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.

Lack of correlation between the response to a proliferation inhibitor and other transformation markers in a mutant liver cell line.

A rat liver factor, which has been found previously to inhibit proliferation of untransformed rat liver cell lines but not of transformed liver cell lines, did not inhibit proliferation of the chemically transformed rat liver cell line, W-8. Moreover, a temperature-sensitive mutant derived from W-8 (TS-223), which exhibits an untransformed phenotype at 39.5-41 degrees and a transformed phenotype at 36 degrees, was not affected by the liver factor at either temperature. Since the factor can be incubated at 41 degrees for 4 days without loss of activity, it would seem that the regulation of cell proliferation is not necessarily linked with the expression of other markers of transformed cells.

Inhibition of N-nitrosodiethylamine-induced respiratory tract carcinogenesis by piperonylbutoxide in hamsters.

The influence of pretreatment with piperonylbutoxide (PIP) on the biological effects of N-nitrosodiethylamine (DEN) in vivo in Syrian golden hamsters was investigated. PIP pretreatment significantly reduced covalent binding of N-[ethyl-1-14C]DEN to tissue macromolecules in trachea, lung, and liver, while it did not change the tissue distribution of the parent compound. In a chronic experiment, hamsters treated with PIP before each DEN injection did not develop any tumors or precancerous changes in the lungs, while 60% of the animals given DEN alone developed lung tumors with the morphology of Clara cells and endocrine cells. Tumor incidence in the trachea was also significantly reduced by PIP, but to a lesser extent than in the lungs.

Specific inhibition of proliferation of nonmalignant rat hepatic cells by a factor from rat liver.

A simple quantitative assay based on the colony-forming ability of cells was used to investigate the effect of partially purified factors isolated from rat liver on the proliferation of nonmalignant and malignant rat liver epithelial cells in culture. Using this assay, which differentiates between the cytostatic and cytotoxic actions of the test materials, we found that the liver factors exerted a dose-dependent cytostatic inhibition of nonmalignant liver cells but had no inhibitory effect on malignant liver cells. However, the crude fractions showed a significant activation of the proliferation of one of the malignant cell lines tested. The inhibitory effect of this material was not due to arginase, thymidine, or thymidine-degrading enzymes. The respective inhibitory and activating effects exerted by very low concentration of the liver fractions could not be resolved by our separation methods. Even though the material may contain a mixture of activators and inhibitors, it is likely that the differential effect on the proliferative capacity of nonmalignant and malignant cells is due to the altered state of the malignant cells.

Metabolic activation and cytotoxicity of 4-ipomeanol in human non-small cell lung cancer lines.

In the normal lungs of many animal species, 4-ipomeanol is transformed to a highly reactive metabolite preferentially in pulmonary bronchiolar Clara cells and to a lesser extent in alveolar type II cells, potentially leading to damage or destruction of these cell types. Since Clara cells and type II cells are suspected sites of origin of certain "non-small cell" lung cancers, the metabolic activation of 4-ipomeanol (measured by the metabolism-dependent covalent binding of 4-ipomeanol to cellular macromolecules) was compared in two human non-small cell carcinoma derived cell lines (NCI-H322 and NCI-H358) and two human small cell carcinoma derived cell lines (NCI-H128 and NCI-H69). Metabolic activation of 4-ipomeanol was evident in the non-small cell lines; the production of covalently bound metabolite was somewhat greater in NCI-H322 (morphology related to Clara cells) compared to NCI-H358 (morphology related to alveolar type II cells), but was entirely undetectable in the small cell lines. The activation pathway was concentration (4-ipomeanol) and time dependent and followed Michaelis-Menten kinetics. Metabolism to the reactive intermediate required oxygen and was strongly inhibited by carbon monoxide. Covalent binding was enhanced in the non-small cell lines by prior incubation with beta-naphthoflavone and by supplementation of the incubate with exogenous reduced nicotinamide adenine dinucleotide phosphate. 4-Ipomeanol was more cytotoxic to the non-small cell lines than to the small cell lines under the in vitro growth conditions used. These studies indicate that certain human non-small cell lung cancers have metabolic characteristics of normal bronchiolar Clara cells and alveolar type II cells; these results would therefore be consistent with an origin of these tumors from Clara cells or type II cells, respectively. The present studies indicate that the further preclinical testing and development of 4-ipomeanol is warranted, with a view toward possible clinical evaluation against human lung cancers.

Metabolism of arachidonic acid in human lung cancer cell lines.

The metabolism of arachidonic acid (AA) was studied in two pulmonary bronchioloalveolar-carcinoma cell lines (NCI-H322 and NCI-H358) and two small cell lung carcinoma cell lines (NCI-H69 and NCI-H128). Exogenous AA was metabolized only in the NCI-H322 and NCI-H358 cells. There was no detectable metabolism of AA in NCI-H69 or NCI-H128 cells, either in the presence or the absence of the calcium ionophore A23187. The major metabolite of AA isolated from both NCI-H322 and NCI-H358 cells was prostaglandin E2 (PGE2). Prostaglandin endoperoxide synthase activities, expressed as immunoreactive PGE2 (pmol/min/mg protein), were 10.3 +/- 0.28 (SD) and 4.8 +/- 0.48 in NCI-H358 and NCI-H322 cells, respectively. The rate of production of PGE2 by both NCI-H358 and NCI-H322 cells was linear up to 10 min. Production of PGE2 in both cell lines was dependent upon substrate concentration and was maximal above 17 microM AA. Moreover, PGE2 did not undergo further metabolism by either the NCI-H358 or the NCI-H322 cells. Aspirin (0.1 mM), a cyclooxygenase inhibitor, decreased PGE2 production by 77 and 60% in NCI-H358 and NCI-H322 cells, respectively. In the presence of exogenous AA the calcium ionophore, A23187 (20 microM), stimulated PGE2 production in NCI-H322 cells by almost 2-fold, although it did not affect PGE2 production in the NCI-H358 cells. In contrast, A23187 stimulated the endogenous production of PGE2 in both NCI-H322 and NCI-H358 cells by 4- and 9-fold respectively. In addition, both the NCI-H358 and NCI-H322 cell lines were susceptible to the cytotoxic effects of the anticancer agent mitoxantrone in both a time and concentration dependent manner. In contrast, the two cell lines lacking detectable prostaglandin synthesis activity, NCI-H69 and NCI-H128 were unaffected by treatment with mitoxantrone. These results illustrate that there are major differences in the abilities of human lung cancer cell lines to biosynthesize and release PGE2. It is conceivable that such differences might have exploitable diagnostic and/or therapeutic implications.

Differential effects of transforming growth factor-beta on proliferation of normal and malignant rat liver epithelial cells in culture.

Transforming growth factors (TGF-betas) have been shown to cause both stimulatory and inhibitory effects on cellular growth in a variety of normal and neoplastic cells. The nature of the inhibitory effects of TGF-beta on proliferation of different cell types is at present unclear. We have used freshly isolated rat hepatocytes, a normal diploid rat liver epithelial cell line (NRLM), and a subline (AFB) derived from it which was transformed in vitro by aflatoxin B1 to study the nature of TGF-beta-induced growth inhibition and its alteration following chemically induced neoplastic transformation. TGF-beta had a vastly different effect on proliferation of normal rat liver epithelial cells (both freshly isolated and NRLM cells) compared to aflatoxin B1-transformed cells. TGF-beta at 20 pg/ml caused 83% inhibition of colony formation of NRLM, whereas the growth of AFB cells was unaffected by TGF-beta at concentrations as high as 10 ng/ml. A parallel dose-dependent inhibition of DNA synthesis by TGF-beta was observed in both primary hepatocytes and NRLM cells at concentrations between 10 pg and 10 ng/ml. No inhibition of DNA synthesis was observed in AFB cells. Furthermore, TGF-beta did neither induce anchorage-independent growth of NRLM cells nor affect the growth of AFB cells in soft agar. TGF-beta-induced inhibition of the NRLM cells was irreversible in nature, since treated cells were unable to proliferate and form colonies upon removal of TGF-beta from the medium. Also, NRLM cells showed, after 4 days in the presence of 20 pg of TGF-beta per ml morphological changes characterized by cytoplasmic hypertrophy and the formation of abundant liposomal derivatives, some of which resemble lipofuscin. The finding that TGF-beta caused a high degree of irreversible inhibition of NRLM cells emphasizes the need for caution in interpreting data from inhibition studies, since most assays presently used are designed for assessing growth stimulation in vitro and do not adequately distinguish between the possible cytotoxic and/or cytostatic action of growth inhibitors.

Arabinosyl-5-azacytosine: mechanisms of native and acquired resistance.

Factors influencing the activity of the nucleoside analogue arabinosyl-5-azacytosine (ara-AC) were studied in P388 murine lymphoblasts in vitro and in vivo, in variants of these cells with artificially acquired resistance, in the naturally resistant colon 38 carcinoma in vivo, and in a panel of six human tumors maintained in continuous culture. Differences were noted not only between the sensitive and artificially developed resistant variants of P388, but also between the naturally sensitive (P388) and naturally resistant (colon 38) tumors. The artificially developed resistant P388 cell lines showed an inhibited capacity to accumulate nucleotides derived from ara-AC and deoxycytidine, whereas the accumulation of cytidine nucleotides remained unchanged. Studies of the initial velocity of facilitated diffusion of ara-AC showed only minor differences between parental and resistant lines, while the nucleotide formation rates from both ara-AC and deoxycytidine were markedly depressed in the latter cells. It is concluded, therefore, that the failure of resistant P388 cells to accumulate these compounds results not from a transport deficit per se but rather from a failure to convert the nucleosides to nondiffusible (i.e., phosphorylated) species inside the cell. This failure was accompanied by a substantial reduction in the incorporation of a radiolabeled product derived from deoxycytidine into the nucleic acids of the resistant clones. The common factor responsible for the resistance of P388 variants toward ara-AC appears to be a markedly decreased level of deoxycytidine kinase activity. The naturally resistant colon 38 carcinoma, on the other hand, in addition to a decrease in the activity of its deoxycytidine kinase, showed a lower level of activity of all its purine and pyrimidine kinases, along with a notably elevated nucleoside triphosphatase activity (with ATP as substrate) when compared to P388. These differences were reflected in lower endogenous nucleoside triphosphate pool sizes in colon 38, and in a lower level of ara-AC-5'-triphosphate accumulation in colon 38 than in P388 after comparable drug exposure. In the six human tumor lines, a positive correlation was established between sensitivity to ara-AC (as determined by its median inhibitory concentration) and cellular content of deoxycytidine kinase. It is concluded that this latter enzyme is a generally important determinant of sensitivity to arabinosyl-5-azacytosine.

Metabolic activation and biological effects of nitrosamines in the mammalian lung.

Nitrosamines and their precursors are among the most common contaminants of our environment, and many of them are highly carcinogenic. Nitrosamines are believed to require metabolic activation in the host organism, and many of them demonstrate a pronounced organ and cell type specificity. This review summarizes recent in vivo and in vitro experiments which focus on the mechanisms of nitrosamine-induced lung carcinogenesis. Currently available in vivo and in vitro data suggest that nitrosamines may be metabolized by cytochrome P-450, prostaglandin endoperoxide synthetase, or monoamine oxidases. The presence of one or the other of these enzyme systems may be partially responsible for the cell type-specific effects of this class of chemicals. Moreover, evidence in vitro suggests selective uptake of nitrosamines by cell type-specific receptors, a phenomenon which offers a more logical explanation than previously published theories for the selectivity of biological effects exerted by nitrosamines.

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.

Inhibition of reverse transcriptase of human immunodeficiency virus type 1 and chimeric enzymes of human immunodeficiency viruses types 1 and 2 by two novel non-nucleoside inhibitors.

We have studied the effects of two non-nucleoside reverse transcriptase inhibitors (NNRTI), nitrophenyl phenyl sulfone (NPPS) and a potent derivative of oxathiin carboxanilide (UC-38), on enzymatically active molecular chimeras composed of complementary segments of the reverse transcriptases (RTs) of human immunodeficiency virus type 1 (HIV-1) and -2 (HIV-2). The substances inhibit only the DNA polymerase activity of HIV-1 RT with no effect on HIV-2 RT. The results suggest that there is a protein segment located between residues 158 and 190 that is critical for the inhibition by both compounds. However, there is probably a second segment that resides between residues 192 and 202, as in the case of NPPS, or residues 203 and 224, as in the case of UC-38, that is also crucial for the sensitivity of HIV-1 RT to both inhibitors.

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.

Sulfonic acid dyes: inhibition of the human immunodeficiency virus and mechanism of action.

Over 50 different commercially available sulfonic acid-containing dyes were analyzed for their ability to prevent HIV-1-induced cell killing and in inhibiting HIV-1 replication. Compounds of remarkably similar structure, but with differing patterns of sulfonic acid group substitutions, had a wide range of potency in inhibiting HIV-1. Chicago sky blue (CSB) was highly effective in the inhibition of HIV-1 with less toxicity to CEM-SS cells than most of the other sulfonated dyes tested. Synthesis of CSB was undertaken to produce a product greater than 98% pure and this compound was used to elucidate the possible mechanisms by which this class of structurally related compounds inhibits HIV-1. Addition of CSB to cells infected at high multiplicity at any time up to 24 h after infection, unlike dideoxycytidine (ddC) or oxathiin carboxanilide (OC), inhibited HIV-1-induced cell killing. Other postinfection time course studies revealed that CSB had to be present for 24 h or longer immediately after infection to be protective. Virus binding to cells occurred in the presence of CSB, but the requirement for virion envelope-cell membrane fusion was delayed. CSB was a potent inhibitor of the reverse transcriptase (RT) of both HIV-1 and HIV-2, although it was less active against HIV-2 in a cell killing-based assay. CSB also inhibited Rauscher and LP-BM5 murine leukemia viruses. CSB appears to disrupt the interaction between viral proteins and cell membranes, both in the fusion step early in the infection cycle and in the development of syncytia in the late stages of virus infection.

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.

Anti-HIV michellamines from Ancistrocladus korupensis.

Here we report details of the isolation and determination of the absolute configurations and comparative anti-HIV activities of novel, atropisomeric naphthylisoquinoline alkaloid dimers, michellamines A, B, and C, from a newly described species of Ancistrocladus from the Korup rainforest of Cameroon. We further provide a more extensive analysis of the range of anti-HIV activity of michellamine B, the most potent and abundant member of the series. Michellamine B inhibited HIV-induced cell killing and viral replication in a variety of human cell lines, as well as in cultures of human peripheral blood leukocytes and monocytes. Michellamine B was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6 and the pyridinone-resistant strain A17; the compound also inhibited several strains of HIV-2.

A nonpromoting phorbol from the samoan medicinal plant Homalanthus nutans inhibits cell killing by HIV-1.

Extracts of Homalanthus nutans, a plant used in Samoan herbal medicine, exhibited potent activity in an in vitro, tetrazolium-based assay which detects the inhibition of the cytopathic effects of human immunodeficiency virus (HIV-1). The active constituent was identified as prostratin, a relatively polar 12-deoxyphorbol ester. Noncytotoxic concentrations of prostratin from greater than or equal to 0.1 to greater than 25 microM protected T-lymphoblastoid CEM-SS and C-8166 cells from the killing effects of HIV-1. Cytoprotective concentrations of prostratin greater than or equal to 1 microM essentially stopped virus reproduction in these cell lines, as well as in the human monocytic cell line U937 and in freshly isolated human monocyte/macrophage cultures. Prostratin bound to and activated protein kinase C in vitro in CEM-SS cells and elicited other biochemical effects typical of phorbol esters in C3H10T1/2 cells; however, the compound does not appear to be a tumor promoter. In skin of CD-1 mice, high doses of prostratin induced ornithine decarboxylase only to 25-30% of the levels induced by typical phorbol esters at doses 1/30 or less than that used for prostratin, produced kinetics of edema formation characteristic of the nonpromoting 12-deoxyphorbol 13-phenylacetate, and failed to induce the acute or chronic hyperplasias typically caused by tumor-promoting phorbols at doses of 1/100 or less than that used for prostratin.

Design, synthesis, and biological evaluation of cosalane, a novel anti-HIV agent which inhibits multiple features of virus reproduction.

Cosalane (3), a novel anti-HIV agent having a disalicylmethane unit linked to C-3 of cholestane by a three-carbon linker, was synthesized from commercially available starting materials by a convergent route. Cosalane proved to be a potent inhibitor of HIV with a broad range of activity against a variety of laboratory, drug-resistant, and clinical HIV-1 isolates, HIV-2, and Rauscher murine leukemia virus. The cytotoxicity of cosalane is relatively low as reflected by an in vitro therapeutic index of > 100. Although cosalane inhibits HIV-1 reverse transcriptase and protease, time of addition experiments indicate that it prevents the cytopathic effect of HIV by acting earlier than reverse transcription in the viral replication cycle. The available evidence indicates that the primary mechanism of action of cosalane involves inhibition of gp120-CD4 binding as well as inhibition of a postattachment event prior to reverse transcription.

HIV-inhibitory natural products. 11. Comparative studies of sulfated sterols from marine invertebrates.

A total of 22 sulfated sterols isolated from marine sponges, ophiuroids (brittle stars), and asteroids (sea stars) were comparatively evaluated for their antiviral activity against HIV-1 and HIV-2. In general, sterols with sulfate groups at position 2, 3, or 6 were the most active, with EC50 values of 3-13 microM against HIV-1 (RF) and 2-8 microM against HIV-2 (CBL20). Those compounds which were sulfated on the sterol D ring were completely inactive against both HIV-1 and HIV-2. Overall, sulfated sterols active against HIV-1 were also active against HIV-2.

The calanolides, a novel HIV-inhibitory class of coumarin derivatives from the tropical rainforest tree, Calophyllum lanigerum.

Eight new coumarin compounds (1-8) were isolated by anti-HIV bioassay-guided fractionation of an extract of Calophyllum lanigerum. The structures of calanolide A (1), 12-acetoxycalanolide A (2), 12-methoxycalanolide A (3), calanolide B (4), 12-methoxycalanolide B (5), calanolide C (6) and related derivatives 7 and 8 were solved by extensive spectroscopic analyses, particularly HMQC, HMBC, and difference NOE NMR experiments. The absolute stereochemistry of calanolide A (1) and calanolide B (4) was established by a modified Mosher's method. Calanolides A (1) and B (4) were completely protective against HIV-1 replication and cytopathicity (EC50 values of 0.1 microM and 0.4 microM, respectively), but were inactive against HIV-2. Some of the related compounds also showed evidence of anti-HIV-1 activity. Studies with purified bacterial recombinant reverse transcriptases (RT) revealed that the calanolides are HIV-1 specific RT inhibitors. Moreover, calanolide A was active not only against the AZT-resistant G-9106 strain of HIV-1 but also against the pyridinone-resistant A17 strain. This was of particular interest since the A17 virus is highly resistant to previously known HIV-1 specific, non-nucleoside RT inhibitors (e.g., TIBO; BI-RG-587; L693,593) which comprise a structurally diverse but apparently common pharmacologic class. The calanolides represent a substantial departure from the known class and therefore provide a novel new anti-HIV chemotype for drug development.

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.