Storm water pollutant removal performance of compost filter socks.

In 2005, the U.S. Environmental Protection Agency (USEPA) National Menu of Best Management Practices (BMPs) listed compost filter socks (FS) as an approved BMP for controlling sediment in storm runoff on construction sites. The objectives of this study were to determine if FS with or without the addition of a flocculation agent to the FS system can significantly remove (i) suspended clay and silt particulates, (ii) ammonium nitrogen (NH(4)-N) and nitrate-nitrite nitrogen (NO(3)-N), (iii) fecal bacteria, (iv) heavy metals, and (v) petroleum hydrocarbons from storm water runoff. Five separate (I-V) 30-min simulated rainfall-runoff events were applied to soil chambers packed with Hartboro silt loam (fine-loamy, mixed, active, nonacid, mesic fluvaquentic Endoaquepts) or a 6-mm concrete veneer on a 10% slope, and all runoff was collected and analyzed for hydraulic flow rate, volume, pollutant concentrations, pollutant loads, and removal efficiencies. In corresponding experiments, runoff was analyzed for (i) size of sediment particles, (ii) NH(4)-N and NO(3)-N, (iii) total coliforms (TC) and Escherichia coli, (iv) Cd, Cr, Cu, Ni, Pb and Zn, and (v) gasoline, diesel, and motor oil, respectively. Results showed that: (i) FS removed 65% and 66% of clay (<0.002 mm) and silt (0.002-0.05 mm), respectively; (ii) FS removed 17%, and 11% of NH(4)-N and NO(3)-N, respectively and when NitroLoxx was added to the FS, removal of NH(4)-N load increased to 27%; (iii) total coliform and E. coli removal efficiencies were 74 and 75%, respectively, however, when BactoLoxx was added, removal efficiency increased to 87 and 99% for TC and 89 and 99% for E. coli, respectively; (iv) FS removal efficiency for Cd, Cr, Cu, Ni, Pb, and Zn ranged from 37 to 72%, and, when MetalLoxx was added, removal efficiency ranged from 47 to 74%; and (v) FS removal efficiency for the three petroleum hydrocarbons ranged from 43 to 99% and the addition of PetroLoxx increased motor oil and gasoline removal efficiency in the FS system.

Large-scale performance and design for construction activity erosion control best management practices.

The National Pollutant Discharge Elimination System (NPDES) Phase II requires construction activities to have erosion and sediment control best management practices (BMPs) designed and installed for site storm water management. Although BMPs are specified on storm water pollution prevention plans (SWPPPs) as part of the construction general permit (GP), there is little evidence in the research literature as to how BMPs perform or should be designed. The objectives of this study were to: (i) comparatively evaluate the performance of common construction activity erosion control BMPs under a standardized test method, (ii) evaluate the performance of compost erosion control blanket thickness, (iii) evaluate the performance of compost erosion control blankets (CECBs) on a variety of slope angles, and (iv) determine Universal Soil Loss Equation (USLE) cover management factors (C factors) for these BMPs to assist site designers and engineers. Twenty-three erosion control BMPs were evaluated using American Society of Testing and Materials (ASTM) D-6459, standard test method for determination of ECB performance in protecting hill slopes from rainfall induced erosion, on 4:1 (H:V), 3:1, and 2:1 slopes. Soil loss reduction for treatments exposed to 5 cm of rainfall on a 2:1 slope ranged from-7 to 99%. For rainfall exposure of 10 cm, treatment soil loss reduction ranged from 8 to 99%. The 2.5 and 5 cm CECBs significantly reduced erosion on slopes up to 2:1, while CECBs < 2.5 cm are not recommended on slopes >or= 4:1 when rainfall totals reach 5 cm. Based on the soil loss results, USLE C factors ranged from 0.01 to 0.9. These performance and design criteria should aid site planners and designers in decision-making processes.

Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway.

In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.

Three new pseudodistomins, piperidine alkaloids from the ascidian Pseudodistoma megalarva.

Bioassay-guided fractionation of the MeOH-CH2Cl2 extract of the Micronesian ascidian Pseudodistoma megalarva yielded three new piperidine alkaloids, pseudodistomins D-F (3-5) and the previously reported pseudodistomins B and C (1 and 2). The structure and stereochemistry of these compounds were established by interpretation of spectral data. Pseudodistomins B-F were found to be active in a cell-based assay for DNA damage induction, but the activity was due to an alternative mechanism.

Two new nitrogenous sesquiterpenes from the sponge Axinyssa aplysinoides.

Bioassay-guided fractionation of the EtOAc extract of the Palauan sponge Axinyssa aplysinoides yielded two novel alkaloids, 1 and 2. The structure of 2-(formylamino)trachyopsane (1) was determined by X-ray analysis; and the structure of N-phenethyl-N'-2-trachyopsanylurea (2), by interpretation of the spectral data.

The yeast FKS1 gene encodes a novel membrane protein, mutations in which confer FK506 and cyclosporin A hypersensitivity and calcineurin-dependent growth.

FK506 and cyclosporin A (CsA) are potent immunosuppressive agents that display antifungal activity. They act by blocking a Ca(2+)-dependent signal transduction pathway leading to interleukin-2 transcription. Each drug forms a complex with its cognate cytosolic immunophilin receptor (i.e., FKBP12-FK506 and cyclophilin-CsA) which acts to inhibit the Ca2+/calmodulin-dependent protein phosphatase 2B, or calcineurin (CN). We and others have defined the Saccharomyces cerevisiae FKS1 gene by recessive mutations resulting in 100-1000-fold hypersensitivity to FK506 and CsA (as compared to wild type), but which do not affect sensitivity to a variety of other antifungal drugs. The fks1 mutant also exhibits a slow-growth phenotype that can be partially alleviated by exogenously added Ca2+ [Parent et al., J. Gen. Microbiol. 139 (1993) 2973-2984]. We have cloned FKS1 by complementation of the drug-hypersensitive phenotype. It contains a long open reading frame encoding a novel 1876-amino-acid (215 kDa) protein which shows no similarity to CN or to other protein phosphatases. The FKS1 protein is predicted to contain 10 to 12 transmembrane domains with a structure resembling integral membrane transporter proteins. Genomic disruption experiments indicate that FKS1 encodes a nonessential function; fks1::LEU2 cells exhibit the same growth and recessive drug-hypersensitive phenotypes observed in the original fks1 mutants. Furthermore, the fks1::LEU2 allele is synthetically lethal in combination with disruptions of both of the nonessential genes encoding the alternative forms of the catalytic A subunit of CN (CNA1 and CNA2). These data suggest that FKS1 provides a unique cellular function which, when absent, increases FK506 and CsA sensitivity by making the CNs (or a CN-dependent function) essential.

Dominant missense mutations in a novel yeast protein related to mammalian phosphatidylinositol 3-kinase and VPS34 abrogate rapamycin cytotoxicity.

Rapamycin is a macrolide antifungal agent that exhibits potent immunosuppressive properties. In Saccharomyces cerevisiae, rapamycin sensitivity is mediated by a specific cytoplasmic receptor which is a homolog of human FKBP12 (hFKBP12). Deletion of the gene for yeast FKBP12 (RBP1) results in recessive drug resistance, and expression of hFKBP12 restores rapamycin sensitivity. These data support the idea that FKBP12 and rapamycin form a toxic complex that corrupts the function of other cellular proteins. To identify such proteins, we isolated dominant rapamycin-resistant mutants both in wild-type haploid and diploid cells and in haploid rbp1::URA3 cells engineered to express hFKBP12. Genetic analysis indicated that the dominant mutations are nonallelic to mutations in RBP1 and define two genes, designated DRR1 and DRR2 (for dominant rapamycin resistance). Mutant copies of DRR1 and DRR2 were cloned from genomic YCp50 libraries by their ability to confer drug resistance in wild-type cells. DNA sequence analysis of a mutant drr1 allele revealed a long open reading frame predicting a novel 2470-amino-acid protein with several motifs suggesting an involvement in intracellular signal transduction, including a leucine zipper near the N terminus, two putative DNA-binding sequences, and a domain that exhibits significant sequence similarity to the 110-kDa catalytic subunit of both yeast (VPS34) and bovine phosphatidylinositol 3-kinases. Genomic disruption of DRR1 in a mutant haploid strain restored drug sensitivity and demonstrated that the gene encodes a nonessential function. DNA sequence comparison of seven independent drr1dom alleles identified single base pair substitutions in the same codon within the phosphatidylinositol 3-kinase domain, resulting in a change of Ser-1972 to Arg or Asn. We conclude either that DRR1 (alone or in combination with DRR2) acts as a target of FKBP12-rapamycin complexes or that a missense mutation in DRR1 allows it to compensate for the function of the normal drug target.

A screen for inhibitors of DNA recombination: identification of two new spirostanol glycosides from Chamaedorea linearis.

Two new glycosides have been isolated from the MeOH extract of the stem wood and stem bark of an Ecuadorian plant Chamaedorea linearis, and their structures have been determined by spectroscopic means and X-ray analysis of the aglycone to be 1-O-[beta-L-fucopyranosyl-(4'-sulfate)]-25R,5 alpha-spirostane-1 beta, 3 beta-diol [1]) and 1-O-[beta-L-fucopyranosyl-(4'-sulfate)]-25R,5 alpha-spirostane-1 alpha, 3 beta-diol [2]. These compounds were identified in a screen for inhibitors of recombinational DNA repair. Cytotoxic activity was also demonstrated.

The tyrosine89 residue of yeast FKBP12 is required for rapamycin binding.

Rapamycin (Rm) is a macrolide antifungal agent related to FK506 that exhibits potent immunosuppressive properties which are mediated through interaction with specific cytoplasmic receptors (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. In Saccharomyces cerevisiae, Rm sensitivity (Rms) is mediated by binding of the drug to RBP1, a homolog of the 12-kDa human FK506-binding protein (FKBP12); null mutations in the yeast RBP1 gene result in a recessive drug resistance phenotype. To identify missense mutations that define amino acid (aa) residues in RBP1 involved in drug sensitivity, we selected and genetically characterized over 250 independent RmR rbp1 mutants and screened them for both RBP1-specific mRNA and protein expression. Whereas all rbp1 mutants expressed abundant levels of RBP1 mRNA, stable RBP1 protein production was detected in only one mutant strain. The RBP1 gene was PCR-generated (in triplicate) from several rbp1 mutants and independent clones were sequenced. Most of the immunoblot-negative alleles were found to contain various types of null mutations; however, some alleles contained specific missense mutations that apparently affect protein stability in vivo. The single immunoblot-positive allele was found to contain a mutation altering a specific residue (Tyr89) which is conserved among the known FKBPs, and which, based on the solution and x-ray structures of human FKBP12, has been proposed to be part of a hydrophobic drug-binding pocket for FK506 and Rm.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein.

Rapamycin is a macrolide antifungal agent with structural similarity to FK506. It exhibits potent immunosuppressive properties analogous to those of both FK506 and cyclosporin A (CsA). Unlike FK506 and CsA, however, rapamycin does not inhibit the transcription of early T-cell activation genes, including interleukin-2, but instead appears to block downstream events leading to T-cell activation. FK506 and CsA receptor proteins (FKBP and cyclophilin, respectively) have been identified and shown to be distinct members of a class of enzymes that possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Despite the apparent differences in their mode of action, rapamycin and FK506 act as reciprocal antagonists in vivo and compete for binding to FKBP. As a means of rapidly identifying a target protein for rapamycin in vivo, we selected and genetically characterized rapamycin-resistant mutants of Saccharomyces cerevisiae and isolated a yeast genomic fragment that confers drug sensitivity. We demonstrate that the resonse to rapamycin in yeast cells is mediated by a gene encoding a 114-amino-acid, approximately 13-kDa protein which has a high degree of sequence homology with human FKBP; we designated this gene RBP1 (for rapamycin-binding protein). The RBP1 protein (RBP) was expressed in Escherichia coli, purified to homogeneity, and shown to catalyze peptidyl-prolyl isomerization of a synthetic peptide substrate. PPIase activity was completely inhibited by rapamycin and FK506 but not by CsA, indicating that both macrolides bind to the recombinant protein. Expression of human FKBP in rapamycin-resistant mutants restored rapamycin sensitivity, indicating a functional equivalence between the yeast and human enzymes.

In vivo development and in vitro characterization of a subclone of murine P388 leukemia resistant to bis(diphenylphosphine)ethane.

Bis(diphenylphosphine)ethane (DPPE) and its gold coordination complexes have demonstrated antitumor activity in transplantable tumor models. This report describes the development of a P388 cell line (P388/DPPEc) that is resistant to DPPE and its analogues and the in vitro characterization of the cross-resistance of this subline to various antitumor and cytotoxic agents. The P388/DPPE tumor cell line was developed by serial transplantation in DPPE-treated mice. Resistance to DPPE was phenotypically stable. The P388/DPPE subline was cross-resistant to DPPE analogues and metal coordination complexes of DPPE. In addition, P388/DPPE cells were resistant to several mitochondrial uncouplers, including rhodamine-123, tetraphenylphosphonium, and carbonylcyanide-p-trifluro-methoxyphenyl hydrazone. P388/DPPE cells were less capable of sequestering and retaining 123Rh than were sensitive (P388/S) cells. Exposure to Au(DPPE)2+, a gold complex of DPPE with increased antitumor activity, resulted in a depletion of cellular ATP; the depletion was more rapid in the sensitive than the resistant cells. The rate of mitochondrial respiration, as measured by 14CO2 evolution from [6-14C]glucose, was greater in P388/S than in P388/DPPE. As with that evidenced for 123Rh, the cellular uptake of radiolabeled DPPE was decreased in P388/DPPEc cells. The results suggest that the basis for the resistance of this cell line may be an alteration in mitochondrial membrane potential. These data and the striking cross-resistance of P388/DPPE to mitochondrial uncouplers support the hypothesis that mitochondria may be one target involved in the cytotoxic or antitumor activities of these compounds. Mitochondria may also be causally related to the cytotoxic or antitumor activities, in that DPPE may be concentrated in cells via the presence of the inner mitochondrial membrane potential. Thus, P388/DPPE cells can serve as a tool to screen for and evaluate drugs that rely on affecting mitochondrial function, either mechanistically or causally, for their antitumor efficacy.

Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity.

Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.

Cytotoxicity and antitumor activity of some tetrahedral bis(diphosphino)gold(I) chelates.

We report the cytotoxicity toward B16 cells and antitumor activity in three transplantable tumor models of a series of ionic, tetrahedral, bischelated gold diphosphine complexes of the type [Au1(R2PYPR2')2]X, where Y = (CH2)2, (CH2)3, or cis-CH = CH. The anion (X = Cl, Br, I, CH3SO3, NO3, PF6) had little effect upon activity. The R = R' = phenyl complexes 1, 7, and 8 [Y = (CH2)2, (CH2)3, cis-CH = CH, X = Cl] were the most active against P388 leukemia, with an increase in lifespan ranging from 83 to 92% and were also active against M5076 sarcoma and B16 melanoma. Complexes with pyridyl or fluorophenyl substituents had reduced activities. For the latter, 19F and 31P NMR were used to verify the formation of bischelated gold(I) complexes in solution. The reduced activity of the complex with R = Et and R' = Ph and inactivity with R = R' = Et are discussed in terms of their increased reactivity as reducing agents. 31P NMR studies show that [AuI(Et2P(CH2)2PPh2)2]Cl readily reacts with serum, albumin, and Cu2+ ions to give oxidized ligand.

Synthesis, tubulin binding, antineoplastic evaluation, and structure-activity relationship of oncodazole analogues.

In an attempt to identify a soluble oncodazole analogue that could be easily formulated, a series of substituted oncodazoles was synthesized and evaluated for tubulin binding affinity, in vitro cytotoxicity against cultured mouse B-16 cells, and ability to prolong lifespan at the maximally tolerated dose in the P388 mouse leukemia model. Biological evaluation of all the isomeric methyloncodazoles demonstrated the thiophene 4'-position to be the only site of significant bulk tolerance, although substitution of this position with polar or charged functional groups abolished biological activity. Simple esters of the 4'-carboxymethyloncodazole were shown to have enhanced antitumor activity and tubulin binding affinity relative to oncodazole. Despite a failure of this study to identify a water-soluble oncodazole with antitumor activity, the structure-activity relationship developed led to a derivative with enhanced activity in the P388 leukemia model and facilitated the preparation of a biologically active photolabile analogue.

Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin.

The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe are both sensitive to camptothecin, an inhibitor of DNA topoisomerase I. An S. cerevisiae DNA repair mutant, rad52, is hypersensitive to the drug. In both species, topoisomerase I mutants totally lacking the enzyme are completely resistant to the drug. A strain with a mutation leading to a temperature-sensitive topoisomerase I exhibits temperature dependence in its in vivo response to camptothecin. A strain carrying a plasmid that overproduces topoisomerase I is hypersensitive to the drug. The rad52 mutant is killed by overproduction of the enzyme, even in the absence of the drug. The response of several of these strains to camptothecin analogs, to DNA topoisomerase II inhibitors, and to other drugs is reported. The cytotoxic effects of camptothecin are discussed in terms of the drug extending the lifetime of a topoisomerase I-DNA covalent intermediate, which is recognized as DNA damage by a DNA repair system.

A murine model to evaluate the ability of in vitro clonogenic assays to predict the response to tumors in vivo.

The use of the human tumor cloning assay as a predictor of clinical response of human tumors to drugs is predicated on the hypothesis that the in vivo response of a tumor to a drug can be correlated with the in vitro response of cells derived from the tumor. To test this hypothesis, we utilized a murine tumor model in which the in vivo and in vitro responses of a tumor can be accurately and reproducibly compared. Drug activity was assessed in P388 leukemia with the standard in vivo antitumor assay (i.p. tumor/i.p. drug administration) and an in vitro assay wherein the ascites tumor cells are removed from mice, treated with a drug, and directly cloned in soft agar to measure clonogenic capacity. The response of P388 cells to analogues within four separate classes of antitumor agents, anthracyclines, anthraquinones, platinum(II) coordination complexes, and phosphinogold(I) complexes was evaluated. The clonogenic assay failed to discriminate between highly active in vivo antitumor agents and analogues with only marginal in vivo efficacy (i.e., doxorubicin and daunorubicin versus rhodomycins A and B, ametantrone versus NSC 276740, cisplatin versus transplatin, [Au(dppe)2]Cl versus [Au(depe)2]PF6. Furthermore, the in vitro clonogenic assay failed to detect carboplatin which was a highly active agent in vivo. The basis for these discrepancies was explored by a more detailed comparison of doxorubicin and rhodomycin B. In vivo or in vitro drug exposure with subsequent measurement of cell kill by the in vitro clonogenic and in vivo tumorigenic assay demonstrated that the in vitro assay overestimated the cytotoxic potency of the drugs relative to the tumorigenic assay. Treatment of tumors in vivo with doxorubicin at doses below the maximally tolerated dose in mice resulted in multiple log cell kill as measured in vitro or in vivo, whereas rhodomycin B was cytotoxic only at dose levels exceeding its maximally tolerated dose. The results indicate that a subset of tumor stem cells capable of forming colonies in soft agar are significantly more sensitive to the cytotoxic effects of anthracyclines than are in vivo tumorigenic stem cells. Cytotoxic potency as measured by an in vitro soft agar clonogenic assay is not an accurate predictor of in vivo antitumor efficacy even in a model in which ascites tumor cells are directly exposed to i.p. drug. The in vitro cytotoxicity assay is useful only as a nonselective prescreen and must be used in combination with other indicators of tumor cell selectivity and dose-limiting organ toxicity.

Pyrrolo[1,4]benzodiazepine antitumor antibiotics: relationship of DNA alkylation and sequence specificity to the biological activity of natural and synthetic compounds.

The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo-[1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease III stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease III which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry at C-11a. These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LD50), and for antitumor activity (ILSmax) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds.

Antimicrobial and anticancer activity of tetrahedral, chelated, diphosphine silver(I) complexes: comparison with copper and gold.

Tetrahedral, bischelated Ag(I) diphosphine complexes [Ag(P-P)2]NO3, where P-P is Ph2P(CH2)2PPh2 (dppe), Et2P(CH2)2PPh2 (depe), and cis-Ph2P(CH = CH)PPh2 (dppey), are potently cytotoxic to B16 melanoma cells in vitro (IC50 4 microM) and exhibit good activity against ip P388 leukemia in mice. The complex [Ag(dppe)2]NO3 is active against M5076 reticulum cell sarcoma. The antibacterial and antifungal activities of Ag(I) diphosphine and related Cu(I) and Au(I) complexes were assessed. The complexes [Au(dppey)2]Cl, [Au(dppp)2]Cl and (CuCl)2(dppe)3 show modest activity against three of the 12 bacterial strains tested, but all complexes exhibit antifungal activity against three strains of C. albicans in a "defined" medium, [Ag(depe)2]NO3 and [Au(dppp)2]Cl having comparable activity to fungizone. Antifungal activity of the complexes is reduced in Sabouraud's broth medium, and lost altogether for the Ag(I) complexes. Reactions of some of the Ag(I) complexes with glutathione and blood plasma were studied by 31P NMR.

Biological characterization and oncogene expression in human colorectal carcinoma cell lines.

To establish well-characterized cellular reagents for the study of colon carcinoma, we have examined 19 human colorectal carcinoma cell lines with regard to morphology, ultrastructure, expression of tumor-associated antigens, proliferative capacity in vitro, anchorage-independent growth, oncogene expression, tumorigenicity and malignant potential. Cell lines examined were cultured under identical conditions, and in vitro and in vivo analyses were performed in parallel on replicate cultures. Three classes of colorectal cell lines were defined according to their tumorigenicity in nude mice. Class-1 lines formed rapidly progressing tumors in nearly all mice at an inoculum of 10(6) cells. Cell lines belonging to class-2 were less tumorigenic, producing tumors later and at a slower growth rate. Class-3 lines were non-tumorigenic under all experimental conditions tested. By Northern analysis, the oncogenes c-myc, H-ras, K-ras, N-ras, myb, fos and p53 were expressed in nearly all cell lines examined. In contrast, transcripts for abl, src and ros were not detected. The best in vitro predictor of tumorigenicity was colony formation in soft agar. There was no detectable correlation between tumorigenicity and metastatic potential, doubling time in vitro, production of tumor-associated markers, xenograft histology or expression of specific oncogenes.

Tumorigenicity of the cyc- variant of the S49 murine lymphoma deficient in the Gs-alpha subunit of adenylate cyclase.

S49 cyc- lymphoma cells contain a mutation resulting in loss of a functional guanine nucleotide regulatory protein rendering their adenylate cyclase refractory to most stimuli. S49 wild-type and cyc- clones were used in the present study to investigate the possible association of altered cAMP metabolism with tumorigenicity and metastatic potential. The S49 clones were implanted i.v., i.p., and intracerebrally in both athymic nude mice and syngeneic, immunocompetent BALB/c mice. Both S49 clones gave rise to tumors when inoculated into athymic mice, and no differences were observed in the tumorigenicity or metastatic potential of S49 wild-type and cyc- cells. Implantation of S49 clones in syngeneic BALB/c mice gave rise to few tumors except when administered intracerebrally, where wild-type cells were more tumorigenic than cyc- cells. This raises the possibility of differences in immunogenicity between the S49 clones. Analysis of cell lines derived from tumors grown in athymic mice showed that they retained the phenotype of the S49 clones used for inoculations. The results indicate that, despite differences in adenylate cyclase responsiveness, S49 wild-type and cyc- cells are both highly tumorigenic and metastatic.