Acridine Orange Technique Research Articles

Apoptosis demonstration of the renal tubules after ischemia and reperfusion by acridine orange technique

Ischemia – reperfusion injury is a complex phenomenon that results in cell damage through a biphasic process. Ischemia initiates the injury by a decrease or complete loss of energy supply needed to maintain homeostasis. Reperfusion increases cellular damage by a variety of proposed mechanisms such as inflammatory reaction and release of oxygen free radicals. I-R resulting in both pathological cell death (necrosis) and programmed cell death (apoptosis) in human and experiments. This study performed to demonstrate the apoptosis and necrosis the renal tubules under the effect of I/R in by florescent acridine orange. Twenty five male rats were divided into six groups: control group, ischemia group (40 minute clamping of renal artery) and ischemia – reperfusion group (the removal of renal clamping after 1, 3, and 6 hours).The samples were prepared for histological technique and the sections stained with acridine orange and examined by florescent microscope. The results observed mild changes in the ischemic group but the damage of the tissue and cells occurred in the reperfusion groups reaches the maximum effect after 1 and 3 hour, the necrosis and apoptosis were observed clearly.After 6 hours reperfusion, emit orange fluorescence as evidence of nuclear fragmentation (apoptosis) in cells.We concluded that the apoptosis and necrosis occurred in the reperfusion after 40 minutes ischemia; also the acridine orange stain is effective stain to investigate the apoptosis and the cellular changes.

Evaluation of sperm head dimensions and chromatin integrity of epididymal sperm from domestic cats using the toluidine blue technique

When using assisted reproductive technologies, there is seldom an evaluation of DNA integrity during sperm analysis, which is an important variable for proper embryo development. The toluidine blue staining technique allows the simultaneous evaluation of sperm chromatin and sperm head dimensions. The objectives of this study were to evaluate the applicability of the toluidine blue staining method for analyzing DNA abnormalities in epididymal sperm (from the caput, corpus, and cauda) of cats and to investigate the correlations among DNA condensation, morphology, and sperm head dimensions. The DNA alteration indexes were obtained using the toluidine blue and acridine orange techniques, and comparisons of these indexes indicated there was a 65.4% (r = 0.654; P < 0.001) correlation. The sperm from the cauda had greater chromatin stability (97.9%) than the sperm from the epididymal head (92.1%; P = 0.0023). There, however, was no difference in chromatin stability between sperm obtained from the corpus and cauda regions, indicating that these sperm were already mature. The sperm head dimension was correlated with chromatin decondensation, and the sperm head size decreased as the sperm were transported through the three epididymal regions (P < 0.0001). In addition, the percentage of sperm that were deficient in chromatin condensation decreased as the sperm were transported through the epididymal caput, corpus and cauda (26.4, 15.7, and 3.4%, respectively; P < 0.0001). Thus, the sperm head size predicts the quality of chromatin condensation in sperm cells.

Ubiquitin-proteasome system (UPS) regulates spermadhesin release during boar sperm capacitation

When using assisted reproductive technologies, there is seldom an evaluation of DNA integrity during sperm analysis, which is an important variable for proper embryo development. The toluidine blue staining technique allows the simultaneous evaluation of sperm chromatin and sperm head dimensions. The objectives of this study were to evaluate the applicability of the toluidine blue staining method for analyzing DNA abnormalities in epididymal sperm (from the caput, corpus, and cauda) of cats and to investigate the correlations among DNA condensation, morphology, and sperm head dimensions. The DNA alteration indexes were obtained using the toluidine blue and acridine orange techniques, and comparisons of these indexes indicated there was a 65.4% (r = 0.654; P < 0.001) correlation. The sperm from the cauda had greater chromatin stability (97.9%) than the sperm from the epididymal head (92.1%; P = 0.0023). There, however, was no difference in chromatin stability between sperm obtained from the corpus and cauda regions, indicating that these sperm were already mature. The sperm head dimension was correlated with chromatin decondensation, and the sperm head size decreased as the sperm were transported through the three epididymal regions (P < 0.0001). In addition, the percentage of sperm that were deficient in chromatin condensation decreased as the sperm were transported through the epididymal caput, corpus and cauda (26.4, 15.7, and 3.4%, respectively; P < 0.0001). Thus, the sperm head size predicts the quality of chromatin condensation in sperm cells.

Ejaculation effect on prostatic Pulsed-Wave Doppler ultrasound in dogs

When using assisted reproductive technologies, there is seldom an evaluation of DNA integrity during sperm analysis, which is an important variable for proper embryo development. The toluidine blue staining technique allows the simultaneous evaluation of sperm chromatin and sperm head dimensions. The objectives of this study were to evaluate the applicability of the toluidine blue staining method for analyzing DNA abnormalities in epididymal sperm (from the caput, corpus, and cauda) of cats and to investigate the correlations among DNA condensation, morphology, and sperm head dimensions. The DNA alteration indexes were obtained using the toluidine blue and acridine orange techniques, and comparisons of these indexes indicated there was a 65.4% (r = 0.654; P < 0.001) correlation. The sperm from the cauda had greater chromatin stability (97.9%) than the sperm from the epididymal head (92.1%; P = 0.0023). There, however, was no difference in chromatin stability between sperm obtained from the corpus and cauda regions, indicating that these sperm were already mature. The sperm head dimension was correlated with chromatin decondensation, and the sperm head size decreased as the sperm were transported through the three epididymal regions (P < 0.0001). In addition, the percentage of sperm that were deficient in chromatin condensation decreased as the sperm were transported through the epididymal caput, corpus and cauda (26.4, 15.7, and 3.4%, respectively; P < 0.0001). Thus, the sperm head size predicts the quality of chromatin condensation in sperm cells.

Anthropogenic Influence on Blood Biomarkers of Stress and Genotoxicity of the Burrowing Owl (Athene Cunicularia)

Anthropogenic activities are putting increasing pressure on ecosystems and raising the need to assess environmental health quickly and accurately. Heterophile/lymphocyte index is accepted as a response to stress factors and the micronucleus test is used as a biomarker to estimate genotoxic damage. To evaluate a model of environmental quality, healthy Burrowing owl (Athene cunicularia) were sampled, and the heterophile/lymphocyte ratio was determined, as well as the frequency of micronucleated erythrocytes and nuclear abnormalities, determined with the Wright-Giemsa and acridine orange technique. The sites with the greatest antropic disturbance recorded the highest frequencies of micronuclei and heterophile/lymphocyte index. The combination of both tests allows the detection of possible acute or chronic exposure to stressors and genotoxic contaminants, both in healthy or altered ecosystems.

Morphological characterization of the gonads of bullfrog tadpoles (Lithobates catesbeianus)

Abstract Introduction: This work describes various aspects of early gonadal development of female and male in pre-metamorphic tadpoles (Lithobates catesbeianus) at Gosner stage 25. Materials and Methods: Ovaries and testicles were prepared for routine light microscopy for morphological study and for acridine orange technique fluroescent microscopy for observation of RNA cytoplasm activity. Results: The results showed that female gonads at Gosner stage 25 predominated primary and secondary oogonias, as well as primary, secondary and tertiary oocytes. The developing testicle presented primary spermatogonia (I or A) and secondary spermatogonia (II or B), and as well as primary and secondary spermatocyte. All this cell phases were morphologically characterized and the cell sizes measured. In pre-metamorphic testes the somniferous duct are not developed and the vasa deferentia is opened. Conclusion: At this point, it was possible differentiate ovary from testes does not for the gonadal cells, but for the general anatomy of the organs, being the ovary a spheroid structure and the testicle an elongated tubule.

The Phosphatidylinositol-3-Kinase Inhibitor BKM120 Impairs Proliferation and Induces Pro-Apoptotic Effects In Acute Myeloid Leukemia

Acute myeloid leukemia (AML) has been characterized by a growing number of recurrent genetic alterations, frequently causing the constitutive activation of signal transduction pathways, which result in enhanced blast proliferation and prolonged survival. Despite the increased understanding of AML biology, prognosis of these patients remains, generally, severe. Therefore, novel therapies targeted on aberrant signaling are now under evaluation. The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a pivotal role in the control of cell growth, proliferation and apoptosis of AML, and is often found constitutively activated. Currently, several inhibitors of this pathway, especially those inhibiting the activity of mTOR, have been investigated showing a limited efficacy due to the reactivation of upstream nodes. Since a novel PI3K inhibitor, BKM120, has been evaluated with encouraging results on solid tumors and lymphoid malignancies, we aimed in this study to investigate the pre-clinical activity of BKM120 on cell lines and primary AML samples. BKM120, kindly provided by Novartis, was tested on myeloid leukemia cell models - U937, MOLM13, OCIAML3 and KG1 - and on 4 primary AML samples at concentrations ranging from 0.5 to 5µM. Cell cycle changes and apoptosis induction were analyzed using the Acridine-Orange technique (AO) and the Annexin V (AnnV) binding assay. Intracellular signaling modulations induced by BKM120 were evaluated at different times (3h and 24h) by Western blot analysis. BKM120 exhibited dose- and time-dependent anti-proliferative and pro-apoptotic effects on the all analyzed leukemia cell models, although with different sensitivity (IC50s of 1.6, 2.4, 2.7 and 3.2μM for the U937, MOLM13, OCIAML3 and KG1 cell lines, respectively). On the most sensitive cell line U937, BKM120 in vitro exposure resulted, at 24h, in a significant cell growth reduction of 48.6% (p=0.006) and 73.9% (p=0.006), at 1 and 2µM, respectively. After 72h of treatment, AnnV positive cells significantly increased from 7.4% ± 0.03 (vehicle) to 43.5% ± 0.08 (p<0.001) and 89.4% ± 0.05 (p<0.001), at 1 and 2μM BKM120, respectively. On the less sensitive cell line KG1, the percentage of apoptotic cells reached 49.6% ± 0.1 and 56.6% ± 0.2 at 2 and 5μM BKM120, respectively. Since our previous data (Ricciardi MR et al., JMM, 2012) have been reported that the U937 cell line is characterized by PI3k/Akt/mTOR hyperactivation while the OCIAML3 cell line relies mostly on the Ras/Raf/MEK/Erk pathway, we evaluated the BKM120-induced intracellular signaling modulations on this two cell lines. After 3h of BKM120 exposure, the analysis of activation status of the PI3K/Akt/mTOR pathway revealed, on the U937, the downregulation of pAkt levels at 0.5µM associated with p70 and 4EBP1 dephosphorylation at 1µM. At variance, on the OCIAML3, both effects were achieved at higher concentrations (1 and 2µM, respectively), reflecting the different sensitivity to apoptosis of this model. A limited cytotoxicity (1.2 fold increase of apoptosis) was observed on normal PBMCs isolated from healthy volunteers following BKM120 exposure. In contrast, a pro-apoptotic activity was seen at 144h on the 4 primary AML cells (5.9, 3.9, 2.2 and 2.1 fold increase of apoptosis). Moreover, a mean increase of AnnV positive cells from 13.0% ± 0.06 (vehicle) to 23.2% ± 0.15, 25.6% ± 0.15, 32.9% ± 0.1 (p=0.02) and 40.3% ± 0.13 (p=0.02) was obtained on this cells following exposure to 0.5, 1, 2 and 5μM, respectively. In conclusion, our study demonstrates, at our knowledge for the first time, that the novel pan-class I PI3K inhibitor BKM120 impairs AML cells growth and proliferation inducing a pro-apoptotic activity and inhibiting the PI3K/Akt/mTOR signaling through dephosphorylation of the key components of this pathway. Disclosures:No relevant conflicts of interest to declare.

Anti-clastogenic Effect of Magnolol-Containing Hange-koboku-to, Dai-joki-to, Goshaku-san, and Magnoliae Cortex on Benzo(a)pyrene-Induced Clastogenicity in Mice

Magnolol was previously shown to inhibit genotoxicity induced by environmental mutagens both in vitro and in vivo. Here, we investigate the effects of the magnolol-containing kampo (traditional) medicines Hange-koboku-to, Dai-joki-to, and Goshaku-san, as well as Magnoliae Cortex, on the clastogenesis induced by benzo(a)pyrene (B(a)P) using the mouse micronucleus test. The mice were first treated with a single intraperitoneal injection of B(a)P, followed by a single oral dose of Hange-koboku-to, Dai-joki-to, Goshaku-san, or Magnoliae Cortex. Peripheral blood specimens were prepared 48 h after B(a)P administration and analyzed using the acridine orange (AO) technique. The anti-clastogenic mechanisms employed by the kampo medicines and Magnoliae Cortex were also investigated by evaluating in vivo CYP1A1 activity using the zoxazolamine paralysis test. Results show that Hange-koboku-to, Dai-joki-to, and Magnoliae Cortex, which contain high levels of magnolol, significantly inhibited the clastogenesis induced by B(a)P and sufficiently inhibited in vivo CYP1A1 activity. In contrast, Goshaku-san, which contains low levels of magnolol, had little inhibitory effect on clastogenicity and in vivo CYP1A1 activity. These findings suggest that magnolol is a major contributor to the inhibition of B(a)P-induced clastogenesis, and that kampo medicines exert significant anti-clastogenic effects.

The clinical significance of sperm DNA damage detection combined with routine semen testing in assisted reproduction

Traditional routine semen analysis and biochemical assays are insufficient for the determination of fertility status in individual men. In recent years, clinical evidence has shown that damaged human sperm DNA may adversely affect assisted reproductive outcomes and that the spermatozoa of infertile men possess substantially more DNA damage than the spermatozoa of fertile men. In this study, we combined these two methods to test their clinical significance in assisted reproductive techniques (ART). A total of 302 in vitro fertilization (IVF) and 67 intracytoplasmic sperm injection (ICSI) couples were included in the study. The acridine orange technique (AOT) was used to detect the DNA integrity of the sperm. Correlation analysis showed that the DNA fragmentation index (DFI) was negatively related to the no. of good quality embryos and the clinical pregnancy rate in IVF patients. No significant correlations were found between DFI and ICSI patients. We then combined the results of the routine semen testing of IVF patients with their DFI values. Statistical analysis revealed notable differences in the no. of good quality embryos (P=0.015) and the clinical pregnancy rate (P=0.015) between subgroups divided according to DFI value in a group with normal semen test results (Group 1). In a group with abnormal semen test results (Group 2), the fertilization rate (P=0.034) and the pregnancy rate (P=0.018) showed remarkable variation between DFI subgroups. In conclusion, the detection of damaged DNA in spermatozoa needs to be conducted along with standard semen analysis. This might prove to be a promising predictor of ART outcome, particularly in IVF.

IgVH Germline and Mutated Chronic Lymphocytic Leukemia (CLL) Cases Exert a Diverse Responsiveness upon BCR Ligation.

Purpose. CLL is an heterogeneous disease with a variable clinical course. In order to investigate the association between specific clinico-biological features and the ability of CLL cells to respond to anti-IgM-mediated signaling, we evaluated the gene expression changes upon BCR stimulation as well as the changes in cell cycle, proliferation and apoptotic rate of IgVH mutated and unmutated samples.Methods. After 24 hours incubation with a F(ab)2 anti-m antibody (10 mg/ml), unstimulated (US) and stimulated (S) CD19+ B cells isolated from untreated CLL patients underwent microarrays analysis using the HGU133 Plus 2.0 Affymetrix arrays. Unsupervised clustering and t-test analyses were performed. In addition, Q-PCR analysis was carried out to evaluate the levels of SYK and ZAP-70 expression in CLL samples at different time points (6 and 24 hours) upon BCR ligation. At 24, 48 and 72 hours from the stimulus, cell cycle distribution changes were evaluated using the Acridine Orange (AO) technique, cell proliferation was measured by 3H-TdR uptake and apoptosis was analyzed by the Annexin-V and/or AO technique.Results. Unsupervised analysis on CLL samples showed that response to BCR stimulation is strictly associated to the IgVH mutational status and IgM expression levels. Based on these findings, to specifically identify the genes that were modulated upon BCR ligation, we performed a t-test to compare US and S samples within germline and mutated cases. In the germline cases, this analysis identified 197 genes differentially expressed; among the more represented functional groups, we found several genes involved in signal transduction, regulation of transcription, cell cycle regulation as well as cytoskeleton. Contrariwise, using the same approach for the mutated cases, no genes were selected in this analysis, suggesting that BCR stimulation induces relevant changes exclusively on IgVH germline patients. To investigate the effects of IgM cross-linking on BCR signaling, we evaluated SYK and ZAP-70 expression in US and S CLL samples by Q-PCR approach. These studies showed that SYK, but not ZAP-70, is down-modulated upon stimulus only in germline cases. Furthermore, cell cycle analysis and proliferation assay documented that IgM cross-linking at 48 hours induced a significant progression into the G1-phase (p=0.037) and a moderate increase of proliferative activity in CLL cells exclusively in germline patients. Moreover, at the same time point we observed only a partial reduction of the percentage of subG0/1 cells without changes in apoptosis in CLL germline cases; contrariwise, increased levels of apoptosis (p=0.04) were observed in S cells from CLL mutated cases.Conclusions. Gene expression profile highlights a different responsiveness to BCR stimulation between IgVH germline and mutated CLL samples. In line with these results, in vitro experiments have shown that differences in cell cycle distribution, proliferative activity and apoptosis levels upon BCR ligation correlate with the IgVH mutational status of the CLL samples, supporting the hypothesis that response to BCR-ligation may play a crucial role in disease progression in IgVH germline cases. * ST and RM equally contributed to the study

Anti-clastogenic effect of magnolol on benzo( a)pyrene-induced clastogenicity in mice

It was previously reported that magnolol strongly inhibited the mutagenicity induced by the indirect mutagens [benzo( a)pyrene (B( a)P), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-aminodipyrido[1,2-a:3′,2′-d]imidazole (Glu-P-2), 2-aminoanthracene (2AA), and 7,12-dimethylbenz[ a]anthracene (DMBA)] in Salmonella typhimurium TA98 and TA100 in the Ames test, and that the mechanism of this anti-mutagenic effect may involve the inhibition of the metabolic activation of indirect mutagen enzymes. In this study, the in vivo anti-clastogenic effect of magnolol against clastogenicity induced by B( a)P was evaluated using the micronucleus test in mice. Animals were treated with an oral administration of magnolol (1, 10, and 100 mg/kg) at −24, 0, 24, 48, 72, and 96 h before a single intraperitoneal injection of B( a)P. Peripheral blood specimens were prepared 48 h after administration of B( a)P, and analyzed by the acridine orange (AO) technique. The results indicated that magnolol inhibited clastogenicity induced by B( a)P at various administration times. In order to elucidate the mechanism behind this effect, we measured the activity of the detoxifying enzymes [UDP-glucuronosyltransferase (UGT) and glutathione-S-transferase (GST)] and antioxidative enzymes [superoxide dismutase (SOD) and catalase] in the liver when treated with an oral administration of magnolol at various administration times. Its effect on clastogenicity created by exposure to oxidative DNA damage-inducing X-ray irradiation was also evaluated using the micronucleus test in mice. Results showed that magnolol increased the activity of both UGT and SOD enzymes, and also inhibited the clastogenicity induced by X-ray irradiation. Magnolol had an anti-clastogenic effect on B( a)P in the micronucleus test as well as an anti-mutagenic effect on indirect mutagens in the Ames test. The anti-clastogenic effect of magnolol was also suggested by the increases in UGT and SOD enzyme activity, and by the attenuation of oxidative damage induced by X-ray irradiation.

Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin

The process of cryopreservation impairs sperm cell function, potentially leading to a reduction in fertility. The objectives of the present study were to evaluate the effects that cryopreservation using two different extenders has on sperm motility and mitochondrial function, as well as on the integrity of plasma membranes, acrosomal membranes and chromatin, using practical and objective techniques. The focus of the present study was to identify correlations between alterations in sperm membranes and sperm motility in cryopreserved bovine spermatozoa. Seven ejaculates were collected from eight Simmental bulls ( n = 56). After collection, semen volume and concentration were assessed for purposes of dilution. Sperm motility was evaluated subjectively and by computer-assisted semen analysis, morphological characteristics were evaluated by differential interference microscopy, the integrity of plasma and acrosomal membranes, as well as mitochondrial function, were determined using a combination of fluorescent probes containing fluorescein isothiocyanate– Pisum sativum agglutinin, propidium iodide or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide. Chromatin integrity was evaluated using the acridine orange technique. The semen was subsequently divided into two aliquots and diluted with one of two extenders (Bioxcell ® or Botu-Bov ®), after which both were packaged in 0.5 mL straws and frozen using an automated system. Two straws of semen from each treatment were thawed, and the semen parameters were evaluated as described above. Cryopreservation of sperm reduced motility, damaging plasma and acrosomal membranes, as well as decreasing mitochondrial function. The Botu-Bov ® extender was more effective in preserving sperm motility and membrane integrity than was the Bioxcell ® extender.

Flow Cytometric Analysis of Micronuclei in Peripheral Blood Reticulocytes: II. An Efficient Method of Monitoring Chromosomal Damage in the Rat

We have evaluated a flow cytometric method that allows assessment of micronucleated reticulocytes (MN-RETs) in microliter quantities of peripheral blood and compared results using this assay with those of established microscopic methods of scoring bone marrow and peripheral blood from rats treated with well-characterized genotoxic agents. Young reticulocytes (RETs) are labeled with FITC-anti-CD71 (transferrin receptor) and micronuclei with propidium iodide (with RNase treatment). Red blood cells parasitized with Plasmodia serve as a calibration standard for DNA content. Microscopic scoring used acridine orange (AO) staining of methanol-fixed slides or supravital AO staining. The effect of the rat spleen on the parameters evaluated was determined by comparing age- and sex-matched normal and splenectomized rats treated with cyclophosphamide, cis-platin, or vinblastine under treatment conditions that established a steady-state frequency of MN-RETs in the bone marrow and peripheral blood compartments. The data demonstrate the sensitivity and reproducibility of the flow cytometric assay in the Sprague-Dawley rat, and comparative studies using identical blinded samples at multiple laboratories show that inter- and intra-laboratory reproducibility is much higher with the flow method than with the microscopic methods currently employed for regulatory studies. A significant effect of splenic selection against genotoxicant-induced MN-RETs was observed with each of the three scoring methodologies, despite the fact that the flow and supravital AO techniques restrict analysis to the youngest fraction of RETs. The high precision of flow-based measurements also demonstrated a slight but statistically significant level of selection against spontaneously arising MN-RET. Despite these spleen effects, assay sensitivity for blood-based analyses was maintained by the flow method as it was shown to have superior counting statistics, lower variability, and higher sensitivity than manual scoring. The data suggest that flow cytometric assessment of micronucleus induction can be integrated into routine toxicity testing, eliminating the need for a separate bioassay.

Effect of chlorophyllin on chromium trioxide-induced micronuclei in polychromatic erythrocytes in mouse peripheral blood

The effect of chlorophyllin on micronucleated polychromatic erythrocytes (MN-PCE) induction by chromium trioxide (CrO 3) exposure in peripheral blood of mice was studied. Animals were treated with a single intraperitoneal dose of chlorophyllin (CHL) (20 mg/kg), CrO 3 (20 mg/kg), and CHL (20 mg/kg) 4 h before (CHL–CrO 3) or 4 h before and 20 h after chromium treatments (20 mg/kg; CHL–CrO 3–CHL). Peripheral blood samples were drawn from the caudal vein at 0, 12 and 48 h, and analyzed by the acridine orange (AO) technique. The results obtained in present study shown that CHL injection did not modify the number of MN-PCE. CrO 3 treatment resulted in a significantly increases 12 and 48 h after the injection, reaching a four-fold increase 48 h after CrO 3 administration. Whereas treatment with 20 mg/kg of CHL prior to chromium, decreased the MN frequency induced by chromium in the 12 h samples. When the samples were analyzed 48 h after CrO 3 injection, no significant differences between CHL–CrO 3 and CHL–CrO 3–CHL in comparison with CrO 3 treatment, were observed. These results indicate that increase of MN-PCE by CrO 3 is CHL-blocked in both protocols used (CHL–CrO 3 and CHL–CrO 3–CHL) at 12 h after treatment, but it was unable to modify the frequency of MN-PCE measured 48 h after CrO 3 injection. The absence of a protective effect by CHL in the MN-PCE induction by CrO 3 at 48 h, show that CHL has action only on one of the times of MN induction and suggests the possible action of CrO 3 by two different mechanisms, and not by CHL time-limited in vivo.

Does Captopril Have a Direct Pro-Apoptotic Effect?

The beneficial effect of ACE inhibitors on cardiovascular remodelling from hypertension and ischemic disease may be due to the effect of these drugs on the proliferation/cell death balance. We therefore investigated the effect of the addition of captopril in vitro, on the onset of apoptosis in human vascular myocytes, by using a propidium iodide fluorescence analysis and a morphological analysis using the acridine orange technique. Captopril (0.23 mM) caused an increase in apoptotic phenomena that was more than 3.5-fold than in controls both at the 24th (7.7 vs. 2%) and the 48th h (10.1 vs. 3.8%). The addition of propranolol strengthened the effect on apoptosis. The induction of apoptotic phenomena may be a mechanism by which ACE inhibitors affect cardiovascular remodelling and it might also explain the favorable effect these drugs have on diseases such as IgA nephropathy and diabetic nephropathy.

Direct acridine orange fluorescence examination of blood slides compared to current techniques for malaria diagnosis

The renewed interest in the use of fluorochromes for malaria diagnosis prompted us to evaluate the acridine orange fluorescence technique on blood slides, and to compare it with established techniques using thick and thin blood films and the QBC ™ malaria test, using the Giemsa-stained thick film technique as our standard method for comparison. We compared 123 positively diagnosed cases and 120 negative cases. For primary samples (day 0), the sensitivity of the thin blood film fluorescence acridine orange technique (AO) was 96·4%, and its specificity was 95·1%. In cases of imported malaria, with a prevalence rate of 16·2%, the positive predictive value was 79·2% and the negative predictive value 99·3%. Sensitivity of AO was significantly higher than that of Giemsa-stained thin blood films for parasitaemias <5000/μL. The potential of AO for species diagnosis of Plasmodium was 85·2%, using Giemsa-stained thin films as the reference technique. Where QBC ™ imposes a cost limitation, especially in developing countries, despite its high performance, the AO diagnostic technique is a valuable alternative, because of its simplicity, almost negligible cost, and its diagnostic reliability. The method may also have potential value in the diagnosis of other microbiological diseases.

Activation of proximal tubular Na(+)-H+ exchange by angiotensin II.

Stimulation of Na(+)-H+ exchange by angiotensin II (ANG II) was characterized in renal proximal tubular cells. Rabbit proximal nephron segments were incubated in the presence or absence of ANG II (5 x 10(-10) M), after which brush-border membrane vesicles (BBMV) were isolated and assayed for Na(+)-H+ antiporter activity using the acridine orange technique. Both the affinity (for sodium) and capacity of the carrier were elevated significantly (P less than 0.05) within 15 min of incubation with ANG II. To determine whether the stimulation of transport capacity involved a change in Na(+)-H+ antiporter density in the luminal membrane, binding of tritiated 5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) was measured in BBMV derived from control and ANG II-treated nephron segments, following maximal stimulation. This demonstrated a significant (P less than 0.05) increase in the maximal specific binding (Bmax) of [3H]MIA binding in the ANG II-treated group compared with control, of a magnitude sufficient to account for the observed change in maximal velocity (Vmax). The data indicate that the Vmax effect is caused by an apparent increase in the number (density) of active Na(+)-H+ carriers present in the luminal membrane. Finally, to test the possibility that the observed kinetic change involves an exocytic mechanism, the effect of colchicine on ANG II-stimulated antiporter activity was examined. The increase in Vmax due to ANG II was blocked by the addition of 0.5 mM colchicine to the incubation medium, whereas colchicine alone had no significant effect on the Vmax of Na(+)-H+ kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Correction of enhanced Na(+)-H+ exchange of rat small intestinal brush-border membranes in streptozotocin-induced diabetes by insulin or 1,25-dihydroxycholecalciferol.

Diabetes was induced in rats by administration of a single i.p. injection of streptozotocin (50 mg/kg body wt). After 7 d, diabetic rats were further treated with insulin or 1,25-dihydroxycholecalciferol [1,25(OH)2D3] for an additional 5-7 d. Control, diabetic, diabetic + insulin, and diabetic + 1,25(OH)2D3 rats were then killed, their proximal small intestines were removed, and villus-tip epithelial cells were isolated and used to prepare brush-border membrane vesicles. Preparations from each of these groups were then analyzed and compared with respect to their amiloride-sensitive, electroneutral Na(+)-H+ exchange activity, using 22Na uptake as well as acridine orange techniques. The results of these experiments demonstrated that (a) H+ gradient-dependent 22Na uptake as well as Na+ gradient-dependent transmembrane H+ fluxes were significantly increased in diabetic vesicles compared to their control counterparts, (b) kinetic studies demonstrated that this enhanced 22Na uptake in diabetes was a result of increased maximal velocity (Vmax) of this exchanger with no change in apparent affinity (Km) for Na+, (c) serum levels of 1,25(OH)2D3 were significantly lower in diabetic animals compared with their control counterparts; and (d) insulin or 1,25(OH)2D3 treatment restored the Vmax alterations to control values, without any significant changes in Km, concomitant with significantly increasing the serum levels of 1,25(OH)2D3 in diabetic animals. These results indicate that Na(+)-H+ activity is significantly increased in proximal small intestinal luminal membranes of streptozotocin-induced diabetic rats. Moreover, alterations in the serum levels of 1,25(OH)2D3 may, at least in part, explain this enhanced antiporter activity and its correction by insulin.

Acridine Orange Detection of Plasmodium Falciparum Malaria: Relationship between Sensitivity and Optical Configuration

Blood samples collected from five volunteers participating in a P. falciparum infectivity trial were examined to determine the efficacy of the acridine orange technique. Several lens configurations were tested for efficiency in the diagnosis of malaria using this system. There was no significant difference in the sensitivity for detecting positive specimens or number of parasites among three lens configurations: a 50x long working distance objective (0.34 mm) with either a 10x ocular (total magnification 500x) or a 12.5x ocular (625x) and a 750x configuration using a 50x objective with a shorter working distance (0.24 mm). All three lens configurations were significantly better than the 1,000x configuration using a commonly available 100x oil immersion objective. The results achieved using this lens still exceeded the sensitivity of the thick blood film.

DNA and RNA flow cytometric study in multiple myeloma. Clinical correlations

Flow cytometric studies of cellular DNA and RNA content using the acridine-orange technique were conducted in 81 patients with multiple myeloma (MM). All patients were treated with the M-2 protocol and clinical response was evaluated according to the criteria of the Chronic Leukemia-Myeloma Task Force. Aneuploid DNA stemlines were found in 38.2% of untreated patients with a median DNA index (DNA-I) of 1.15 in marrow aspirates and 1.22 in biopsy specimens. The median percentage of cells with abnormal DNA content was 31.5 (aspirates) and 35 (biopsy specimens) and a positive correlation with the percentage of bone marrow plasma cells was observed. Significantly higher proliferation (S-phase) was found in marrow biopsy specimens as compared with marrow aspirates. Significantly higher RNA content (RNA index [RNA-I]) was observed in aneuploid versus diploid patients in biopsy material. There was no difference in response to the Memorial Hospital M-2 protocol between diploid and aneuploid patients. In patients with DNA-I greater than 1.15 remission duration was shorter as compared with DNA-I less than or equal to 1.15. Furthermore, no difference in cellular RNA content was noted between responders and nonresponders. This study demonstrates no correlation between cellular RNA content and response, as previously described for patients treated with vincristine, Adriamycin, and dexamethasone (VAD), but DNA aneuploidy appears to be an adverse prognostic factor in MM patients treated with the M-2 protocol. It also demonstrates that prognostic models for MM are not universal but depend on the chemotherapeutic regimen used.