Fluorochrome Acridine Orange Research Articles

Laboratory techniques for diagnosing Giardia parasite in dogs in Mosul City, Iraq

The results of the current study showed that the different techniques of microscopic examination recorded the highest rate of infection and diagnosis of Giardia in dogs with significant differences at the level of probability p<0.05 It reached 73.4%, followed by the rapid test technique and indirect enzyme linked immunosorbent assay (ELISA) with a rate of 14.2% and 12.2% respectively. With regard to determining the sensitivity and specificity of the rapid test and microscopic examination, a sensitivity of 19% and a specificity of 100% were recorded. The results, using the indirect ELISA showed an infection rate of 12.2% of the total samples. The results showed that the number of positive samples with this technique amounted to 12 samples positive for Giardia lamblia out of the total samples positive for microscopic examination. To confirm the diagnosis of giardiasis, the results using the direct immunofluorescence technique using acridine orange fluorochrome showed the appearance of a glowing brilliance, as the cysts appeared oval green glowing and the trophozoites appeared glowing green pear.

Bedbug salivation patterns during hematophagy in the skin of a mammalian host

Cimex lectularius (Hemiptera:Cimicidae) infestations have increased over the past decades in several parts of the world, constituting a major urban pest with no reversion signs. The impact on human health caused by these insects, commonly known as bedbugs, is associated with their obligatory hematophagous habit. Allergies induced by hematophagous arthropod bites are related to the deposition of salivary molecules in the host tissues. Many reports of humans developing severe allergic reactions due to bedbug bites have been recorded, however, there is limited information on the salivation of bedbugs on the host, which was the objective of this study. C. lectularius females were fed on blood containing acridine orange fluorochrome, which labeled the principal salivary glands content. The salivation pattern of bedbugs was investigated using intravital microscopy during its blood meal on the ear skin of hairless mice. Saliva deposition occurred during all insect blood-feeding phases, beginning as soon as the mouthpart touched the host skin. During the probing phase, saliva was deposited in large quantities in the host dermis. In contrast, during the engorgement phase (which represents the largest blood meal of the insects), saliva was released at a much slower rate. The apparent release of saliva into the cannulated vessel and/or adjacent tissue occurs only sporadically during insect blood ingestion. However, a small area (spot) of fluorescence was detected around the proboscis tip during this feeding phase. An interesting feature of bedbugs is that they release saliva inside and outside the vessels without removing their mouthparts from the vessel lumen. This is an effective feeding strategy because it does not interrupt blood ingestion and decreases the mouthparts movements on the host’s skin, minimizing the damage to tissues and contact time with the host (feeding time).

Reproductive Aspects of Chagas Disease Vectors: Evidence of Transcriptional Activity during the Nucleolar Persistence Phenomenon in the Spermatogenesis of Triatomines.

The subfamily Triatominae currently consists of 154 species, most of them being of great importance for public health because they are considered potential vectors of the protozoan Trypanosoma cruzi, the etiologic agent of Chagas disease. In addition to their epidemiological importance, these insects are considered important biological models for cell studies because they have peculiar characteristics in their cells, for example, persistence of the nucleolus during spermatogenesis. This phenomenon is characterized by the presence of the nucleolus or nucleolar corpuscles during all phases of meiosis. To date, all knowledge is restricted to the study of the presence/absence of the nucleolus during the triatomine meiosis, so the present work aimed to analyze if this persistent nucleolar material has transcriptional activity. Analysis of the meiotic metaphases of Rhodnius montenegrensis and Panstrongylus megistus by using fluorochrome acridine orange made it possible to characterize the presence of RNA in the nucleolar material. Thus, it was demonstrated, for the first time, that the persistent nucleolar material during triatomine meiosis is transcriptionally active, supporting the hypothesis of the relationship between nucleolar persistence during meiosis of these insects and the formation of the chromoid body, an organelle responsible for the support of all transcriptional activities during spermiogenesis.

Concurrent detection of lysosome and tissue transglutaminase activation in relation to cell cycle position during apoptosis induced by different anticancer drugs.

Described is the new cytometric approach do detect either stimulation or a collapse of lysosomal proton pump (lysosomes rupture) combined with activation of transglutaminase 2 (TG2) during induction of apoptosis. Apoptosis of human lymphoblastoid TK6 cells was induced by combination of 2-deoxyglucose with the isoquinoline alkaloid berberine, by DNA topoisomerase I inhibitor camptothecin, its analog topotecan, topoisomerase II inhibitors etoposide or mitoxantrone, as well as by the cytotoxic anticancer ribonuclease ranpirnase (onconase). Activity of the proton pump of lysosomes was assessed by measuring entrapment and accumulation of the basic fluorochrome acridine orange (AO) resulting in its metachromatic red luminescence (F>640 ) within these organelles. Activation of TG2 was detected in the same cell subpopulation by the evidence of crosslinking of cytoplasmic proteins revealed by the increased intensity of the side light scatter (SSC) as well as following cell lysis by detergent, by its red fluorescence after staining by sulforhodamine 101. Because at low AO concentration nuclear DNA of the lysed cells was stoichiometrically stained green (F530 ) its quantity provided information on effects of the drug treatments on cell cycle in relation to activation of TG2. The data reveal that activation of lysosomal proton pump was evident in subpopulations of cells treated with 2-deoxyglucose plus berberine, topotecan, etoposide and mitoxantrone but not with ranpirnase. The collapse of lysosomal proton pump possibly reporting rupture of these organelles was observed in definite cell subpopulations after treatment with each of the studied drugs. Because regardless of the inducer of apoptosis TG2 activation invariably was correlated with lysosomes rupture it is likely that it was triggered by calcium ions or protons released from the ruptured lysosomes. This new methodological approach offers the means to investigate mechanisms and factors affecting autophagic lysosomes proton pump activity vis-à-vis TG2 activation that are common in several pathological states. © 2019 International Society for Advancement of Cytometry.

Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H+ homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.

Immunocytochemical markers of neuronal maturation in human diagnostic neuropathology.

Histological descriptions of morphogenesis in human fetal brain and in malformations and tumours can now be supplemented by the timing and sequence of the maturation of individual neurons. In human neuropathology, this is principally achieved by immunocytochemical reactivities used as maturational markers of neuronal properties denoted by molecules and cell products. Cytological markers can appear early and then regress, often being replaced by more mature molecules, or might not exhibit the onset of immunoreactivity until a certain stage of neuronal differentiation is achieved, some early, others intermediate and some late during the maturational process. Inter-specific differences occur in some structures of the brain. The classification of markers of neuronal maturation can be based, in addition to those mentioned above, on several criteria: cytological localisation, water solubility, biochemical nature of the antigen, specificity and various technical factors. The most useful immunocytochemical markers of neuronal maturation in human neuropathology are NeuN, synaptophysin, calretinin and other calcium-binding molecules, various microtubule-associated proteins and chromogranins. Non-antibody histochemical stains that denote maturational processes include luxol fast blue for myelination, acridine orange fluorochrome for nucleic acids, mitochondrial respiratory chain enzymes and argentophilic impregnations. Neural crest derivatives of the peripheral nervous system, including chromaffin and neuroendocrine cells, have special features that are shared and others that differ greatly between lineages. Other techniques used in human diagnostic neuropathology, particularly as applied to tumours, include chromosomal and genetic analyses, the mTOR signalling pathway, BRAF V600E and other tumour-suppressor gene products, transcription products of developmental genes and the proliferation index of the tumour cells and of mitotic neuroepithelial cells.

Bacterial and Archaeal direct counts: A faster method of enumeration, for enrichment cultures and aqueous environmental samples

A new presence/absence method has been developed to count fluorochrome-stained bacterial and archaeal cells on membrane filters using epifluorescence microscopy. This approach was derived from the random distribution of cells on membranes that allowed the use of the Poisson distribution to estimate total cell densities. Comparison with the standard Acridine Orange Direct Count (AODC) technique shows no significant difference in the estimation of total cell populations, or any reduction in the precision of these estimations. The new method offers advantages over the standard AODC in considerably faster counting, as there is no need to discriminate between every potential cell visible on a field and fluorescent detritus, it is only necessary to confirm the presence of one cell. Additionally, the new method requires less skill, so has less reliance on expert counters, and that should reduce inter-counter variability. Although this work used the fluorochrome Acridine Orange, clearly the results are applicable to any fluorochrome used to count bacterial and archaeal cells. This method was developed using enrichment cultures for use with enrichment cultures and aqueous environmental samples where interfering detrital and mineral particles are minimal e.g., freshwater/seawater, therefore, it is not suitable for estimating total cells from sediment samples. This method has the potential for use in any situation where counts of randomly distributed items are made using a grid or quadrat system.

Phenomenon of selective staining of pericentromeric heterochromatin regions in chromosomes from spontaneously dividing cells by the acridine orange fluorochrome

A novel phenomenon of unusual selective acridine orange (AO) staining of pericentromeric heterochromatin regions (HRs) in chromosomal preparations from tissue with known spontaneous mitotic activity (chorionic villi, placenta, embryonic tissues, bone marrow, and testes), as well as embryonic stem cells, is described. Staining with 0.01% AO in a citric-phosphate (pH 5.5) or sodium phosphate (pH 7.0) buffer solution allows the HRs of human chromosomes (1q12, 9q12, 13p11.2, 14p11.2, 15p11.2, 16q11.2, 21p11.2, 22p11.2, and Yq12) and pericentromeric HRs of mouse chromosomes to be reliably detected by the red fluorescence of AO. This method of AO staining does not require any pretreatment. Explanations for metachromatic AO staining of polymorphic pericentromeric HRs in chromosomes of spontaneously dividing cells are suggested. A high reproducibility of the specific AO staining makes it possible to suggest its using as a reliable quick method for detection of polymorphic HRs of human chromosomes in cytogenetic prenatal diagnosis and oncohematology.

Antitumor Activity of Metal-Chelating Compound Dp44mT Is Mediated by Formation of a Redox-Active Copper Complex That Accumulates in Lysosomes

The metal-chelating compound Dp44mT is a di-2-pyridylketone thiosemicarbazone (DpT) which displays potent and selective antitumor activity. This compound is receiving translational attention, but its mechanism is poorly understood. Here, we report that Dp44mT targets lysosome integrity through copper binding. Studies using the lysosomotropic fluorochrome acridine orange established that the copper-Dp44mT complex (Cu[Dp44mT]) disrupted lysosomes. This targeting was confirmed with pepstatin A-BODIPY FL, which showed redistribution of cathepsin D to the cytosol with ensuing cleavage of the proapoptotic BH3 protein Bid. Redox activity of Cu[Dp44mT] caused cellular depletion of glutathione, and lysosomal damage was prevented by cotreatment with the glutathione precursor N-acetylcysteine. Copper binding was essential for the potent antitumor activity of Dp44mT, as coincubation with nontoxic copper chelators markedly attenuated its cytotoxicity. Taken together, our studies show how the lysosomal apoptotic pathway can be selectively activated in cancer cells by sequestration of redox-active copper. Our findings define a novel generalized strategy to selectively target lysosome function for chemotherapeutic intervention against cancer.

Cytometric detection of chromatin relaxation, an early reporter of DNA damage response

One of the early events of the DNA damage response (DDR), particularly if the damage involves induction of DNA double-strand breaks, is remodeling of chromatin structure characterized by its relaxation (decondensation). The relaxation increases accessibility of the damaged DNA sites to the repair machinery. We present here a simple cytometric approach to detect chromatin relaxation based on the analysis of the proclivity of DNA in situ to undergo denaturation after treatment with acid. DNA denaturation is probed by the metachromatic fluorochrome acridine orange (AO) which differentially stains single-stranded (denatured) DNA by fluorescing red and the double-stranded DNA by emitting green fluorescence. DNA damage was induced in both human leukemic TK6 cells and mitogen-stimulated human peripheral blood lymphocytes by exposure to UV light or by treatment with H2O2. Chromatin relaxation was revealed by diminished susceptibility of DNA to denaturation, likely reflecting decreased DNA torsional stress, seen as soon 10 min after subjecting cells to UV or H2O2. While cells in all phases of the cell cycle showed a comparable extent of chromatin relaxation upon UV or H2O2 exposure, H2AX was phosphorylated on Ser139 predominantly in S-phase cells. The data are consistent with the notion that chromatin relaxation is global, affects all cells with damaged DNA, and is a prerequisite to the subsequent steps of DDR that can be selective to cells in a particular phase of the cell cycle. The method offers a rapid and simple means of detecting genotoxic insult on cells.

Evidence of sperm nuclear chromatin heterogeneity in ex-cryptorchid subjects.

Ex-cryptorchid subjects are frequently affected by infertility, even if the usually determined seminal parameters are still within the normal range. We evaluated in this study, in 14 ex-unilateral cryptorchid adult males (22-28 years), with normal sperm concentration, morphology and viability, the resistance of sperm nuclear chromatin to denaturation, using the fluorochrome acridine orange, which fluorescences green when bound to native DNA (double stranded) and red when bound to denaturated DNA (single stranded). Our findings demonstrate that the percentage of green-stained cells with acridine orange was significantly lower in our patients than in controls (p less than 0.001). We therefore suggest that the chromatin of ex-cryptorchid subjects' spermatozoa has a decreased resistance to denaturation and we hypothesize that this finding may explain the infertility of these patients, when the common seminal parameters are still within the normal range.

Morphological aspects ofPassiflora edulisf.flavicarpachromosomes using acridine orange banding and rDNA-FISH tools

Abstract Passiflora edulis f. flavicarpa is considered the most important species of the genus Passiflora, mainly because of its botanical and commercial importance, as well as for crop breeding and genomic programs. However, its chromosome characterization has been conflicting on what concerns the number and localization of secondary constrictions (SC) and nucleolus organizer regions (NORs). In this context, conventional and molecular cytogenetic methodologies have been adapted to resolve these morphology problems. Cellular dissociation, airdrying techniques and Giemsa staining, acridine orange fluorochrome and fluorescent in situ hybridization (FISH) methodologies were used. We identified four secondary constrictions, in the subterminal portion of the long arm of chromosomes 1 and 8 and in the subterminal portion of short arm of chromosomes 2 and 7. Regions emitting yellowish fluorescence were observed on chromosomes 7 and 8 after acridine orange staining. The same pairs were evidenced with the rDNA 18S...

Study of the nucleolar cycle and ribosomal RNA distribution during meiosis in triatomines (Triatominae, Heteroptera)

Aspects of nucleolar activity during spermatogenesis were assessed in three triatomine species ( Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans) using cytochemical and fluorescent staining techniques. Toluidine blue and a variant of critical electrolytic concentration (CEC) allowed the discrimination of rRNA providing structural details of the nucleolus and RNA distribution during meiotic cell division. Acridine orange fluorochrome staining permitted the differentiation of nucleic acids, and silver-ion impregnation made possible the observation of pre-nucleolar bodies (PNBs). Our results support the phenomenon known as “persistence of the nucleolar material”, and the hypothesis of post-meiotic reactivation of rRNA genes. Nucleolar organizer regions (NORs) were observed in some metaphasic spermatogonial chromosomes in P. megistus and T. infestans. In P. megistus at diplotene–diakinesis, NORs were also detected in one of the sex chromosomes and in an autosome. Therefore, it may be inferred that, in triatomines, the nucleolus does not completely disappear, but persists in the form of small bodies that get together to form the next nucleolar cycle which, in the case of meiosis, will be completed if fertilization occurs and a new zygote is formed.

2,4-Dichlorophenoxyacetic acid: Effects on Syrian hamster embryo (SHE) cell transformation, c- Myc expression, DNA damage and apoptosis

2,4-Dichlorophenoxyacetic acid (2,4-D) is a selective, systemic auxin-type herbicide extensively used throughout the world. The present research was aimed at studying effects of low and non-cytotoxic concentrations of 2,4-D on SHE cells in relation with carcinogenicity. Effects were studied on Syrian hamster morphological cell transformation, c-Myc expression – both at the gene and protein level – DNA damage and apoptosis. 2,4-D significantly induced cell transformation at 11.5 μM and 23 μM (i.e. 2.5μg/mL and 5 μg/mL). An increase in the expression of the transcription factor c-Myc, measured by use of RT-PCR with respect to mRNA level and by Western blotting for protein level was registered at these concentrations, as well as genotoxic effects evaluated with the single-cell gel electrophoresis (Comet) assay. Consequences for apoptosis of 2,4-D treatment were also investigated. The fluorochrome acridine orange was used to study DNA fragmentation as a marker of apoptosis. No effect on apoptosis was found at 2,4-D concentrations that induced cell transformation. This was confirmed by the unchanged expression of Bcl-2 and Bax, two regulator genes of the mitochondrial pathway of apoptosis. Our results demonstrate the transforming and genotoxic effects of low concentrations of 2,4-D in mammalian cells. This information contributes to a better understanding of the mechanism of 2,4-D toxicity in mammalian cells and demonstrates that 2,4-D should be considered as potentially hazardous to humans.

Re‐evaluating acridine orange for rapid flow cytometric enumeration of parasitemia in malaria‐infected rodents

Methods facilitating research in malaria are of pivotal relevance. Flow cytometry offers the possibility of rapid enumeration of parasitemia. It relies on staining the parasite DNA to distinguish between infected and non-infected red blood cell (RBC) populations. Unfortunately, in rodents abundant reticulocyte RNA interferes with the application of the method. This results in time-consuming sample preparation protocols that offer no clear advantage over microscopic counting. We re-evaluated the use of the DNA/RNA discriminating vital fluorochrome acridine orange (AO) for rapid flow cytometric enumeration of parasitemia in rodents. Whole blood from rodents infected with Plasmodium berghei and Plasmodium yoelii was stained with AO and analyzed by flow cytometer. A newly developed two-channel (FL1/FL3) detection method was compared with conventional one-channel (FL1) detection and microscopic counting. The new AO two-channel detection method clearly discriminated between infected and non-infected RBC populations. It showed to be linear above parasitemias of 0.3%. Sample processing time amounted to approximately 5 min. It is shown that AO can be used for rapid, precise, and accurate enumeration of parasitemia in rodents. Due to its ease of handling the method might find widespread application in malaria research.

Use of the fluorchrome vital dye acridine orange to determine viability and germination of Tilletia indica teliospores in soil

A vital staining method using the fluorochrome acridine orange (AO) was developed for staining teliospores and primary sporidia (basidiospores) of Tilletia indica the causal agent of Karnal bunt of wheat. This procedure allows determination of the viability of teliospores in soil. Also the method makes it possible to monitor germination of teliospores in different soil types under different soil moisture levels. The primary sporidia produced from germinated teliospores could be easily detected in small soil samples (10 g). This is the first report on evaluation of germination of viable teliospores directly in different soil types. This method makes it possible qualitatively to determine the germination of teliospores in nonsterile soil taken directly from the field. The AO procedure, as a supplement to the quarantine inspection of seed lots, could be used for routine testing of the viability of teliospores of T. indica extracted from seed lots.

Intercellular cell adhesion molecule-1 regulates lymphocyte movement into intestinal microlymphatics of rat Peyer's patches

The objective of this study was to determine whether specific adhesion molecules modulate lymphocyte movement from Peyer's patches into intestinal microlymphatics. The fluorochrome acridine orange was injected via a micropipette into Peyer's patches to fill lymphatics. The flux of labeled lymphocytes into intestinal microlymphatics was monitored with intravital fluorescence microscopy. The lymphatic microvessels in the perifollicular area of Peyer's patches were filled with lymphocytes, most of which remained within the lymphatics. Some lymphocytes became detached and were drained into intestinal lymph. Administration of antibodies directed against ICAM-1 significantly increased lymphocyte flux into interfollicular lymphatics. The immunohistochemical study showed intense ICAM-1 expression on the lymphocytes densely packed in the lymphatics surrounding follicles in Peyer's patches. A large number of lymphocytes are normally sequestered in the lymphatic network of Peyer's patches. This sequestration of lymphocytes is largely mediated by ICAM-1-dependent cell-cell interactions.

Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter.

The cytometric methods of bivariate analysis of cellular RNA versus DNA content have limitations. The method based on the use of metachromatic fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument that provides two-laser excitation, including the ultraviolet (UV) light wavelength. Phytohemagglutinin (PHA)-stimulated human lymphocytes were deposited on microscope slides and fixed. DNA and double-stranded (ds) RNA were stained with propidium iodide (PI) and protein was stained with BODIPY 630/650-X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file-merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and after the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted from the fluorescence intensity of the same cell measured prior to RNase treatment. This RNase-specific differential value of fluorescence (differential fluorescence [DF]) was plotted against the cell fluorescence measured after RNase treatment or against the protein-associated BODIPY 630/650-X or FITC fluorescence. The scattergrams were characteristic of the RNA versus DNA bivariate distributions where DF represented cellular ds RNA content and fluorescence intensity of the RNase-treated cells, their DNA content. The distributions were used to correlate cellular ds RNA content with the cell cycle position or with protein content. One advantage of this novel approach based on the recording and plotting of DF is that only the RNase -specific fraction of cell fluorescence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell constituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wavelength detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same-wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents.

Sperm chromatin integrity of bucks transgenic for the WAP bGH gene

The aim of the study was to compare sperm chromatin structure of transgenic and non-transgenic rabbits. In addition, the effect of chromatin structure on semen fertility was determined. Twenty male rabbits transgenic (TG) for WAP bGH gene (Edison Biotechnology Institute Ohio University, USA) and nine non-transgenic (NTG) males were used. Both TG and NTG rabbits were 13–18 months old. Semen was collected at 1-week intervals and 3–7 ejaculates from each rabbit were examined in total.Sperm chromatin abnormalities were measured flow cytometrically according to the Sperm Chromatin Structure Assay method: after chromatin denaturation by low pH, sperm cells were stained with metachromatic fluorochrome acridine orange. Spermatozoa with abnormal chromatin structure and, subsequently, higher degree of denaturation, showed a shift in red fluorescence. Two different methods of semen fertility estimation were used: (1) for TG rabbits, AI of superovulated does and calculation of percentages of fertilised eggs and embryos developing in vitro to the blastocyst stage; (2) for NTG rabbits, AI of non-stimulated does and calculation of percentages of pregnant does and mean litter sizes.The mean value of COMPαt was 3.71 for TG rabbits and 2.89 for NTG rabbits (no significant difference, t-test). The mean values of S.D.αt for the TG and NTG rabbits were 10.94 and 10.40 (no significant difference, t-test), respectively. There were no significant correlations between sperm chromatin structure of TG males and the percentages of fertilised eggs or embryos developing to the blastocyst stage. A statistically significant correlation (−0.68, P<0.05) was found between S.D.αt of NTG males and percentages of pregnant does.The results showed chromatin stability was not different for sperm obtained from TG versus NTG bucks. The presence of WAP bGH gene construct in the genome of transgenic rabbits did not cause any spermatogenesis process disturbances leading to the production of spermatozoa with damaged chromatin structure. This suggests that the mere presence of the introduced gene construct does not lead to any abnormalities in DNA and chromatin proteins interaction. The possible chromatin damages in transgenic animals should be attributed to the activity of the introduced gene.The relationships between chromatin structure and fertility are only significant for sperm from NTG bucks.

Localization and Identification of Populations of Phosphatase-Active Bacterial Cells Associated with Activated Sludge Flocs.

Abstract The majority of phosphatase (PO(4)ase) activity detected in fresh aerobic activated sludge from a municipal wastewater treatment plant was associated with suspended floc material. PO(4)ase activity appeared to be localized in discrete bacteria-containing areas of the floc matrix based on the distribution of nucleic acid-stained cells and precipitated fluorescent crystals produced as a result of reaction of the enzyme(s) with the artificial substrate ELFtrade mark-PO(4). Of the total floc-associated bacterial cells that stained positive with the nucleic acid-binding fluorochrome acridine orange (AO), 8.8 +/- 1.2% displayed PO(4)ase activity based on the proximity of AO-stained cells to precipitated ELF crystals. Using a 16S rRNA oligonucleotide probe specific for the cytophaga-flavobacteria group, it was determined that 17-20% of the floc-associated bacteria that probed positive also displayed PO(4)ase activity. Furthermore, 35-45% of the ELF fluorescence was associated with bacterial cells that probed positive for the cytophaga-flavobacteria group. The results suggest that the cytophaga-flavobacteria, as a group, is important in mediating the liberation of inorganic orthophosphate (P(i)) from phosphomonoesters of detrital organic phosphate (organic-P) in the aerobic activated sludge process of wastewater treatment.http://link.springer-ny.com/link/service/journals/00248/bibs/38n3p201.html</hea