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PubMed1982

Fast axonal transport in squid giant axon.

Video-enhanced contrast-differential interference contrast microscopy has revealed new features of axonal transport in the giant axon of the squid, where no movement had been detected previously by conventional microscopy. The newly discovered dominant feature is vast numbers of "submicroscopic" particles, probably 30- to 50-nanometer vesicles and other tubulovesicular elements, moving parallel to linear elements, primarily in the orthograde direction but also in a retrograde direction, at a range of steady velocities up to +/- 5 micrometers per second. Medium (0.2 to 0.6 micrometer) and large (0.8 micrometer) particles move more slowly and more intermittently with a tendency at times to exhibit elastic recoil. The behavior of the smallest particles and the larger particles during actual translocation suggests that the fundamental processes in the mechanisms of organelle movement in axonal transport are not saltatory but continuous.

Animals↗
PubMed2003

Killing activity of micafungin against Aspergillus fumigatus hyphae assessed by specific fluorescent staining for cell viability.

We studied the anti-Aspergillus activity of micafungin by using two fluorescent dyes to detect cell viability. Micafungin induced flattened hyphae, caused by the bursting of cells, which had lost their viability. Micafungin has killing activity against actively growing hyphae, even though it is not fungicidal against the whole burden of Aspergillus fumigatus.

Antifungal Agents↗
PubMed2006

Induction of autophagy by second-fermentation yeasts during elaboration of sparkling wines.

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.

Autophagy↗
PubMed1994

Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment.

This study reports improvements in two of the key steps, lysis of indigenous cells and DNA purification, required for achieving a rapid nonselective protocol for extracting nucleic acids directly from sodium dodecyl sulfate (SDS)-treated sediment rich in organic matter. Incorporation of bead-mill homogenization into the DNA extraction procedure doubled the densitometrically determined DNA yield (11.8 micrograms of DNA.g [dry weight] of sediment-1) relative to incorporation of three cycles of freezing and thawing (5.2 micrograms of DNA.g [dry weight] of sediment-1). The improved DNA extraction efficiency was attributed to increased cell lysis, measured by viable counts of sediment microorganisms which showed that 2 and 8%, respectively, survived the bead-mill homogenization and freeze-thaw procedures. Corresponding measurements of suspensions of viable Bacillus endospores demonstrated that 2 and 94% of the initial number survived. Conventional, laser scanning epifluorescence phase-contrast, and differential interference-contrast microscopy revealed that small coccoid bacterial cells (1.2 to 0.3 micron long) were left intact after combined SDS and bead-mill homogenization of sediment samples. Estimates of the residual fraction of the fluorescently stained cell numbers indicated that 6% (2.2 x 10(8) cells.g [dry weight] of sediment-1) of the original population (3.8 x 10(9) cells.g [dry weight] of sediment-1) remained after treatment with SDS and bead-mill homogenization. Thus, lysis of total cells was less efficient than that of cells which could be cultured. The extracted DNA was used to successfully amplify nahR, the regulatory gene for naphthalene catabolism in Pseudomonas putida G7, by PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriolysis↗
PubMed1998

Detection of Cyclospora cayetanensis in wastewater.

Cyclospora cayetanensis causes diarrheal disease worldwide without a confirmed mode of transmission. Wastewater was examined for the presence of this organism. Oocysts were detected microscopically, and their identity was confirmed by molecular techniques. These findings verify that current techniques can isolate Cyclospora oocysts and suggest that fecally contaminated water may act as a vehicle of transmission.

Animals↗
PubMed2003

Development and application of a monoclonal-antibody technique for counting Aureococcus anophagefferens, an alga causing recurrent brown tides in the Mid-Atlantic United States.

A method was developed for the rapid detection and enumeration of Aureococcus anophagefferens, the cause of harmful algal blooms called "brown tides" in estuaries of the Mid-Atlantic United States. The method employs a monoclonal antibody (MAb) and a colorimetric, enzyme-linked immunosorbent assay format. The MAb obtained exhibits high reactivity with A. anophagefferens and very low cross-reactivities with a phylogenetically diverse array of other protists and bacteria. Standard curves are constructed for each 96-well microtiter plate by using known amounts of a preserved culture of A. anophagefferens. This approach allows estimation of the abundance of the alga in natural samples. The MAb method was compared to an existing method that employs polyclonal antibodies and epifluorescence microscopy and to direct microscopic counts of A. anophagefferens in samples with high abundances of the alga. The MAb method provided increased quantitative accuracy and greatly reduced sample processing time. A spatial survey of several Long Island estuaries in May 2000 using this new approach documented a range of abundances of A. anophagefferens in these bays spanning nearly 3 orders of magnitude.

Animals↗
PubMed1972

Physiological and biochemical changes associated with macroconidial germination in Microsporum gypseum.

A study was made of the metabolic processes associated with macroconidial germination in Microsporum gypseum. The optimum conditions for stimulation of endogenous respiration, changes in chemical composition as germination proceeds, and the uptake and synthetic fates of amino acids, glucose, and uracil were investigated. The assimilation and conversion of (14)C-glucose, (14)C-amino acids, and (14)C-uracil into the cell pool and into trichloroacetic acid-precipitable material were studied during the early stages of germination (i.e., prior to germ-tube emergence). The macroconidia were not metabolically inert for any significant period of time after exposure to germination conditions. Rather, the spores rapidly assimilated all metabolites and slowly converted them into macromolecules. Investigations of the effect of inhibitors of nucleic acid and protein synthesis prior to germ-tube emergence and during early germ-tube elongation suggested significant changes in metabolism and cell permeability may be correlated with the emergence of germ tubes. Radioactivity of incorporated glucose was found to be associated largely with the lipid fractions of the macroconidia early in germination.

Amino Acids↗
PubMed2004

A WD40 repeat protein regulates fungal cell differentiation and can be replaced functionally by the mammalian homologue striatin.

Fruiting body development in fungi is a complex cellular differentiation process that is controlled by more than 100 developmental genes. Mutants of the filamentous fungus Sordaria macrospora showing defects in fruiting body formation are pertinent sources for the identification of components of this multicellular differentiation process. Here we show that the sterile mutant pro11 carries a defect in the pro11 gene encoding a multimodular WD40 repeat protein. Complementation analysis indicates that the wild-type gene or C-terminally truncated versions of the wild-type protein are able to restore the fertile phenotype in mutant pro11. PRO11 shows significant homology to several vertebrate WD40 proteins, such as striatin and zinedin, which seem to be involved in Ca2+-dependent signaling in cells of the central nervous system and are supposed to function as scaffolding proteins linking signaling and eukaryotic endocytosis. Cloning of a mouse cDNA encoding striatin allowed functional substitution of the wild-type protein with restoration of fertility in mutant pro11. Our data strongly suggest that an evolutionarily conserved cellular process controlling eukaryotic cell differentiation may regulate fruiting body formation.

Amino Acid Sequence↗
PubMed1983

Effect of human leukocyte interferon on invasiveness of Salmonella species in HEp-2 cell cultures.

The effect of human leukocyte interferon on the invasiveness of Salmonella and Shigella species in HEp-2 cell cultures was examined. The intracellular and extracellular bacteria were identified by a combination of Nomarski differential interference contrast microscopy and UV incident light microscopy applied on the same microscope. Pretreatment of HEp-2 cells with human leukocyte interferon reduced the number of Salmonella typhimurium and Salmonella paratyphi-B bacteria per cell and the proportion of cells containing bacteria in a dose-dependent manner. Maximum inhibitory effect was observed with ca. 100 U of interferon per ml. The inhibitory effect was neutralized with anti-human interferon globulin. Murine fibroblast interferon did not influence the invasiveness of Salmonella species. Invasiveness of Shigella flexneri was not influenced by treatment of cells with human interferon.

Cells, Cultured↗
PubMed1994

Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans.

Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.

Antigens, Fungal↗
PubMed2002

Fc-dependent and Fc-independent opsonization of Cryptococcus neoformans by anticapsular monoclonal antibodies: importance of epitope specificity.

Monoclonal antibodies (MAbs) reactive with glucuronoxylomannan (GXM), the major capsular polysaccharide of the yeast Cryptococcus neoformans, produce distinct capsular reactions when viewed by differential interference contrast microscopy. These reactions depend on the epitope specificity of the antibody. Opsonic activities of immunoglobulin G1 (IgG1) MAbs that produce patterns termed rim and puffy were examined. Rim-pattern MAbs are reactive with an epitope shared by GXM serotypes A, B, C, and D. Puffy-pattern MAbs are reactive only with serotypes A and D. In phagocytosis assays, using serotype A cells and resident murine peritoneal macrophages, rim-pattern MAbs were markedly more opsonic than puffy-pattern MAbs. F(ab')(2) fragments of rim-pattern MAbs were synergistic with heat-labile factors in normal human serum for opsonization of the yeast. F(ab')(2) fragments of puffy-pattern MAbs were also synergistic with normal serum in opsonization but at a much lower level than fragments of rim-pattern MAbs. Normal serum alone was not opsonic. F(ab')(2) fragments of rim-pattern MAbs, but not puffy-pattern MAbs, stimulated phagocytosis of encapsulated cryptococci in the absence of serum. This serum-independent opsonic action of F(ab')(2) fragments was abrogated by pretreatment of macrophages with purified GXM, suggesting the involvement of a phagocyte GXM receptor. The results indicate that (i) there are multiple mechanisms by which anticapsular IgG MAbs facilitate phagocytosis of encapsulated cryptococci, (ii) some anti-GXM antibodies are opsonic in an Fc-independent manner, and (iii) opsonic activity correlates with the capsular reaction and occurs in an epitope-specific manner.

Animals↗
PubMed1970

Fine structure of Thermus aquaticus, an extreme thermophile.

Electron microscopic studies using thin sections revealed that Thermus aquaticus has a structure similar to that of most other gram-negative bacteria. The cell envelope is tripartite: plasma membrane, thin middle layer, and a thicker and irregular outer layer. The outer layer appears to be joined to the plasma membrane by a series of connections and, when seen in tangential section, the outer layer appears as a series of parallel bands. The cell division mechanism resembles that of typical gram-negative bacteria. Large spherical bodies designated "rotund bodies" are formed as a result of the association of a number of separate cells. In this association the outer envelope layers of the cells fuse and pull away from the middle layer. The rotund body thus appears as a series of rods, usually lying in parallel around the periphery of the sphere, completely connected by means of the fused outer layer.

Bacteria↗
PubMed1976

Effect of cis-platinum(II)diamminodichloride on cell division of Hyphomicrobium and Caulobacter.

Low concentrations of the radiomimetic agent cis-platinum(II)diamminodichloride (PDD) inhibited cell division in Caulobacter crescentus (0.1 mug/ml) and Hyphomicrobium sp. strain B-522 (1.0 mug/ml) without altering the length of prosthecae. After exposure, cells of C. crescentus appeared as long filaments, whereas only the bud portion of Hyphomicrobium underwent elongation. PDD-treated cells of both species were multinucleated. After the removal of PDD by washing, filaments of C. crescentus fragmented unequally and then normal growth resumed. In Hyphomicrobium (where division involves release of swarmer cells that arise as buds on the distal ends of hyphae), potential septation sites formed in the presence of PDD remained inactive after washing. Reinitiation of cell division in this species was dependent upon the synthesis of new hyphae that could arise from either end of the elongated bud. This finding suggests that the PDD-induced lesion at a given septation site is irreversible and, upon removal of this compound, alternate sites must be synthesized for the subsequent occurrence of cell division.

Bacteria↗
PubMed1999

Recruitment of ZipA to the septal ring of Escherichia coli is dependent on FtsZ and independent of FtsA.

Cell division in prokaryotes is mediated by the septal ring. In Escherichia coli, this organelle consists of several essential division proteins, including FtsZ, FtsA, and ZipA. To gain more insight into how the structure is assembled, we studied the interdependence of FtsZ, FtsA, and ZipA localization using both immunofluorescence and Gfp tagging techniques. To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted. Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ. Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass. In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa. It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.

Amino Acid Sequence↗
PubMed2000

Real-time imaging of fluorescent flagellar filaments.

Bacteria swim by rotating flagellar filaments that are several micrometers long, but only about 20 nm in diameter. The filaments can exist in different polymorphic forms, having distinct values of curvature and twist. Rotation rates are on the order of 100 Hz. In the past, the motion of individual filaments has been visualized by dark-field or differential-interference-contrast microscopy, methods hampered by intense scattering from the cell body or shallow depth of field, respectively. We have found a simple procedure for fluorescently labeling cells and filaments that allows recording their motion in real time with an inexpensive video camera and an ordinary fluorescence microscope with mercury-arc or strobed laser illumination. We report our initial findings with cells of Escherichia coli. Tumbles (events that enable swimming cells to alter course) are remarkably varied. Not every filament on a cell needs to change its direction of rotation: different filaments can change directions at different times, and a tumble can result from the change in direction of only one. Polymorphic transformations tend to occur in the sequence normal, semicoiled, curly 1, with changes in the direction of movement of the cell body correlated with transformations to the semicoiled form.

Escherichia coli↗
PubMed1989

Phialophora repens, an emerging agent of subcutaneous phaeohyphomycosis in humans.

A 63-year-old Japanese man had phaeohyphomycosis that occurred as a solitary subcutaneous nodule on the dorsal aspect of his left hand. In the nodule there were foci of mixed granulomatous and suppurative infiltrations circumscribed by thick fibrous tissue reaction. The foci contained short septate hyphae and occasionally small rounded aggregates of irregularly branched septate hyphae, both of which were nonpigmented or rarely weakly pale brown. Fungal culture from the nodule was positive for a dematiaceous mold. The mycologic features of the mold were typical of Phialophora repens. The infection was successfully treated by excision of the nodule. This is the second reported case of infection due to P. repens.

Dermatomycoses↗
PubMed1995

Effects of select medium supplements on in vitro development of Cryptosporidium parvum in HCT-8 cells.

Surface-sterilized oocysts of Cryptosporidium parvum were applied to subconfluent monolayers of human adenocarcinoma (HCT-8) cells grown on coverslips in six-well cluster plates. Parasite-infected cultures were then incubated in RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, and antibiotics at 37 degrees C in a 5% CO2-95% air incubator for 2 h to allow sporozoites to excyst and enter cells. After cultures were washed free of debris, fresh cell culture media containing select supplements were added and cultures were reincubated. Parasite growth was assessed 66 h later by counting the number of parasite developmental stages in 25 random x 100 oil fields by Nomarski interference-contrast microscopy. Four vitamin supplements, calcium pantothenate, L-ascorbic acid, folic acid, and 4-(para)-aminobenzoic acid, each resulted in a significant increase in parasite numbers in vitro. The addition of insulin and the sugars glucose, galactose, and maltose also had a positive effect on parasite growth, although the effect was less pronounced than with any of the vitamins. Using the above information, we developed a supplemental medium formulation consisting of RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES, 50 mM glucose, and 35 micrograms of ascorbic acid, 1.0 micrograms of folic acid, 4.0 micrograms of 4-aminobenzoic acid, 2.0 micrograms of calcium pantothenate, 0.1 U of insulin, 100 U of penicillin G, 100 micrograms of streptomycin, and 0.25 microgram of amphotericin B (Fungizone) per ml (pH 7.4). The growth of c. parvum in this medium was found to be enhanced approximately 10-fold compared with that in control medium without additional glucose, insulin, or vitamins.

Animals↗
PubMed1997

Culture of the causative organism of donovanosis (Calymmatobacterium granulomatis) in HEp-2 cells.

We report successful culture of Calymmatobacterium granulomatis by standard cell culture methods. Swabs were obtained from lesions in three patients with a clinical diagnosis of donovanosis. For two patients, there was histological confirmation of the disease (i.e., the presence of Donovan bodies in Giemsa-stained smears). Specimens were inoculated onto cycloheximide-treated HEp-2 cell monolayers in RPMI 1640 medium (supplemented with fetal calf serum, NaHCO3, vancomycin hydrochloride, and benzylpenicillin). At 48 h, organisms resembling Donovan bodies were identified in monolayer cultures from all three specimens. The organisms appeared as pleomorphic bacilli with characteristic bipolar staining and "safety pin" appearance. Using a PCR designed to differentiate C. granulomatis from the Klebsiella species (which have a high degree of molecular homology), we were able to demonstrate that the cultured organisms produced a PCR product identical to that obtained from the original swab specimens. It is now possible to test in vitro susceptibility of C. granulomatis to antibiotics and to provide a ready source of DNA and antigenic material to enable the development of serological tests and, possibly in the future, a vaccine.

Cycloheximide↗