Measurement of DNA content in single cells morphologically identified on smears

A method was developed for measuring the nuclear DNA content in single cells previously identified on a bone marrow smear stained by the Wright-Giemsa method. The smear was first photographed and the location of individual cells, identified by morphology, was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained by the Feulgen method in 0.05% pararosaniline Schiff's reagent (pH 2.3) at 7 degrees C for 10 min. Nuclear red fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA after prior irradiation of smears with green light for 9 hr. The method is useful for measuring cell DNA content in heterogeneous cell populations when morphological cell identification is required.     

Voltage-gated inward currents of morphologically identified cells of the frog taste disc

We used the patch clamp technique to record from taste cells in vertical slices of the bullfrog (Rana catesbeiana) taste disc. Cell types were identified by staining with Lucifer yellow in a pipette after recording their electrophysiological properties. Cells could be divided into the following three groups: type Ib (wing) cells with sheet-like apical processes, type II (rod) cells with single thick rod-like apical processes and type III (rod) cells with thin rod-like apical processes. No dye-coupling was seen either between cells of the same type or between cells of different types. We focused on the voltage-gated inward currents of the three types of cells. Type Ib and type II cells exhibited tetrodotoxin (TTX)-sensitive voltage-gated Na+ currents. Surprisingly, type III cells showed TTX-resistant voltage-gated Na+ currents and exhibited a lack of TTX-sensitive Na+ currents. TTX-resistant voltage-gated Na+ currents in taste cells are reported for the first time here. The time constant for the inactivating portion of the voltage-gated inward Na+ currents of type III cells was much larger than that of type Ib and type II cells. Therefore, slow inactivation of inward Na+ currents characterizes type III cells. Amplitudes of the maximum peak inward currents of type III cells were smaller than those of type Ib and type II cells. However, the density (pA/pF) of the maximum peak inward currents of type III cells was much higher than that of type Ib cells and close to that of type II cells. No evidence of the presence of voltage-gated Ca2+ channels in frog taste cells has been presented up to now. In this study, voltage-gated Ba2+ currents were observed in type III cells but not in type Ib and type II cells when the bath solution was a standard Ba2+ solution containing 25 mM Ba2+. Voltage-gated Ba2+ currents were blocked by addition of 2 mM CoCl2 to the standard Ba2+ solution, suggesting that type III cells possess the voltage-gated Ca2+ channels and they do classical (calcium-influx) synaptic transmission. It appears that type III cells are taste receptor cells.     

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

OBJECTIVE: To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. STUDY DESIGN: Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. RESULTS: Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. CONCLUSION: There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.     

Instruments for rapid cytophotometric analysis of morphologically identified clinical cell material

Two instruments for cytophotometric analysis are described. The first is a generally applicable object stage scanning microspectrophotometer for the wavelength region 240-700 nm. The microscopic image is shown on a monitor screen and delineation of the analyzed objects is made with the aid of a light pen. The second instrument is a high speed microscope photometer specially adapted for the measurement of DNA, nuclear protein, and projected nuclear area following combined Feulgen and Naphthol Yellow S staining. A two-dimensional CCD sensor measures the light intensity in the microscopic image with a resolution of 0.3 micron. The objects are selected by the operator pointing the light pen at the image screen. The integrated absorbance at two different wavelengths, together with the projected area, is automatically measured for each object and presented in histogram form. Cells in clusters or sections can be measured under operator control with the help of the light pen. The time required for measuring 100 cells is 5-10 min in an ordinary preparation. The application of these instruments to the grading of cellular atypia and in the prognostic evaluation of malignant tumors is demonstrated on material from developing squamous bronchial carcinoma and in invasive mammary and prostatic adenocarcinoma.     

Ribosomal and RPB2 DNA sequence analyses suggest that Sporidesmium and morphologically similar genera are polyphyletic

Sporidesmium and morphologically similar dematiaceous, hyphomycetous genera are characterised by holoblastic phragmoconidia produced on proliferating or non-proliferating conidiophores. They include a number of asexual (anamorphic) genera taxonomically segregated from Sporidesmium sensu lato and are similar in having schizolytic conidial secession. The taxonomy of these ubiquitous asexual fungi and their affinities with known Ascomycetes are, however, still obscure. This study incorporates a phylogenetic investigation, based on the LSU nu-rDNA and RNA polymerase II second largest subunit (RPB2) gene sequence, to assess the possible familial placement of Ellisembia, Linkosia, Repetophragma, Sporidesmiella, Sporidesmium and Stanjehughesia, and justify whether anamorphic characters are proper phylogenetic indicators. Phylogenies provide conclusive evidence to suggest that Sporidesmium is not monophyletic and species are phylogenetically distributed in two major ascomycete classes, Dothideomycetes and Sordariomycetes. Morphologies currently used in their classification have undergone convergent evolution and are not phylogenetically reliable. The possible teleomorphic affinities of these anamorphic genera are discussed in light of morphology and molecular data. As these anamorphs, in most cases, are the sole known morph of the holomorph, it is proposed that in the absence of or failure to detect their teleomorphic phase, the anamorph names should be used for the holomorph.     

Two immunologically distinct types of protofilaments can be identified in Natrialba magadii flagella

A rabbit antiserum to the 15-kDa acetylcholinesterase toxin neutralised the lethal effect of the 15-kDa toxin of Aeromonas hydrophila when injected into trout. However, immunisation of fish with the 15-kDa toxoid failed to induce an antibody response, and a higher molecular mass form of this toxin was purified from the extracellular products with the aim of inducing an immune response in fish. The optimal conditions for production of extracellular products by A. hydrophila strain B32 were studied to increase the concentration of this protoxin. The extracellular products were fractionated by molecular exclusion chromatography to yield a purified protoxin with an estimated molecular mass of 45 kDa by SDS-PAGE and which gave a positive reaction in Western blotting with the rabbit anti-15-kDa toxin serum. Since the 45-kDa protoxin showed lower specific acetylcholinesterase activity than the active 15-kDa toxin, the behaviour of the active site was studied using specific inhibitors. This 45-kDa protoxin was 13.3-fold less toxic than the 15-kDa toxin and induced antibody production in fish.     

Flow cytometric DNA content in Kaposi's sarcoma by histologic stage. Comparison with angiosarcoma

Kaposi's sarcoma occurs as a multicentric proliferation of endothelial cells. A lesion may progress through several histologic stages, culminating in a lesion consisting of spindle cells with marked nuclear atypia that may be indistinguishable from angiosarcoma. To assess the relationship between the nuclear DNA content and the stage, 29 paraffin-embedded biopsy specimens from 25 cases of Kaposi's sarcoma were classified according to their histologic stage and flow cytometric DNA ploidy status. The findings were compared with those in 14 angiosarcomas (5 postmastectomy angiosarcomas, 6 other cutaneous angiosarcomas and 3 angiosarcomas of deep tissues). The Kaposi's sarcoma specimens studied included samples with irregular lymphatic-like channels (stage 1), transition to spindle cells (stage 1t2), nodular spindle-cell aggregates (stage 2), scattered atypical spindle cells (stage 2t3) and histologic features indistinguishable from those of angiosarcoma (stage 3). Of the 25 Kaposi's sarcoma specimens of stage 2 or less, 17 had a diploid DNA distribution while an additional 8 had broad diploid G0G1 peaks (peridiploid, with a coefficient of variation greater than 7.5%, present in similar proportions in stages 1, 1t2 and 2). One of three stage 2t3 lesions showed tetraploidy while the single stage 3 specimen (from the leg) was aneuploid, with a DNA index (DI = 1.16) similar to that of four of the five postmastectomy angiosarcomas (DI = 1.14 to 1.20). An additional three angiosarcomas also showed nondiploid distributions (DI = 1.16, 1.98 and 2.13, respectively); the remainder were diploid or peridiploid. These results support previous cytogenetic data suggesting a normal karyotype in Kaposi's sarcoma up to stage 2, with atypia beginning as cells acquire numerical and structural chromosomal aberrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal occurrence of planktotrophic bivalve larvae identified morphologically and by single step nested multiplex PCR

We report the application of a recently developed molecularmethod, single step nested multiplex PCR (SSNM-PCR) assay andmicroscopy to identify and investigate temporal patterns ofbivalve larvae in a Danish estuary, Isefjord. All samples werecollected during the SUSTAINEX program from June to November2001. Using the molecular assay, larvae could be categorizedinto six groups: the blue mussel, Mytilus edulis, Ensis spp.,species of the Myoidae superfamily (Mya spp.), the common cockle(Cardiidae family), members of the Abra and Macoma genera ofthe Tellinoidae superfamily and members of the surf clam genera,Spisula spp. A seventh group was composed of unknown larvae.Greater resolution was possible by microscopy, but only forrelatively large and intact individuals (>150–200 µm).The molecular approach was capable of differentiating betweenlarvae regardless of shell size. Where it was possible to directlycompare identifications based on both methods, concordance washigh for M. edulis, Macoma balthica/Abra alba and E. americanus,whereas identification of Myoidae spp. and Cardiids was lessconsistent. Over the course of the study, two patterns of larvaloccurrence were observed. Larvae from species known to exhibita protracted annual spawning period (M. edulis, Myoidae spp.,Mysella bidentata and Cardiids) were present in the water columnthroughout the sampling period, whereas larvae of Abra alba,Barnea candida, E. americanus, Macoma balthica, Musculus marmoratusScrobicularia plana and Tapes pullastra appeared at clearlydefined periods.     

Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Flow cytometric estimation of nuclear DNA content was performed in six plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the species-specific differences in binding of these fluorochromes to DNA. It was concluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalting dyes (e.g. propidium iodide) is recommended for this purpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.     

DNA analysis of malignant effusions. Comparison with cytologic diagnosis and carcinoembryonic antigen content.

Twenty-three consecutive malignant effusions from 19 patients submitted for cytologic examination were analyzed for carcinoembryonic antigen (CEA) content and for DNA analysis by flow cytometry. The study was undertaken to determine if the addition of DNA analysis would improve the sensitivity of cytologic diagnosis and CEA assay. CEA examination was performed on Papanicolaou-stained smears and hematoxylin-and-eosin-stained cell blocks. Final diagnoses were correlated with histologic examination (four patients), clinical and radiologic studies, and follow-up. The malignant effusions in 19 patients were secondary to carcinoma of the breast (5), lung (5), ovary (1), endometrium (1), mucinous carcinoma of the colon (1), unknown primary (1), extraovarian papillary carcinoma (1), mesothelioma (2) and large cell lymphoma (2). The sensitivity of cytologic diagnosis was 100% and specificity 100%. DNA aneuploidy, defined as the presence of two separate peaks in the histogram, was present in 7 of 23 fluids (sensitivity, 30%). Four fluids had insufficient cells for analysis, and one histogram showed debris (following chemotherapy). DNA aneuploidy was detected in effusions secondary to carcinoma of the breast (4), lung (1) and lymphoma (2). Using 5 ng/mL as the cutoff, the sensitivity of CEA was 68%. DNA analysis of cells in malignant effusions is less sensitive than cytologic diagnosis, and CEA assay and is not recommended for routine use in the diagnosis of malignant effusions.     

In vitro aging of articular chondrocytes identified by analysis of DNA and tubulin content and relationship to cell size and protein content

In vitro senescence of chondrocytes, characterized by a decline in the proliferation rate during late passages, resulted from a rapid growth rate in early subcultures to a complete loss of division after seven to nine passages. One senescent-associated phenotypic change was the apparent increase in the density of cytoplasmic cytoskeletal proteins. We examined the relationship between tubulin content and growth (measured by DNA and total protein contents and cell volume), using flow cytometry, in the assessment of cytoskeleton analysis during in vitro aging. In contrast with previous microscopic observations of tubulin organization, flow cytometry revealed a tubulin content that was modulated as a function of protein content and/or cell volume.     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

Intracellular activity of morphologically identified neurons of the grass frog's optic tectum in response to moving configurational visual stimuli

Summary In the grass frogRana temporaria, various classes of tectal neurons were identified by means of intracellular recording and iontophoretic staining using potassium-citrate/Co³⁺-lysine-filled micropipettes, which have been defined previously by extracellular recording methods. Class T5(1) neurons had receptive fields (RF) of 33°±5° diameter. In response to a moving 8°×8° square (S), a 2°×16° worm-like (W), or a 16°×2° antiworm-like (A) moving stripe, these cells showed excitatory postsynaptic potentials (EPSPs) and spikes which were interrupted occasionally by small inhibitory postsynaptic potentials (IPSPs). The excitatory responses (R) were strongest towards the square (R_S) and less to the worm (R_W). For the antiworm (R_A) the responses were smallest or equal to the worm stimulus yielding the relationship R_S>R_WR_A. Some of these cells were identified as pear-shaped or large ganglionic neurons, whose somata were located in the tectal cell layer 8. The somata of other large ganglionic neurons were found in layer 7 and the somata of other pear-shaped neurons at the top of layer 6, both displaying T5(1) properties. Class T5(2) neurons (RF=34°±3°) responded with large EPSPs and spikes, often interrupted by small IPSPs, when their RF was traversed by the square stimulus. The excitatory activity was somewhat less to the worm stimulus, whereas no activity at all, or only IPSPs, were recorded in response to the antiworm-stimulus; thus yielding the relationship for the excitatory activity R_S>R_W>R_A 0. Such a cell was identified as pyramidal neuron; the soma was located at the top of layer 6, with the long axon travelling into layer 7 to the medulla oblongata. Class T5(3) neurons (RF=29°±6°) showing EPSPs and spikes according to the relationship R_S>R_A>R_W have been identified as large ganglionic neurons. Their somata were located in layer 8. Class T5(4) neurons (RF=24±7°) responded only to the square stimulus with EPSPs and spikes, sometimes interrupted by IPSPs and yielding the relationship R_S>R_AR_W0. The somata of these large ganglionic or pear-shaped neurons were located in layer 8. Class T1(1) neurons (RF=30°–40°) were most responsive to stimuli moving at a relatively long distance in the binocular visual field, and have been identified as pear-shaped neurons. Their somata were located in layer 6.Further neurons are described and morphologically identified which have not yet been classified by extracellular recording methods. For example,IPSP neurons (RF=20°–30°) responded (R) with IPSPs only according to the relationship R_S>R_A R_W. The somata of these pear-shaped neurons were located in layer 6.The properties of tectal cells in response to electrical stimulation of the optic tract and to brisk changes of diffuse illumination suggest certain neuronal connectivity patterns. The results support the idea ofintegrative functional units (assemblies) of connected cells which are involved in various perceptual processes, such as configurational prey selection expressed by T5(2) prey-selective neurons.Abbreviations A antiworm-like 16°×2° stripe stimulus with long axis perpendicular to the direction of movement - W wormlike 2°×16° stripe stimulus with long axis oriented parallel to the direction of movement - S square 8°×8° moving stimulus - ERF excitatory receptive field - IRF inhibitory receptive field - RF receptive field - EPSP excitatory postsynaptic potential - IPSP inhibitory postsynaptic potential     

Cytophotometric measurement of the cellular DNA content of [3H]thymidine-labelled spheroids. Demonstration that some non-labelled cells have S and G2 DNA content

Spheroids from the V279-171b and MCa-11 cell lines were incubated continuously for 24 hr in [3H]thymidine for labelling of the outer cells of the viable rim. The spheroids were dispersed into single cells, and the DNA content of photomapped cells was measured by absorption cytophotometry. Autoradiographs were then prepared from which we ascertained cellular labelling. For spheroids of both cell lines, we found a larger proportion of cells with a G0/G1 DNA content among the non-labelled inner spheroid cells than among the labelled outer cells (P less than 0.001). This block of non-labelled spheroid cells in G0/G1 was not a cell cycle perturbation caused by the isotope for the MCa-11 spheroids. Approximately 8% of non labelled MCa-11 spheroid cells had S/G2 DNA content, suggesting that non-cycling cells in spheroids may be blocked in S and G2 as well as in the G0/G1 phase of the cell cycle.     

Comparison of nuclear DNA content in primary and metastatic malignant melanoma.

The DNA patterns obtained from 23 primary malignant melanomas and 35 corresponding metastases were compared and found to differ in many cases. In eight cases the primary tumors and their metastases had a ploidy type I ("euploid") DNA pattern. One case had a type I primary tumor and both type I and type II metastases. Five cases had type I primary tumors and ploidy type II ("aneuploid") DNA pattern metastases. In five cases the primary tumors and corresponding metastases were type II, and in another four cases the primary tumors were type II, whereas the metastases were type I. We interpret these data as indicating that malignant melanomas (more often than adenocarcinomas) are composed of genetically heterogeneous tumor sublines that frequently give rise to heterogeneously composed metastases. Since we sometimes observed a change in the DNA content in malignant melanomas, it seems to be more difficult to obtain prognostic information from DNA analysis in malignant melanoma as compared to the more stable adenocarcinomas.