Electrochemical sensors based on biomimetic magnetic molecularly imprinted polymer for selective quantification of methyl green in environmental samples.

A new biomimetic sensor was prepared on carbon paste with magnetic molecularly imprinted polymer (mag-MIP) for sensitive and selective detection of methyl green dye. The mag-MIP was synthesized using a functional monomer that was selected before by computational simulation. A mag-NIP (magnetic non-imprinted polymer) control material was also prepared for comparative purposes. Modeling adsorption studied revealed that the dye-polymer interface followed pseudo-first order kinetics and that maximum adsorption (Qm) of the dye on mag-MIP was 3.13 mg g-1, while the value for mag-NIP was 1.58 mg g-1. The selective material was used as a sensing spot in fabrication of an electrochemical sensor based on modified carbon paste. For electrochemical analysis, the best achievement of the sensor was acquire by tack together a paste with 6.7% (w/w) of mag-MIP and using square-wave adsorptive anodic stripping voltammetry (SWAdASV) in 0.1 mol L-1 phosphate buffer (pH 7.0), with an applied potential (Eappl) of 0.3 V vs. Ag|AgClsat during an adsorption time (Tads) of 120 s. The results were obtained under optimized conditions in which sensor provided a linear concentration range of methyl green from 9.9 × 10-8 to 1.8 × 10-6 mol L-1, with a limit of detection (LOD) of 1.0 × 10-8 mol L-1 and a satisfactory relative standard deviation (RSD) of 4.3% (n = 15). The proposed sensor was applying using two spiked river water samples, obtaining recoveries ranging from 93% to 103%. The proposed method exhibits excellent precision also high reliability and proved to be an alternative method for the quantification of methyl green in real samples.

The application of Compont gel in chronic obstructive jaundice rats model.

PURPOSE: To establish a new rat model, the pathogenesis of which is closer to the clinical occurrence of chronic obstructive jaundice with liver fibrosis. METHODS: 90 SD rats were randomly divided into 3 groups. Group A common bile duct ligation, group B common bile duct injection compont and group C injection saline. The serum of three groups was extracted, and the liver function was detected by ELISA. HE staining, Masson staining and immunohistochemistry were used to detect liver pathology. RESULTS: Group B showed a fluctuant development of jaundice, obstructive degree reached a peak at 2 weeks, and decreased from 3 weeks. HA, LA and PCIII were significantly higher than control group. 3 weeks after surgery, liver tissue fibrosis occurred in group B, and a wide range of fiber spacing was formed at 5 weeks. Immunohistochemistry showed that hepatic stellate cells were more active than the control group. CONCLUSION: Intra-biliary injection of Compont gel is different from the classic obstructive jaundice animal model caused by classic bile duct ligation, which can provide an ideal rat model of chronic obstructive jaundice with liver fibrosis.

The application of Compont gel in chronic obstructive jaundice rats model

Abstract Purpose: To establish a new rat model, the pathogenesis of which is closer to the clinical occurrence of chronic obstructive jaundice with liver fibrosis. Methods: 90 SD rats were randomly divided into 3 groups. Group A common bile duct ligation, group B common bile duct injection compont and group C injection saline. The serum of three groups was extracted, and the liver function was detected by ELISA. HE staining, Masson staining and immunohistochemistry were used to detect liver pathology. Results: Group B showed a fluctuant development of jaundice, obstructive degree reached a peak at 2 weeks, and decreased from 3 weeks. HA, LA and PCIII were significantly higher than control group. 3 weeks after surgery, liver tissue fibrosis occurred in group B, and a wide range of fiber spacing was formed at 5 weeks. Immunohistochemistry showed that hepatic stellate cells were more active than the control group. Conclusion: Intra-biliary injection of Compont gel is different from the classic obstructive jaundice animal model caused by classic bile duct ligation, which can provide an ideal rat model of chronic obstructive jaundice with liver fibrosis.

Histological characterization of umbilical cord in alpaca (Vicugna pacos).

The histomorphometric features of umbilical cord constituents in seven foetuses of alpaca (Vicugna pacos) from Cerro de Pasco, Department, Peru, were determined. Sections of 2-5 cm of umbilical cord were fixed in 10% neutral buffered formalin and processed for light microscopy. Standard histological slides stained with haematoxylin and eosin, Masson's trichrome and Van Gieson's trichrome were obtained. Histologically, common features of umbilical artery and vein were observed as well as mucous connective tissue, some cell features that compound this tissue constituted by cells presented features of myofibroblasts. Among most important findings that were observed, the lumen of umbilical vein was obliterated into star-shaped form with the thinner umbilical artery wall; the smooth muscles and fibroblast were comparatively more in number in umbilical artery than that of umbilical vein, and the tunica media was larger in dimension than the tunica adventitia in umbilical vein. Conclusively, this histological study features an observation of the umbilical cord of alpaca foetuses and shows the similarity between them and those of other mammal species, including dromedaries and South American camelids.

Influence of transcutaneous electrical stimulation on heterotopic ossification: an experimental study in Wistar rats

Heterotopic ossification (HO) is a metaplastic biological process in which there is newly formed bone in soft tissues, resulting in joint mobility deficit and pain. Different treatment modalities have been tried to prevent HO development, but there is no consensus on a therapeutic approach. Since electrical stimulation is a widely used resource in physiotherapy practice to stimulate joint mobility, with analgesic and anti-inflammatory effects, its usefulness for HO treatment was investigated. We aimed to identify the influence of electrical stimulation on induced HO in Wistar rats. Thirty-six male rats (350-390 g) were used, and all animals were anesthetized for blood sampling before HO induction, to quantify the serum alkaline phosphatase. HO induction was performed by bone marrow implantation in both quadriceps of the animals, which were then divided into 3 groups: control (CG), transcutaneous electrical nerve stimulation (TENS) group (TG), and functional electrical stimulation (FES) group (FG) with 12 rats each. All animals were anesthetized and electrically stimulated twice per week, for 35 days from induction day. After this period, another blood sample was collected and quadriceps muscles were bilaterally removed for histological and calcium analysis and the rats were killed. Calcium levels in muscles showed significantly lower results when comparing TG and FG (P<0.001) and between TG and CG (P<0.001). Qualitative histological analyses confirmed 100% HO in FG and CG, while in TG the HO was detected in 54.5% of the animals. The effects of the muscle contractions caused by FES increased HO, while anti-inflammatory effects of TENS reduced HO.

Influence of transcutaneous electrical stimulation on heterotopic ossification: an experimental study in Wistar rats.

Heterotopic ossification (HO) is a metaplastic biological process in which there is newly formed bone in soft tissues, resulting in joint mobility deficit and pain. Different treatment modalities have been tried to prevent HO development, but there is no consensus on a therapeutic approach. Since electrical stimulation is a widely used resource in physiotherapy practice to stimulate joint mobility, with analgesic and anti-inflammatory effects, its usefulness for HO treatment was investigated. We aimed to identify the influence of electrical stimulation on induced HO in Wistar rats. Thirty-six male rats (350-390 g) were used, and all animals were anesthetized for blood sampling before HO induction, to quantify the serum alkaline phosphatase. HO induction was performed by bone marrow implantation in both quadriceps of the animals, which were then divided into 3 groups: control (CG), transcutaneous electrical nerve stimulation (TENS) group (TG), and functional electrical stimulation (FES) group (FG) with 12 rats each. All animals were anesthetized and electrically stimulated twice per week, for 35 days from induction day. After this period, another blood sample was collected and quadriceps muscles were bilaterally removed for histological and calcium analysis and the rats were killed. Calcium levels in muscles showed significantly lower results when comparing TG and FG (P<0.001) and between TG and CG (P<0.001). Qualitative histological analyses confirmed 100% HO in FG and CG, while in TG the HO was detected in 54.5% of the animals. The effects of the muscle contractions caused by FES increased HO, while anti-inflammatory effects of TENS reduced HO.

A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green.

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.

Analysis of the frequency and nature of hyaline ring granulomas in inflammatory odontogenic cysts.

AIM: To determine the prevalence of hyaline ring granulomas (HRGs) in a large case series of inflammatory odontogenic cysts, and to investigate the nature of these structures. METHODOLOGY: All records from the patients diagnosed with inflammatory odontogenic cysts between January 1970 and April 2009 were reviewed. Histologic sections were evaluated by light microscopy and cases with HRGs for which sufficient biological material was available were submitted to histochemical analysis (Masson's trichrome) and immunohistochemistry (CD34, CD68 and collagen IV). RESULTS: Twenty-two (3.3%) of the 661 cases of inflammatory odontogenic cysts diagnosed during the study period presented HRGs. The relative frequency of HRGs was higher amongst residual radicular cysts (6.1%), followed by paradental cysts (5.6%) and radicular cysts (3.0%). HRGs appeared as roughly circular homogeneous/fibrillar masses in 14 (63.6%) cases and as round structures enclosing amorphous material in 3 (13.6%) cases. Most (77.8%) roughly circular homogeneous/fibrillar masses were positive for collagen, whereas all (100.0%) round structures enclosing amorphous material were negative for this protein. Immunohistochemistry showed that most mononucleated cells and all multinucleated giant cells were positive for CD68, but negative for CD34, in all cases. In addition, collagen IV immunostaining was negative in amorphous structures and weakly positive in homogeneous/fibrillar masses. CONCLUSIONS: The present results suggest a very low frequency of HRGs in inflammatory odontogenic cysts and support the hypothesis that these structures arise from the implantation of foreign material, most likely food particles of plant or vegetable origin. The diverse microscopic features of HRG possibly represent different developmental stages of this structure.

Radioprotective effect of sodium selenite on bone repair in the tibia of ovariectomized rats.

This study evaluated protection by selenium (Se) in the bone repair process in ovariectomized rats after irradiation. For such purpose, 80 ovariectomized female Wistar rats were randomly divided into 4 experimental groups: ovariectomized (Ov), Ov/Se, Ov/irradiated (Irr) and Ov/ Se/Irr. A bone defect was created on the tibia of all animals 40 days after ovariectomy. Two days after surgery, only the Ov/Se and Ov/Se/Irr rats received 0.8 mg Se/kg. Three days after surgery, only the Ov/Irr and Ov/Se/Irr rats received 10 Gy of x-rays on the lower limb region. The animals were euthanized at 7, 14, 21 and 28 days after surgery to assess the repair process, which was evaluated by analysis of trabecular bone number (Masson Trichrome) and birefringence analysis (Picrosirius). It was possible to observe a delay in the bone repair process in the ovariectomized/irradiated group and similarity between the ovariectomized, Ov/Se and Ov/Se/Irr groups. In conclusion, sodium selenite exerted a radioprotective effect in the bone repair of tibia of ovariectomized rats without toxicity.

Radioprotective effect of sodium selenite on bone repair in the tibia of ovariectomized rats

This studyevaluated protection by selenium (Se) in the bone repair process in ovariectomized rats after irradiation. For such purpose, 80 ovariectomized female Wistar rats were randomly divided into 4 experimental groups: ovariectomized (Ov), Ov/Se, Ov/irradiated (Irr) and Ov/ Se/Irr. A bone defect was created on the tibia of all animals 40 days after ovariectomy. Two days after surgery, only the Ov/Se and Ov/Se/Irr rats received 0.8 mg Se/kg. Three days after surgery, only the Ov/Irr and Ov/Se/Irr rats received 10 Gy of x-rays on the lower limb region. The animals were euthanized at 7, 14, 21 and 28 days after surgery to assess the repair process, which was evaluated by analysis of trabecular bone number (Masson Trichrome) and birefringence analysis (Picrosirius). It was possible to observe a delay in the bone repair process in the ovariectomized/irradiated group and similarity between the ovariectomized, Ov/Se and Ov/Se/Irr groups. In conclusion, sodium selenite exerted a radioprotective effect in the bone repair of tibia of ovariectomized rats without toxicity.

Esse estudo avaliou a proteção do selênio no processo de reparação óssea em ratas ovariectomizadas após irradiação. Para isso, 80 ratas Wistar foram divididas aleatoriamente em 4 grupos experimentais: ovariectomizado, ovariectomizado/selênio, ovariectomizado/irradiado e ovariectomizado/selênio/irradiado. Foi realizado um defeito ósseo na tíbia de todos os animais 40 dias após ovariectomia. Dois dias após essa cirurgia, os animais dos grupos ovariectomizado/selênio e ovariectomizado/selênio/irradiado receberam 0,8 mg Se/kg. Três dias após a cirurgia, os animais dos grupos ovariectomizado/irradiado e ovariectomizado/selênio/irradiado receberam 10 Gy de radiação X na região de membros inferiores. Os animais foram sacrificados 7, 14, 21 e 28 dias após a cirurgia para avaliação do processo de reparo ósseo, que foi realizado pela análise do número de trabéculas ósseas (coloração Tricrômico de Masson) e pela análise de birrefringência (coloração de Picrosirius). Foi observado atraso no processo de reparo ósseo no grupo ovariectomizado/irradiado e semelhança entre os grupos ovariectomizado, ovariectomizado/selênio e ovariectomizado/selênio/irradiado. Foi possível concluir que o selenito de sódio exerceu efeito radioprotetor no processo de reparação de tíbias em ratas ovariectomizadas sem toxicidade.

Exposed collagen in aged resin-dentin bonds produced on sound and caries-affected dentin in the presence of chlorhexidine.

PURPOSE: To evaluate the effect of chlorhexidine on the presence of collagen in aged resin-dentin bonds produced on sound and caries-affected dentin. MATERIALS AND METHODS: Flat dentin surfaces were obtained from 16 sound molars, from which 8 were microbiologically processed for induction of caries. Single Bond 2 was applied to both sound and caries-affected substrates. In half of the teeth assigned for 6-month storage in water, the phosphoric acid demineralized dentin was impregnated with 2% chlorhexidine before the application of the adhesive. Specimens (2 x 2 x 5 mm) were produced and stored in water for 24 h, or 6 months in either water or mineral oil. The specimens were subjected to histological processing and sections were stained with Goldner's Trichrome. The thickness of the zone of exposed collagen was measured by optical microscopy and the data were subjected to two-way ANOVA and Tukey's test (α = 0.05). RESULTS: There was no statistically significant difference (p > 0.05) between sound and caries-affected dentin regardless of the storage condition. For both substrates, significantly greater collagen exposure was observed after 6 months in water. Chlorhexidine-treated groups resulted in similar collagen exposure to that of the control and 6 months in water groups (p > 0.05), while no increase of the exposed collagen zone was observed after mineral oil storage. CONCLUSION: Aging in water resulted in degradation of the resin-dentin bond, as demonstrated by the increase of the zone of exposed collagen. However, the degradation of the exposed collagen was decelerated in the presence of chlorhexidine.

Morphology of collagen fibers and elastic system fibers in actinic cheilitis.

BACKGROUND: Actinic cheilitis (AC) is a premalignant condition intimately related to exposure of the lips to sun rays. AIM: The objective of this study was to evaluate the elastic and collagen fibers in the lamina propria of AC. The degree of epithelial atypia was correlated with the quantity of elastic and collagen fibers. MATERIALS AND METHODS: Fifty-one cases were investigated. One slide was stained with hematoxylin-eosin for the evaluation of atypia, the second was stained with Weigert's resorcin-fuchsin for the assessment of elastic fibers, and the third slide was stained with Mallory's trichrome for the analysis of collagen fibers. RESULTS: Ordinal logistic regression analysis revealed a significant correlation between the presence of atypia and collagen fibers (P<0.05). CONCLUSIONS: It was concluded that there seems to be a reduction in the quantity of collagen fibers in cases of moderate and severe atypia. No correlation was observed between the degradation of elastic system fibers and the grade of dysplasia.

Contagem total e diferencial dos leucócitos presentes no leite de cabras sadias utilizando-se a coloração de verde de metil e pironina e a citocentrifugação / Total and differential leukocyte counts in the milk of healthy goats, using methyl green-pyronin stain and cytocentrifugation

As particularidades da secreção láctea do tipo apócrina, na espécie caprina, tornam necessárias técnicas específicas para a determinação da quantidade e da qualidade das células presentes no leite desta espécie. Dentre estas particularidades, pode-se citar a presença dos corpúsculos citoplasmáticos. De acordo com estas características, o objetivo do presente trabalho foi determinar a contagem total de leucócitos no leite de cabras sadias, por meio da contagem microscópica direta com verde de metil e pironina-Y, e a contagem celular automática por citometria de fluxo, assim como determinar os tipos leucocitários, através da técnica de citocentrifugação. Foram analisadas 102 de leite de cabras sadias, das raças Saanen, Parda Alpina e Toggenburg. O valor mediano obtido pela contagem microscópica direta e automática foi de 142.840 e 406.000 células somáticas/mL de leite, respectivamente. Os valores médios obtidos na citocentrifugação foram de 73,24 ± 18,35% de neutrófilos, 3,55 ± 3,06% de linfócitos e 24,33 ± 18,89% de monócitos e células epiteliais. De acordo com os resultados, pode-se concluir que o valor obtido pela coloração de verde de metil e pironina-Y é significativamente menor, sendo um importante método para a determinação da celularidade presente no leite de cabras, pois exclui os corpúsculos citoplasmáticos característicos desta espécie.

The characteristics of the apocrine milk secretion observed in goats make it necessary to use specific techniques to determine the quantity and quality of cells found in this kind of milk. Among these characteristics is the presence of cytoplasmic bodies. The objective of the present study was to determine total leukocyte counts in the milk of healthy goats using direct microscopy and methyl green-pyronin Y and automatic cell count by flow cytometry as well as to determine leukocyte types by means of cytocentrifugation. A total of 102 milk samples from healthy Saanen, Brown Alpine and Toggenburg goats were analyzed. The median value of direct microscopic and automatic counts was 142,840 and 406,000 somatic cells/mL of milk, and mean values obtained in cytocentrifugation were 73.24 ± 18.35% neutrophils, 3.55 ± 3.06% lymphocytes and 24.33 ± 18.89% monocytes and epithelial cells. According to the results obtained, it was concluded that methyl green-pyronin Y is an important method, perhaps the most precise one, for determining cell counts in goat milk, because it excludes cytoplasmic bodies. Moreover, the different cell types found in milk are important defenses of the mammary gland against mastitis-causing pathogens.

Microscopic and ultrastructural evaluation of the saphenous vein endothelium for CABG prepared by the no touch technique.

OBJECTIVE: Saphenous vein grafts (SV) used in coronary artery bypass grafting have a limited life and vein occlusion may be the final adverse effect. Efforts to develop new techniques to harvest the saphenous vein may improve the viability of the graft. METHODS: Twenty patients were randomly divided into two groups with the objective of evaluating the vascular endothelium. The No Touch (NT) technique consists in removing the saphenous vein with perivascular tissue. The conventional technique consists in harvesting with "in situ" removal of the perivascular tissue. The standard saphenous vein harvesting procedure used bridged incisions. Characteristics of the vein were considered. Evaluation of the endothelium was achieved by electron microscopy and histologic analysis using hematoxylin eosin staining. The Picrosirius and Masson Trichrome methods were used to analyze subendothelial collagen. RESULTS: Electron microscopy demonstrated that the NT Group had larger non-denudated endothelial areas as well as a smaller number of degraded cells. Histological analysis showed the form and integrity of the saphenous vein layers. A larger amount of collagen fibers were identified in the NT Group. CONCLUSIONS: The NT technique better preserves the saphenous vein endothelium suggesting a more viable graft in the long term.

Avaliação microscópica e ultra-estrutural do endotélio de veia safena preparada pela técnica "no touch" / Microscopic and ultrastructural evaluation of the saphenous vein endothelium for CABG prepared by the no touch technique

OBJETIVO: O enxerto de veia safena (VS) utilizado em revascularização miocárdica possui uma vida útil, sendo o estágio final a oclusão do vaso. Esforços em adquirir novas técnicas de coleta da VS podem possibilitar uma viabilidade maior do enxerto. MÉTODOS: Vinte pacientes foram randomizados e divididos em dois grupos com o objetivo de avaliação do endotélio vascular. A técnica "no touch" (NT) consiste em retirar o segmento de VS com o tecido perivascular. A técnica convencional consiste em retirar a VS, com remoção "in situ" do tecido perivascular e conseqüente vasoespasmo. Houve um padrão de retirada das VS com incisões longitudinais escalonadas. Características da VS foram consideradas. A avaliação do endotélio das VS foi realizada usando microscópio eletrônico (ME) pelo método de varredura e de transmissão. Cortes histológicos das VS foram corados em Hematoxilina-Eosina (HE). O colágeno subendotelial foi analisado pelos métodos de Picro-Sirius e Tricrômio de Masson. RESULTADOS: A ME evidenciou que o Grupo NT possui maiores áreas endoteliais não desnudadas, além de um menor número de células degradadas. A coloração em HE nos permitiu verificar a forma e a integridade das camadas das VS. Há um predomínio maior de fibras colágenas coradas no Grupo NT. CONCLUSÕES: A técnica NT permite uma melhor preservação endotelial da VS, sugerindo um enxerto mais viável em longo prazo.

OBJECTIVE: Saphenous vein grafts (SV) used in coronary artery bypass grafting have a limited life and vein occlusion may be the final adverse effect. Efforts to develop new techniques to harvest the saphenous vein may improve the viability of the graft. METHODS: Twenty patients were randomly divided into two groups with the objective of evaluating the vascular endothelium. The No Touch (NT) technique consists in removing the saphenous vein with perivascular tissue. The conventional technique consists in harvesting with "in situ" removal of the perivascular tissue. The standard saphenous vein harvesting procedure used bridged incisions. Characteristics of the vein were considered. Evaluation of the endothelium was achieved by electron microscopy and histologic analysis using hematoxylin eosin staining. The Picrosirius and Masson Trichrome methods were used to analyze subendothelial collagen. RESULTS: Electron microscopy demonstrated that the NT Group had larger non-denudated endothelial areas as well as a smaller number of degraded cells. Histological analysis showed the form and integrity of the saphenous vein layers. A larger amount of collagen fibers were identified in the NT Group. CONCLUSIONS: The NT technique better preserves the saphenous vein endothelium suggesting a more viable graft in the long term.

Histomorphologic characterization and bond strength evaluation of caries-affected dentin/resin interfaces: effects of long-term water exposure.

OBJECTIVES: To evaluate the longevity of sound (SD) and caries-affected dentin (CAD) bonds made with etch-and-rinse and self-etching adhesives after a 6-month water-storage period, using bond strength and morphological evaluations. METHODS: Extracted human molars with coronal carious lesions were selected. Flat surfaces of CAD surrounded by SD were bonded with etch-and-rinse (Adper Scotchbond 1) or with self-etching (Clearfil Protect Bond and AdheSE) adhesives. Trimmed resin-dentin bonded interfaces (1mm2) were stored in distilled water for 24h or 6 months and subjected to microtensile bond strength (microTBS) evaluation. The quality of the dentin beneath fractured specimens was measured by Knoop microhardness (KHN). ANOVA and multiple comparisons tests were used (P<0.05). Fractographic analysis and interfacial nanoleakage evaluation were performed by scanning electron microscopy (SEM). Resin-dentin bonded sections (10microm thick) were stained with Masson's trichrome and examined using light microscopy. Collagen exposure and adhesive penetration were examined qualitatively. RESULTS: microTBS to SD was significantly higher than that to CAD for all bonding agents. Bonds made with AdheSE were weaker than the other adhesives after 6-months storage regardless of the dentin substrate. CAD bonded specimens presented a significant muTBS decrease over time. Lower KHN was recorded in CAD compared to SD. An increase in the exposed collagen zone and a decrease in the quality of the adhesive infiltration were observed in CAD interfaces. SIGNIFICANCE: CAD bonded interfaces are more prone to hydrolytic degradation than SD bonds. Additionally, as compared to SD, there were remarkable differences in depth of demineralization, adhesive infiltration and interfacial bond strength with CAD.

Evaluation of the biological activity of crude extracts from plants used in Yucatecan traditional medicine. Part II. DNA-interacting activity.

Extracts from leaves, stems, and roots of twelve plants used commonly in Yucatecan traditional medicine were evaluated in the DNA-methyl green assay. Twenty one extracts showed DNA-interacting activity, and nine of them, belonging to five plant species, presented a displacement activity of 5% or higher. The highest activity (17.6%) was detected in the leaf extract of Heliotropium angiospermum.

Modes of cell death in the hypopharyngeal gland of the honey bee (Apis mellifera l).

Different modes of cell death have been revealed in the regressing hypopharyngeal glands of worker honey bees. The hypopharyngeal gland, which is well developed in young nursing bees to produce protein for larval food, was seen to regress naturally in foraging adult worker bees. A range of techniques including histology, cytochemistry, in situ TUNEL, Annexin V and Comet assays indicated that cells within the gland demonstrate progressive symptoms of apoptosis, necrosis and a vacuolar form of programmed cell death. The latter mode of cell death did not display chromatin margination, but was accompanied by an enhanced level of autophagic and hydrolytic activity in which a cytosolic source of acid phosphatase became manifest in the extra-cisternal spaces. Normal and annexin-positive cells were found to occur in the younger nursing bees, whilst necrosis and an aberrant vacuolar type of apoptosis predominated in the older foraging bees. The relevance of these results to the classification of programmed cell death is discussed.

Specific heterochromatic banding of metaphase chromosomes using nuclear yellow.

The bis-benzimidazole compound nuclear yellow (NY) belongs to the same chemical family as the DNA binding fluorochromes Hoechst 33258 and Hoechst 33342. Spectroscopic studies of NY alone and in the presence of calf thymus DNA show high DNA binding affinity and behavior similar to the Hoechst fluorochromes above. Mitotic metaphase chromosomes from Balb/c mice stained with NY show C-banding and weak G/Q-banding, both of them disappearing after distamycin A (DA) or methyl green (MG) counterstaining. The same staining of human metaphase chromosomes from lymphocyte cultures, however, reveal only faint G/Q-banding (NY) and a characteristic DA-DAPI-like banding (NY-DA, NY-MG). Image analysis of NY stained human chromosomes, confirms that NY is suitable for studying polymorphisms affecting size in the pericentromeric heterochromatin of pairs 1, 9 and 16, and shows significant enhancement of NY fluorescence induced by DA in DA-DAPI heterochromatin. Our spectroscopic and cytological results show that NY, either alone or counterstained with DA or MG, can be used for DNA cytochemistry and chromosome banding. Possible mechanisms for the banding patterns induced by NY are discussed.

Cytotoxic and DNA interaction activities of extracts from medicinal plants used in Argentina.

Eight crude extracts from seven Argentine plants with cancer-related ethnobotanical uses have been subjected to a bioscreening study to detect cytotoxic activity. The plants studied were: Aristolochia triangularis, Baccharis grisebachii, Bolax gummifera, Eupatorium hecatanthum, Erythrina crista-galli, Pterocaulon polystachium and Salpichroa origanifolia. Crown gall tumour inhibition, DNA interaction and cytotoxicity towards KB cells were assayed using the potato disc, the DNA-methyl green (DNA-MG) and the KB cells cytotoxicity bioassays respectively. The results obtained indicate that A. triangularis (ED50=47 microg/ml), B. gummifera (ED50=32 microg/ml) and E. hecatanthum (ED50=35 microg/ml) contained cytotoxic compounds against KB cells. All of the plants studied inhibited the growth of crown gall tumours, showing correlation between the experimental data and the uses reported for these plants. Moreover, the results obtained for the extracts of E. hecatanthum and P. polystachium indicate the presence of compounds that interact with DNA (48 and 22% of absorbance decrease, respectively). The results obtained suggest that cytotoxicity could play an important role in the activities claimed for the plants under study.