Correlation between adult transformation and aldehyde oxidase staining pattern in wing discs ofApterous genotypes inDrosophila melanogaster

Summary The aldehyde oxidase staining pattern in wing discs ofDrosophila melanogaster bearing the genotypesap ^blt /ap ^blt andap ^blt andap ^blt /ap ⁷³ⁿ showns changes from the wild-type pattern. Extensive areas of the presumptive dorsal posterior wing blade, which are normally unstained, have enzyme activity in these mutants. In wings of these genotypes, dorsal posterior structures are replaced by dorsal anterior wing structures. A strong correlation has been found between the frequencies of various staining patterns in the discs and the extent of transformation in the cuticular structures of the wing, which is consistent with the idea that aldehyde oxidase activity can be used as an indicator in the wing disc of this transformation. Unlike the homoeotic mutationengrailed, apterous has not been interpreted as a selector gene yet the work reported here shows thatapterous alleles can cause changes resembling those of theengrailed phenotype both in aldehyde oxidase staining behaviour and in the cuticular transformation.     

Pyridine nucleotide metabolism in imaginal discs of Drosophila melanogaster

The pyridine nucleotide metabolism of imaginal discs of Drosophila melanogaster has been studied in vitro by incubating discs with labeled nicotinic acid in the presence and absence of ecdysterone. The major labeled compounds found within the discs are NAD, NADP, and nicotinic acid. There is preferential uptake of nicotinamide over nicotinic acid, although the Priess-Handler pathway is used exclusively. The presence of ecdysterone produces a small increase in the NADP/NAD ratio, and an increase in NAD synthesis, probably to compensate for increased NAD turnover.Supported by Grant GB 43569 from the National Science Foundation.     

6-gingerol protects nucleus pulposus-derived mesenchymal stem cells from oxidative injury by activating autophagy

BACKGROUNDTo date, there has been no effective treatment for intervertebral disc degeneration (IDD). Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) showed encouraging results in IDD treatment, but the overexpression of reactive oxygen species (ROS) impaired the endogenous repair abilities of NPMSCs. 6-gingerol (6-GIN) is an antioxidant and anti-inflammatory reagent that might protect NPMSCs from injury.AIMTo investigate the effect of 6-GIN on NPMSCs under oxidative conditions and the potential mechanism.METHODSThe cholecystokinin-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of 6-GIN. ROS levels were measured by 2´7´-dichlorofluorescin diacetate analysis. Matrix metalloproteinase (MMP) was detected by the tetraethylbenzimidazolylcarbocyanine iodide assay. TUNEL assay and Annexin V/PI double-staining were used to determine the apoptosis rate. Additionally, autophagy-related proteins (Beclin-1, LC-3, and p62), apoptosis-associated proteins (Bcl-2, Bax, and caspase-3), and PI3K/Akt signaling pathway-related proteins (PI3K and Akt) were evaluated by Western blot analysis. Autophagosomes were detected by transmission electron microscopy in NPMSCs. LC-3 was also detected by immunofluorescence. The mRNA expression of collagen II and aggrecan was evaluated by real-time polymerase chain reaction (RT-PCR), and the changes in collagen II and MMP-13 expression were verified through an immunofluorescence assay.RESULTS6-GIN exhibited protective effects against hydrogen peroxide-induced injury in NPMSCs, decreased hydrogen peroxide-induced intracellular ROS levels, and inhibited cell apoptosis. 6-GIN could increase Bcl-2 expression and decrease Bax and caspase-3 expression. The MMP, Annexin V-FITC/PI flow cytometry and TUNEL assay results further confirmed that 6-GIN treatment significantly inhibited NPMSC apoptosis induced by hydrogen peroxide. 6-GIN treatment promoted extracellular matrix (ECM) expression by reducing the oxidative stress injury-induced increase in MMP-13 expression. 6-GIN activated autophagy by increasing the expression of autophagy-related markers (Beclin-1 and LC-3) and decreasing the expression of p62. Autophagosomes were visualized by transmission electron microscopy. Pretreatment with 3-MA and BAF further confirmed that 6-GIN-mediated stimulation of autophagy did not reduce autophagosome turnover but increased autophagic flux. The PI3K/Akt pathway was also found to be activated by 6-GIN. 6-GIN inhibited NPMSC apoptosis and ECM degeneration, in which autophagy and the PI3K/Akt pathway were involved.CONCLUSION6-GIN efficiently decreases ROS levels, attenuates hydrogen peroxide-induced NPMSCs apoptosis, and protects the ECM from degeneration. 6-GIN is a promising candidate for treating IDD.     

Optimization of Drug Loading Procedures and Characterization of Liposomal Formulations of Two Novel Agents Intended for Boron Neutron Capture Therapy (BNCT)

Abstract

The characterization of two liposomal formulations of boronated DNA-interacting agents has been performed. It is shown that the two boronated drugs, WSA-Water Soluble Acridine and WSP-Water Soluble Phenantridine, can be encapsulated within unilamellar sterically stabilized liposomes with high drug-to-lipid ratios (up to 0.50:1 (mol:mol)), using transmembrane pH gradients. The steric stabilization of the liposomes was accomplished by the addition of DSPE-PEG(2000) (PEG-lipid) to DSPC/Cho lipid mixtures and the composition used was DSPC: Cho: DSPE-PEG 55:40:5 (moI%). The loading of the drugs resulted in drug precipitation in the liposomal aqueous core as observed by cryo-transmission electron microscopy (c-TEM). Moreover, it is shown that when pH gradients across the bilayer were used for remote loading of WSP or when ammonium sulfate gradients were used for remote loading of WSA, the formation of small bilayer fragments (discs) was induced. We present compelling evidence that the formation of discs is a consequence of precipitate growth in the liposomal interior. The precipitate growth causes some of the liposomes to rupture resulting in the above mentioned disc-formation and a substantial decrease in trapping efficiency. The in vitro stability of the drug loaded liposomes was excellent, both in buffer and in 25% human serum. For most of the formulations, the release of the drugs was below or around 10% after 24 hours at 37^oC. Furthermore, the influence of initial internal pH and internal buffering capacity on release properties of WSA and WSP were investigated. It is shown that the release profiles of the drugs can be controlled, to a large extent, by varying the composition of the internal liposomal aqueous phase.     

Computational biomechanics of a lumbar motion segment in pure and combined shear loads

Anterior shear has been implicated as a risk factor in spinal injuries. A 3D nonlinear poroelastic finite element model study of a lumbar motion segment L4-L5 was performed to predict the temporal shear response under various single and combined shear loads. Effects of nucleotomy and facetectomy as well as changes in the posture and facet gap distance were analyzed as well.     

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.     

Lumbar spine angles and intervertebral disc characteristics with end-range positions in three planes of motion in healthy people using upright MRI

Understanding changes in lumbar spine (LS) angles and intervertebral disc (IVD) behavior in end-range positions in healthy subjects can provide a basis for developing more specific LS models and comparing people with spine pathology. The purposes of this study are to quantify 3D LS angles and changes in IVD characteristics with end-range positions in 3 planes of motion using upright MRI in healthy people, and to determine which intervertebral segments contribute most in each plane of movement. Thirteen people (average age = 24.4 years, range 18–51 years; 9 females; BMI = 22.4 ± 1.8 kg/m²) with no history of low back pain were scanned in an upright MRI in standing, sitting flexion, sitting axial rotation (left, right), prone on elbows, prone extension, and standing lateral bending (left, right). Global and local intervertebral LS angles were measured. Anterior-posterior length of the IVD and location of the nucleus pulposus was measured. For the sagittal plane, lower LS segments contribute most to change in position, and the location of the nucleus pulposus migrated from a more posterior position in sitting flexion to a more anterior position in end-range extension. For lateral bending, the upper LS contributes most to end-range positions. Small degrees of intervertebral rotation (1–2°) across all levels were observed for axial plane positions. There were no systematic changes in IVD characteristics for axial or coronal plane positions.     

DNA synthesis in the imaginal wing discs of the American bollwormHelicoverpa armigera (Hübner)

The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instarHelicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of incubation up to 8 h and decreased later without the addition of moulting hormone. Addition of 20-hydroxyecdysone supported long term acquisition of competence for DNA synthesis in the wing discs. Both DNA synthesis and protein content were drastically reduced in plumbagin and azadirachtin-treated insects. Underin vitro conditions, plumbagin had a more pronounced inhibitory effect than azadirachtin. All the ecdysteroids tested, viz. makisterone A, 20-hydroxyecdysone and the ecdysteroidal fraction from the silver fernCheilanthes farinosa enhanced DNA synthesis     

Ultrastructural changes in the muscles,midgut, hemopoietic organ,imaginal discs and Malpighian tubules of the mosquito Aedes aegypti larvae infected by the fungus Coelomomyces stegomyiae

Fungi belonging to the genus Coelomomyces can infect mosquito larvae and develop within the larval hemocoel. To examine fungal development, Aedesaegypti larvae infected with Coelomomyces stegomyiae Keilin were fixed, embedded and sectioned for both light and electron microscopy. While fungal hyphae of C. stegomyiae did not invade cells other than the cuticular epithelial cells, they did penetrate a number of tissues including muscles, midgut, hemopoietic organ, imaginal discs, and Malpighian tubules. This revised version was published online in July 2006 with corrections to the Cover Date.