NEUROSECRETORY CELL PROTEIN METABOLISM IN THE LAND SNAIL,OTALA LACTEA

—Protein synthesis in an identified molluscan neurosecretory cell of the land snail, Otala lactea was examined using three different types of polyacrylamide gel electrophoresis. Cells taken from active snails synthesized specific low molecular weight proteins while those from aestivated snails did not. Most of the newly synthesized low molecular weight proteins in the active snails were lost from the cell body when the preparations was chased for 19 h in label-free enriched medium in the presence of anisomycin, an inhibitor of protein synthesis. If colchicine, a blocker of axonal transport, was included in the chase medium, the proteins present following a pulse were largely replaced by smaller molecular weight species. The results suggest that specific low molecular weight proteins are converted to smaller species and then transported from the cell body.     

Ataxin1L Is a Regulator of HSC Function Highlighting the Utility of Cross-Tissue Comparisons for Gene Discovery

Hematopoietic stem cells (HSCs) are rare quiescent cells that continuously replenish the cellular components of the peripheral blood. Observing that the ataxia-associated gene Ataxin-1-like (Atxn1L) was highly expressed in HSCs, we examined its role in HSC function through in vitro and in vivo assays. Mice lacking Atxn1L had greater numbers of HSCs that regenerated the blood more quickly than their wild-type counterparts. Molecular analyses indicated Atxn1L null HSCs had gene expression changes that regulate a program consistent with their higher level of proliferation, suggesting that Atxn1L is a novel regulator of HSC quiescence. To determine if additional brain-associated genes were candidates for hematologic regulation, we examined genes encoding proteins from autism- and ataxia-associated protein–protein interaction networks for their representation in hematopoietic cell populations. The interactomes were found to be highly enriched for proteins encoded by genes specifically expressed in HSCs relative to their differentiated progeny. Our data suggest a heretofore unappreciated similarity between regulatory modules in the brain and HSCs, offering a new strategy for novel gene discovery in both systems.     

Comprehensive profiling of accessible surface glycans of mammalian sperm using a lectin microarray

It is well known that cell surface glycans or glycocalyx play important roles in sperm motility, maturation and fertilization. A comprehensive profile of the sperm surface glycans will greatly facilitate both basic research (sperm glycobiology) and clinical studies, such as diagnostics of infertility. As a group of natural glycan binders, lectin is an ideal tool for cell surface glycan profiling. However, because of the lack of effective technology, only a few lectins have been tested for lectin-sperm binding profiles. To address this challenge, we have developed a procedure for high-throughput probing of mammalian sperm with 91 lectins on lectin microarrays. Normal sperm from human, boar, bull, goat and rabbit were collected and analyzed on the lectin microarrays. Positive bindings of a set of ~50 lectins were observed for all the sperm of 5 species, which indicated a wide range of glycans are on the surface of mammalian sperm. Species specific lectin bindings were also observed. Clustering analysis revealed that the distances of the five species according to the lectin binding profiles are consistent with that of the genome sequence based phylogenetic tree except for rabbit. The procedure that we established in this study could be generally applicable for sperm from other species or defect sperm from the same species. We believe the lectin binding profiles of the mammalian sperm that we established in this study are valuable for both basic research and clinical studies.     

Ultraviolet photoelectron studies of 5-halouracils

Ultravoilet photoelectron spectroscopy has been employed to exmamin the valence electronic structure of 5-fluorouracil, 5-chlorouracil, 5-bromouracil, and 5-iodouracil. Photoelectron bands associted with the three highest π orbitals and the two oxygen atom lone-pair orbitals were assigned by a comparison to similar bands observed in the photoelectron spectrum of uraciul. Bands arising from the halogen atom lone-pair orbitals were assigned by comparing the present results with photoelectron spectra measured for halobenzenes, and by considering the linear dependence of halogen atom lone-pair ionization potentials upon halogen atom electronegativities. The present spectroscopic results have been compared with results from studies of association constants of 5-halouracil–adenine complexes. This examination in dicates that the complex association constants incresase as the ionization potentials of the highest occupied π orbital and the halogen atom lone-pair orbitals of th halouracils decrease.     

LncRNA LEF1-AS1 regulates the migration and proliferation of vascular smooth muscle cells by targeting miR-544a/PTEN axis

Long noncoding RNAs (lncRNAs) play important roles in endothelium development. A lncRNA, LEF1-AS1, is recently emerging as a potent mediator of the proliferation and migration of a number of cells, including smooth muscle cells. However, the effects of LEF1-AS1 in atherosclerosis remains largely unknown. Specimens from patients with coronary artery atherosclerosis were collected. The quantitative real-time polymerase chain reaction was used to analyze levels of LEF1-AS1 and microRNA-544a (miR-544a). Western blot analysis was used to assess PTEN, P-Akt, and T-Akt protein expression. Proliferation, migration, and invasion of cells were analyzed by cell counting kit-8 assay, scratch wound assay, and transwell assay, respectively. The interaction between LEF1-AS1, miR-544a, and PTEN was probed using bioinformatical analysis and dual-luciferase assay. In plasma and tissue of patients with coronary artery atherosclerosis, LEF1-AS1 was upregulated and miR-544a was downregulated. A negative correlation was found between LEF1-AS1 and miR-544a. miR-544a overexpression reversed the inhibition of LEF1-AS1 in smooth muscle cell proliferation and invasion, which were mediated through the PTEN pathway. LEF1-AS1 regulates smooth muscle cell proliferation and migration through the miR-544a/PTEN axis, indicating that LEF1-AS1 may be a potential therapeutic target in atherosclerosis.     

The Unique GGA Clathrin Adaptor of Drosophila melanogaster Is Not Essential

The Golgi-localized, γ-ear-containing, ARF binding proteins (GGAs) are a highly conserved family of monomeric clathrin adaptor proteins implicated in clathrin-mediated protein sorting between the trans-Golgi network and endosomes. GGA RNAi knockdowns in Drosophila have resulted in conflicting data concerning whether the Drosophila GGA (dGGA) is essential. The goal of this study was to define the null phenotype for the unique Drosophila GGA. We describe two independently derived dGGA mutations. Neither allele expresses detectable dGGA protein. Homozygous and hemizygous flies with each allele are viable and fertile. In contrast to a previous report using RNAi knockdown, GGA mutant flies show no evidence of age-dependent retinal degeneration or cathepsin missorting. Our results demonstrate that several of the previous RNAi knockdown phenotypes were the result of off-target effects. However, GGA null flies are hypersensitive to dietary chloroquine and to starvation, implicating GGA in lysosomal function and autophagy.     

A Biodegradable,Sustained-Released,Prednisolone Acetate Microfilm Drug Delivery System Effectively Prolongs Corneal Allograft Survival in the Rat Keratoplasty Model

Frequent and long-term use of topical corticosteroids after corneal transplantation is necessary to prevent graft rejection. However, it relies heavily on patient compliance, and sustained therapeutic drug levels are often not achieved with administration of topical eye drops. A biodegradable drug delivery system with a controlled and sustained drug release may circumvent these limitations. In this study, we investigated the efficacy of a prednisolone acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC) microfilm drug delivery system on promoting the survival of allogeneic grafts after penetrating keratoplasty (PK) using a rat model. The drug release profiles of the microfilms were characterized (group 1). Subsequently, forty-eight PK were performed in four experimental groups: syngeneic control grafts (group 2), allogeneic control grafts (group 3), allogeneic grafts with subconjunctivally-implanted PA microfilm (group 4), and allogeneic grafts with PA eye drops (group 5; n = 12 in each). PA-loaded microfilm achieved a sustained and steady release at a rate of 0.006–0.009 mg/day, with a consistent aqueous drug concentration of 207–209 ng/ml. The mean survival days was >28 days in group 2, 9.9±0.8 days in group 3, 26.8±2.7 days in group 4, and 26.4±3.4 days in group 5 (P = 0.023 and P = 0.027 compared with group 3). Statistically significant decrease in CD4+, CD163+, CD 25+, and CD54+ cell infiltration was observed in group 4 and group 5 compared with group 3 (P<0.001). There was no significant difference in the mean survival and immunohistochemical analysis between group 4 and group 5. These results showed that sustained PA-loaded microfilm effectively prolongs corneal allograft survival. It is as effective as conventional PA eye drops, providing a promising clinically applicable alternative for patients undergoing corneal transplantation.