Site-directed mutagenesis of human endothelial cell ecto-ADPase/soluble CD39: requirement of glutamate 174 and serine 218 for enzyme activity and inhibition of platelet recruitment

Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasis. It rapidly metabolizes ADP released from stimulated platelets, thereby preventing further platelet activation and recruitment. We recently developed a recombinant, soluble form of human CD39, solCD39, with enzymatic and biological properties identical to CD39. To identify amino acids essential for enzymatic/biological activity, we performed site-directed mutagenesis within the four highly conserved apyrase regions of solCD39. Mutation of glutamate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in complete and approximately 90% loss of solCD39 enzymatic activity, respectively. Furthermore, compared to wild-type, S57A exhibited a 2-fold increase in ADPase activity without change in ATPase activity, while the tyrosine 127 to alanine (Y127A) mutant lost 50-60% of both ADPase and ATPase activity. The ADPase activity of wild-type solCD39 and each mutant, except for R135A, was greater with calcium as the required divalent cation than with magnesium, but for ATPase activity generally no such preference was observed. Y127A demonstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold higher than that of wild-type, even though its enzyme activity was greatly reduced. SolCD39 mutants were further characterized by correlating enzymatic with biological activity in an in vitro platelet aggregation system. Each solCD39 mutant was similar to wild-type in reversing platelet aggregation, except for E174A and S218A. E174A, completely devoid of enzymatic activity, failed to inhibit platelet responsiveness, as anticipated. S218A, with 91% loss of ADPase activity, could still reverse platelet aggregation, albeit much less effectively than wild-type solCD39. Thus, glutamate 174 and serine 218 are essential for both the enzymatic and biological activity of solCD39.     

Nick recognition by DNA ligases

Phage T7 DNA ligase seals nicked DNA substrates and is a representative member of the ATP-dependent class of DNA ligases. Although the catalytic mechanism of DNA ligases has been delineated, little is known about the nature of nick recognition by these enzymes. Here, we show that T7 ligase discriminates, at the nick-binding step, between nicks containing either a 5'-phosphate or a 5'-OH. T7 ligase binds preferentially to phosphorylated nicks and catalyses the sealing reaction. We also show using DNA footprinting studies, that T7 ligase binds asymmetrically to nicks as a monomer, with the protein interface covering between 12 and 14 bp of DNA. Based on molecular modelling studies we propose a structural model of the ligase-DNA complex consistent with these and other data. Using photo-crosslinking and site-directed mutagenesis we have identified two residues, K238 and K240, critical for the transadenylation and nick-sealing reactions. Sequence conservation and structural analysis supports the premise that these two lysine residues are critical for both nucleotide binding and DNA nick recognition. The implications of these results on the ligation mechanism are discussed.     

Profound protection against respiratory challenge with a lethal H7N7 influenza A virus by increasing the magnitude of CD8(+) T-cell memory

The recall of CD8(+) T-cell memory established by infecting H-2(b) mice with an H1N1 influenza A virus provided a measure of protection against an extremely virulent H7N7 virus. The numbers of CD8(+) effector and memory T cells specific for the shared, immunodominant D(b)NP(366) epitope were greatly increased subsequent to the H7N7 challenge, and though lung titers remained as high as those in naive controls for 5 days or more, the virus was cleared more rapidly. Expanding the CD8(+) memory T-cell pool (<0.5 to >10%) by sequential priming with two different influenza A viruses (H3N2-->H1N1) gave much better protection. Though the H7N7 virus initially grew to equivalent titers in the lungs of naive and double-primed mice, the replicative phase was substantially controlled within 3 days. This tertiary H7N7 challenge caused little increase in the magnitude of the CD8(+) D(b)NP(366)(+) T-cell pool, and only a portion of the memory population in the lymphoid tissue could be shown to proliferate. The great majority of the CD8(+) D(b)NP(366)(+) set that localized to the infected respiratory tract had, however, cycled at least once, though recent cell division was shown not to be a prerequisite for T-cell extravasation. The selective induction of CD8(+) T-cell memory can thus greatly limit the damage caused by a virulent influenza A virus, with the extent of protection being directly related to the number of available responders. Furthermore, a large pool of CD8(+) memory T cells may be only partially utilized to deal with a potentially lethal influenza infection.     

Diminished primary and secondary influenza virus-specific CD8(+) T-cell responses in CD4-depleted Ig(-/-) mice

Optimal expansion of influenza virus nucleoprotein (D(b)NP(366))-specific CD8(+) T cells following respiratory challenge of naive Ig(-/-) microMT mice was found to require CD4(+) T-cell help, and this effect was also observed in primed animals. Absence of the CD4(+) population was consistently correlated with diminished recruitment of virus-specific CD8(+) T cells to the infected lung, delayed virus clearance, and increased morbidity. The splenic CD8(+) set generated during the recall response in Ig(-/-) mice primed at least 6 months previously showed a normal profile of gamma interferon production subsequent to short-term, in vitro stimulation with viral peptide, irrespective of a concurrent CD4(+) T-cell response. Both the magnitude and the localization profiles of virus-specific CD8(+) T cells, though perhaps not their functional characteristics, are thus modified in mice lacking CD4(+) T cells.     

Endogenous opiates: 1997

This paper is the twentieth installment of our annual review of research concerning the opiate system. It summarizes papers published during 1997 that studied the behavioral effects of the opiate peptides and antagonists, excluding the purely analgesic effects, although stress-induced analgesia is included. The specific topics covered this year include stress; tolerance and dependence; eating and drinking; alcohol; gastrointestinal, renal, and hepatic function; mental illness and mood; learning, memory, and reward; cardiovascular responses; respiration and thermoregulation; seizures and other neurologic disorders; electrical-related activity; general activity and locomotion; sex, pregnancy, and development; immunologic responses; and other behaviors.     

Milk-Based Nutraceutical for Treating Autoimmune Arthritis via the Stimulation of IL-10- and TGF-β-producing CD39+ Regulatory T Cells

Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39⁺ Tregs producing TGF-β and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I.     

Point-of-Care CD4 Testing to Inform Selection of Antiretroviral Medications in South African Antenatal Clinics: A Cost-Effectiveness Analysis

Background

Many prevention of mother-to-child HIV transmission (PMTCT) programs currently prioritize antiretroviral therapy (ART) for women with advanced HIV. Point-of-care (POC) CD4 assays may expedite the selection of three-drug ART instead of zidovudine, but are costlier than traditional laboratory assays.

Methods

We used validated models of HIV infection to simulate pregnant, HIV-infected women (mean age 26 years, gestational age 26 weeks) in a general antenatal clinic in South Africa, and their infants. We examined two strategies for CD4 testing after HIV diagnosis: laboratory (test rate: 96%, result-return rate: 87%, cost: $14) and POC (test rate: 99%, result-return rate: 95%, cost: $26). We modeled South African PMTCT guidelines during the study period (WHO “Option A”): antenatal zidovudine (CD4 ≤350/μL) or ART (CD4>350/μL). Outcomes included MTCT risk at weaning (age 6 months), maternal and pediatric life expectancy (LE), maternal and pediatric lifetime healthcare costs (2013 USD), and cost-effectiveness ($/life-year saved).

Results

In the base case, laboratory led to projected MTCT risks of 5.7%, undiscounted pediatric LE of 53.2 years, and undiscounted PMTCT plus pediatric lifetime costs of $1,070/infant. POC led to lower modeled MTCT risk (5.3%), greater pediatric LE (53.4 years) and lower PMTCT plus pediatric lifetime costs ($1,040/infant). Maternal outcomes following laboratory were similar to POC (LE: 21.2 years; lifetime costs: $23,860/person). Compared to laboratory, POC improved clinical outcomes and reduced healthcare costs.

Conclusions

In antenatal clinics implementing Option A, the higher initial cost of a one-time POC CD4 assay will be offset by cost-savings from prevention of pediatric HIV infection.     

Drebrin Regulates Neuroblast Migration in the Postnatal Mammalian Brain

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.     

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.     

Building the Foundation for International Conservation Planning for Breeding Ducks across the U.S. and Canadian Border

We used publically available data on duck breeding distribution and recently compiled geospatial data on upland habitat and environmental conditions to develop a spatially explicit model of breeding duck populations across the entire Prairie Pothole Region (PPR). Our spatial population models were able to identify key areas for duck conservation across the PPR and predict between 62.1 – 79.1% (68.4% avg.) of the variation in duck counts by year from 2002 – 2010. The median difference in observed vs. predicted duck counts at a transect segment level was 4.6 ducks. Our models are the first seamless spatially explicit models of waterfowl abundance across the entire PPR and represent an initial step toward joint conservation planning between Prairie Pothole and Prairie Habitat Joint Ventures. Our work demonstrates that when spatial and temporal variation for highly mobile birds is incorporated into conservation planning it will likely increase the habitat area required to support defined population goals. A major goal of the current North American Waterfowl Management Plan and subsequent action plan is the linking of harvest and habitat management. We contend incorporation of spatial aspects will increase the likelihood of coherent joint harvest and habitat management decisions. Our results show at a minimum, it is possible to produce spatially explicit waterfowl abundance models that when summed across survey strata will produce similar strata level population estimates as the design-based Waterfowl Breeding Pair and Habitat Survey (r² = 0.977). This is important because these design-based population estimates are currently used to set duck harvest regulations and to set duck population and habitat goals for the North American Waterfowl Management Plan. We hope this effort generates discussion on the important linkages between spatial and temporal variation in population size, and distribution relative to habitat quantity and quality when linking habitat and population goals across this important region.