FOURIER TRANSFORM INFRARED SPECTROSCOPY OF MICROALGAE AS A NOVEL TOOL FOR BIODIVERSITY STUDIES,SPECIES IDENTIFICATION,AND THE ASSESSMENT OF WATER QUALITY1

Fourier transform infrared (FTIR) spectrometry was used to study the spectral features of 12 eukaryotic and two prokaryotic species of microalgae. The algae were cultured in liquid media containing either NO₃^? or NH₄⁺ as the sole N‐source; for the NH₄⁺ treatment, the algae were subjected to short‐term (24 h) or long‐term (1 month) incubations; for the hypersaline species, cells were also grown in the presence of 2 M NaCl. Over 500 spectra, acquired from at least three distinct cultures for each species, in each growth regime, were subjected to hierarchical cluster analysis (HCA) and were successfully separated according to their taxonomy, showing that the overall spectra were characteristic of each species and that this technique could be fruitfully employed to separate microalgal species living in a similar condition (as would be the case for a natural assemblage). In addition, in most cases, it was possible to differentiate between algae subjected to different growth treatments although belonging to the same species. We also demonstrated that it is possible to accurately identify species and determine the nutritional status of their environment of origin (e.g., N‐source), provided that suitable FTIR spectral libraries are available. This study aims to provide the basis for the development of rapid, easy, and inexpensive methods for the evaluation of biodiversity in natural phytoplankton samples and to monitor the water quality of natural environments.     

Determinants of Vaccine Immunogenicity in HIV-Infected Pregnant Women: Analysis of B and T Cell Responses to Pandemic H1N1 Monovalent Vaccine

Influenza infections have high frequency and morbidity in HIV-infected pregnant women, underscoring the importance of vaccine-conferred protection. To identify the factors that determine vaccine immunogenicity in this group, we characterized the relationship of B- and T-cell responses to pandemic H1N1 (pH1N1) vaccine with HIV-associated immunologic and virologic characteristics.pH1N1 and seasonal-H1N1 (sH1N1) antibodies were measured in 119 HIV-infected pregnant women after two double-strength pH1N1 vaccine doses. pH1N1-IgG and IgA B-cell FluoroSpot, pH1N1- and sH1N1-interferon γ (IFNγ) and granzyme B (GrB) T-cell FluoroSpot, and flow cytometric characterization of B- and T-cell subsets were performed in 57 subjects.pH1N1-antibodies increased after vaccination, but less than previously described in healthy adults. pH1N1-IgG memory B cells (Bmem) increased, IFNγ-effector T-cells (Teff) decreased, and IgA Bmem and GrB Teff did not change. pH1N1-antibodies and Teff were significantly correlated with each other and with sH1N1-HAI and Teff, respectively, before and after vaccination. pH1N1-antibody responses to the vaccine significantly increased with high proportions of CD4+, low CD8+ and low CD8+HLADR+CD38+ activated (Tact) cells. pH1N1-IgG Bmem responses increased with high proportions of CD19+CD27+CD21- activated B cells (Bact), high CD8+CD39+ regulatory T cells (Treg), and low CD19+CD27-CD21- exhausted B cells (Bexhaust). IFNγ-Teff responses increased with low HIV plasma RNA, CD8+HLADR+CD38+ Tact, CD4+FoxP3+ Treg and CD19+IL10+ Breg.In conclusion, pre-existing antibody and Teff responses to sH1N1 were associated with increased responses to pH1N1 vaccination in HIV-infected pregnant women suggesting an important role for heterosubtypic immunologic memory. High CD4+% T cells were associated with increased, whereas high HIV replication, Tact and Bexhaust were associated with decreased vaccine immunogenicity. High Treg increased antibody responses but decreased Teff responses to the vaccine. The proportions of immature and transitional B cells did not affect the responses to vaccine. Increased Bact were associated with high Bmem responses to the vaccine.     

A NEWLY ANTI-Streptococcus suis BACTERIOCIN PRODUCING STRAIN FROM UNWEANED PIGLETS FECAL MATTER: ISOLATION, PRELIMINARY IDENTIFICATION, AND OPTIMIZATION OF MEDIUM COMPOSITION FOR ENHANCED BACTERIOCIN PRODUCTION

A newly isolated anti-Streptococcus suis bacteriocin-producing strain LPL1-5 was obtained from healthy unweaned piglets' fecal matter, and was designated as Lactobacillus pentosus LPL1-5 based on morphology, biochemical properties, and 16S rDNA sequencing analysis. The medium composition for enhanced bacteriocin production by L. pentosus LPL1-5 was optimized by statistical methodology. Yeast extract, K(2)HPO(4)?·?3H(2)O, and MnSO(4)?·?H(2)O were identified as significant components influencing pentocin LPL1-5 production using the Plackett-Burman method. Response surface methodology was applied for further optimization. The concentrations of medium components for enhanced pentocin LPL1-5 production were as follows (g/L): lactose 20.00, tryptone 10.00, beef extract 10.00, yeast extract 14.00, MnSO(4)?·?H(2)O 0.84, K(2)HPO(4)?·?3H(2)O 4.92, triammonium citrate 2.00, Na-acetate 5.00, MgSO(4)?·?7H(2)O 0.58, Tween 80 1.00. Under the optimized condition, a value of 3154.65?±?27.93 IU/mL bacteriocin activity was achieved, which was 4.2-fold that of the original medium.     

A NEWLY ANTI-Streptococcus suis BACTERIOCIN PRODUCING STRAIN FROM UNWEANED PIGLETS FECAL MATTER: ISOLATION,PRELIMINARY IDENTIFICATION,AND OPTIMIZATION OF MEDIUM COMPOSITION FOR ENHANCED BACTERIOCIN PRODUCTION

A newly isolated anti-Streptococcus suis bacteriocin-producing strain LPL1-5 was obtained from healthy unweaned piglets' fecal matter, and was designated as Lactobacillus pentosus LPL1-5 based on morphology, biochemical properties, and 16S rDNA sequencing analysis. The medium composition for enhanced bacteriocin production by L. pentosus LPL1-5 was optimized by statistical methodology. Yeast extract, K₂HPO₄ · 3H₂O, and MnSO₄ · H₂O were identified as significant components influencing pentocin LPL1-5 production using the Plackett–Burman method. Response surface methodology was applied for further optimization. The concentrations of medium components for enhanced pentocin LPL1-5 production were as follows (g/L): lactose 20.00, tryptone 10.00, beef extract 10.00, yeast extract 14.00, MnSO₄ · H₂O 0.84, K₂HPO₄ · 3H₂O 4.92, triammonium citrate 2.00, Na-acetate 5.00, MgSO₄ · 7H₂O 0.58, Tween 80 1.00. Under the optimized condition, a value of 3154.65 ± 27.93 IU/mL bacteriocin activity was achieved, which was 4.2-fold that of the original medium.     

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES1

In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high‐resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two‐step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1‐5.8S‐ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species.     

Discovery of subtype-selective NMDA receptor ligands: 4-benzyl-1-piperidinylalkynylpyrroles, pyrazoles and imidazoles as NR1A/2B antagonists.

4-Benzyl-1-[4-(1H-imidazol-4-yl)but-3-ynyl]piperidine (8) has been identified as a potent antagonist of the NR1A/2B subtype of the NMDA receptor. When dosed orally, this compound potentiates the effects of L-DOPA in the 6-hydroxydopamine-lesioned rat, a model of Parkinson's disease.     

Cationic Lipid-Formulated DNA Vaccine against Hepatitis B Virus: Immunogenicity of MIDGE-Th1 Vectors Encoding Small and Large Surface Antigen in Comparison to a Licensed Protein Vaccine

Currently marketed vaccines against hepatitis B virus (HBV) based on the small (S) hepatitis B surface antigen (HBsAg) fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L) protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals.     

Pyrido[2,3-d]pyrimidines: Discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A inhibitors

DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy. From a data-mining effort, the team has discovered analogues of pyrido[2,3-d]pyrimidines as potent enantio-selective inhibitors of DYRK1B. Cells treated with a tool compound from this series showed the same cellular effects as down regulation of DYRK1B with siRNA. Such effects are consistent with the proposed mechanism of action. Progress of the SAR study is presented.     

Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin: IDENTIFICATION OF THE HEPARIN-BINDING MOTIF OF p17 AS A TARGET FOR THE DEVELOPMENT OF MULTITARGET ANTAGONISTS*

Once released by HIV⁺ cells, p17 binds heparan sulfate proteoglycans (HSPGs) and CXCR1 on leukocytes causing their dysfunction. By exploiting an approach integrating computational modeling, site-directed mutagenesis of p17, chemical desulfation of heparin, and surface plasmon resonance, we characterized the interaction of p17 with heparin, a HSPG structural analog, and CXCR1. p17 binds to heparin with an affinity (K_d = 190 nm) that is similar to those of other heparin-binding viral proteins. Two stretches of basic amino acids (basic motifs) are present in p17 N and C termini. Neutralization (Arg→Ala substitution) of the N-terminal, but not of the C-terminal basic motif, causes the loss of p17 heparin-binding capacity. The N-terminal heparin-binding motif of p17 partially overlaps the CXCR1-binding domain. Accordingly, its neutralization prevents also p17 binding to the chemochine receptor. Competition experiments demonstrated that free heparin and heparan sulfate (HS), but not selectively 2-O-, 6-O-, and N-O desulfated heparins, prevent p17 binding to substrate-immobilized heparin, indicating that the sulfate groups of the glycosaminoglycan mediate p17 interaction. Evaluation of the p17 antagonist activity of a panel of biotechnological heparins derived by chemical sulfation of the Escherichia coli K5 polysaccharide revealed that the highly N,O-sulfated derivative prevents the binding of p17 to both heparin and CXCR1, thus inhibiting p17-driven chemotactic migration of human monocytes with an efficiency that is higher than those of heparin and HS. Here, we characterized at a molecular level the interaction of p17 with its cellular receptors, laying the basis for the development of heparin-mimicking p17 antagonists.     

NUCLEIC ACID COMPONENTS AND THEIR ANALOGUES: A NOVEL AND EFFICIENT METHOD FOR THE SYNTHESIS OF A NEW CLASS OF BIPYRIDYL AND BIHETEROCYCLIC-NITRO GEN THIOGLYCOSIDES FROM PYRIDINE-2(1H)-THIONES

ABSTRACT

A novel synthesis of a new class of bipyridyl and biheterocyclic-nitrogen thioglycosides utilizing the reactions of heterocyclic substituted pyridine-2(1H)-thiones and α-bromoglucose or α-bromogalactose tetraacetate as starting components is described.     

The PRY/SPRY/B30.2 Domain of Butyrophilin 1A1 (BTN1A1) Binds to Xanthine Oxidoreductase: IMPLICATIONS FOR THE FUNCTION OF BTN1A1 IN THE MAMMARY GLAND AND OTHER TISSUES*

Butyrophilin 1A1 (BTN1A1) and xanthine oxidoreductase (XOR) are highly expressed in the lactating mammary gland and are secreted into milk associated with the milk fat globule membrane (MFGM). Ablation of the genes encoding either protein causes severe defects in the secretion of milk lipid droplets, suggesting that the two proteins may function in the same pathway. Therefore, we determined whether BTN1A1 and XOR directly interact using protein binding assays, surface plasmon resonance analysis, and gel filtration. Bovine XOR bound with high affinity in a pH- and salt-sensitive manner (K_D = 101 ± 31 nm in 10 mm HEPES, 150 mm NaCl, pH 7.4) to the PRY/SPRY/B30.2 domain in the cytoplasmic region of bovine BTN1A1. Binding was stoichiometric, with one XOR dimer binding to either two BTN1A1 monomers or one dimer. XOR bound to BTN1A1 orthologs from mice, humans, or cows but not to the cytoplasmic domains of the closely related human paralogs, BTN2A1 or BTN3A1, or to the B30.2 domain of human RoRet (TRIM 38), a protein in the TRIM family. Analysis of the protein composition of the MFGM of wild type and BTN1A1 null mice showed that most of the XOR in mice lacking BTN1A1 was released from the MFGM in a soluble form when the milk lipid droplets were disrupted to prepare membrane, compared with wild-type mice, in which most of the XOR remained membrane-bound. Thus BTN1A1 functions in vivo to stabilize the association of XOR with the MFGM by direct interactions through the PRY/SPRY/B30.2 domain. The potential significance of BTN1A1/XOR interactions in the mammary gland and other tissues is discussed.Members of the butyrophilin (BTN)³ gene family are attracting increasing attention because they may play multifunctional roles in diverse physiologies, including lactation (1, 2), selection and regulation of T-cells in the immune system (3–6), and modulation of autoimmune disease (7–9). BTN proteins have the canonical structures of cell surface receptors, which, after an N-terminal signal sequence, generally comprise two exoplasmic Ig folds (10, 11), a membrane anchor and a cytoplasmic domain consisting of a stem region, a PRY/SPRY/B30.2 domain (12, 13), and a cytoplasmic tail at the C terminus (14).The eponymous BTN1A1 protein has been linked to the secretion of milk lipid droplets because it is highly expressed in the mammary epithelium during lactation and is incorporated into the surface membrane coat surrounding cytoplasmic lipid droplets (the milk fat globule membrane (MFGM)) as they bud into milk from the apical surface (15). Furthermore, ablation of the Btn1a1 gene disrupts lipid secretion, causing the accumulation of large pools of triacylglycerol in the cytoplasm of Btn1a1 null mice (1). In a different context, dietary exposure to BTN1A1 in dairy products has been associated with modulation of the autoimmune disease multiple sclerosis because of structural similarities between the IgI fold of BTN1A1 (16) and the IgV fold of myelin oligodendrocyte glycoprotein (MOG) (17) an antigen on the myelin nerve sheath that is a target for autoantibodies in multiple sclerosis patients (8–10).Potential interactions between the exoplasmic Ig folds of several BTN proteins, and putative receptors on immune cells are postulated to regulate positive selection of epidermal γδ-T cells in the case of Skint1 (6) and suppress T-cell activation in the case of BTNL2 (4, 5). In addition, BTN2A1 binds to the C-type lectin, DC-SIGN, on immature dendritic cells (18), and proteins in the BTN3A1–3 subfamily bind to an unidentified ligand on various immune cells (19).Interactions between the cytoplasmic domain of BTN and intracellular proteins have not been investigated in any detail. The intracellular region of most BTNs is dominated by the B30.2 or the PRY/SPRY domain, which comprises two sheets of antiparallel β-strands folded into a β- sandwich, which in some proteins is contiguous at the N terminus with one or two α-helices (20–24) (for a discussion of the relationship between PRY, SPRY, and B30.2 domains, see Ref. (25)). This domain (here abbreviated as B30.2),⁴ is postulated to serve as a protein-binding module, by which proteins interact through the extended surface loops that adjoin individual β-strands (22).One protein that may bind to the cytoplasmic region of BTN proteins (and the B30.2 domain) is the redox enzyme, xanthine oxidoreductase (XOR),⁵ because it was shown to bind to the cytoplasmic domain of mouse BTN1A1 in an in vitro binding assay (26). Furthermore, one XOR-deficient mouse strain (Xdh^+/−) (27) displayed a lactation phenotype similar to that of Btn1a1 null mice (1), suggesting that the two proteins may be functionally linked by direct interaction. These conclusions, however, have been challenged, because XOR does not co-localize with BTN1A1 in immunolabeled freeze-fractured replicates of secreted milk lipid droplets (28), and a second mouse strain deficient in XOR does not appear to have an altered lactation phenotype (29).In this paper, we devise in vivo and in vitro assays to show that the cytoplasmic domain of BTN1A1 binds to XOR via the B30.2 domain and that BTN1A1 is required for the stable association of XOR with the MFGM in vivo. Furthermore, interaction with XOR appears to be limited to BTN1A1 orthologs. These results are discussed in the context of potential functions of BTN1A1 in the mammary gland and other tissues and the relationship of BTN1A1 to other BTN family members.     

Identification of an Htm1 (EDEM)-dependent,Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) Pathway in Saccharomyces cerevisiae: APPLICATION OF A NOVEL ASSAY FOR GLYCOPROTEIN ERAD*

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related α-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.     

Discovery of marinopyrrole A (maritoclax) as a selective Mcl-1 antagonist that overcomes ABT-737 resistance by binding to and targeting Mcl-1 for proteasomal degradation

The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-X(L) and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-X(L) with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-X(L), and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-X(L)-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ～60- to 2000-fold at 1-2 μM. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation.     

Ceramide Synthase Inhibition by Fumonisin B1 Causes Accumulation of 1-Deoxysphinganine: A NOVEL CATEGORY OF BIOACTIVE 1-DEOXYSPHINGOID BASES AND 1-DEOXYDIHYDROCERAMIDES BIOSYNTHESIZED BY MAMMALIAN CELL LINES AND ANIMALS*

Fumonisin B₁ (FB₁) is a mycotoxin that inhibits ceramide synthases (CerS) and causes kidney and liver toxicity and other disease. Inhibition of CerS by FB₁ increases sphinganine (Sa), Sa 1-phosphate, and a previously unidentified metabolite. Analysis of the latter by quadrupole-time-of-flight mass spectrometry assigned an m/z = 286.3123 in positive ionization mode, consistent with the molecular formula for deoxysphinganine (C₁₈H₄₀NO). Comparison with a synthetic standard using liquid chromatography, electrospray tandem mass spectrometry identified the metabolite as 1-deoxysphinganine (1-deoxySa) based on LC mobility and production of a distinctive fragment ion (m/z 44, CH₃CH=NH ⁺₂) upon collision-induced dissociation. This novel sphingoid base arises from condensation of alanine with palmitoyl-CoA via serine palmitoyltransferase (SPT), as indicated by incorporation of l-[U-¹³C]alanine into 1-deoxySa by Vero cells; inhibition of its production in LLC-PK₁ cells by myriocin, an SPT inhibitor; and the absence of incorporation of [U-¹³C]palmitate into 1-[¹³C]deoxySa in LY-B cells, which lack SPT activity. LY-B-LCB1 cells, in which SPT has been restored by stable transfection, however, produce large amounts of 1-[¹³C]deoxySa. 1-DeoxySa was elevated in FB₁-treated cells and mouse liver and kidney, and its cytotoxicity was greater than or equal to that of Sa for LLC-PK₁ and DU-145 cells. Therefore, this compound is likely to contribute to pathologies associated with fumonisins. In the absence of FB₁, substantial amounts of 1-deoxySa are made and acylated to N-acyl-1-deoxySa (i.e. 1-deoxydihydroceramides). Thus, these compounds are an underappreciated category of bioactive sphingoid bases and “ceramides” that might play important roles in cell regulation.Fumonisins (FB)² cause diseases of horses, swine, and other farm animals and are regarded to be potential risk factors for human esophageal cancer (1) and, more recently, birth defects (2). Studies of this family of mycotoxins, and particularly of the highly prevalent subspecies fumonisin B₁ (FB₁) (reviewed in Refs. 1 and 2), have established that FB_1, is both toxic and carcinogenic for laboratory animals, with the liver and kidney being the most sensitive target organs (3, 4). Other FB are also toxic, but their carcinogenicity is unknown.FB are potent inhibitors of ceramide synthase(s) (CerS) (5), the enzymes responsible for acylation of sphingoid bases using fatty acyl-CoA for sphingolipid biosynthesis de novo and recycling pathways (6). As a consequence of this inhibition, the substrates sphinganine (Sa) and, usually to a lesser extent, sphingosine (So), accumulate and are often diverted to sphinganine 1-phosphate (Sa1P) and sphingosine 1-phosphate (S1P), respectively (7), while the product N-acylsphinganines (dihydroceramides), N-acylsphingosines (ceramides, Cer), and more complex sphingolipids decrease (5, 7). This disruption of sphingolipid metabolism has been proposed to be responsible for the toxicity, and possibly carcinogenicity, of FB, based on mechanistic studies with cells in culture (5, 7–9). This has been borne out by a number of animal feeding studies that have correlated the elevation of Sa in blood, urine, liver, and kidney with liver and kidney toxicity (4, 7, 10, 11).Most of the mechanistic studies have focused on the accumulation of free Sa and other sphingoid bases, because these compounds are highly cytotoxic, although the large number of bioactive metabolites in this pathway make it likely that multiple mediators may participate (7, 9). Nonetheless, inhibition of serine palmitoyltransferase (SPT), the initial enzyme of de novo sphingolipid biosynthesis, reverses the increased apoptosis and altered cell growth induced by FB₁ treatment (12–19). Therefore, it is likely that these effects of FB₁ are due to the accumulation of cytotoxic intermediate(s) rather than depletion of downstream metabolites, because the latter also occurs when SPT is inhibited.In studies of the effects of FB₁ on the renal cell line LLC-PK₁ (20),³ we have noted that in addition to the elevation of Sa and So, there is a large increase in an unidentified species that appears to be a sphingoid base, because it is extracted by organic solvents, derivatized with ortho-phthalaldehyde (OPA), and eluted from reverse-phase liquid chromatography (LC) in the sphingoid base region. Herein we report: (i) the isolation and characterization of this novel sphingoid base as 1-deoxysphinganine (1-deoxySa); (ii) that its origin is the utilization of alanine instead of serine by SPT as well as that the N-acyl-derivatives of 1-deoxySa (1-deoxydihydroceramides (1-deoxyDHCer)) are normally found in mammalian cells; (iii) that 1-deoxySa has cytotoxicity comparable to other sphingoid bases elevated by FB₁; and (iv) that 1-deoxySa is not only elevated in cells in culture but also in tissues of animals exposed to dietary FB and, therefore, might contribute to diseases caused by these mycotoxins.     

Modulation of A1 and A2B adenosine receptor activity: a new strategy to sensitise glioblastoma stem cells to chemotherapy

Therapies that target the signal transduction and biological characteristics of cancer stem cells (CSCs) are innovative strategies that are used in combination with conventional chemotherapy and radiotherapy to effectively reduce the recurrence and significantly improve the treatment of glioblastoma multiforme (GBM). The two main strategies that are currently being exploited to eradicate CSCs are (a) chemotherapeutic regimens that specifically drive CSCs toward cell death and (b) those that promote the differentiation of CSCs, thereby depleting the tumour reservoir. Extracellular purines, particularly adenosine triphosphate, have been implicated in the regulation of CSC formation, but currently, no data on the role of adenosine and its receptors in the biological processes of CSCs are available. In this study, we investigated the role of adenosine receptor (AR) subtypes in the survival and differentiation of CSCs isolated from human GBM cells. Stimulation of A₁AR and A_2BAR had a prominent anti-proliferative/pro-apoptotic effect on the CSCs. Notably, an A₁AR agonist also promoted the differentiation of CSCs toward a glial phenotype. The differential effects of the two AR agonists on the survival and/or differentiation of CSCs may be ascribed to their distinct regulation of the kinetics of ERK/AKT phosphorylation and the expression of hypoxia-inducible factors. Most importantly, the AR agonists sensitised CSCs to the genotoxic activity of temozolomide (TMZ) and prolonged its effects, most likely through different mechanisms, are as follows: (i) by A_2BAR potentiating the pro-apoptotic effects of TMZ and (ii) by A₁AR driving cells toward a differentiated phenotype that is more sensitive to TMZ. Taken together, the results of this study suggested that the purinergic system is a novel target for a stem cell-oriented therapy that could reduce the recurrence of GBM and improve the survival rate of GBM patients.Glioblastoma multiforme (GBM), classified as grade IV on the World Health Organization scale,¹ is the most common type of primary malignant brain tumour.² The current therapeutic strategy includes surgery followed by radiation and chemotherapy using temozolomide (TMZ). This therapeutic approach slightly improves the survival rate of GBM patients, but their prognosis remains poor and most patients die of tumour recurrence.³ The causes of the recurrence of GBM are complex and include the high proliferative index of the tumour cells and their resistance to chemotherapy and radiotherapy, particularly in the case of the cancer stem cells (CSCs). These cells have been proposed to not only initiate the genesis of GBM and contribute to its highly proliferative nature, but to also be the basis for its recurrences following treatment. Moreover, it has been reported that the most aggressive or refractory cancers contain the highest number of CSCs.^{4, 5, 6}These findings suggest that innovative stem cell-orientated therapy may be an effective strategy to reduce tumour recurrence and significantly improve GBM treatment outcomes.^{7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18} This type of therapy may not be easy to implement because CSCs have been shown to have a low level of reactive oxygen species¹⁹ and to be more resistant to ionising radiation,²⁰ vincristine,²¹ hypoxia and other chemotherapeutics²² compared with non-CSCs. In contrast, the preferential elimination of the CSC population may contribute to the effectiveness of TMZ, which is the most effective pharmacologic agent used in glioma treatment;²³ however, the activity of TMZ appears to be short lived because the drug causes the reversible blockage of the cell cycle of CSCs.²⁴ Moreover, long-term TMZ therapy results in the occurrence of drug-resistant GBM cells,²⁵ indicating the need to develop distinct strategies to overcome this resistance.Extracellular purines have been implicated in several aspects of GBM biology, such as proliferation,²⁶ migration,²⁷ invasion²⁸ and death.²⁹ The concentration of adenosine in the extracellular fluid of glioma tissue was reported to be in the low micromolar range,³⁰ which is sufficiently high to stimulate all the four of the adenosine receptor (AR) subtypes (A₁, A_2A, A_2B and A₃).³¹ Each of the ARs have a pivotal role in the control of tumour growth and invasiveness^{32, 33, 34} but to date, no data on their role in CSC biology are available. Recently, it was demonstrated that treatment with adenosine triphosphate reduced the rate of sphere formation by glioma cells and that purinergic receptors are differentially expressed in spheres of tumour cells and adherent cells.³³ In this study, we investigated the role of AR subtypes in the survival and differentiation of CSCs. Globally, our data clarified the role of each AR subtype in CSC functionality and suggested that the purinergic system is a novel pharmacological target for the development of new anti-CSC therapies, particularly those aimed at the treatment of GBM recurrences.     

Engineering glycoprotein B of bovine herpesvirus 1 to function as transporter for secreted proteins: a new protein expression approach

Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1) is essential for BHV-1 replication and is required for membrane fusion processes leading to virus penetration into the target cell and direct spreading of BHV-1 from infected to adjacent noninfected cells. Like many of the herpesvirus gB homologs, BHV-1 gB is proteolytically processed by furin, an endoproteinase localized in the trans-Golgi network. Cleavage by furin is a common mechanism for the activation of a number of viral fusion (F) proteins. Among these, the F proteins of both human and bovine respiratory syncytial virus (RSV) have the so-far unique feature that cleavage of the respective F protein precursors occurs at two furin recognition sites, resulting in the release of a 27-amino-acid intervening peptide which is secreted into the extracellular space. We showed recently that the intervening peptide of bovine RSV can be replaced by bovine interleukins which are secreted into the medium of cells infected with the respective bovine RSV recombinants (P. Konig, K. Giesow, K. Schuldt, U. J. Buchholz, and G. M. Keil, J. Gen. Virol. 85:1815-1824, 2004). To elucidate whether the approach to transport heterologous proteins as furin-excisable polypeptides functions in principle also in glycoproteins which are cleaved by furin only once, we inserted a second furin cleavage site into BHV-1 gB and integrated a 16-amino-acid peptide sequence, the 246-amino-acid green fluorescent protein (GFP), or the 167 amino acids for mature bovine alpha interferon (boIFN-alpha) as an intervening polypeptide. The resulting gB variants rescued gB-negative BHV-1 mutants, the resulting BHV-1 recombinants were fully infectious, and infected cells secreted biologically active GFP and boIFN-alpha, respectively. In contrast to the gB2Fu and gB2FuGFP precursor molecules, which were efficiently cleaved at both furin sites, the majority of pgB2FuIFN-alpha was not cleaved at the site between the amino-terminal (NH2) subunit and boIFN-alpha, whereas cleavage at the newly introduced site was normal. This resulted in virus particles that also contain the NH2-subunit/boIFN-alpha fusion protein within their envelopes. Our results demonstrate that BHV-1 gB can be used as a transporter for peptides and proteins which could be important for development of novel vaccines. In addition, the general principle might be useful for other applications, e.g., in gene therapy and also in nonviral systems.     

1-naphthol-pyronin B as a novel substrate for silver intensification: application to light and electron microscopic immunocytochemistry of neuroendocrine systems

We describe a modification of silver intensification of immunoperoxidase end-product using 1-naphthol (1N) and 1N enhanced by pyronin B after suppressing nonspecific tissue argyrophilia with a solution of penicillamine and merthiolate buffered near neutral pH. This approach facilitates the preservation of a second antigen sequentially labeled in the same tissue section for light microscopic double immunolabeling experiments and also allows retention of ultrastructural detail. Using this protocol, we obtained rapid and uniform silver intensification of somatostatin (SRIF)-immunoreactive (IR) neuronal perikarya and processes in the rat hypothalamic paraventricular nucleus (PVN). Ultrastructurally, 1N- and 1N-pyronin B-silver intensified reaction product was clearly recognized by the presence of a coarse intracellular precipitate of high electron density. Light microscopic double-immunolabeling studies demonstrated the association between SRIF- and thyrotropin-releasing hormone (TRH)-IR neuronal systems in the PVN. We propose that silver intensification of 1N and 1N-pyronin B is a useful alternative to standard methods of silver intensification of immunoperoxidase reaction product at both light and ultrastructural levels and may be particularly amenable for double-immunolabeling studies.