Ontogeny of the Spitzenkörper in germlings of Neurospora crassa

The Spitzenkörper (Spk) is a highly dynamic and pleomorphic complex located at the hyphal apex of filamentous fungi. Most studies revealing the structure and behavior of the Spk have been conducted on mature vegetative hyphae of filamentous fungi, including both main leading hyphae and branches. However, these reports do not address whether the observations can be extended to germ tubes. By enhanced phase-contrast video-microscopy and laser scanning confocal microscopy we have analyzed the intracellular changes prior to the appearance of a Spk in germlings of Neurospora crassa. Observations began at the early stages of spore germination and were carried out until a conspicuous Spk could be observed at the apex of germ tubes. Before a Spk could be observed, young germ tubes (<150 μm) displayed a uniform distribution of organelles such as nuclei, mitochondria, and cytoplasmic granules along the length of the cells. Once the germlings started reaching lengths of more than approximately 150 μm, visible organelles experienced a displacement towards the subapical region of the cell and a small exclusion zone free of organelles (0.6 ± 0.3 μm) formed at the apex. The position of this exclusion zone within the apex seemed to determine the germling growth direction, which was highly erratic. Few minutes after it first appeared, upon growth of the germling, the exclusion zone started to become occupied by an accumulation of material that gradually concentrated into a light gray body that we describe as an immature Spk. During this phase the presence of a Spk in the apical dome was not constant. Approximately 30 min later, the immature Spk became more robust and gradually acquired its typical phase-dark appearance, while the growth direction of the germ tube became less wavering. The formation of a mature phase-dark Spk coincided with the stabilization of the growth direction of the germling, therefore suggesting that it is at this stage when the transition from germling to vegetative hypha occurs.     

Location and contribution of individual β-glucosidase from Neurospora crassa to total β-glucosidase activity

This study investigated the cellular location and the contribution of individual β-glucosidase (BGL) to total BGL activity in Neurospora crassa. Among the seven bgl genes, bgl3, bgl5, and bgl7 were transcribed at basal levels, whereas bgl1, bgl2, bgl4, and bgl6 were significantly up-regulated when the wild-type strain was induced with cellulose (Avicel). BGL1 and BGL4 were found to be contributors to intracellular BGL activity, whereas the activities of BGL2 and BGL6 were mainly extracellular. Sextuple bgl deletion strains expressing one of the three basally transcribed bgls did not produce any detectable BGL activity when they were grown on Avicel. BGL6 is the major contributor to overall BGL activity, and most of its activity resides cell-bound. The sextuple bgl deletion strain containing only bgl6 utilized cellobiose at a rate similar to that of the wild type, while the strain with only bgl6 deleted utilized cellobiose much slower than that of the wild type.     

Chitosomes from the wall-less “slime” mutant of Neurospora crassa

Cell-free extracts from the wall-less slime mutant of Neurospora crassa and the mycelium of wild type exhibit similar chitin synthetase properties in specific activity, zymogenicity and a preferential intracellular localization of chitosomes. The yield of chitosomal chitin synthetase from sline cells was essentially the same irrespective of cell breakage procedure (osmotic lysis or ballistic disruption) —an indication that chitosomes are not fragments of larger membranes produced by harsh (ballistic) disruption procedures. The plasma membrane fraction, isolated from slime cells treated with concanavalin A, contained only a minute portion of the total chitin synthetase of the fungus. Most of the activity was in the cytoplasmic fraction; isopycnic sedimentation of this fraction on a sucrose gradient yielded a sharp band of chitosomes with a buoyant density=1.125 g/ cm³. Approximately 76% of the total chitin synthetase activity of the slime mutant was recovered in the chitosome band. Because of their low density, chitosomes could be cleanly separated from the rest of the membranous organelles of the fungus. Apparently, the lack of a cell wall in the slime mutant is not due to the absence of either chitosomes or zymogenic chitin synthetase.Abbreviations Con A concanavalin A - d buoyant density in g/cm³ - GlcNAc N-acetyl-D-glucosamine - MES 2-[N-morpholino]ethanesulfonic acid - UDP-GlcNAc uridine diphosphate N-acetyl-D-glucosamine     

Change of the Structure of Cell Wall β-l,3-d-Glucan with the Growth of Neurospora crassa Cells

The chemical structure of cell wall β-d-glucans as well as the activities of lytic enzymes such as β-1,3-d-glucanase and β-1,6-d-glucanase changed during the growth of Neurospora crassa.

A dramatic change in the cell wall β-d-glucan structure was observed between cells of the middle logarithmic phase and ones of the late logarithmic phase. The ratio of 1,3-linked glucose residues to non reducing terminal glucose residues decreased from 85 to 55 and the ratio of gentiobiose as a hydrolysis product with exo-β-1,3-d-glucanase increased significantly between the two phases.

Two prominent peaks of β-1,3-d-glucanase as well as the β-1,6-d-glucanase activities appeared in the culture filtrate at different growth stages, the early logarithmic phase and the stationary phase. In the cell wall, β-d-glucosidase activity instead of the β-l,6-d-glucanase and β-1,3-d-glucanase activities was observed in the late logarithmic phase.     

Comparative study of K channel behavior inβ cell lines with different secretory responses to glucose

Summary The patch-clamp technique was used to identify and investigate two K channels in the cell membrane of the HIT cell, an insulin secreting cell line with glucose-sensitive secretion. Channel characteristics were compared with those of glucose-modulated K channels in the RINm5F cell, an insulin secreting cell line in which secretion is largely glucose insensitive. A 65.7 pS channel, identified with the ATP-sensitive K channel was seen in HIT cell-attached patches. Channel activity was dose-dependently inhibited by glucose, by 50 and 100% at 450 m and 8mm glucose, respectively, similar to the values previously reported for RIN cells. In inside-out patches channel activity was 50% inhibited by 56 m ATP and completely blocked between 500 m and 1mm, again, similar to the values reported for RIN cells.As in RIN cells a second, considerably larger (184 pS), K channel was glucose sensitive; the glucose sensitivity was similar to that in RIN cells with 50 and 100% channel inhibition at 7.5 and 25mm, respectively. After patch excision the mean channel conductance increased from 184 to 215 pS. Under these conditions activity was strongly calcium dependent in the rangepCa 5–7, identifying this as a calcium- and voltage-dependent K (K(Ca,V)) channel; the calcium sensitivity was similar to that of the adult rat cell K(Ca,V) channel. In inside-out RIN cell patches, the large K channel was less abundant but displayed a similar conductance (223 pS). However, its calcium sensitivity was more than 10 times lower than in HIT cells, similar to that of the K(Ca,V) channel in the neonatal rat cell, which also displays a reduced secretory response to glucose. Based on these observations, it is proposed that the low calcium sensitivity of the K(Ca,V) channel may be causally associated with secretory deficiency in RIN cells and the immature secretory response of the neonatal cell.     

Biotechnological production of ethanol from renewable resources by Neurospora crassa: an alternative to conventional yeast fermentations?

Microbial production of ethanol might be a potential route to replace oil and chemical feedstocks. Bioethanol is by far the most common biofuel in use worldwide. Lignocellulosic biomass is the most promising renewable resource for fuel bioethanol production. Bioconversion of lignocellulosics to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/distillation. Conventional bioethanol processes for lignocellulosics apply commercial fungal cellulase enzymes for biomass hydrolysis, followed by yeast fermentation of resulting glucose to ethanol. The fungus Neurospora crassa has been used extensively for genetic, biochemical, and molecular studies as a model organism. However, the strain's potential in biotechnological applications has not been widely investigated and discussed. The fungus N. crassa has the ability to synthesize and secrete all three enzyme types involved in cellulose hydrolysis as well as various enzymes for hemicellulose degradation. In addition, N. crassa has been reported to convert to ethanol hexose and pentose sugars, cellulose polymers, and agro-industrial residues. The combination of these characteristics makes N. crassa a promising alternative candidate for biotechnological production of ethanol from renewable resources. This review consists of an overview of the ethanol process from lignocellulosic biomass, followed by cellulases and hemicellulases production, ethanol fermentations of sugars and lignocellulosics, and industrial application potential of N. crassa.     

Cytogenetic characterization of ataxia telangiectasia (AT) heterozygotes using lymphoblastoid cell lines and chronic γ-irradiation

Summary Lymphoblastoid cell lines (LCLs) derived from two patients identified as ataxia telangiectasia (AT), two obligate AT heterozygotes and two controls (healthy subjects with no known genetic disease or relationship to AT patients) were compared with respect to the induction of chromosomal breaks by acute and chronic -irradiation. Although there was a considerable increase in the frequency of chromosomal breaks per cell in the LCLs of AT patients resulting from acute irradiation, the small increase occurring in the LCLs of the AT heterozygotes made it difficult to distinguish them from the controls. Following chronic -irradiation, however, the frequency of chromosomal breaks per cell in the LCLs of the AT heterozygotes occupied a significantly distinct position from that of the controls. These observations suggested that the use of chronic irradiation may be a better choice in the cytogenetic characterization of AT heterozygotes.     

Separation of chitosomes and secretory vesicles from the “slime” variant of Neurospora crassa

Cells from the slime variant of Neurospora crassa were broken in isotonic conditions by use of triethanolamine buffer plus EDTA. After removal of large membranous structures by low-speed centrifugation, chitosomes and secretory vesicles were separated by means of gel filtration, precipitation of membranous contaminants with Concanavalin A, and centrifugation in sucrose or glycerol gradients. Polypeptidic composition of fractions enriched in secretory vesicles or chitosomes was found to be distinct. By these criteria we concluded that chitosomes and secretory vesicles represent different populations of microvesicles. Both microvesicular populations appeared free of endoplasmic reticulum and vacuolar contaminants as demonstrated by determination of appropriate enzymatic markers.Abbreviations ER Endoplasmic reticulum - UDP-GlcNAc uridine-5-diphosphate N-acetyl glucosamine - GlcNAc N-acetyl glucosamine - SDS sodium dodecyl sulfate - PMSF phenyl methyl sulfonyl fluoride - EDTA ethylene diamino tetraacetic acid Investigador Nacional de Mexico. On leave from the Centro de Investigacion y Estudios Avanzados (IPN), and the Universidad de Guanajuato, Gto., Mexico     

Purification and Some Properties of an Endo-β,1,6-glucanase from Neurospora crassa

An endo-β-1,6-glucanase (E.C. 3.2.1.75) was purified from the culture filtrate of Neurospora crassa IFO-6O68 by chromatographies on CM-cellulofine, Con-A Sepharose 4B, and Sepharose Cl-6B followed by preparative affinity gel electrophoresis. The purified enzyme had an apparent molecular weight of 47,000. The pH and temperature optima for the activity were 5.0 and 50°C. The enzyme acted on β-1,6-glucan (Pustulan) and yielded a series of gentio-oligosaccharides with endo- type action, and finally, glucose and gentiobiose were produced. The enzyme was also able to act on N. crassa cell wall β-glucan, and a small amount of hydrolysis fragments were liberated without apparent change of the cell wall glucan molecules.     

Synchronization of DNA synthesis in Neurospora crassa by 2′-deoxyadenosine and spore selection

2-Deoxyadenosine (2 mM), a DNA inhibitor, was used to synchronize DNA synthesis in cultures of Neurospora crassa lys 3. The cultures recovered spontaneously from the inhibitor which had little or no effect on the synthesis of RNA, protein or carbohydrate or on the specific growth rate of the mould. The degree of synchrony of DNA synthesis obtained with 2-deoxyadenosine varied directly with the organism's specific growth rate when the latter was altered by temperature changes. A direct relationship was observed between the rate of synthesis of DNA during the S period and the organism's specific growth rate.Conidia of Neurospora crassa lys 3 were separated into different density classes using urografin gradients; the separation treatment did not have an appreciable effect on the subsequent germination or growth of conidia. Populations of large, less dense conidia produced germ tubes more rapidly and more synchronously than populations of small, dense conidia. Cultures inoculated with the large conidia displayed continuous synthesis of RNA and protein but discontinuous synthesis of DNA.     

Advantages of AlaGln as an additive to cell culture medium: use with anti-CD20 chimeric antibody-producing POTELLIGENT™ CHO cell lines

l-alanyl-l-glutamine (AlaGln) is dipeptide that has better solubility and stability than Glutamine (Gln). In this study, we evaluated the utility of this dipeptide during culture of POTELLIGENT™ Chinese hamster ovary (CHO) cells expressing anti-CD20 chimeric antibody. Although AlaGln in the culture medium lowered the specific growth rate, the MAb titer was maximized when Gln was completely replaced by AlaGln in both the basal and feed media. Moreover, AlaGln augmented production of antibody not only at flask scale but also at spinner scale, although the extent of this effect was dependent on the cell clone. To explore the mechanism responsible for the effect of AlaGln on cell growth, we measured apoptosis in the early phase of cell culture on days 8, 9, and 10. The apoptotic ratio was reduced in medium containing AlaGln. Ammonia was generated in medium containing Gln when it was maintained at 37 °C, which impeded the growth and productivity of the cells. In contrast, AlaGln produced less ammonia under these conditions, which may have been one of the properties associated with its beneficial effects. We conclude that certain dipeptides can serve as superior alternative sources of amino acids in cell culture and antibody production.     

Are carotenoids the blue-light photoreceptor in the photoinduction of protoperithecia in Neurospora crassa?

A triple albino mutant of Neurospora crassa with a measured content of carotenoids absorbing at 470 nm less than 0.5% of that of the wild type (calculated value less than 8·10^-4%) had the same threshold for photoinduction of protoperithecia as the wild type when illuminated with monochromatic light at 471 nm. This is strong evidence against the hypothesis that the bulk of carotenoids are the blue-light photoreceptor for this phenomenon. However, it is impossible to exclude traces of carotenoids acting as the photoreceptor at less than 3·10^-12 M in a very efficient sensory transduction chain.Abbreviations A absorbance - al albino mutant - WT wild type     

A cyclic adenosine 3′,5′-monophosphate-dependent protein kinase mutant of Neurospora crassa

A cpk mutant of Neurospora crassa with morphological alteration was obtained spontaneously during the cross between the wild-type and a glycerol utilizing cr-l strain. The growth rate of cpk was intermediate between the wild-type and cr-1 mutant strains. The cpk conidia contained a reduced level of carotenoid pigments as compared to the wild-type conidia. The cpk mutant had no detectable amount of cyclic adenosine 3,5-monophosphate (cAMP)-binding protein at all stages of growth tested. On a DEAE-Sephacel column chromatogram, protein kinase activity of the wild type was eluted at two peaks; the first peak was cAMP-dependent, and the second one was not. In contrast, the cpk strain had two peaks of cAMP-independent enzymes. It is suggested that cAMP-dependent protein kinase may be altered in the cpk mutant into a cAMP-independent type by an alteration of the regulatory subunit of this enzyme.Abbreviations cAMP Cyclic adenosine 3,5-monophosphate - 8-N₃-[³H] cAMP 8-azido-[³H]cyclic adenosine 3,5-monophosphate     

δ-Aminolaevulinate dehydratase, the regulatory enzyme of the haem-biosynthetic pathway in Neurospora crassa

The activity of delta-aminolaevulinate dehydratase is very low in the mould Neurospora crassa compared with the activities detected in bacterial and animal systems. The enzyme is inducible in iron-deficient cultures by addition of iron and is repressed by protoporphyrin. The properties of the purified enzyme indicate its allosteric nature and susceptibility to feedback inhibition by coproporphyrinogen III. Neurospora extracts also contain a protein inhibitor of the enzyme and a small-molecule activator, which appears to be associated with the enzyme. The regulatory function of this enzyme in vivo is correlated with the accumulation of delta-aminolaevulinic acid in normal cultures of N. crassa. The decay curve of the iron-induced enzyme in vivo shows a biphasic pattern, with one of the components showing a half-life of 4-5 min.     

Self-assembly properties of the proteinaceous coat secreted by the “slime” variant of Neurospora crassa

The proteinaceous extracellular material (PEM) synthesized by the cells of the slime strain of Neurospora crassa (see Martinez et al. 1989) was solubilized by treatment with urea or guanidine. Removal of these chemicals by dialysis, caused reassembly of the solubilized proteins into material with the same microscopic appearance as the original PEM. Polypeptide patterns from both native and reassembled structures were identical. Dialysis-mediated reassembly of the solubilized proteins appeared to be dependent on both concentration of the soluble macromolecules and time. Gel chromatography of PEM solubilized with different agents revealed two discrete populations of complexes with molecular masses of 1,500 and 500 kDa respectively. These were able to reassemble into lamellar structures with a variable degree of efficiency.Abbreviations ConA Concanavalin A - Fe-ConA ferritin-labeled Concanavalin A - Endo H endo--N-acetylglucosaminidase H - PMSF phenyl methyl sulphonyl fluoride     

Characterization of a proteinaceous extracellular coat synthesized by the “slime” variant of Neurospora crassa

Cells of the slime strain of Neurospora crassa synthesize a coherent extracellular material which remains attached to the cell surface, but is released into the liquid medium by shaking. The material was purified and studied by different criteria. By electron microscopy it appears as long wavy sheets which strongly bind concanavalin A, but not wheat germ agglutinin, and maintain their integrity in the absence of structural polysaccharides. Analysis of the purified material revealed that it was free of contaminating membranes; it contained more than 70% protein, 1% neutral sugars (glucose, mannose, fucose and galactose), less than 2% lipids and ca. 4% not-characterized hexosaminelike compounds. Its polypeptide pattern as determined by PAGE was complex. The significance of this material is discussed.Abbreviations used Au-WGA colloidal gold-wheat germ agglutinin - Endo H endo--N-acetylglucosaminidase H - Fe-Con A ferritin labcled concanavalin A - FITC-Con A fluorescent concanavalin A - PEM proteinaceous extracellular material - PMSF phenylmethyl sulfphonyl fluoride - BSDA bovine serum albumin - CTAB cetyltrimethyl-ammonium bromide - DOC-Na sodium deoxychlate - DTT dl-dithiothreitol - EDTA ethylene diamine tetracetic acid     

Transcriptomic “portraits” of canine mammary cancer cell lines with various phenotypes

In light of the high incidence of mammary cancer in dogs and completion of the canine genome sequencing, the new possibilities of gene profiling by using DNA microarrays give hope to veterinary oncology. The cell lines isolated from mammary tumors are a valuable tool in developing and testing new pathway-specific cancer therapeutics. Differential cytometric analysis of 6 canine mammary cancer cell lines was performed. We divided cell lines into 3 groups based on their phenotype: 2 lines with high proliferative potential, 2 lines with high antiapoptotic potential, and 2 lines with high metastatic potential. DNA microarray analysis revealed common genes for cell lines of each group. We found that genes encoding the receptors for growth hormone and ghrelin are related to high proliferation rate, whileABR (active BCR-related) andTMD1 (TM2 domain containing 1) genes are related to a high antiapoptotic potential of the cancer cells. Metastatic properties of mammary cancer cells seem to be associated with elevated expression ofPGP (P glycoprotein),SEMA3B (semaphorin 3B), andSTIM1 (stromal interaction molecule 1).