Dull Fluorescence Research Articles

264 EFFECT OF SEASON ON CRYOCAPACITATION OF BUFFALO (BUBALUS BUBALIS) SEMEN

Buffalo are short-day breeders; at our latitudes, reproductive activity improves during autumn. Although extensive studies have been conducted on the female, seasonal variations were also reported on post-thaw motility and membrane integrity of buffalo sperm (Andrabi 2009 Reprod. Domest. Anim. 44, 552–569). It was reported that cryopreservation induces capacitation-like changes in buffalo spermatozoa, assessed by both chlortetracycline (CTC) fluorescent and protein tyrosine phosphorylation assays (Kadirvel et al. 2011 Theriogenology 75, 1630–1639; Elkhawagah et al. 2014 J. Buffalo Sci. 3, 3–11). The aim of this study was to evaluate the effect of season on cryocapacitation of buffalo semen. At least two ejaculates were collected from 4 bulls during 2 seasons with different daylength: spring (low season) and autumn (peak season). Each ejaculate was diluted at 37°C with BioXcell extender to a final concentration of 30 × 106 spermatozoa per mL. After 4 h at 4°C, straws were frozen in an automated system. Immediately after thawing, sperm motility was evaluated by phase-contrast microscopy and viability, as well as capacitation status, were assessed by CTC fluorescent staining, as reported (Kadirvel et al. 2011 Theriogenology 75, 1630–1639). Briefly, sperm suspensions were first stained with 0.1 µg mL–1 Hoechst 33258 for 2 min. Then, equal volumes of sperm suspension and CTC solution (750 mM CTC, 5 mM cysteine in 130 mM NaCl, and 20 mM Tris-HCl) were mixed at room temperature, and glutaraldehyde (12.5%) was added. Sperm suspensions were mounted on slides and stored at 4°C overnight (in the dark). Each sample was assessed twice under a microscope equipped with phase contrast and epifluorescent optics. At least 100 spermatozoa per slide were evaluated and classified into 3 CTC staining patterns: 1) uniform bright fluorescence over the entire head (uncapacitated spermatozoa, pattern F); 2) fluorescence-free band in the post-acrosomal region (capacitated spermatozoa, pattern B); and 3) dull fluorescence over the entire head, except for a thin punctuate band of fluorescence along the equatorial segment (acrosome-reacted spermatozoa, pattern AR). Data were analysed by chi-square. There were no differences in sperm viability between seasons (78.4 and 76.4%, respectively, in autumn and spring). However, post-thaw motility increased (P < 0.05) in autumn (60.0%) compared with spring (50.0%). The percentage of sperm displaying CTC pattern F increased in autumn compared with spring (40.5 and 27.3%, respectively; P < 0.01), whereas the percentage of sperm with both pattern B (57.9 and 66.6%, respectively; P < 0.01) and AR (1.6 and 6.1%, respectively; P < 0.01) decreased. The number of bulls and ejaculates used in this study was too low to draw definitive conclusions. However, these findings suggested that capacitation-like changes after sperm cryopreservation may be reduced during the favourable season in buffalo.

An intriguing Y chromosome in Heteromys anomalus (Rodentia, Heteromyidae).

The mitotic chromosomes of the pocket mouse Heteromys anomalus (Rodentia, Heteromyidae) from Venezuela were analyzed by means of conventional staining and various banding techniques. The karyotype of this species is distinguished by a Y chromosome with exceptional banding properties. The very large heterochromatic Y long arm shows a remarkable bright quinacrine fluorescence, but only moderate or dull fluorescence when labeled with other AT base pair-specific fluorochromes. The implications of these findings are discussed.

Fluorescent antibody tagging of colon cancer in the nude mouse; a model for improved surgical accuracy in the oncologic patient

13501 Background: Induced fluorescence of malignant tumors has the potential to improve the surgeon’s ability to accurately identify and excise all malignant tissue. Tumors can be made fluorescent using fluorophore-tagged anti-tumor antigen antiboies. We present here our results using a human colon cancer cell line in the nude mouse model. Methods: HCT 116 colon cancer cells were subcutaneously or orthotopically implanted in 16 nude mice (12 study mice and 4 control mice). Two to 8 weeks after injection, tumor nodules were easily detectable. Using the tail vein method, all mice were injected with fluorophore-tagged anti-CEA or fluorophore-tagged IgG. Mice were examined using a small animal imaging system with a 470 nm light source and appropriate filters. They were also examined using a simple blue LED flashlight fitted with a fixed 470 nm band pass filter for illumination and were observed through filtered goggles. Results: All tumor nodules in the study mice demonstrated green fluorescence when visualized through the skin. On dissection and exposure of the tumor nodules, this fluorescence was intense and clearly distinguishable from the surrounding normal tissue using either the imaging system or the blue LED. Very small (<0.5mm) metastatic nodules were easily identified. Bright tumor fluorescence remained visible up to 5 days after injection. Control mice injected with fluorophore-tagged IgG and examined in a similar manner revealed no tumor fluorescence. Minimal non-specific dull fluorescence was occasionally observed in gut mucosa and ovarian tissue but was easily distinguished from the bright tumor fluorescence. Conclusions: When tumor surface antigens are known and antibodies to those antigens are available, this technology is simple, easy to perform, requires no technically complex equipment or operator expertise and could be readily adapted for cancer surgery in the academic or community hospital setting. Major indications for this technology would be in those patients where tumor resection offers an excellent chance for cure or significantly improves survival. The implications for increased accuracy of procedures such as resection of primary colorectal cancer or resection of solitary hepatic or pulmonary metastases are clear. No significant financial relationships to disclose.

Chromosome CPD(PI/DAPI)- and CMA/DAPI-banding patterns in Allium cepa L

Chromosome banding patterns of Allium cepa L. were obtained by using fluorochrome combinations chromomycin A3 (CMA) + 4',6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa, telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (95 degrees C for 1-3 min) followed by renaturation in the 2 x SSC buffer (37 degrees C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of the NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of DAPI fluorescence in GR-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.

Cold-sensitive chromosome regions and their relation to constitutive heterochromatin in Cestrum parqui (Solanaceae)

The structural cause for cold-induced undercondensation of certain chromosome segments is still unknown, and no consistent cytochemical correlations have emerged as yet. Cestrum parqui belongs to one of the few plant genera having chromosomes with such cold-sensitive regions. In the present study, four heterochromatin classes were distinguished in the karyotype of this species by sequential fluorochrome staining with chromomycin A3, DAPI, and D 287/170, and C-banding, N-banding, and silver impregnation. (i) Cold-sensitive regions (CSRs): Banding behaviour indicates AT-rich constitutive heterochromatin. Silver impregnation occurs after cold-treatment from interphase to metaphase. CSRs are subject to polymorphisms. Quantitatively they are only a minor component of the band endowment. (ii) Nucleolar organizers: Two pairs were identified. Banding behaviour indicates GC-rich constitutive heterochromatin. The nucleolar constrictions proper are morphologically indistinct at mitotic metaphase and lose silver impregnation capability at that stage. (iii) Non-nucleolar CMA-positively fluorescent C-bands: There is only one polymorphic paracentromeric band in this class, (iv) Low-fluorescence N-bands. These show quenched fluorescence with all fluorochromes and occur as clusters of small bands scattered over all chromosome arms. This class of bands is quantitatively predominating. Dull fluorescence with both the "GC-specific" chromomycin and the "AT-specific" DAPI is an anomaly but not completely unknown in plants.Key words: Cestrum parqui, cold-sensitive regions, heterochromatin, chromosome banding, silver impregnation.

Characterization of a monoclonal antibody enhancing porcine natural killer cell activity (PNK-E)

A monoclonal antibody, termed PNK-E, that functionally enhances porcine natural killer (NK) cell activity but not antibody-dependent cellular cytotoxicity (ADCC) is investigated in this report. When PNK-E and K562 target cells were simultaneously added to effector cells, killing of target cells could be detected as early as 30 min, and a dramatic enhancement of killing activity was observed in short term 51Cr-release assays. When a panel of five NK-sensitive targets were tested, PNK-E enhanced the killing of K562, MOLT-4, and U937 cells, but not the killing of CEM and YAC-1. F(ab)′ 2 fragments of PNK-E did not enhance NK activity, indicating a requirement for the Fc portion of PNK-E to elicit enhancement of NK. Immunofluorescence analysis shows that PNK-E antigen is expressed on approximately 15% of peripheral blood lymphocytes with a relatively dull fluorescence staining pattern. PNK-E-positive sorted cells were enriched for large granular lymphocytes (LGL) and contained all detectable NK activity as compared to the PNK-E-negative sorted cells. When analyzed by polyacrylamide gel electrophoresis, PNK-E antibody immunoprecipitated a protein from 125I-labeled peripheral blood lymphocyte (PBL) cell lysates that resolved as a single band of approximately 205 kDa under nonreducing conditions and as two bands of approximately 50 kDa and 47 kDa under reducing conditions. The present data demonstrate a functional association between PNK-E antigen and NK cell activation.

Localisation of non-replicating heterochromatin in polytene cells of Drosophila nasuta by fluorescence microscopy.

A simple fluorescence technique is decribed to localise in situ the non-replicating alpha heterochromatin in the chromocentre region of Drosophila nasuta polytene nuclei. After incorporating 5-bromodeoxyuridine in larval salivary gland cells for one or two cycles of replication, the polytene nuclei are examined for Hoechst 33258 flourescence at pH 7.0. The non-replicating alpha heterochromatin remains brightly fluorescing as it does not incorporate any 5-bromodeoxyuridine while the rest of the replicating chromatin shows dull fluorescence due to the quenching of Hoechst 33258 fluorescence by the bromodeoxyuridine substituted DNA.

Optical Studies of the interaction of 4'-6'-diamidino-2-phenylindole with DNA and metaphase chromosomes.

The optical absorption and fluorescence characteristics of 4'-6-diamidino-2-phenylindole (DAPI) with DNA and chromosomes were studied. There is a decrease in extinction coefficient and chift in the absorption spectra to a higher wavelength when the dye binds to DNA. The fluorescence of DAPI is enhanced by both A-T and G-C base-pairs. The enhancement by A-T rich is significantly greater than by G-C rich DNA. The chromosomes and the constrictions of human chromosomes 1 and 16; these regions are known to contain A-T rich DNA and show dull fluorescence when treated with quinacrine. This dye may be useful for identifying A-T rich region in chromosomes. The fluorescence of DAPI bound to polynucleotides or chromosomes is partially quenched by the introduction of BrdU. This suppression of dye fluorescence allows optical detection of sister chromatid exchanges and chromosome region containing DNA with an unequal distribution of thymidine between polynucleotide chains after BrdU incorporation.

Localization and significance of the Q bands observed on mitotic chromosomes of the amphibian Pleurodeles waltlii Michah. after coloration by quinacrine mustare (author's transl)

Metaphase plates from tailbuds of Pleurodeles waltlii embryos (stage 34) with or without preceding cold treatment were obtained by squashing followed by quinacrine mustard staining. In both cases, caryograms were established and sites of Q bands located.--Most of the secondary constrictions exhibit very high fluorescence. In general, it is the same for most centromeric parts, but their fluorescence is quenched very strongly by cold treatment.--The proximal part of the long arm of chromosome VII and satellites of chromosomes III and XI exhibit dull fluorescence. All these sites are compared with heterochromatin localization. The relation between banding and base composition or non-histone proteins interactions is discussed.

Fluorescent staining of L cell centromeres and chromocenters with 1-dimethylaminonaphthalene-5-sulfonyl chloride and G-bandings

Fluorescent staining patterns of L cell chromosomes with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride) were studied. Ordinary air-dried L cell metaphase chromosomes exhibited relatively uniform and bright yellowish green fluorescence by dansyl-staining under the fluorescence microscope. However, after the chromosome preparations were treated with 10 mM NaCl for 24 h at 4 °C, which produced distinctive G-bands with Giemsa-staining, the centromeric regions and several interstitial regions of some particular chromosomes were clearly fluorescent but other regions showed only dull fluorescence. After the treatment of chromosome slides with cupric sulfite reagent, which converts sulfhydryls and disulfides to thiosulfates chromosomes showed clear G-bands which were indistinguishable from those after 10 mM NaCl treatment. By dansyl-staining, however, the cupric sulfite-treated chromosomes exhibited very faint fluorescence on their contour alone, and neither centromeric regions nor some interstitial regions of marker chromosomes had distinctly bright fluorescence. Although Giemsa-staining disclosed dark chromocenters in approx. 75% of interphase nuclei irrespective of pretreatments, dansyl-staining revealed bright chromocenters in approx. 60% of interphase nuclei in control slides, in about 40% of nuclei in 10 mM NaCl-treated slides, and in only about 30% of nuclei in cupric sulfite-treated preparations. These observations indicated that in the air-dried chromosome preparations, the distribution of protein over the metaphase chromosome is relatively uniform along its length, and that G-bands in the chromosome and Giemsa-staining of chromocenters in interphase nuclei are not significantly affected by apparent loss of protein from the preparations. It was also suggested that particular protein may be associated with the centromeric regions of L cell chromosomes. Some technical details of dansyl fluorochroming and the significance of the observations were discussed.

The relationship between patterns of DNA replication and of quinacrine fluorescence in the human chromosome complement.

Cultured human peripheral blood lymphocytes were labelled with 3H-thymidine in the early or late S phase prior to mitosis. Quinacrine fluorescence patterns in metaphase chromosomes were then recorded photographically and the slides reprocessed for autoradiography so that the same metaphase cells were examined with the two techniques. The intensity and distribution of 3H-thymidine labelling was compared with the intensity and distribution of Q fluorescence with particular reference to chromosomes 1, 13, 14, 15, 17, 18, 19, 20, 21 and 22. It was found that chromosome regions showing bright fluorescence were also late replicating and that, in general, patterns of late replications reflected the patterns of fluorescence. Exceptions to this generalisation included the late labelling X chromosome in cells of female origin and areas near the centromeres on chromosomes 1, 9, 16 and 22. These centromeric regions show a dull fluorescence but, with exception of chromosome 9, are strongly Giemsa-positive in the ASG staining technique. On the basis of staining reaction, late replicating heterochromatic regions fall into five categories, the relationships and functional significance of these categories is discussed.

人癩の病理組織学的研究(第1報)

The all lymph nodes and lymphoid tissues of the 15 dead leprosy patients were collected at autopsies and their 603 specimens were histologically investigated.The tissue reactions were devided into 4 types. 1) the change occured chiefly in reticulum and contained migrating simple large cells (large cell type), 2) foamy cells stained with Sudan III and in serious cases diffuse fatty infiltration were observed (foamy type), 3) in which, accomapnied with fatty infiltration, giant vacuoles suggestive of fatty masses and giant cells of foreign bodies were recognized (fat accumulating type) and 4) which produced necrosis (necrosed type).In macular and neural leprosy, findings observed were similar to those of simple lymphadenitis, while in tuberous leprosy fatty infiltration is interpreted as a chief histological change.Fatty infiltration is consisted by the so-called foamy cells derived from the reticulum system. In the lesions with giant vacuoles and foreign body giant cells, fatty tissue debris from the skin or the peripheral nerves are carried through the lymph stream and caught by the lymphnodes of the filter activity, the reticuloendothelial system and the foreign body giant cells with strong phagocytic ability accumulate fatty substances.Fatty infiltration is recognized most intense in the superficial lymph nodes, especially in the inguinal nodes, becomes weaker in the profound nodes and no sign of it in the visceral nodes. But the lymph nodes of the liver port and pylorus present fatty infiltration. In the case after erythema nodosum leprosum remarkable necrosis was observed in the liver port.In macular and neural leprosy no appreciable change was observed in the lymphoid tissues. Foamy cells appeared in the lymph follicles of the spleen in tuberous leprosy, but other tissues presented no change.The result of bacillus research by the fluorescence microscope was extremely low. In general, bacilli were recognized in a small number as brilliant granules in the cells with dull fluorescence (a kind of foamy cells).

Fluorescence of Soil Constituents in Ultra-violet Light

THE only record of which I am aware of the use of the screened ultra-violet lamp for the detection of soil constituents is by H. L. Richardson1. Richardson examined air-dried samples from a very comprehensive collection of soil profiles. He noted that soils rich in organic matter displayed a uniform coffee-brown or even blackish colour. A distinct fluorescence was observed in soils which were heavily impregnated with salts, while a dull fluorescence was observed where gleying had occurred. He noted that non-suberized rootlets fluoresced brightly, and it was suggested that screened ultra-violet light might prove serviceable in studies of root distribution.