Immunotherapy of neoplastic diseases with neuraminidase: Contradictions,new aspects,and revised concepts

Summary The divergent experimental results in immunotherapy of spontaneous, chemically induced or virus-induced solid tumors or leukemias with neuraminidase are reviewed and analyzed under the various aspects of the possible modes and conditions of action of the enzyme: Immunocompetence of the host, animal residual tumor volume, enzymatic activity of the neuraminidase, and identity of the antigenic specificity within the tumor system are well-known prerequisites for an effective tumor immunotherapy. In addition, there seems to be evidence that the number of tumor cells used for vaccination and the dose of enzymatically active VCN, whether bound to VCN-treated tumor cells or injected intratumorally, may be decisive in the negative or positive outcome. Moreover, there are indications that a preexistent sensitization against the so-called Thomsen-Friedenreich antigen, which seems to be unmasked after VCN treatment of cells, may influence the tumor therapeutic success. The effect of nonspecific immunostimulators given in addition to neuraminidase or to neuraminidase-treated cells is controversial. Thus, this combination cannot be recommended unless it is fully explored. To overcome the problem of the dependence of the tumor therapeutic effect on the dose of cells and the amount of neuraminidase with respect to different tumors and different adjuvant treatments, a new immunization concept, named chessboard vaccination, has been proposed. The data obtained so far in vitro and in vivo with this chessboard vaccination are briefly reviewed. They show that chessboard vaccination might be of diagnostic as well as of therapeutic interest.     

Cargo binding and regulatory sites in the tail of fungal conventional kinesin

Here, using a quantitative in vivo assay, we map three regions in the carboxy terminus of conventional kinesin that are involved in cargo association, folding and regulation, respectively. Using C-terminal and internal deletions, point mutations, localization studies, and an engineered 'minimal' kinesin, we identify five heptads of a coiled-coil domain in the kinesin tail that are necessary and sufficient for cargo association. Mutational analysis and in vitro ATPase assays highlight a conserved motif in the globular tail that is involved in regulation of the motor domain; a region preceding this motif participates in folding. Although these sites are spatially and functionally distinct, they probably cooperate during activation of the motor for cargo transport.     

The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis,Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P₂. In N. crassa hyphae, PtdIns(4,5)P₂ localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P₂ that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.     

The N-terminal portion of the main cytosolic loop mediates K+ sensitivity in the retinal rod Na+/Ca2+-K+-exchanger.

Two types of Na+/Ca2+-exchangers have been characterized in the literature: The first is the cardiac, skeletal muscle and brain type, which exchanges 1 Ca2+ for 3 Na+, the second, found in retinal photosensor cells, transports 1 Ca2+ and 1 K+ in exchange for 4 Na+. The present work describes the properties of chimeric constructs of the two exchanger types. Ca2+ gel overlay experiments have identified a high affinity (Kd in the 1 microM range) Ca2+-binding domain between Glu601 and Asp733 in the main cytosolic loop of the retinal protein, just after transmembrane domain 5. Insertion of the retinal Ca2+-binding domain in the cytosolic loop of the cardiac exchanger conferred K+-dependence to the Ca2+ uptake activity of the chimeric constructs expressed in HeLa cells. The apparent Km of the K+ effect was about 1 mM. Experiments with C-terminally truncated versions of the retinal insert indicated that the sequence between Leu643 and Asp733 was critical in mediating K+ sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+-binding domain in the main cytosolic loop of the retinal exchanger may regulate the activity of the retinal protein by binding Ca2+, and by conferring to it K+ sensitivity.     

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