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.

During the M in equilibrium with N----BR reaction sequence in the bacteriorhodopsin photocycle, proton is exchanged between D96 and the Schiff base, and D96 is reprotonated from the cytoplasmic surface. We probed these and the other photocycle reactions with osmotically active solutes and perturbants and found that the M in equilibrium with N reaction is specifically inhibited by withdrawing water from the protein. The N----BR reaction in the wild-type protein and the direct reprotonation of the Schiff base from the cytoplasmic surface in the site-specific mutant D96N are much less affected. Thus, it appears that water is required inside the protein for reactions where a proton is separated from a buried electronegative group, but not for those where the rate-limiting step is the capture of a proton at the protein surface. In the wild type, the largest part of the barrier to Schiff base reprotonation is the enthalpy of separating the proton from D96, which amounts to about 40 kJ/mol. We suggest that in spite of this D96 confers an overall kinetic advantage because when this residue becomes anionic in the N state its electric field near the cytoplasmic surface lowers the free energy barrier of the capture of a proton in the next step. In the D96N protein, the barrier to the M----BR reaction is 20 kJ/mol higher than what would be expected from the rates of the M----N and N----BR partial reactions in the wild type, presumably because this mechanism is not available.

Lithium is one of the most widely used mood-stabilizing agents for the treatment of bipolar disorder. Although the underlying mechanism(s) of this mood stabilizer remains controversial, recent evidence linking lithium to neurotrophic/neuroprotective effects (Choi and Sung (2000) 1475, 225-230; Davies et al. (2000) 351, 95-105) suggests novel benefits of this drug in addition to mood stabilization. Here, we report that both lithium as well as valproic acid (VPA) inhibit beta-amyloid peptide (Abeta) production in HEK293 cells stably transfected with Swedish amyloid precursor protein (APP)(751) and in the brains of the PDAPP (APP(V717F)) Alzheimer's disease transgenic mouse model at clinically relevant plasma concentrations. Both lithium and VPA are known to be glycogen synthase kinase-3 (GSK3) inhibitors. Our studies reveal that GSK3beta is a potential downstream kinase, which modulates APP processing because inhibition of GSK3 activity by either a dominant negative GSK3beta kinase-deficient construct or GSK3beta antisense oligonucleotide mimics lithium and VPA effects. Moreover, lithium treatment abolished GSK3beta-mediated Abeta increase in the brains of GSK3beta transgenics and reduced plaque burden in the brains of the PDAPP (APP(V717F)) transgenic mice.

The NAD(+)-dependent protein deacetylase SIRT1 is linked to cellular survival pathways by virtue of keeping the tumor suppressor gene p53 and members of the forkhead transcription factor family deacetylated. To validate SIRT1 as a therapeutic anti-cancer target, we performed immunohistochemistry experiments to study the in vivo expression of SIRT1 in cancer specimens. We show that human SIRT1 is highly expressed in cancer cell lines as well as in tissue samples from colon carcinoma patients. Interestingly, there is a strong cytosolic component in the SIRT1 expression pattern. We further characterized SIRT1 in p53-wild-type and -mutant cell lines and show that SIRT1 mRNA-knockdown leads to a p53-independent decrease of cell proliferation and induction of apoptosis. In addition, SIRT1 expression has been found to be inducible upon DNA damage. A previously discovered small molecule SIRT1 inhibitor with nanomolar in vitro activity has been tested in cancer relevant assays. The SIRT1 inhibitory compound showed no potent anti-proliferative activity despite hitting its molecular target within tumor cells. From these studies we conclude that it may not be sufficient to block the catalytic function of SIRT1, and that its survival effects may be mainly brought about by means other then the deacetylase function. The increased cytosolic expression of SIRT1 in cancer cells could be an indicator of such novel functions.

Neurotoxicity induced by overstimulation of N-methyl-D-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca 2؉ ; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration-and time-dependent activation of the interleukin 1␤-converting enzyme (ICE)͞CED-3-related protease, CPP32͞Yama͞apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamateinduced apoptosis of CGNs is mediated by a posttranslational activation of the ICE͞CED-3-related cysteine protease caspase 3.