Intervenções psicológicas necessárias: a prática como residente no serviço de medicina fetal / Necessary psychological interventions: the practice as a resident in the fetal medicine service / Intervenciones psicológicas necesarias: la práctica como residente en el servicio de medicina fetal

Com os avanços tecnológicos e o aprimoramento da prática médica via ultrassonografia, já é possível detectar possíveis problemas no feto desde a gestação. O objetivo deste estudo foi analisar a prática do psicólogo no contexto de gestações que envolvem riscos fetais. Trata-se de um estudo qualitativo sob formato de relato de experiência como psicólogo residente no Serviço de Medicina Fetal da Maternidade Escola da Universidade Federal do Rio de Janeiro (UFRJ). Os registros, feitos por observação participante e diário de campo, foram analisados em dois eixos temáticos: 1) intervenções psicológicas no trabalho em equipe em consulta de pré-natal, exame de ultrassonografia e procedimento de amniocentese; e 2) intervenções psicológicas em casos de bebês incompatíveis com a vida. Os resultados indicaram que o psicólogo nesse serviço é essencial para atuar de forma multiprofissional na assistência pré-natal para gravidezes de alto risco fetal. Ademais, a preceptoria do residente é relevante para sua formação e treinamento para atuação profissional no campo da psicologia perinatal.(AU)

Face to the technological advances and the improvement of medical practice via ultrasound, it is already possible to detect possible problems in the fetus since pregnancy. The objective of this study was to analyze the psychologist's practice in the context of pregnancies which involve fetal risks. It is a qualitative study based on an experience report as a psychologist trainee at the Fetal Medicine Service of the Maternity School of UFRJ. The records, based on the participant observation and field diary, were analyzed in two thematic axes: 1) psychological interventions in the teamwork in the prenatal attendance, ultrasound examination and amniocentesis procedure; and 2) psychological interventions in cases of babies incompatible to the life. The results indicated that the psychologist in this service is essential to work in a multidisciplinary way at the prenatal care for high fetal risk pregnancies. Furthermore, the resident's preceptorship is relevant to their education and training for professional performance in the field of Perinatal Psychology.(AU)

Con los avances tecnológicos y la mejora de la práctica médica a través de la ecografía, ya se puede detectar posibles problemas en el feto desde el embarazo. El objetivo de este estudio fue analizar la práctica del psicólogo en el contexto de embarazos de riesgos fetal. Es un estudio cualitativo basado en un relato de experiencia como residente de psicología en el Servicio de Medicina Fetal de la Escuela de Maternidad de la Universidade Federal do Rio de Janeiro (UFRJ). Los registros, realizados en la observación participante y el diario de campo, se analizaron en dos ejes temáticos: 1) intervenciones psicológicas en el trabajo en equipo, en la consulta prenatal, ecografía y los procedimientos de amniocentesis; y 2) intervenciones psicológicas en casos de bebés incompatibles con la vida. Los resultados señalaron como fundamental la presencia del psicólogo en este servicio trabajando de forma multidisciplinar en la atención prenatal en el contexto de embarazos de alto riesgo fetal. Además, la tutela del residente es relevante para su educación y formación para el desempeño profesional en el campo de la Psicología Perinatal.(AU)

Aviculin Isolated from Lespedeza cuneata Induce Apoptosis in Breast Cancer Cells through Mitochondria-Mediated Caspase Activation Pathway.

The global incidence of breast cancer has increased. However, there are many impediments to the development of safe and effective anticancer drugs. The aim of the present study was to evaluate the effect of aviculin isolated from Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae) on MCF-7 human breast cancer cells and determine the underlying mechanism. Using the bioassay-guided isolation by water soluble tetrazolium salt (WST-1)-based Ez-Cytox assay, nine compounds (four lignan glycosides (1-4), three flavonoid glycosides (5-7), and two phenolic compounds (8 and 9)) were isolated from the ethyl acetate (EA) fraction of the L. cuneata methanolic extract. Of these, aviculin (2), a lignan glycoside, was the only compound that reduced metabolic activity on MCF-7 cells below 50% (IC50: 75.47 ± 2.23 µM). The underlying mechanism was analyzed using the annexin V Alexa Fluor 488 binding assay and Western blotting. Aviculin (2) was found to induce apoptotic cell death through the intrinsic apoptosis pathway, as indicated by the increased expression of initiator caspase-9, executioner caspase-7, and poly (ADP-ribose) polymerase (PARP). Aviculin (2)-induced apoptotic cell death was accompanied by an increase in the Bax/Bcl-2 ratio. These findings demonstrated that aviculin (2) could induce breast cancer cell apoptosis through the intrinsic apoptosis pathway, and it can therefore be considered an excellent candidate for herbal treatment of breast cancer.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization.

Flowering plants have a unique sexual reproduction system called 'double fertilization', in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes' nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Ligustilide attenuates nitric oxide-induced apoptosis in rat chondrocytes and cartilage degradation via inhibiting JNK and p38 MAPK pathways.

Ligustilide (LIG) is the main lipophilic component of the Umbelliferae family of pharmaceutical plants, including Radix angelicae sinensis and Ligusticum chuanxiong. LIG shows various pharmacological properties associated with anti-inflammation and anti-apoptosis in several kinds of cell lines. However, the therapeutic effects of LIG on chondrocyte apoptosis remain unknown. In this study, we investigated whether LIG had an anti-apoptotic activity in sodium nitroprusside (SNP)-stimulated chondrocyte apoptosis and could delay cartilage degeneration in a surgically induced rat OA model, and elucidated the potential mechanisms. In vitro studies revealed that LIG significantly suppressed chondrocyte apoptosis and cytoskeletal remodelling, which maintained the nuclear morphology and increased the mitochondrial membrane potential. In terms of SNP, LIG treatment considerably reduced the expression levels of cleaved caspase-3, Bax and inducible nitric oxide synthase and increased the expression level of Bcl-2 in a dose-dependent manner. The LIG-treated groups presented a significantly suppressed expression of activating transcription factor 2 and phosphorylation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). The inhibitory effect of LIG was enhanced by the p38 MAPK inhibitor SB203580 or the JNK inhibitor SP600125 and offset by the agonist anisomycin. In vivo studies demonstrated that LIG attenuated osteoarthritic cartilage destruction by inhibiting the cartilage chondrocyte apoptosis and suppressing the phosphorylation levels of activating transcription factor 2, JNK and p38 MAPK. This result was confirmed by histological analyses, micro-CT, TUNEL assay and immunohistochemical analyses. Collectively, our studies indicated that LIG protected chondrocytes against SNP-induced apoptosis and delayed articular cartilage degeneration by suppressing JNK and p38 MAPK pathways.

Volatile oil from alpinia officinarum promotes lung cancer regression in vitro and in vivo.

The anti-lung cancer activity of volatile oil from Alpinia officinarum (VOAO) and the underlying mechanism has not been studied. Herein, VOAO was extracted by steam distillation and its components were identified by GC-MS analysis. Cells viability was measured by an MTT assay and the cell survival capacity was tested via a colony formation assay. Apoptosis cells were detected using the Annexin V-FITC/PI method. Hoechst 33342 cell unclear staining was employed to evaluate the nuclear morphology change. The mitochondrial membrane potential was detected by a JC-1 staining assay. Bcl-2, Mcl-1 and cleaved caspase-3 proteins were quantified by immune blotting. Furthermore, VOAO anti-cancer activity was evaluated on an A549 cell xenograft nude mice model. Our results have revealed that VOAO could inhibit the cell viability of lung carcinoma cells and the colony formation capacity. VOAO downregulates Bcl-2 and Mcl-1 and triggers dysfunction of the mitochondrial membrane potential. VOAO further activates caspase-3 cleavage and induces lung cancer cells apoptosis. In addition, VOAO administration significantly suppresses lung cancer growth in xenograft mice without obvious hepatotoxicity. We conclude that VOAO could be an effective, low cytotoxicity natural component for treatment of lung carcinoma.

Nuclear shape and orientation features from H&E images predict survival in early-stage estrogen receptor-positive breast cancers.

Early-stage estrogen receptor-positive (ER+) breast cancer (BCa) is the most common type of BCa in the United States. One critical question with these tumors is identifying which patients will receive added benefit from adjuvant chemotherapy. Nuclear pleomorphism (variance in nuclear shape and morphology) is an important constituent of breast grading schemes, and in ER+ cases, the grade is highly correlated with disease outcome. This study aimed to investigate whether quantitative computer-extracted image features of nuclear shape and orientation on digitized images of hematoxylin-stained and eosin-stained tissue of lymph node-negative (LN-), ER+ BCa could help stratify patients into discrete (<10 years short-term vs. >10 years long-term survival) outcome groups independent of standard clinical and pathological parameters. We considered a tissue microarray (TMA) cohort of 276 ER+, LN- patients comprising 150 patients with long-term and 126 patients with short-term overall survival, wherein 177 randomly chosen cases formed the modeling set, and 99 remaining cases the test set. Segmentation of individual nuclei was performed using multiresolution watershed; subsequently, 615 features relating to nuclear shape/texture and orientation disorder were extracted from each TMA spot. The Wilcoxon's rank-sum test identified the 15 most prognostic quantitative histomorphometric features within the modeling set. These features were then subsequently combined via a linear discriminant analysis classifier and evaluated on the test set to assign a probability of long-term vs. short-term disease-specific survival. In univariate survival analysis, patients identified by the image classifier as high risk had significantly poorer survival outcome: hazard ratio (95% confident interval) = 2.91(1.23-6.92), p = 0.02786. Multivariate analysis controlling for T-stage, histology grade, and nuclear grade showed the classifier to be independently predictive of poorer survival: hazard ratio (95% confident interval) = 3.17(0.33-30.46), p = 0.01039. Our results suggest that quantitative histomorphometric features of nuclear shape and orientation are strongly and independently predictive of patient survival in ER+, LN- BCa.

Dietary ascorbic acid reduced micronucleus and nuclear abnormalities in Clarias gariepinus (Burchell 1822) exposed to hospital effluent.

Hospital effluents contain myriad of mutagens and genotoxins capable of increasing DNA damage in aquatic biota. African mudfish, Clarias gariepinus, are exposed to genotoxins when cultured in swamps and derelict water bodies often contaminated by effluents. Moreover, its DNA is susceptible to xenobiotic-induced lesions since it lacks L-gulonolactone oxidase and hence cannot synthesize L-ascorbic acid. This study investigated 96-h acute toxicity and protective effects of dietary ascorbic acid (AA) against micronucleus (MN) and abnormal nuclear (NAs) formation in C. gariepinus exposed to sub-lethal concentrations of hospital effluent. Six concentrations (0.5-3.0%) of the effluent were selected to determine the 96-h acute toxicity of the effluent in C. gariepinus, after range finding test. Fish were exposed to sub-lethal concentrations (0.08-1.30%) of the 96 h LC50. Two other groups were exposed to the 96 h LC50 (1.30%) of the effluent +50 and +100 mg/kg of dietary ascorbic for 7 days, and MN and NAs assessed in peripheral erythrocytes. The 96 h LC50 (1.30%) was 1.18 times more toxic than the 24 h LC50 (1.54%), indicating that the toxicity of the effluent increased with exposure duration. MN, nuclear bud, enucleated, fragmented nucleus (apoptosis), and necrotic erythrocytes significantly increase in effluent treated fish. Dietary AA reduced MN from 6.35-fold (1.30% treated group) to 3.72-fold (1.30% + 50 mg AA) and 3.54-fold (1.30% + 100 mg AA). Also, AA reduced total NAs from 2.26-fold (1.30%) to 1.40-fold (1.30% + 50 mg AA) and 1.06-fold (1.30% + 100 mg AA) compared to the control. Heavy metals and physicochemical parameters analyzed in the tested effluent possibly induced the mortality and cytogenotoxicity in C. gariepinus, and this was ameliorated by dietary AA.

Editor's Highlight: Multiparametric Image Analysis of Rat Dorsal Root Ganglion Cultures to Evaluate Peripheral Neuropathy-Inducing Chemotherapeutics.

Chemotherapy-induced peripheral neuropathy (CIPN) is a major, dose-limiting adverse effect experienced by cancer patients. Advancements in mechanism-based risk mitigation and effective treatments for CIPN can be aided by suitable in vitro assays. To this end, we developed a multiparametric morphology-centered rat dorsal root ganglion (DRG) assay. Morphologic alterations in subcellular structures of neurons and non-neurons were analyzed with an automated microscopy system. Stains for NeuN (a neuron-specific nuclear protein) and Tuj-1 (ß-III tubulin) were used to identify neuronal cell nuclei and neuronal cell bodies/neurites, respectively. Vimentin staining (a component of Schwann cell intermediate filaments) was used to label non-neuronal supporting cells. Nuclei that stained with DAPI, but lacked NeuN represented non-neuronal cells. Images were analyzed following 24 h of continuous exposure to CIPN-inducing agents and 72 h after drug removal to provide a dynamic measure of recovery from initial drug effects. Treatment with bortezomib, cisplatin, eribulin, paclitaxel or vincristine induced a dose-dependent loss of neurite/process areas, mimicking the 'dying back' degeneration of axons, a histopathological hallmark of clinical CIPN in vivo. The IC50 for neurite loss was within 3-fold of the maximal clinical exposure (Cmax) for all five CIPN-inducing drugs, but was >4- or ≥ 28-fold of the Cmax for 2 non-CIPN-inducing agents. Compound-specific effects, eg, neurite fragmentation by cisplatin or bortezomib and enlarged neuronal cell bodies by paclitaxel, were also observed. Collectively, these results support the use of a quantitative, morphologic evaluation and a DRG cell culture model to inform risk and examine mechanisms of CIPN.

Chemical composition and cytotoxic properties of Clinacanthus nutans root extracts.

CONTEXT: Clinacanthus nutans Lindau (Acanthaceae) is a medicinal plant that has been reported to have anti-inflammatory, antiviral, antimicrobial and antivenom activities. In Malaysia, it has been widely claimed to be effective in various cancer treatments but scientific evidence is lacking. OBJECTIVE: This study investigates the chemical constituents, anti-proliferative, and apoptotic properties of C. nutans root extracts. MATERIALS AND METHODS: The roots were subjected to solvent extraction using methanol and ethyl acetate. The anti-proliferative effects of root extracts were tested at the concentrations of 10 to 50 µg/mL on MCF-7 and HeLa by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay for 72 h. Morphological changes were observed under light microscope. Pro-apoptotic effects of root extracts were examined using flow cytometric analysis and RT-PCR. The chemical compositions of root extracts were detected using GC-MS. RESULTS: The proliferation of MCF-7 cells was inhibited with the IC50 values of 35 and 30 µg/mL, respectively, for methanol and ethyl acetate root extracts. The average inhibition of HeLa cells was ∼25%. Induction of apoptosis in MCF-7 was supported by chromatin condensation, down-regulation of BCL2 and unaltered expression of BAX. However, only ethyl acetate extract caused the loss of mitochondrial membrane potential. GC-MS analysis revealed the roots extracts were rich with terpenoids and phytosterols. DISCUSSION AND CONCLUSIONS: The results demonstrated that root extracts promote apoptosis by suppressing BCL2 via mitochondria-dependent or independent manner. The identified compounds might work solely or cooperatively in regulating apoptosis. However, further studies are required to address this.

Protective effect of Cistanchis A on ethanol-induced damage in primary cultured mouse hepatocytes.

Cistanoside A (C. A) was one of phenylethanol glycosides isolated from Cistanche deserticola, a tonic in traditional Chinese medicine. In our previous research, we demonstrated that Cistanoside A (C. A) possess the protective activities on CCl4 induced hepatotoxicity in mice, such as increasing free radicals clearing activities, alleviating lipid-overoxidation damage, and improving respiratory chain function in mitochondria. Meanwhile, our previous research also demonstrated C.A possess protective activities on alcohol induced hepatotoxicity in mice, shown in ameliorate the hepatic function indices, lightening steatosis and inflammatory infiltration, increasing free radicals clearing activities, alleviating lipid-overoxidation damage, and alleviating energy metabolism in mitochondria. The aim of this research was to evaluate the effects of Cistanoside A (C. A) on ethanol-induced damage in primary cultured mouse hepatocytes, and probe into the mechanism related. Using fluorescent staining, flow cytometer, immunohistochemistry analysis, and Western blotting, we demonstrated that C.A could enhance the survival rate of the primary cultured hepatocytes, alleviate apoptosis and necrosis, the mechanism was involved with enhance the expression of apoptosis inhibition factor bcl-2, and inhibition the expression of immediate early genes c-fos.

Olive leaf extract activity against Candida albicans and C. dubliniensis - the in vitro viability study.

Olive leaf extract is characterized by a high content of polyphenols (oleuropein, hydroxytyrosol and their derivatives), which is associated with its therapeutic properties. The objective of the present research was to evaluate the antifungal activity of olive leaf extract against Candida albicans ATCC 10231 and C. dubliniensis CBS 7987 strains. Minimum inhibitory concentrations (MIC) of the extract were determined by several in vitro assays. The extract showed a concentration depended effect on the viability of C. albicans with MIC value of 46.875 mg mL-1 and C. dubliniensis with MIC value 62.5 mg mL-1. Most sensitive methods for testing the antifungal effect of the extracts were the trypan blue exclusion method and fluorescent dye exclusion method while MIC could not be determined by the method according to the EUCAST recommendation suggesting that herbal preparations contain compounds that may interfere with this susceptibility testing. The fluorescent dye exclusion method was also used for the assessment of morphological changes in the nuclei of treated cells. According to the obtained results, olive leaf extract is less effective against the tested strains than hydroxytyrosol, an olive plant constituent tested in our previous study.

Mitochondria-dependent apoptogenic activity of the aqueous root extract of Croton membranaceus against human BPH-1 cells.

Croton membranaceus aqueous root extract (CMARE) is among the widely used phytotherapeutics in Ghana for the management of benign prostatic hyperplasia (BPH) and prostate cancer. However, the mechanism of action of CMARE remains to be elucidated. This study aimed to establish whether apoptosis is involved in the antiproliferative effect of CMARE on human BPH-1 cells. We determined the effect of treatment with 0, 1, 3, and 5 mg/mL CMARE for 24, 48, and 72 h on the viability and morphology of BPH-1 cells using the MMT assay and phase-contrast microscopy, respectively. We examined the apoptosis-inducing effects of CMARE after 48 h at the cellular level using Hoescht 33258 and JC-1 dye staining and flow cytometry analysis. We performed reverse transcription polymerase chain reaction and Western blotting to confirm the apoptotic effects of CMARE at the molecular level. CMARE induced a significant dose-dependent inhibition in the proliferation of BPH-1 cells (P < 0.05) and an alteration in their morphology and a reduction their density. Furthermore, CMARE induced dose-dependent staining of the nuclear chromatin, significant DNA fragmentation with G0/G1 sub-diploid cells (P < 0.01), and loss of the mitochondrial membrane potential in the treated cells compared to the controls after 48 h (P < 0.01). Additionally, while CMARE induced a significant upregulation of the mRNA and protein levels of Bax, those of Bcl2 did not change significantly. Therefore, induction of mitochondria-dependent apoptosis of BPH-1 cells may be a possible mechanism of action of CMARE.

Chromatin alterations during pollen development in Hordeum vulgare.

The dynamics of posttranslational histone modifications in relation to nuclear architecture has been analyzed during pollen development in Hordeum vulgare L. cv. Igri. Notwithstanding the asymmetry of cytokinesis associated with pollen mitosis I, immunolabeling revealed that the vegetative and generative nuclei initially display identical chromatin modification patterns. Yet, differential chromatin modification patterns between vegetative and generative nuclei emerge with the development of conspicuous differences in nuclear morphology as visualized by 4',6-diamidino-2-phenylindole staining. The temporal and spatial distribution of most histone modifications observed is in agreement with reduced gene activity in the generative nucleus and increased expression in the vegetative nucleus as indicated by immunolabeling of active RNA polymerase II. Signals of trimethylation of histone H3 lysine 27 proved to be particularly enriched in euchromatic domains of subtelomeric regions. In the context of nuclear differentiation in bicellular pollen, this modification became restricted to the vegetative nucleus, indicating a role in activating rather than suppressing gene expression. The presence of acetylated histone H3 at lysine 9 in the cytoplasm of the generative cell is indicative of a more complex, still unknown function of this particular modification.

Madecassoside, a triterpenoid saponin isolated from Centella asiatica herbs, protects endothelial cells against oxidative stress.

This study aimed to investigate the effect of madecassoside against oxidative stress-induced injury of endothelial cells. Hydrogen peroxide (H(2)O(2), 500 µmol/L) was employed as an inducer of oxidative stress in human umbilical vein endothelial cells (HUVECs). Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry. Caspase-3 activity and mitochondria membrane potential were further examined. As a result, madecassoside (10, 30, 100 µmol/L) could reverse morphological changes, elevate cell viability, increase glutathione levels, and decrease lactate dehydrogenase and malondialdehyde levels caused by H(2)O(2) in a concentration-dependent manner. It attenuated apoptosis, preventing the activation of caspase-3 and the loss of mitochondria membrane potential, as well as the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in HUVECs. These data suggested that madecassoside could protect HUVECs from oxidative injury, which was probably achieved by inhibiting cell apoptosis via protection of mitochondria membranes and downregulation of the activation of caspase-3 and p38 MAPK.

Aqueous fraction of Nephelium ramboutan-ake rind induces mitochondrial-mediated apoptosis in HT-29 human colorectal adenocarcinoma cells.

The aim of this study was to investigate the cytotoxic and apoptotic effects of Nephelium ramboutan-ake (pulasan) rind in selected human cancer cell lines. The crude ethanol extract and fractions (ethyl acetate and aqueous) of N. ramboutan-ake inhibited the growth of HT-29, HCT-116, MDA-MB-231, Ca Ski cells according to MTT assays. The N. ramboutan-ake aqueous fraction (NRAF) was found to exert the greatest cytotoxic effect against HT-29 in a dose-dependent manner. Evidence of apoptotic cell death was revealed by features such as chromatin condensation, nuclear fragmentation and apoptotic body formation. The result from a TUNEL assay strongly suggested that NRAF brings about DNA fragmentation in HT-29 cells. Phosphatidylserine (PS) externalization on the outer leaflet of plasma membranes was detected with annexin V-FITC/PI binding, confirming the early stage of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis, and the results clearly suggested that NRAF led to collapse of mitochondrial transmembrane potential in HT-29 cells. This attenuation of mitochondrial membrane potential (Δψm) was accompanied by increased production of ROS and depletion of GSH, an increase of Bax protein expression, and induced-activation of caspase-3/7 and caspase-9. These combined results suggest that NRAF induces mitochondrial-mediated apoptosis.

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem.

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.

[Effects of oil-refining microbes (genus Acinetobacter) on cytogenetical structures of human lymphocytes in cell cultures].

The objective of this study was to assess ability of oil-refining bacteria Acinetobacter calcoaceticus and A. valentis to induce karyopathological abnormalities and chromosomal aberrations in human lymphocyte cultures. It was found that the cultures infected with A. calcoaceticus showed significantly high frequencies of cytogenetical effects and chromosomal aberrant cells as compared to the intact cultures and cultures infected with A. valentis. The most of chromosomal aberrations, mainly chromatid aberrations, were located in 1 and 2 chromosomes. Moreover, the aberrations were detected in some specific chromosome areas. Abnormalities of mitotic cell division and nucleus morphology were determined in lymphocyte cultures infected with A. calcoaceticus. There were found significantly high frequencies of cells with micronuclei, nucleus protrusions, anaphase or metaphase chromosome and chromosomal fragments lagging as well as multipolar and C-mitoses. Thus, the oil-refining bacteria A. calcoaceticus in contrast to A. valentis demonstrated strong genotoxic effects in human lymphocyte cultures in vitro.

Protective effects of aucubin on H2O2-induced apoptosis in PC12 cells.

The present study investigated the neuroprotective effects of aucubin on hydrogen peroxide (H2O2)-induced apoptosis in PC12 cells. Exposure of PC12 cells to 0.25 mm H2O2 induced a leakage of lactate dehydrogenase and decreased cell viability, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In a dose over 0.1 mm, aucubin increased PC12 cellular viability and markedly attenuated H2O2-induced apoptotic cell death. Quantitation of apoptosis by flow cytometry indicated that aucubin inhibited H2O2-induced apoptosis in PC12 cells. Nuclear damage was alleviated by aucubin, as shown by Hoechst staining. In addition, the levels of malondialdehyde were reduced and the activity of superoxide dismutase, catalase and glutathione peroxidase was augmented in these cells. These results indicated that aucubin inhibited H2O2-induced apoptosis in PC12 cells through regulation of the endogenous oxidant-antioxidant balance. Our results suggest that aucubin is a potential protective agent for the treatment of oxidative-stress-induced neurodegenerative disease.

Natural plant extract tubeimoside I promotes apoptosis-mediated cell death in cultured human hepatoma (HepG2) cells.

Tubeimoside I (TBMS I), an extract from Chinese herbal medicine Bolbostemma paniculatum (MAXIM.) FRANQUET (Cucurbitaceae) has been shown as a potent anti-tumor agent for a variety of human cancers, but yet to be evaluated for hepatoma that is highly prevalent in Eastern Asian countries including China. Here, we examined in vitro the cytotoxic effects of TBMS I on human hepatoma (HepG2) and normal liver (L-02) cell lines. We also investigated TBMS I-induced molecular events related to apoptosis in HepG2 cells. The results show that TBMS I inhibited the proliferation of both HepG2 and L-02 cells in a dose- and time-dependent manner, but HepG2 cells appeared more sensitive to the agent. When exposed to TBMS I for 24, 48 and 72 h, IC50 for HepG2 cells versus L-02 cells were 15.5 vs. 23.1, 11.7 vs. 16.2, 9.2 vs. 13.1 (µM, p<0.01), respectively. TBMS I induced cell shrinkage, nuclear condensation and fragmentation, cell cycle arrest at the G2/M phase, mitochondrial membrane disruption, release of cytochrome c from the mitochondria, activation of caspase 3 and 9, and shifting Bax/Bcl-2 ratio from being anti-apoptotic to pro-apoptotic, all indicative of initiation and progression of apoptosis involving mitochondrial dysfunction. Taken together, these results indicate for the first time that TBMS I potently inhibited growth in HepG2 cells by mediating a cascade of apoptosis signaling pathways. Considering its sensitivity of HepG2 cells, preferential distribution in the liver and natural product origin, TBMS I therefore may have a great potential as a chemotherapeutic drug candidate for hepatoma.

Sperm dimorphism in terms of nuclear shape and microtubule accumulation in Cyrtanthus mackenii.

Pollen tubes of Cyrtanthus mackenii, a species with bicellular pollen, were cultured in vitro to investigate nuclear phase changes during generative cell division and male germ unit (MGU) formation, using flow cytometric analysis. Results revealed that sperm cells were formed after 12 h of culture. During sperm maturation, the nuclei of sperm cells were not associated with the vegetative nucleus (unassociated sperm cells; Sua) and became longer than those of sperm cells associated with the vegetative nucleus (Svn). These findings indicate that the pair of sperm cells in the C. mackenii MGU is dimorphic in terms of nuclear shape. Dimorphism coincides with anti-alpha-tubulin antibody immunofluorescence, which was higher in the Sua than in Svn. Following treatment with oryzalin, triggering microtubule depolymerization, differences between nuclear shapes in the two sperm nuclei disappeared, suggesting that microtubule accumulation between sperm cells in the MGU correlates with differences in the nuclear shape.