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PubMed1983

Procedure for isolation of neuron- and astrocyte-enriched fractions from chick brain of different ages.

A method for the isolation of neurons and astrocytes from chick brain of different ages is described. In brief, the procedure involves incubation of the gray matter with trypsin, passage of the tissue successively through nylon/stainless steel screens of decreasing pore size, and further separation and purification of cell-enriched fractions by centrifugation on Ficoll discontinuous gradient. Phase-contrast microscopy revealed intact morphology of the cells. The cells were viable by trypan blue exclusion test.

Age Factors↗
PubMed1987

Hypothalamo-neurohypophysial neurons in vitro: developmental potentials depend on the donor rat stock.

Primary dispersed cell cultures were established from fetal diencephalic tissue derived from two stocks of outbred rats. The overall growth pattern, as followed by phase-contrast microscopy and visualized by immunocytochemistry for neurofilament proteins and neuron specific enolase, depended on the age of the donor animal. Stock differences were not revealed by these methods. In contrast, when the cultures were immunostained in order to assess the yield of a specific type of cell, i.e., magnocellular hypothalamic neurons, striking differences were observed between the two stocks of rats. Whereas neurophysin-immunoreactive cells could be grown in cultures established from fetal Chbb: Thom rats at embryonic days 14, 15, and 18, no such cells could be detected in cultures derived from Crl: CD (SD) BR rats older than embryonic day 14. These findings may reflect differences in the differentiation schedule and/or requirements for certain epigenetic and trophic factors between magnocellular neuroblasts derived from the two stocks of rats.

Animals↗
PubMed1990

Toxicity of 6-hydroxydopamine and dopamine for dopaminergic neurons in culture.

Toxicity of 6-hydroxydopamine (6-OHDA) and dopamine were studied in cultures of dissociated fetal rat mesencephalic cells. To assess survival and function of dopaminergic cells we quantified the number of tyrosine hydroxylase-positive cells and measured dopamine uptake. Non-dopaminergic cells were monitored by counting the number of cells visible with phase-contrast microscopy and measuring GABA uptake. 6-OHDA, in contrast to MPP+, which selectively destroyed dopaminergic neurons, was found to be a non-selective neurotoxin in this culture system. Between 10 and 100 microM, dopaminergic and non-dopaminergic cells were destroyed. At concentrations higher than 100 microM, i.e., concentrations frequently used to lesion catecholaminergic neurons in vivo, 6-OHDA resulted in structural fixation and loss of viability of dopaminergic and non-dopaminergic cells. Dopamine produced the same actions at slightly higher concentrations. One hundred to 300 microM was toxic for all cell types, and concentrations above 300 microM resulted in fixation. The findings suggest that 6-OHDA cannot be considered a selective toxin for catecholaminergic neurons in vitro. The demonstrated toxicity of dopamine tends to support speculations that processes related to dopamine metabolism may play a role in the pathogenesis of Parkinson's disease.

1-Methyl-4-phenylpyridinium↗
PubMed1990

Phagocytosis of peripheral nerve myelin in vitro: effect of antibody.

We have previously shown that antisera to whole CNS myelin, whole PNS myelin, galactocerebroside (GC), and myelin basic protein (MBP) promote the uptake of CNS myelin by cultured macrophages, and stimulate the conversion of myelin lipids to cholesterol ester and triglycerides. Here we report the results of similar studies using PNS myelin purified from the rat sciatic nerve. Antisera to whole CNS myelin, whole PNS myelin, GC, and MBP preincubated with 14C-labeled PNS myelin increased the production of radioactive cholesterol ester by macrophages in culture to a level about twice that with preimmune serum, and five to six times that of untreated myelin. The amounts of [14C]triglyceride were similarly increased with these antisera, whole P0 and P2 antisera had little or no effect. IgG prepared from the antisera stimulated lipid metabolism to almost the same extent, while heating the antisera did not decrease the stimulatory effect, indicating that myelin was opsonized by IgG, but not likely by complement. With a few exceptions, the four active sera and their IgGs promoted the macrophage metabolism of CNS and PNS myelin almost equally. The cultured macrophages converted about 3% of untreated CNS myelin and about 6% PNS myelin cholesterol to cholesterol ester. Under phase contrast microscopy it was noted that vesicles of CNS myelin appeared to bind individually to macrophages, whereas PNS myelin vesicles tended to self-associate to form large clumps which were found to macrophages. Binding studies showed PNS myelin to be bound more firmly to macrophages than CNS myelin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Hyperphosphorylation of Tau and filopodial retraction following microinjection of protein kinase C catalytic subunits.

Limited proteolysis of protein kinase C (PKC) by calcium-activated proteolysis cleaves the regulatory and catalytic subunits of PKC, generating a free, constitutively activated kinase ("PKM") that, unlike the intact parent enzyme, is not calcium-dependent, and is not restricted to the plasma membrane. These latter properties leave open the possibility that PKM may have access to, and may therefore phosphorylate, substrates normally unavailable to intact PKC. We examined the potential involvement of such aberrant phosphorylation in certain aspects of the neurodegeneration accompanying Alzheimer's disease by microinjecting PKC and PKM, along with a rhodamine-conjugated dextran tracer, into undifferentiated NB2a/d1 mouse neuroblastoma cells. After 4 hr, cultures were fixed and processed for immunofluorescence with monoclonal antibodies (PHF-1, ALZ-50, Tau-1, AT8) directed against tau in various phosphorylation states followed by fluorescein-conjugated secondary antibodies. Microinjected cells were localized via co-injected rhodamine-conjugated dextran tracer under rhodamine illumination, after which antibody immunoreactivity was examined under fluorescein illumination. Microdensitometric analyses indicated that microinjection of PKC did not increase basal immunofluorescent intensities of the antibodies; by contrast, microinjection of PKM induced three- and twofold increases in PHF-1 and ALZ-50 levels, respectively. By contrast, no significant alteration was observed in AT8 and Tau-1 immunofluorescence following either PKC or PKM microinjection. Whereas undifferentiated NB2a/d1 cells typically elaborate short, filopodia-like neurites, phase-contrast microscopy revealed the absence of filopodia or neurites on PKM-injected cells, while a similar percentage of PKC-injected cells. Cell-free analyses confirmed the ability of PKC, in the presence of necessary co-factors, and PKM to increase PHF-1 and ALZ-50 immunoreactivity; no change was observed in AT8 or Tau-1 immunoreactivity. These findings underscore the possibility that an abnormal amplification in limited PKC proteolysis to generate PKM could, under certain pathological conditions, contribute to neuronal degeneration.

Animals↗
PubMed1976

[Microscopic studies of Streptomyces hygroscopicus growth kinetics].

Growth kinetics, branch formation, and cytological properties of mycelial growth of Streptomyces hygroscopicus on solid media were investigated by phase-contrast microscopy using a microculture method. Measurements were made on taken photographs of the growing hyphae from the beginning of spore germination up to maximal 18 hours. The specific growth rate of the germ tube was much higher than the specific growth rate of the mycelium. The doubling time of the total length of the mycelial hyphae and the doubling time of branch formation was quite the same for the period investigated. After a short time of outgrowth each individual hypha grows at a constant rate, i.e. the length increases linearly, but the growth kinetics of the whole mycelium becomes exponentially by branching. It seems, that nucleoids only divide in that part of a hypha between the tip and the nearest branch. A cell unit (1.4-1.9 mum on different media) could be calculated by the length of a hypha and the number of nucleoids. A hypha is growing with the cell unit at the tip or with the polar cap only. No interkalary growth in length could be found. Branches were formed only up to about 100 mum from the tip on complex medium and up to about 50 mum from the tip on mineral salt medium. A simple method of mycelial growth has been developed. Some properties showing the connection between Streptomycetes and prokaryotic organisms on one hand and hyphal growing fungi on the other hand are discussed.

Culture Media↗
PubMed1978

Kinetics of vegetative growth of Thermoactinomyces vulgaris.

The growth of Thermoactinomyces vulgaris on solid media was studied by microculture technique. Starting from a germinating spore the further development was followed by phase contrast microscopy. From serially taken photographs the kinetics of growth and branching of the mycelium was calculated. Also some observations on morphological behaviour were made. Regarding the total mycelium developing from one spore the growth occurred witha constant exponential growth rate, at least for the time tested (about ten doublings). The constancy of the mycelial growth rate was not attributed to constant rates of hyphal growth or branching. The hyphal growth rate increased during the development of the mycelium, so that the length produced by a hypha within a given time was smallest after germination and largest at the end of the experiment. Between the first and the 10th doubling of the mycelium the mean hyphal growth rate increased by the factor 27, in some cultures even by the factor 40. While hyphal growth changed from exponential to linear, branching was first linear and then approached exponential kinetics. Under different conditions of cultivation mycelia could have identical doubling times, but different hyphal growth rates and vice versa. Yeast extract caused a reduction of the hyphal growth rate and an enhancement of branching. Likewise the diminution of the supply of oxygen did not reduce the mycelial growth rate, but markedly decreased the hyphal growth rate, especially of the germ tube. The data obtained concerning the kinetics of growth and branching can be explained by a special form of cooperation of cytoplasmic and cell wall synthesis. With respect to the synthesis of cytoplasma a T. vulgaris mycelium behaves as one individual, which is not differentiated into replicating and non replicating areas (as recently was shown to be likely for Streptomyces hygroscopicus by Schuhmann and Bergter 1976). The mycelial growth rate thus reflects the rate of the synthesis of cytoplasm. The hyphal growth rate is moreover determined by the rate of cell wall synthesis at the tip of each hypha. The observed increase of the hyphal growth rate is explainable by an acceleration of the cell wall synthesis during the development of the mycelium. Probably the branching is regulated indirectly. According to the different manner of cytoplasmic and cell wall synthesis, within a hypha a balance between both processes is maintainable only for a limited time. As soon as the momentary apical capacity for cell wall synthesis is exhausted, the balance is attained anew by originating a new site for wall synthesis, i.e. by branching. Branching was often defective. Especially in the absence of yeast extract a lot of lateral branches arose, which ceased further growth at an average length of 2.5 micron. It is suggested that yeast extract supports the transport of the nucleoids into the lateral branches, which otherwise is often infeasible. Reduction of the supply of oxygen favoured apical branching, so that dichotomous forms resulted.

Culture Media↗
PubMed1983

[Protoplast liberation and regeneration in the ascomycete Hypomyces ochraceus (Pers.) Tul].

A rapid and convenient method for producing protoplasts from 3 d old mycelium of the ascomycete Hypomyces ochraceus is described. The procedure involves a Helix pomatia enzyme preparation and sucrose (20%) for stabilization. Pretreatment with disulfide bond reducing agents reduced the amount of viable protoplasts. Formation of protoplasts and different stages of regeneration were observed by phase contrast microscopy. There was only one type of true regeneration from protoplasts to hyphae in 15-30% gelatine medium by direct forming a germ tube from the original protoplast. Cytological events and physiological conditions are discussed.

Animals↗
PubMed1994

Cyclic stretching of human osteoblasts affects proliferation and metabolism: a new experimental method and its application.

We developed an experimental system to stimulate cell cultures by uniform and cyclic biaxial strain of the cell culture surface. The studies reported here were designed to determine the uniformity of the strain distribution, the suitability of the surface for the growth of human osteoblasts, and the effects of strain magnitude on cell proliferation and alkaline phosphatase (AP) activity. Subconfluent cell cultures were grown in rectangular silicone dishes that were stretched cyclically (1 Hz) in the long axis by an electromechanical apparatus that controlled peak stretch and cycle frequency. We applied cyclic strains (1.0, 2.4, 5.3, and 8.8% surface strains) for 15 minutes per day on 3 consecutive days. Phase contrast microscopy confirmed the transfer of dish surface strain to the cells. Stretching of the dish resulted in a homogeneous strain distribution that deviated approximately 0.05% from the applied strain. In comparison with plastic dishes, there was a 20% reduction of cell proliferation on the silicone substrate whereas morphology, AP activity, and total protein content of the cells were similar. The proliferation of human osteoblasts was increased significantly (16.4-100%) by 1% strains, although higher strain magnitudes had lesser (nonsignificant) effects or decreased the mitotic activity of the cells. AP and lactate dehydrogenase activities were not influenced significantly by cyclic strains. This study demonstrates that the cell stretching system is suitable for the investigation of the effects of well defined cyclic strains.

Cell Division↗
PubMed1998

Differentiated cellular function in fetal chondrocytes cultured with insulin-like growth factor-I and transforming growth factor-beta.

This study examined fetal chondrocyte proliferation and function following exposure to transforming growth factor-beta and insulin-like growth factor-I. Fetal equine articular chondrocytes of the early third-trimester were isolated and cultured in monolayer conditions, then exposed to 0, 1, 5, or 10 ng/ml transforming growth factor-beta or 0, 10, 50, or 100 ng/ml insulin-like growth factor-I for 48 hours. Proliferative responses were assessed by cell counts and [3H]thymidine uptake into precipitable DNA. Differentiated chondrocyte metabolic activity was determined by sulfated glycosaminoglycan quantitation, 35[SO4] incorporation into precipitable glycosaminoglycan, and proteoglycan molecular sizing by CL-2B column chromatography. Morphological changes seen on phase-contrast microscopy included a larger proportion of rounded cells in monolayer cultures supplemented with insulin-like growth factor-I and cytotoxic changes in cells treated with transforming growth factor-beta. Both insulin-like growth factor-I and transforming growth factor-beta resulted in significant elevations of [3H]thymidine uptake; however, cell numbers did not rise sufficiently over the 48-hour culture period to reach significant levels. Maximum mitogenic responses were evident at 50 and 100 ng/ml insulin-like growth factor-I and 5 ng/ml transforming growth factor-beta. The production of proteoglycan was also enhanced (435%) by exposure to 50 ng/ml insulin-like growth factor-I, and an increased proportion of larger proteoglycan monomer species was evident in cultures treated with 50 and 100 ng/ml insulin-like growth factor-I. A similar dose-response was also evident in cultures treated with transforming growth factor-beta (maximal 164% increase with 5 ng/ml), although the presence of serum in the culture medium altered the pattern of enhanced proteoglycan synthesis to favor the lower concentration of 1 ng/ml (191%). Additionally, larger proteoglycan molecules were synthesized in response to high concentrations of transforming growth factor-beta in serum-free cultures. Significant biochemical changes resulted from the addition of transforming growth factor-beta to fetal chondrocyte cultures; however, monolayer cultures that were treated with transforming growth factor-beta and supplemented with serum began to develop cellular toxicity, including nuclear pyknosis and cytoplasmic fragmentation. Degenerative cellular changes were not evident in cultures treated with insulin-like growth factor-I, and significant differentiated metabolic activity resulted from the presence of insulin-like growth factor-I in the culture medium. These data suggest that the responses of fetal chondrocytes to insulin-like growth factor-I and transforming growth factor-beta were enhanced compared with the responses of chondrocytes derived from postnatal animals and that these metabolically active cells can be primed by endogenous or exogenous growth factors to provide enhanced articular function and repair.

Animals↗
PubMed2002

Photothermal time-resolved imaging of living cells.

BACKGROUND AND OBJECTIVES: Thermal effects of laser radiation at cell level play very important role in cell functioning and in many laser applications. The aim of this study was to evaluate a new method of photothermal imaging (PTI) for monitoring short-time nano-scale thermal effects in individual living cells. STUDY DESIGN/MATERIALS AND METHODS: PTI is based on the irradiation of a cell with a short laser pump pulse (8 nanoseconds, 532 nm) and on registration of the laser-induced local thermal effects using time-resolved phase-contrast imaging with a pulsed probe laser. RESULTS: PT images of lymphocytes, lympholeukemia cells in vitro were obtained at different laser energies. PTI in time-resolved mode allowed visualizing the structures with size less than diffraction limit (90-nm liposomes). The photodamage process was visualized for a single human leukocyte in suspension. CONCLUSIONS: PTI in non-invasive mode offered better contrast of living cell image than conventional optical phase-contrast microscopy. The data obtained showed that PTI is in perspective for studies of live non-fluorescent cells.

Burns↗
PubMed1987

Thermal injury by laser pulses: protection by heat shock despite failure to induce heat-shock response.

Pulsed lasers produce a variety of therapeutically useful effects in tissues by thermal mechanisms. In contrast with conventional hyperthermia, pulsed lasers rapidly induce extremely high temperatures confined to small areas within the tissues. It is not established, however, how cellular damage and repair responses differ between conventional and pulsed laser-induced hyperthermia. We have addressed this question by examining the induction of cellular heat-shock proteins (HSPs) and protection by heat-shock response against pulsed laser vs conventional hyperthermia. Cultured human dermal fibroblasts at confluency were exposed to single microsecond, 10.6-microns-wavelength pulses from a pulsed CO2 laser at radiant exposures between 0 and 1.3 J/cm2. Cell monolayers were directly exposed after removal of overlying medium. Positive control for the ability of the cells to synthesize of HSPs was obtained by heating the cells for 20 min in a water bath at 45 degrees C or 47 degrees C. The cultures were then labeled with L-[35S] methionine (Met), and proteins analyzed by sodium dodecyl sulfate polyacrylamide and two-dimensional gel electrophoresis 2 hr after exposure. The morphological alterations induced by laser or heat were similar under phase contrast microscopy. Conventional heating induced a heat-shock response, with the appearance of HSPs 2 hours after heating. In contrast, synthesis of HSPs could not be detected following laser irradiation, even at exposures decreasing both cell viability and incorporation of [35S] Met into cellular protein. Pre-exposure of cells to temperatures which induced synthesis of HSPs, however, significantly decreased cell death induced by subsequent laser irradiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗
PubMed1991

Preparation of spermatogenic cell populations at specific stages of differentiation in the human.

Studying biochemical events in human spermatogenesis requires separated populations of spermatogenic cells. Dissociation of these cells was performed by a Trypsin-DNAse method adapted from the technique used for rodents. Cell separation was performed by centrifugal elutriation. Seven populations were collected, one further purified by Percoll gradient centrifugation, giving nine different cell populations. The efficiency of the cell separation was evaluated by phase contrast microscopy, flow cytometric DNA analysis, and electron microscopy. Five populations were enriched in spermatids: two in round spermatids (87% and 73%), another in round (52%) and elongating (44%) spermatids, another constituted by 80% elongating spermatids, and the last by 90% elongated spermatids. Two of the four remaining populations were enriched in primary spermatocytes (74% and 54%); another population was the upper part of the Percoll gradient and constituted cytoplasmic lobes and residual bodies (89%); the last population was made up of various cells, with no specific enrichment. Electron microscopic observations revealed good preservation of the separated cells; only the flagella from elongated spermatids were lost. Furthermore, an unusual pattern of nucleoplasm distribution during stages 2-4 of spermatid differentiation was observed and its signification is discussed with regard to the shape of the human spermatozoon.

Aged↗
PubMed1987

Morphology and fertilizability of frozen human oocytes.

Human oocytes were frozen and thawed by four methods previously used for cryopreservation of human embryos. Most of these oocytes were inseminated after thawing to assess their capacity to fertilize and form pronuclear ova. Their morphology was assessed by phase-contrast microscopy used in routine IVF. Twenty-three oocytes were examined by electron microscopy to critically evaluate the effects of cooling and cryopreservation and to confirm fertilization. Morphological survival was observed in more than 60% of the oocytes examined after freeze-thawing. The main features of cryoinjury were cracks in the zona pellucida, disruption of the plasma membrane and extensive disorganization of the ooplasm. Subtle changes in the cytosol of cumulus cells was also observed. Cooling to 0 degrees C or -6 degrees C had little effect on cytoplasmic structure. Spindles were damaged in two frozen oocytes. Cumulus cell activity, sperm binding to the zona, sperm penetration of the zona seem to be largely unaffected by freeze-thawing. Fertilization was observed in eight oocytes after postthaw insemination and three embryos (8-cell to morula stages) were developed from pronuclear ova on further culture. Both monospermic and polyspermic fertilization were confirmed by electron microscopy and micronuclei were detected in three pronuclear ova. The genetic implications of these nuclear aberrations are discussed. These preliminary studies indicate that oocyte freezing needs to be integrated cautiously with clinical IVF by further assessment of embryos developed from frozen oocytes.

Female↗
PubMed1991

Recurrent respiratory infections in a child with fucosidosis: is the mucus too thin for effective transport?

Fucosidosis is caused by a deficiency of the lysosomal enzyme alpha-L-fucosidase (ALF) leading to an accumulation of glycoproteins in a variety of cells. Infants and young children with this disorder are prone to recurrent sinus and pulmonary infections and often die of pneumonia. We studied the mucociliary and systemic immune function in a 6 year old girl with fucosidosis and recurrent respiratory infections. All measurements of systemic immune function were normal. Sweat chloride was normal when measured on angiokeratotic skin but was greater than 65 mg/L on uninvolved areas. During the placement of tympanic ventilation tubes, tracheal mucus was gently aspirated and a mucosal biopsy was taken. Tracheal mucus transport was not measured. The biopsy material was examined under phase contrast microscopy and revealed ciliated cells with apparently normal beating. TEM of these cells showed a characteristic pattern of vacuoles in the cytoplasm as described in other tissues from patients with fucosidosis. Ciliary ultrastructure was normal. Mucus viscoelasticity was measured in a magnetic microrheometer. The loss tangent was 2 SD above the mean for normal mucus and mechanical impedance was about 2 SD below the mean. These changes are similar in direction but double in magnitude to what has been described with methacholine administration in dogs. The high compliance of the mucus may be due to incomplete assembly of mucus glycoprotein or to decreased secretion of glycoproteins in respiratory secretions. This leads to mucus that is abnormally watery and thus difficult to clear from the airway.

Child↗
PubMed1981

Cell separation and characterization of epithelial cells from human benign prostatic hyperplasia.

Epithelial cells were isolated from human prostatic hyperplasia (BPH) after mechanical disruption of the tissue. The tissue was cut into small pieces, then mechanical pressure was applied. Stroma and epithelium were separated by a sequence of sedimentation steps. The recovery rate of epithelial cells was around 20 million cells per gram of tissue and more than 95% of the cells did exclude trypan blue. Epithelial cells can be identified by phase contrast microscopy and by the acid phosphatase content of the cells. In more than 95% of the cells the presence of acid phosphatase could be shown cytochemically with phosphorylcholine as a substrate. This finding indicates that the contamination with other cell types is very small. Histological study of the remaining stroma indicates that most of the epithelial cells are removed. Also, the acid phosphatase content of this fraction was found to be very low. The problem of obtaining large quantities of stromal cells in suspension has not yet been resolved. However, the technique described may be more suitable than others for the separate study of stroma and epithelium from BPH.

Acid Phosphatase↗
PubMed1983

Primary explant culture: an in vitro model of the human prostate.

Human prostate tissue gave rise to essentially pure cultures of basal cells in vitro. Careful analysis by phase-contrast microscopy and photography suggested that these cells attempt to differentiate into secretory acinar cells. This was confirmed by immunocytochemical analysis for prekeratin which is present only in basal cells in situ and for prostatic acid phosphatase, prostate-specific antigen, and prostate fluid antigen, which are present only in secretory acinar cells in situ.

Acid Phosphatase↗
PubMed1996

Thiophosphate induces apoptosis in human leukemia cell lines.

Thiophosphate induced apoptosis in human HL-60 cells. HL-60 cell proliferation was inhibited with an IC50 of about 60 microM. Typical morphological changes of apoptosis were observed by phase contrast microscopy and DNA laddering was observed after agarose gel electrophoresis. Thiophosphate-induced DNA fragmentation was time and concentration-dependent. After exposure to thiophosphate (100 microM) apoptosis occurred as early as 4 h after treatment and 90% of cells were apoptotic by 24 h. dbcAMP-differentiated HL-60 cells as well as undifferentiated HL-60 cells were susceptible. Thiophosphate was also effective in inducing apoptosis in other leukemia cell lines including CEM and K562 and a lymphoma cell line, Raji.

Apoptosis↗