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PubMed2004

Acanthocytes in the urine: useful tool to differentiate diabetic nephropathy from glomerulonephritis?

OBJECTIVE: The presence of hematuria has been suggested to indicate nondiabetic nephropathy in diabetic patients with proteinuria. However, hematuria is frequently found in patients with biopsy-proven diabetic glomerulosclerosis without nondiabetic nephropathy. Urine microscopy allows discrimination of glomerular hematuria, which is defined as acanthocyturia (urinary excretion of acanthocytes, which are dysmorphic erythrocytes with vesicle-like protrusions), from nonglomerular hematuria. We hypothesized that acanthocyturia is an uncommon finding in diabetic nephropathy, which suggests the presence of a nondiabetic nephropathy in diabetic patients with proteinuria. RESEARCH DESIGN AND METHODS: Urine samples of patients with the clinical diagnosis of diabetic nephropathy (n = 68), of patients with biopsy-proven glomerulonephritis (n = 43), and of age-matched healthy control subjects (n = 20) were examined by phase-contrast microscopy for the presence of hematuria (>/=8 erythrocytes/ micro l) and acanthocyturia. Acanthocyturia of >/=5% (5 acanthocytes among 100 excreted erythrocytes) was classified as glomerular hematuria; acanthocyturia of 2-4% was classified as suspected glomerular hematuria. RESULTS: Hematuria was found in 62% of patients with the clinical diagnosis of diabetic nephropathy, in 84% of patients with glomerulonephritis, and in 20% of the healthy control subjects upon a single urine examination. In contrast, glomerular hematuria occurred in 4% of patients with diabetic nephropathy and in 40% of patients with glomerulonephritis (P < 0.001). CONCLUSIONS: In contrast to hematuria, acanthocyturia is uncommon in patients with the clinical diagnosis of diabetic nephropathy. In diabetic patients with proteinuria, the finding of acanthocyturia points to nondiabetic glomerulopathies, and renal biopsy should be considered.

Acanthocytes↗
PubMed2001

Primary ciliary dyskinesia: diagnosis in children with inconclusive ultrastructural evaluation.

The purpose of this study was to distinguish between acquired and genetically determined ciliary abnormalities in children with severe chronic respiratory diseases. Samples of nasal ciliated epithelium from 50 subjects (25 male, 25 female; age-range 2-19 years) with severe chronic respiratory diseases were examined using transmission electron microscopy (TEM). Based on TEM findings, patients were divided into two groups: A and B. Group A comprised 39 children with ciliary alterations compatible with a condition probably occurring secondary to chronic inflammation (alterations of peripheral pairs, swollen cilia, and compound cilia). The other 11 patients, Group B, exhibited a greater number of alterations of the central pair and dynein arms (p< 0.001), which were qualitatively similar to, but less numerous than, those observed in primary ciliary dyskinesia (PCD). In both groups, analysis of ciliary beat frequency and waveform was performed by phase contrast microscopy (PCM). All the children with a ciliary beat frequency of < 7 Hz were treated with daily physiotherapy and with antibiotics, as recommended for PCD, for a 6-month period. After this treatment, the children were reexamined by PCM. Almost 50% of the children from Group B (i.e. those with a small proportion of specific ultrastructural defects) showed permanence of low ciliary beat frequency. This was also observed in two children of Group A. These children were considered to be affected by PCD. Our study describes a method for the diagnosis of PCD in the absence of specific ultrastructural defects or when these defects are present in only a small proportion of the cilia.

Adolescent↗
PubMed2003

Organotypic slices in vitro: repeated, same-cell, high-resolution tracking of nuclear and cytoplasmic fluorescent signals in live, transfected cerebellar neurons by confocal microscopy.

The culture of organotypic slices for the purposes of tracking dynamic cellular events within the same live cell at high resolution, as a function of development in vitro has not been previously reported. The present study was undertaken to define the conditions most suitable for both the in vitro organotypic development of Purkinje neurons in cerebellar slices of neonatal mice, and the repeated visualization of nuclear signals within such cells. Slices of cerebella were maintained on 25 mm diameter, collagen-coated Anodisc membranes, placed in six-well plates and raised to the air-medium interface by use of glass fibre filter supports. This system permits cultures to be repeatedly observed both by phase contrast microscopy and, upon biolistic transfection, by laser confocal microscopy using 40x, 60x, and 100x water-immersion objectives, at high resolution. Upon co-transfection with two plasmids, differentiation of the same transfected Purkinje neurons was followed across in vitro development for periods of up to 10 days. Despite the relative thickness of the slice culture, even small, punctate, nuclear signals, were detectable. The results show that Purkinje neurons in cerebellar slices explanted from postnatal day 2 mice, developed cytotypically, although some were ectopically located. In contrast, Purkinje neurons in slices from postnatal day 6 cerebella developed in an organotypic manner. It is concluded that this culture system serves as an ideal tool for applications in experimental biology where high resolution tracking of cellular signals, over extended time periods, is of interest.

Animals↗
PubMed1991

Mapping trajectories of Pgp-1 membrane protein patches on surfaces of motile fibroblasts reveals a distinct boundary separating capping on the lamella and forward transport on the retracting tail.

Patches of aggregated membrane proteins on motile fibroblasts are transported from the surfaces of the leading lamella to a site just ahead of the nucleus in the phenomenon known as capping. A major cell surface glycoprotein, Pgp-1 (GP80), was tagged with a monoclonal IgG and then aggregated with fluorescent secondary antibodies. Correlated digitized fluorescence and phase-contrast microscopy were used to map the trajectories of fluorescent Pgp-1 patches located in various regions of the cell surface. The response of patches located in lamellar and nonlamellar regions to spontaneous retraction of the trailing cell margin, or tail was examined in detail. During capping, Pgp-1 patches accumulated at a morphologically distinct site on the cell surface, the 'null border', corresponding to the boundary between lamelloplasm and endoplasm and the posterior edge of the dorsal cortical F-actin sheath. Posterior to this site, gradual forward movement of patches accompanied the gradual narrowing phase of the trailing edge retraction that occurs prior to abrupt detachment of the tail, but patches did not actually accumulate at the null border. The rate of forward patch movement was generally greater at positions further behind the boundary. Patch movement correlated approximately with forward organelle movement in the trailing region of the cell. The boundary was also apparent during simultaneous capping and retraction when forward patch transport on the trailing edge and rearward transport of patches across the lamellar surface appeared to converge on the null border. Forward patch transport was strictly confined to regions behind the boundary while retrograde patch transport was confined to the lamellar region ahead of the boundary. Patches are thought to be linked to the cortical cytoskeleton and their transport is discussed in terms of the very different cortical cytoskeletal dynamics occurring in the leading and trailing edges of locomoting cells.

Actins↗
PubMed1991

Distribution, morphology and epithelial interactions of bovine spermatozoa in the oviduct before and after ovulation: a scanning electron microscope study.

In cows undergoing spontaneous oestrous cycles and mated during the first 6 hours of oestrus, the distribution of spermatozoa in the oviduct isthmus and changes in their surface membranes and neighbouring epithelium have been examined shortly before and after ovulation. In agreement with previous histological studies, relatively few spermatozoa were detected in the oviduct lumen: most were located in the caudal isthmus before ovulation, frequently among folds and in the presence of a viscous secretion. A majority of spermatozoa in this region showed strands and droplets of secretory material distributed over the anterior portion of an intact head before ovulation, whereas distribution of material over the post-nuclear cap of spermatozoa close to vesiculation or already acrosome-reacted was characteristic of the post-ovulatory situation. These changes in sperm head membranes were viewed as an expression of the completion of capacitation, and seemingly permit microvillous engagement with the rostral tip of the head. In conjunction with a narrow lumen and viscous secretions in the caudal isthmus, microvilli may thus serve to regulate periovulatory sperm progression towards the site of fertilisation, and be the basis of intermittent phases of adhesion to the oviduct epithelium as seen by phase-contrast microscopy. Although cilia do not similarly engage the heads of bull spermatozoa (cf. boar spermatozoa), they may act to regulate progression of capacitated spermatozoa by contacting the principal piece of the flagellum. In the light of these observations, changes in the molecular composition of sperm surface domains during the process of capacitation in vivo now require specific definition.

Animals↗
PubMed2002

Preservation of urine for flow cytometric and visual microscopic testing.

BACKGROUND: Preservatives that could prevent destruction of cells, casts, and bacteria in urine are of great practical importance because they allow centralization and improvement of accuracy of urine particle counting. We compared two in-house mixtures and one commercial solution, as well as refrigeration, for their ability to preserve urine for both automated analysis (flow cytometry) and visual microscopy. METHODS: Urine specimens were preserved by refrigeration at 4 degrees C without preservatives (procedure 1); in a lyophilized solution intended to preserve specimens for bacterial culture (Urine C&S tubes; BD Preanalytical Solutions; procedure 2); in 10 mL/L formalin-0.15 mol/L NaCl (procedure 3); in 80 mL/L ethanol-20 g/L polyethylene glycol (procedure 4); and by storage at 20 degrees C without preservatives (procedure 5). Test strip measurements were used to select specimens positive for leukocyte esterase, hemoglobin, albumin, or nitrite. For 106 consecutive strip-positive specimens, urinalysis was performed by UF-100 (Sysmex) and by phase-contrast microscopy after Sternheimer supravital staining. Automated analysis was performed at arrival in the morning, on the same day in the afternoon, and after 1 and 3 days. Visual microscopy was performed at arrival and 3 days later. RESULTS: Urine bacterial counts were well preserved with procedures 1-3, with a false-positive rate of 0.0-3.4% at day 3 vs 28% without preservation (procedure 5). Erythrocytes were poorly preserved for 3 days (kappa coefficients, 0.24-0.61); after 1 day, fair preservation was seen with procedure 2 (kappa = 0.78), compared with less favorable preservation with procedure 1 (kappa = 0.61) or procedure 5 (kappa = 0.66). Leukocytes were well preserved by all five procedures in the acidic adult urines investigated. Counts of casts and large epithelial cells were artifactually increased by procedure 3. Procedure 2 performed at least as well as refrigeration for specimens analyzed with visual microscopy. CONCLUSIONS: Urine specimens from adults can be stabilized at room temperature for both automated particle analysis and visual microscopy.

Adult↗
PubMed1994

Tumor cell-derived 12(S)-hydroxyeicosatetraenoic acid induces microvascular endothelial cell retraction.

Our previous work demonstrated that the 12-lipoxygenase metabolite of arachidonic acid, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] induced a nondestructive and reversible retraction of cultured endothelial cells. In the current study we tested the hypothesis that tumor cells produce 12(S)-HETE during their interactions with endothelial cells which in turn induces endothelial cell retraction. Coincubation of Lewis lung carcinoma cells or elutriated B16 amelanotic melanoma (B16a) cells but not 3T3 fibroblasts with microvascular endothelial cells (CD3) resulted in a time- and concentration-dependent retraction of the CD3 monolayers as revealed by quantitative binding assays and phase contrast microscopy. Lewis lung carcinoma cell-induced endothelial cell retraction was blocked by specific lipoxygenase inhibitors but not by cyclooxygenase inhibitors, suggesting the involvement of a lipoxygenase metabolite(s). Radioimmunoassay and high-performance liquid chromatography analysis of tumor cell extracts identified 12(S)-HETE as the major lipoxygenase metabolite of arachidonic acid and tumor cell generation of 12(S)-HETE was specifically blocked by a select 12-lipoxygenase inhibitor N-benzyl-N-hydroxy-5-phenyl-pentamide. The identity and stereochemistry of tumor cell-derived 12-HETE was substantiated by gas chromatography-mass spectrometry analysis and chiral phase high-performance liquid chromatography, respectively. Lewis lung carcinoma cell adhesion to CD3 monolayers was accompanied by an enhanced 12(S)-HETE biosynthesis by tumor cells, which paralleled the tumor cell-induced endothelial cell retraction in a cell number-dependent manner. Pretreatment of tumor cells with N-benzyl-N-hydroxy-5-phenylpentamide inhibited both increased 12(S)-HETE biosynthesis and tumor cell-induced endothelial cell retraction. Highly metastatic variants of elutriated B16a cells which had been shown to produce large quantities of 12(S)-HETE induced significant CD3 cell retraction, while low metastatic subpopulations of B16a cells which synthesized no or little 12(S)-HETE did not induce endothelial cell retraction. These results suggest that 12(S)-HETE synthesis during tumor cell-endothelial cell interactions may represent a key contributory factor in cancer metastasis.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1985

A versatile and quantitative computer-assisted photoelectronic technique used for the analysis of ciliary beat cycles.

The beat cycles of rabbit tracheal cilia in culture and Mytilus laterofrontal cirri were recorded using a phototransistor, transillumination, video, and phase-contrast microscopy. The photoelectronic signal and video image of the ciliary activity were simultaneously recorded as a composite image. The photoelectronic signal was converted into a digital signal by a data acquisition system for further computer processing. By the selection of a small detector area and accurate detector alignment, a simple, repetitive photoelectronic signal representing ciliary activity was obtained. This signal records the ciliary beat frequency and demonstrates the triphasic nature of the beat cycle. The photoelectronic signal can be precisely correlated with the ciliary activity by analysis of the composite video recordings to provide the duration of the effective, recovery, and rest phases of the beat cycle. The video-photoelectronic signal correlations were verified by high-speed cinematography. High-speed films of ciliary activity were digitized, and the image density of selected pixels was analyzed by computer with respect to time and ciliary motion. These studies indicate that duration of the phases of the beat cycle are differentially reduced with increased beat frequency; the effective phase duration was quickly reduced to a minimum. This was followed by the reduction of the duration of the recovery phase to a minimum. The rest phase continues to be reduced without reaching a minimum, over the range of beat frequencies observed. These results suggest that ciliary beat frequency may be regulated either by modifying the rates of dynein-microtubule interactions or the rate of transition from one beat phase to the next.

Animals↗
PubMed1995

Semiquantitative analysis by scanning electron microscopy of cochlear hair cell damage by ototoxic drugs.

The ototoxicity of cisplatin and carboplatin in the organ of Corti of the guinea pig was evaluated semiquantitatively. Damage of the stereocilia of outer hair cells (OHCs) observed by scanning electron microscopy (SEM) was classified into normal, grade 1 (10-50% loss of stereocilia), grade 2 (less than 50% remaining stereocilia), or grade 3 (missing stereocilia). The OHCs observed by light microscopy (LM) were classified as remaining or missing cells. Fifty OHCs of each row in the middle part of each turn of the cochlea were counted (a total of 150 cells per turn). Guinea pigs were administered 5 mg/kg of cisplatin or 50 mg/kg of carboplatin intraperitoneally for three consecutive days. In groups 1 and 2, in which both cochlea were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide (OsO4) and observed by SEM, the percentages of damage of the OHC stereocilia were similar in each cochlear turn bilaterally. In group 3, the right cochleae were fixed in OsO4 and observed by phase contrast microscopy as surface preparations. Left cochleae were submitted for SEM observation. Missing and grade 3 cells were observed at similar percentages in each row of each turn. In group 4, succinate dehydrogenase staining was performed in the right cochleae and observed by LM. The degree of damage in the right cochleae was compared with that of the left cochleae which was observed by SEM. On average, the mean numbers of missing cells and cells showing grade 3 damage were similar in each row of each turn. From these similarities of evaluation of ototoxicity at LM and SEM levels, it was concluded that semiquantitative analysis by SEM only is appropriate for the assessment of ototoxicity.

Animals↗
PubMed2005

[Expression of SLP-2 mRNA in endometrial cancer and its significance].

OBJECTIVE: To characterize the differential expression of SLP-2 in endometrial cancer, and to study the effect of human SLP-2 gene on human endometrial cancer cell line. METHODS: The expression of SLP-2 gene in 32 cases of endometrial cancer and 28 cases of normal endometrial tissues was examined by semi-quantitative RT-PCR. Eukaryotic expression vectors of sense and antisense SLP-2 were constructed and transfected into HEC-1B cell line using lipofectamine 2000 respectively. The morphological changes of the cell were observed by phase contrast microscopy. The cell growth was detected by methyl thiazolyl tetrazolium (MTT) assay, and the cell cycles were analyzed by flow cytometry. RESULTS: The expression of SLP-2 mRNA in endometrial cancer tissues was higher than that in normal endometrial tissues (1.6 +/- 0.7 vs 0.7 +/- 0.3, P < 0.05). Sense and antisense human SLP-2 constructs were transfected into HEC-1B cell line respectively. After being transfected with sense SLP-2, the expression of SLP-2 mRNA in HEC-1B cell line was increased by about 2.4 times that of the control group, the cell growth was accelerated, and the number of cells in G(1) phase was decreased by 12.5%, S phase was increased by 8.0%. After being transfected with antisense SLP-2, the expression of SLP-2 mRNA was declined 50%. The transfected cells showed slower growth, and the number of cells in G(1) phase was significantly increased by 10.5%, and S phase was declined by 9.8%. CONCLUSION: SLP-2 mRNA shows up-regulation in endometrial cancer tissues, and it may have some relationship with carcinogenesis of endometrial cancer.

Blood Proteins↗
PubMed2000

Characterization of vernix caseosa: water content, morphology, and elemental analysis.

Recent studies have prompted interest in the use of epidermal barrier creams as protective biofilms for very low birthweight preterm infants. The key to understanding the role of epidermal barrier films is an elucidation of their interaction with water and a basic knowledge of their composition. In this study, we investigated the morphologic properties and elemental composition of the naturally occurring biofilm, vernix caseosa. This biofilm is typically lacking in preterm infants and its production coincides in utero with terminal differentiation of the epidermis and formation of the stratum corneum. Significantly, vernix (80.5+/-1.0% H2O) had a much higher water content than other barrier creams (Eucerin: 17.1+/-0.6%, Aquaphor: 0.33+/-0.03%, Ilex: 0.19+/-0.02%, petrolatum: 0.03+/-0.01%; all p<0.05). Phase contrast microscopy of vernix showed multiple cellular elements with nucleic "ghosts" embedded in a putative lipid matrix. Transmission electron microscopy revealed flattened structures approximately 1-2 microm in thickness with distinct cellular envelopes indicative of differentiated corneocytes. Compared with mature corneocytes in adult stratum corneum, vernix corneocytes appeared swollen, the density of the keratin filaments was less, and there was a relative lack of tonofilament orientation. Cryofractured specimens were examined by cryoscanning electron microscopy with subsequent elemental localization by X-ray beam analysis. The findings indicate the high water content of vernix is largely compartmentalized within fetal corneocytes. These results are consistent with the novel view of vernix as a "fluid phase" stratum corneum consisting of a hydrophobic lipid matrix with embedded fetal corneocytes possessing unique biomechanical and water-binding properties.

Elements↗
PubMed1986

Flicker spectroscopy of erythrocytes. A sensitive method to study subtle changes of membrane bending stiffness.

Frequency analysis of thermally excited surface undulations of erythrocytes leading to the flicker phenomenon is applied to determine biochemically and physically induced modulations of the membrane curvature elasticity. Flicker spectra of individual cells fixed to the window of a flow chamber by polylysine are taken by phase contrast microscopy, enabling investigations of the reversibility of the structural modifications. The spectra may be approximated by Lorentzian lines in most cases. By measuring the amplitude (at zero frequency) and the line width, effects of the structural changes on the curvature elastic constant, Kc, and the wavelength distribution of the undulations may be studied separately. Effect of physically induced modifications: The temperature dependence of the flicker spectra are taken from 10 degrees C to 37 degrees C. Above 20 degrees C, Kc decreases with increasing temperature whereas the reverse holds below this limit. The latter anomalous behaviour is explained in terms of a conformational change associated with protein and lipid lateral phase separation. The bending stiffness increases when the cells swell osmotically, owing to surface tension effects. The dependence of the flicker spectra on the viscosity of the suspension medium agrees with the theoretical prediction. Biochemically and drug induced modifications: 5 vol% of ethanol leads to a pronounced and reversible suppression of the long wavelength undulations without altering the discoid cell shape and without affecting the bending stiffness appreciably. Adsorption of dextran to the glycocalix increases Kc by a factor of 1.6 at saturation. The bending stiffness is increased by a factor of 1.3 after cross-linking the proteins with the SH-oxidizing agent diamid. Injection of Ca++ into the cell via ionophores evokes (within 10 min) the formation of fine--probably spectrin free--spicules. This leads to an increase in Kc by a factor of 1.3 which is explained in terms of a lateral condensation of the spectrin/actin network. The spicule formation and Kc change is completely reversible (within 2 min) after perfusion with Ca++-free buffer. Cholesterol depletion leads first to a continuous increase in Kc without change of the cell shape whereas a sudden discocyte- to echinocyte transformation sets in below a critical steroid content. The latter transition is also observed in cell suspensions and is reminiscent of a phase transition. The anti-tumor drug actinomycin D evokes an increase in the bending stiffness Kc by a factor of two, suggesting that its effect is at least partially due to a modulation of the membrane structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗
PubMed1996

Nuclear (DNA, RNA, histone and non-histone protein) and nucleolar changes during growth and senescence of may apple leaves.

Quantitative interference microscopy was used to determine changes in nuclear and nucleolar indices (dry mass and cross-sectional area) in upper and lower epidermal cells and adjacent leaf-margin hair cells of the May apple (Podophyllum peltatum L.) leaves over a 42-day period (after leaves emerged above the ground litter). These indices decreased in a highly correlated manner. A ploidy variation may exist between epidermal cells and leaf-margin hair cells. Using the leaf-margin hair cells model, six nuclear macromolecule indices (total nucleic acid, DNA, RNA, total nuclear protein, histone and non-histone protein), nuclear volume, nucleolar volume and perinucleolar volume (measured using quantitative epifluorescence-phase contrast microscopy) all declined with age (42-day study) in a highly correlated manner. The degeneration of the nucleus and nucleolus in the three leaf locations studied followed the patterns observed for programmed cellular senescence and death (necrosis) in epidermal cells of onion leaf bases (stored tissue; leaf bases did not contain chlorophyll) and human epithelial cells (buccal; cervical). We conclude that the epidermal cells and leaf-margin hair cells from green leaves of the May Apple are ideal for the study of programmed cell senescence and death in plants, especially for the partitioning of this process into the study of: the point-of-no-return (solubilization of the karyoskeleton and loss of non-histone proteins and RNA associated with the karyoskeleton from the nucleus); nuclear pycnosis (loss of nuclear dry mass and volume and loss of nuclear internal support structure); chromatin condensation, margination along the inner nuclear envelope; and DNA-histone degeneration; degeneration of the nucleolus and loss of the perinucleolar zone of exclusion. The characterization of chlorenchyma cells during the 42-day period should now be undertaken (leaf senescence as indicated by the beginning of yellowing about 35 days after emergence) to determine whether these cells with functional chloroplasts undergo nuclear changes like those lacking functional chloroplasts.

Allium↗
PubMed1997

Effect of IL-6 on tumor cell invasion of vascular endothelial monolayers.

The effect of interleukin-6 (IL-6) on the invasive capacity of B16-F1 mouse melanoma cells into vascular endothelial monolayers was examined, and an in vitro assay system for the quantitative determination of tumor cell invasiveness, using confocal microscopy with a fluorescence image analyzer, was developed. First, the invasive capacity of B16-F1 mouse melanoma cells against bovine vascular endothelial monolayers was estimated; then, the gap junctional intercellular communication (GJIC) of endothelial cells was examined. Treatment of endothelial cells with IL-6 resulted in a remarkable increase in the invasion of tumor cells into the endothelial monolayer, which was found to be significant from 25 ng/ ml, and peaked at levels of more than 50 ng/ml. This stimulatory effect of IL-6, which was observed from 3 h after the initiation of treatment and lasted for up to 24 h, was abolished by the addition of the anti-IL-6 antibody. Although phase-contrast microscopy did not reveal any morphological changes in the endothelial cells following treatment with 25-200 ng/ml IL-6 for 24 h, the GJIC was observed to be significantly decreased. These findings indicate that the invasive capacity of tumor cells into endothelial cells is affected by IL-6.

Analysis of Variance↗
PubMed1992

Preparation and examination of flea larvae (Siphonaptera) by light and electron microscopy.

Techniques are described for making permanent slide-mounted specimens. Larvae were thoroughly macerated in KOH to remove soft internal tissue, yielding specimens with enhanced transparency and allowing examination of most of the exoskeleton in a single larva. Fatty material was removed in acetone to eliminate confusion of fat globules with sensilla in the cuticle. The more sclerotized exoskeletal elements, such as integumentary scales and the body sclerites together with their setae and sensilla, were highlighted by staining with carbol fuchsin. Resolution of numbers and arrangement of setae, especially those on the dorsum of the head capsule, in the anal comb, and on the anal mounds, was greatly facilitated by mounting larvae in dorsal orientation. Clearer views of the antennae and mouthparts were obtained, in orientations seldom achieved in whole mounts, by dissecting the head capsule. Discrimination of many taxonomically valuable features was enhanced by the use of phase-contrast microscopy. Interpretation of structures noted in light microscopy was complemented using scanning electron microscopy. The latter allowed differentiation between surface and internal cuticular structures.

Animals↗
PubMed1994

Airborne concentrations of asbestos in non-occupational environments.

Concentrations of asbestos in air were determined from analysis of samples collected in over 300 buildings involved in litigation. Samples were collected by certified industrial hygienists and analysed in certified laboratories by transmission electron microscopy. Building group mean concentrations of asbestos in building air inhaled by occupants were generally less than 0.0005 f ml-5 > 5 microns (90th percentile). At these concentrations the risk from asbestos exposure would be very low for building occupants. Another data set was obtained from the maintenance logs kept by owners of buildings containing asbestos fireproofing and subject to Operations and Maintenance Programmes to evaluate asbestos inhalation risk to maintenance workers. The logs were kept to document protective measures and maintenance personnel exposures during 1991-1992. Data are presented for one commercial building, which is typical of data for three additional commercial buildings and a medical centre. All samples were evaluated by the NIOSH 1400 protocol for sampling and analysis by phase contrast microscopy. Operations and maintenance precautions to reduce dust emission were modest; they included spraying of ceiling tiles with amended water, HEPA vacuuming tile edges before entry and after tile replacement, respirator usage and careful work. Negative pressure containment was not used. In this building personal exposures in electrical/plumbing work ranged from 0.000 to 0.035 f ml-1 > 5 microns in length (average work time of one job was 118 min); the 8-h TWA was 0.0149 f ml-1 > 5 microns.(ABSTRACT TRUNCATED AT 250 WORDS)

Air Pollutants↗
PubMed1986

Rat spermatogenesis in vitro traced by quantitative flow cytometry.

In vitro differentiation of germ cells in rat seminiferous tubule segments at stages II-III of the epithelial cycle was studied. DNA flow cytometry was used for quantitation of absolute cell numbers from the cultured tubule segments that were compared to freshly isolated stages of the cycle, as identified by transillumination stereomicroscopy of the seminiferous tubules and phase-contrast microscopy of live cell squashes. Spermatogonia and spermatocytes from stages II-III showed normal morphological differentiation during 7 days in vitro. Round spermatids differentiated to Step 7 of spermiogenesis but Step 16 spermatids failed to develop. Acid phosphatase activity in the spermatogenic cells changed normally during the culture. As compared with freshly isolated control tubule segments, 35% of round spermatids and 42% of pachytene spermatocytes were present in culture after 7 days. The cell numbers recovered from defined stages by DNA flow cytometry were close to those found in morphometric studies. Flow cytometry is an efficient quantitation method for cells liberated from seminiferous epithelium. Spermatogonia, spermatocytes, and early spermatids are able to differentiate in vitro, but spermatids approaching the elongation (acrosome) phase, and particularly the maturation phase, fail to differentiate under present culture conditions.

Acid Phosphatase↗
PubMed2001

Pharmacological characteristics of solid-phase von Willebrand factor in human platelets.

1. The pharmacological characteristics of solid-phase von Willebrand factor (svWF), a novel platelet agonist, were studied. 2. Washed platelet suspensions were obtained from human blood and the effects of svWF on platelets were measured using aggregometry, phase-contrast microscopy, flow cytometry and zymography. 3. Incubation of platelets with svWF (0.2 - 1.2 microg ml(-1)) resulted in their adhesion to the ligand, while co-incubations of svWF with subthreshold concentrations of ADP, collagen and thrombin resulted in aggregation. 4. 6B4 inhibitory anti-glycoprotein (GP)Ib antibodies abolished platelet adhesion stimulated by svWF, while aggregation was reduced in the presence of 6B4 and N-Acetyl-Pen-Arg-Gly-Asp-Cys, an antagonist of GPIIb/IIIa. 5. Platelet adhesion stimulated with svWF was associated with a concentration-dependent increase in expression of GPIb, but not of GPIIb/IIIa. 6. In contrast, collagen (0.5 - 10.0 microg ml(-1)) caused down-regulation of GPIb and up-regulation of GPIIb/IIIa in platelets. 7. Solid-phase vWF (1.2 microg ml(-1)) resulted in the release of MMP-2 from platelets. 8. Inhibition of MMP-2 with phenanthroline (10 microM), but not with aspirin or apyrase, inhibited platelet adhesion stimulated with svWF. 9. In contrast, human recombinant MMP-2 potentiated both the effects of svWF on adhesion and up-regulation of GPIb. 10. Platelet adhesion and aggregation stimulated with svWF were reduced by S-nitroso-n-acetyl-penicillamine, an NO donor, and prostacyclin. 11. Thus, stimulation of human platelets with svWF leads to adhesion and aggregation that are mediated via activation of GPIb and GPIIb/IIIa, respectively. 12. Mechanisms of activation of GPIb by svWF involve the release of MMP-2, and are regulated by NO and prostacyclin.

Biological Transport↗