Specific binding of dehydroepiandrosterone to the N terminus of the microtubule-associated protein MAP2

The effect of neurosteroids is mediated through their membrane or nuclear receptors. However, no dehydroepiandrosterone (DHEA)-specific receptors have been evidenced so far in the brain. In this paper, we showed by isothermal titration calorimetry that the DHEA specifically binds to the dendritic brain microtubule-associated protein MAP2C with an association constant of 2.7 x 10(7) m-1 and at a molar ratio of 1:1. By partial tryptic digestions and mass spectrometry analysis, we found that the binding involved the N-terminal region of MAP2C. Interestingly, MAP2C displays homologies with 17 beta-hydroxysteroid dehydrogenase 1, an enzyme required for estrogen synthesis. Based on these sequence homologies and on the x-ray structure of the DHEA-binding pocket of 17 beta-hydroxysteroid dehydrogenase 1, we modeled the complex of DHEA with MAP2C. The binding of DHEA to MAP2C involved specific hydrogen bonds that orient the steroid into the pocket. This work suggests that DHEA can directly influence brain plasticity via MAP2C binding. It opens interesting ways for understanding the role of DHEA in the brain.     

Contribution of plasma proteins to splanchnic and total anabolic utilization of dietary nitrogen in humans

Splanchnic tissues are largely involved in the postprandial utilization of dietary amino acids, but little is yet known, particularly in humans, about the relative contributions of different splanchnic protein pools to splanchnic and total postprandial anabolism. Our aim was to develop a compartmental model that could distinguish dietary nitrogen (N) incorporation among splanchnic constitutive, plasma (splanchnic exported), and peripheral proteins after a mixed-protein meal in humans. Eight healthy subjects were fed a single mixed meal containing 15N-labeled soy protein, and dietary N postprandial kinetics were measured in plasma free amino acids, proteins, and urea and urinary urea and ammonia. These experimental data and others previously obtained for dietary N kinetics in ileal effluents under similar experimental conditions were used to develop the compartmental model. Six hours after the mixed-meal ingestion, 31.5, 7.5, and 21% of ingested N were predicted to be incorporated into splanchnic constitutive, splanchnic exported, and peripheral proteins, respectively. The contribution of splanchnic exported proteins to total splanchnic anabolism from dietary N was predicted to be approximately 19% and to remain steady throughout the simulation period. Model behavior and its predictions were strongly in line with current knowledge of the system and the scarce, specific data available in the literature. This model provides the first data concerning the anabolism of splanchnic constitutive proteins in the nonsteady postprandial state in humans. By use of only slightly invasive techniques, this model could help to assess how the splanchnic anabolism is modulated under different nutritional or pathophysiological conditions in humans.     

Extending the scope of the controlled logical clock

Event traces are helpful in understanding the performance behavior of parallel applications since they allow the in-depth analysis of communication and synchronization patterns. However, the absence of synchronized clocks on most cluster systems may render the analysis ineffective because inaccurate relative event timings may misrepresent the logical event order and lead to errors when quantifying the impact of certain behaviors or confuse the users of time-line visualization tools by showing messages flowing backward in time. In our earlier work, we have developed a scalable algorithm called the controlled logical clock that eliminates inconsistent inter-process timings postmortem in traces of pure MPI applications, potentially running on large processor configurations. In this paper, we first demonstrate that our algorithm also proves beneficial in computational grids, where a single application is executed using the combined computational power of several geographically dispersed clusters. Second, we present an extended version of the algorithm that—in addition to message-passing event semantics—also preserves and restores shared-memory event semantics, enabling the correction of traces from hybrid applications.     

“Reverse” Carbocyclic Fleximers: Synthesis of a New Class of Adenosine Deaminase Inhibitors

A series of flexible carbocyclic pyrimidine nucleosides has been designed and synthesized. In contrast to previously reported “fleximers” from our laboratory, these analogues have the connectivity of the heterocyclic base system “reversed”, where the pyrimidine ring is attached to the sugar moiety, rather than the five membered imidazole ring. As was previously seen with the ribose fleximers, their inherent flexibility should allow them to adjust to enzyme binding site mutations, as well as increase the affinity for atypical enzymes. Preliminary biological screening has revealed surprising inhibition of adenosine deaminase, despite their lack of resemblance to adenosine.     

The Golgi puppet master: COG complex at center stage of membrane trafficking interactions

The central organelle within the secretory pathway is the Golgi apparatus, a collection of flattened membranes organized into stacks. The cisternal maturation model of intra-Golgi transport depicts Golgi cisternae that mature from cis to medial to trans by receiving resident proteins, such as glycosylation enzymes via retrograde vesicle-mediated recycling. The conserved oligomeric Golgi (COG) complex, a multi-subunit tethering complex of the complexes associated with tethering containing helical rods family, organizes vesicle targeting during intra-Golgi retrograde transport. The COG complex, both physically and functionally, interacts with all classes of molecules maintaining intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, vesicular coats, and molecular motors. In this report, we will review the current state of the COG interactome and analyze possible scenarios for the molecular mechanism of the COG orchestrated vesicle targeting, which plays a central role in maintaining glycosylation homeostasis in all eukaryotic cells.     

Toxic activity from cultures of Karlodinium micrum (=Gyrodinium galatheanum) (Dinophyceae)—a dinoflagellate associated with fish mortalities in an estuarine aquaculture facility

The goal of this study was to test for, and partially characterize, toxic activity associated with the dinoflagellate Karlodinium micrum. Since 1996, three fish kill events associated with blooms of K. micrum have occurred at HyRock Fish Farm, an estuarine pond aquaculture facility raising hybrid striped bass on the Chesapeake Bay, MD, USA. Using an assay based on the lysis of rainbow trout erythrocytes, cultures of a Chesapeake Bay isolate of K. micrum have been shown to produce toxic substances which are released upon cell disturbance or damage. The LC₅₀ for hemolysis of a sonicated cell suspension was 2.4×10⁴ cells ml⁻¹, well within the range of cell concentrations observed associated with fish kills. The toxic activity from K. micrum cells and culture filtrates was traced to two distinct fractions that co-elute with polar lipids. The LC₅₀ for hemolysis of the larger of these two fractions (Tox A) was 284 ng ml⁻¹ while the LC₅₀ of the second, smaller, fraction (Tox B) was 600 ng ml⁻¹. For comparison, the LC₅₀ for the standard hemolysin saponin was 3203 ng ml⁻¹. At concentrations of 800 and 2000 ng ml⁻¹, respectively, Tox A was further shown to be ichthyotoxic to zebrafish (Danio rerio) larvae (80% mortality), and cytotoxic to a mammalian GH(4)C(1) cell line (100% LDH release). At a concentration of 600 ng ml⁻¹ Tox B was shown to be cytotoxic to a mammalian GH(4)C(1) cell line (>30% LDH release), but not ichthyotoxic to zebrafish (D. rerio) larvae up to a concentration of 250 ng ml⁻¹. Although treatment with either algicidal copper or potassium permanganate caused significant lysis of K. micrum cells (>70%), toxic activity was released after treatment with copper and eliminated following treatment with potassium permanganate. This observation in cultures is consistent with observations made at HyRock Fish Farm where significantly higher mortality was observed following treatment of a K. micrum bloom with copper sulfate compared to treatment with potassium permanganate. This study represents the first direct evidence of the toxicity of K. micrum isolated from the Chesapeake Bay.     

Isolation and transformation of rice aleurone protoplasts

Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.     

Methotrexate in rheumatoid arthritis is frequently effective, even if re-employed after a previous failure

Effectiveness of therapy with individual disease-modifying antirheumatic drugs (DMARDs) in rheumatoid arthritis (RA) is limited, and the number of available DMARDs is finite. Therefore, at some stage during the lengthy course of RA, institution of traditional DMARDs that have previously been applied may have to be reconsidered. In the present study we investigated the effectiveness of re-employed methotrexate in patients with a history of previous methotrexate failure (original course). A total of 1,490 RA patients (80% female, 59% rheumatoid factor positive) were followed from their first presentation, yielding a total of 6,470 patient-years of observation. We identified patients in whom methotrexate was re-employed after at least one intermittent course of a different DMARD. We compared reasons for discontinuation, improvement in acute phase reactants, and cumulative retention rates of methotrexate therapy between the original course of methotrexate and its re-employment. Similar analyses were peformed for other DMARDs. Methotrexate was re-employed in 86 patients. Compared with the original courses, re-employment was associated with a reduced risk for treatment termination because of ineffectiveness (P = 0.02, by McNemar test), especially if the maximum methotrexate dose of the original course had been low (<12.5 mg/week; P = 0.02, by logistic regression). In a Cox regression model, re-employed MTX was associated with a significantly reduced hazard of treatment termination compared with the original course of methotrexate, adjusting for dose and year of employment (hazard ratio 0.64, 95% confidence interval 0.42-0.97; P = 0.04). These findings were not recapitulated in analyses of re-employment of other DMARDs. Re-employment of MTX despite prior inefficacy, but not re-employment of other DMARDs, is an effective therapeutic option, especially in those patients in whom the methotrexate dose of the original course was low.     

Light-dependent hypersensitive response and resistance signaling against Turnip Crinkle Virus in Arabidopsis

Resistance to Turnip Crinkle Virus (TCV) in Arabidopsis ecotype Dijon (Di)-17 is conferred by the resistance gene HRT and a recessive locus rrt. In Di-17, TCV elicits a hypersensitive response (HR), which is accompanied by increased expression of pathogenesis-related (PR) genes and high levels of salicylic acid (SA). We have previously shown that HRT-mediated resistance to TCV is dependent on SA-mediated signal transduction and that increased levels of SA confer enhanced resistance to TCV via upregulation of the HRT gene. Here we show that HRT-mediated HR and resistance are dependent on light. A dark treatment immediately following TCV inoculation suppressed HR, resistance and activation of the majority of the TCV-induced genes. However, the absence of light did not affect either TCV-induced elevated levels of free SA or the expression of HRT. Interestingly, in the dark, transgenic plants overexpressing HRT showed susceptibility, but overexpression of HRT coupled with high levels of endogenous SA resulted in pronounced resistance. Consistent with these results is the finding that exogenous application of SA prior to TCV inoculation partially overcame the requirement for light. Light was also required for N gene-mediated HR and resistance to Tobacco Mosaic Virus, suggesting that it is an important factor which may be generally required during defense signaling.     

Crystal structure of a Cbf5-Nop10-Gar1 complex and implications in RNA-guided pseudouridylation and dyskeratosis congenita

H/ACA RNA-protein complexes, comprised of four proteins and an H/ACA guide RNA, modify ribosomal and small nuclear RNAs. The H/ACA proteins are also essential components of telomerase in mammals. Cbf5 is the H/ACA protein that catalyzes isomerization of uridine to pseudouridine in target RNAs. Mutations in human Cbf5 (dyskerin) lead to dyskeratosis congenita. Here, we describe the 2.1 A crystal structure of a specific complex of three archaeal H/ACA proteins, Cbf5, Nop10, and Gar1. Cbf5 displays structural properties that are unique among known pseudouridine synthases and are consistent with its distinct function in RNA-guided pseudouridylation. We also describe the previously unknown structures of both Nop10 and Gar1 and the structural basis for their essential roles in pseudouridylation. By using information from related structures, we have modeled the entire ribonucleoprotein complex including both guide and substrate RNAs. We have also identified a dyskeratosis congenita mutation cluster site within a modeled dyskerin structure.