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PubMed1979

The rostral level of origin of sympathetic neurons in the chick embryo, studied in tissue culture.

Groups of three consecutive somites from the first to the eleventh somite from chick embryos of stages 17-18 were grown in tissue culture for seven days. Sympathetic neurons, identified both by phase contrast microscopy and FIF histochemistry, occurred only in cultures which included the sixth, or more caudal, somites. If it is assumed that sympathetic precursor cells (neural crest cells) have not undergone a caudal shift prior to stages 17-18, and taking into account the loss of one or two rostral somites, then the anterior sympathetic ganglia are derived from neural crest caudal to the sixth or seventh somite. Thus, the vagal zone (level with somites 1-7) contributes little to the sympathetic nervous system.

Animals↗

PubMedDate not supplied

Immunogold-labeled microtubule crossbridges in platinum-carbon replicas of the marginal band of erythrocyte cytoskeletons.

Cytoskeletons of erythrocytes from the toad Bufo marinus are composed of a surface-associated cytoskeleton that encapsulates the annular bundle of microtubules known as the marginal band (MB) and the centrally located nucleus. As seen by phase-contrast microscopy, the microtubules (MTs) of the MB remain tightly bundled after cell lysis without the need for added stabilizing factors. The integrity of this structure suggested that in addition to MTs other components were present in the MB and were responsible for its stability. Thin (less than 18 nm) platinum-carbon (Pt-C) replicas of freeze-dried cytoskeletons prepared by using a modified Balzers 300 system provided a novel method of sample preparation for a high-resolution study of the ultrastructure of the MB. Electron micrographs of replicas revealed that, the MTs of the MB displayed numerous filamentous projections which, when viewed in stereo, appear as side-arm connections between adjacent MTs. Immunofluorescence data show that monospecific antibodies to tubulin and to MT-associated protein 2 (MAP2) from brain each detect cross-reactive material in the MB. The combination of immunogold cytochemistry with Pt-C replication provided the increased resolution required to identify the individual structures recognized by antibodies to tubulin and MAP2. As expected, antitubulin labeled the MTs of the MB. However, anti-MAP2 antibodies were localized specifically to the cross-bridging filaments between adjacent MTs. Thus, a MAP2-like protein was identified in situ as the crossbridging filament that bundles MTs to form a stable MB.

Animals↗

PubMed1978

Acute lymphoblastic leukemia--hand mirror cell variant: a detailed cytological and ultrastructural study with an analysis of the immunologic surface markers.

Acute lymphoblastic leukemia was observed in a 22-year-old white female patient and was manifested by normal palelet counts, 50--60% hand mirror cells (HMC) in the bone marrow, and prolonged survival without treatment. The characteristic neoplastic cell had a nucleus within the "mirror" portion and a cytoplasmic uropod forming the "handle" portion. The presence of acid phosphatase and beta-glucuronidase activity suggested that the cell was a T cell lymphoblast. However, extensive immunologic surface marker studies indicated the cells were non-T, non-B. Terminal transferase activity further supported the lymphoid nature of the cell. The hand mirrow cells were considered a real phenomenon since they were demonstrated on phase contrast microscopy, scanning electron microscopy, and transmission electron microscopy. The cells did not grow in tissue culture and cytogenetics revealed a normal female karyotype. From the above observations, the HMC is a lymphoblast with morphological, cytochemical, and immunological features which may differentiate it from the usual cell in acute lymphoblastic leukemia. Therefore, cases involving increased numbers of hand mirror cells in the bone marrow and acute lymphoblastic leukemia require further investigation to elucidate the full importance of this cell. This study represents the first attempt to investigate this cell in detail.

Binding Sites↗

PubMed1980

Detection of platelet associated IgG in immune thrombocytopenia: a new assay employing protein A and peroxidase anti-peroxidase (PROA-PAP).

Immune thrombocytopenia is frequently encountered in medical practice and is generally accepted as being caused by an IgG antibody. The capability of detecting platelet-bound IgG as a diagnostic and therapeutic modality is critical for appropriate care and management of patients with idiopathic thrombocytopenic purpura (ITP), as well as other immune thrombocytopenias. We have modified our previous assay (Br J Haematol 37:265, 1977) by employing protein A and PAP as a labeled antibody. Surface bound platelet IgG was quantitated by phase contrast microscopy after incubation with PAP, graded per 100 platelets and expressed as a reactive index (RI). Controls (n=13) had RIs ranging from 0.49 to 0.72 (mean 0.63 +/- 0.02 SE). The nonimmune thrombocytopenic group (n=7) had an RI ranging from 0.58 to 0.72 (mean 0.64 +/- 0.01 SE). In contrast, the immune thrombocytopenic group (n=28) had RIs ranging from 1.04 to 1.75 (mean 1.43 +/- ;0.03 SE). Platelet-associated IgG was evaluated further by absorbing representative sera samples from each group against washed granulocytes, red cells and platelets. Only when sera from the immune thrombocytopenic group were absorbed against platelets did the reactive indices of pre- and postabsorption samples change significantly. These findings suggest that our assay is clinically applicable in detecting platelet-associated IgG in immune thrombocytopenia and has the advantage of being rapid, reproducible and easy to perform in a clinical laboratory.

Adolescent↗

PubMed1986

Asbestos dust exposure during brake repair.

About 10,000 tons of chrysotile per year are used in the Federal Republic of Germany for the production of friction materials. During brake repair an unknown number of approximately 300,000 mechanics in automobile service stations are exposed to asbestos dust. In a field study, asbestos fiber concentrations during brake repair were measured. Occupational histories and chest X-rays of brake service mechanics are being examined. Ninety dust measurements in 76 service stations were made by phase contrast microscopy and by scanning transmission electron microscopy. By electron microscopy, extremely fine chrysotile fibers with lengths less than 5 microns were identified in brake drum dust. Fibers with lengths greater than or equal to 5 microns constituted less than 1% of all chrysotile fibers counted in brake drum dust. Short-term asbestos dust exposures were measured by light microscopy in 101 personal samples during blowing out of brakes, and grinding and turning of brake linings. During blowing out of car brakes, as well as during grinding of brake linings, the product of fiber concentration with length greater than 5 microns and sampling time amounted to about 4-5 fibers/ml X min corresponding to a concentration of 10(6) fibers/m3 over 4-5 min. For trucks and buses higher amounts of 5-10 X 10(6) fibers/m3 X min were observed during these operations. From occupational histories of 210 vehicle mechanics, an average duration of employment of mean +/- s = 21 +/- 10 years and a mean cumulative fiber dose of mean +/- s = (0.54 +/- 1.1) X 10(6) fibers/m3 X years were calculated.

Air Pollutants, Occupational↗

PubMed1995

Occupational exposures to fibers and quartz at 19 crushed stone mining and milling operations.

From 1979 to 1982, the National Institute for Occupational Safety and Health (NIOSH) conducted a cross-sectional exposure assessment and mortality study of selected crushed stone facilities in the United States. This study was undertaken in part to address concerns that asbestos exposures could be occurring in some crushed stone operations due to the presence of amphibole and serpentine minerals. The investigation was also designed to characterize exposures to crystalline silica and other mineral compounds. Nineteen crushed stone operations, mining limestone, granite, or traprock were surveyed to assess exposures to respirable and total dusts, mineral compounds including crystalline silica, asbestos, and mineral fibers. At the initiation of the study, crushed stone operations were selected from a Mine Safety and Health Administration (MSHA) listing of the active industry in 1978. With the exception of requiring inclusion of the traprock operation in Maryland where asbestos fibers were initially discovered, a stratified sample of operations was randomly selected by rock type (granite, limestone, traprock, or sandstone). However, because of reluctance or refusal of some companies to participate and because of the closures of some of the selected operations, replacements were randomly selected. Some replacement selections were likewise replaced due to lack of cooperation from the companies. The studied sample included only 10 of the 27 randomly selected operations in the original sample. Asbestos fibers were detected at one traprock facility, the Maryland operation where asbestos was originally found. Measured personal exposures to fibers exceeded the NIOSH Recommended Exposure Limit (REL) for two out of 10 samples. All of the samples were below the MSHA Permissible Exposure Limit (PEL), which was in effect at the time of the survey. However, due to the presence of nonasbestos mineral fibers in the environment, it could not be stated with certainty that all of the fibers counted by phase contrast microscopy were asbestos. A variety of silicate mineral fibers (other than those classified by NIOSH as asbestos) were detected in the traprock operations and at one granite operation. Crystalline silica was detected at 17 of the 19 surveyed crushed stone operations. Overexposures to crystalline silica were measured at 16 of the crushed stone operations; approximately one in seven personal-respirable dust samples (14%) exceeded the MSHA PEL for crystalline silica. Approximately 25% of the respirable dust samples exceeded the NIOSH REL for crystalline silica. Mill operators and mill laborers consistently had the highest and most frequent overexposures to crystalline silica.

Asbestos↗

PubMed1991

Potentiation of cochlear hair cell loss by acoustic stimulus and gentamicin in the guinea pig.

We explored the possibility of synergism between a pure-tone stimulus and gentamicin in causing cochlear injury by analyzing hair cell loss. Guinea pigs receiving daily injections of gentamicin (200 mg/kg body wt.) for 1 week were exposed to a 2 kHz tone (95 dB SPL, 2 hours daily). Surface preparations of the spiral organ were studied by phase contrast microscopy, and the extent of hair cell loss in the entire organ of Corti was recorded in cytocochleograms. Gentamicin by itself was slightly ototoxic, damaging the innermost row of outer hair cells, whereas exposure to sound alone caused no hair cell loss. Combined antibiotic and acoustic exposure produced extensive cochlear damage. A few animals showed massive hair cell degeneration and collapse of the organ of Corti, except in the apical turn. The site of damage was possibly determined by the frequency of the sound stimulus. Thus, an intermittent tonal stimulus such as that used in the present experiment can be harmless by itself, but causes injury to cochlear hair cells in guinea pigs when administered in combination with gentamicin.

Acoustic Stimulation↗

PubMed1990

T-1, a mitotic arrester, alters centrosome configurations in fertilized sea urchin eggs.

T-1 induces modifications in the shape of the centrosome at division in fertilized eggs of the North American sea urchin, Lytechinus pictus. Phase contrast microscopy observations of mitotic apparatus isolated from T-1-treated (1.7-8.5 microM) eggs at first division shows that the centrosomes already begin to spread or to separate by prophase and that the mitotic spindle is barrel-shaped. When eggs are fertilized with sperm that have been preteated with T-1, the centrosomes become flattened; the spindles are of normal length. Immunofluorescence microscopy using an anti-centrosomal monoclonal antibody reveals that T-1 modifies the structure of the centrosome so that barrel-shaped spindles with broad centrosomes are observed at metaphase, rather than the expected focused poles and fusiform spindle. Higher concentrations of T-1 induce fragmentation of centrosomes, causing abnormal accumulation of microtubules in polar regions. These results indicate that T-1 directly alters centrosomal configuration from a compact structure to a flattened or a spread structure. T-1 can be classified as a new category of mitotic drugs that may prove valuable in dissecting the molecular nature of centrosomes.

Animals↗

PubMed1988

DNA flow cytometric analysis of mouse seminiferous epithelium.

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide. DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.

Animals↗

PubMed1991

Clinical prevalence of glomerular hematuria: a nine-year retrospective study.

The authors evaluated red blood cell (RBC) morphology by phase contrast microscopy and identified casts and lipidic material in 4,448 urine sediments (US). Microhematuria was discovered on systematic examination in 1,186 apparently healthy patients (group I). Another 4,362 patients (group II) with microscopic or gross hematuria were being treated or evaluated for various renal or extrarenal diseases (renal transplantation excepted). Glomerular hematuria (GH) was observed in 93.1% of group I patients and in 58.0% of group II patients. GH and non-GH were observed together in 0.6% of cases. The origin of hematuria remained uncertain in 11.6% of cases. In GH, the other cytological findings were as follows: RBC casts were seen in 5.3% of group I and 20.5% of group II; granular and/or cellular casts were seen in 12.8% of group I and 39.6% of group II; and lipidic material was seen in 0.6% of group I and 8.8% of group II. Both groups showed a higher frequency of RBC casts, granular and/or cellular casts, and lipidic material in patients under 20. This method of investigation of urine, which is simple, rapid, inexpensive, and noninvasive, provides useful information for the clinician and avoids unnecessary investigations.

Adolescent↗

PubMed1989

Human thymic dendritic cell-thymocyte association: ultrastructural cell phenotype analysis.

In rodent thymus, associations between dendritic cells (DC) and thymocytes have been suggested to be implicated in differentiation and/or maturation processes. In this study, we report intimate associations formed between human thymic DC and thymocytes in culture and we analyze their ultrastructural cell phenotype. Observations by phase contrast microscopy showed that DC present long and thin dendrites and bind many thymocytes. Transmission (TEM) and scanning electron microscopy (SEM) revealed that both cellular populations were in close connection and tight membrane contact could be observed. The phenotype of DC and attached thymocytes was characterized with a series of monoclonal antibodies by protein A-gold TEM and SEM immunolabelings. Quantitative evaluation of immunolabeling (number of gold granules/microns of cellular membrane) suggests the presence of two subpopulations of CD1+ thymic DC (strong and weak), whereas this discrepancy is not observed in DR+ and CD4+ DC populations. On the other hand, the majority of thymocytes bound to DC strongly express the CD1, CD4, CD8 and CD2 antigens and weakly the CD3 antigen, indicating that they represent double-positive immature thymocytes. Uniform distribution of DC and thymocytes membrane antigens was confirmed with a backscattered SEM study. This morphological and immunolabeling TEM and SEM analysis demonstrates that human thymic DC may form associations with CD4+CD8+CD3weak thymocytes and raises questions about their physiological relationship.

Antigens, CD1↗

PubMed1991

Quisqualate-induced excitotoxic death of glial cells: transient vulnerability of cultured astrocytes.

The cytotoxic effect of quisqualate, a potent excitatory amino acid (EAA), was examined in purified astrocyte cultures obtained from neonatal rat cerebral cortex and grown in a serum-free, defined medium (G5). Excitotoxicity was evaluated by phase contrast microscopy and quantified by the measurement of lactic dehydrogenase (LDH) activity released from the damaged cells into the culture medium. Quisqualate evoked not only osmotic swelling, but also cytotoxic cell death of cultured astrocytes. The vulnerability of cells was restricted to a period between day 4 and 8 in subculture, with a maximal sensitivity on day 7. The present findings indicate that not only neurons, but also glial cells may exhibit excitotoxic vulnerability. The vulnerability to quisqualate appears to be a transient phenomenon, at least in cultured astrocytes.

Animals↗

PubMedDate not supplied

Sinusoidal endothelial cells from guinea pig liver synthesize and secrete cellular fibronectin in vitro.

Endothelial liver cells were obtained from guinea pig by enzymatic digestion and centrifugal elutriation. Cells were cultured on gelatin and fibronectin pretreated culture vessels. Endothelial cells were characterized by phase-contrast microscopy, electron microscopy and the presence of Factor VIII-related antigen. Fibronectin secretion was determined in cell-free supernatants by a sensitive and specific ELISA and localized on fixed cultured cells by immunofluorescence. [35S]Methionine endogeneously labeled fibronectin was immunoprecipitated from supernatants and cellular lysates and displayed on sodium dodecyl sulfate polyacrylamide slab gel electrophoresis. After attachment to the culture vessel, one day after plating, endothelial cells start to produce fibronectin as measured by ELISA and demonstrated by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Secretion of fibronectin increases as cells proliferate to form a confluent monolayer. By immunofluorescence, fibronectin is visualized inside permeabilized cells and as a fibrillar network on the cell surface. Underneath the cell bodies, fibronectin-positive material is present as short strands. From supernatants and cellular lysates, fibronectin is immunoprecipitated with an apparent Mr of about 235,000 obviously larger than plasma fibronectin with an Mr of 220,000, which behaves electrophoretically like fibronectin isolated from early hepatocyte cultures. As endothelial cells incorporate [3H]fucose in fibronectin, whereas hepatocytes do not, we conclude that endothelial cells in contrast to hepatocytes produce cellular fibronectin. Endothelial cells, therefore, are probably the cellular source of the fibronectin present in the space of Disse. The significance of this finding with respect to fibrotic liver disease is discussed.

Animals↗

PubMed2006

3-Nitropropionic acid toxicity in hippocampus: protection through N-methyl-D-aspartate receptor antagonism.

The over-activation of glutamate receptors can lead to excitotoxic cell death and is believed to be involved in the progression of neurodegenerative events in the vulnerable hippocampus. Here, we used an in vitro slice model to study toxicity produced in the hippocampus by the mitochondrial toxin 3-nitropropionic acid (3-NP). The organotypic slice cultures exhibit native cellular organization as well as dense arborization of neuronal processes and synaptic contacts. The hippocampal slices were exposed to 3-NP for 2-20 days, causing calpain-mediated breakdown of the spectrin cytoskeleton, a loss of pre- and postsynaptic markers, and neuronal atrophy. The N-methyl-D-aspartate (NMDA) receptor antagonist memantine reduced both the cytoskeletal damage and synaptic decline in a dose-dependent manner. 3-NP-induced cytotoxicity, as determined by the release of lactate dehydrogenase, was also reduced by memantine with EC50 values from 1.7 to 2.3 microM. Propidium iodide fluorescence and phase contrast microscopy confirmed memantine neuroprotection against the chronic toxin exposure. In addition, the protected tissue exhibited normal neuronal morphology in the major hippocampal subfields. These results indicate that antagonists of NMDA-type glutamate receptors are protective during the toxic outcome associated with mitochondrial dysfunction. They also provide further evidence of memantine's therapeutic potential against neurodegenerative diseases.

Animals↗

PubMed1995

Dendritic antigen-presenting cells from the peripheral blood of renal-cell-carcinoma patients.

Dendritic cells are considered to be the initiators of immune responses, including those directed against tumors. Clinical research on dendritic cells was long hampered by the limited availability of these cells. The recent identification of cytokine combinations that mobilize dendritic cells with potent antigen-presenting cell function from peripheral blood represented a major progress. We show in this study that substantial numbers of dendritic cells can be obtained from the peripheral blood of patients with renal-cell carcinoma. The procedure requires a relatively small blood sample (40 ml) and avoids both priming of the patient with granulocyte-colony stimulating factor and leukapheresis. Approximately 2 to 8 million cells with the characteristics of dendritic cells could be obtained: phase-contrast microscopy revealed the typical cytoplasmic processes or veils; phenotypic analysis confirmed expression of dendritic-cell-associated molecules, including MHC class II, CD1a, CD4, ICAM-1 (CD54), LFA-3 (CD58), B7-1 (CD80) and B7-2 (CD86), and absence of T-cell, B-cell and monocyte markers; in addition, these cells rapidly attached to and migrated on collagen-type-1-coated surfaces. Interestingly, attachment was accompanied by acquisition of the CD14 antigen; functionally, cultured dendritic cells proved to be very potent co-stimulators of the phytohemagglutinin-induced proliferation of autologous tumor-infiltrating lymphocytes. The reproducible growth of functional dendritic cells from cancer patients is encouraging for the design of immunotherapy protocols.

Adult↗

PubMed1994

A mutant cysteinyl-tRNA synthetase affecting timing of chromosomal replication initiation in B. subtilis and conferring resistance to a protein kinase C inhibitor.

A Bacillus subtilis mutant spnA95 was isolated as resistant at 30 degrees C to the protein kinase C (PKC) inhibitor, sphinganine, and temperature sensitive for growth. As deduced by flow cytometry measurements, the mutant has a 35% reduced initiation mass at permissive temperature, resulting in initiation of DNA replication much earlier in the cell cycle than in the wild type. This modification is accompanied by a change in cell size, as determined by phase-contrast microscopy and flow cytometry. Therefore, this strain displays the characteristics of a novel cell clock mutant. spnA is a newly identified gene in B.subtilis and was shown to encode a cysteinyl-tRNA synthetase. At non-permissive temperature, the mutant was defective in the synthesis of P70, a protein with several characteristics of PKC (a cysteine-rich protein). As one possibility, we propose that the altered timing of replication may be due to the reduced synthesis of specific cysteine-rich proteins normally involved in controlling chromosomal replication initiation in B. subtilis.

Amino Acid Sequence↗

PubMedDate not supplied

Leydig cells do not have Fc receptors.

It has been reported that Leydig cells have Fc receptors, which traditionally have been considered markers specific for macrophages and polymorphonuclear leukocytes. The purpose of this investigation was to study further this phenomenon and also to determine if Leydig cells and macrophages could be separated from each other either by density gradient centrifugation using Percoll or by differential detachment with trypsin treatment of cultures of crude interstitial preparations. Interstitial cells were obtained by collagenase digestion of rat testis and established in culture. These cultures were reacted for 3 beta-steroid dehydrogenase and Fc receptor and viewed with phase contrast microscopy. No individual cells were positive for both steroid dehydrogenase activity and Fc receptors. The order in which the cells were stained for these two markers did not influence the results. Trypsin treatment of these crude interstitial cultures removed over 90% of the Leydig cells and approximately 20% of the macrophages. Macrophages were located in the same portion of Percoll gradients as the less dense (Population I) Leydig cells, while Leydig cells found in the dense area of the gradient (Population II) were not contaminated with macrophages. These studies indicate that Leydig cells do not have Fc receptors and that a subpopulation of Leydig cells can be isolated free of macrophages using density gradient centrifugation.

Animals↗

PubMed1993

Corneal endothelium cell adhesion on intraocular lenses in vitro.

An in vitro evaluation was conducted of the adhesion capabilities of rabbit corneal endothelial cells on intraocular lenses (IOLs) made of heparin-coated polymethylmethacrylate (HSM-PMMA). The concave endothelial surfaces of albino rabbit corneas were placed in contact in vitro with the convex surfaces of the optical side of HSM-PMMA IOLs in 4-day cultures. PMMA IOLs served as controls. After an incubation period, the preparations were examined via phase-contrast microscopy and via inverted microscopy after staining with vital dye (neutral red), both with and without the cornea in place. After fixation and staining with Giemsa, the cells adherent on the lens were counted on five different microscopic fields. It was observed that the corneal endothelial cells adhered equally to heparin-coated and untreated PMMA IOLs.

Animals↗