Control of nucleotide and erythroascorbic acid pools by cyclic AMP in Neurospora crassa

UDPglucuronic acid and erythroascorbic acid were identified in extracts of the fungus Neurospora crassa. The concentrations of these two compounds are estimated, in growing wild type N. crassa, to be about 0.10 and 0.28 μmol/ml of cell water, respectively. The pools of these two compounds are regulated by cyclic AMP in Neurospora, both being elevated in the cr-1, adenylate cyclase deficient mutant and both being lowered by exogenous cyclic AMP. The pools of these two compounds are also elevated on nitrogen deprivation. The pools of a large number of other nucleotides are not influenced by cyclic AMP. Possible relationships between the metabolism of UDPglucuronic acid and erythroascorbic acid are discussed. It was found that exogenous cyclic AMP was much more effective in influencing cultures grown at 30–37°C than those grown at 25°C. We suggest that higher temperatures may render Neurospora more permeable to a variety of different compounds.     

Regulation of lactate/pyruvate ratios by cyclic AMP in Neurospora crassa

Cyclic AMP is thought to have a general role in stimulating the breakdown of carbohydrate reserves and subsequent glycolytic activity. This would be expected to increase the availability of reducing equivalents in the form of cytoplasmic NADH. The current study examines another potential reaction controlling cytoplasmic NADH in the fungus Neurospora crassa, that of lactate dehydrogenase, to determine whether it is also regulated by cyclic AMP. The cr-1, adenylate cyclase and cyclic AMP-deficient mutant, grown with and without exogenous cyclic AMP was compared with an isogenic wild type. The results show that cyclic AMP raises pyruvic acid pools and lowers both lactic acid pools and lactate/pyruvate ratios. It does that, in part or in whole, by lowering lactate dehydrogenase activity. The possibility that cytoplasmic NAD+/NADH is a major target of cyclic AMP control is discussed. The high performance liquid chromatography procedures used in these studies are applicable to the measurement of intracellular pools of tricarboxylic acid cycle and other organic acids.     

Deficient cyclic adenosine 3'',5''-monophosphate control in mutants of two genes of Neurospora crassa.

Strains of Neurospora crassa mutant in either of two genes, Crisp-1 (cr1) and Frost (fr), showed no increase of cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels when subjected to several treatments which produce large increases of cyclic AMP in wild-type Neurospora. Evidently, the previously reported deficiencies of adenylate cyclase in these mutants were sufficient to block the normal increases. This fact suggests that both mutants could be used to help determine which control phenomena involve cyclic AMP and to interrupt the control of established cyclic AMP-regulated functions. Earlier studies had suggested an interdependence of the cyclic AMP level and the electric potential difference across the plasma membrane of Neurospora. Present experiments, therefore, employed several strains with the cr1 mutation to test for possible roles of cyclic AMP in recovery and oscillatory behavior of the Neurospora membrane potential. The results showed all such phenomena to be normal in the adenylate cyclase-defective strains, which demonstrates that variations of cyclic AMP are not obligatorily involved in the apparent control processes. Evidence is also presented that the induction of both glucose transport system II and the alternative oxidase do not require elevated cyclic AMP levels.     

In vivo control of gluconeogenesis in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp) mutant.

The rate of cycloheximide-resistant incorporation of carbon from [14C]alanine and [14C]acetate into polysaccharidic material was used to study gluconeogenic activity in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp-1) mutant. The wild-type efficiently utilized alanine and acetate as gluconeogenic substrates, whereas the mutant used acetate efficiently but was unable to use alanine. Cycloheximide-resistant 14C-incorporating activity was sensitive to carbon catabolite effects (repression and inactivation) in the two strains, which suggested that cyclic AMP metabolism was not involved in these regulatory responses. In the wild type, gluconeogenesis was induced by incubation of the cells in the absence of a carbon source. In contrast, cr-1 required supplementation with acetate. This finding suggested that induction of gluconeogenesis in N. crassa could be mediated by metabolites formed in carbon-starved cells. The cr-1 mutant seemed to be deficient in this process and to depend on an exogenous effector to induce gluconeogenesis. Incubation of cr-1 with cyclic AMP partially overcame the acetate requirement for induction of gluconeogenesis.     

Control of cyclic adenosine 3'',5''-monophosphate levels by depolarizing agents in fungi.

It has been reported that diverse treatments which depolarize the plasma membrane of Neurospora crassa produce rapid increases in cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels. In the current study, membrane active antibiotics, which are known or putative depolarizing agents, were found to produce similar cyclic AMP increases, not only in N. crassa, but also in the distantly related fungi Saccharomyces cerevisiae and Mucor racemosus. Uncouplers of oxidative phosphorylation, which have been found to depolarize Neurospora, also produced cyclic AMP increases in all three fungi. The time course of the cyclic AMP response to these various treatments was similar in all three fungi. The fungal studies and studies on depolarized central nervous tissue suggest that cyclic AMP increases may be produced in response to plasma membrane depolarization in diverse eucaryotic cells. A model is proposed for eucaryotic microorganisms in which membrane depolarization serves as a signal of breakdown of the plasma membrane integrity. The subsequent cyclic AMP increase, in turn, may mediate cellular response to help protect the plasma membrane from chemical and mechanical threats to its integrity.     

Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases of Neurospora crassa.

A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. N,N'-Dicyclohexylcarbodiimide, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, N-1-napthylphthalamate, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H+ transport across their respective membranes.     

Characterization of Neurospora crassa cyclic AMP phosphodiesterase activated by calmodulin.

Activation of cyclic AMP phosphodiesterase I by brain or Neurospora calmodulin was studied. The stimulation required micromolar concentrations of Ca2+, and it was observed at cyclic AMP concentrations between 0.1 and 500 microM. Activation was blocked by EDTA and some neuroleptic drugs such as chlorpromazine and fluphenazine. These drugs inhibit the elongation of N. crassa wild-type aerial hyphae. These results reinforce the evidence towards the recognition of Ca2+-calmodulin as one of the systems controlling cyclic nucleotide concentrations in Neurospora.     

Role of lipids in the Neurospora crassa membrane. I. Influence of fatty acid composition on membrane lipid phase transitions.

The relationship between lipid composition and phase transition was investigated by differential scanning calorimetry for intact and membrane phospholipid extracts of wild-type (w/t) and the cel-(Tw 40) mutant of Neurospora crassa. The cel-(Tw 40) mutant (grown on minimal, sucrose medium supplemented with Tween 40 at approximately 34 degrees C) had approximately twice the saturated fatty acid content of w/t organisms grown at approximately 22 degrees C. The gel-liquid crystal phase transitions of ergosterol-free extracts derived from w/t and cel-(Tw 40) occur at -31 and -11 degrees C, respectively. The heats of transition (delta H) of these extracts were 1 and 13 cal/g, respectively. The addition of ergosterol (the predominant sterol in Neurospora) to the phospholipid extracts decreased the observed heats of transition, but did not alter the transition temperature. Intact Neurospora, whether w/t or cal-(Tw 40) did not manifest similar gel-liquid crystal phase transitions in the differential scanning calorimeter. However, an endothermic peak at approximately 30 degrees C was observed in intact cells and extracted phospholipids of both w/t and cel-(Tw 40) organisms. This peak was insensitive to the addition of ergosterol, had a low heat content (delta H congruent to 1 cal/g), and was reversible.     

Effects of heat shock on the level of trehalose and glycogen, and on the induction of thermotolerance in Neurospora crassa.

Neurospora crassa conidiospore germlings exposed to a heat shock (30-45 C) rapidly accumulated trehalose and degraded glycogen, even in the presence of cycloheximide. This phenomenon was also rapidly reversible upon return of the cells at 30 degrees C. Trehalose accumulation at 45 degrees C demanded an exogenous source of carbon and either glucose or glycerol fulfilled such requirement. Experiments with the cyclic AMP-deficient cr-1 mutant suggested that the effects of temperature shifts on trehalose level were independent of cAMP metabolism. Cells exposed at 45 degrees C under conditions permissive for trehalose accumulation (i.e. in the presence of an assimilable carbon source) also acquired thermotolerance.     

Regulation of fructose 2,6-bisphosphate levels in Neurospora crassa

Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-bisphosphate at levels of 27 nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-bisphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-bisphosphate. Thus, the above-described regulation of fructose 2,6-bisphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-bisphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-bisphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-bisphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-bisphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.     

Genes for elongation factor EF-1 alpha in the brine shrimp Artemia

Neurospora crassa had a heat-stable (up to 95 degrees C), soluble cyclic nucleotide phosphodiesterase (PDE). Both unheated and heat-stable PDE activities were inhibited by micromolar concentrations of Ca2+. This inhibition was reversed by EGTA or EDTA in molar excess of the Ca2+ concentration. Calmodulin was not involved in the Ca2+ inhibition, nor was Ca2+ inhibition of the heat-stable PDE due to cleavage inactivation of the enzyme by a Ca2+-stimulated protease. In addition to Ca2+, several other cations inhibited the activity of the heat-stable enzyme. Cyclic AMP and cGMP, but not 2'3' cAMP were substrates for both unheated and heat-stable PDEs. This is the first report of a PDE which is inhibited by micromolar concentrations of Ca2+.     

ATP-induced protyrosinase synthesis and carboxyl-terminal processing in Neurospora crassa

The effects of 3'-5' cyclic AMP and ATP upon tyrosinase induction in Neurospora crassa were examined. Northern analysis of total cellular RNA revealed rapid de novo synthesis of protyrosinase after addition of these substances to stationary-phase mycelia. The maturation of protyrosinase in crude extracts of mycelia was followed by Western analysis. Polyclonal rabbit antiserum directed against the denatured carboxyl-terminal extension of protyrosinase does recognize the proform and several intermediate forms of different molecular weight but not mature tyrosinase. Disruption of ATP-induced mycelia in sodium phosphate buffer (pH 6.0) demonstrate processing at the carboxyl-terminal end of protyrosinase. The activity assays revealed that protyrosinase is an inactive precursor and that at least two active forms of slightly different molecular weight are present in crude extracts. Maturation of protyrosinase thus involves specific and sequential proteolytic cleavage at the carboxyl-terminus. These results suggest the presence of a tyrosinase activator in Neurospora crassa mycelia, which is kept apart from protyrosinase in the intact mycelium.     

Properties of two cyclic nucleotide-deficient mutants of Neurospora crassa.

Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.     

Metabolism and excretion of exogenous adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat.

Isolated rat kidneys were perfused with a recirculating medium containing exogenous adenosine 3':5'-monophosphate (cyclic AMP) or guanosine 3':5'-monophosphate (cyclic GMP) at an initial concentration of 0.1 mM. Both cyclic nucleotides were rapidly removed from the perfusate. Urinary excretion accounted for about 20% and 40% of the respective cyclic AMP and cyclic GMP lost from the perfusate. The metabolism of the cyclic nucleotides was studied by 14C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [14C]AMP was metabolized to renal [14C]adenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [14C]uric acid. Similarly, 20% of cyclic[14C]GMP was metabolized to renal [14C]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [14C]uric acid. Urine contained principally unchanged 14C-labeled cyclic nucleotide. Addition of 0.1 mM cyclic AMP to the perfusate elevated the renal ATP and ADP contents 2-fold. Addition of 0.1 mM of either cyclic AMP or cyclic GMP to the perfusate also elevated the renal production of uric acid 2- to 3-fold. The production and distribution of metabolites of exogenous cyclic nucleotides were also studied in the intact rat. Within 60 min after injection, 3.3 mumol of either 14C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for 14C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [14C]AMP and cyclic [14C]GMP lost from the plasma, respectively. The 14C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [14C]allantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged 14C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [14C]uric acid was not a significant metabolite of the 14C-labeled cyclic nucleotides in these in vivo experiments.     

The effects of altered levels of phosphatidylcholine and phosphatidylethanolamine on fatty acid desaturase activity and sterol metabolism during temperature acclimation in a choline auxotroph of Neurospora crassa

Experiments were conducted to determine the effects of choline deprivation on levels of phospholipid fatty acids in a choline auxotroph (chol-1; chol-2) of Neurospora crassa with respect to high (37 degrees C) and low (15 degrees C) growth temperatures and during acclimation following a shift from high to low temperature conditions. Although grossly altered levels of phosphatidylethanolamine and phosphatidylcholine were observed at both temperatures, phospholipid fatty acid levels remained virtually identical to those found in a phenotypically wild-type and maximally supplemented chol-1; chol-2 strains grown under the same conditions. Deprivation of choline from supplemented cultures of the mutant followed by a shift from high to low growth temperatures did not significantly affect the level of fatty acid desaturation with respect to control cultures. Free sterols did not significantly affect the level of fatty acid desaturation with respect to control cultures. Free sterols were reduced, however, and sterol ester levels were elevated in choline-deprived cultures, suggesting that sterol interconversions may be closely tied to aspects of phospholipid biosynthesis. These experiments suggest that although major modifications in membrane fluidity may be brought about by thermally induced changes in fatty acid desaturase activity, it seems probable that additional cellular mechanisms may be involved if fluidity is under precise control.     

Pyrophosphatase and glucuronosyltransferase in microsomal UDPglucuronic-acid metabolism in the rat liver.

1. A radiochemical method for the studies on the microsomal UDPglucuronic acid metabolism has been developed. 2. The rat liver microsomes caused a rapid hydrolysis of UDPglucuronic acid to D-glucuronic acid 1-phosphate and further although much slower to free D-glucuronic acid. In Tris-HCl buffer (pH 7.4) they were produced in ratio 72 : 1. No other metabolites were found in measurable amounts. The pyrophosphatase splitting UDPglucuronic acid showed a pH optimum at 8.9, but the liberation of D-glucuronic acid from UDPglucuronic acid had two pH maxima (pH 3.5 and 8.5). EDTA appeared to be less powerful inhibitor of pyrophosphatase than previously suggested. About 25 per cent of the UDPglucuronic acid hydrolyzing activity was still remaining in the presence of 10 mM EDTA. D-Glucaro-1,4-lactone was found to have a slight inhibitory action on the pyrophosphatase activity. Citrate inhibited powerfully the hydrolysis of UDPglucuronic acid and the liberation of free D-glucuronic acid. Phosphate was also inhibitory. 3. In the presence of an exogenous UDPglucuronosyltransferase substrate, 4-nitrophenol, the formation of D-glucuronic acid 1-phosphate and free D-glucuronic acid were slightly reduced, and D-glucuronic acid 1-phosphate, 4-nitrophenylglucuronide and free D-glucuronic acid were produced in ratio 78 : 23 : 1. When 10 mM EDTA was added to diminish the hydrolytic consumption of the glucuronyl donor substrate, the corresponding ratio was still as unfavorable as 19 : 2.6 : 1. The measurable activity of UDPglucuronosyltransferase was lower in the presence of phosphate or citrate than in Tris-HCl buffer, although they protected the glucuronyl donor substrate against hydrolysis. 4. The results indicate that even in the presence of added glucuronyl acceptor substrate the hydrolysis of UDPglucuronic acid predominates the conjugation in rat liver microsomes. The rate of the hydrolysis of UDPglucuronic acid is quite considerable even in the presence of EDTA, and it is recommended to control the UDPglucuronic acid pyrophosphatase activity when UDPglucuronosyltransferase and glucuronidation reactions are studied. Free D-glucuronic acid appears to be produced from UDPglucuronic acid for further use via D-glucuronic acid 1-phosphate, the rate-limiting step being the hydrolysis of this intermediate. UDP-glucuronosyltransferase, glucuronides of either endogenous or exogenous aglycones and beta-glucuronidase have only a minor role in this respect in rat liver microsomes.     

Adenylyl cyclase deficient cr-1 (Crisp) mutant of Neurospora crassa: Cyclic AMP-dependent nutritional deficiencies

The inability to synthesize cyclic AMP drastically affects the nutritional metabolism of Neurospora crassa. The adenylyl cyclase-less mutant cr-1 (crisp) did not utilize several carbon sources, including glycerol, mannitol, arabinose, and casaminoacids. However, in glucose or acetate it grew as well as the wild type. The following evidence suggested that these nutritional deficiencies were a direct result of the cr-1 mutation: (i), in crosses to wild type they segregated together with the crisp morphological marker; (ii), cyclic AMP added to the cr-1 mutant growth medium overcame the nutritional deficiencies; (iii), the cyclic AMP effect was specific for the crisp mutant, for it was not observed with the wild type, nor with a spontaneous glycerol-utilizing cr-1 strain.     

Cyclic nucleotide phosphodiesterase activities in Neurospora crassa.

Cyclic nucleotide phosphodiesterase activities in soluble Neurospora crassa mycelial extracts were resolved into two peaks, phosphodiesterase I and II, by chromatography on DEAE-cellulose columns. Phosphodiesterase I hydrolysed cyclic AMP and cyclic GMP equally well. Phosphodiesterase II was active on cyclic GMP but scarcely active on cyclic AMP. Phosphodiesterase I was resolved by gel filtration and sucrose-density-gradient centrifugation into three peaks having molecular weights of about 57 000, 125 000 and 225 000. This suggests that this enzyme activity has at least three aggregation forms, tentatively defined as monomeric, dimeric and tetrameric. Similarly, phosphodiesterase II was resolved into two forms, having molecular weights of about 170 000 and 320 000. Evidence on the interconversion between phosphodiesterase I forms was obtained.     

Biochemical comparison of the Neurospora crassa wild type and the temperature-sensitive and leucine-auxotroph mutant leu-5. Purification of the cytoplasmic and mitochondrial leucyl-tRNA synthetases and comparison of the enzymatic activities and the degradation patterns

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 degrees C) and mutant (grown at 28 degrees C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28 degrees C and transferred then to 37 degrees C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37 degrees C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish and difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 degrees C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of approximately 1:600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme.     

The cellulase complex of Neurospora crassa: activity, stability and release

The temperature and pH optima, and the temperature and pH stability, of crude and purified enzymes of the cellulase complex of the cellulolytic ascomycete fungus Neurospora crassa were investigated. The effects of some non-ionic surfactants and fatty acids on the production/release of enzymes of cellulase complex were also examined. For the different enzymes of the complex, activity maxima occurred between pH 4.0 and 7.0, with pH 5.0 being close to optimal for stability of all. Temperature optima for activity ranged between 45 and 65 degrees C, with the stability optimum between 45 and 50 degrees C. The presence of C18 fatty acids and surfactants resulted in increased production of both endoglucanase and exoglucanase in the medium. Oleic acid was the most effective fatty acid tested, and Tween 80 the most effective surfactant. Oleic acid had no detectable effect on production of beta-glucosidase, and Tween 80 actually reduced its production.