Identification and function of the centrosome centromatrix

Centrosomes direct the organization of microtubules in animal cells. However, in the absence of centrosomes, cytoplasm has the potential to organize microtubules and assemble complex structures such as anastral spindles. During cell replication or following fertilization, centrioles that are incapable of organizing microtubules into astral arrays are introduced into this complex cytoplasmic environment. These centrioles become associated with pericentriolar material responsible for centrosome-dependent microtubule nucleation, and thus the centrosome matures to ultimately become a dominant microtubule organizing center that serves as a central organizer of cell cytoplasm. We describe the identification of a novel structure within the pericentriolar material of centrosomes called the centromatrix. The centromatrix is a salt-insoluble filamentous scaffold to which subunit structures that are necessary for microtubule nucleation and abundant in the cytoplasm bind. We propose that the centromatrix serves to concentrate and focus these subunits to form the microtubule organizing center. Since binding of these subunits to the centromatrix does not require nucleotides, we propose a model for centrosome assembly which predicts that the assembly of the centromatrix is a rate-limiting step in centrosome assembly and maturation.     

Some observations on the effect of Daflon (micronized purified flavonoid fraction of Rutaceae aurantiae) in bancroftian filarial lymphoedema

BACKGROUND: Morbidity management is a core component of the global programme for the elimination of lymphatic filariasis. In a double-blind clinical trial, the tolerability and efficacy of Daflon (500 mg) + DEC (25 mg) or DEC (25 mg) alone, twice daily for 90 days, was studied in 26 patients with bancroftian filarial lymphoedema. RESULTS: None of the patients in either drug group reported any adverse reaction throughout the treatment period (90 days). Haematological and biochemical parameters were within normal limits and there was no significant difference between the pre-treatment (day 0) and post-treatment (day 90) values. The group receiving Daflon showed significant reduction in oedema volume from day 90 (140.6 PlusMinus; 18.8 ml) to day 360 (71.8 PlusMinus; 20.7 ml) compared to the pre-treatment (day 0, 198.4 PlusMinus; 16.5 ml) value. This accounted for a 63.8% reduction in oedema volume by day 360 (considering the pre-treatment (day 0) as 100%). In the DEC group, the changes in oedema volume (between day 1 and day 360) were not significant when compared to the pre-treatment (day 0) value. The percentage reduction at day 360 was only 9%, which was not significant (P > 0.05). CONCLUSION: This study has shown that Daflon (500 mg, twice a day for 90 days) is both safe and efficacious in reducing oedema volume in bancroftian filarial lymphoedema. Further clinical trials are essential for strengthening the evidence base on the role of this drug in the morbidity management of lymphatic filariasis.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.     

The beta-agonist isoproterenol attenuates EGF-stimulated wound closure in human airway epithelial cells

Asthma is a disease characterized by reversible airway obstruction. An additional hallmark of chronic asthma is altered wound healing that leads to airway remodeling. Although beta-agonists are effective in treating the bronchospasm associated with asthma, their effects on airway wound healing, which are related to airway remodeling, are unknown. It has been demonstrated that beta-agonists can alter the signaling of epidermal growth factor (EGF) receptors, which are important in timely wound healing. Therefore, we hypothesized that the beta-agonist isoproterenol would affect wound healing. Using an in vitro scrape wound assay, we demonstrated that isoproterenol attenuates EGF-stimulated wound healing in 16HBE airway epithelial cell cultures. Through experiments with forskolin and cells overexpressing beta2-adrenergic receptor-yellow fluorescent protein, we show that attenuation is due to the accumulation of cAMP and the involvement of at least one additional pathway. Furthermore, attenuation is not due to a direct effect on the EGF receptor or to an alteration of the ERK/MAPK signaling cascade. Based on these results, we propose that isoproterenol may exert its effects through other MAPK signaling pathways (JNK and/or p38) or through parallel mechanisms. These results also demonstrate a problem of potential therapeutic relevance in which a commonly prescribed medication may alter wound healing and contribute to the remodeling of asthmatic airways.     

Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.     

Amino acid sequence, crystallization and structure determination of reduced and oxidized cytochrome c6 from the green alga Scenedesmus obliquus.

Cytochrome c6from the unicellular green alga Scenedesmus obliquus was sequenced, crystallized in its reduced and oxidized state and the three-dimensional structure of the protein in both redox states was determined by X-ray crystallography. Reduced cytochrome c6crystallized as a monomer in the space group P 21212, whereas the oxidized protein crystallized as a dimer in the space group P 3121. The structures were solved by molecular replacement and refined to 1. 9 and 2.0 A, respectively.Comparison of the structures of both redox states revealed only slight differences on the protein surface, whereas a distortion along the axis between the heme iron and its coordinating Met61 residue was observed. No redox-dependent movement of internal water molecules could be detected. The high degree of similarity of the surfaces and charge distributions of both redox states, as well as the dimerization of cytochrome c6as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c6and its redox partners. The dimer of oxidized cytochrome c6may represent a molecular structure occurring in a binary complex with cytochrome b6f. This assembly might be required for the correct orientation of cytochrome c6with respect to its redox partner cytochrome b6f, facilitating the electron transfer within the complex. If the dimerization is not redox-dependent in vivo, the almost identical surfaces of both redox states do not support a long range differentiation between reduced and oxidized cyt c6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.     

Evaluation of NMR spectral data of urine in conjunction with measured clinical chemistry and histopathology parameters to assess the effects of liver and kidney toxicants

Single low and high doses of several compounds with known renal toxic effects (para-aminophenol, puromycin aminonucleoside, sodium chromate, and hexachlorobutadiene,) or known liver toxic effects (galactosamine, allyl alcohol, and thioacetamide) were administered to male Wistar rats in groups of 4 or 8 for each compound. Predose urine samples (Day 0) and samples from post-dosing (Days 1–4) were collected for each rat and monitored by 1D ¹H NMR. Principal component analysis (PCA) of the NMR spectra was used to investigate differences between dose levels for each compound individually. The findings from PCA at both dose levels for each compound were examined in the context of the corresponding clinical chemistry and pathology data collected during the study. The PCA clustering of NMR spectra from rats dosed with each individual compound were shown to be associated with the measured levels of creatinine, BUN, AST, ALT and histopathology findings. Finally, scaled-to-maximum, aligned, and reduced trajectories (SMART) analysis was applied to compare the temporal metabolic trajectories obtained for each animal at each dose level of the administered compounds. By day 4, the SMART trajectories for allyl alcohol and hexachlorobutadiene had returned to predose levels indicating a recovery response, however, the high dose SMART trajectories for para-aminophenol, puromycin aminonucleoside, sodium chromate, and galactosamine did not appear to return to predose levels indicating a prolonged toxic effect.