Failure of highly immunogenic filarial proteins to provide host-protective immunity

In areas that are endemic for lymphatic filariasis, there are individuals who are parasite free and who appear not to have experienced symptoms attributable to filarial infection. These "putatively immune" individuals may recognize immunogens that could be important in host protection. We have immunoscreened expression libraries expressing epitopes encoded by filarial open reading frames and have identified three antigens that are differentially recognized by the two polar clinical groups-endemic normals and asymptomatic microfilaremics. Pre-immunization of susceptible hosts (Meriones unguiculatus) with these antigens revealed that none was able to elicit consistent host protective immunity. Our data are consistent with Waksman's conjecture that highly immunogenic antigens of parasite origin may be inappropriate candidates for prophylactic immunization.     

Sucrose synthase activity does not restrict glycolysis in roots of transgenic potato plants under hypoxic conditions

The effect of hypoxia on root development and carbon metabolism was studied using potato (Solanum tuberosum L.) plants as a model system. Hypoxia led to a cessation of root elongation, and finally to the death of meristematic cells. These changes were accompanied by a 4- to 5-fold accumulation of hexoses, suggesting that insufficient carbohydrate supply was not the cause of cell death. In addition, prolonged hypoxia (96 h) resulted in a 50% increase in activity of most glycolytic enzymes studied and the accumulation of glycerate-3-phosphate and phosphoenolpyruvate. This indicates that endproduct utilisation may restrict metabolic flux through glycolysis. As expected, the activities of alcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.17) increased during hypoxia. Apart from the enzymes of ethanolic fermentation the activity of sucrose synthase (SuSy; EC 2.4.1.13) was enhanced. To investigate the in-vivo significance of this increase, transgenic plants with reduced SuSy activity were analysed. Compared to untransformed controls, transgenic plants showed a reduced ability to resume growth after re-aeration, emphasising the crucial role of SuSy in the toleration of hypoxia. Surprisingly, analysis of glycolytic intermediates in root extracts from SuSy antisense plants revealed no change as compared to wildtype plants. Therefore, limitation of glycolysis is most likely not responsible for the observed decreased ability for recovery after prolonged oxygen starvation. We assume that the function of SuSy during hypoxia might be to channel excess carbohydrates into cell wall polymers for later consumption rather than fuelling glycolysis. Received: 17 February 1999 / Accepted: 10 June 1999     

Pharmacochaperones post-translationally enhance cell surface expression by increasing conformational stability of wild-type and mutant vasopressin V2 receptors

Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V2 receptor GFP fusion protein (mV2R.GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC50 values similar to their KD values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V2Rs (hV2Rs, three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TM I and TM VII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a three-dimensional homology model of the antagonist-bound hV2R.     

Human BACE forms dimers and colocalizes with APP

Beta-site APP-cleaving enzyme (BACE) is a membrane-bound aspartyl protease with no strict primary preference for cleavage. The molecular mechanisms that link the gamma-secretase multicomponent amyloid precursor protein (APP) processing complex to biochemical properties of BACE generating the N terminus of the amyloid beta-peptide have not, as yet, been identified. We found that in human brain tissue, BACE occurred as a dimer. The overall stability of the BACE homodimer was based on intermolecular interactions that were not affected by high salt, nonionic detergents or reducing conditions. BACE homodimers could only partially be separated even under strong denaturing conditions and revealed dramatic differences in the surface charge distribution compared with the monomer. In contrast, the soluble ectodomain of truncated BACE revealed a seemingly lower avidity to the prototypic aspartate protease inhibitor pepstatin and exclusively occurred in the monomeric form. Immunocytochemical studies colocalized APP and BACE in the plasma membrane of cells expressing endogenous levels of BACE and overexpressing APP. In cells that were cotransfected with APP and a putative active site D289A mutant of BACE, colocalization persisted. Remaining enzyme activity was found to be attributable to the mutant protease. Accordingly, inactivation of the carboxyl-terminal active site motif of BACE without an impairment of overall enzyme activity suggests that the enzyme may act as a dimer. Thus, homodimerization of BACE may help the enzyme to acquire specific mechanisms to associate with its substrates to exert catalytic activity.     

Modulation of the M2 muscarinic acetylcholine receptor activity with monoclonal anti-M2 receptor antibody fragments

Antibodies directed against the second extracellular loop of G protein-coupled receptors are known to have functional activities. From a partial agonist monoclonal antibody directed against the M2 muscarinic receptor, we constructed and produced a single chain variable fragment with high affinity for its target epitope. The fragment is able to recognize its receptor on Chinese hamster ovary cells transfected with the M2 muscarinic acetylcholine receptor to block the effect of carbachol on this receptor and to exert an inverse agonist activity on the basal activity of the receptor. The antibody fragment is also able to increase the basal rhythm of cultured neonatal rat cardiomyocytes and to inhibit in a non-competitive manner the negative chronotropic effect of carbachol. This antibody fragment is able to exert its inverse agonist activity in vivo on mouse heart activity. The immunological strategy presented here could be useful to develop specific allosteric inverse agonist reagents for G protein-coupled receptors.     

Presynaptic regulation of dopaminergic neurotransmission

The development of electrochemical recordings with small carbon-fiber electrodes has significantly advanced the understanding of the regulation of catecholamine transmission in various brain areas. Recordings in vivo or in slice preparations monitor diffusion of catecholamine following stimulated synaptic release into the surrounding tissue. This synaptic 'overflow' is defined by the amount of release, by the activity of reuptake, and by the diffusion parameters in brain tissue. Such studies have elucidated the complex regulation of catecholamine release and uptake, and how psychostimulants and anti-psychotic drugs interfere with it. Moreover, recordings with carbon-fiber electrodes from cultured neurons have provided analysis of catecholamine release and its plasticity at the quantal level.     

Expression pattern and raft association of NIPSNAP3 and NIPSNAP4, highly homologous proteins encoded by genes in close proximity to the ATP-binding cassette transporter A1

The highly homologous genes NIPSNAP3 and NIPSNAP4, with 87% amino acid identity, are members of the NIPSNAP family with putative roles in vesicular trafficking. NIPSNAP3 mRNA and NIPSNAP4 mRNA and protein were detected in multiple tissues and cells at varying degrees. Interestingly, NIPSNAP3 is most highly expressed in skeletal muscle, where NIPSNAP4 has a low mRNA abundance. NIPSNAP4 was found associated with membranes and partly localized in rafts. The ubiquitous expression of the highly conserved NIPSNAPs and their association with membranes further support an important cellular function of these proteins probably linked to vesicular trafficking. The NIPSNAP3 and NIPSNAP4 genes are located in close proximity to the 3' end of the ATP-binding cassette transporter A1 (ABCA1), whose mutations cause familial high-density lipoprotein deficiency syndromes. The adjacent genomic location and the finding that ABCA1 is a regulator of vesicular trafficking may indicate a functional relation of these proteins, even though NIPSNAP4 does not interact directly with ABCA1 nor is its expression altered in cells with mutated ABCA1.