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.     

Apolar growth of Neurospora crassa leads to increased secretion of extracellular proteins

Protein secretion in filamentous fungi has been shown to be restricted to actively growing hyphal tips. To determine whether an increase in the amount f growing surface area of a fungus can lead to an increase in the amount of protein secretion, we examined secretion in a temperature-sensitive Neurospora crassa mcb mutant that shows a loss of growth polarity when incubated at restrictive temperature. Incubation of the mcb mutant at restrictive temperature results in a three- to fivefold increase in the level of extracellular protein and a 20-fold increase in carboxymethyl cellulase activity relative to a wild-type strain. A mutation in the cr-1 gene has been shown previously to suppress the apolar growth phenotype of the mcb mutant, and we find that the level of extracellular protein produced by a mcb; cr-1 double mutant was reduced to that of the wild-type control. Immunolocalization of a secreted endoglucanase revealed that proteins are secreted mainly at hyphal tips in hyphae exhibiting polar growth and over the entire surface area of bulbous regions of hyphae that are produced after a shift of the mcb mutant to restrictive temperature. These results support the hypothesis that secretion of extracellular protein by a filamentous fungus can be significantly increased by mutations that alter growth polarity.     

Photoregulation of cot-1, a kinase-encoding gene involved in hyphal growth in Neurospora crassa

Lattice-like perineuronal accumulations of extracellular-matrix proteoglycans have been shown to develop during postnatal maturation and to persist throughout life as perineuronal nets (PNs) in many brain regions. However, the dynamics of their reorganization in adults are as yet unknown. The aim of the present study was to examine the capability of PNs for reconstitution after experimental destruction and to search for possible consequences of extracellular-matrix degradation for neurons and glial cells. The changes were induced by single intracortical injections of Proteus vulgaris chondroitinase ABC and studied after postinjection periods of 1 day to 5 months. The N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA), an antibody against chondroitin-sulphate proteoglycans, three antibodies recognizing initial chondroitin or chondroitin-sulphate moieties ('stubs') of proteoglycan core proteins, an antibody against the hyaluronan-binding protein component of versican, and biotinylated hyaluronectin, which binds to hyaluronan, were used as cytochemical markers. One day postinjection, the WFA-binding sites and hyaluronan were shown to be almost completely removed within a circumscribed digestion zone. The staining of different core-protein components revealed only fragments of PNs. These changes were found to be partly compensated 4 weeks after injection of chondroitinase ABC. After 8 and 12 weeks postinjection, the cytochemical and structural characteristics as well as the area-specific distribution patterns of PNs were progressively reconstituted. At 5 months postinjection, they could not be distinguished from those in untreated tissue. In contrast to such transient changes, a diffuse chondroitin-sulphate proteoglycan immunoreactivity persisted in the neuropil. Loss of neurons or alterations of their structure as well as reactions of glial cells were not observed. We conclude from this study that PNs, enzymatically destroyed in the adult rat brain, can be completely reconstituted, but the restoration of their extracellular-matrix components needs several months.     

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.     

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.     

Expression theory and the preference reversal phenomena.

Previous research has confounded judgment versus choice with the response scale used to express preference. When these factors are crossed in a 2?×?2 design, 6 pairs of preference reversals are possible. Experiment 1 demonstrates that all of these reversals exist in one-outcome gambles. We then develop expression theory, which assumes that the basic evaluation of a gamble is expressed on various scales via a subjective interpolation process. A quantitative model is developed that accounts for both the magnitude and direction of the various reversals. In Experiment 2, the model correctly predicts that the strength of reversals is weakened using two-outcome gambles. Nevertheless, 5 of the 6 reversals are still significant. In Experiment 3, empirical violations of dominance are demonstrated and the results are shown to be consistent with expression theory. In Experiment 4, the model is used to predict ratings from prices and vice versa. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

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.     

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities.     

Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient

The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction of the target function had been achieved in antisense transformants with multiple construct integrations. Induction conditions caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the distinctive tip-high Ca2+ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise regulation of apical growth, a common form of cellular proliferation. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype very similar to that of the cna-1 antisense mutants is consistent with this idea.     

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.     

Genetic interactions among cytoplasmic dynein, dynactin, and nuclear distribution mutants of Neurospora crassa

Cytoplasmic dynein is a multisubunit, microtubule-associated, mechanochemical enzyme that has been identified as a retrograde transporter of various membranous organelles. Dynactin, an additional multisubunit complex, is required for efficient dynein-mediated transport of vesicles in vitro. Recently, we showed that three genes defined by a group of phenotypically identical mutants of the filamentous fungus Neurospora crassa encode proteins that are apparent subunits of either cytoplasmic dynein or dynactin. These mutants, designated ropy (ro), display abnormal hyphal growth and are defective in nuclear distribution. We propose that mutations in other genes encoding dynein/dynactin subunits are likely to result in a ropy phenotype and have devised a genetic screen for the isolation of additional ro mutants. Cytoplasmic dynein/dynactin is the largest and most complex of the cytoplasmic motor proteins, and the genetic system described here is unique in its potentiality for identifying mutations in undefined genes encoding dynein/dynactin subunits or regulators. We used this screen to isolate > 1000 ro mutants, which were found to define 23 complementation groups. Unexpectedly, interallelic complementation was observed with some allele pairs of ro-1 and ro-3, which are predicted to encode the largest subunits of cytoplasmic dynein and dynactin, respectively. The results suggest that the Ro1 and Ro3 polypeptides may consist of multiple, functionally independent domains. In addition, approximately 10% of all newly isolated ro mutantsdisplay unlinked noncomplementation with two or more of the mutants that define the 23 complementation groups. The frequent appearance of ro mutants showing noncomplementation with multiple ro mutants having unlinked mutations suggests that nuclear distribution in filamentous fungi is a process that is easily disrupted by affecting either dosage or activity of cytoplasmic dynein, dynactin, and perhaps other cytoskeletal proteins or regulators.     

Constitutive modelling of brain tissue: experiment and theory

Recent developments in computer-integrated and robot-aided surgery--in particular, the emergence of automatic surgical tools and robots--as well as advances in virtual reality techniques, call for closer examination of the mechanical properties of very soft tissues (such as brain, liver, kidney, etc.). The ultimate goal of our research into the biomechanics of these tissues is the development of corresponding, realistic mathematical models. This paper contains experimental results of in vitro, uniaxial, unconfined compression of swine brain tissue and discusses a single-phase, non-linear, viscoelastic tissue model. The experimental results obtained for three loading velocities, ranging over five orders of magnitude, are presented. The applied strain rates have been much lower than those applied in previous studies, focused on injury modelling. The stress-strain curves are concave upward for all compression rates containing no linear portion from which a meaningful elastic modulus might be determined. The tissue response stiffened as the loading speed increased, indicating a strong stress-strain rate dependence. The use of the single-phase model is recommended for applications in registration, surgical operation planning and training systems as well as a control system of an image-guided surgical robot. The material constants for the brain tissue are evaluated. Agreement between the proposed theoretical model and experiment is good for compression levels reaching 30% and for loading velocities varying over five orders of magnitude.