Defining fibronectin's cell adhesion synergy site by site-directed mutagenesis.

Fibronectin's RGD-mediated binding to the alpha5beta1 integrin is dramatically enhanced by a synergy site within fibronectin III domain 9 (FN9). Guided by the crystal structure of the cell-binding domain, we selected amino acids in FN9 that project in the same direction as the RGD, presumably toward the integrin, and mutated them to alanine. R1379 in the peptide PHSRN, and the nearby R1374 have been shown previously to be important for alpha5beta1-mediated adhesion (Aota, S., M. Nomizu, and K.M. Yamada. 1994. J. Biol. Chem. 269:24756-24761). Our more extensive set of mutants showed that R1379 is the key residue in the synergistic effect, but other residues contribute substantially. R1374A decreased adhesion slightly by itself, but the double mutant R1374A-R1379A was significantly less adhesive than R1379A alone. Single mutations of R1369A, R1371A, T1385A, and N1386A had negligible effects on cell adhesion, but combining these substitutions either with R1379A or each other gave a more dramatic reduction of cell adhesion. The triple mutant R1374A/P1376A/R1379A had no detectable adhesion activity. We conclude that, in addition to the R of the PHRSN peptide, other residues on the same face of FN9 are required for the full synergistic effect. The integrin-binding synergy site is a much more extensive surface than the small linear peptide sequence.

Regionally specific neuronal expression of human APOE gene in transgenic mice.

Apolipoprotein E (APOE, gene; apoE, protein) is a susceptibility gene for late-onset Alzheimer's disease (AD). To examine whether neurons can synthesize apoE, we performed in situ hybridization on brain tissue of transgenic mice carrying genomic constructs for the three major human APOE alleles. We find human APOE mRNA in glial cells of cerebellum, striatum and cerebral cortex and also in neurons of cerebral cortex, corresponding to apoE localization in humans. Synthesis of apoE by neurons implies that models of AD may need to consider intrinsic apoE production in addition to uptake. Inclusion of human regulatory sequences may result in more realistic transgenic models of human disease.

Tenascin-C knockout mouse has no detectable tenascin-C protein.

A recent study by Mitrovic and Schachner (J Neurosci Res 42:710-717, 1995) reported the detection of a small amount of truncated tensacin-C (TN-C) in the nervous system of the TN-C knockout mice created by Saga et al. (Genes Dev 6:1821-1831, 1992). The authors suggested that the truncated protein might be responsible for the failure to detect any phenotypic abnormalities in the knockout mice. We have reexamined the knockout mice in our laboratories by Western blot and immunocytochemistry, and have not detected any full-length or truncated TN-C protein. In addition, we note that the construction of the knockout gene deleted the signal sequence, so if any residual truncated protein were produced it would be trapped in the cytoplasm, and therefore inaccessible to extracellular ligands or receptors. We therefore conclude that the TN-C knockout created by Saga et al. is a valid TN-C null.

Tenascin-C expression in dystrophin-related muscular dystrophy.

The mdx mouse has a mutated dystrophin gene and is used as a model for the study of Duchenne muscular dystrophy (DMD). We investigated whether regenerating mdx skeletal muscle contains the extracellular matrix protein tenascin-C (TN-C), which is expressed in wound healing and nerve regeneration. Prior to the initiation of muscle degeneration, both normal and mdx mice displayed similar weak staining for TN-C in skeletal muscle, but by 3 weeks of age the mice differed substantially. TN-C was undetectable in normal muscle except at the myotendinous junction, while in dystrophic muscle, TN-C was prominent in degenerating/regenerating areas, but absent from undegenerated muscle. With increasing age, TN-C staining declined around stable regenerated mdx myofibers. TN-C was also observed in muscle from dogs with muscular dystrophy and in human boys with DMD. Therefore, in dystrophic muscle, TN-C expression may be stimulated by the degenerative process and remain upregulated unless the tissue undergoes successful regeneration.

Glucose feedings and exercise in rats: glycogen use, hormone responses, and performance.

This study compared the effects of glucose feeding and water on endurance performance, glycogen utilization, and endocrine responses to exhaustive running in rats. Forty-eight trained rats ran at approximately 70% peak O2 consumption (VO2) while receiving, via gavage, 1 ml of an 18% glucose solution or water every 30 min. Glucose- (GF) and water-fed rats (WF) were pair matched and killed at rest, at 25 or 50% of their previously determined run time to exhaustion, or at exhaustion. Run times to exhaustion were 4.6 +/- 1.0 and 3.0 +/- 0.9 h in GF and WF rats, respectively. In WF rats, plasma glucose declined continuously from a resting value of 7.4 +/- 0.5 to 1.8 +/- 0.5 mM at exhaustion and was lower than in GF rats at all exercise time points. In GF rats, glucose was maintained at 7.4 +/- 0.5 mM for 3 h before dropping to 3.9 +/- 0.6 mM at exhaustion. In both groups, liver and muscle glycogen decreased dramatically during the 1st h and changed only slightly thereafter. During the 3rd h, glycogen levels were maintained in GF rats but continued to decrease in WF rats (P less than 0.05). Insulin decreased during exercise and was not significantly different between groups. Glucagon, epinephrine, norepinephrine, and corticosterone increased to a greater extent in WF than in GF rats during the first 3 h of exercise.(ABSTRACT TRUNCATED AT 250 WORDS)