Laboratory cultivation of prochloron, a tryptophan auxotroph.

Laboratory cultures have been established of the didemnid symbiont Prochloron, a unique prokaryotic alga that synthesizes chlorophylls a and b but no phycobilin pigments. Cell division in Prochloron cultures occurs under acidic conditions (pH 5.5) in the presence of tryptophan. The alga is a naturally occurring tryptophan auxotroph that survives in nature by close association with the host, Diplosoma similis. The metabolic dysfunction that renders Prochloron auxotrophic may involve only the initial step of the tryptophan biosynthetic pathway.

AIDS-antiviral sulfolipids from cyanobacteria (blue-green algae).

A recently developed tetrazolium-based microculture assay was used to screen extracts of cultured cyanobacteria (blue-green algae) for inhibition of the cytopathic effects of the human immunodeficiency virus (HIV-1), which is implicated as a causative agent of AIDS. A number of extracts were found to be remarkably active against the AIDS virus. A new class of HIV-1-inhibitory compounds, the sulfonic acid-containing glycolipids, was discovered through the use of the microculture assay to guide the fractionation and purification process. The pure compounds were active against HIV-1 in cultured human lymphoblastoid CEM, MT-2, LDV-7, and C3-44 cell lines in the tetrazolium assay as well as in p24 viral protein and syncytium formation assays.

Scytophycins, novel microfilament-depolymerizing agents which circumvent P-glycoprotein-mediated multidrug resistance.

Cells demonstrating the multidrug resistance phenotype because of overexpression of P-glycoprotein, a drug efflux pump, are resistant to the cytotoxic effects of most natural product drugs. To determine if P-glycoprotein confers resistance to the syctophycins, a family of natural cytotoxic macrolides recently isolated from cyanobacteria of the family Syctone-mataceae, we have characterized the effects of these compounds on drug-sensitive (SKOV3) and drug-resistant (SKVLB1) human ovarian carcinoma cells. While SKVLB1 cells demonstrated > 150- and 10,000-fold decreases in sensitivity to Adriamycin and vinblastine, respectively, they were equally sensitive as SKOV3 cells to the antiproliferative effects of tolytoxin and certain related scytophycins. The SKVKB1 cells were 4- to 11-fold resistant to other scytophycins and were 14-fold resistant to cytochalasin B. Microfilaments in SKOV3 and SKVLB1 cells were depolymerized by similar concentrations of tolytoxin, while cytochalasin B was less potent toward SKVLB1 cells than SKOV3 cells. Both tolytoxin and cytochalasin B enhanced the cytotoxicity of vinblastine toward SKVLB1 cells; however, neither compound affected the sensitivity to Adriamycin or cisplatin. Verapamil markedly increased the accumulation of [3H]vinblastine by SKLVB1 cells, while cytochalasin B caused only modest increase, and tolytoxin had no effect on [3H]vinblastine accumulation. These results suggest that some of the scytophycins, including tolytoxin, are not subject to P-glycoprotein-mediated efflux from cells exhibiting multidrug resistance due to overexpression of this transport protein. These compounds may therefore be useful for killing drug-resistant tumor cells.

Cryptophycin: a new antimicrotubule agent active against drug-resistant cells.

Cryptophycin is a cytotoxic dioxadiazacyclohexadecenetetrone isolated from cyanobacteria of the genus Nostoc. Incubation of L1210 leukemia cells with cryptophycin resulted in dose-dependent inhibition of cell proliferation in parallel with increases in the percentage of cells in mitosis (half-maximal effects at < 10 pM). Indirect immunofluorescence studies demonstrated that treatment of A-10 vascular smooth muscle cells with cryptophycin results in marked depletion of cellular microtubules and reorganization of vimentin intermediate filaments, similar to the effects of vinblastine. Cytochalasin B caused the depolymerization of microfilaments in these cells, while neither vinblastine nor cryptophycin affected this cytoskeletal component. Pretreatment of cells with taxol prevented microtubule depolymerization in response to either vinblastine or cryptophycin. While microtubule depolymerization in response to vinblastine was rapidly reversed by removal of the drug, cells treated with cryptophycin remained microtubule depleted for at least 24 h after removal of the compound. Combinational treatments with vinblastine and cryptophycin resulted in additive cytotoxicity. Ovarian carcinoma and breast carcinoma cells which are multiply drug resistant due to overexpression of P-glycoprotein are markedly less resistant to cryptophycin than they are to vinblastine, colchicine, and taxol. Therefore, cryptophycin is a new antimicrotubule compound which appears to be a poorer substrate for P-glycoprotein than are the Vinca alkaloids. This property may confer an advantage to cryptophycin in the chemotherapy of drug-resistant tumors.

In vitro bioassays for anticancer drug screening: effects of cell concentration and other assay parameters on growth inhibitory activity.

In vitro growth inhibition assays were performed using human cancer cell lines at various concentrations with experimental anticancer drugs such as the cryptophycins and other cytotoxins. The effect of variations in assay parameters on the observed growth inhibition of these anticancer therapeutic agents was determined. The results demonstrated that the observed inhibitory activity of these compounds varied inversely with the cell concentrations used. The observed differences in activity between different cytotoxins were not necessarily proportionate. Thus, the relative activities of two toxins also varied with cell concentration. Furthermore, the sensitivity of these cell lines to the cytostatic purine analog, 6-mercaptopurine (used as a control), varied with cell concentration as well. The activity of this compound was dependent on the medium used for cell growth, yielding good activity in Eagle's minimum essential medium, but not in Ham's F-12 (Kaigin) medium. Moreover, growth inhibition by cryptophycin as well as 6-mercaptopurine was also dependent on the serum concentration in the medium. Finally, the sensitivity of the cancer cell lines to various organic solvents commonly used as drug vehicles for in vitro testing, such as ethanol, dimethylformamide, and dimethylsulfoxide, was likewise found to vary inversely with cell concentration.

Physiologic definitions of obliterative bronchiolitis in heart-lung and double lung transplantation: a comparison of the forced expiratory flow between 25% and 75% of the forced vital capacity and forced expiratory volume in one second.

BACKGROUND AND METHODS: A comparison of the forced expiratory flow between 25% and 75% of the forced vital capacity (FEF25-75) and forced expiratory volume in 1 second (FEV1) was conducted for the detection of obstructive airway disease as an early manifestation of obliterative bronchiolitis. Pulmonary function tests performed on heart-lung and double lung transplant recipients between March 1981 and March 1983 were reviewed. Thirty patients were identified who showed progressive deterioration in pulmonary function after transplantation. Ratios determining proportionate decreases were calculated from measurements of absolute values for the FEF25-75 and FEV1 at the point when the FEF25-75 reached < 70% and < or = 30% of predicted, divided by baseline values obtained before the decline in function. Similar ratios were obtained for FEV1 and FEF25-75 at the point the FEV1 declined > or = 20% from its baseline value. RESULTS: Comparison of the ratios for the FEF25-75 and FEV1 at FEF25-75 values < 70% and < or = 30% of predicted and a similar comparison when the FEV1 declined > or = 20% from baseline showed a greater proportional decrease in FEF25-75 than FEV1 (p < 0.01). With the use of the FEF25-75, declines in airway function were detected earlier. After transplantation a decline in FEF25-75 to < 70% of predicted occurred approximately 112 days before a 20% decline a FEV1. CONCLUSION: The FEF25-75 is more sensitive than the FEV1 for the early detection of obliterative bronchiolitis. A presumptive diagnosis of obliterative bronchiolitis can be made with physiologic criteria, providing infection or acute rejection has been ruled out. When conducting epidemiologic studies or for vital statistics we propose that a decline in FEF25-75 to < 70% be used to define the onset of obliterative bronchiolitis.

The diagnosis of obliterative bronchiolitis after heart-lung and lung transplantation: low yield of transbronchial lung biopsy.

Obliterative bronchiolitis is the most significant long-term complication of lung and heart-lung transplantation characterized by the rapid development of obstructive airway disease. It is thought to be a manifestation of chronic rejection and has been treated, with limited success, with augmentation of immunosuppression. Early detection of obliterative bronchiolitis and prompt initiation of therapy may result in an improved outcome. The role of transbronchial biopsy has been reported in the diagnosis of acute rejection and infection but not for obliterative bronchiolitis. To study this problem we retrospectively reviewed the transbronchial biopsy results of patients with advanced clinical obliterative bronchiolitis, as defined physiologically. Between January 1, 1988, and December 31, 1991, 46 "sets" of adequate transbronchial biopsy specimens were obtained from 16 patients (15 heart-lung recipients and one double lung recipient). Seven sets of transbronchial biopsy specimens (15.2%) showed obliterative bronchiolitis by pathologic study. In four patients with severe clinical obliterative bronchiolitis, only one transbronchial biopsy specimen of seven (14.3%) showed obliterative bronchiolitis. The pathologic diagnosis of obliterative bronchiolitis was confirmed in three of these patients at the time of autopsy or retransplantation. Twelve patients were still alive at the end of the study period, and all experienced further deterioration of lung function typical for obliterative bronchiolitis. We conclude that the sensitivity of transbronchial biopsy for obliterative bronchiolitis is poor. Possible explanations for these results are explored.

Preclinical anticancer activity of cryptophycin-8.

Cryptophycin-8 was prepared by the conversion of the epoxide group on cryptophycin-1 to a chlorohydrin. In the studies reported here, cryptophycin-8 was evaluated for preclinical activity against subcutaneous tumors of both mouse and human origin. At the highest non-toxic single course treatment, the following results were obtained (Table A). Cryptophycin-8 was less potent than cryptophycin-1 by approximately 4-fold; however, it was both more water soluble and had greater therapeutic efficacy, as demonstrated by % T/C, tumor cell log kill values, range of dose effectiveness and host cures.

Reversal of multiple drug resistance by tolyporphin, a novel cyanobacterial natural product.

The effects of a novel porphyrin, tolyporphin, on P-glycoprotein-mediated multiple drug resistance in human ovarian and breast cell lines were characterized. Compared with parental SKOV3 and MCF-7 cells, the P-glycoprotein-overexpressing sublines SKVLB1 and MCF-7/ADR were 5- and 1.3-fold less sensitive to the cytotoxic effects of tolyporphin. Subtoxic doses of tolyporphin increased the sensitivity of the SKVLB1 and MCF-7/ADR cells to P-glycoprotein-transported drugs, but did not increase the antiproliferative effects of nontransported drugs. Tolyporphin also enhanced the accumulation of [3H]-vinblastine in SKVLB1 and MCF-7/ADR cells at doses approximately 10-fold lower than those required for similar responses to verapamil. In contrast, tolyporphin did not affect drug accumulation in SKOV3 or MCF-7 cells. Tolyporphin reduced [3H]-vinblastine efflux from SKVLB1 cells, reduced [3H]-vinblastine binding to membranes from SKVLB1 cells, and blocked the ability of [3H]-azidopine to photoaffinity-label P-glycoprotein in these membranes. These results indicate that tolyporphin binds to P-glycoprotein and inhibits the transport of cytotoxic natural product drugs. This novel natural product, and related compounds, may be useful for the reversal of multiple drug resistance and for further definition of the drug binding site(s) of P-glycoprotein.

Antifungal cyclic peptides from the terrestrial blue-green alga Anabaena laxa. II. Structures of laxaphycins A, B, D and E.

Laxaphycins A and B are the major components in an antifungal mixture of cyclic peptides from the terrestrial blue-green alga Anabaena laxa FK-1-2. NMR and MS spectral studies coupled with amino acid analysis indicate that the gross structures of laxaphycins A and B are cyclic (Aoc-Hse-E-Dhb-Hyp-Hse-Phe-Leu-Ile-Ile-Leu-Gly) where Aoc is a 3-aminooctanoic acid residue and cyclic (Ala-Hleu-Gln-N-MeIle-Hasn-Thr-Pro-Leu-Thr-Ade-Val- Hleu) where Ade is a 3-aminodecanoyl unit, respectively. Laxaphycin E, a minor cyclic undecapeptide, differs in gross structure from laxaphycin A in possessing a 3-aminodecanoic acid unit (Ade) in lieu of Aoc, whereas laxaphycin D, a minor cyclic dodecapeptide, differs from laxaphycin B in possessing a 3-aminooctanoyl unit (Aoc) instead of an Ade unit.

Synthesis and in vitro activity of some methylenomycin analogs.

A cationic cyclopentannelation reaction has afforded a very simple route to a wide variety of methylenomycin analogs. The antibacterial activity of these readily accessible synthetic compounds parallels that of naturally occurring methylenomycin A. The in vitro antitumor activity of the synthetics assayed using human nasopharyngeal carcinoma (KB) cells is particularly promising.

Cytotoxic, antiviral indolocarbazoles from a blue-green alga belonging to the Nostocaceae.

6-Cyano-5-methoxy-12-methylindolo[2,3-alpha]carbazole and 6-cyano-5-methoxyindolo[2,3-alpha]carbazole are responsible for most of the cytotoxicity and antiviral activity associated with the blue-green alga Nostoc sphaericum EX-5-1. The compounds are active against HSV II and show weak cytotoxicity against KB and LoVo human carcinoma cell lines.

Antifungal cyclic peptides from the terrestrial blue-green alga Anabaena laxa. I. Isolation and biological properties.

Laxaphycins are responsible for the antifungal and cytotoxic activity of crude ethanolic extracts from the cultured blue-green alga Anabaena laxa. These cyclic peptides exhibit an unusual biological synergism when tested for antifungal or cytotoxic effects. The isolation procedure for the peptides, their characterization and biological activities are described here along with experiments demonstrating synergism between the two major laxaphycins.

Cytotoxic, fungicidal nucleosides from blue green algae belonging to the Scytonemataceae.

Tubercidin, toyocamycin, and the corresponding 5'-alpha-D-glucopyranose derivatives of the nucleosides are frequently responsible for much of the cytotoxicity and antimycotic activity associated with extracts of cultured cyanophytes belonging to the family Scytonemataceae. The 5'-alpha-D-glucopyranoses of tubercidin and toyocamycin, for example, are the major cytotoxic and fungicidal nucleosides in Fijian Plectonema radiosum and Hawaiian Tolypothrix tenuis, respectively.

Scytophycin production by axenic cultures of the cyanobacterium Scytonema ocellatum.

Nonaxenic clones prepared from the cyanobacterium Syctonema ocellatum Lyngbye ex Bornet et Flahault strain FF-66-3 exhibited a high degree of heterogeneity with respect to tolytoxin titer. Thirty-four of 114 clones (29.8%) isolated by fragmentation of Scytonema filaments did not produce detectable amounts of tolytoxin in culture. One clone (designated SO127) produced approximately twice as much tolytoxin as the parental culture and continued to produce tolytoxin after repeated subculture. Three axenic clones were prepared from SO127 by a combination of antibiotic treatment and mechanical separation. Although axenic cultures yielded slightly greater biomass, tolytoxin content did not significantly differ between axenic and nonaxenic cultures, indicating that bacteria do not play a role in tolytoxin biosynthesis. Bacterial cultures derived from S. ocellatum did not produce detectable amounts of tolytoxin.

Regulation of scytophycin accumulation in cultures of Scytonema ocellatum. II. Nutrient requirements.

The effects of optimal sources and concentrations of major nutrients (supplying N, S, P, K+, Na+, Ca2+, Mg2+, and inorganic carbon) and organic buffers on growth and secondary metabolite accumulation in Scytonema ocellatum strain FF-66-3 were determined. Nitrate, phosphate, magnesium, and sulfur had no specific stimulatory or inhibitory effects on scytophycin accumulation within the range of concentrations that supported optimal growth. Calcium concentrations greater than those required for growth (0.1 mM) stimulated scytophycin accumulation. Sodium carbonate concentrations in excess of 0.25 mM strongly inhibited growth. Ammonium (2.5 mM) inhibited both growth and product formation. 3-[N-Morpholino]propanesulfonic acid at 3-5 mM effectively controlled pH and facilitated both growth and product formation.

Biological effects of tolytoxin (6-hydroxy-7-O-methyl-scytophycin b), a potent bioactive metabolite from cyanobacteria.

Tolytoxin, a macrocyclic lactone, is a potent antifungal antibiotic, exhibiting MICs in the range of 0.25 to 8 nanomolar. Tolytoxin also inhibits the growth of a variety of mammalian cells at similar doses, without specific inhibition of macromolecular synthesis. The effects in mammalian cells are primarily cytostatic, with cell death being time- and dose-dependent. Tolytoxin is highly toxic to mice, exhibiting an LD50 (ip) of 1.5 mg/kg. No antibacterial, antiviral, or hemolytic activities were observed.

beta-Carbolines from the blue-green alga Dichothrix baueriana.

Three new chlorine-containing beta-carbolines, bauerines A-C (1-3), have been isolted from the terrestrial blue-green alga Dichothrix baueriana GO-25-2, and identifying mass and nmr spectral analysis. The alkaloids show activity against herpes simplex virus type 2.

New actin mutants allow further characterization of the nucleotide binding cleft and drug binding sites.

We have generated 9 site-specific mutations in Saccharomyces cerevisiae actin. These mutants display a variety of phenotypes when expressed in vivo, including slow actin filament turnover, slow fluid-phase endocytosis, and defects in actin organization. Actin mutation D157E confers resistance to the actin-sequestering drug, latrunculin A. Latrunculin A inhibits nucleotide exchange on wild-type yeast actin but not on D157E actin, suggesting that this residue is part of the latrunculin A binding site. We have refined our earlier map of the phalloidin binding site on actin, demonstrating a requirement for residue G158 in addition to D179 and R177. The nine new actin mutants as well as a large collection of existing actin mutants were also used to identify the putative binding site of another actin binding drug, tolytoxin, on actin. The actin alleles that result in decreased sensitivity to this drug cluster at a site near the nucleotide-binding pocket. Actin purified from one of these mutants has a reduced affinity for tolytoxin. In addition, tolytoxin causes a 2.4-fold increase in the t1/2 of ATP exchange, further suggesting that this drug binds near the nucleotide-binding pocket of actin. We note that the binding sites for latrunculin A, phalloidin, and tolytoxin all map close to the actin nucleotide binding pocket.