Gene amplification at a locus encoding a putative Na+/H+ antiporter confers sodium and lithium tolerance in fission yeast.

We have identified a new locus, sodium 2 (sod2) based on selection for increased LiCl tolerance in fission yeast, Schizosaccharomyces pombe. Tolerant strains have enhanced pH-dependent Na+ export capacity and sodium transport experiments suggest that the gene encodes an Na+/H+ antiport. The predicted sod2 gene product can be placed in the broad class of transporters which possess 12 hydrophobic transmembrane domains. The protein shows some sequence similarity to the human and bacterial Na+/H+ antiporters. Overexpression of sod2 increased Na+ export capacity and conferred sodium tolerance. Osmotolerance was not affected and sod2 cells were unaffected for growth in K+. In a sod2 disruption strain cells were incapable of exporting sodium. They were hypersensitive to Na+ or Li+ and could not grow under conditions that approximate pH7. The sod2 gene amplification could be selected stepwise and the degree of such amplification correlated with the level of Na+ or Li+ tolerance.     

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.     

Brain somatostatin receptors in spontaneously hypertensive rats: an autoradiographic study.

The apparent densities of brain somatostatin (SRIF) receptor sites were compared in adult spontaneously hypertensive rats (SH) and their normotensive genetic counterparts (Wistar-Kyoto; WKY) using quantitative receptor autoradiography. Globally, the distribution of brain [125I][Tyr0, D-Trp8]SRIF14 binding sites was very similar in both strains. However, apparent densities of specific labeling were either higher (subfornical organ, 3.2 x; locus coeruleus, 1.9 x; lateroanterior hypothalamic nucleus, 1.3 x) or lower (basolateral amygdaloid nucleus, 0.8 x; spinal trigeminal sensory nucleus, 0.6 x) in SH than WKY rats in areas especially relevant to CNS cardiovascular integration. This provides further evidence for the possible involvement of brain SRIF neurons in cardiovascular regulation.     

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.