Increased rates of sugar transport in Saccharomyces cerevisiae a result of sugar metabolism

Preincubation of yeast cells with glucose or other metabolic energy sources increased the rate of sorbose efflux 2- or 3-fold. Stimulated rates persisted for several h, decreasing slowly. They were approximately halved by including K_m concentrations of highly competitive sugars such as deoxyglucose, glucose, fructose and mannose in sorbose efflux suspensions, and were greatly slowed at reduced temperatures. Inhibitors of energy metabolism blocked the rate stimulation, as did cycloheximide; added nitrogen sources increased the rate additionally. The rate of sorbose uptake was also increased, whereas that of dimethylsulfoxide, which enters the cell by simple diffusion, was not changed. Transport of arabinose and fucose also occurred at increased rates. The data indicate a change in the sorbose transport system rather than in membrane permeability. The change, apparently the synthesis of a transport system component, requires metabolic energy and involves protein synthesis.     

“SORBOSE TOXICITY” IN NEUROSPORA

Brockman, II. E., and F. J. de Serres. (Oak Ridge Natl. Lab., Oak Ridge, Tenn.) “Sorbose toxicity” in Neurospora . Amer. Jour. Bot. 50(7): 709–714. Illus. 1963.—The effect of “sorbose toxicity” on Neurospora conidia or ascospores was compared in sorbose-fructose-glucose (S-F-G) and sorbose-sucrose (S-S) media. Many frequently encountered and difficultly controlled experimental variables may strongly affect viability in S-S media but are essentially without effect in S-F-G media. The viabilities of different mutant strains are affected to varying extents by autoclave exposure time of the S-S media but not of the S-F-G media. Ascospores are more sensitive than conidia to “sorbose kill” in S-S media. An over plating method described by New meyer (1954) for increasing ascospore “viability” is without effect when sucrose is replaced with fructose and glucose. In all experiments in which sorbose was added to induce colonial growth, “sorbose toxicity” or “sorbose kill” was eliminated or minimized by replacing sucrose with a mixture of fructose and glucose as the carbon source for Neurospora media.     

A new coryneform hydrogen bacterium: Corynebacterium autotrophicum strain 7 C

A slime-free mutant was isolated from Corynebacterium autotrophicum strain 7 C. The mutant appeared in a substrate (fructose) limited chemostat culture. No significant differences between wildtype and mutant were detected with respect to pigmentation, to growth rate, and respiratory rate at varying substrate (fructose) concentrations. During growth in chemostat culture the wildtype/mutant ratio present in the inoculum was maintained for weeks. The results do not explain the selection of the mutant in the substrate limited chemostat culture.The slime is a polysaccharide containing glucose, mannose and galactose; the uronic acid present was not identified. Copious amounts are produced from fructose, less from lactate or during autotrophic growth. The slime is not reutilized.     

beta-Adrenergic receptor desensitization stimulates glucose uptake in C6 rat glioma cells

When C₆ glioma cells were stimulated by β -adrenergic ligands, [³H]-deoxyglucose uptake by the cells decreased in the first 30 min, followed by its acceleration. The stimulation of deoxyglucose uptake was attributable to desensitization of β-adrenergic receptor-adenylate cyclase system. When the cells were treated with quinacrine or tetracaine, phospholipase inhibitors, the stimulation of deoxyglucose uptake by isoproterenol was diminished without changing the basal rate. On the other hand, when C₆ glioma cells were treated with melittin or phorbol ester, phospholipase A₂ activators, the deoxyglucose uptake increases even in the absence of isoproterenol. Since these compounds inhibit or enhance phospholipase A₂ as well as the desensitization of β -adrenergic receptors (Proc. Natl. Acad. Sci. USA 77, 1341–1345, 1980), these results suggest that turnovers of phospholipids in the vicinity of β -adrenergic receptors modify the glucose uptake of C₆ glioma cells.     

Analysis of sorbose resistance in Neurospora crassa in heterokaryons of sorbose resistant mutants; contribution to the genetics of active transport

Summary Suitable auxotrophic markers were introduced into sorbose-resistant mutants and the sorbose-sensitive wildtype strain. Pairwise combinations of one resistant and one sensitive strain each as well as of two sensitive strains were then grown on minimal-agar to obtain forced heterocaryons. The growth behaviour of these on minimal-agar with and without added sorbose was compared.Of seven resistant mutants, representing six separate genes, among which were genes A and B, six mutants were recessive to the wildtype. The seventh, representing gene C, was recessive only with regard to colony-size, but intermediate with regard to germination counts. Heterocaryons forced between pairs of 2 closely linked mutants (intragenic case of the type A^– ₁+A^– ₂) were resistant, as were the separate mutants. However two heterocaryons forced between pairs of unlinked mutants (intergenic case of the type A^–+B^–) were sorbose sensitive. Heterocaryons forced between A or B-mutants and the C-mutant mentioned, unlinked to either A or B (intergenic cases of the type A^–+C^– and B^–+C^–) were more sensitive than the separate mutants but more resistant than the wildtype.It follows that sorbose-resistant mutants in heterocaryons of the intergenic types can complement each others defects (no growth complementation), but can not do so in heterocaryons of the intragenic type. Their complementation is considered to be the result of the activity of the intact wildtype genes homologous to the defective ones that are contained together in the multinucleate cells of the heterocaryons. This complementation may be taken as evidence for the recessiveness resp. intermediate expression of the different resistant mutants.Since none of the mutants checked so far were dominant compared to the wildtype, none of them can be a regulator-mutant. The possibility of explaining them as suppressor mutants is restricted by their recessiveness to mechanisms of suppression giving a recessive phenotype. An alternative explanation suggests that the respective wildtype genes may contain structural information for the synthesis of permeases involved in sorbose transport. The mutants would then be resistant due to defective permeases. Their recessiveness is in full accord with this suggestion.

---

---

II. Teil einer Habilitationsschrift bei der Naturwissenschaftlichen Fakultät der Universität München.     

Analysis of sorbose resistance in Neurospora crassa double mutants; contribution to the genetics of active transport. II

Summary 4 sorbose-resistant Neurospora crassa mutants, whose resistance was found to be due to a decreased rate of sorbose uptake, were crossed in two pairs with each other. In both crosses mutants were combined which contained mutational changes at sites not closely linked with each other, and therefore representing separate genes. Among the ascosporeisolates, double mutant recombinants were selected. This was facilitated by suitable markers in one of the crosses.Growth of these double mutants with and without added sorbose was compaired with that of a number of parental- and wildtype-reisolates from the same crosses. In spite of the fact that the double mutant sor ^r A-10/sor ^r C-17 without sorbose grows more slowly than the parental- and wildtype-reisolates, it proved much more resistant than these in the presence of sorbose. The same was true for the second double mutant sor ^r A-2/sor ^r -6.Since it can be shown that probably none of the two A-mutants used (sor ^r A-10 and sor ^r A-2) forms any active A-protein, it seems unlikely that the respective pairs of genes in the wildtype specify proteins catalyzing a reaction sequence. Another possibility envisages a compound-permease made up of subunits specified by the respective genes, but is not very likely either. A third possibility, which explains satisfactorily all earlier as well as the present results, is that the resp. genes code for identical or different proteins which as isopermeases catalyze one and the same reaction step in the course of the uptake of sorbose.

---

---

III. Teil einer Habilitationsschrift bei der Naturwissenschaftlichen Fakultät der Universität München.     

Effects of some carbon sources on growth and nitrogen fixation in the cyanobacteriumNostoc linckia

Glucose, fructose, sucrose, maltose, and lactose stimulated photoheterotrophic growth ofNostoc linckia (Roth.)Born. as well as its heterocyst frequency, chlorophyll and protein contents, ammoniacal nitrogen uptake and nitrogenase activities. Glucose, fructose and sucrose also supported slow chemoheterotrophic growth. α-ketoglutarate, pyruvate, ribose, succinate, acetate, sorbose and formate were inhibitory.     

Edeine inhibition and resistance in Neurospora

To obtain data on the biochemical effects of edeine in the fungus Neurospora crassa, in vivo protein synthesis, in vitro protein synthesis, as well as in vivo RNA and DNA synthesis of the wildtype and an edeine resistant mutant were measured.—Incorporation of ³H leucine into conidia of both strains, which served as a measure for in vivo protein synthesis, was inhibited by 200 g edeine/ml as follows: Wildtype approx. 40%, mutant approx. 6%.—Incorporation of ¹⁴C phenylalanine into polyphenylalanine in a cell free system with ribosomes from either the wildtype or the mutant, was inhibited between 74 and 95% by edeine at a ratio of 2 molecules edeine per ribosome.—Incorporation of ³H adenosine into conidia, serving as a measure for in vivo RNA synthesis, was inhibited in the wild-type (approx. 30% inhibition by 200 g edeine/ml). It was, however, not influenced in the ed ^r mutant. Similarly, in vivo DNA synthesis was decreased in the wildtype, but not in the mutant.—These results suggest that edeine acts at more than one site. The resistance of the mutant ed ^r -29 (ed ^r -2 locus) is tentatively interpreted as due to a block in edeine uptake.     

Role of vacuolar ion pool in Saccharomyces carlsbergensis: potassium efflux from vacuoles is coupled with manganese or magnesium influx.

Saccharomyces carlsbergensis cells accumulated Mn2+ (or Mg2+) ions in the presence of glucose, fructose, or mannose, but not of deoxyglucose, 3-O-methylglucose, and sorbose. Accumulation of one equivalent of Mn/2+ was coupled with the efflux of two equivalents of K+ from the cells. Mg/2+ did not exit during Mn2+ uptake. Preliminary treatment of cells with various proton conductors or glucose led to the loss of K+ and to the proportional inhibition of Mn2+ uptake. Polyene antibiotic candicidin together with glucose elicited rapid efflux of K+ and completely inhibited Mn2+ accumulation. Exogenous K+ (more than 1 mM), 100 microM N,N'-dicyclohexylcarbodiimide, and 30 mM sodium arsenate inhibited both K+ efflux and Mn2+ influx. K+ efflux from S. carlsbergensis cells affected the vacuolar pool of K+ both during the accumulation of Mn2+ or Mg2+ and during glucose uptake.     

Selektion Actidion-resistenter Mutanten bei Neurospora crassa sowie ihre genetische und biochemische Analyse

Summary Conidia of an actidione-sensitive wildtype strain of Neurospora crassa were irradiated with UV-light. They were then plated into nutrient-agar, either with or without actidione. The latter plates were incubated for several hours, before nutrient agar containing actidone was layered onto the plates. Colonies formed in both sets of plates were isolated as actidione-resistent. They were studied further by genetic and biochemical means.Pre-incubation of the irradiated conidia before subjecting them to the action of actidione increased the mutant yield considerably, as compared to immediate plating with the drug. E.g. a 13 hours pre-incubation gave ca. 100 times more resistent colonies than were obtained without pre-incubation (Fig. 2). Their resistent phenotype was stable on vegetative propagation.17 mutants were mapped by crossing them with suitable tester-strains. Of them, 14 were found to belong to linkage group I, the remaining to linkage group V. The mutants are, therefore, considered as characterizing resp. genes act-1 and -2 of Hsu (1963). Act-1 and -2 mutants were crossed with suitable auxotrophic strains to obtain auxotrophic, actidione-resistent isolates. These were combined on minimal medium with auxotrophic, actodione-sensitive strains of the same mating type. Conidia of the arising heterokaryotic mycelia were tested on minimal medium with and without actidione. In these tests resistence of act-1 and -2 mutants was found to be dominant over the sensitivity of the wildtype. However, an analysis of nuclear ratios in the conidial populations by differential plating does not exclude incomplete dominance of act-1.Incorporation of 14-C-leucine into protein of conidia of the wildtype was strongly inhibited by 1 actidione/ml. Resistence in two mutants, representing the two separate genes, was accompanied by a marked decrease of this inhibition. No significant differences in the amount of inhibition were found between the two mutants. It is suggested that cytoplasmic ribosomes may be the cellular components influenced by actidione. In the case of the mutant cells the actidione is no longer effective in this capacity, possibly because of changes in the ribosomal proteins.     

Crossing analysis of sorbose resistant mutants of Neurospora crassa

Summary After treatment of the conidia of Neurospora crassa with nitrous acid 19 of the resulting mutants were selected for their resistance to sorbose, an agent which has a toxic effect on the wild type. The selected sorbose resistant mutants were analyzed genetically by crossing them with tester strains containing known markers. Mutational sites were thus found to be located on at least 5 of the full complement of 7 linkage groups. 9 sites were found to be located on the left arm of linkage group VI near the ylo-marker. These were so far not separable by recombinational analysis and therefore seem to represent a single gene-locus (A). Other sorbose resistant sites were mapped on linkage groups I, III (gene C), V and VII (gene B). Another site which has not yet been definitely located is not closely linked to any of the aforementioned. One site each on linkage groups I and VI were mapped more precisely by means of 3-point crosses. These crosses permitted the location of these sites of sorbose resistance in regard to neighbouring markers. From these crosses it is concluded that the mutation of any 1 of at least 6 different chromosomal genes can cause the formation of a sorbose resistant phenotype. The mutation of any 1 of 4 of these genes could result in a mutant with an altered sorbose permease system or containing a suppressor which decreases the efficiency of the permease system.

---

---

I. Teil einer Habilitationsschrift bei der Naturwissenschaftlichen Fakultät der Universität München.     

Sugar transport in Neurospora conidia: Influence of pH and starvation

Summary Uptake of ¹⁴C-sorbose and ¹⁴C-3-O-methylglucose by ungerminated conidia of Neurospora crassa was measured by means of the millipore filter technique.Initial rates of uptake of both sorbose and 3-O-methylglucose exemplify a marked dependence on pH of the incubation medium in the range between pH 3.5 and 6.5. The optimal pH for uptake of both sugars is close to 4.75.When ungerminated conidia are starved with buffer for prolonged periods of time prior to assaying their transport capacity, in contrast to findings for germinated conidia and mycelia no de-repression of the glucose-repressible sugar transport system is effectuated.     

Membrane Functions and Tolerance to Aromatic Hydrocarbon Fungitoxicants in Conidia of Fusarium solani

With the use of ³²P-labeled phosphate and ⁴²K₂CO₃ the effect of diphenyl on permeability and uptake properties of the cytoplasmic membrane in wild type and diphenyl-tolerant mutant conidia of Fusarium solani f. cucurbitae was studied. No general damage to the membrane with unspecific leakage of cell constituents was demonstrated under conditions in which diphenyl prevents germination of wild type conidia. The fresh conidia do not require exogenous supply of energy for the uptake of phosphate or of potassium. In the wild type the entry of ³²P is inhibited but that of ⁴²K strikingly stimulated by diphenyl. Independently of the tolerant mutant gene present, the mutant conidia are significantly less sensitive to the phosphate uptake inhibition and not affected at all by diphenyl with respect to the uptake of potassium. The latter difference from the wild type seems to indicate genetic control of some property of the potassium transport system in this fungus.     

Nerve Terminal Degeneration Is Independent of Muscle Fiber Genotype in SOD1G93A Mice

Background

Motor neuron degeneration in SOD1^G93A transgenic mice begins at the nerve terminal. Here we examine whether this degeneration depends on expression of mutant SOD1 in muscle fibers.

Methodology/Principal Findings

Hindlimb muscles were transplanted between wild-type and SOD1^G93A transgenic mice and the innervation status of neuromuscular junctions (NMJs) was examined after 2 months. The results showed that muscles from SOD1^G93A mice did not induce motor terminal degeneration in wildtype mice and that muscles from wildtype mice did not prevent degeneration in SOD1^G93A transgenic mice. Control studies demonstrated that muscles transplanted from SOD1^G93A mice continued to express mutant SOD1 protein. Experiments on wildtype mice established that the host supplied terminal Schwann cells (TSCs) at the NMJs of transplanted muscles.

Conclusions/Significance

These results indicate that expression of the mutant protein in muscle is not needed to cause motor terminal degeneration in SOD1^G93A transgenic mice and that a combination of motor terminals, motor axons and Schwann cells, all of which express mutant protein may be sufficient.     

Preblastoderm mosaics of mutants of the bithorax-complex

Summary InDrosophila melanogaster, segmental specification takes place in groups of cells around the blastoderm stage. This segmental specification requires the function of the genes of the bithorax-complex. We have studied preblastoderm mosaics (gynandromorphs) of mutant (bx ³,pbx, Ubx, Ubx ⁸⁰) and wildtype (heterozygotes for these alleles) cells. The results show a total cell autonomy in the differentiation of both wildtype and homoeotially transformed cells. However, several unexpected phenotypes were found. They are discussed in terms of the function of the bithorax genes and early interactions between mutant and wildtype territories.     

Trophic dependencies of some larval chironomidae (Diptera) and fish species in the River Thames

Experiments are described to characterise the heterotrophic potential of Westiellopsis prolifica Janet, which fixes nitrogen under light and dark conditions. The growth of the organism in terms of dry weight increase, was more in fructose, lactose, sucrose, sorbose, galactose, glucose, sodium acetate, mannitol, sorbitol, glycerol, ethyl alcohol and butyl alcohol, when the alga was pretreated with light and subsequently incubated with the substrates in light. Mannose, xylose, acetic acid, propionic acid, fructose 1,6 di Po₄, pyruvic acid, dihydroxyacetone and succinic acid decreased the growth of the organism in the same condition. In dark incubation after pretreatment with light, as well as in the dark, Westiellopsis showed a better growth response to almost all the exogenous substrates. However, after pretreatment either with light or dark, the test organism utilised exogenous substrates quicker in light than in dark incubations. These experiments would suggest that the substrate specificity and efficiency of substrate utilisation by the alga during its heterotrophic growth are governed by the growth conditions.     

Glucose-induced Release of Amino Acids from Saccharomyces carlsbergensis by Action on the Cytoplasmic Membrane

When washed yeast cells grown under appropriate conditions were suspended in glucose solution there was a sudden release of α-amino nitrogen into the medium. This released material was of low molecular weight, and its composition was closely similar to that of the intracellular free amino acid pool. During the leakage of amino acids, the yeast did not efficiently absorb labeled amino acids added to the test medium, despite the rapid uptake and metabolism of glucose. Uptake of a labeled amino acid and reabsorption of the released α-amino nitrogen occurred almost simultaneously. When these yeast cells were exposed to glucose in the presence of calcium ions, leakage was strongly inhibited. Butanol under the same conditions increased glucose-induced leakage of cell contents. The adenosine triphosphatase activity of intact yeast cells exposed to glucose was greater than that of cells exposed to water. Yeast cells treated with glucose prior to equilibration with sorbose exhibited less ability to retain the sorbose when washed at 0 C than did cells pretreated with water. It was concluded that glucose-induced leakage of amino acids was the result of two factors acting together. These were (i) a change in membrane permeability associated with glucose uptake, and (ii) a temporary shortage of energy for amino acid uptake or retention.     

Deoxyglucose mapping of nervous activity induced inDrosophila brain by visual movement

Summary The pattern of visually induced local metabolic activity in the optic lobes of two structural mutants ofDrosophila melanogaster is compared with the corresponding wildtype pattern which has been reported in Part I of this work (Buchner et al. 1984b). Individualoptomotor-blind ^H31 (omb) flies lacking normal giant HS-neurons were tested behaviourally, and those with strongly reduced responses to visual movement were processed for 3H-deoxyglucose autoradiography. The distribution of metabolic activity in the optic lobes ofomb apparently does not differ substantially from that found in wildtype. In the mutantlobula plate-less ^N684 (lop) the small rudiment of the lobula plate which lacks many small-field input neurons does not show any stimulus-specific labelling. The data provide further support for the hypothesis that small-field input neurons to the lobula plate are the cellular substrate of the direction-specific labelling inDrosophila (see Buchner et al. 1984b).Abbreviations DG deoxyglucose - omb optomotor blind^H31 - lop lobula plate-less^N684 - WT wildtype     

Combined tazemetostat and MAPKi enhances differentiation of papillary thyroid cancer cells harbouring BRAFV600E by synergistically decreasing global trimethylation of H3K27

Clinical efficacy of differentiation therapy with mitogen-activated protein kinase inhibitors (MAPKi) for lethal radioiodine-refractory papillary thyroid cancer (RR-PTC) urgently needs to be improved and the aberrant trimethylation of histone H3 lysine 27 (H3K27) plays a vital role in BRAF^V600E-MAPK-induced cancer dedifferentiation and drug resistance. Therefore, dual inhibition of MAPK and histone methyltransferase (EZH2) may produce more favourable treatment effects. In this study, BRAF^V600E-mutant (BCPAP and K1) and BRAF-wild-type (TPC-1) PTC cells were treated with MAPKi (dabrafenib or selumetinib) or EZH2 inhibitor (tazemetostat), or in combination, and the expression of iodine-metabolizing genes, radioiodine uptake, and toxicity were tested. We found that tazemetostat alone slightly increased iodine-metabolizing gene expression and promoted radioiodine uptake and toxicity, irrespective of the BRAF status. However, MAPKi induced these effects preferentially in BRAF^V600E mutant cells, which was robustly strengthened by tazemetostat incorporation. Mechanically, MAPKi-induced decrease of trimethylation of H3K27 was evidently intensified by tazemetostat in BRAF^V600E-mutant cells. In conclusion, tazemetostat combined with MAPKi enhances differentiation of PTC cells harbouring BRAF^V600E through synergistically decreasing global trimethylation of H3K27, representing a novel differentiation strategy.     

Response of last instar Heliothis zea larvae to carbohydrates: stimulation of biting, nutritional value

Several carbohydrates were tested for their ability to stimulate last instar Heliothis zea larvae to bite and, as a measure of nutritional value, for their ability to prolong the lives of H. zea larvae in the absence of a protein. Sucrose, fructose and glucose strongly stimulated biting. Maltose and sorbose were weakly stimulating, and mannitol, rhamnose, galactose, and lactose did not stimulate biting at the concentrations tested. Sucrose, fructose, mannitol, maltose, glucose, galactose, corn starch, and lactose all prolonged life, but larval longevity varied among these carbohydrates. Rhamnose did not prolong life, nor did sorbose, which was slightly toxic. When fructose, glucose, or mannitol replaced sucrose in a nutritionally complete defined diet containing protein, utilization efficiencies were minimally affected. When sorbose was substituted for sucrose, it did not cause mortality, but it did depress the efficiency of utilization significantly below that of a control diet that lacked any carbohydrate except cellulose powder.

---

Résumé L'étude a porté sur l'apitude de plusieurs carbohydrates à stimuler les morsures des chenilles du dernier stade de H. zea et à prolonger leur vie, (mesure de la valeur nutritive), en absence de protéines. Sucrose, fructose et glucose stimulent fortement les morsures. Maltose et sorbose ont été faiblement stimulants, et rhamnose, galactose, lactose et mannitol n'ont pas stimulé aux concentrations examinées. Sucrose, fructose, mannitol, maltose, glucose, galactose, amidon de maïs et lactose ont tous prolongé la vie, mais d'une façon différente suivant les carbohydrates.La rhamnose n'a pas prolongé la vie, tout comme le sorbose, qui s'est révélé légèrement toxique. Quand le fructose, le glucose ou le mannitol ont remplacé le sucrose dans un régime complètement défini contenant des protéines, la perturbation de l'efficacité d'utilisation a été minimale, mais la substitution du sorbose par le sucrose, bien qu'elle n'ait pas provoqué de mortalité, a donné une efficacité d'utilisation significativement plus faible qu'un témoin négatif ayant perdu tous les carbohydrates, à l'exception de poudre de cellulose.