The seven-transmembrane spanning topography of the Alzheimer disease-related presenilin proteins in the plasma membranes of cultured cells

To ascertain the membrane topography of the multi-transmembrane spanning presenilin proteins PS-1 and PS-2, anti-peptide antibodies were raised to several specific amino acid sequences in the two proteins, and, after their specificity was ascertained, the anti-peptide antibodies were used in immunofluorescent labeling of live PS-transfected, cultured DAMI cells, which are impermeable to the antibodies, as well as of their fixed and permeabilized counterparts. In such experiments, antibodies that specifically stain the intact live cells must label epitopes of the PS proteins that are on the exterior face of the plasma membrane whereas those antibodies that do not stain the live cells but do stain the fixed and permeabilized cells must label epitopes that face the cytoplasmic side of the membrane. The results obtained were entirely in accord with the predictions of the seven-transmembrane spanning topography (like that of rhodopsin and the β-adrenergic receptor) and were totally inconsistent with the expectations for either the six- or eight-transmembrane topographies that have been proposed.

Control of gating mode by a single amino acid residue in transmembrane segment IS3 of the N-type Ca2+ channel

N-type Ca2+ channels can be inhibited by neurotransmitter-induced release of G protein βγ subunits. Two isoforms of Cav2.2 α1 subunits of N-type calcium channels from rat brain (Cav2.2a and Cav2.2b; initially termed rbB-I and rbB-II) have different functional properties. Unmodulated Cav2.2b channels are in an easily activated “willing” (W) state with fast activation kinetics and no prepulse facilitation. Activating G proteins shifts Cav2.2b channels to a difficult to activate “reluctant” (R) state with slow activation kinetics; they can be returned to the W state by strong depolarization resulting in prepulse facilitation. This contrasts with Cav2.2a channels, which are tonically in the R state and exhibit strong prepulse facilitation. Activating or inhibiting G proteins has no effect. Thus, the R state of Cav2.2a and its reversal by prepulse facilitation are intrinsic to the channel and independent of G protein modulation. Mutating G177 in segment IS3 of Cav2.2b to E as in Cav2.2a converts Cav2.2b tonically to the R state, insensitive to further G protein modulation. The converse substitution in Cav2.2a, E177G, converts it to the W state and restores G protein modulation. We propose that negatively charged E177 in IS3 interacts with a positive charge in the IS4 voltage sensor when the channel is closed and produces the R state of Cav2.2a by a voltage sensor-trapping mechanism. G protein βγ subunits may produce reluctant channels by a similar molecular mechanism.

Three-dimensional reconstruction of the recombinant type 3 ryanodine receptor and localization of its amino terminus

Recombinant type 3 ryanodine receptor (RyR3) has been purified in quantities sufficient for structural characterization by cryoelectron microscopy and three-dimensional (3D) reconstruction. Two cDNAs were prepared and expressed in HEK293 cells, one encoding the wild-type RyR3 and the other encoding RyR3 containing glutathione S-transferase (GST) fused to its amino terminus (GST-RyR3). RyR3 was purified from detergent-solubilized transfected cells by affinity chromatography using 12.6-kDa FK506-binding protein in the form of a GST fusion as the affinity ligand. Purification of GST-RyR3 was achieved by affinity chromatography by using glutathione-Sepharose. Purified recombinant RyR3 and GST-RyR3 proteins exhibited high-affinity [3H]ryanodine binding that was sensitive to activation by Ca2+ and caffeine and to inhibition by Mg2+. 3D reconstructions of both recombinant RyR3 and GST-RyR3 appeared very similar to that of the native RyR3 purified from bovine diaphragm. Comparison of the 3D reconstructions of RyR3 and GST-RyR3 revealed that the GST domains and, hence, the amino termini of the RyR3 subunits are located in the “clamp” structures that form the corners of the square-shaped cytoplasmic region of homotetrameric RyR3. This study describes the 3D reconstruction of a recombinant ryanodine receptor and it demonstrates the potential of this technology for characterizing functional and structural perturbations introduced by site-directed mutagenesis.

Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae

In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments. We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme. This is the first structure of any component of a V-type ATPase. The H-subunit is not required for assembly but plays an essential regulatory role. Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins. A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself. The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins. A cleft between the amino- and carboxyl-terminal domains also suggests another possible site of interaction with other factors.

Nonnucleoside reverse transcriptase inhibitors are chemical enhancers of dimerization of the HIV type 1 reverse transcriptase

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV type 1 (HIV-1) reverse transcriptase (RT). Yeast grown in the presence of many of these drugs exhibited dramatically increased association of the p66 and p51 subunits of the HIV-1 RT as reported by a yeast two-hybrid assay. The enhancement required drug binding by RT; introduction of a drug-resistance mutation into the p66 construct negated the enhancement effect. The drugs could also induce heterodimerization of dimerization defective mutants. Coimmunoprecipitation of RT subunits from yeast lysates confirmed the induction of heterodimer formation by the drugs. In vitro-binding studies indicate that NNRTIs can bind tightly to p66 but not p51 and then mediate subsequent heterodimerization. This study demonstrates an unexpected effect of NNRTIs on the assembly of RT subunits.

Initial Steps in the Assembly of the Vacuole-Type H+-ATPase1

The plant vacuole is acidified by a complex multimeric enzyme, the vacuole-type H+-ATPase (V-ATPase). The initial association of ATPase subunits on membranes was studied using an in vitro assembly assay. The V-ATPase assembled onto microsomes when V-ATPase subunits were supplied. However, when the A or B subunit or the proteolipid were supplied individually, only the proteolipid associated with membranes. By using poly(A+) RNA depleted in the B subunit and proteolipid subunit mRNA, we demonstrated A subunit association with membranes at substoichiometric amounts of the B subunit or the 16-kD proteolipid. These data suggest that poly(A+) RNA-encoded proteins are required to catalyze the A subunit membrane assembly. Initial events were further studied by in vivo protein labeling. Consistent with a temporal ordering of V-ATPase assembly, membranes contained only the A subunit at early times; at later times both the A and B subunits were found on the membranes. A large-mass ATPase complex was not efficiently formed in the absence of membranes. Together, these data support a model whereby the A subunit is first assembled onto the membrane, followed by the B subunit.

Monoclonal antibodies inhibit in vitro fibrillar aggregation of the Alzheimer beta-amyloid peptide.

The beta-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study beta-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against beta-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the beta-amyloid peptide chain, respectively. In vitro aggregation of beta-amyloid peptide was induced by incubation for 3 h at 37 degrees C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAs prevent the aggregation of beta-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibody-binding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the beta-amyloid peptide aggregation that is associated with Alzheimer disease.

Truncated WT1 mutants alter the subnuclear localization of the wild-type protein.

WT1 encodes a zinc-finger protein, expressed as distinct isoforms, that is inactivated in a subset of Wilms tumors. Both constitutional and somatic mutations disrupting the DNA-binding domain of WT1 result in a potentially dominant-negative phenotype. In generating inducible cell lines expressing wild-type isoforms of WT1 and WT1 mutants, we observed dramatic differences in the subnuclear localization of the induced proteins. The WT1 isoform that binds with high affinity to a defined DNA target, WT1(-KTS), was diffusely localized throughout the nucleus. In contrast, expression of an alternative splicing variant with reduced DNA binding affinity, WT1 (+KTS), or WT1 mutants with a disrupted zinc-finger domain resulted in a speckled pattern of expression within the nucleus. Although similar in appearance, the localization of WT1 variants to subnuclear clusters was clearly distinct from that of the essential splicing factor SC35, suggesting that WT1 is not directly involved in pre-mRNA splicing. Localization to subnuclear clusters required the N terminus of WT1, and coexpression of a truncated WT1 mutant and wild-type WT1(-KTS) resulted in their physical association, the redistribution of WT1(-KTS) from a diffuse to a speckled pattern, and the inhibition of its transactivational activity. These observations suggest that different WT1 isoforms and WT1 mutants have distinct subnuclear compartments. Dominant-negative WT1 proteins physically associate with wild-type WT1 in vivo and may result in its sequestration within subnuclear structures.

Characterization of the wild-type form of 4a-carbinolamine dehydratase and two naturally occurring mutants associated with hyperphenylalaninemia.

The characterization of 4a-carbinolamine dehydratase with the enzymatically synthesized natural substrate revealed non-Michaelis-Menten kinetics. A Hill coefficient of 1.8 indicates that the dehydratase exists as a multisubunit enzyme that shows cooperativity. A mild form of hyperphenylalaninemia with high 7-biopterin levels has been linked to mutations in the human 4a-carbinolamine dehydratase gene. We have now cloned and expressed two mutant forms of the protein based on a patient's DNA sequences. The kinetic parameters of the mutant C82R reveal a 60% decrease in Vmax but no change in Km (approximately 5 microM), suggesting that the cysteine residue is not involved in substrate binding. Its replacement by arginine possibly causes a conformational change in the active center. Like the wild-type enzyme, this mutant is heat stable and forms a tetramer. The susceptibility to proteolysis of C82R, however, is markedly increased in vitro compared with the wild-type protein. We have also observed a decrease in the expression levels of C82R protein in transfected mammalian cells, which could be due to proteolytic instability. The 18-amino acid-truncated mutant GLu-87--> termination could not be completely purified and characterized due to minute levels of expression and its extremely low solubility as a fusion protein. No dehydratase activity was detected in crude extracts from transformed bacteria or transfected mammalian cells. Considering the decrease in specific activity and stability of the mutants, we conclude that the patient probably has less than 10% residual dehydratase activity, which could be responsible for the mild hyperphenylalaninemia and the high 7-biopterin levels.

The experimental stress analysis of the Firestone type R-5⁰, 20 x 75 truck rim.

Vols. 2-3 have title: The experimental stress analysis of truck rims.

Observations on fevers : especially those of the continued type and on the scarlet fever attended with ulcerated sore-throat /

Mode of access: Internet.

Police sergeants manual; the essential principles of handling police personnel and the basic duties of supervisory officers, sergeants, lieutenants, captains, detectives, and peace officers generally. With appendix, containing selected and recommended questions and answers of the approved type. Now used in police civil service promotional examinations. Suggested reading list for sergeants' training. Sergeants' check list for instructing recruits. Bibliography.

Mode of access: Internet.

Reclamation of the Fresno type of black-alkali soil /

Mode of access: Internet.

Chronic administration of an aluminium-enriched diet impairs spatial orientation in a sub-group of C57BL6 female mice

Disturbances of spatial orientation are an early clinical component of senile dementia of the Alzheimer type (SDAT). since it has been suggested that an elevated aluminium intake associated with chronic nutritional deficiencies of calcium and magnesium may play an important role in the aetiology of SDAT, we have investigated the effect of such a dietary regime on the spatial orientation abilities of female C57BL6 mice using the Morris swimming pool test. Statistical analysis of the performances of control and experimental groups indicate that the ability to orientate towards a submerged and thus invisible platform is conistently and markedly impaired in the experimental group. The ability to orientate towards a visible platform is also significantly impaired although to a lesser extent. Analysis of the performances of individual animals demonstrate that this impairment of orientation in the experimental group only occurs in a sub-group of animals: the remainder display normal orientational ability.