Heterogenic Final Cell Cycle by Chicken Retinal Lim1 Horizontal Progenitor Cells Leads to Heteroploid Cells with a Remaining Replicated Genome

Retinal progenitor cells undergo apical mitoses during the process of interkinetic nuclear migration and newly generated post-mitotic neurons migrate to their prospective retinal layer. Whereas this is valid for most types of retinal neurons, chicken horizontal cells are generated by delayed non-apical mitoses from dedicated progenitors. The regulation of such final cell cycle is not well understood and we have studied how Lim1 expressing horizontal progenitor cells (HPCs) exit the cell cycle. We have used markers for S- and G2/M-phase in combination with markers for cell cycle regulators Rb1, cyclin B1, cdc25C and p27Kip1 to characterise the final cell cycle of HPCs. The results show that Lim1+ HPCs are heterogenic with regards to when and during what phase they leave the final cell cycle. Not all horizontal cells were generated by a non-apical (basal) mitosis; instead, the HPCs exhibited three different behaviours during the final cell cycle. Thirty-five percent of the Lim1+ horizontal cells was estimated to be generated by non-apical mitoses. The other horizontal cells were either generated by an interkinetic nuclear migration with an apical mitosis or by a cell cycle with an S-phase that was not followed by any mitosis. Such cells remain with replicated DNA and may be regarded as somatic heteroploids. The observed heterogeneity of the final cell cycle was also seen in the expression of Rb1, cyclin B1, cdc25C and p27Kip1. Phosphorylated Rb1-Ser608 was restricted to the Lim1+ cells that entered S-phase while cyclin B1 and cdc25C were exclusively expressed in HPCs having a basal mitosis. Only HPCs that leave the cell cycle after an apical mitosis expressed p27Kip1. We speculate that the cell cycle heterogeneity with formation of heteroploid cells may present a cellular context that contributes to the suggested propensity of these cells to generate cancer when the retinoblastoma gene is mutated.     

New cytologic clues in localized Leishmania lymphadenitis

OBJECTIVE: To describe new cytologic clues to diagnose localized leishmania lymphadenitis (LLL). STUDY DESIGN: The study examined cytologic smears of 170 cases of LLL referred to our department from November 1989 to October 2004. A total of 120 cases were confirmed by detecting Leishman-Donovan (LD) bodies in at least 1 of the cytologic smears and 50 cases, which were histologically confirmed. For comparison we studied cytologic smears of 20 cases of tuberculous lymphadenitis, 20 cases of toxoplasma lymphadenitis and 20 cases of granulomatous lymphadenitis of unspecified causes. RESULTS: Cases were divided into 4 major groups. Cytologic findings in these groups were studied to find highly suggestive clues. Cytologic findings present in most of these groups, but absent or very rare in other granulomatous lymphadenitis, were LD kinetoplasts, plasma cells with different shapes of inclusions and lymphogranular bodies. Rare findings not reported previously were: intraneutrophilic LD bodies, hematoxylin body-like inclusions, fibroblasts, cytoplasmic blebbing and floating parasitophorous vacuoles. CONCLUSION: Despite previous reports emphasizing detecting LD bodies in diagnosing LLL, we present cytologic clues highly suggestive of this self-limited disease when LD bodies cannot be detected or are very few on the smears.     

Investigation of Critical Core Formulation and Process Parameters for Osmotic Pump Oral Drug Delivery

Push–pull osmotic pump (PPOP) tablets of a practically insoluble model drug were developed and the effect of various formulation and process parameters on tablet performance was evaluated in order to identify critical factors. The formulation factors such as the viscosity grade of polyethylene oxide as the primary polymer as well as the level and location of osmogen within the bilayer tablets led to a difference in performance of osmotic tablets and hence should be critically evaluated in the design of such dosage forms. Modification of granulation process, i.e., the granulating liquid composition or drying method of granules, did not impact the drug release from the osmotic tablets at the evaluated scale of this study. The influence of varying dose and aqueous solubility of other model drugs (i.e., theophylline, acetaminophen, and verapamil HCl) on the developed PPOP template was also investigated. Results showed that irrespective of the perceived complexity of development and manufacturing of osmotic pumps, the osmotic tablets in this study demonstrated a robust and yet flexible platform in accommodating different types of drug candidates, regardless of solubility, for the dose levels below 25% w/w of the pull layer formulation.     

The heterogenic final cell cycle of chicken retinal Lim1 horizontal cells is not regulated by the DNA damage response pathway

Cells with aberrations in chromosomal ploidy are normally removed by apoptosis. However, aneuploid neurons have been shown to remain functional and active both in the cortex and in the retina. Lim1 horizontal progenitor cells in the chicken retina have a heterogenic final cell cycle, producing some cells that enter S-phase without proceeding into M-phase. The cells become heteroploid but do not undergo developmental cell death. This prompted us to investigate if the final cell cycle of these cells is under the regulation of an active DNA damage response. Our results show that the DNA damage response pathway, including γ-H2AX and Rad51 foci, is not triggered during any phase of the different final cell cycles of horizontal progenitor cells. However, chemically inducing DNA adducts or double-strand breaks in Lim1 horizontal progenitor cells activated the DNA damage response pathway, showing that the cells are capable of a functional response to DNA damage. Moreover, manipulation of the DNA damage response pathway during the final cell cycle using inhibitors of ATM/ATR, Chk1/2, and p38MAPK, neither induced apoptosis nor mitosis in the Lim1 horizontal progenitor cells. We conclude that the DNA damage response pathway is functional in the Lim1 horizontal progenitor cells, but that it is not directly involved in the regulation of the final cell cycle that gives rise to the heteroploid horizontal cell population.     

Exercise restores coronary vascular function independent of myogenic tone or hyperglycemic status in db/db mice

Regulation of coronary function in diabetic hearts is an important component in preventing ischemic cardiac events but remains poorly studied. Exercise is recommended in the management of diabetes, but its effects on diabetic coronary function are relatively unknown. We investigated coronary artery myogenic tone and endothelial function, essential elements in maintaining vascular fluid dynamics in the myocardium. We hypothesized that exercise reduces pressure-induced myogenic constriction of coronary arteries while improving endothelial function in db/db mice, a model of type 2 diabetes. We used pressurized mouse coronary arteries isolated from hearts of control and db/db mice that were sedentary or exercised for 1 h/day on a motorized exercise-wheel system (set at 5.2 m/day, 5 days/wk). Exercise caused a approximately 10% weight loss in db/db mice and decreased whole body oxidative stress, as measured by plasma 8-isoprostane levels, but failed to improve hyperglycemia or plasma insulin levels. Exercise did not alter myogenic regulation of arterial diameter stimulated by increased transmural pressure, nor did it alter smooth muscle responses to U-46619 (a thromboxane agonist) or sodium nitroprusside (an endothelium-independent dilator). Moderate levels of exercise restored ACh-simulated, endothelium-dependent coronary artery vasodilation in db/db mice and increased expression of Mn SOD and decreased nitrotyrosine levels in hearts of db/db mice. We conclude that the vascular benefits of moderate levels of exercise were independent of changes in myogenic tone or hyperglycemic status and primarily involved increased nitric oxide bioavailability in the coronary microcirculation.     

Chaperone Potential of Pulicaria undulata Extract in Preventing Aggregation of Stressed Proteins

This study examined the effect of an aqueous extract of Pulicaria undulata on the 1,4-dithiothreitol (DTT)-induced aggregation of proteins. The effects of the chaperone properties of P. undulata extract on protein aggregation were determined by measuring light scattering absorption, fluorescence, and circular dichroism (CD) spectroscopy. The aqueous extract of P. undulata possesses good chaperone properties but the protection effect was varied in different protein. The extract showed a higher level of protection in high molecular weight proteins than in those of low molecular weight. Using a fluorescence study, the present study provides information on the hydrophobic area of proteins interacting with the P. undulata extract. In fact, by increasing the concentration of the P. undulata extract, the hydrophic area of the protein decreased. CD spectroscopy also revealed that DTT caused changes in both the tertiary and the secondary structure of the proteins, while in the presence of P. undulata extract, there was little change. Our finding suggests the possibility of using P. undulata extract for the inhibition of aggregation and the deposition of protein in disease.     

The Effect of Quercetin Nanosuspension on Prostate Cancer Cell Line LNCaP via Hedgehog Signaling Pathway

Background:Prostate cancer (PCa) is the second leading cause of cancer death in American population. In this manner, novel therapeutic approaches for identification of therapeutic targets for PCa has significant clinical implications. Quercetin is a potent cancer therapeutic agent and dietary antioxidant present in fruit and vegetables.Methods:To investigate the underlying mechanism by which the PCa was regulated, nanoparticles of quercetin were administrated to cells. For in vitro experiments, human PCa cell line LNCaP were involved. Cell viability assay and quantitative RT-PCR (qRT-PCR) for hedgehog signaling pathway genes were used to determine the key signaling pathway regulated for PCa progression.Results:The cell viability gradually decreased with increased concentration of quercetin nanoparticles. At 48 h, 40 mM concentration of quercetin treatment showed near 50% of viable cells. Quercetin nanoparticles upregulates Su(Fu) mRNA expressions and downregulates gli mRNA expressions in the LNCaP cells.Conclusion:The results showed that the hedgehog signaling targeted inhibition may have important implications of PCa therapeutics. Additionally, the outcomes provided new mechanistic basis for further examination of quercetin nanoparticles to discover potential treatment strategies and new targets for PCa inhibition.Key Words: Hedgehog, Prostate cancer, Proliferation, Quercetin nanoparticles, Signaling pathway     

TRAPPC2L is a Novel, Highly Conserved TRAPP-Interacting Protein

Mutations in the trafficking protein particle complex C2 protein (TRAPPC2), a mammalian ortholog of yeast Trs20p and a component of the trafficking protein particle (TRAPP) vesicle tethering complex, have been linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT). Intriguingly, the X-linked TRAPPC2 is just one of a complement of Trs20-related genes in humans. Here we characterize TRAPPC2L, a novel, highly conserved TRAPP-interacting protein related to TRAPPC2 and the uncharacterized yeast open reading frame YEL048c . TRAPPC2L and TRAPPC2 genes are found in pairs across species and show broad and overlapping expression, suggesting they are functionally distinct, a notion supported by yeast complementation studies and biochemical characterization. RNA interference-mediated knockdown of either TRAPPC2L or TRAPPC2 in HeLa cells leads to fragmentation of the Golgi, implicating both proteins in Golgi dynamics. Gradient fractionation of cellular membranes indicates that TRAPPC2L is found with a portion of cellular TRAPP on very low-density membranes whereas the remainder of TRAPP, but not TRAPPC2L, is found associated with Golgi markers. YEL048c displays genetic interactions with TRAPP II-encoding genes and the gene product co-fractionates with and interacts with yeast TRAPP II. Taken together these results indicate that TRAPPC2L and its yeast ortholog YEL048c are novel TRAPP-interacting proteins that may modulate the function of the TRAPP II complex.     

C4orf41 and TTC-15 are mammalian TRAPP components with a role at an early stage in ER-to-Golgi trafficking

TRAPP is a multisubunit tethering complex implicated in multiple vesicle trafficking steps in Saccharomyces cerevisiae and conserved throughout eukarya, including humans. Here we confirm the role of TRAPPC2L as a stable component of mammalian TRAPP and report the identification of four novel components of the complex: C4orf41, TTC-15, KIAA1012, and Bet3L. Two of the components, KIAA1012 and Bet3L, are mammalian homologues of Trs85p and Bet3p, respectively. The remaining two novel TRAPP components, C4orf41 and TTC-15, have no homologues in S. cerevisiae. With this work, human homologues of all the S. cerevisiae TRAPP proteins, with the exception of the Saccharomycotina-specific subunit Trs65p, have now been reported. Through a multidisciplinary approach, we demonstrate that the novel proteins are bona fide components of human TRAPP and implicate C4orf41 and TTC-15 (which we call TRAPPC11 and TRAPPC12, respectively) in ER-to-Golgi trafficking at a very early stage. We further present a binary interaction map for all known mammalian TRAPP components and evidence that TRAPP oligomerizes. Our data are consistent with the absence of a TRAPP I-equivalent complex in mammalian cells, suggesting that the fundamental unit of mammalian TRAPP is distinct from that characterized in S. cerevisiae.     

Quantitative characterization of postnatal growth trends in proximal pulmonary arteries in rats by phase-contrast magnetic resonance imaging

Malformations of the pulmonary arteries can increase right heart workload and result in morbidity, heart failure, and death. With the increased use of murine models to study these malformations, there is a pressing need for an accurate and noninvasive experimental technique that is capable of characterizing pulmonary arterial hemodynamics in these animals. We describe the growth trends of pulmonary arteries in 13 male Sprague-Dawley rats at 20, 36, 52, 100, and 160 days of age with the introduction of phase-contrast MRI as such a technique. PCMRI results correlated closely with cardiac output measurements by ultrasound echocardiography and with fluorescent microspheres in right-left lung flow split (flow partition). Mean flow, average cross-sectional area, distensibility, and shear rates for the right and left pulmonary arteries (RPA and LPA) were calculated. The RPA was larger and received more flow at all times than the LPA (P < 0.0001). Right-left flow split did not change significantly with age, and arterial distensibility was not significantly different between RPA and LPA, except at 160 days (P < 0.01). Shear rates were much higher for the LPA than the RPA (P < 0.0001) throughout development. The RPA and LPA showed different structure-function relationships but obeyed similar allometric scaling laws, with scaling exponents comparable to those of the main pulmonary artery. This study is the first to quantitatively describe changes in RPA and LPA flows and sizes with development and to apply phase-contrast MRI techniques to pulmonary arteries in rats.