The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.

Converging lines of evidence implicate the beta-amyloid peptide (Ab) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce Ab production by functionally inhibiting g-secretase, the activity responsible for the carboxy-terminal cleavage required for Ab production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Ab production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-di¯uoro-phenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP V717F reduces brain levels of Ab in a dose-dependent manner within 3 h. These studies represent the ®rst demonstration of a reduction of brain Ab in vivo. Development of such novel functional g-secretase inhibitors will enable a clinical examination of the Ab hypothesis that Ab peptide drives the neuropathology observed in Alzheimer's disease.

We have isolated a brain-specific cDNA that encodes a Na+-dependent inorganic phosphate (Po) Subtractive Cloning. Subtractive hybridization was used to identify genes differentially expressed after NMDA exposure (12 hr) of cerebellar granule cells. Rat cerebellar granule cells (P8) were cultured and exposed to NMDA as described (6). Poly(A)+ RNA was isolated by using oligo(dT)-cellulose columns (9) from NMDA-treated (12 hr) (tester) and control (untreated) cerebellar granule cells (driver). The tester mRNA (1 I.g) was used to generate[32P]dCTP-labeled first-strand cDNA (6000 Ci/mmol; NEN; 1 Ci = 37 GBq). Driver mRNA (10 pg) was photobiotinylated with a 300 W Sylvania clear bulb and was used to hybridize to tester cDNAs. Subtractive hybridization was done by using subtractor 1, as described by the manufacturer (Invitrogen). After hybridization, common sequences were removed by adding streptavidin and extracting with phenol/chloroform. Resulting subtracted cDNA was used to screen a Zap II cDNA library constructed from NMDA-treated cerebellar granule cells. Positive clones were rescued, replated individually, and rescreened with labeled, single-stranded cDNAs derived from driver and tester mRNAs. Those cDNAs that were detected only by tester probes, and were not detected by driver probes, were isolated for further analysis. Plasmids were excised from A phage, as described by the manufacturer (Stratagene), digested with EcoRI, and electrophoresed in a 1% agarose gel before DNA blotting. cDNA inserts that were specifically detected by tester probes were sequenced.DNA Sequencing and Sequence Analysis. The nucleotide sequence of the BNPI cDNA clone was determined for both strands. Sequence reactions were done by using doublestranded DNA templates, sequence-specific oligonucleotide primers, fluorescently labeled dideoxynucleotide terminators (Applied Biosystems) and Ampli-Taq polymerase in cyclesequencing reactions modified as described (10). Individual sequences were assembled with an Applied Biosystems Abbreviations: NMDA, N-methyl-D-aspartate; BNPI, brain Na+-dependent inorganic phosphate cotransporter I. tPresent address:

A subset of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to preferentially reduce the secretion of the highly amyloidogenic, 42-residue amyloid-beta peptide Abeta42. We found that Rho and its effector, Rho-associated kinase, preferentially regulated the amount of Abeta42 produced in vitro and that only those NSAIDs effective as Rho inhibitors lowered Abeta42. Administration of Y-27632, a selective Rock inhibitor, also preferentially lowered brain levels of Abeta42 in a transgenic mouse model of Alzheimer's disease. Thus, the Rho-Rock pathway may regulate amyloid precursor protein processing, and a subset of NSAIDs can reduce Abeta42 through inhibition of Rho activity.