Legacy of Multiple Stressors: Responses of Gastropod Larvae and Juveniles to Ocean Acidification and Nutrition.

Ocean acidification poses a significant threat to calcifying invertebrates by negatively influencing shell deposition and growth. An organism's performance under ocean acidification is not determined by the susceptibility of one single life-history stage, nor is it solely controlled by the direct physical consequences of ocean acidification. Shell development by one life-history stage is sometimes a function of the pH or pCO2 levels experienced during earlier developmental stages. Furthermore, environmental factors such as access to nutrition can buffer organismal responses of calcifying invertebrates to ocean acidification, or they can function as a co-occurring stressor when access is low. We reared larvae and juveniles of the planktotrophic marine gastropod Crepidula fornicata through combined treatments of nutritional stress and low pH, and we monitored how multiple stressors endured during the larval stage affected juvenile performance. Shell growth responded non-linearly to decreasing pH, significantly declining between pH 7.6 and pH 7.5 in larvae and juveniles. Larval rearing at pH 7.5 reduced juvenile growth as a carryover effect. Larval rearing at pH 7.6 reduced subsequent juvenile growth despite the absence of a negative impact on larval growth, demonstrating a latent effect. Low larval pH magnified the impact of larval nutritional stress on competence for metamorphosis and increased carryover effects of larval nutrition on juvenile growth. Trans-life-cycle effects of larval nutrition were thus modulated by larval exposure to ocean acidification.

Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation.

Withanolide E, a steroidal lactone from Physalis peruviana, was found to be highly active for sensitizing renal carcinoma cells and a number of other human cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. Withanolide E, the most potent and least toxic of five TRAIL-sensitizing withanolides identified, enhanced death receptor-mediated apoptotic signaling by a rapid decline in the levels of cFLIP proteins. Other mechanisms by which TRAIL sensitizers have been reported to work: generation of reactive oxygen species (ROS), changes in pro-and antiapoptotic protein expression, death receptor upregulation, activation of intrinsic (mitochondrial) apoptotic pathways, ER stress, and proteasomal inhibition proved to be irrelevant to withanolide E activity. Loss of cFLIP proteins was not due to changes in expression, but rather destabilization and/or aggregation, suggesting impairment of chaperone proteins leading to degradation. Indeed, withanolide E treatment altered the stability of a number of HSP90 client proteins, but with greater apparent specificity than the well-known HSP90 inhibitor geldanamycin. As cFLIP has been reported to be an HSP90 client, this provides a potentially novel mechanism for sensitizing cells to TRAIL. Sensitization of human renal carcinoma cells to TRAIL-induced apoptosis by withanolide E and its lack of toxicity were confirmed in animal studies. Owing to its novel activity, withanolide E is a promising reagent for the analysis of mechanisms of TRAIL resistance, for understanding HSP90 function, and for further therapeutic development. In marked contrast to bortezomib, among the best currently available TRAIL sensitizers, withanolide E's more specific mechanism of action suggests minimal toxic side effects.

Griffithsin, a potent HIV entry inhibitor, is an excellent candidate for anti-HIV microbicide.

BACKGROUND: The predominant mode of HIV-1 transmission is by heterosexual contact. The cervical/vaginal mucosa is the main port of HIV entry in women. A safe and effective topical microbicide against HIV is urgently needed to prevent sexual transmission. Hence, we evaluated griffithsin (GRFT), a 12.7 kDa carbohydrate-binding protein, both native and recombinant GRFT, potently inhibited both CXCR4-and CCR5-tropic HIV infection and transmission in vitro. METHODS: The antiviral efficacy of native and recombinant GRFT against CXCR4-and CCR5-tropic HIV and SHIV strains and SIVmac251 was evaluated by in vitro assays. We also evaluated the time course of antiviral activity and stability of GRFT in cervical/vaginal lavage as a function of pH 4-8. RESULTS: Griffithsin blocked CXCR4-and CCR5-tropic viruses at less than 1 nm concentrations and exhibited a high potency. GRFT was stable in cervical/vaginal lavage fluid and maintained a similar potency of anti-HIV activity. GRFT is not only a highly potent HIV entry inhibitor, but also prevents cell fusion and cell-to-cell transmission of HIV. CONCLUSIONS: The in vitro efficacy of GRFT revealed low cytotoxicity, high potency, rapid onset of antiviral activity and long-term stability in cervical/vaginal lavage. GRFT is an excellent candidate for anti-HIV microbicide development.

Cyanovirin-N defines a new class of antiviral agent targeting N-linked, high-mannose glycans in an oligosaccharide-specific manner.

Herein we report that the novel HIV-inactivating protein cyanovirin-N (CV-N) targets specific, N-linked high-mannose oligosaccharides found on the viral envelope of HIV-1. First, we released the oligosaccharides by PnGase-treatment of HIV-gp120 (containing high-mannose, hybrid-type and complex-type oligosaccharides) or HSV-1 gC (containing only complex-type). Then, in an affinity chromatographic system, we found that CV-N bound to the free oligosaccharides from gp120 but not from gC-1, suggesting that high-mannose oligosaccharides constitute a target structure for CV-N. This was supported by the affinity of CV-N for high-mannose glycans released from gp120 by endo-H as well as high-mannose glycans released from castanospermine-treated HSV-1 gC. Furthermore, free Man-8 or Man-9 oligosaccharides partially inhibited the binding of CV-N to gp120, although neither oligosaccharides smaller than Man-7 nor monosaccharides interfered with CV-N/gp120 interaction, thereby establishing the oligosaccharide-specific affinity of CV-N to high-mannose glycans. This affinity for high-mannose oligosaccharides may explain the broad antiviral activity of CV-N against human and primate immunodeficiency retroviruses as well as certain other viruses that carry these oligosaccharides.

Development of a cyanovirin-N-HIV-1 gp120 binding assay for high throughput screening of natural product extracts by time-resolved fluorescence.

The unique, high-affinity binding of cyanovirin-N (CV-N), a potent anti-human immunodeficiency virus (HIV) protein, to the HIV envelope glycoprotein gp120, was exploited to develop an HTS assay in an attempt to discover small-molecule mimetics of CV-N. A competition binding assay was developed using CV-N labeled with europium (Eu(3+)). The labeling protocol did not significantly alter the gp120 binding properties or the antiviral activity of CV-N. This report describes the assay development, validation, and results of screening a large library of aqueous and organic natural product extracts. The extracts were incubated with immobilized recombinant gp120 in 96-well plates prior to the addition of Eu(3+)-labeled CV-N. Following a wash step, bound CV-N was measured by dissociation-enhanced time-resolved fluorometry of Eu(3+). The assay proved to be robust, rapid, and reproducible, and was used to screen over 50,000 natural product extracts, and has resulted in the identification of several aqueous natural product extracts that inhibited CV-N-gp120 binding and also had anti-HIV activity.

The HIV-inactivating protein, cyanovirin-N, does not block gp120-mediated virus-to-cell binding.

Concentrations of the potent, HIV(human immunodeficiency virus) inactivating protein, cyanovirin-N (CV-N), which completely inhibit HIV-1 infectivity, do not block the binding of soluble CD4-receptor (sCD4) to HIV-1 lysates nor the attachment of intact HIV-1 virions to several target T-cell lines. Furthermore, in contrast to the known disassociative effects of sCD4 on viral envelope glycoproteins, treatment of HIVRF with high concentrations of CV-N results in complete viral inactivation but without apparent shedding of gp120 or other ultrastructural changes. These results are consistent with the view that the virucidal effects of CV-N result from interference with step(s) in the fusion process subsequent to the initial binding of the virus to target cells.

Isolation and characterization of adociavirin, a novel HIV-inhibitory protein from the sponge Adocia sp.

Aqueous extracts of the New Zealand sponge Adocia sp. (Haplosclerida) displayed potent anticytopathic activity in CEM-SS cells infected with HIV-1. Protein fractions of the extract bound both to the viral coat protein gp120 and to the cellular receptor CD4, but not to other tested proteins. The purified active protein, named adociavirin, was characterized by isoelectric focusing, amino acid analysis, MALDI-TOF mass spectrometry and N-terminal sequencing. Adociavirin, a disulfide-linked homodimer with a native molecular weight of 37 kDa, was active against diverse strains and isolates of HIV-1, as well as HIV-2, with EC50 values ranging from 0.4 nM to > 400 nM. The anti-HIV potency of adociavirin appears dependent on host cell type, with macrophage cultures being the most sensitive and peripheral blood lymphocytes the most resistant.

Recombinant production of cyanovirin-N, a potent human immunodeficiency virus-inactivating protein derived from a cultured cyanobacterium.

Here we describe the recombinant production and purification of a novel anti-human immunodeficiency virus (HIV) protein, cyanovirin-N (CV-N), in Escherichia coli. Initial attempts to express CV-N using a vector containing an ompA signal peptide sequence resulted in production of an intractable mixture of the full-length (101 amino acid residue) protein and a truncated form lacking the first two N-terminal amino acids. The truncated protein was observed regardless of the host cell line, culture conditions, or induction time. These observations suggested that an as yet unidentified protease or peptidase was responsible for proteolytic cleavage between the second and third N-terminal amino acids of CV-N when presented as an ompA-CV-N fusion protein. When the ompA signal peptide sequence was replaced by a pelB signal peptide sequence, CV-N was produced in high yield as a single, homogeneous protein. This was confirmed by electrospray ionization mass spectrometry and N-terminal sequencing. This expression system provides a basis for large-scale production of clinical grade CV-N for further research and development as an anti-HIV microbicide.

Construction and enhanced cytotoxicity of a [cyanovirin-N]-[Pseudomonas exotoxin] conjugate against human immunodeficiency virus-infected cells.

Cyanovirin-N (CV-N) is a novel 11-kDa anti-HIV(human immunodeficiency virus) protein that binds with high affinity to the viral envelope glycoprotein gp120. In contrast to soluble CD4 and most known neutralizing antibodies that bind gp120, CV-N exerts potent anti-viral activity against primary clinical HIV isolates as well as laboratory-adapted strains of HIV. Here we describe the recombinant production, purification, and characterization of a chimeric toxin molecule, FLAG-CV-N-PE38, that contains CV-N as a gp120-targeting moiety linked to the translocation and cytotoxic domains of Pseudomonas exotoxin A. FLAG-CV-N-PE38 showed enhanced cytotoxicity to HIV-infected, gp120-expressing H9 cells compared to uninfected H9 cells. Competition experiments with free CV-N provided further support that the enhanced FLAG-CV-N-PE38-induced cytotoxicity was due to interactions of the CV-N moiety with cell surface gp120. This study establishes the feasibility of use of CV-N as a gp120-targeting sequence for construction and experimental therapeutic investigations of unique new chimeric toxins designed to selectively destroy HIV-infected host cells.

Analysis of sequence requirements for biological activity of cyanovirin-N, a potent HIV (human immunodeficiency virus)-inactivating protein.

Site-directed mutagenesis of DNA constructs coding for the novel, HIV-inactivating proteins cyanovirin-N (CV-N) and FLAG-cyanovirin-N (F-CV-N) was performed using mutagenic oligonucleotide primers in the polymerase chain reaction or by a restriction site elimination maneuver. The mutant constructs were expressed in Escherichia coli and the recombinant protein products were tested for binding to the HIV surface envelope glycoprotein gp 120 and for antiviral activity against infectious HIV. Results showed an overall very high correlation (r2 > 0.9) between the relative gp120 binding affinities and the anti-HIV activities of CV-N, F-CV-N, and the various mutants. An outlier, however, was a mutant which lacked one of the internal disulfide linkages normally present in CV-N and which showed modest gp120 binding but no antiviral activity against HIV. These findings are consistent with the view that gp120 binding is a necessary but not sufficient requirement for the HIV-inactivating activity of CV-N and related proteins; the sequence specificities for gp120 binding and anti-HIV activity are not identical.

Isolation, primary sequence determination, and disulfide bond structure of cyanovirin-N, an anti-HIV (human immunodeficiency virus) protein from the cyanobacterium Nostoc ellipsosporum.

A novel anti-HIV protein, cyanovirin-N (CV-N), was isolated from an aqueous cellular extract of the cultured cyanobacterium (blue-green alga) Nostoc ellipsosporum, purified by reverse-phase HPLC, and sequenced by N-terminal Edman degradation of the intact protein and peptide fragments produced by endoproteinase digestions. CV-N consists of a single 101 amino acid chain which exhibits significant internal sequence duplication, but no significant homology to previously described proteins or to the transcription products of known nucleotide sequences. Alignment of residues 1-50 with residues 51-101 reveals 13 conservative amino acid changes as well as direct homology between 16 amino acid residues. CV-N contains four cysteines which form two intrachain disulfide bonds. The positions of the disulfide linkages were established by fast atom bombardment mass spectral studies of peptide fragments generated by a tryptic digestion of the native protein. Reductive cleavage of these crosslinks resulted in loss of anti-HIV activity.

Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp120: potential applications to microbicide development.

We have isolated and sequenced a novel 11-kDa virucidal protein, named cyanovirin-N (CV-N), from cultures of the cyanobacterium (blue-green alga) Nostoc ellipsosporum. We also have produced CV-N recombinantly by expression of a corresponding DNA sequence in Escherichia coli. Low nanomolar concentrations of either natural or recombinant CV-N irreversibly inactivate diverse laboratory strains and primary isolates of human immunodeficiency virus (HIV) type 1 as well as strains of HIV type 2 and simian immunodeficiency virus. In addition, CV-N aborts cell-to-cell fusion and transmission of HIV-1 infection. Continuous, 2-day exposures of uninfected CEM-SS cells or peripheral blood lymphocytes to high concentrations (e.g., 9,000 nM) of CV-N were not lethal to these representative host cell types. The antiviral activity of CV-N is due, at least in part, to unique, high-affinity interactions of CV-N with the viral surface envelope glycoprotein gp120. The biological activity of CV-N is highly resistant to physicochemical denaturation, further enhancing its potential as an anti-HIV microbicide.

Michellamines D-F, new HIV-inhibitory dimeric naphthylisoquinoline alkaloids, and korupensamine E, a new antimalarial monomer, from Ancistrocladus korupensis.

New monomeric (korupensamine E, 6) and dimeric (michellamines D-F, 7-9) naphthylisoquinoline alkaloids have been isolated from exracts of the tropical liana Ancistrocladus korupensis. Structures were determined by spectroanalytical methods, and stereochemistry was defined through NOE correlations, chemical degradation, and CD spectroscopy. Michellamines D-F exhibited in vitro HIV-inhibitory activity comparable to michellamine B, and korupensamine E exhibited in vitro antimalarial activity comparable to korupensamines A-D.

Isolation and characterization of niphatevirin, a human-immunodeficiency-virus-inhibitory glycoprotein from the marine sponge Niphates erecta.

Anti-human immunodeficiency virus (HIV)-bioassay-guided fractionation of aqueous extracts of the Caribbean sponge Niphates erecta led to isolation of a novel anti-HIV protein, named niphatevirin. The protein was purified to homogeneity by ethanol precipitation, ammonium sulfate precipitation, gel-permeation chromatography and concanavalin-A-Sepharose affinity chromatography. Niphatevirin potently inhibited the cytopathic effects of HIV-1 infection in cultured human lymphoblastoid (CEM-SS) cells; the effective concentration of drug that results in 50% protection of the cells through inhibition of cell lethality, cell-cell fusion and syncytium formation was approximately 10 nM. Delay of addition of niphatevirin to infected cultures by two hours markedly decreased (approximately 50%) cytoprotection; delay of addition by eight hours resulted in no antiviral activity. Niphatevirin bound to CD4 in a manner that prevented the binding of gp120, but did not directly bind gp120. Niphatevirin (6.5 microM) was inactive in both hemagglutination and hemolysis assays. Niphatevirin had a molecular mass of about 19 kDa by matrix-assisted laser-desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and a native molecular mass of approximately 18 kDa by gel-filtration chromatography. The protein had an acidic isoelectric point of 4.2-4.6, and was shown by periodate acid Schiff's staining to be glycosylated.

Antireplicative and anticytopathic activities of prostratin, a non-tumor-promoting phorbol ester, against human immunodeficiency virus (HIV).

Prostratin, a non-tumor-promoting phorbol ester, inhibited human immunodeficiency virus (HIV)-induced cell killing and viral replication in a variety of acutely-infected cell systems. The potency and degree of cytoprotection was dependent on both viral strain and host cell type. Prostratin activated viral expression in two latently-infected cell lines, but had little or no effect on chronically-infected cell lines. Prostratin caused a dose-dependent, but reversible, decrease in CD4 expression in the CEM-SS and MT-2 cell lines. This down-regulation of CD4 was inhibited in a dose-dependent manner by the protein kinase C (PKC) antagonist, staurosporine. In addition, the cytoprotective and cytostatic effects of prostratin in CEM-SS cells acutely infected with HIV-1RF were reversed by bryostatin-1, a PKC agonist. Prostratin had no effect on reverse transcriptase or HIV-1 protease, nor did it inhibit the binding of gp120 to CD4. We conclude that prostratin inhibits HIV cytopathicity and replication through mechanism(s) involving PKC enzyme(s).

Antiviral activity and mechanism of action of calanolide A against the human immunodeficiency virus type-1.

Calanolide A, recently discovered in extracts from the tropical rainforest tree, Calophyllum lanigerum, is a novel inhibitor of the human immunodeficiency virus (HIV) type 1. The compound is essentially inactive against strains of the less common HIV type 2. The present study focused on the further characterization of the selective antiviral activity and mechanism of action of calanolide A. The compound inhibited a wide variety of laboratory strains of HIV type 1, with EC50 values ranging from 0.10 to 0.17 microM. The compound similarly inhibited promonocytotropic and lymphocytotropic isolates from patients in various stages of HIV disease, as well as drug-resistant strains. Viral life-cycle studies indicated that calanolide A acted early in the infection process, similar to the known HIV reverse transcriptase (RT) inhibitor 2', 3'-dideoxycytidine. In enzyme inhibition assays, calanolide A potently and selectively inhibited recombinant HIV type 1 RT but not cellular DNA polymerases or HIV type 2 RT within the concentration range tested. Serial passage of the virus in host cells exposed to increasing concentrations of calanolide A yielded a calanolide A resistant virus strain. RT from the resistant virus was not inhibited by calanolide A but retained sensitivity to other nonnucleoside as well as nucleoside RT inhibitors, including 3'-azido-2',3'-dideoxythymidine triphosphate and nevirapine. The study substantially supports the conclusion that calanolide A represents a novel subclass of nonnucleoside RT inhibitor which merits consideration for anti-HIV drug development.

Kinetic analysis of inhibition of human immunodeficiency virus type-1 reverse transcriptase by calanolide A.

Calanolide A, first isolated from the tropical rain forest tree Calophyllum lanigerum, is a potent human immunodeficiency virus type-1 (HIV-1) specific reverse transcriptase (RT) inhibitor, broadly active against diverse HIV-1 strains, including nucleoside and nonnucleoside-resistant variants. We examined the biochemical mechanism of inhibition of HIV-1 RT by calanolide A. Two template/primer systems were examined: ribosomal RNA and homopolymeric rA-dT 12-18. Calanolide A inhibited HIV-1 RT by a complex mechanism involving two calanolide A binding sites. With respect to either deoxynucleotide triphosphate (dNTP) or template/primer binding, one site was competitive and the other was uncompetitive. The data indicated that calanolide A bound near the active site of the enzyme and interfered with dNTP binding. Calanolide A inhibited HIV-1 RT in a synergistic fashion with nevirapine, further distinguishing it from the general class of nonnucleoside RT inhibitors. At certain concentrations, calanolide A bound HIV-1 RT in a mutually exclusive fashion with respect to both the pyrophosphate analog, phosphonoformic acid and the acyclic nucleoside analog 1-ethoxymethyl-5-ethyl-6-phenylthio-2-thiouracil. This indicates that calanolide A shares some binding domains with both phosphonoformic acid and 1-ethoxymethyl-5-ethyl-6-phenylthio-2-thiouracil, presumably reflecting that it interacts with RT near both the pyrophosphate binding site and the active site of the enzyme.

Structure-activity modifications of the HIV-1 inhibitors (+)-calanolide A and (-)-calanolide B.

The delta 7,8 olefinic linkages within (+)-calanolide A(1) and (-)-calanolide B(2) were catalytically reduced to determine impact on the anti-HIV activity of the parent compounds. In addition, a series of structure modifications of the C-12 hydroxyl group in (-)-calanolide B was made to investigate the importance of that substituent to the HIV-1 inhibitory activity of these coumarins. A total of 14 analogs were isolated or prepared and compared to (+)-calanolide A and (-)-calanolide B in the NCI primary anti-HIV assay. While none of the compounds showed activity superior to the two unmodified leads, some structure-activity requirements were apparent from the relative anti-HIV potencies of the various analogs.

New pyranocoumarins isolated from Calophyllum lanigerum and Calophyllum teysmannii.

During a chemotaxonomic survey of Calophyllum extracts present in the National Cancer Institute's natural product repository, four new pyranocoumarins were isolated from extracts of C. lanigerum var. austrocoriaceum and C. teysmannii var. inophylloide (King.) P. F. Stevens (Clusiaceae). The structure elucidation and anti-HIV activity of calanolide E2 (4), cordatolide E (5), pseudocordatolide C (6), and calanolide F (9), along with a simple prenylated coumarin precursor (11), are described here.

Biological and biochemical anti-human immunodeficiency virus activity of UC 38, a new non-nucleoside reverse transcriptase inhibitor.

UC 38, a simple analog of oxathiin carboxanilide, UC 84, lacking the oxathiin ring, was found to be a potent inhibitor of human immunodeficiency virus (HIV)-1-induced cell killing and HIV replication in a variety of human cell lines, as well as in human peripheral blood lymphocytes and macrophages. UC 38 was active against a wide range of biologically diverse laboratory and clinical strains of HIV-1. However, UC 38 was inactive against HIV-2 and both nevirapine- and pyridinone-resistant strains of HIV-1. UC 38 selectively inhibited HIV-1 reverse transcriptase (RT), but not HIV-2 RT. Combination of UC 38 with 3'-azido-3'-deoxythymidine synergistically inhibited HIV-induced cell killing. An HIV-1 isolate resistant to UC 38 was selected in cell culture, and the mutations in the RT nucleotide sequences were determined. Comparison with the wild-type RT sequence revealed an amino acid change at position 181 (Tyr to Cys). The UC 38-resistant virus was found to be cross-resistant to a variety of structurally diverse non-nucleoside RT inhibitors. UC 38 was susceptible to rapid degradation in vitro and in vivo; yet, nontoxic in vivo concentrations of UC 38 many-fold in excess of the in vitro effective concentrations could be achieved and maintained after s.c. or p.o. administration in hamsters. These results establish UC 38 as a new chemotype within the general class of HIV-1-specific RT inhibitors. The favorable physical characteristics, lack of toxicity, potency and bioavailability of UC 38 may make it a candidate for combination chemotherapy of acquired immune deficiency syndrome.