An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

The Høylandet area – patterns and processes of a pristine boreal-subalpine ecosystem – a synopsis

The research activities on soil–freshwaterinteractions and freshwater biotic structure andprocesses in the Høylandet area during the years 1986–89are presented in this issue ofHydrobiologia. The 9 papers cover a range of topicsfrom physiochemical gradients and processes in soilsand freshwaters to selected aspects of freshwaterbiotic community structure and temporal variability.This synopsis aims (a) to present an overview of majorfindings, (b) to interpret the results in the contextof air pollution effects and (c) to outlinesuggestions for future research activities inHøylandet. The area has a sub-oceanic climate varyingfrom 1100 to 2200 mm of precipitation normallyoriginating from unpolluted airmasses from the NorthAtlantic. It is chemically dominated by sea salts, haslow concentrations and low deposition of mineralacids. The catchment geology is variable and complexand consists mainly of various granites and gneisses.The soils are undisturbed ancient soils,primarily iron podzols associated with forests, orintact histosols with strong organic characterassociated with mires, often quite acidic.They are sensitive because occurrence of predominantlyinsoluble unstable Al-complexes may leach out ifexposed to anthropogenically acidified soil drainagewater and turn up as toxic inorganic aqueous Al insurface runoff. Small amounts of locally generatednitrogen compounds are mostly retained in thecatchment. The water qualities of these Høylandetsites are run by inputs of sea salts, hydrologicalprocesses including dilution of the base flow, orlateral surface or overland flow and in-catchmentprocesses. Relative to affected areas, it appears thatthe inputs and catchment processes are in balance,i.e., that the inputs of strong acid anions, hydrogenions and sea salts are equilibrated by internalweathering and other processes and integrated withinapparently intact biogeochemical cycles. The overallresult is that these soils remain a net source ofalkalinity to the runoff. These results are confirmedby regional studies of water quality both at low andhigh flow which show that the humus and ionic contentof the water decreased with elevation, headwatersbeing more dominated by sea salts and dilutionprocesses. Excess sulphate concentrations are low, andfew if any sites appear anthropogenically acidified,the most acid pHs reflecting presence of natural weakorganic acids.The resulting surface water quality allows a diverseaquatic fauna and flora with representatives of manyacid-sensitive groups. The fish fauna consists ofself-reproducing salmonid populations with normaldemography and ecological interactions. Despite highin-lake mortality, both brown trout and Arctic charrexhibited normal age-class compositions, with noindication of the recruitment failure or highpost-spawning mortality commonly observed inacid-stressed situations. The zooplankton and benthos in the lakesare inhabited by populations of several acid sensitivespecies, like Daphnia spp and Gammaruslacustris, and with dominance of mayfly species ofthe genus Baetis in the stream fauna. Thediatom flora includes many species which are rare orabsent in acidified waters. The observed gradients indiversity and biomass seem dominated by localenvironmental conditions which reflect natural ratherthan man-made processes. The pristine nature of thearea also emerges from the paleoecological studies inLake Røyrtjønna which show that the low acidity ofthis lake evironment has persisted from preindustrialtill present times. Suggestion for future researchactivities in Høylandet are outlined.     

Surface water chemistry characteristics in the Lake Storgrønningen drainage area, Høylandet, during periods of high and low discharge

The surface water chemistry of Høylandet has beenstudied by performing two synoptic surveys, duringhigh runoff in the autumn of 1986 and during a lowflow period in the summer of 1988. Based on watersamples of up to 38 chemical variables from 75 sites,analyses show considerable variation in the chemicalcomposition. There is a strong altitude gradient, i.e.very dilute poorly buffered waters dominate at higherelevations near the timberline while progressivelyhigher salt content and alkalinities arecharacteristic at lower altitudes more dominated byforests. The influence of mires is less pronounced.The overall water quality is of oligotrophic naturewith low concentrations of strong acid anions andmetals known to be enriched under acidifiedconditions. The natural pH gradients are considerablebut with no indication of anthropogenic acidification.The data provide little support for the hypothesisthat in-catchment production of organically derivedacidity leads to acid runoff, which in thesecatchments appears compensated by increasedweathering. The findings are in general accordancewith other Høylandet catchment studies. It isconcluded that this area may serve as a representativepristine surface runoff analogue for catchmentscurrently affected by atmospheric deposition of strongmineral acids.     

Characterization of monoclonal antibodies against voltage-dependent K+ channels raised using alpha-dendrotoxin acceptors purified from bovine brain.

Seven monoclonal antibodies raised against alpha-dendrotoxin-sensitive K+ channels, purified from bovine cerebral cortex, recognize these proteins in their native or denatured states, via interaction with the alpha- but not the beta-subunit. This finding, together with a similar observation made with polyclonal antibodies, shows that the latter is a distinct protein and not a proteolytic fragment of the larger subunit. Also, coimmunoprecipitation of alpha- and beta-subunits provides further evidence that both are tightly associated constituents of the K+ channel complexes. At least three isoforms of the K+ channel alpha-subunit are distinguishable by immunoblotting of a detergent extract of synaptic membranes with mAb 5. Likewise, multiple forms are also detectable in the purified protein with mAb 5 although deglycosylation, which does not alter reactivity with any of the mAbs, was required to achieve adequate electrophoretic resolution. These results confirm the proposal that variants of this K+ channel group, known to exist in the nervous system, are heterooligomeric complexes of alpha- and beta-subunits. Although different areas of rat brain contain proteins of similar sizes reactive with mAb 5, these are absent from heart, liver, pancreas, kidney, testes, and spleen, highlighting the selectivity of this antibody.     

The citrus plant pathogen Xanthomonas citri has a dual polyamine-binding protein

ATP-Binding Cassette transporters (ABC transporters) are protein complexes involved in the import and export of different molecules, including ions, sugars, peptides, drugs, and others. Due to the diversity of substrates, they have large relevance in physiological processes such as virulence, pathogenesis, and antimicrobial resistance. In Xanthomonas citri subsp. citri, the phytopathogen responsible for the citrus canker disease, 20% of ABC transporters components are expressed under infection conditions, including the putative putrescine/polyamine ABC transporter, PotFGHI. Polyamines are ubiquitous molecules that mediate cell growth and proliferation and play important role in bacterial infections. In this work, we characterized the X. citri periplasmic-binding protein PotF (XAC2476) using bioinformatics, biophysical and structural methods. PotF is highly conserved in Xanthomonas sp. genus, and we showed it is part of a set of proteins related to the import and assimilation of polyamines in X. citri. The interaction of PotF with putrescine and spermidine was direct and indirectly shown through fluorescence spectroscopy analyses, and experiments of circular dichroism (CD) and small-angle X-ray scattering (SAXS), respectively. The protein showed higher affinity for spermidine than putrescine, but both ligands induced structural changes that coincided with the closing of the domains and increasing of thermal stability.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.