Molecular cloning of a cDNA complementary to a UDP-glucose pyrophosphorylase mRNA of Dictyostelium discoideum

Uridine diphosphoglucose pyrophosphorylase (UTP: -alpha-D-glucose-1-phosphate uridyltransferase, EC 2.7.7.9) is an essential enzyme for normal development of Dictyostelium discoideum and its specific activity increases 3- to 10-fold by the later stages of development. Previous experiments have shown that additional forms of the enzyme appear concomitantly with this increase and that two uridine diphosphoglucose pyrophosphorylase (UDPGP) polypeptides are immunoprecipitated from the in vitro translation products of total cellular RNA at any stage of development (B. F. Fishel, R. E. Manrow and R. P. Dottin, 1982, Dev. Biol. 92, 175-187). Using an in vitro translation-immunoprecipitation assay of UDPGP mRNA, we show that an increase in the amount of translatable mRNA is correlated with the accumulation of enzyme during development. A cDNA bank was constructed from a mRNA population that had been enriched for UDPGP mRNA by size fractionation on sucrose gradients containing methylmercuric hydroxide (C. W. Schweinfest, R. W. Kwiatkowski, and R. P. Dottin, 1982, Proc. Natl. Acad. Sci. USA 79, 4997-5000). A 1.8-Kb cDNA complementary to a UDPGP mRNA was identified after screening the bank by hybridization selection and translation. Only the mRNA encoding the higher molecular weight in vitro translation product is hybrid selected by this cDNA. In hybrid-arrested translation experiments, the coding strand of this cDNA selectively inhibits the translation of only one of the two in vitro translation products. Therefore, there are two distinct UDPGP mRNAs.     

Early Exposure to Ketamine Impairs Axonal Pruning in Developing Mouse Hippocampus

Mounting evidence suggests that prolonged exposure to general anesthesia (GA) during brain synaptogenesis damages the immature neurons and results in long-term neurocognitive impairments. Importantly, synaptogenesis relies on timely axon pruning to select axons that participate in active neural circuit formation. This process is in part dependent on proper homeostasis of neurotrophic factors, in particular brain-derived neurotrophic factor (BDNF). We set out to examine how GA may modulate axon maintenance and pruning and focused on the role of BDNF. We exposed post-natal day (PND)7 mice to ketamine using a well-established dosing regimen known to induce significant developmental neurotoxicity. We performed morphometric analyses of the infrapyramidal bundle (IPB) since IPB is known to undergo intense developmental modeling and as such is commonly used as a well-established model of in vivo pruning in rodents. When IPB remodeling was followed from PND10 until PND65, we noted a delay in axonal pruning in ketamine-treated animals when compared to controls; this impairment coincided with ketamine-induced downregulation in BDNF protein expression and maturation suggesting two conclusions: a surge in BDNF protein expression “signals” intense IPB pruning in control animals and ketamine-induced downregulation of BDNF synthesis and maturation could contribute to impaired IPB pruning. We conclude that the combined effects on BDNF homeostasis and impaired axon pruning may in part explain ketamine-induced impairment of neuronal circuitry formation.     

Experimental Data in Support of a Direct Displacement Mechanism for Type I/II l-Asparaginases

Bacterial l-asparaginases play an important role in the treatment of certain types of blood cancers. We are exploring the guinea pig l-asparaginase (gpASNase1) as a potential replacement of the immunogenic bacterial enzymes. The exact mechanism used by l-asparaginases to catalyze the hydrolysis of asparagine into aspartic acid and ammonia has been recently put into question. Earlier experimental data suggested that the reaction proceeds via a covalent intermediate using a ping-pong mechanism, whereas recent computational work advocates the direct displacement of the amine by an activated water. To shed light on this controversy, we generated gpASNase1 mutants of conserved active site residues (T19A, T116A, T19A/T116A, K188M, and Y308F) suspected to play a role in hydrolysis. Using x-ray crystallography, we determined the crystal structures of the T19A, T116A, and K188M mutants soaked in asparagine. We also characterized their steady-state kinetic properties and analyzed the conversion of asparagine to aspartate using NMR. Our structures reveal bound asparagine in the active site that has unambiguously not formed a covalent intermediate. Kinetic and NMR assays detect significant residual activity for all of the mutants. Furthermore, no burst of ammonia production was observed that would indicate covalent intermediate formation and the presence of a ping-pong mechanism. Hence, despite using a variety of techniques, we were unable to obtain experimental evidence that would support the formation of a covalent intermediate. Consequently, our observations support a direct displacement rather than a ping-pong mechanism for l-asparaginases.     

Developing biotechnology tools for ‘beautiful’ vavilovia (<Emphasis Type="Italic">Vavilovia formosa</Emphasis>), a legume crop wild relative with taxonomic and agronomic potential

Beautiful vavilovia, the closest species to the common now extinct ancestor of the whole tribe Fabeae holds significant taxonomical interest and also for breeding within this group of species, which includes the most cultivated leguminous pulses in the world. In spite of this, vavilovia has attracted very scarce research to date and is in danger of complete extinction. Thus, as a part of the research carried out by an informal international group of researchers from various countries, we report here various experiments for the development and exploitation of a range of biotechnology tools for vavilovia, ranging from standard in vitro propagation, to plant regeneration from explant-derived callus, and also from protoplasts. Plants were successfully recovered following propagation from nodes, and by regeneration through organogenesis from callus derived from internodes (which provided the best responses) and leaves. Also, protoplasts were isolated from leaves and stems from in vitro shoots and from callus derived from these two explants, with the latter undergoing sustained division. Subsequently, protoplasts isolated from internode callus proliferated and also underwent organogenesis coupled with whole plant recovery at a low frequency, while protoplasts from leaf callus origin followed both organogenesis and embryogenesis simultaneously but failed to yield viable plants. Flow cytometry assessments permitted to ascertain the genetic fidelity of both propagated and regenerated plants irrespectively of the source tissue from which they were derived (i.e., either callus from explants or from protoplasts). Finally, flow cytometry also permitted us to provide the first record on the relative nuclear DNA content and genome size for Vavilovia formosa.     

The effect of positive pressure breathing on the cerebral circulation and the content of catecholamines in hypothalamus and adrenals

Arterial pressures in the Circle of Willis were measured in dogs subjected to positive pressure breathing. Catecholamines in hypothalamus and adrenals were measured in sacrificed animals. Pathologic changes in brain tissue were demonstrated. Pressure changes and catecholamine variations are discussed.     

3D organization and function of the cell: Golgi budding and vesicle biogenesis to docking at the porosome complex

Insights into the three-dimensional (3D) organization and function of intracellular structures at nanometer resolution, holds the key to our understanding of the molecular underpinnings of cellular structure-function. Besides this fundamental understanding of the cell at the molecular level, such insights hold great promise in identifying the disease processes by their altered molecular profiles, and help determine precise therapeutic treatments. To achieve this objective, previous studies have employed electron microscopy (EM) tomography with reasonable success. However, a major hurdle in the use of EM tomography is the tedious procedures involved in fixing, high-pressure freezing, staining, serial sectioning, imaging, and finally compiling the EM images to obtain a 3D profile of sub-cellular structures. In contrast, the resolution limit of EM tomography is several nanometers, as compared to just a single or even sub-nanometer using the atomic force microscope (AFM). Although AFM has been hugely successful in 3D imaging studies at nanometer resolution and in real time involving isolated live cellular and isolated organelles, it has had limited success in similar studies involving 3D imaging at nm resolution of intracellular structure-function in situ. In the current study, using both AFM and EM on aldehyde-fixed and semi-dry mouse pancreatic acinar cells, new insights on a number of intracellular structure-function relationships and interactions were achieved. Golgi complexes, some exhibiting vesicles in the process of budding were observed, and small vesicles were caught in the act of fusing with larger vesicles, possibly representing either secretory vesicle biogenesis or vesicle refilling following discharge, or both. These results demonstrate the power and scope of the combined engagement of EM and AFM imaging of fixed semi-dry cells, capable of providing a wealth of new information on cellular structure-function and interactions.     

Nuclear geometry: mitotic nucleus flares out when arrested

Studies of budding yeast arrested in mitosis outline a set of rules for nuclear envelope expansion during closed nuclear division.     

Regulation of ecto-5'-nucleotidase (CD73) in cultured cortical astrocytes by different inflammatory factors

Ecto-5'-nucleotidase (e-5NT) is a cell-surface located, rate-limiting enzyme in the extracellular metabolism of ATP, catalyzing the final step of the conversion of AMP to adenosine. Since this enzyme shifts the balance from pro-inflammatory ATP to anti-inflammatory adenosine, it is considered to be an important regulator of inflammation. Although up-regulation of e-5NT was repeatedly reported in several in vivo models of brain injury, the regulation of its expression and function remains largely unknown. We have studied effects of several pro-inflammatory factors, namely, bacterial endotoxin lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), glutamate (Glu) and hydrogen peroxide (H(2)O(2)) on e-5NT (i) activity, (ii) mRNA expression and (iii) membrane protein abundance in primary cultured cortical astrocytes. We are clearly able to demonstrate a stimulus-specific regulation of the e-5NT pathway. IFN-γ, LPS, Glu and H(2)O(2) decrease, while TNF-α increases e-5NT activity. The analysis of e-5NT gene expression and e-5NT membrane protein levels revealed that tested factors regulate e-5NT at different levels and by employing different mechanisms. In summary, we provide evidence that e-5NT activity is tightly regulated in a stimulus-specific manner.