The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.     

Structural basis for the red light induced repolarization of tip growth in caulonema cells ofCeratodon purpureus

Summary Two dynamic changes are associated with the phytochrome-regulated phototropic response in tip cells of the mossCeratodon purpureus: a tip-located gradient shift of chlortetracy-cline (CTC)-stained calcium and a structural reorganization of apical microfilaments (MFs). We examined the interdependence of these processes. Cells were treated with the antimicrotubule drug oryzalin, the antimicrofilament drug cytochalasin-D, and the calcium channel blocker nifedipine. respectively. The effects on phototropic growth, on the structural alignment of the cytoskeleton (microtubules, MTs; microfilaments) and on the distribution of CTC-stained calcium were studied under each of these conditions. In gravitropically growing tip cells the apical MFs form a cortical collar-like structure, consisting of actin bundles with a parallel axial alignment. These MFs point towards the presumptive growing point, a weakly stained region in the tip of the cell from which bundles are absent. MTs are present in the cortex and in the endoplasm of the tip, predominantly oriented longitudinally. The MTs converge within the central apex. The cells show a steep tip-to-base CTC-calcium gradient with its highest signal in the central apex. Destruction of MTs by 1 M oryzalin induces several translocational effects: (i) the growing zone and phototropic outgrowth shift from the apex to subapical parts of the cell; (ii) the structural integrity of the apical MFs and the tip-to-base alignment of the CTC-calcium gradient are disturbed; and (iii) the red light induced gradient shift and the reorientation of MFs proceed in an expanded area spanning from the tip to subapical parts of the cell. Cytochalasin-D (10 g/ml) destroys the MFs. Under these conditions tip growth stops and the phototropic outgrowth is suppressed. The apical MT-structure and the CTC-calcium gradient are not influenced by the agent. Unilateral red light still induces the light-directed translocation of the gradient. Tip cells memorize a unilateral irradiation applied during growth inhibition with cytochalasin-D. After recovery in darkness the cells start to grow in the former light direction. The restoration of the MFs precedes the outgrowth. The structural alignment of the rebuilt actin bundles indicates the future growth direction. The calcium channel blocker nifedipine (10 M) also inhibits tip growth and concurrently phototropic outgrowth. Nifedipine destroys the CTC-calcium gradient and apical MFs; MTs are not influenced by the channel blocker.Abbreviations CTC chlortetracycline - DIC differential interference contrast - DMSO dimethyl sulfoxide - EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N-N-tetraacetic acid - MBS 3-maleimidobenzoic acid N-hydroxysuccimide ester - MF microfilament - MT microtubule - MTSB microtubule stabilizing buffer - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - RP rhodamine labeled phalloidin     

Biometrics,biomathematics and the morphometric synthesis

At the core of contemporarymorphometrics—the quantitative study of biological shape variation—is a synthesis of two originally divergent methodological styles. One contributory tradition is the multivariate analysis of covariance matrices originally developed as biometrics and now dominant across a broad expanse of applied statistics. This approach, couched solely in the linear geometry of covariance structures, ignores biomathematical aspects of the original measurements. The other tributary emphasizes the direct visualization of changes in biological form. However, making objective the biological meaning of the features seen in those diagrams was always problematical; also, the representation of variation, as distinct from pairwise difference, proved infeasible. To combine these two variants of biomathematical modeling into a valid praxis for quantitative studies of biological shape was a goal earnestly sought though most of this century. That goal was finally achieved in the 1980s when techniques from mathematical statistics, multivariate biometrics, non-Euclidean geometry and computer graphics were combined in a coherent new system of tools for the complete regionalized quantitative analysis oflandmark points together with the biomedical images in which they are seen. In this morphometric synthesis, correspondence of landmarks (biologically labeled geometric points, like “bridge of the nose”) across specimens is taken as a biomathematical primitive. The shapes of configurations of landmarks are defined as equivalence classes with respect to the Euclidean similarity group and then represented as single points in David Kendall'sshape space, a Riemannian manifold with Procrustes distance as metric. All conventional multivariate strategies carry over to the study of shape variation and covariation when shapes are interpreted in the tangent space to the shape manifold at an average shape. For biomathematical interpretation of such analyses, one needs a basis for the tangent space compatible with the reality of local biotheoretical processes and explanations at many different geometric scales, and one needs graphics for visualizing average shape differences and other statistical contrasts there. Both of these needs are managed by thethin-plate spline, a deformation function that has an unusually helpful linear algebra. The spline also links the biometrics of landmarks to deformation analysis of the images from which the landmarks originally arose. This article reviews the history and principal tools of this synthesis in their biomathematical and biometrical context and demonstrates their usefulness in a study of focal neuroanatomical anomalies in schizophrenia.     

Enhanced stability in vivo of a thermodynamically stable mutant form of yeast iso-1-cytochrome c

Previous work has established that the N57I amino acid replacement in iso-1-cytochrome c from the yeast Saccharomyces cerevisiae causes an unprecedented increase in thermodynamic stability of the protein in vitro, whereas the N57G replacement diminishes stability. Spectrophotometric measurements of intact cells revealed that the N57I iso-l-cytochrome c is present at higher than normal levels in vivo. Although iso-1-cytochrome c turnover is negligible during aerobic growth, transfer of fully derepressed, aerobically grown cells to anaerobic growth conditions leads to reduction in the levels of all of the cytochromes. Pulsechase experiments carried out under these anaerobic conditions demonstrated that the N57I iso-l-cytochrome c has a longer half-life than the normal protein. This is the first report of enhanced stability in vivo of a mutant form of a protein that has an enhanced thermodynamic stability in vitro. Although the N57I protein concentration is higher than the normal level, reduced growth in lactate medium indicated that the specific activity of this iso-l-cytochrome c in vivo is diminished relative to wild-type. On the other hand, the level of the thermodynamically labile N57G iso-1-cytochrome c was below normal. The in vivo levels of the N57I and N57G iso-l-cytochrome c suggest that proteins in the mitochondrial intermembrane space can be subjected to degradation, and that this degradation may play a role in controlling their normal levels.     

Contribution of structural elements to Thermus thermophilus ribonuclease P RNA function.

We have performed a deletion and mutational analysis of the catalytic ribonuclease (RNase) P RNA subunit from the extreme thermophilic eubacterium Thermus thermophilus HB8. Catalytic activity was reduced 600-fold when the terminal helix, connecting the 5' and 3' ends of the molecule, was destroyed by deleting 15 nucleotides from the 3' end. In comparison, the removal of a large portion (94 nucleotides, about one quarter of the RNA) of the upper loop region impaired function only to a relatively moderate extent (400-fold reduction in activity). The terminal helix appears to be crucial for the proper folding of RNase P RNA, possibly by orientating the adjacent universally conserved pseudoknot structure. The region containing the lower half of the pseudoknot structure was shown to be a key element for enzyme function, as was the region of nucleotides 328-335. Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetranucleotide sequence or a single cytidine reduced catalytic activity only 6-fold, whereas a simultaneous mutation of the five highly conserved nucleotides in the region of nucleotides 328-335 reduced catalytic activity by > 10(5)-fold. The two strictly conserved adenines 244 and 245 (nucleotides 248/249 in Escherichia coli RNase P RNA) were not as essential for enzyme function as suggested by previous data. However, additional disruption of two helical segments (nucleotides 235-242) adjacent to nucleotides 244 and 245 reduced activity by > 10(4)-fold, supporting the notion that nucleotides in this region are also part of the active core structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Germ-Line Mutations in the von Hippel–Lindau Tumor-Suppressor Gene Are Similar to Somatic von Hippel–Lindau Aberrations in Sporadic Renal Cell Carcinoma

von Hippel–Lindau (VHL) disease is a hereditary tumor syndrome predisposing to multifocal bilateral renal cell carcinomas (RCCs), pheochromocytomas, and pancreatic tumors, as well as angiomas and hemangioblastomas of the CNS. A candidate gene for VHL was recently identified, which led to the isolation of a partial cDNA clone with extended open reading frame, without significant homology to known genes or obvious functional motifs, except for an acidic pentamer repeat domain. To further characterize the functional domains of the VHL gene and assess its involvement in hereditary and nonhereditary tumors, we performed mutation analyses and studied its expression in normal and tumor tissue. We identified germ-line mutations in 39% of VHL disease families. Moreover, 33% of sporadic RCCs and all (6/6) sporadic RCC cell lines analyzed showed mutations within the VHL gene. Both germ-line and somatic mutations included deletions, insertions, splice-site mutations, and missense and nonsense mutations, all of which clustered at the 3' end of the corresponding partial VHL cDNA open reading frame, including an alternatively spliced exon 123 nt in length, suggesting functionally important domains encoded by the VHL gene in this region. Over 180 sporadic tumors of other types have shown no detectable base changes within the presumed coding sequence of the VHL gene to date. We conclude that the gene causing VHL has an important and specific role in the etiology of sporadic RCCs, acts as a recessive tumor-suppressor gene, and appears to encode important functional domains within the 3' end of the known open reading frame.