Isolation and characterization of a copper containing protein from blue cell walls of Neurospora crassa

Culturing Neurospora crassa in presence of toxic amounts of copper (0.63 mM) resulted in blue coloured mycelia and cell walls. Significant amounts (approximately 45%) of total mycelial copper were associated with cell wall isolates under conditions of copper toxicity. Hence, such blue cell walls were analysed to identify specific ligands involved in copper binding. While decuprification of the blue cell walls with 8-hydroxy quinoline (8 HQ) did not alter their copper binding abilities, similar treatment with EDTA (10 mM) decreased such abilities indicating that EDTA treatment lead to loss of copper binding ligands from cell walls. Treatment of blue cell walls with 8 HQ followed by EDTA resulted in the solubilization of a copper binding protein (relative MW approximately 14 kDa) which was associated with phosphate and carbohydrate moieties. On amino acid analysis, this protein was found to be devoid of free thiol groupings but enriched in acidic and basic amino acids, distinguishing it from classical intracellular metal binding proteins such as metallo-thioneins and phytochelatins that are inducively synthesized under conditions of metal toxicity. The biological significance of the isolated wall-bound copper binding protein, which appears to be a normal constituent of cell walls, is discussed in relation to cytoplasmic metal binding proteins and mechanism(s) adapted by fungi in countering metal toxicity.     

Energy and metabolic intercellular interaction in the growth region of Neurospora crassa

A major group of systemic autoimmune diseases is associated with abnormal lymphoproliferation, as a result of defects in the termination of lymphocyte activation and growth. Recent progress has been made in understanding the causes and consequences of these abnormalities. At the molecular level, the defects in CD95 and its ligand are only the most obvious reasons for the breakdown of 'clonal contraction' which in fact requires the participation of multiple gene products, including the IL-2-IL-2-receptor system, to set up a functional apoptotic machinery.     

Two distinct protein-protein interactions between the NIT2 and NMR regulatory proteins are required to establish nitrogen metabolite repression in Neurospora crassa

Unrecognized and untreated depression occurs frequently in Alzheimer's disease (AD) patients, adding to the agony already experienced by patient and caregiver. The authors screened AD patients living with a family caregiver for depression. Twelve patients with confirmed depression were treated in an open-label study with antidepressant medication, with dosage adjustment by a psychiatrist at Weeks 2, 4, 8, and 16. Data collection occurred at baseline, Week 4, and Week 16. Depression decreased significantly (p < .01). Contrary to expectations, patient functional capacity declined (p = .045). Cognition remained unchanged (p > .05). Caregiver burden, caregiver depressive symptomatology, and quality of life of patient and caregiver remained unchanged (p > .05). The authors conclude that depression in AD can be detected if a collateral source, such as the caregiver, is available. The depression can and should be treated. More research is needed to determine the impact on patient functioning, caregiver burden, caregiver depressive symptomatology, and quality of life of patient and caregiver.     

Purification and characterization of the catalytic subunit of protein phosphatase 1 from Neurospora crassa

Protein phosphorylation is a universal regulatory mechanism in eukaryotic cells. The phosphorylation state of proteins is affected by the antagonistic activities of protein kinases and phosphatases. Protein phosphatases (PPs) can be classified as serine/threonine and tyrosine specific phosphatases. Ser/Thr phosphatases are divided into four subclasses (PP1, PP2A, PP2B, PP2C) on the basis of their substrate specificity, metal ion dependence and inhibitor sensitivity. We were able to detect the activities of all four Ser/Thr protein phosphatases in the mycelial extract of Neurospora crassa. The catalytic subunit of PP1 was purified 1500-fold with a yield of 1.3% using ammonium sulfate-ethanol precipitation, DEAE-Sephacel, heparin-Sepharose and MonoQ FPLC chromatography. The protein product was nearly homogenous, as judged by SDS-polyacrylamide gel electrophoresis. The most important properties of the enzyme were the following: /1/ its molecular mass proved to be 35 kD, /2/ it was completely inhibited by inhibitor-2, microcystin and okadaic acid, /3/ it was bound to heparin-Sepharose, and /4/ its specific activity was 2000 mU/mg. These biochemical properties are very similar to those of the homologous enzyme from rabbit muscle and indicate a high level of conservation of PP1 structure during evolution.     

Structure of the chromosome VII centromere region in Neurospora crassa: degenerate transposons and simple repeats

DNA from the centromere region of linkage group (LG) VII of Neurospora crassa was cloned previously from a yeast artificial chromosome library and was found to be atypical of Neurospora DNA in both composition (AT rich) and complexity (repetitive). We have determined the DNA sequence of a small portion (approximately 16.1 kb) of this region and have identified a cluster of three new retrotransposon-like elements as well as degenerate fragments from the 3' end of Tad, a previously identified LINE-like retrotransposon. This region contains a novel full-length but nonmobile copia-like element, designated Tcen, that is only associated with centromere regions. Adjacent DNA contains portions of a gypsy-like element designated Tgl1. A third new element, Tgl2, shows similarity to the Ty3 transposon of Saccharomyces cerevisiae. All three of these elements appear to be degenerate, containing predominantly transition mutations suggestive of the repeat-induced point mutation (RIP) process. Three new simple DNA repeats have also been identified in the LG VII centromere region. While Tcen elements map exclusively to centromere regions by restriction fragment length polymorphism analysis, the defective Tad elements appear to occur most frequently within centromeres but are also found at other loci including telomeres. The characteristics and arrangement of these elements are similar to those seen in the Drosophila centromere, but the relative abundance of each class of repeats, as well as the sequence degeneracy of the transposon-like elements, is unique to Neurospora. These results suggest that the Neurospora centromere is heterochromatic and regional in character, more similar to centromeres of Drosophila than to those of most single-cell yeasts.     

Analysis of electrical phenomena accompanying the growth of Neurospora crassa hyphae: theory and experiment

To describe electrical phenomena observed in growth of Neurospora crassa hyphae, a theoretical model was developed considering the hypha as a one-dimensional electric cable with non-uniform longitudinal distribution of current sources reflecting the activity of proton pumps. A profile of the density of the pump current along the hypha is proposed, at which the results of simulation quantitatively coincide with the results of physiological experiments. The model values of energy coupling in the growth zones were estimated. The experimental dependence of the elongation rate of regenerating apical hypha fragments on their lengths was determined. Based on the comparison of these experimental results with the results of analysis of the model, the contribution of the axial metabolite transport, from the distal parts of the hypha to the apical part, to the dynamics of the apical cell growth was estimated. The possibility of evaluating the intensity of high-molecular-weight syntheses and/or accumulation of substances in granules was demonstrated. The growth rate of the regenerating hypha fragments was shown to correlate with the electric current flowing into the apical fragment 0.2-mm in length.     

Indications for the occurrence of nitric oxide synthases in fungi and plants and the involvement in photoconidiation of Neurospora crassa

The structure of the complex of Aeromonas proteolytica aminopeptidase, a two-zinc exopeptidase, with the inhibitor p-iodo-D-phenylalanine hydroxamate has been determined by X-ray crystallography. Refinement of the structure, which includes 220 water molecules, using data at 0.80-0.23-nm resolution resulted in a crystallographic residual R value of 16%. The hydroxamate group adopts a planar conformation whereby the two oxygen atoms interact with the zinc ions. The N-hydroxyl group of the inhibitor is located between the two zinc ions, a position which is close to that occupied by a water molecule in the native structure. The carbonyl oxygen of the inhibitor binds to Zn1, which becomes pentacoordinated while Zn2 remains tetracoordinated, in contrast to the native protein where both zinc ions were shown to be tetracoordinated and structurally equivalent. Interactions of the carboxylate oxygens of Glu151 with the hydroxamate group play an important role in the stabilization of the complex.     

M-glycogenin, the protein moiety of Neurospora crassa proteoglycogen, is an auto- and transglucosylating enzyme

Neurospora crassa proteoglycogen was purified and its protein moiety, M-glycogenin, was released by amylolytic treatment. The released protein was capable of autoglucosylation from UDP-glucose forming glucosyl-alpha 1,4-glucosyl linkage. The kinetics of autoglucosylation suggested an intramolecular mechanism of reaction. M-glycogenin was also able to glucosylate dodecyl-beta-maltoside and autoglucosylate, simultaneously and independently. Both auto- and transglucosylation reactions were dependent on Mn2+. Thus, M-glycogenin, which has also been described as the constituent of Escherichia coli proteoglycogen (A. Goldraij and J. A. Curtino. 1993, Biochem. Mol. Biol. Int. 30, 453-458), is a glucosyltransferase that bears similar catalytic properties with mammalian glycogenin. This is the first report on the enzymatic character of the protein constituent of proteoglycogen in primitive organisms, which suggest that the mechanism for the de novo biosynthesis of glycogen was conserved over a very long period of evolution.     

Comparison of the sequence-selective DNA binding by peptide dimers with covalent and noncovalent dimerization domains

Sequence-specific DNA binding proteins generally consist of more than two DNA-contacting regions to ensure the selectivity of recognition. The multiple DNA binding modules are connected either through the covalent linker or through the noncovalent dimerization domain. We have compared the DNA binding of peptide dimers with covalent and noncovalent dimerization domains to explore the potential advantage of each linkage on the sequence-specific DNA binding. Three sets of head-to-tail peptide dimers were synthesized by using the same basic region peptide to target the same DNA sequence; one dimer was assembled with a bridged biphenyl derivative as a covalent dimerization domain, and two other dimers were assembled with the cyclodextrin guest noncovalent dimerization domains. One of the noncovalent dimers was a heterodimer that consisted of cyclodextrin and guest peptides, while the other was a homodimer that consisted of peptides bearing both cyclodextrin and the guest molecule within the same chain. Both noncovalent dimers formed the specific DNA complexes within narrower ranges of peptide concentrations and showed higher sequence selectivity than the covalent dimer did. Among the three dimers, the noncovalent homodimer that can form an intramolecular inclusion complex showed the highest sequence selectivity. Because the noncovalent homodimer with the higher stability of the circular intramolecular inclusion complex exhibited the higher sequence selectivity, it was concluded that an equilibrium involving a conformational transition of a monomeric peptide effectively reduced the stability of its nonspecific binding complex, hence increasing the efficacy of cooperative dimer formation at the specific DNA sequence.     

Quantitative and qualitative comparison of spontaneous and chemical-induced specific-locus mutation in the ad-3 region of heterokaryon 12 of Neurospora crassa

The data from forward-mutation experiments to obtain specific-locus mutations at two closely linked loci in the adenine-3 (ad-3) region of heterokaryon 12 (H-12) of Neurospora crassa have been tabulated to determine the relative frequencies and mutational spectra of ad-3 mutants occurring spontaneously and those induced by 22 different chemical treatments. Previous studies have demonstrated that specific-locus mutations at these two loci result from 5 major genotypic classes, namely two classes of gene/point mutations (ad-3AR and ad-3BR), and 3 classes of multilocus deletion mutations ([ad-3A]IR, [ad-3B]IR and [ad-3A ad-3B]IR). In addition, prior studies have demonstrated that some chemical mutagens induced ad-3 mutants exclusively, or almost exclusively, by gene/point mutation and other chemical mutagens by gene/point mutation and multilocus deletion mutation. In the latter cases, there was wide variation in the percentages of ad-3 mutants in these 5 major genotypic classes. Two comparative methods of analysis that also were used to compare spontaneous and chemical-induced ad-3 mutational spectra included X2-tests on the numbers of ad-3 mutants resulting in the following two sets of ratios: (1) gene/point mutations and multilocus deletion mutations; and (2) complementing and non-complementing ad-3BR, mutants. Combination of the p-values from X2-tests for these two methods of comparison demonstrated that all 22 chemicals induce a spectrum of ad-3 mutants that is qualitatively different from that occurring spontaneously. In addition, these same two methods of comparison have been used to compare the mutagenic effects of each of the 22 chemical treatments with each other. Combination of the data from these two methods of comparison has demonstrated that 93.1% (215/231) of the pairwise combinations of these 22 chemicals were different from each other. The implication of these experimental data on the induction of specific-locus mutations in somatic cells of Neurospora for genetic risk assessment exercises is discussed.     

Functional analysis by site-directed mutagenesis of individual amino acid residues in the flavin domain of Neurospora crassa nitrate reductase

Nitrate reductase of Neurospora crassa is a complex multi-redox protein composed of two identical subunits, each of which contains three distinct domains, an amino-terminal domain that contains a molybdopterin cofactor, a central heme-containing domain, and a carboxy-terminal domain which binds a flavin and a pyridine nucleotide cofactor. The flavin domain of nitrate reductase appears to have structural and functional similarity to ferredoxin NADPH reductase (FNR). Using the crystal structure of FNR and amino acid identities in numerous nitrate reductases as guides, site-directed mutagenesis was used to replace specific amino acids suspected to be involved in the binding of the flavin or pyridine nucleotide cofactors and thus important for the catalytic function of the flavin domain. Each mutant flavin domain protein was expressed in Escherichia coli and analyzed for NADPH: ferricyanide reductase activity. The effect of each amino acid substitution upon the activity of the complete nitrate reductase reaction was also examined by transforming each manipulated gene into a nit-3- null mutant of N. crassa. Our results identify amino acid residues which are critical for function of the flavin domain of nitrate reductase and appear to be important for the binding of the flavin or the pyridine nucleotide cofactors.     

Tissue-specific repression of starvation and stress responses of the Neurospora crassa con-10 gene is mediated by RCO1

Abstract-This study presents an evaluation of the effectiveness of the AIDS Community-Based Outreach/Intervention projects implemented as part of the National Institute on Drug Abuse (NIDA) Cooperative Agreement (CA), which began in 1990 and is currently ongoing. Participants in the CA were randomly assigned to one of two interventions: a NIDA/CA-developed standard intervention (SI); or the SI plus a site-specific enhanced intervention (EI). Analyses of drug use and needle-related risk behaviors were conducted among injection drug users (IDUs) in eight participating cities where follow-up rates of at least 60% were obtained (N=3,743). Results indicated that IDUs significantly reduced their needle-related risk behaviors following delivery of the interventions and that a substantial portion entered substance abuse treatment. However, there was relatively little to support the effectiveness of more expensive and involved enhanced interventions. A number of factors associated with increasing or maintaining high risk behaviors, including an HIV negative serostatus and a greater perceived chance of acquiring AIDS, were also observed. Continued outreach to drug injectors is recommended, as well as the development of new and creative interventions targeting individuals who are HIV negative and those who are aware of their high risk status but have not changed their behaviors in response to risk-reduction interventions.     

Synthesis and activity of bivalent FKBP12 ligands for the regulated dimerization of proteins

The total synthesis and in vitro activities of a series of chemical inducers of dimerization (CIDs) is described. The use of small-molecule CIDs to control the dimerization of engineered FKBP12-containing fusion proteins has been demonstrated to have broad utility in biological research as well as potential medical applications in gene and cell therapies. The facility and flexibility of preparation make this new class of wholly synthetic compounds exceptionally versatile tools for the study of intracellular signaling events mediated by protein-protein interactions or protein localization. While some congeners possess potency comparable to or better than the first generation natural product-derived CID, FK1012, structure-activity relationships are complex and underscore the need for application-specific compound optimizations.     

Purification, kinetic characterization and involvement of tryptophan residue at the NADPH binding site of xylose reductase from Neurospora crassa

Xylose reductase (XR) from Neurospora crassa was purified to homogeneity and was found to be specific to NADPH (nicotinamide adenine dinucleotide phosphate). The purified enzyme showed M(r) of 60 and 29 kDa by gel filtration and SDS-PAGE indicating the presence of two subunits. The kinetic mechanism of xylose reductase is 'iso-ordered bi bi'. Inactivation of XR by N-bromosuccinimide (NBS) was found to be biphasic with second-order rate constants of 2.5 x 10(2) and 80 M-1S-1 for the fast (kf) and slow phase (ks), respectively. NADPH protected 90% of XR activity against inhibition by NBS. The fluorescence and circular dichroism (CD) studies revealed that inactivation was not due to gross conformational change in the enzyme. Analysis of the modified Stern-Volmer plot indicated that 49% of the tryptophanyl fluorescence was available for quenching which was completely abolished in the presence of NADPH confirming the involvement of tryptophan at the coenzyme binding site. Experimental evidence presented here serves to implicate the involvement of a tryptophan residue at the low-affinity NADPH binding site and the nature of this site has been assessed by using the hydrophobic probe ANS.     

Anion channels and the stimulation of anthocyanin accumulation by blue light in Arabidopsis seedlings

The effects of endothelin 1 (ET-1) on hemodynamics and acute liver damage were studied using perfused livers of rats treated with D-galactosamine. In control liver perfused in situ with constant pressure, infusion of ET-1 into the portal vein at a concentration of 0.1 nmol/L decreased the flow rate without a significant leakage of lactate dehydrogenase (LDH) or aspartate transaminase (AST) into the effluent. In contrast, in similarly perfused liver 24 hours after treatment with D-galactosamine (800 mg/kg intraperitoneally), ET-1 caused rapid and remarkable increases in the leakage of LDH and AST from the liver accompanied by the reduction of perfusion flow to the extent similar to that observed in control livers. In addition, ET-1 decreased oxygen uptake and bile secretion in galactosamine-treated livers. The potentiating effects of ET-1 on enzyme leakage were also observed under constant flow conditions. Moreover, infusion of the thromboxane A2 analogue at a concentration of 10 nmol/L decreased the flow rate markedly, yet the rapid increases in enzyme leakage were not observed. Infusion of ET-3 induced the responses of flow reduction and the potentiation of rapid enzyme leakage similar to those obtained with ET-1. Neither the endothelin A-receptor antagonist BQ485 nor the endothelin B-receptor antagonist BQ788 could inhibit the acute liver damage caused by ET-1; instead they exaggerated its effects. The combination of both antagonists together, however, almost completely suppressed the flow reduction and the potentiation of enzyme leakage caused by ET-1. These results indicate that ET-1 is capable of aggravating acute liver damage not merely through reduction of the flow rate but through direct action on liver cells. They also suggest that both the endothelin A and endothelin B receptors are involved in this action of ET-1.     

Roles of individual kringle domains in the functioning of positive and negative effectors of human plasminogen activation

In order to identify the individual contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1Pg] ([D139-->N]r-Pg), [K4Pg] ([D413-->N]r-Pg), and [K5Pg] ([D515--N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu1]Pg and, to a lesser extent, the [K5Pg] mutant, [D518-->N/Glu1]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C50) occurred at 3.3 mM for wtr-[Glu1]Pg and at 0.7 mM for [D518-->N/Glu1]r-Pg. Subsequent inhibition by EACA occurred with a C50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during the activation. Similar initial activation rates of both [D139-->N]r-Pg and [D413N]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C50 for this process that was similar to wtr-[Glu1]Pg and [D518-->N/Glu1]r-Pg.(ABSTRACT TRUNCATED AT 250 WORDS)