Immunofluorescence analysis of (A) Bax and (B) Bcl-2 protein expression in HT-29 cells.

<p>Cells were treated with annomuricin E at the IC₅₀ concentration for 12, 24 and 48 h and were stained with DAPI and Bax/Bcl-2 antibodies conjugated to FITC. Cells treated with 0.1% vehicle DMSO were employed as the control treatment. As the number of cells reduced in a time-dependent manner, the fluorescent intensity showed a marked upregulation and down-regulation for Bax and Blc-2 proteins, respectively.</p

Katamnestische Untersuchungen zur suprakondylären und y-kondylären Humerusfraktur im Kindesalter

Die Arbeit beschäftigt sich mit der suprakondylären und y-kondylären Humerusfraktur im Kindesalter. Die suprakondyläre Humerusfraktur ist die häufigste Ellenbogenfraktur beim Kind, die y-kondyläre Fraktur ist der suprakondylären ähnlich, tritt aber nur selten auf. Es wurden 172 suprakondyläre und 6 y-kondyläre Humerusfrakturen retrospektiv untersucht. Dazu wurden die Daten von 55 ambulant und 123 stationär behandelten Kindern bezüglich Unfallursache, Therapie und Komplikationen ausgewertet. Die Röntgenbilder der stationären Patienten wurden nachbefundet. 32,5% der stationär behandelten Kinder konnten nachuntersucht werden. Die häufigste Unfallursache war ein Sturz aus der Höhe. Im Patientengut waren Frakturen nach Baumann Klassifikation I, II und III zu ähnlichen Teilen vorhanden. Häufigste Behandlungsverfahren waren, ggf. nach geschlossener Reposition, Ruhigstellung in Gipslonguetten oder Blountscher Schlinge, und geschlossene oder offene Reposition und anschließende Fixation mittels Kirschner Drähten. Die häufigsten Komplikationen waren primäre und sekundäre Nervenstörungen, Abweichung der Ellenbogenachse oder Bewegungseinschränkung nach Therapie. 68,5% der nachuntersuchten Patienten hatten ein exzellentes oder gutes Behandlungsergebnis. Die Analyse der Ergebnisse und deren Vergleich mit den Ergebnissen anderer Autoren zeigen Folgendes: Nicht dislozierte Extensionsfrakturen sollten in der Blountschen Schlinge ruhiggestellt werden. Dislozierte Frakturen müssen exakt und schonend, möglichst geschlossen, reponiert werden. Gekreuzte Kirschner Drähte bieten gute Stabilität. Primäre und sekundäre Nervenstörungen haben eine gute Prognose. Radiologische Hilfsmittel wie Baumannwinkel, Rotationsfehlerquotient und Epiphysenachsenwinkel geben nur eine Orientierung bei der Vermeidung von Abweichungen der Ellenbogenachse oder Bewegungseinschränkungen. Y-kondyläre Frakturen mit nur geringer Dislokation der Kondylenfragmente können primär wie suprakondyläre Frakturen behandelt werden.This paper deals with supracondylar and y-condylar humerus fractures in childhood. The supracondylar humerus fracture is the most frequent elbow fracture in children, the y-condylar fracture is similar to the supracondylar one, but occurs only rarely. 172 supracondylar and 6 y-condylar humerus fractures have been analysed retrospectively. For that purpose data of 55 outpatients and 123 in-house treated children have been analysed with regard to the cause of accident, therapy and complications. The radiographs of the in-patients have been revaluated. 32.5% of the in-patients have been seen for follow-up. Most frequent cause of accident was a fall from a height. Within the group of patients, fractures that were classified to Baumann I, II and III occurred to equal rates. The treatments which were used mostly are immobilization in plaster casts or Blount´s loop, after closed reduction if necessary, and closed or open reduction followed by fixation by Kirschner wires. The most frequent complications were primary and secondary nerval disorders, deviation of the axis of the elbow or limitation of elbow function after therapy. 68.5% of the follow-up patients had an excellent or good outcome. The analysis of the results including their comparison to the results of other authors have led to the following conclusion: Nondisplaced extension fractures should be immobilized by Blount`s method. Displaced fractures have to be reduced accurately and sparing, if possible by closed reduction. Crossed Kirschner wires provide a good stability. Primary and secondary nerval disorders have got a good prognosis. Radiological tools like angle of Baumann, rotation error quotient and angle of epiphyseal axis only provide an orientation in avoiding deviation of elbow axis or limitation of elbow function. Y-condylar fractures with only slight dislocation of the condylar fragments can primary be treated like supracondylar fractures

Effect of annomuricin E on Bax and Bcl-2 mRNA expression was assessed using Q-PCR analysis.

<p>The housekeeping gene β-actin was used for the normalization of the mRNA expression. The result depicted a time-dependent upregulation of Bax and down-regulation of Bcl-2 after treatment with annomuricin E at the IC₅₀ concentration. The data represent the means ± SEM of three independent experiments. *<i>P</i><0.05 compared with the control.</p

The experimental design and specifications.

<p>The experimental design and specifications.</p

Images of HT-29 cells treated with annomuricin E at the IC₅₀ concentration for 24 h.

<p>The treated cells were stained with different and specific dyes for the detection of total nuclear intensity, cell membrane permeability, MMP and cytochrome <i>c</i> release. Cells treated with 0.1% vehicle DMSO were employed as the control treatment.</p

Effect of annomuricin E on cell cycle distribution in HT-29 cells.

<p>Cells were treated with (A) 0.1% vehicle DMSO (control) for 48 h and annomuricin E at the IC₅₀ concentration for (B) 12, (C) 24 and (D) 48 h. After staining the cells with PI, the DNA contents were monitored using flow cytometry. (E) The representative bar chart shows the significant induction of G₁ cell cycle arrest by annomuricin E after 12 h of treatment. The data represent the means ± SEM of three independent experiments. *<i>P</i><0.05 compared with the control.</p

Effects of annomuricin E on LDH leakage formation in HT-29 cells.

<p>Cells were exposed to 0.1% vehicle DMSO (control) and annomuricin E at different concentrations for 24 h. The treated HT-29 cells showed a significant LDH release at 4 to 16 μg/ml concentrations compared with the control. The data represent the means ± SEM of three independent experiments. *<i>P</i><0.05 compared with the control.</p

The Chemopotential Effect of <i>Annona muricata</i> Leaves against Azoxymethane-Induced Colonic Aberrant Crypt Foci in Rats and the Apoptotic Effect of Acetogenin Annomuricin E in HT-29 Cells: A Bioassay-Guided Approach

<div><p><i>Annona muricata</i> has been used in folk medicine for the treatment of cancer and tumors. This study evaluated the chemopreventive properties of an ethyl acetate extract of <i>A</i>. <i>muricata</i> leaves (EEAML) on azoxymethane-induced colonic aberrant crypt foci (ACF) in rats. Moreover, the cytotoxic compound of EEAML (Annomuricin E) was isolated, and its apoptosis-inducing effect was investigated against HT-29 colon cancer cell line using a bioassay-guided approach. This experiment was performed on five groups of rats: negative control, cancer control, EEAML (250 mg/kg), EEAML (500 mg/kg) and positive control (5-fluorouracil). Methylene blue staining of colorectal specimens showed that application of EEAML at both doses significantly reduced the colonic ACF formation compared with the cancer control group. Immunohistochemistry analysis showed the down-regulation of PCNA and Bcl-2 proteins and the up-regulation of Bax protein after administration of EEAML compared with the cancer control group. In addition, an increase in the levels of enzymatic antioxidants and a decrease in the malondialdehyde level of the colon tissue homogenates were observed, suggesting the suppression of lipid peroxidation. Annomuricin E inhibited the growth of HT-29 cells with an IC50 value of 1.62 ± 0.24 μg/ml after 48 h. The cytotoxic effect of annomuricin E was further substantiated by G1 cell cycle arrest and early apoptosis induction in HT-29 cells. Annomuricin E triggered mitochondria-initiated events, including the dissipation of the mitochondrial membrane potential and the leakage of cytochrome <i>c</i> from the mitochondria. Prior to these events, annomuricin E activated caspase 3/7 and caspase 9. Upstream, annomuricin E induced a time-dependent upregulation of Bax and downregulation of Bcl-2 at the mRNA and protein levels. In conclusion, these findings substantiate the usage of <i>A</i>. <i>muricata</i> leaves in ethnomedicine against cancer and highlight annomuricin E as one of the contributing compounds in the anticancer activity of <i>A</i>. <i>muricata</i> leaves.</p></div

Chemical structure of annomuricin E.

<p>Chemical structure of annomuricin E.</p

¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectral data of annomuricin E in CDCl₃ (δ in ppm, <i>J</i> in Hz).

<p>¹H NMR (500 MHz) and ¹³C NMR (125 MHz) spectral data of annomuricin E in CDCl₃ (δ in ppm, <i>J</i> in Hz).</p