Background: Listeria monocytogenes CdaA is an essential diadenylate cyclase. Results: CdaA activity depends on manganese and cobalt ions. Conclusion: CdaA has an unusual requirement for metal cofactors. Significance: Characterization of essential enzymes is important for developing novel antibiotics.

In plasma, von Willebrand factor (vWf) associates with Factor VIII (FVIII); however, the site at which these proteins first interact has not been defined. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) causes a rapid, concomitant elevation in plasma levels of both vWf and FVIII, suggesting the existence of a DDAVP-releasable storage pool for both proteins. To determine whether vWf and FVIII can associate intracellularly and colocalize to storage vesicles, we transfected AtT-20 cells with vWf and FVIII expression plasmids. FVIII alone was not detectable within storage granules; however, transfection of vWf cDNA into the same cell caused FVIII to alter its intracellular trafficking and to undergo granular storage, colocalizing to the vWf-containing granules. In contrast, colocalization of FVIII was not observed when these cells were transfected with plasmids encoding defective FVIII-binding vWf mutants. Transfection of bovine endothelial cells with FVIII further demonstrated vesicular storage of FVIII with vWf in Weibel-Palade bodies. Since gene therapy of hemophilia A may ultimately target endothelium or hematopoietic stem cells, the interaction between vWf and FVIII within a secretory cell is important. Thus, vWf can alter the intracellular trafficking of FVIII from a constitutive to a regulated secretory pathway, thereby producing an intracellular storage pool of both proteins.

Immunotherapy is a promising treatment for drug addiction. However, insufficient immune responses to vaccines in most subjects pose a challenge. In this study, we tested the efficacy of a new cocaine vaccine (dAd5GNE) in antagonizing cocaine addiction-related behaviors in rats. This vaccine used a disrupted serotype 5 adenovirus (Ad) gene transfer vector coupled to a third-generation cocaine hapten, termed GNE (6-(2R,3S)-3-(benzoyloxy)-8-methyl-8-azabicyclo [3.2.1] octane-2-carboxamido-hexanoic acid). Three groups of rats were immunized with dAd5GNE. One group was injected with 3 H-cocaine, and radioactivity in the blood and brain was determined. A second group was tested for cocaine-induced locomotor sensitization. A third group was examined for cocaine self-administration, extinction, and reinstatement of responding for cocaine. Antibody titers were determined at various time-points. In each experiment, we added a control group that was immunized with dAd5 without a hapten. The vaccination with dAd5GNE produced long-lasting high titers (410 5 ) of anti-cocaine antibodies in all of the rats. The vaccination inhibited cocaine-induced hyperlocomotor activity and sensitization. Vaccinated rats acquired cocaine self-administration, but they showed less motivation to self-administer cocaine under a progressive-ratio schedule than control rats. When cocaine was not available in a session, control rats exhibited 'extinction burst' responding, whereas vaccinated rats did not. Moreover, when primed with cocaine, vaccinated rats did not reinstate responding, suggesting a blockade of cocaine-seeking behavior. These data strongly suggest that our dAd5GNE vector-based vaccine may be effective in treating cocaine abuse and addiction.

Based on the concept that anticocaine antibodies could prevent inhaled cocaine from reaching its target receptors in the brain, an effective anticocaine vaccine could help reverse cocaine addiction. Leveraging the knowledge that E1(-)E3(-) adenovirus (Ad) gene transfer vectors are potent immunogens, we have developed a novel vaccine platform for addictive drugs by covalently linking a cocaine analog to the capsid proteins of noninfectious, disrupted Ad vector. The Ad-based anticocaine vaccine evokes high-titer anticocaine antibodies in mice sufficient to completely reverse, on a persistent basis, the hyperlocomotor activity induced by intravenous administration of cocaine.

While studying patient plasma containing an unusual pattern of von Willebrand factor (VWF) multimers, we discovered a previously unreported phenomenon: heavy predominance of dimeric VWF. Genomic analysis revealed a new congenital mutation (Tyr87Ser) that altered the final stages of VWF biosynthesis. This mutation in the propeptide (VWFpp) resulted in synthesis of dimeric VWF with an almost complete loss of N-terminal multimerization. The multimer pattern in patient plasma appears to result from separate alleles' synthesizing wild-type or mutant (dimeric) VWF, with homodimers composing the predominant protomeric species. We have expressed VWF protein containing the Tyr87Ser mutation and analyzed the intracellular processing and resulting VWF biological functions. The expressed dimeric VWF displayed a loss of several specific functions: collagen binding, factor VIII binding, and ristocetin-induced platelet binding. However, granular storage of dimeric VWF was normal, demonstrating that the lack of multimerization does not preclude granular storage. Although the tertiary structure of the VWFpp remains unknown, the mutant amino acid is located in a region that is highly conserved across several species and may play a major role in the multimerization of VWF. Our data suggest that one function of the highly cysteine-rich VWFpp is to align the adjacent subunits of VWF into the correct configuration, serving as an intramolecular chaperone. The integrity of the VWFpp is essential to maintain the proper spacing and alignment of the multiple cysteines in the VWFpp and N-terminus of the mature

Summary Leukodystrophies are rare white matter genetic disorders of the central nervous system (CNS) with progressive neurologic deterioration. One approach to the treatment of leukodystrophies is by gene therapy. Using metachromatic leukodystrophy (MLD), a leukodystrophy resulting from deficiency of a lysosomal catabolic enzyme arylsulfatase A (ARSA) as the example, this review is focused on the current status of preclinical and clinical development of gene therapy as a viable treatment option for leukodystrophies. In MLD, mutations in the ARSA gene result in excess buildup of sulfatides, which triggers apoptosis of glia and neurons. The disease is characterized by severe cerebral demyelination and atrophy, with progressive loss of oligodendrocytes, neurons and Schwann cells. The optimal therapy for MLD would provide persistent and high level expression of ARSA in the CNS. Gene therapy using adeno-associated virus (AAV) is an ideal choice for clinical development as it provides the best balance of potential for efficacy with a reduced safety risk profile. In this review, we have summarized preclinical data that support the use of a gene therapy with the AAVrh.10 serotype for clinical development as a treatment for MLD.

Summary. von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within a-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to traf®cking and storage of FVIII with VWF in platelet a-granules. Isolex 1 selected CD34 cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIIItransduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immuno¯uorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the a-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2±6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.

Abstract-Although it is known that factor VIII (FVIII) plasma levels increase rapidly in response to a number of stimuli, the biological stimuli behind this release is less clear.