugar-binding pockets with three HMTGs of gp120 via the 'cluster effect' of lectin. It remains to be seen which residues of the sugar-binding pockets are essential for acceptance of Man-α(1-2)-Man. To identify the amino acid residues critical for anti-HIV effect, we performed mutational analysis. Mutant AHs were subjected to enzyme-linked immunosorbent assay testing for gp120-binding activity and to syncytium formation assay. As a result, it was revealed that Asp15, Tyr23, Leu25, Asn28 and Tyr32 in segment 1, Tyr61 in segment 2 and Tyr99 in segment 3 are essential for anti-HIV activity. The conserved residues, Asp53, Leu63, Asn66 and Tyr70, in segment 2 and, Asp91, Leu101, Asn104 and Tyr108, in segment 3 are also necessary. Furthermore, aromatic residues at positions 23 and 32 are required for creation of potency. These data will be useful for predicting the detailed mechanism of AH-Man-α(1-2)-Man/HMTG/gp120 interaction by computational analysis and for possible development of more potent microbicides for prevention of HIV transmission.

Inhibition of Wnt signaling pathway decreases chemotherapy-resistant side-population colon cancer cells.

BACKGROUND: The prognosis of advanced or recurrent colorectal cancer is still poor. Dye-effluxing side population (SP) colon cancer cells are reportedly resistant to chemotherapeutic agents. Most sporadic colorectal cancers involve constitutive activation of the Wnt signaling pathway. In this study, we examined the effect of the Wnt signaling on SP cells and the possibility that inhibition of Wnt signaling may decrease the resistance to chemotherapeutic drugs in the human colon cancer cells. MATERIALS AND METHODS: Drug resistance of SP cells to 5-fluorouracil (5-FU) and irinotecan, decrease of SP cells by the Wnt signaling inhibition and activation of the Wnt signaling of the sorted SP cells were examined using the SW480 colon cancer cell line. mRNA expressions of ATP-binding cassette (ABC) transporters when Wnt signaling was inhibited were evaluated with real-time PCR using colon cancer cell lines (SW480, DLD-1, HCT116, HT29 and LOVO). The sensitivity to irinotecan and paclitaxel when the Wnt signaling was inhibited was investigated using SW480. Inhibition of Wnt signaling was performed by siRNA of beta-catenin. RESULTS: SP cells showed more resistance to 5-FU and irinotecan, and higher activation of the Wnt signaling pathway, than non-SP cells. Silencing of beta-catenin decreased significantly more SP cells than non-SP cells. Expression of ABC transporter genes, such as ABCB1 and ABCG2, was significantly higher in SP cells than non-SP cells. Silencing of beta-catenin decreased transcription of these ABC transporter genes; beta-catenin-silenced cells became relatively sensitive to paclitaxel and irinotecan. CONCLUSION: These results indicate that inhibiting the Wnt signaling pathway may be a fruitful strategy for targeting chemotherapy-resistant colon cancer cells, including SP cells.

HIV-1 neutralization profile and plant-based recombinant expression of actinohivin, an Env glycan-specific lectin devoid of T-cell mitogenic activity.

The development of a topical microbicide blocking the sexual transmission of HIV-1 is urgently needed to control the global HIV/AIDS pandemic. The actinomycete-derived lectin actinohivin (AH) is highly specific to a cluster of high-mannose-type glycans uniquely found on the viral envelope (Env). Here, we evaluated AH's candidacy toward a microbicide in terms of in vitro anti-HIV-1 activity, potential side effects, and recombinant producibility. Two validated assay systems based on human peripheral blood mononuclear cell (hPBMC) infection with primary isolates and TZM-bl cell infection with Env-pseudotyped viruses were employed to characterize AH's anti-HIV-1 activity. In hPMBCs, AH exhibited nanomolar neutralizing activity against primary viruses with diverse cellular tropisms, but did not cause mitogenicity or cytotoxicity that are often associated with other anti-HIV lectins. In the TZM-bl-based assay, AH showed broad anti-HIV-1 activity against clinically-relevant, mucosally transmitting strains of clades B and C. By contrast, clade A viruses showed strong resistance to AH. Correlation analysis suggested that HIV-1's AH susceptibility is significantly linked to the N-glycans at the Env C2 and V4 regions. For recombinant (r)AH expression, we evaluated a tobacco mosaic virus-based system in Nicotiana benthamiana plants as a means to facilitate molecular engineering and cost-effective mass production. Biochemical analysis and an Env-mediated syncytium formation assay demonstrated high-level expression of functional rAH within six days. Taken together, our study revealed AH's cross-clade anti-HIV-1 activity, apparent lack of side effects common to lectins, and robust producibility using plant biotechnology. These findings justify further efforts to develop rAH toward a candidate HIV-1 microbicide.