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PubMed1982

Morphological and electrophysiological mapping of giant neurons in the suboesophageal ganglia of Helix pomatia.

1. To reveal the morphology of the suboesophageal ganglia of Helix pomatia, the connective tissue was completely removed and the preparations stained whole-mount with methyl green-pyronin G to display the relative locations of the neurons. 2. Fifteen large cells which could be recognized as individuals in at least 75% of the preparations investigated, were morphologically identified by size, position and color. 3. The cells were electrophysiologically characterized with respect to spontaneous activity, synaptic input from peripheral nerves, and response to application of drugs (e.g. ACh, DA and 5-HT). 4. The peripheral axonal projections of eight of the major identified cells were investigated by intracellular CoCl2 injection and by cobalt backfilling of the peripheral nerves.

Animals↗

PubMed1983

The maturation of Theileria annulata in Hyalomma anatolicum anatolicum stimulated by incubation or feeding to produce sporozoites.

Adult Hyalomma anatolicum anatolicum ticks infected with Theileria annulata (Hissar strain) were incubated at 36 degrees C or fed on rabbits. Tick salivary glands were stained whole with methyl green pyronin or ground up and deposited on microscope slides and stained with Giemsa's solution. Separate batches of ticks from both treatments were ground up, centrifuged and filtered to produce sporozoite suspensions. The suspensions were examined as deposits on microscope slides stained with Giemsa's solution. The Theileria in the salivary glands of the fed ticks matured more completely and rapidly than in the incubated ticks. The peak numbers of sporozoites from the fed ticks was greater by at least tenfold than the peak from the incubated ticks. This peak was on the third day of feeding or on the fourth day of incubation. It was confirmed that fed ticks will be more suitable for sporozoite production for infection of cattle and production of stabilates.

Animals↗

PubMed1989

Relative role of male and female Hyalomma anatolicum anatolicum ticks in Theileria transmission.

The relative role of male and female Hyalomma anatolicum anatolicum ticks in the epidemiology of Theileria transmission was studied by detecting Theileria sporozoites in the dissected salivary glands of 568 ticks by the methyl green pyronin staining method. Detailed frequency distribution of Theileria-positive acini in the salivary glands of the 264 (46.48%) positive ticks from a field collection in Haryana indicated that the number of infected salivary acini per positive tick was greater in females than in males. This suggests that female ticks have a more important role in Theileria transmission than male ticks. This finding assumes greater significance in the light of the observation that the natural male:female ratio is also in favour of female ticks.

Animals↗

PubMed1991

Effect of temperature on transtadial transmission of Theileria annulata in Hyalomma anatolicum anatolicum ticks.

The effect of temperature on the transtadial transmission of Theileria annulata in Hyalomma anatolicum anatolicum was studied. Variation in temperature (4-40 degrees C) had a significant effect on moulting rate of the ticks and transmission of theilerial parasites from nymphs to resultant adults. The temperatures above 40 degrees C and below 12 degrees C prevented moulting. Maximum infection levels were obtained in salivary glands of adult ticks when the infected engorged nymphs were incubated at 24-28 degrees C. The infection rate in salivary glands was assessed using a methyl green pyronin technique.

Animals↗

PubMed1993

Immunological and histological study of T- and B-lymphocyte activity in canine visceral leishmaniosis.

Lymphocyte activity during canine leishmaniosis was studied by histological and immunological methods in experimental and natural infections. Eight dogs were inoculated with 5 x 10(7) promastigotes of Leishmania infantum, LEM 75, zimodeme MON-1, and maintained for 110 days post-infection. Another eight dogs with advanced natural infection were identified by parasitological and serological methods. Three techniques were used: the lymphocyte transformation test (LTT) to study T-cell activity, immunofluorescence assay and enzyme-linked immunosorbent assay to measure antibody production (B-cell activity), and methyl green-pyronin staining to assess tissue responses of lymphocytes. There was a noticeable B-cell response, reflected both histologically and immunologically. High percentages of activated lymphocytes and plasma cells were evident in lymphoid organs and production of specific antibodies was seen throughout the infection. LTT results showed a T-cell unresponsiveness during canine leishmaniosis. These same immunohistological features were observed, although to different degrees, in both experimental and natural infections.

Animals↗

PubMed2005

Differential subcellular distribution of estrogen receptor isoforms: localization of ERalpha in the nucleoli and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines.

The localization of estrogen receptors alpha (ERalpha) and beta (ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In both cell types, ERalpha was localized mainly in the nucleus, particularly concentrated on nuclear structures, which on the basis of their staining with pyronin and with antibodies against the nucleoli-specific Ki67 antigen and C23-nucleolin, were characterized as nucleoli. A faint, diffuse ERalpha staining was also observed in the cytoplasm. ERbeta was specifically enriched at the site of the mitochondria, visualized by labelling with the vital dye CMX and antibody against the mitochondrial-specific cytochrome oxidase subunit I. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of ERalpha and ERbeta. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of ERalpha in nucleolar-related processes.

Blotting, Western↗

PubMed2004

Chrysin and its phosphate ester inhibit cell proliferation and induce apoptosis in Hela cells.

To improve the biological activities of chrysin (CR), we synthesize Diethyl Chysin-7-yl phosphate (CPE: C(19)H(19)O(7)P) and tetraethyl bis-phosphoric ester of chrysin (CP: C(23)H(28)O(10)P(2)) through a simplified Atheron-Todd reaction. The interactions of the CR and CPE with lysozyme were explored by electrospray ionization mass spectrometry (ESI) and fluorescence spectrometry method. Experimental results indicate that CPE could form the noncovalent compound with lysozyme, while the interaction of the CR with lysozyme was not detected. In addition, whether and how the compounds CPE and CP affect proliferation and apoptosis in human cervical cancer Hela cells were investigated. Moreover, the effects of CPE and CP in Hela cells were compared with that of the nonmodified CR compound. The Hela cells were co-cultured with CR, CP, and CPE as experimental groups, respectively, and corresponding control groups treated without CR, CP, and CPE. The proliferation and apoptosis were detected using MTT assay, HCl denatured-methyl green-pyronin staining, PCNA immunohistochemistry and TUNEL techniques. The cell growth IC(50), relative absorbance (RA), proliferating index (PI), PCNA-IR (immunoreactivity IR) integration value (IV), and apoptosis index (AI) were calculated and their correlation was analyzed in each group. The results show that all CR, CP, and CPE could inhibit proliferation and induce apoptosis in Hela cells. Moreover, the effects of CP and CPE were more potent than that of CR. The CP and CPE were proved to be a kind of stronger apoptosis inducers than nonphosphated CR. There was a negative correlation between proliferation and apoptosis. In conclusion, the CR, CP, and CPE could effectively inhibit growth by down-regulated expression of PCNA, and induce apoptosis in Hela cells. The efficiency of the modified CP and CPE preceded nonmodified CR compounds. The CP and CPE may be a new potential anti-cancer drug for therapy of human cervical carcinoma.

Antineoplastic Agents↗

PubMed2005

The calyx fluid of Microplitis rufiventris parasitoid and growth of its host Spodoptera littoralis larvae.

The morphology of the female reproductive system of pupal and adult stages of Microplitis rufiventris and the ultrastructure of the ovaries are described and illustrated. Two morphologically distinct types of particles of nuclear origin, i.e., polydnavirus (PDV) and a virus-like filamentous particle (VLFP) were detected in the ovarian calyx fluid of the female wasp. It is likely that these particles are injected into the host during oviposition. PDV initiated replication in the calyx of mid-aged pupae and in pharate adults and were present throughout adult life. VLFP were only seen in the calyx fluid of newly emerged adults, and therefore observed after the PDV. Feulgen and methyl-green pyronin staining revealed the presence of DNA in both types of particles. The effects of injection of Spodoptera littoralis larvae with a combination of parasitoid viruses and venom of M. rufiventris females (CxFV) were investigated and the results were compared with two control groups, i.e., larvae injected with Pringle's saline (PS) and naturally parasitized larvae. CxFV-larvae showed significant declines (P<0.05) in food consumption, weight of ejected faeces and weight gain when compared with PS-larvae. However, naturally parasitized larvae (parasitoid egg+CxFV+ovarian protein) displayed a high significance score (P<0.01) in comparison with those of PS-larvae. The approximate digestibility (AD) values of S. littoralis larvae were positively affected as early as day 2 post-treatment by either injection of CxFV or parasitization. However, a reduction in AD was observed in both PS- and CxFV-larvae on day 3-7 in comparison with naturally parasitized larvae. Other indices of food utilization were unchanged in CxFV-larvae when compared to saline treated or parasitized controls.

Animals↗

PubMed2006

Mass spectrometrical identification of brain proteins including highly insoluble and transmembrane proteins.

Conventional two-dimensional electrophoresis (2DE) is the main technique used for protein profiling of tissues and cells, however separation of strongly acidic, basic or highly insoluble proteins is still limited. A series of methods have been proposed to cope with this problem and the use of discontinuous gel electrophoresis in an acidic buffer system using the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC) with subsequent SDS-PAGE followed by mass spectrometry showed that results from 2DE can be complemented by this approach. It was the aim of this study to separate and identify proteins from whole mouse brain that were not demonstrated by 2DE. For this purpose samples were homogenised, soluble proteins were removed by ultracentrifugation and the water-insoluble pellet was resuspended in a mixture containing urea, 16-BAC, glycerol, pyronine Y and dithiothreitol. Electrophoresis was run in the presence of 16-BAC, the strip from the gel containing separated proteins was cut out and was re-run on SDS-PAGE. Protein spots were analyzed by MALDI-TOF-TOF mass spectrometry. One hundred and six individual proteins represented by 187 spots were unambiguously identified consisting of 42 proteins with predicted pI values of pI>8.0, 25 with a 6.0<pI<8.0 and 39 with a pI<6.0. Twelve proteins with transmembrane domains (ranging from 1 to 8) including channels and carriers were identified. The generated map revealed a series of important brain proteins that were not separated and identified previously. Therefore, this system may be relevant for protein chemical determination of channels and carriers independent of antibody availability and specificity. The fact that transmembrane, basic, acidic as well as hydrophobic proteins with a positive Gravy Index can be resolved warrants work on further improvement of this analytical tool.

Animals↗

PubMed2004

Assessment of contact allergens by dissociation of irritant and sensitizing properties.

The human organotypic skin explant culture (hOSEC) model is a promising alternative in vitro model for screening contact allergens. In this model, the chemical-induced migration of Langerhans cells (LCs) out of the epidermis, evaluated after a 24-h exposure period, is used as a measure of sensitizer potential. As skin irritants can also induce LC migration it is essential that concentrations of test chemicals are used that are not even weakly irritant. Using the hOSEC irritation model chemicals are classified as weak irritants if they are toxic after a 48-h exposure period. Toxicity is determined by methyl green-pyronine (MGP) staining of hOSEC. We studied three frequently used non-sensitizing skin irritants and six potent or frequent human sensitizers in a dose-response. A complete discrimination between non-sensitizers and contact sensitizers was obtained for the chemicals tested when the concentrations used were lower than the weak irritant concentrations. Frequency of positive allergen reactions in patch test of human populations correlated with the difference between weak irritant concentrations and the lowest concentration inducing significant LC migration. Sensitizer potency correlated with chemical irritancy as determined by keratinocyte death. For the compounds tested, the hOSEC model predicted allergenicity in humans better than the guinea pig maximization test and the mouse local lymph node assay.

Allergens↗

PubMed2006

Infectivity rate and transmission potential of Hyalomma anatolicum anatolicum ticks for Babesia equi infection.

The infectivity rate of Babesia equi in the salivary glands of Hyalomma anatolicum anatolicum was assessed. The hungry nymphs were fed on a donkey experimentally infected with B. equi. The engorged dropped-off nymphs were collected at different levels of parasitaemia and kept in BOD incubator. After ecdysis, the hungry adults were prefed on rabbits for different time intervals, thereafter the salivary glands were dissected out and acini were examined after methyl green pyronin (MGP) staining. A total of 134 male and 139 female ticks were dissected out. Average infected acini per tick were found to be significantly higher (p<0.05) in male as compared to the female ticks. Further, maximum infected acini in both male and female ticks were found at 24h of prefeeding on rabbits and overall infected acini per tick increased with rise in parasitaemia. The release of infected ticks on susceptible donkeys resulted in development of clinical babesiosis.

Animals↗

PubMed1989

Inhibition of the bovine-heart mitochondrial F1-ATPase by cationic dyes and amphipathic peptides.

The bovine heart mitochondrial F1-ATPase is inhibited by a number of amphiphilic cations. The order of effectiveness of non-peptidyl inhibitors examined as assessed by the concentration estimated to produce 50% inhibition (I0.5) of the enzyme at pH 8.0 is: dequalinium (8 microM), rhodamine 6G (10 microM), malachite green (14 microM), rosaniline (15 microM) greater than acridine orange (180 microM) greater than rhodamine 123 (270 microM) greater than rhodamine B (475 microM), coriphosphine (480 microM) greater than safranin O (1140 microM) greater than pyronin Y (1650 microM) greater than Nile blue A (greater than 2000 microM). The ATPase activity was also inhibited by the following cationic, amphiphilic peptides: the bee venom peptide, melittin; a synthetic peptide corresponding to the presence of yeast cytochrome oxidase subunit IV (WT), and amphiphilic, synthetic peptides which have been shown (Roise, D., Franziska, T., Horvath, S.J., Tomich, J.M., Richards, J.H., Allison, D.S. and Schatz, G. (1988) EMBO J. 7, 649-653) to function in mitochondrial import when attached to dihydrofolate reductase (delta 11.12, Syn-A2, and Syn-C). The order of effectiveness of the peptide inhibitors as assessed by I0.5 values is: Syn-A2 (40 nM), Syn-C (54 nM) greater than melittin (5 microM) greater than WT (16 microM) greater than delta 11,12 (29 microM). Rhodamines B and 123, dequalinium, melittin, and Syn-A2 showed noncompetitive inhibition, whereas each of the other inhibitors examined (rhodamine 6G, rosaniline, malachite green, coriphosphine, acridine orange, and-Syn-C) showed mixed inhibition. Replots of slopes and intercepts from Lineweaver-Burk plots obtained for dequalinium were hyperbolic indicating partial inhibition. With the exception of Syn-C, for which the slope replot was hyperbolic and the intercept replot was parabolic, steady-state kinetic analyses indicated that inhibition by the other inhibitors was complete. The inhibition constants obtained by steady-state kinetic analyses were in agreement with the I0.5 values estimated for each inhibitor examined. Rhodamine 6G, rosaniline, dequalinium, melittin, Syn-A2, and Syn-C were observed to protect F1 against inactivation by the aziridinium of quinacrine mustard in accord with their experimentally determined I0.5 values.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMedDate not supplied

Immunohistochemical study on follicular dendritic cell of conjunctiva-associated lymphoid tissue.

We performed an immunohistochemical study of follicular dendritic cells (FDC) in the follicular area of the conjunctiva-associated lymphoid tissue (CALT). Hartley guinea pigs were sensitized with a topical application of an emulsion of ovalbumin and Freund's complete adjuvant in the eye. They were divided into four groups. The control (group A) underwent no sensitization. The sensitized animals were studied at 1 week (group B1) or 2 weeks (group B2) after the sensitization. Additional sensitization at 1 week after the initial sensitization was also performed (group C). Histological methods included methylgreen pyronine staining, alpha-naphthylacetate esterase staining, and enzyme-antibody method against S-100 protein. The uptake of topically applied peroxidase-anti-peroxidase (PAP) in CALT was also examined histologically. In each group, positive reticular patterns by alphanaphthyl acetate esterase staining and immunoperoxidase staining with anti-S-100 protein antiserum were found in the CALT follicular area. The positively stained cells were found to be dendrite cells by immunoelectronmicroscopy. An uptake of PAP was found in the CALT follicular area, suggesting the function of trapping and retaining antigen-antibody complex by FDC. It was concluded that dendritic cells in the CALT follicular area were identified to be FDC.

Animals↗

PubMed1990

A study of immunoglobulin-containing and other cells in ovine aural squamous cell carcinoma.

The peroxidase-antiperoxidase method was used to quantify immunoglobulin (Ig)-containing cells at the host-tumour interface in ovine aural squamous cell carcinoma lesions at various stages of development. IgM-containing cells were never observed; only scattered, but strongly staining, IgA-containing cells were present. IgG-containing cells predominated; their prevalence increased from 0.17 +/- 0.34 cells per high power field (hpf; mean +/- standard deviation) in precursor lesions to a maximum of 13.6 +/- 2.6 cells per hpf in overt tumours which had not metastasized. Their prevalence in overt tumours which had metastasized was 4.6 +/- 3.9 cells per hpf. Quantitation of pyroninophils in sections stained by the methyl green-pyronin method showed a greater prevalence of plasma cells than IgG-containing cells, but a similar pattern of change was apparent. Mast cells were most prevalent in early stages, but no significant change in eosinophil numbers was observed.

Animals↗

PubMed1976

Histochemical observation of the mycetomes of Idiocerus clypealis Leth.

Histochemical tests on the mycetomes and mycetocytes of Idiocerus clypealis show positive PAS and Congo red reactions for the presence of glycogen and glycoprotein (amyloid). The mycetocytes failed to take alcian blue and methyl green pyronin Y stains showing the absence of mucopolysaccharides and RNA respectively. Positive millions and bromophenol blue reactions prove that they contain tyrosine and general proteins. The presence of lipids and lipoprotein is shown by a positive stain with sudan black B.

Animals↗

PubMed1980

The morphology of the bone marrow of the mouse.

For an investigation on the morphology of the cells of the bone marrow of the mouse, use was made of 2 stains: MAY-GRUNWALD-GIEMSA (MGG) and methyl-green-pyronine-stilbene (MPS). The results are reported, and are compared with each other and with those obtained with the LENDRUM stain. To compare results in the same cell, its coordinates were determined with a computer-controlled scanning microscope after staining with the first method, and the preparation was then destained and restained for evaluation of the second method. The advantages and disadvantages of the stains are discussed, and it is concluded that for the evaluation of the morphology of the eosinophilic leukocyte, the MPS stain seems to be superior.

Animals↗

PubMed1989

Tungsten and molybdenum heteropolyacids as staining and contrasting agents: reactivity with epoxyresin-embedded cell and tissue structures.

In this work, we carry out a further approach to the knowledge of the reaction mechanism of phosphotungstic and phosphomolybdic acids (PTA and PMA), as well as some derivatives, with cell structures from epoxyresin-embedded materials. Applied on thin sections from glutaraldehyde-fixed tissues, PTA and PMA induced a strong electron contrasting reaction in spermatid acrosomes, goblet cell mucin, callose and plant cell walls, endexine, intine and starch granules. In light microscopy, the localization of heteropolyacids on these structures was achieved by treatments of semithin sections with suitable reducing agents (titanous sulfate, stannous chloride, sodium borohydride, or p-phenylenediamine) to form the mixed-valence heteropolyblues, or with Schiffs's reagent. The use of PTA-dye complexes (pyronin-PTA and Mallory's PTA-hematoxylin) also showed the same staining pattern. Taking into account the chemical characteristics of the PTA- and PMA-reactive tissue elements, the present results indicate that heteropolyacids selectively enter into the highest hydrophilic structures from non-polar epoxy-embedded sections; after brief washing, they appear predominantly retained in tissue structures containing a great amount of carbohydrate components.

Animals↗

PubMed1996

A new monoclonal antibody for specific immunocytochemical staining of nucleoli.

We have isolated a hybridoma cell line (clone 1E10) producing a monoclonal antibody which specifically recognizes nucleoli. The antibody (IgM, k isotype) was found to react in a nucleolar pattern with a variety of cell types. Specific staining was only obtained on cryostat sections of unfixed tissues. Paraffin embedding destroyed the epitope. Tissue specificity or species specificity was not observed. Nucleoli in neoplastic cells were highly reactive, presumably due to the larger size of nucleoli in these cells. Immunoelectron-microscopy (using a pre-embedding as well as a post-embedding technique) confirmed the specific nucleolar localization of the immunoreactivity. Immunoreactivity was confined to the granular component of the nucleolus. The intensity of the immunoreactivity increased after cell or tissue pretreatment with DNase, pronase or trypsin, indicating that the target epitope is not DNA or a protein. On Western blots of immunoreactive cells no specific signal was obtained, which supports the non-protein nature of the epitope. Acid hydrolysis and RNase digestion abolished the immunoreactivity. Parallel staining experiments with methylgreen pyronin and acridin orange confirmed the RNA nature of the epitope. In spot blots, immunoreactivity was not found with tRNA or mRNA. These observations indicate that 1E10 recognizes a conformational RNA epitope which occurs only in the nucleolus.

Animals↗