Chemical recovery and browning of Nova Scotia surface waters in response to declining acid deposition.

Declining emissions of sulfur and nitrogen have curtailed acid deposition across large areas of North America and Europe. This has allowed many lakes to recover from acidification, with decreases in sulfate, increases in pH, and increases in alkalinity. But reduced acid deposition has not always coincided with chemical lake recovery. Surface waters in Nova Scotia did not exhibit clear evidence of recovery as recently as 2007, due in part to increasing organic acidity and slow replenishment of base cations. In an updated assessment with data collected as recently as 2019, we analyze water chemistry representing 81 lakes and rivers and two precipitation monitoring stations over up to 41 years. We find that Nova Scotia surface waters are now exhibiting signs of chemical recovery. We estimated the linear decrease in precipitation sulfate and nitrate yield at up to 0.31 and 0.18 kg ha-1 year-2, respectively, and the linear increase in precipitation pH at up to 0.014 year-1. Sulfate decreased in 60 of 62 lakes and 14 of 17 rivers (-0.0051 to -0.23 mg L-1 year-1), while pH increased in 55 of 64 lakes and 11 of 17 rivers (0.0015-0.072 year-1). Apparent colour increased in 54 of 62 lakes and 13 of 17 rivers (0.0026-3.9 Pt-Co year-1). We identified increasing aluminum trends in 46 of 61 lakes, and we show using size-exclusion chromatography that binding to organic and iron-based colloids may help to explain these trends. To the extent that increases in apparent colour are explained by chromophoric dissolved organic matter (DOM), they imply greater binding capacity for metals in surface waters, and greater capacity for DOM to stabilize metal (oxyhydr)oxide colloids.

Phenotypic instability of rat mammary tumor epithelial cells.

The spontaneous production of elongated derivatives by cuboidal rat mammary epithelial cells was examined with the use of a series of single-cell clones grown in tissue culture. Four representative cell lines derived from a 7,12-dimethylbenz[a]anthracene-induced mammary tumor in an inbred WF rat were examined for morphologic stability, chromosome number, presence of immunoreactive fibronectin, laminin, prekeratin, and milk fat globule membrane (MFGM) antigens, ultrastructural characteristics, and tumorigenicity in syngeneic hosts. Conversion of cuboidal to elongated cells occurred by way of apparent morphologic intermediates, examples of which were isolated and cloned. Levels of immunoreactive fibronectin and laminin were greater in the elongated than the cuboidal clones, whereas the converse was true of prekeratin. MFGM antigens were present to a variable extent in all 4 clones. When grown on 0.3% collagen gels, cells of Rama 37 CL-A3 and Rama 37CS-A2 cuboidal clones exhibited surface microvilli and desmosomes. A minority of elongated cells contained microfilamental structures and pinocytotic vesicles similar to those seen in myoepithelial cells; the remainder lacked distinguishing ultrastructural features. After injection into syngeneic recipients, Rama 37 CL-A3 cuboidal line gave rise to glandular tumors consisting of cuboidal cells arranged in acinar structures, Rama 37 E5 elongated line induced spindle cell tumors, and Rama 37 CS-A2 and Rama 37 E8 lines induced tumors containing nests of mixed spindle and cuboidal cells. The majority of these tumors failed to metastasize.

Loss of myoepithelial cell characteristics in metastasizing rat mammary tumors relative to their nonmetastasizing counterparts.

A series of WF rat mammary tumors comprising one transplantable nonmetastasizing line (MT-W9), two predominantly lymphatic (SMT-2A and SMT-077) and one lymphatic and hematogenous (MT-450) metastasizing transplantable lines, and 7,12-dimethylbenz[a]anthracene (DMBA)-induced nonmetastasizing primary tumors was examined for the presence of epithelial and myoepithelial cell characteristics with the use of immunocytochemical techniques. Tumor cells staining for myosin were only occasionally observed in a basal orientation in glandular structures in sections of DMBA-induced and MT-W9 tumors; anti-laminin serum stained the peripheries of the glandular structures in the DMBA-induced and MT-W9 tumors but failed to stain the SMT-2A and SMT-077 tumor cells. In the nonmetastasizing tumors immunologically detectable keratin occurred mainly in the outer cellular layer of glandlike structures, whereas milk fat globule membrane immunoreactivity occurred primarily in the luminal cells. Both these types of immunoreactivity were observed in MT-450 tumor cells, but the pattern of keratin staining was random. No such immunoreactivity occurred in SMT-2A or SMT-077 tumors. No tumor cell-associated staining for fibronectin was seen in any of the tumors examined, although host stromal components stained intensely. The nonmetastasizing tumors contained cuboidal epithelial cells with lumen formation, surface microvilli, and intercellular junctional complexes, together with a relatively undifferentiated elongated cell component. Other elongated cells showed hemidesmosomes, pinocytotic vesicles, tonofilaments, and small bundles of cytoplasmic filaments, suggesting gradations toward a myoepithelial phenotype. The MT-450 tumors were ultrastructurally similar to the nonmetastasizing tumors, but no features of myoepithelial cells were seen, although some cuboidal epithelial cells exhibited prominent tonofilaments. The SMT-2A and SMT-077 tumors consisted of nests of cuboidal-like cells with highly pleomorphic nuclei and much intercellular collagen. The results indicate a progressive loss of cellular differentiation characteristics, particularly those of the myoepithelial cell with increasing malignancy in this system.

Dedifferentiation of rat mammary myoepithelial-like cell lines after passage in vivo or cloning in vitro.

Myoepithelial-like cell lines from normal mammary glands of neonatal Ludwig Wistar rats, rat mammary (Rama) 401 and Rama 704E, were injected into fat pads of syngeneic animals or were single-cell cloned in vitro. Rama 401 produced tumors that were predominantly composed of elongated cells, while the subclones of both cell lines yielded multilayered structures of elongated cells when grown on floating 0.3% collagen gels in vitro. Immunocytochemical analysis of histologic sections for markers of myoepithelial cells revealed that anti-actin-myosin and human keratin sera failed to stain the Rama 401 tumor cells or subclones of both cell lines on collagen gels, but both were stained with antilaminin serum. Immunofluorescent analysis of cultures of Rama 401 tumors showed that the resulting elongated cells failed to stain with antikeratin serum, but abundant staining was observed with antilaminin and antivimentin sera, as in the tumors. Ultrastructural analysis of the Rama 401 tumor cells identified intermediate junctions and extracellular basement membrane-like material in the vicinity of plasma membrane-associated pinocytotic vesicles, but neither true desmosomes nor myofilamental bundles were observed. Thus growth of rat mammary myoepithelial-like cells as tumors in syngeneic animals or as subclones in vitro can lead to selective loss of myofilaments and prekeratin-containing intermediate filaments. Similar relatively undifferentiated elongated cells may be responsible for some of the cellular heterogeneity observed in certain carcinogen-induced rat mammary tumors.

Production of skeletal muscle elements by cell lines derived from neoplastic rat mammary epithelial stem cells.

Single-cell-cloned cell lines intermediate in morphology between the cuboidal epithelial and fully elongated myoepithelial-like cells have been isolated from the single-cell-cloned epithelial stem cell lines Rama 25 and Rama 37 originally obtained from dimethylbenz(a)anthracene-induced mammary tumors from Sprague-Dawley and Wistar-Furth rats, respectively. These are designated Rama 25-l1, Rama 25-l2, Rama 25-l4 (Sprague-Dawley) and Rama 50-55, Rama 59, and Rama 60 (Wistar-Furth), respectively. When growing as tumors in nude mice or syngeneic Wistar-Furth rats, respectively, many of the newly cloned cell lines give rise to spindle and giant, multinucleated cells which stain immunocytochemically with antisera to myoglobin and myosin and contain longitudinal fibrils, some of which contain phosphotungstic acid-hematoxylin-staining cross-striations. Ultrastructural analysis demonstrates the presence of A-, l-, and H-bands and Z-discs and the hexagonal arrangement of thick and thin filaments characteristic of skeletal muscle. Similar results are obtained with selected cloned cell lines growing on floating collagen gels in vitro. Thus, a developmentally committed mammary epithelial cell can give rise, under suitable conditions, to a well-differentiated mesenchymal lineage, that of skeletal muscle. It is suggested that such cells may be responsible for the generation of the well-differentiated mesenchymal elements seen in the mixed (epithelial and myoepithelial) tumors of glandular origin.

Lack of production of myoepithelial variants by cloned epithelial cell lines derived from the TMT-081 metastasizing rat mammary tumor.

A series of cell lines was isolated from the metastasizing rat mammary tumor cell strain TMT-081. MS by single-cell cloning. Feeder cells were required for development of single tumor cells into clonal colonies. The rate, pattern, and incidence of metastases following injection of cells into the mammary fat pads of syngeneic rats were relatively similar for the various cell lines, with dissemination to the lungs and axillary and paraaortic lymph nodes. When a representative cell line termed Rama 800 was subcloned, one subline was nontumorigenic, and another gave a lower incidence of lung metastases, but the remainder had similar in vivo properties to the parental Rama 800 cells. The metastatic properties of Rama 800 cells were not affected by passage in vitro through 60 cell generations. No production of myoepithelial-like variants from Rama 800 cells was observed at the ultrastructural level. Antisera to keratin, actin, laminin, and fibronectin, which normally stain myoepithelial cells and basement membrane, failed to stain Rama 800 cells, either in cultures or in tumor sections. Heterogeneous staining of Rama 800 tumor cells with antiserum to epithelial cell-specific milk fat globule membrane antigens was seen in tumor sections but not in culture. Abundant microvilli and membrane blebs were observed on the surface of cultured Rama 800 cells, but no lumen formation, desmosomes, or tonofilaments were seen, either in vivo or in vitro. The results suggest that the metastatic epithelial-derived cell lines lack the ability to express features of myoepithelial cells, in contrast to cell lines isolated previously from nonmetastasizing rat mammary tumors.

Structure-activity studies of rapamycin analogs: evidence that the C-7 methoxy group is part of the effector domain and positioned at the FKBP12-FRAP interface.

BACKGROUND: Rapamycin is an immunosuppressant natural product, which blocks T-cell mitogenesis and yeast proliferation. In the cytoplasm, rapamycin binds to the immunophilin FKBP12 and the complex of these two molecules binds to a recently discovered protein, FRAP. The rapamycin molecule has two functional domains, defined by their interaction with FKBP12 (binding domain) or with FRAP (effector domain). We previously showed that the allylic methoxy group at C-7 of rapamycin could be replaced by a variety of different substituents. We set out to examine the effects of such substitutions on FKBP12 binding and on biological activity. RESULTS: Rapamycin C-7-modified analogs of both R and S configurations were shown to have high affinities for FKBP12, yet these congeners displayed a wide range of potencies in splenocyte and yeast proliferation assays. The X-ray crystal structures of four rapamycin analogs in complexes with FKBP12 were determined and revealed that protein and ligand backbone conformations were essentially the same as those observed for the parent rapamycin-FKBP12 complex and that the C-7 group remained exposed to solvent. We then prepared a rapamycin analog with a photoreactive functionality as part of the C-7 substituent. This compound specifically labeled, in an FKBP12-dependent manner, a protein of approximately 250 kDa, which comigrates with recombinant FRAP. CONCLUSIONS: We conclude that the C-7 methoxy group of rapamycin is part of the effector domain. In the ternary complex, this group is situated in close proximity to FRAP, at the interface between FRAP and FKBP12.

Antiserum to a novel peptide sequence reacts selectively with epithelial subpopulations.

A synthetic peptide corresponding to a novel protein sequence isolated from bovine kidney was used to immunize rabbits. When applied to Western blots of bovine kidney extracts, antiserum to this peptide recognizes proteins with molecular weights of 23 and 18 KD. Immunohistochemical examination of a variety of bovine and rat tissues with this antiserum revealed a unique distribution of immunoreactivity with the intermediate layers of a variety of stratified epithelia, in addition to renal glomeruli. The pattern of reactivity differed from previously described epithelial markers such as cytokeratins. These results indicate that this antiserum may be useful as a tool for the identification of cells of the intermediate layer of stratified epithelia and, as such, may aid in the study of this differentiating/proliferating tissue compartment.

An ultra-high throughput screening approach for an adenine transferase using fluorescence polarization.

We have developed a novel assay for measuring the activity of an enzyme that transfers multiple adenine-containing groups to an acceptor protein. The assay is based on fluorescence polarization (FP) technology in a 1536-well plate format. In the assay, a long wavelength fluorescence tracer, Texas Red (Rhodamine), was covalently conjugated to adenine of the donor substrate through a C(6) spacer arm. As a result of the transfer of the adenine-containing moieties to the acceptor protein substrate, the rotational correlation time of the Texas Red conjugate increased, hence increasing the degree of fluorescence polarization. The pharmacological profile and kinetics of the enzyme measured according to the FP method were consistent with those determined previously by conventional analysis. We have successfully executed a 250,000-compound high throughput screening program based on the FP assay method. The quality and validity of the assay were verified by a variety of statistical analyses.

Development and evaluation of high throughput functional assay methods for HERG potassium channel.

Three functional hERG channel assay methods have been developed and evaluated. The methods were tested against five known hERG channel inhibitors: dofetilide, terfenadine (Seldane), sertindole (Serdolect), astemizole (Hismanal), and cisapride (Propulsid). The DiBAC4(3)-based assays were found to be the most economical but had high false-hit rates as a result of the interaction of dye with the test compounds. The membrane potential dye assay had fewer color-quenching problems but was expensive and still gave false hits. The nonradioactive Rb+ efflux assay was the most sensitive of all the assays evaluated and had the lowest false-hit rate.

Automated assay optimization with integrated statistics and smart robotics.

The transition from manual to robotic high throughput screening (HTS) in the last few years has made it feasible to screen hundreds of thousands of chemical entities against a biological target in less than a month. This rate of HTS has increased the visibility of bottlenecks, one of which is assay optimization. In many organizations, experimental methods are generated by therapeutic teams associated with specific targets and passed on to the HTS group. The resulting assays frequently need to be further optimized to withstand the rigors and time frames inherent in robotic handling. Issues such as protein aggregation, ligand instability, and cellular viability are common variables in the optimization process. The availability of robotics capable of performing rapid random access tasks has made it possible to design optimization experiments that would be either very difficult or impossible for a person to carry out. Our approach to reducing the assay optimization bottleneck has been to unify the highly specific fields of statistics, biochemistry, and robotics. The product of these endeavors is a process we have named automated assay optimization (AAO). This has enabled us to determine final optimized assay conditions, which are often a composite of variables that we would not have arrived at by examining each variable independently. We have applied this approach to both radioligand binding and enzymatic assays and have realized benefits in both time and performance that we would not have predicted a priori. The fully developed AAO process encompasses the ability to download information to a robot and have liquid handling methods automatically created. This evolution in smart robotics has proven to be an invaluable tool for maintaining high-quality data in the context of increasing HTS demands.

Mechanism of action of bicyclic imidazoles defines a translational regulatory pathway for tumor necrosis factor alpha.

Expression of tumor necrosis factor alpha (TNF) by lipopolysaccharide-treated human monocytic cells is inhibited by bicyclic imidazoles. We studied the mechanism of action of a representative inhibitor, SK&F 86002, on synthesis of TNF by THP-1 cells. Levels of TNF protein were lowered by SK&F 86002 under conditions where TNF mRNA accumulation was unaffected, suggesting a post-transcriptional action. No effect of SK&F 86002 was detected on the rate of induction of TNF mRNA or steady state levels over a 5 hr period. The kinetics of SK&F 86002 inhibition of TNF protein synthesis coincided with those of anisomycin, not with actinomycin, suggesting an effect of SK&F 86002 on TNF mRNA translation. By using sucrose gradient sedimentation, we showed that quiescent THP-1 cells contained a substantial amount of TNF mRNA which was primarily associated with 43S pre-ribosomal complexes. Activation of the cells with lipopolysaccharide caused an elevation of the TNF mRNA level and increased the proportion associated with polyribosomes. Treatment with lipopolysaccharide plus SK&F 86002 led to a marked accumulation of TNF mRNA in the 43S complex-containing fractions and a concomitant reduction of polysome-associated TNF message. Neither lipopolysaccharide nor SK&F 86002 affected the amount or distribution of cyclophilin mRNA in the same fractions. The results suggest that lipopolysaccharide activates TNF translation at the initiation step and that SK&F 86002 inhibits this activation.

Identification of a low molecular weight form of epithelial transforming growth factor.

We have purified a novel form of epithelial transforming growth factor (TGFe) from bovine kidney. Acid ethanol extracts of kidney were fractionated by size exclusion, reverse phase and cation exchange chromatography and activity was monitored by measuring growth of SW13 adrenocortical carcinoma cells in soft agar. The purified material was highly cationic, bound weakly to heparin and gave a band at 13-15000 Mr by SDS-PAGE following Bolton-Hunter iodination. This band correspond to the migration of biological activity extractable from gel slices. The results suggest that we have isolated a truncated form of TGFe which nonetheless retains biological activity.

Growth factors as novel therapeutic targets in neoplastic disease.

The emerging concept of autocrine/paracrine control of tumour cell proliferation coupled with the identification of several polypeptide mitogens has created new opportunities for the discovery of novel classes of antineoplastic drugs. Growth factors (for example, transforming growth factor alpha, fibroblast growth factor and platelet-derived growth factor) and, in certain cases, their receptors have been identified in a number of human tumours and their expression may contribute to unregulated cell proliferation. Selective antagonists that block the activity of these mediators may have important therapeutic utility in the management of cancer patients, particularly in metastatic disease where patterns of tumour cell dissemination may be strongly influenced by paracrine mediators. However, since many, if not all, of these growth factors are polypeptides with molecular weights over 5 kDa, the discovery of potent antagonists represents an important pharmacological challenge. Advances in understanding protein structure-function relationships will be essential in guiding rational attempts at generating growth factor antagonists through site-directed mutagenesis and peptide synthesis, while better insights into the mechanisms by which growth factors are synthesized, processed, released and exert their mitogenic effects may also reveal new sites for pharmacological assault. The availability of (low molecular weight) potent growth factor antagonists will allow the autocrine/paracrine hypothesis to be clinically tested and in the process will throw considerable light on the function of these growth regulatory molecules in vivo.

Stimulation of anchorage independent proliferation of human adrenocortical carcinoma cells by inhibition of cholesterol biosynthesis.

Proliferation of SW13 human adrenocortical carcinoma cells under anchorage independent conditions was stimulated in a dose-dependent manner by treatment with the cholesterol biosynthesis inhibitor mevinolin. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was observed in mevinolin treated cultures. The growth stimulatory effect of mevinolin, but not that of epithelial transforming growth factor, a polypeptide growth factor for SW13 cells, was reversed by exogenous mevalonic acid. However, neither dolichol nor low density lipoprotein supplementation affected the response of SW13 cells to mevinolin. The results suggest that mevalonic acid metabolites may participate in the regulation of anchorage independent growth of SW13 cells.

Pyridinyl imidazoles inhibit IL-1 and TNF production at the protein level.

The mechanism by which SK&F 86002 and other pyridinyl imidazoles inhibit the production of IL-1 and TNF from LPS-stimulated human monocytes was examined. Inhibition of IL-1 and TNF production was found to depend on the time of addition of SK&F 86002, with diminishing effect when added more than 2 h after LPS stimulation. Analysis of Western blots confirmed that both intracellular IL-1 beta and extracellular TNF were significantly reduced in response to SK&F 86002, but these reductions were not paralleled by changes in IL-1 and TNF mRNA. 35S methionine pulse and pulse-chase studies on IL-1 biosynthesis suggest that significant inhibition by SK&F 86002 and related compounds occurs at the translational level.

Characterization of an animal model of metastatic colon carcinoma.

Although numerous animal tumor models have been used to study colon carcinoma, few display metastatic properties. We have characterized an animal tumor model that has 3 properties essential for the study of metastasis of colon carcinoma cells: epithelial cell origin; a reproducible pattern of metastatic behavior and the ability to be propagated both in vitro and in vivo to facilitate identification of biochemical correlates of metastasis. The K12/TR cell line was derived from a transplantable colon carcinoma induced by dimethylhydrazine in the BD-1X rat strain. Transmission electron microscopy of K12/TR cells demonstrated junctional complexes, desmosomes and surface microvilli characteristic of gastrointestinal epithelial cells. The epithelial cell origin of K12/TR was confirmed by demonstrating the presence of keratin, a marker of epithelial cells, but not vimentin, a constituent of mesenchymal cells. Secretion of CEA and Ca19-9 antigens by K12/TR cells in vitro was below the sensitivity of the assays (1 ng/ml and 6 U/ml respectively). K12/TR cells produced tumors following s.c. injection into syngeneic BD-1X rats, allogeneic RNU/rnuDF rats and xenogeneic CRL:nu/nuBR mice. Macroscopic lung metastases were observed in animals from all 3 groups. Distal lymph node metastases were more frequent in BD-1X rats than in nude rats or mice. The histological appearances of all tumors and metastases were similar, showing a moderate to poorly differentiated glandular carcinoma. Intrasplenic injections of K12/TR cells in nude mice resulted in liver colonization. Preferential growth of tumor cells at sites of trauma was also observed. The results show that the K12/TR system can be used as a model to study metastasis of colon carcinoma cells and may find utility in the testing of chemotherapeutic agents against metastatic lesions.