In vitro toxicology of fumonisins and the mechanistic implications

The effects of fumonisins B₁FB₁, B₂(FB{2}), and the backbone of fumonisin B₁ remaining after hydrolysis of the tricarballylic groups with base (HFB₁) on sphingolipid biosynthesis were studied in both primary rat hepatocytes and pig kidney epithelial cells (LLC-PK₁). Fumonisins were potent inhibitors of sphingolipid biosynthesis in hepatocytes (IC₅₀ of FB₁=0.1 M), but overt toxicity was not observed. In renal cells, fumonisins also inhibited sphingosine biosynthesis (IC₅₀ for FB₁=35 M), and caused decreased cell proliferation as well. Higher doses (70 M) killed renal cells after exposure for 3 days. The inhibition of de novo sphingolipid biosynthesis was specific, and appeared to be at the site of ceramide synthase, which catalyzes the formation of dihydroceramide or ceramide by the addition of the amide-linked fatty acid to sphinganine or sphingosine. These results may account for the ability of fumonisins to cause equine leucoencephalomalacia and to promote tumor formation.     

Expression of developmentally relevant proteins by rodent embryo CNS cells in vivo and in vitro: Proto-oncogene pp60c-src and high molecular weight neurofilament protein

The expression of proteins that play a role in neuronal differentiation was examined in central nervous system (CNS) micromass embryo cell cultures and compared to expression at comparable developmental stages in vivo. The protein product of the src proto-oncogene (pp60^c-src) has been postulated to have a specific role in development because, although it is expressed in many tissues, marked increases in amount and activity of pp60^c-src occur in neurons at the time of differentiation. Another protein of interest, high molecular weight neurofilament (NF) protein, is found in differentiated neurons. In the present study, changes over time in the expression of these two proteins in vitro and in vivo were examined. In the micromass cell cultures, primary cells from day 12 rat embryo CNS are plated at high density and differentiate into neurons during five days in culture. Tissues from embryos grown in vivo were assessed at 12 and 17 days post-coitum. Proteins were quantified by PAGE separation of equal amounts of total protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Increases in the amount of both proteins with neuronal differentiation was shown. Protein kinase activity of immunoprecipitated pp60^c-src also increased in cell cultures and in embryos. Similarity in patterns of expression between in vitro and in vivo tissue samples provides further evidence that the cultures closely simulate in vivo differentiation and are a useful system for examining expression of developmental genes in vitro.Abbreviations BCIP 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt - CNS central nervous system - DPBS Dulbecco's phosphate-buffered saline - GAM-AP goat anti-mouse IgG alkaline phosphatase conjugate - LB limb bud - NF high molecular weight neurofilament protein - NBT nitroblue tetrazolium chloride - SDS-PAGE polyacrylamide gel electrophoresis - PVDF polyvinylidene difluoride - RIPA radioimmunoprecipitation - TBS Tris-buffered saline - TTBS TBS with 0.05% Tween-20 Presented in part at the 1989 and 1990 Teratology Society Meetings and the 1990 Society of Toxicology Meetings.     

The preparation in vitro of chrysanthemum for transplantation to soil

Plantlets of Dendranthema grandiflora Pennine Reel were grown from nodal sections on Sorbarods saturated with liquid medium containing 0–3 mg 1^–1 of various growth retardants. After 4 weeks they were transferred to compost and maintained at a relative humidity of 42% at 27.5°C. Wilting was assessed over a period of 3 h. Plantlets treated with paclobutrazol, flurprimidol, triapenthenol, chlorphonium chloride, uniconazol and ancymidol showed dose-related reductions in wilting up to a concentration of 3 mg 1^–1. Responses to tetcyclacis and mepiquat chloride were weaker, and no responses to chlormequat chloride, BTS 44584 or diaminozide could be detected. These observations are compatible with an hypothesis that resistance to wilting derives from inhibited synthesis of gibberellins.     

Immunochemical characterisation of parathyroid hormone-related protein from tumor and non-tumor cells

The molecular forms of parathyroid hormone-related protein (PTHRP) in conditioned media from the BEN human lung cancer cell line, rat parathyroid cells (PT-r) and human keratinocytes were studied by gel-filtraton chromatography with assay of PTHRP by immunoassays and bioassay. Immunoreactivity (1–86 and 1–34) and bioactivity (1–34) in conditioned media eluted as a coincident major peak (approx. molecular mass 19–22 kDa) and there was evidence of amino-terminal species in the molecular mass range 10–16 kDa in BEN and keratinocyte media. Western blotting of PTHRP affinity purified by monoclonal antibodies directed at regions 1–34 or 37–67, identified a major species in all cell cytosols and media with an apparent molecular mass of 24–25 kDa, consistently slighty larger than recombinant PTHRP(1–141) (mobility of 21 kDa) which may represent an intact or native form of PTHRP. Additional amino-terminal species were identified in medium from keratinocytes (16 and 7 kDa), BEN cells (18 and 14 kDa) and PT-R cells (17 kDa), suggesting that processing occurs at the C-terminus and within the mid-region to form a range of amino-terminal fragments.     

Autolysis-resistant peptidoglycan of anomalous composition in amino-acid-starved Escherichia coli.

Nongrowing Escherichia coli deprived of an essential amino acid continued to produce peptidoglycan at a rate approximately 30% of that of growing cells. The composition of this peptidoglycan was very different from that of growing cells and resembled that of peptidoglycan left undegraded during partial autolysis of the bacteria. Synthesis of this peptidoglycan of anomalous composition began at once upon the removal of the amino acid from the medium. Fifteen minutes of amino acid deprivation was sufficient to virtually completely prevent penicillin-induced autolytic wall degradation in vivo. During this time, although the specific activities of soluble and membrane-bound hydrolytic transglycosylases and endopeptidases remained high, the peptidoglycan produced showed decreased sensitivity to degradation in vitro. After more extensive (2-h) starvation, triggering of autolysis by chaotropic agents was also blocked. Autolysis in growing cells may be selective for peptidoglycan representing the cylindrical portion of the sacculus. It is suggested that at least part of the mechanism of the well-known lysis resistance of nongrowing E. coli is related to the deposition of structurally anomalous and relatively autolysin-resistant peptidoglycan at some strategically located sites on the bacterial surface.     

The rate of killing of Escherichia coli by beta-lactam antibiotics is strictly proportional to the rate of bacterial growth

Nongrowing bacteria evade the bactericidal activity of beta-lactam antibiotics. We sought to determine if slow growth rate also alters bactericidal activity. The bactericidal activity of two beta-lactams on Escherichia coli grown in glucose limited chemostats was compared for generation times ranging from 0.7 to 12 h. The degree of killing varied with drug structure and with E. coli strain. However, all killing rates were a constant function of the bacterial generation time: slowly growing bacteria became progressively more phenotypically tolerant to beta-lactam antibiotics as the generation time was extended.     

Quantitative field investigations of feeding and territorial behaviour of young-of-the-year brook charr,Salvelinus fontinalis

Synopsis Direct observations of young-of-the-year brook charr, Salvelinus fontinalis, in a second-order woodland stream indicated that most of their feeding effort was directed toward sub-surface, drifting prey (83% of feeding time). Feeding from the substrate and water surface was much less frequent (17% of feeding time). Comparisons of gut contents to drift net and substrate fauna samples corroborated that the most commonly consumed prey (chironomid and trichopteran larvae, ostracods, and ephemeropteran nymphs) were captured primarily from sub-surface, invertebrate drift. The disproportionate numbers of some prey species in the guts of several fish indicate that some prey selection occurred. Territories appeared to be cardioid-shaped, and were often contiguous, with dominance hierarchies evident among the residents. Agonistic interactions were frequent. Charges and chases predominated (91% of interactions) while lateral displays were infrequent (9% of interactions). Overall, these fish spent most of the daylight hours station-holding (77%) and feeding (18%). While only 3% of total time was spent in aggression, this amounted to 14% of the time a fish spent away from its station. There was some indication that territories were defended at a cost of feeding time.     

Hepes may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites

Summary Zwitterion buffers are often used to modulate the pH of cell culture medium but their effect on cultured cells is controversial. We found that addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) caused superoxide dismutase (SOD) inhibitable increases in nitroblue tetrazolium dye reduction and SOD and catalase inhibitable decreases in the growth of cultured bovine pulmonary artery endothelial cells. The findings suggest that HEPES stimulates endothelial cells to make toxic oxygen metabolites that contribute to decreased cell growth. This work was supported in part by the National Institutes of Health, Colorado and American Lung Associations, Colorado and American Heart Associations, the Council for Tobacco Research, and the Kroc, Hill, Swan and Kleberg Foundations. Dr. Bowman is a Clinician Scientist Awardee of the American Heart Association.     

Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: I. Receptor Characterisation

Characterisation of receptor-mediated breakdown of inositol phospholipids in rat cortical slices has been performed using a direct assay which involves prelabelling with [3H]inositol. When slices were preincubated with [3H]inositol, lithium was found to greatly amplify the capacity of receptor agonists such as carbachol, noradrenaline, and 5-hydroxytryptamine to increase the amount of radioactivity appearing in the inositol phosphates. Using a large variety of agonists and antagonists it could be shown that cholinergic muscarinic, alpha 1-adrenoceptor, and histamine H1 receptors appear to be linked to inositol phospholipid breakdown in cortex. The large responses produced by receptor agonists allowed a clear discrimination between full and partial agonists as well as quantitative analysis of competitive antagonists for each receptor. Whereas carbachol and acetylcholine (in the presence of a cholinesterase inhibitor) were full agonists, oxotremorine and arecoline were only partial agonists. Very low concentrations of atropine shifted the carbachol dose-response curve to the right and allowed inhibition constants for the antagonist to be easily calculated. The nicotinic antagonist, mecamylamine, was ineffective. Noradrenaline adrenaline were full agonists at alpha 1-adrenoceptors, but phenylephrine and probably methoxamine were partial agonists. Prazosin, but not yohimbine, potently and competitively antagonised the noradrenaline inositol phospholipid response. Mepyramine but not cimetidine competitively antagonised the histamine response. These data provide strong confirmation for the potentiating effect of lithium on neurotransmitter inositol phospholipid breakdown and emphasise the ease with which functional responses at a number of cortical receptors can be characterised.