Multiple sex pheromones and receptors of a mushroom-producing fungus elicit mating in yeast.

The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors. Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis. A complex set of pheromone-receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone. The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein. Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor. The specificity of interaction among pheromone-receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone-receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms.

Differential and inefficient splicing of a broadly expressed Drosophila erect wing transcript results in tissue-specific enrichment of the vital EWG protein isoform.

In this report, we document an unusual mode of tissue-enriched gene expression that is primarily mediated by alternative and inefficient splicing. We have analyzed posttranscriptional regulation of the Drosophila erect wing gene, which provides a vital neuronal function and is essential for the formation of certain muscles. Its predominant protein product, the 116-kDa EWG protein, a putative transcriptional regulator, can provide all known erect wing-associated functions. Moreover, consistent with its function, the 116-kDa protein is highly enriched in neurons and is also observed transiently in migrating myoblasts. In contrast to the protein distribution, we observed that erect wing transcripts are present in comparable levels in neuron-enriched heads and neuron-poor bodies of adult Drosophila. Our analyses shows that erect wing transcript consists of 10 exons and is alternatively spliced and that a subset of introns are inefficiently spliced. We also show that the 116-kDa EWG protein-encoding splice isoform is head enriched. In contrast, bodies have lower levels of transcripts that can encode the 116-kDa protein and greater amounts of unprocessed erect wing RNA. Thus, the enrichment of the 116-kDa protein in heads is ensured by tissue-specific alternative and inefficient splicing and not by transcriptional regulation. Furthermore, this regulation is biologically important, as an increased level of the 116-kDa protein outside the nervous system is lethal.

Plasmodium vivax, P. cynomolgi, and P. knowlesi: identification of homologue proteins associated with the surface of merozoites.

We have identified a Plasmodium vivax merozoite surface protein (MSP) that migrates on SDS-polyacrylamide gels at a Mr of about 185 kDa. This protein was recognized by a P. vivax monoclonal antibody (mAb) that localizes the protein by immunofluorescence to the surface of merozoites and also immunoprecipitates this protein from NP-40 detergent extracts of [35S]methionine metabolically radiolabeled P. vivax schizonts. The P. vivax MSP does not become biosynthetically radiolabeled with [3H]glucoamine, [3H]myristate, [3H]palmitate, or [3H]mannose, indicating that this P. vivax MSP is not posttranslationally modified and bound to the merozoite membrane by a glycosylphosphatidylinositol (GPI) lipid anchor. Thus, in this respect, this protein is different from members of the MSP-1 protein family and from MSP-2 and MSP-4 of P. falciparum. The mAb cross-reacts with and outlines the surface of P. cynomolgi merozoites and immunoprecipitates a 150-kDa P. cynomolgi homologue. The mAb was used as an affinity reagent to purify the native homologous MSP from NP-40 extracts of P. cynomolgi mature schizonts in order to develop a specific polyclonal antiserum. The resulting anti-PcyMSP rabbit antiserum cross-reacts strongly with the P. vivax 185-kDa MSP and also recognizes an analogous 110-kDa protein from P. knowlesi. We have determined via an immunodepletion experiment that the 110-kDa P. knowlesi MSP corresponds to the PK 110 protein partially characterized earlier (Perler et al. 1987). The potential of P. vivax MSP as a vaccine candidate was addressed by conducting in vitro inhibition of erythrocyte invasion assays, and the IgG fraction of both the P. vivax MSP mAb and the P. cynomolgi MSP rabbit antiserum significantly inhibited entry of P. vivax merozoites. We denote, on a preliminary basis, these antigenically related merozite surface proteins PvMSP-185, PcyMSP-150, and PkMSP-110.

IL-4 inhibits mouse mast cell Fc epsilonRI expression through a STAT6-dependent mechanism.

Mast cell activation by IgE-mediated stimuli is a central event in atopic disease. The regulation of the mast cell high affinity receptor, Fc epsilonRI, is poorly understood. We show that IL-4 can inhibit Fc epsilonRI expression on mouse bone marrow-derived mast cells and fetal liver-derived mast cell progenitors. This effect could be observed at 2.5 ng/ml IL-4 and was dose dependent. IL-4-mediated inhibition of cultured BMMC required 4 days of stimulation and was sustained at maximum levels for at least 21 days. The inhibition of Fc epsilonRI expression resulted in decreased sensitivity to IgE-mediated stimulation, as measured by serotonin release, and the induction of mRNA for IL-4, IL-5, IL-6, and IL-13. Additionally, IL-4 could abrogate the IgE-mediated increase in Fc epsilonRI expression. Lastly, IL-4-mediated inhibition was dependent upon expression of the STAT6 transcription factor, as STAT6-deficient bone marrow-derived mast cells did not decrease Fc epsilonRI levels in response to IL-4. These data argue for a homeostatic role of IL-4 in the regulation of Fc epsilonRI expression, a role that could be critical to understanding atopic disease.

Switch-domain mutations in the Saccharomyces cerevisiae G protein alpha-subunit Gpa1p identify a receptor subtype-biased mating defect.

The response to pheromone in Saccharomyces cerevisiae involves a heterotrimeric G protein composed of Gpa1p (alpha subunit), Ste4p (beta) and Ste18p (gamma). The switch II region of G alpha subunits is involved in several protein-protein interactions and an intrinsic GTPase activity. To investigate the role of this region of Gpa1p, we have analyzed the effect of switch II mutations. The Q323 analog in G alpha subunits and Ras is implicated in GTP hydrolysis. Mutation of the Q323 residue of Gpa1p resulted in constitutive activation of the pheromone response pathway and eliminated the ability to interact with Ste4p, consistent with a defect in GTPase activity. Mutation of residue A59 of Ras and the analogous G alphas residue has had quite different effects. The analogous Gpa1p G321T mutation resulted in phenotypes consistent with a less severe GTPase defect, but also led to an unexpected mating phenotype: mating was decreased in both mating types, but the defect was 1000-fold more severe in alpha cells than in a cells. In addition the G321T mutation resulted in an unusual pheromone response phenotype. We discuss the possibility that these phenotypes may reflect a differential role for the switch II region in activation by the a- and alpha-factor receptors.

ERECT WING, the Drosophila member of a family of DNA binding proteins is required in imaginal myoblasts for flight muscle development.

The erect wing locus of the fruit fly Drosophila melanogaster encodes a protein, EWG, that shares extensive homology with the P3A2 DNA binding protein of sea urchin and a recently identified mammalian transcription factor. Loss-of-function erect wing alleles result in embryonic lethality. Viable alleles of erect wing cause severe abnormalities of the indirect flight muscles. We have analyzed the spatial pattern of erect wing expression in the developing indirect flight muscles during postembryonic development. EWG is detected, 10 hours after puparium formation, in myoblasts that will form the indirect flight muscles. The early events of muscle development are normal in ewg mutants. However, a few hours after the onset of erect wing expression in myoblasts, defects are seen in the developing indirect flight muscles which subsequently degenerate. We present results that show that the normal development of the indirect flight muscles requires erect wing expression in the progenitor myoblasts themselves. Finally, we examine the role of target muscles in the arborization of motor axons by studying the developing innervation to the flight muscle in erect wing mutants. Our study demonstrates, for the first time, a role for a regulatory gene expressed in imaginal myoblasts in Drosophila.

The Drosophila erect wing gene, which is important for both neuronal and muscle development, encodes a protein which is similar to the sea urchin P3A2 DNA binding protein.

The erect wing (ewg) locus of Drosophila melanogaster encodes a vital function important for the development of the nervous system and the indirect flight muscles. In order to understand the ewg function at a molecular level, cDNA clones were isolated. Sequence analysis of cDNAs revealed a single open reading frame (ORF) encoding a protein of 733 residues. The translational start for this ORF is a CTG codon. A 225-amino-acid region of this protein is 71% identical to the DNA binding region of the Strongylocentrotus purpuratus P3A2 DNA binding protein. Additionally, the ORF contains large acidic and basic domains characteristic of those in proteins involved in nuclear regulatory functions. Immunoblot analysis using polyclonal anti-EWG antisera generated against a bacterial fusion protein reveals a single, 116-kDa protein present throughout development, beginning at approximately stage 12 of embryogenesis, which is enriched in adult heads and absent from embryos carrying certain ewg alleles. Additionally, we show that EWG is localized specifically to the nuclei of virtually all embryonic neurons. Finally, a minigene consisting of an ewg cDNA under control of the hsp70 promoter can provide the ewg function in transgenic ewg mutant flies.

Molecular isolation and analysis of the erect wing locus in Drosophila melanogaster.

The molecular study of the erect wing (ewg) locus was initiated by isolating DNA in the 1A8-1B1 interval of the X chromosome. Previous developmental genetic analyses of the mutant alleles at the ewg locus have demonstrated that the wild-type ewg product is essential during embryogenesis and is required postembryonically at least for the development of the indirect flight muscle. To define the ewg-encoding DNA, chromosomal breakpoints that genetically flank the ewg locus were used. P-element-mediated transformation followed by subsequent rescue of the ewg-lethal alleles has defined a 11.5-kilobase genomic fragment as encoding the ewg locus. Northern blot analysis of transcription from this DNA has revealed a complex pattern of transcription with respect to both size and developmental profile. Tissue distribution of putative ewg transcription was examined by in situ hybridization to 6- to 14-h-old embryonic sections. These sections revealed that the expression of putative ewg messages is limited to the central nervous system-derived structures and not observed within the mesoderm during this developmental stage.

Ion transport across the frog olfactory mucosa: the action of cyclic nucleotides on the basal and odorant-stimulated states.

The action of cyclic nucleotides on the short-circuit current across the isolated bullfrog olfactory mucosa was studied both in the absence and presence of odorants. 8-Bromo-cAMP applied to the ciliated side of the mucosa caused a concentration-dependent, reversible increase in the basal short-circuit current, but not when it was applied to the submucosal side. The current had a sigmoidal concentration dependence described by the Hill equation. The magnitude of the odorant-evoked current was enhanced after bathing the ciliated side with cAMP analogs or modulators of intracellular cAMP. GTP gamma S added to the ciliated side increased the odorant-evoked current, while GDP beta S caused a decrease. Current transients induced by stimulating the ciliated side with either pulses of odorant or 8-bromo-cAMP were partially suppressed by amiloride, but only when amiloride and stimulant were presented simultaneously. Pulses of 8-bromo-cAMP and odorant presented simultaneously resulted in currents that added nonlinearly. In the absence of odorant, 8-bromo-cGMP caused a concentration-dependent decrease in net inward current that was reversed by 8-bromo-cAMP. Odorant-evoked currents were also reduced by 8-bromo-cGMP, and these could not be reversed by 8-bromo-cAMP. The results indicate that one type of olfactory transduction process involves the activation by cAMP of an inward current through an amiloride-sensitive apical ion channel and that this mechanism is mediated by a stimulatory G-protein.

Sugar-activated ion transport in canine lingual epithelium. Implications for sugar taste transduction.

There is good evidence indicating that ion-transport pathways in the apical regions of lingual epithelial cells, including taste bud cells, may play a role in salt taste reception. In this article, we present evidence that, in the case of the dog, there also exists a sugar-activated ion-transport pathway that is linked to sugar taste transduction. Evidence was drawn from two parallel lines of experiments: (a) ion-transport studies on the isolated canine lingual epithelium, and (b) recordings from the canine chorda tympani. The results in vitro showed that both mono- and disaccharides in the mucosal bath stimulate a dose-dependent increase in the short-circuit current over the concentration range coincident with mammalian sugar taste responses. Transepithelial current evoked by glucose, fructose, or sucrose in either 30 mM NaCl or in Krebs-Henseleit buffer (K-H) was partially blocked by amiloride. Among current carriers activated by saccharides, the current response was greater with Na than with K. Ion flux measurements in K-H during stimulation with 3-O-methylglucose showed that the sugar-evoked current was due to an increase in the Na influx. Ouabain or amiloride reduced the sugar-evoked Na influx without effect on sugar transport as measured with tritiated 3-O-methylglucose. Amiloride inhibited the canine chorda tympani response to 0.5 M NaCl by 70-80% and the response to 0.5 M KCl by approximately 40%. This agreed with the percent inhibition by amiloride of the short-circuit current supported in vitro by NaCl and KCl. Amiloride also partially inhibited the chorda tympani responses to sucrose and to fructose. The results indicate that in the dog: (a) the ion transporter subserving Na taste also subserves part of the response to K, and (b) a sugar-activated, Na-preferring ion-transport system is one mechanism mediating sugar taste transduction. Results in the literature indicate a similar sweet taste mechanism for humans.

Species-specific oligonucleotide probes for the identification of human filarial parasites.

Species-specific oligonucleotide probes have been constructed for the filarial parasites Brugia malayi and Brugia pahangi. Both parasites contain a 322 base pair repeated DNA sequence that is cleaved once by the restriction endonuclease HhaI. A consensus repeat sequence was determined from the DNA sequence of 15 cloned isolates of each species. Although the two repeats have an average homology of 89%, half the differences are clustered in a region of 66 nucleotides that has a homology of only 72%. Within this region, two probes, a 29-mer that is B. malayi specific and a 21-mer that is B. pahangi specific, were constructed. The sequence of both probes was chosen to obtain the maximum difference between the consensus sequences of the two species. The probes were also selected to be GC rich to increase their stability as a DNA hybrid. In a filter hybridization assay, the B. malayi probe has a 500-fold preference for B. malayi DNA versus B. pahangi DNA and a sensitivity of 200 pg. The B. pahangi probe has similar specificity and sensitivity for B. pahangi DNA. A rapid lysis procedure allows the probes to detect 1-2 third stage larvae of either B. malayi or B. pahangi in a filter hybridization assay.

Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi.

A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross-hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved by Alu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% homologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes.

The identity of the current carriers in canine lingual epithelium in vitro.

Ion transport across the lingual epithelium has been implicated as an early event in gustatory transduction. The fluxes of isotopically labelled Na+ and Cl- were measured across isolated canine dorsal lingual epithelium under short-circuit conditions. The epithelium actively absorbs Na+ and to a lesser extent actively secretes Cl-. Under symmetrical conditions with Krebs-Henseleit buffer on both sides, (1) Na+ absorption accounts for 46% of the short-circuit current (Isc); (2) there are two transcellular Na+ pathways, one amiloride-sensitive and one amiloride-insensitive; (3) ouabain, added to the serosal solution, inhibits both Isc and active Na+ absorption. When hyperosmotic (0.25 M) NaCl is placed in the mucosal bath, both Isc and Na+ absorption increase; net Na+ absorption is at least as much as Isc. Ion substitution studies indicate that the tissue may transport a variety of larger ions, though not as effectively as Na+ and Cl-. Thus we have shown that the lingual epithelium, like other epithelia of the gastrointestinal tract, actively transports ions. However, it is unusual both in its response to hyperosmotic solutions and in the variety of ions that support a transepithelial short-circuit current. Since sodium ion transport under hyperosmotic conditions has been shown to correlate well with the gustatory neural response, the variety of ions transported may likewise indicate a wider role for transport in taste transduction.

The active ion transport properties of canine lingual epithelia in vitro. Implications for gustatory transduction.

The electrophysiological properties of the dorsal and ventral canine lingual epithelium are studied in vitro. The dorsal epithelium contains a special ion transport system activated by mucosal solutions hyperosmotic in NaCl or LiCl. Hyperosmotic KCl is significantly less effective as an activator of this system. The lingual frenulum does not contain the transport system. In the dorsal surface it is characterized by a rapid increase in inward current and can be quantitated as a second component in the time course of either the open-circuit potential or short-circuit current when the mucosal solution is hyperosmotic in NaCl or LiCl. The increased inward current (hyperosmotic response) can be eliminated by amiloride (10(-4) M). The specific location of this transport system in the dorsal surface and the fact that it operates over the concentration range characteristic of mammalian salt taste suggests a possible link to gustatory transduction. This possibility is tested by recording neural responses in the rat to NaCl and KCl over a concentration range including the hyperosmotic. We demonstrate that amiloride specifically blocks the response to NaCl over the hyperosmotic range while affecting the KCl response significantly less. The results suggest that gustatory transduction for NaCl is mediated by Na entry into the taste cells via the same amiloride-sensitive pathway responsible for the hyperosmotic response in vitro. Further studies of the in vitro system give evidence for paracellular as well as transcellular current paths. The transmural current-voltage relations are linear under both symmetrical and asymmetrical conditions. After ouabain treatment under symmetrical conditions, the short-circuit current decays to zero. The increase in resistance, though significant, is small, which suggests a sizeable shunt pathway for current. Flux measurements show that sodium is absorbed under symmetrical conditions. Mucosal solutions hyperosmotic in various sugars also induce an amiloride-sensitive inward current. In summary, this work provides evidence that the sodium taste receptor is most probably a sodium transport system, specifically adapted to the dorsal surface of the tongue. The transport paradigm of gustation also suggests a simple model for electric taste and possible mechanisms for sweet taste.

Active ion transport in dog tongue: a possible role in taste.

An in vitro preparation of the dorsal epithelium of the dog tongue actively transports ions, producing a transepithelial potential difference characteristic of the ions and their concentration. Hypertonic sodium chloride solutions generally cause increased potentials and short-circuit currents and reduced resistances when placed on the mucosal surface. This hypertonic flux is eliminated by ouabain and is not found in ventral lingual epithelia. When either sodium acetate or tetramethylammonium chloride is substituted for sodium chloride in the mucosal medium, the currents are diminished but their sum at a given concentration approximates that for sodium chloride at the same concentration. This result suggests a current composed of inward sodium ion movement and outward chloride ion movement. Actively regulated potentials and currents, whether generated in the taste buds or in supporting cells, may be important in both normal chemotransduction and in taste responses evoked by currents passing through the tongue.

Senile macular degeneration and alteration of the metabolism of the lipids.

An accurate study on the alterations of lipid metabolism was made on a sample of 30 patients affected by senile macular degeneration and 13 patients affected by senile macular degeneration complicated by retinopathy due to hyperlipidemia. The authors came to the conclusion that hyperlipidemia can complicate simple macular degeneration, even if a close correlation between the two phenomena can be excluded.