We reported earlier that the levels of Ca2+-dependent metalloproteinases are increased in Alzheimer's disease (AD) specimens, relative to control specimens. Here we show that these enzymes are forms of the matrix metalloproteinase MMP-9 (EC3.4.24. 35) and are expressed in the human hippocampus. Affinity-purified antibodies to MMP-9 labeled pyramidal neurons, but not granular neurons or glial cells. MMP-9 mRNA is expressed in pyramidal neurons, as determined with digoxigenin-labeled MMP-9 riboprobes, and the presence of this mRNA is confirmed with reverse transcriptase PCR. The cellular distribution of MMP-9 is altered in AD because 76% of the total 100 kDa enzyme activity is found in the soluble fraction of control specimens, whereas only 51% is detectable in the same fraction from AD specimens. The accumulated 100 kDa enzyme from AD brain is latent and can be converted to an active form with aminophenylmercuric acetate. MMP-9 also is detected in close proximity to extracellular amyloid plaques. Because a major constituent of plaques is the 4 kDa beta-amyloid peptide, synthetic Abeta1-40 was incubated with activated MMP-9. The enzyme cleaves the peptide at several sites, predominantly at Leu34-Met35 within the membrane-spanning domain. These results establish that neurons have the capacity to synthesize MMP-9, which, on activation, may degrade extracellular substrates such as beta-amyloid. Because the latent form of MMP-9 accumulates in AD brain, it is hypothesized that the lack of enzyme activation contributes to the accumulation of insoluble beta-amyloid peptides in plaques.

Recently, a single gene, DYSF, has been identified which is mutated in patients with limb-girdle muscular dystrophy type 2B (LGMD2B) and with Miyoshi myopathy (MM). This is of interest because these diseases have been considered as two distinct clinical conditions since different muscle groups are the initial targets. Dysferlin, the protein product of the gene, is a novel molecule without homology to any known mammalian protein. We have now raised a monoclonal antibody to dysferlin and report on the expression of this new protein: immunolabelling with the antibody (designated NCL-hamlet) demonstrated a polypeptide of approximately 230 kDa on western blots of skeletal muscle, with localization to the muscle fibre membrane by microscopy at both the light and electron microscopic level. A specific loss of dysferlin labelling was observed in patients with mutations in the LGMD2B/MM gene. Furthermore, patients with two different frameshifting mutations demonstrated very low levels of immunoreactive protein in a manner reminiscent of the dystrophin expressed in many Duchenne patients. Analysis of human fetal tissue showed that dysferlin was expressed at the earliest stages of development examined, at Carnegie stage 15 or 16 (embryonic age 5-6 weeks). Dysferlin is present, therefore, at a time when the limbs start to show regional differentiation. Lack of dysferlin at this critical time may contribute to the pattern of muscle involvement that develops later, with the onset of a muscular dystrophy primarily affecting proximal or distal muscles.

C. M. Steele and J. Aronson (1995) showed that making race salient when taking a difficult test affected the performance of high-ability African American students, a phenomenon they termed stereotype threat. The authors document that this research is widely misinterpreted in both popular and scholarly publications as showing that eliminating stereotype threat eliminates the African American-White difference in test performance. In fact, scores were statistically adjusted for differences in students' prior SAT performance, and thus, Steele and Aronson's findings actually showed that absent stereotype threat, the two groups differ to the degree that would be expected based on differences in prior SAT scores. The authors caution against interpreting the Steele and Aronson experiment as evidence that stereotype threat is the primary cause of African American-White differences in test performance.

Gain-of-function mutations in fibroblast growth factor-23 (FGF23) are responsible for autosomal dominant hypophosphatemic rickets, a disorder of isolated renal phosphate wasting. Patients with the disorder display hypophosphatemia with normocalcemia as well as inappropriately normal 1,25-dihydroxyvitamin D [1,25(OH)2D3] concentrations. Reciprocally tumoral calcinosis (TC) patients are often hyperphosphatemic with inappropriately normal or elevated serum 1,25(OH)2D3 levels and have ectopic and vascular calcifications, a phenotype similar to that of Fgf23 null mice. Therefore, the goal of the present studies was to test whether FGF23 was a candidate gene for TC. Two sisters in a consanguineous TC family had hyperphosphatemia and normal 1,25(OH)2D3 levels with characteristic ectopic and vascular calcifications. Interestingly, these patients had low-normal intact serum FGF23 levels but demonstrated FGF23 concentrations approximately 40 times normal when assessed with a C-terminal FGF23 serum assay. Mutational analyses identified a homozygous S71G mutation in FGF23 in the TC patients, which was not found in control alleles. Finally, modeling demonstrated that the S71G mutation most likely destabilizes full-length FGF23. In summary, recessive FGF23 mutations can lead to TC. Additionally, our findings indicate that FGF23 may adopt an unstable conformation in some TC patients, possibly leading to nonfunctional FGF23 protein.

SUMMARYInterlobular ducts were isolated from the pancreas of copper-deficient rats and maintained in culture on polycarbonate filter rafts. Within 8 h the ends of the ducts had sealed. This was followed by a marked dilatation of the lumen, a flattening of the epithelium against the surrounding connective tissue layer and an over-all swelling of the duct. Apart from a reduction in their height, a fall in the number of intracellular fat droplets and a widening of intercellular spaces, epithelial cells within the cultured ducts retained all the ultrastructural characteristics of those in freshly isolated preparations. The basal concentration of adenosine 3',5'-phosphate (cyclic AMP) in the cultured ducts was 43 3 +6 8 ,umol .1-1 duct epithelium (n = 5) and was increased to 1889 + 55-2 ,umol . 1-1 duct epithelium (n = 5) in the presence of 10-8 M secretin. The basal rate of fluid secretion, measured using micropuncture techniques, was 0-16 + 0 03 nl. h-. nl-l duct epithelium (n = 12). This was increased 14-fold by 10-8 M secretin while the dose of the hormone required for half-maximal secretion was about 2 x 10-11 mol . 1-1. The concentration of chloride ions in secreted fluid and perifusion buffer were similar. Variation in culture time up to 52 h had no effect on fluid secretion, and the response to secretin was dependent on the presence of bicarbonate ions in the perifusion fluid. N6,02-dibutyryl adenosine 3',5'-phosphate (dibutyryl cyclic AMP) also increased fluid secretion but caerulein had no effect. We suggest that isolated ducts secrete fluid at comparable rates to ducts in situ within the pancreas of copper-replete rats.

To examine the generalizability of stereotype threat theory findings from laboratory to applied settings, the authors developed models of the pattern of relationships between cognitive test scores and outcome criteria that would be expected if the test scores of women and minority group members were affected by stereotype threat. Two large data sets were used to test these models, one in an education setting examining SAT-grade relationships by race and gender and the other in a military job setting examining Armed Services Vocational Aptitude Battery-job performance relationships by race. Findings were not supportive of the predictions arising from stereotype threat theory, suggesting caution in positing threat as a key determinant of subgroup mean test score differences in applied settings.

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Background-Genetic mutations of dystrophin and associated glycoproteins underlie cell degeneration in several inherited cardiomyopathies, although the precise physiological role of these proteins remains under discussion. We studied the distribution of dystrophin in relation to the force-transducing vinculin-rich costameres in left ventricular cardiomyocytes from normal and failing human hearts to further elucidate the function of this protein complex. Methods and Results-Single-and double-label immunoconfocal microscopy and parallel high-resolution immunogold fracture-label electron microscopy were used to localize dystrophin and vinculin in human left ventricular myocytes from normal (nϭ6) and failing hearts (idiopathic dilated cardiomyopathy, nϭ7, or ischemic heart disease, nϭ5). In control cardiomyocytes, dystrophin had a continuous distribution at the peripheral sarcolemma, with concentrated bands corresponding to the vinculin-rich costameres. Intracellular labeling extended along transverse (T) tubule membranes. Fracture-label confirmed this distribution, showing significantly greater label on plasma membrane fractures overlying I-bands (I-band 4.1Ϯ0.3 gold particles/m; A-band 3.3Ϯ0.2 gold particles/m: meanϮSE, Pϭ0.02). Hypertrophied myocytes from failing hearts showed maintenance of this surface distribution except in degenerating cells; there was a clear increase in intracellular dystrophin label reflecting T-tubule hypertrophy. Conclusions-Dystrophin partially colocalizes with costameric vinculin in normal and hypertrophied myocytes, a distribution lost in degenerating cells. This suggests a primarily mechanical role for dystrophin in maintenance of cell membrane integrity in normal and hypertrophied myocytes. The presence of dystrophin in the cardiac T-tubule membrane, in contrast to its known absence in skeletal muscle T-tubules, implies additional roles for dystrophin in membrane domain organization.