Phosphorylation of eIF4E at a conserved serine in Aplysia

We have cloned eIF4E from the marine mollusk, Aplysia californica. The sequence of eIF4E from Aplysia is more similar to vertebrate eIF4Es than to other invertebrate sequences. Aplysia eIF4E is encoded by two tissue-specific RNAs. Antibodies raised to the carboxyl terminus of eIF4E recognize a 29-kDa protein that can bind to 7-methyl-GTP caps. The phosphorylation site identified in mammalian eIF4E is conserved in the Aplysia homologue, and an Aplysia eIF4E fusion protein is phosphorylated well by both Aplysia protein kinase C isoforms. However, protein kinase C phosphorylates both Ser-207 and Thr-208 in vitro, while only Ser-207 is phosphorylated in vivo. We have confirmed that Ser-207 is phosphorylated in vivo by raising a phosphopeptide antibody to this site. This antibody will be useful in determining the signal transduction pathways leading to eIF4E phosphorylation in Aplysia.     

Effects of injury on the biokinetics of the lumbar spine

In vitro experiments used to investigate the three-dimensional kinetics of the intact, normal ligamentous human lumbar spine and the changes due to injuries are described. A major objective was to assess the extent of injury and the necessity for surgery. The analysis of the data is described, and the results are discussed. The utility of the method is assessed and compared with that of in vivo studies. The experimental technique has enabled the injury types that are likely to induce spinal instability to be identified.     

Forced vital capacity, slow vital capacity, or inspiratory vital capacity: which is the best measure of vital capacity?

To assess whether evoked changes in arterial pressure after stimulation of the bed nucleus of the stria terminalis (BST) are opposed by baroreceptor input to the central nervous system, the rostral BST of sinoaortic-denervated (SAD), urethane- (1.3 g/kg) anesthetized, male Sprague-Dawley rats was probed for cardiovascular reactive sites. Electrical stimuli (50 microA, 50 Hz), delivered through stereotaxically placed glass semimicroelectrodes, were directed to the rostral medial BST. Sham-operated animals served as controls. Stimulation sites were correlated with cytoarchitecturally distinct areas within the rostral BST, and changes in mean arterial pressure (MAP) were subjected to statistical analysis. Consistent with our previous observations, stimulation of the rostral BST produced changes (p < 0.05) in MAP in both sham-operated and SAD rats. Medial stimulation produced pressor responses; lateral stimulation produced depressor responses. In contrast, neither the magnitude nor the duration of the stimulation-evoked changes in MAP were affected by SAD. Thus, in the urethane-anesthetized rat, the rostral medial BST influence on cardiovascular function is not affected by changes in baroreceptor activity.     

Cell binding sequences in mouse laminin alpha1 chain

Laminin-1, a multifunctional glycoprotein of the basement membrane, consists of three different subunits, alpha1, beta1, and gamma1 chains. Previously, we used synthetic peptides to screen for biologically active sequences in the laminin alpha1 chain C-terminal globular domain (G domain) and identified several cell binding sequences (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Song, S. Y., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). Here, we identify new cell binding sequences on the remainder of the laminin alpha1 chain by systematic peptide screening, using 208 overlapping synthetic peptides encompassing the central and N-terminal portions of the alpha1 chain. HT-1080 cell attachment activity to the peptides was evaluated using peptide-coated plastic substrates and peptide-conjugated Sepharose beads. Twenty five peptides showed cell attachment activities on either the peptide-coated plastic substrates and/or the peptide-conjugated Sepharose beads. A-13 (RQVFQVAYIIIKA) showed strongest cell attachment activity in both the assays. Cell attachment to 14 of the peptides was inhibited by heparin. EDTA and integrin antibodies inhibited cell adhesion to two of the peptides, A-13 and A-25, suggesting that these sites likely bind to integrins. These peptides inhibited cell attachment to laminin-1 but not to collagen I, suggesting these active sites are available on the intact molecule. Most of active sequences were localized on globular domains suggesting that these structures play a critical role in binding to cell-surface receptors.     

Genetic dissection of sperm individualization in Drosophila melanogaster

The morphogenesis of spermatids generally takes place within a syncytium, in which all spermatid nuclei descended from a primary spermatocyte remain connected via an extensive network of cytoplasmic bridges. A late step in sperm maturation therefore requires the physical resolution of the syncytium, or cyst, into individual cells, a process sometimes referred to as sperm individualization. Despite the identification of specialized machinery involved in the individualization of Drosophila spermatids (Tokuyasu, K. T., Peacock, W. J. and Hardy, R. W. (1972) Z. Zellforsch 124, 479-506), and of many Drosophila genes mutable to male-sterile phenotypes, little is known of the mechanisms by which this extensive remodeling of the cyst is accomplished. Here, the identification of a major cytoskeletal component of the individualization complex as actin is confirmed with a simple fluorescence assay. Using rhodamine-phalloidin as a probe, the individualization complex is readily visualized forming around bundles of spermatid nuclei at one end of highly elongated cysts, then translocating along the length of the cysts. The structure of the individualization complex in a male-sterile clathrin heavy chain (Chc) mutant is observed to be reduced or disrupted relative to wild-type, consistent with the individualization-deficient phenotype of this mutant. Using the fluorescence assay, a sampling of male-sterile mutant phenotypes in which spermatogenesis proceeds to the assembly of highly elongated cysts distinguishes at least four different phenotypic classes: (1) mutations (nanking class) that block or significantly retard the assembly of the actin-based individualization complex around the nuclear bundle, (2) mutations (dud class) in which the individualization complex assembles in/around the nuclear bundle, but fails to translocate down the cyst, (3) mutations (mulet class) that allow the assembly of a morphologically normal individualization complex around the nuclear bundle, but result in a breakdown in the complex after it begins to translocate down the cyst, and (4) mutations (purity of essence class) that allow the assembly of a motile but morphologically altered or reduced individualization complex. Individualization also fails in a number of mutants with altered nuclear shape, consistent with the hypothesis that spermatid nuclei provide a physical scaffolding for the assembly of the individualization complex. Genetic analysis suggests that a substantial number of additional loci with phenotypes distinguishable with this assay remain to be identified. The large proportion of male-sterile mutations resulting in a late block to spermatogenesis, in which highly elongated cysts fail to be individualized, suggest a substantial susceptibility of this process to a broad range of cellular perturbations. The massive reorganization of cyst cytoplasm required at individualization is expected to be a correspondingly complex function requiring exquisite coordination of multiple cytoplasmic functions, and may account for the previously noted high frequency with which Drosophila genes are mutable to male-sterile phenotypes.     

Comparison of methods to identify outliers observed in health services small area variation studies

Small area variation analysis (SAV) is an established methodology in health services and epidemiological research. The goal is to demonstrate that rates differ across areas, and to explain these differences by differences in physician practice styles or patient characteristics. While the SAV statistics provide an overall variation estimate, they do not provide a statistical means to identify significant outliers. We compared the chi-square (chi2) test with three approaches in determining significant outliers in SAV. We used data from the Canadian Institute for Health Information (CIHI) for Ontario residents discharged between 1989 and 1991. Coronary artery bypass surgery, hysterectomy and hip replacement data were used to compare four statistics in determining outliers: the chi2 test, Swift's approximate bootstrap confidence interval (ABC), Carriere's T2 (T2) with simultaneous confidence intervals (SCI), and Gentleman's normalized scores (GNS). Both the ABC and SCI correct the skewness of the distribution of the adjusted rates. With large data, confidence intervals calculated by the normal or the ABC methods are indistinguishable. The T2 can be applied to also nonbinary events. For binary events, it is asymptotically the same as the chi2. The GNS ranks the rates, but the distribution of these ranks does not differ significantly from that of the adjusted rates. We concluded that when using large data with binary events, there is little advantage in using the ABC, SCI or GNS over the commonly known chi2. The chi2 remains a useful tool in small area variation analysis to 'screen' or flag potential differences beyond chance alone.