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At least 217 records · Page 12Linked to original sources
PubMed1986

Electrophysiological properties of Achlya hyphae: ionic currents studied by intracellular potential recording.

The electrical properties of the water mold Achlya bisexualis were investigated using intracellular microelectrodes. Hyphae growing in a defined medium maintained a membrane potential (Vm) of -150 to -170 mV, interior negative. Under the conditions used here, this potential was insensitive to changes in the inorganic ion composition of the medium. Changes in external pH did affect Vm, but only outside the physiological pH range. By contrast, the addition of respiratory inhibitors caused a rapid depolarization without affecting the conductance of the plasma membrane. Taken together these findings strongly suggest that the membrane potential is governed by an electrogenic ion pump rather than by an ionic diffusion potential. Previous work from this laboratory showed that Achlya hyphae generate a transcellular proton current that enters the growing tip, flows along the hyphal length, and exits distally from the trunk. These initial experiments used an extracellular vibrating electrode, and I now report intracellular electrical recordings which support the hypothesis that protons enter the tip by symport with amino acids and are expelled distally by a proton-translocating ATPase. Most significantly, current flowing intracellularly along the hyphal length is associated with a cytoplasmic electric field of 0.2 V/cm or greater. Conditions that inhibit the current also abolish the internal field, suggesting that these two phenomena are closely linked.

Amino Acids↗
PubMed1999

Mutational activation of a Galphai causes uncontrolled proliferation of aerial hyphae and increased sensitivity to heat and oxidative stress in Neurospora crassa.

Heterotrimeric G proteins, consisting of alpha, beta, and gamma subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Galpha protein, GNA-1, that is a member of the Galphai superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Deltagna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1(R178C) and gna-1(Q204L) strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1(R178C) and gna-1(Q204L) strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1(R178C) and gna-1(Q204L) strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Deltagna-1 mutants are more resistant. In contrast to Deltagna-1 mutants, gna-1(R178C) and gna-1(Q204L) strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gbetagamma-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.

Alleles↗
PubMed1983

Mechanisms of destruction of Aspergillus fumigatus hyphae mediated by human monocytes.

Normal monocytes attached to and destroyed Aspergillus hyphae by morphologic and metabolic criteria. Inhibition by anaerobiosis, azide, cyanide, halide-free conditions, catalase, histidine, and tryptophan suggested mediation of hyphal damage primarily through the myeloperoxidase system. However, myeloperoxidase-independent oxidative or nonoxidative mechanisms appeared active in hyphal damage by monocytes from patients with myeloperoxidase deficiency or chronic granulomatous disease (CGD). Moreover, hyphae were damaged by lysates and granule-enriched fractions of monocytes from patients with CGD, whereas comparable fractions of normal monocytes were active only with added halide and H2O2. Hyphal damage by both whole monocytes and granule-enriched fractions from patients with CGD was inhibited by polyanions, a result suggesting that cationic proteins may be involved. Hyphal damage by normal monocytes or monocytes from patients with CGD was inhibited by cells that lacked antihyphal activity (chlorpromazine-treated normal neutrophils or neutrophils from patients with CGD, respectively). This effect may predispose patients with CGD to chronic, invasive aspergillosis.

Aspergillosis↗
PubMed1986

In vitro killing of spores and hyphae of Aspergillus fumigatus and Rhizopus oryzae by rabbit neutrophil cationic peptides and bronchoalveolar macrophages.

The fungicidal activity of six rabbit neutrophil cationic peptides (NP) against resting (dormant) spores, preincubated (swollen) spores, and hyphae of Aspergillus fumigatus and Rhizopus oryzae was examined. Whereas resting spores of both fungi were totally resistant to killing by up to 500 micrograms of NP/ml, preincubated spores and hyphae were sensitive, under optimal conditions, to less than 1 microgram /ml. NP-1 and NP-2, also found in rabbit bronchoalveolar macrophages, were the most active of the peptides. Activity was inhibited with increasing ionic strength, calcium but not magnesium, incubation at 4 C, and the chitin fragments chitobiose and chitotriose. 125I-labeled NP-1 bound to all three forms of A. fumigatus. Finally, killing of A. fumigatus conidia by rabbit bronchoalveolar macrophages was faster and greater if the conidia were first preincubated in RPMI 1640 medium. Thus, although NP did not kill the resting A. fumigatus conidia, a role for cationic peptides in macrophage-mediated killing is not precluded, as fungicidal activity of both isolated cationic peptides and intact bronchoalveolar macrophages appeared to occur only after the conidia became activated.

Animals↗
PubMed1993

Impairment of neutrophil antifungal activity against hyphae of Aspergillus fumigatus in children infected with human immunodeficiency virus.

Human immunodeficiency virus (HIV)-infected patients may acquire invasive aspergillosis without previously recognized risk factors, such as neutropenia or corticosteroid therapy. Because neutrophils (PMNL) are an important component of host defense in aspergillosis, the antifungal activity of PMNL against hyphae of Aspergillus fumigatus in 31 HIV-infected children was assessed. Hyphal damage was unaffected in 15 HIV-infected children with age-adjusted CD4 cell counts > or = 25% of the normal median value; it was decreased in 16 with CD4 cell counts < 25% (both vs. 20 healthy controls, P = .001. Incubation with sera from 12 of 14 HIV-infected children but not with the recombinant HIV proteins gp120, gp41, and p24 suppressed antifungal activity of normal PMNL compared with normal serum (P = .002). Pretreatment of defective PMNL from 5 patients with granulocyte colony-stimulating factor (G-CSF) partially corrected the defect (P = .002). These findings suggest that impaired serum-mediated antifungal activity against Aspergillus hyphae exists in PMNL of HIV-infected patients with low CD4 cell counts; G-CSF may improve this activity.

Adolescent↗
PubMed1995

Defective killing of Candida albicans hyphae by neutrophils from beige mice.

The candidacidal activity of neutrophils from BALB/c, C.B-17 +/+, C.B-17 scid/scid, scid/scid-bg/bg, C57BL/6 bg/+, C57BL/6 bg/bg, NIH III bg/bg-nu/+, and bg/bg-nu/nu mice was assessed. Killing of blastoconidia was moderately deficient with neutrophils from 2 strains of homozygous beige mice; however, neutrophils from all homozygous beige strains had a significantly decreased capacity to kill Candida albicans hyphae. This is the first demonstration of a decreased capacity of beige mouse neutrophils to kill C. albicans hyphae. The latter defect could be related to the enhanced susceptibility of beige mice to candidiasis.

Animals↗
PubMed1995

Cytoplasts generate oxidants but require added neutrophil granule constituents for fungicidal activity against Candida albicans hyphae.

Killing of Candida albicans hyphae requires oxidant generation by neutrophils (PMNL), but it is uncertain whether hyphal killing is mediated by PMNL oxidants alone or requires contributions by granule constituents. This was assessed using U-cytoplasts (U-CYT), anucleate PMNL fragments depleted of cytoplasmic granules but retaining motility and respiratory burst activity. Granule-depleted U-CYT killed Staphylococcus aureus, but hyphae remained viable despite targeted generation of putatively fungicidal oxidants by avidly adherent U-CYT. Hyphal killing occurred by combining U-CYT with sublethal concentrations of purified PMNL granule extracts approximating those present in equivalent numbers of intact PMNL. Contributions of granule constituents were not entirely attributable to purified granule constituents with known antimicrobial activity (lactoferrin, cathepsin G, myeloperoxidase, and human neutrophil peptide defensins 1-3) individually or combined. Thus, oxidant generation by intact PMNL may be necessary but not always sufficient to mediate hyphal killing without complementary nonoxidative mechanisms.

Candida albicans↗
PubMed1984

Growing hyphae of Achlya bisexualis generate a longitudinal pH gradient in the surrounding medium.

Growing hyphae of Achlya bisexualis were found to generate a longitudinal pH gradient in the surrounding medium; the medium adjacent to the tip was slightly more alkaline than the bulk phase, while that near distal parts was acidic. The profile of external pH paralleled that of electric current, as measured with a vibrating probe; the apical alkaline zone corresponded to the region of current inflow. In organisms grown in complete medium, both current flow and apical alkalinization were inhibited when amino acid uptake was blocked, either by removing amino acids from the medium or by raising the external pH to 8.5. Achlya could, however, adapt to a medium deficient in organic nutrients; elongating hyphae again generated both the pH profile and the transcellular electric current. It is proposed that both the pH profile and the electric current are manifestations of a transcellular proton current, which arises from the segregation of proton pumps from proton leaks. Symport of protons with amino acids may be one mechanism by which protons enter the hyphal apex.

Adaptation, Physiological↗
PubMed1984

Interactions of Candida albicans yeast cells, germ tubes and hyphae with human polymorphonuclear leucocytes in vitro.

Suspensions of Candida albicans yeast cells, germ tubes and hyphae with biomass standardized by ATP measurement were compared for their relative susceptibilities to phagocytosis and intracellular killing by human polymorphonuclear leucocytes. All three forms were ingested to a similar extent, but significantly fewer yeast cells were killed intracellularly after ingestion than were filamentous forms of the fungus. Ketoconazole pretreatment significantly enhanced the susceptibility of hyphae, but not of germ tubes, to phagocytosis and intracellular killing. The opsonic requirements of the yeasts and filamentous forms for efficient phagocytosis and killing differed.

Candida albicans↗
PubMed1986

Development of Candida albicans hyphae in different growth media--variations in growth rates, cell dimensions and timing of morphogenetic events.

In six liquid culture media, all of which stimulated Candida albicans to grow in the hyphal form, the rates of hyphal extension and increase in cellular ATP concentration, hyphal diameters, times of evagination of hyphae, times of septum formation and positions of septa in the hyphae appeared to vary independently. There were no discernible associations between properties such as length or volume of hyphal compartments at the time of septation and temporal parameters of hyphal growth. The results suggest that growth environment influences many of the processes contributing to hyphal development, but that these processes are not necessarily interrelated.

Adenosine Triphosphate↗
PubMed1997

Amplification of a Streptomyces lividans 4.3 kb DNA element causes overproduction of a novel hypha- and vesicle-associated protein.

The wild-type Streptomyces lividans 66 genome contains a 4.3 kb amplifiable DNA unit (AUD), and its four ORFs encode proteins that could not be identified by sequence comparison with databases. One of the gene products (encoded by orf-2) was purified and determined to be a novel 23 kDa protein. This protein is synthesized by the wild-type strain, absent in a variant lacking the AUD and overproduced in a variant in which the AUD is amplified (ADS). Immunological studies and analyses by confocal laser microscopy showed that the 23 kDa protein is associated with the substrate hyphae of the wild-type and the ADS-containing variant. Examination by microscopy revealed that the strain carrying the ADS forms bulges within the substrate hyphae and apical vesicles. These bulges have high levels of associated 23 kDa protein and contain storage-like material.

Amino Acid Sequence↗
PubMed1998

A hydrophobin (ABH3) specifically secreted by vegetatively growing hyphae of Agaricus bisporus (common white button mushroom).

Aerial mycelium and hyphal strands of Agaricus bisporus, strain U1, exhibited a rodlet pattern at their surfaces characteristic for assembled class I hydrophobins. An SDS-insoluble/trifluoroacetic-acid-soluble fraction from strands was found to contain one abundant protein with an apparent molecular mass on gel of 19 kDa. Two sequences for this protein (ABH3), typical of class I hydrophobins, could be deduced by sequencing cDNA clones obtained by RT-PCR. The two forms of the protein could be assigned to different alleles present in the two homokaryons that constitute the heterokaryotic U1 strain. ABH3 displays all the in vitro properties of a typical class I hydrophobin such as SC3 from Schizophyllum commune but is not glycosylated or otherwise post-translationally modified because the molecular mass values deduced from the amino acid sequence (9228 and 9271 Da) and derived from mass spectrometry were in good agreement. The ABH3 transcript was found to be present in the vegetative mycelium of both primary and secondary mycelium but not in the fruiting bodies, whereas the reverse was found for the ABH1 hydrophobin. Using an S. commune mutant with a disrupted SC3 gene it was found that ABH3 can substitute for SC3 in inducing formation of aerial hyphae, suggesting a role of ABH3 in the emergence of aerial hyphae and strands in A. bisporus.

Agaricus↗
PubMed1975

Morphology and growth kinetics of hyphae of differentiated and undifferentiated mycelia of Neurospora crassa.

A comparison was made of the morphology and growth kinetics of hyphae of differentiated and undifferentiated mycelia of Neurospora crassa. Undifferentiated mycelia were formed during exponential growth on solid media or submerged culture. Hyphae at the margin of differentiated mycelia (colonies) differed from undifferentiated mycelia in diameter, extension rate, extension zone length, and intercalary and apical compartment length. The mean hyphal extension rate (E) of an undifferentiated mycelium was a function of the length of the mycelium's hyphal growth unit (G) and the organism's specific growth rate (alpha). Thus, E=Galpha.

Culture Media↗
PubMed1981

Tissue responses to the blastospores and hyphae of Candida albicans in the mouse.

Separate groups of mice challenged intravenously with either the blastospore or hyphal forms of the same strain of Candida albicans were examined for comparative mortality rates, organ localisation, and tissue and cellular response to the organisms. Blastospores were more rapidly and consistently fatal to mice than the hyphae. Relatively more hyphal elements than blastospores were initially localised in the lungs but more blastospores than hyphal elements were trapped in the liver. The cells of both forms were more effectively killed in the lungs than in other organs. Blastospores initially found in the kidneys increased rapidly in numbers, but hyphal inocula either grew slowly in the kidneys or were eliminated. After mice were challenged with either hyphae or blastospores the initial inflammatory response in the lungs and liver was predominantly of polymorphonuclear leukocytes, but macrophages were the first inflammatory cells to be seen in kidney sections. Peripheral blood counts showed a leukocytosis in mice of both groups although only blastospores resulted in increased numbers of circulating atypical lymphocytes. The results indicated that lungs may play a more important role than other reticuloendothelial organs in innate resistance to vascular invasion by either of the morphological forms of C. albicans, and macrophages may be crucial to host resistance to renal invasion by this fungus.

Animals↗
PubMed1985

Quantitative Localization of the Phytoalexin Glyceollin I in Relation to Fungal Hyphae in Soybean Roots Infected with Phytophthora megasperma f. sp. glycinea.

A radioimmunoassay specific for glyceollin I was used to quantitate this phytoalexin in roots of soybean (Glycine max [L.] Merr. cv Harosoy 63) after infection with zoospores of either race 1 (incompatible) or race 3 (compatible) of Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Erwin. The sensitivity of the radioimmunoassay and an inmmunofluorescent stain for hyphae permitted quantitation of phytoalexin and localization of the fungus in alternate serial cryotome sections from the same root. The incompatible interaction was characterized by extensive fungal colonization of the root cortex which was limited to the immediate vicinity of the inoculation site. Glyceollin I was first detected in extracts of whole roots 2 hours after infection, and phytoalexin content rose rapidly thereafter. Significant concentrations of glyceollin I were present at the infection site in cross-sections (42 micrometers thick) of such roots by 5 hours, and exceeded 0.6 micromoles per milliliter (EC(90)in vitro for glyceollin I) by 8 hours after infection. Longitudinal sectioning (14 micrometers thick) showed that glyceollin I accumulated particularly in the epidermal cell layers, but also was present in the root cortex at inhibitory concentrations. No hyphae were observed in advance of detectable levels of the phytoalexin and, in most roots, glyceollin I concentrations dropped sharply at the leading edge of the infection. In contrast, the compatible interaction was characterized by extensive unchecked fungal colonization of the root stele, with lesser growth in the rest of the root. Only small amounts of glyceollin I were detected in whole root extracts during the first 14 hours after infection. Measurable amounts of glyceollin I were detected only in occasional cross-sections of such roots 11 and 14 hours after infection. The phytoalexin was present at inhibitory concentrations in the epidermal cell layers, but the inhibitory zone did not extend appreciably into the cortex. Altogether, these data support the hypothesis that the accumulation of glyceollin I is an important early response of soybean roots to infection by P. megasperma, but may not be solely responsible for inhibition of fungal growth in the resistant response.

Journal Article↗
PubMed2001

An evaluation of semiautomatic approaches to contour segmentation applied to fungal hyphae.

Semiautomatic image analysis techniques are particularly useful in biological applications, which commonly generate very complex images, and offer considerable flexibility. However, systematic study of such methods is lacking; most research develops fully automatic algorithms. This paper describes a study to evaluate several different semiautomatic or computer-assisted approaches to contour segmentation within the context of segmenting degraded images of fungal hyphae. Four different types of contour segmentation method, with varying degrees and types of user input, are outlined and applied to hyphal images. The methods are evaluated both quantitatively and qualitatively by comparing results obtained by several test subjects segmenting simulated images qualitatively similar to the hyphal images of interest. An active contour model approach, using control points, emerges as the method to be preferred to three more traditional approaches. Feedback from the image provider indicates that any of the methods described have something useful to offer for segmentation of hyphae.

Algorithms↗
PubMed1992

Monoclonal antibodies bind identically to both spores and hyphae of Aspergillus fumigatus.

Immunoelectron microscopy (IEM) was used to determine the binding of six monoclonal antibodies (MoAbs) produced against Aspergillus fumigatus antigens present on or within the conidia and hyphae of the fungus. Antigen-antibody complexes were demonstrated in EM using labelled colloidal gold particles (15 nm). Three out of 6 MoAbs (C9, F12 and H10) reacted only with the cytoplasmic components of A. fumigatus while the remaining three (B12, F6G5 and D6E6) showed reactivity to both cytoplasm and cell wall of the conidia and hyphae. The results indicate that IEM is of considerable value in determining and selecting monoclonal antibodies having specific reactivity with diverse antigenic components.

Antibodies, Fungal↗
PubMed1996

The positions of the sigma-factor genes, whiG and sigF, in the hierarchy controlling the development of spore chains in the aerial hyphae of Streptomyces coelicolor A3(2).

whiG and sigF encode RNA polymerase sigma factors required for sporulation in the aerial hyphae of Streptomyces coelicolor. Their expression was analysed during colony development in wild-type and sporulation-defective whi mutant strains. Each gene was transcribed from a single promoter. Unexpectedly, whiG mRNA was present at all time points, including those taken prior to aerial mycelium formation; this suggests that whiG may be regulated post-transcriptionally. Transcription of whiG did not depend upon any of the six known 'early' whi genes required for sporulation septum formation (whiA, B, G, H, I and J), placing it at the top of the hierarchy of whi loci. sigF expression appeared to be regulated at the level of transcription; sigF transcripts were detected transiently when sporulation septa were observed in the aerial hyphae. Transcription of sigF depended upon all six of the early whi genes, including whiG. The sigF promoter does not resemble the consensus sequence established for sigma WhiG-dependent promoters and E sigma WhiG did not transcribe from the sigF promoter in vitro. Consequently, the genetic dependence of sigF upon whiG is very likely to be indirect. These results show that there is a hierarchical relationship between sigma factors required for Streptomyces sporulation and also that at least five other genes are involved in this transcriptional network.

Bacterial Proteins↗