Pharmacogenetic features of cathepsin B inhibitors that improve memory deficit and reduce β-amyloid related to Alzheimer's disease

Beta-amyloid (Aβ) in the brain is a major factor involved in Alzheimer's disease (AD) that results in severe memory deficit. Our recent studies demonstrate pharmacogenetic differences in the effects of inhibitors of cathepsin B to improve memory and reduce Aβ in different mouse models of AD. The inhibitors improve memory and reduce brain Aβ in mice expressing the wild-type (WT) β-secretase site of human APP, expressed in most AD patients. However, these inhibitors have no effect in mice expressing the rare Swedish (Swe) mutant amyloid precursor protein (APP). Knockout of the cathepsin B decreased brain Aβ in mice expressing WT APP, validating cathepsin B as the target. The specificity of cathepsin B to cleave the WT β-secretase site, but not the Swe mutant site, of APP for Aβ production explains the distinct inhibitor responses in the different AD mouse models. In contrast to cathepsin B, the BACE1 β-secretase prefers to cleave the Swe mutant site. Discussion of BACE1 data in the field indicate that they do not preclude cathepsin B as also being a β-secretase. Cathepsin B and BACE1 could participate jointly as β-secretases. Significantly, the majority of AD patients express WT APP and, therefore, inhibitors of cathepsin B represent candidate drugs for AD.

Multiple Pro197Substitutions in the Acetolactate Synthase of Corn Poppy (Papaver rhoeas) Confer Resistance to Tribenuron

Variations in the acetolactate synthase (ALS) gene sequence were determined from 28 populations of corn poppy resistant (R) to tribenuron and from 6 populations susceptible (S) to this herbicide. TheALSgene fragment (634 bp) sequence revealed in R populations five point mutations at the codon Pro197, and among them the substitution of Pro197by Ala was the most common. The sequencing chromatograms revealed that nine R individuals had only the mutantALSgene and were homozygous (RR), 18 R individuals had both the wild type and the mutantALSgene and were heterozygous (RS), whereas one R individual was heterozygous but with two different mutantALSalleles (R1R2). The use of restriction digestion profile analysis to verify the DNA sequence results by detecting the existence of point mutations at the codon 197 managed to distinguish the R and S alleles and confirmed the results obtained from the sequencing chromatograms analysis. The secondary protein structure prediction suggested the formation of novelβ-strands for each of the five mentioned amino acid substitutions that was not present in wild type ALS around the mutant site. These findings support the hypothesis that the substitution of Pro197by Ser, Thr, Ala, Arg, or Leu resulted in altered secondary structure, which stabilizes an ALS tertiary conformation that prevents tribenuron binding and thus confers resistance to this herbicide.

Further Studies with Lipoamide Dehydrogenase Mutants of Escherichia coli k12

The immunological properties of ten lipoamide dehydrogenase mutants of Escherichia coli were investigated with antiserum raised against purified lipoamide dehydrogenase. Seven mutants were CRM+ (cross-reacting material present) as they contained lipoamide dehydrogenase proteins exhibiting either complete or partial immunological identity with the wild-type protein. This indicates that at least seven of the mutations affect the lipoamide dehydrogenase structural gene (lpd). The remaining three mutants (CRM-) contained no detectable cross-reacting protein. None of the lpd mutations were sensitive to any of three different amber-suppressors. Genetic analysis by P1-transduction showed that all the lpd mutant sites were clustered very near the distal gene (aceF) of the ace region which specifies the dehydrogenase (aceE) and transacetylase (aceF) components of the pyruvate dehydrogenase multienzyme complex. Calculations based on the recombination frequency between an aceF mutant and the nearest lpd mutant site support the conclusion that apart from the possible presence of a regulatory element, the aceF and lpd genes are contiguous.

Sequence analysis of mutations arising during prolonged starvation of Salmonella typhimurium.

We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA). Both of these mutants can revert to His+ via intragenic and extragenic mechanisms. Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site. Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites. Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs. A/T sites as well as on intragenic sites vs. extragenic suppressor sites. Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants. Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations. The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium. Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.

ORGANIZATION OF THE qa GENE CLUSTER IN NEUROSPORA CRASSA: DIRECTION OF TRANSCRIPTION OF THE qa-3 GENE

ABSTRACT In Neurospora crassa, the enzyme quinate (shikimate) dehydrogenase catalyzes the first reaction in the inducible quinic acid catabolic pathway and is encoded in the qa-3 gene of the qa cluster. In this cluster, the order of genes has been established as qa-I qa-3 qa-4 qa-2. Amino-terminal sequences have been determined for purified quinate dehydrogenase from wild type and from UV-induced revertants in two different qa-3 mutants. These two mutants (M16 and M45) map at opposite ends of the qa-3 locus. In addition, mapping data (CASE et al. 1978) indicate that the end of the qa-3 gene specified by M45 is closer to the adjacent qa-1 gene than is the end specified by the MI6 mutant site. In one of the revertants (R46 from qa-3 mutant M45), the amino-terminal sequence for the first ten amino acids is identical to that of wild type. The other revertant (R1 from qa-3 mutant M16) differs from wild type at the amino-terminal end by a single altered residue at position three in the sequence. The observed change involves the substitution of an isoleucine in M16-Rl for a proline in wild type. This substitution requires a two-nucleotide change in the corresponding wild-type codon.——The combined genetic and biochemical data indicate that the qa-3 mutants M16 and M45 carry amino acid substitutions near the amino-terminal and carboxyl-terminal ends of the quinate dehydrogenase enzyme, respectively. On this basis we conclude that transcription of the qa-3 gene proceeds from the end specified by the MI6 mutant site in the direction of the qa-1 gene. It appears probable that transcription is initiated from a promoter site within the qa cluster, possibly immediately adjacent to the qa-3 gene.

THE SUPPRESSION OF AD-3B MUTANTS BY SUPERSUPPRESSORS IN NEUROSPORA CRASSA

ABSTRACT The action of eight suppersuppressors has been tested on 76 ad-3B mutants which were induced by base-pair transition mutagens. Thirteen mutants were found to be suppressible by at least one ssu gene. Most of the suppressible mutants were found to belong to a class in which one would expect to find nonsense mutants (polar complementing or noncomplementing classes, with AT at the mutant site). However, suppression was observed in other classes, including those containing presumed missense mutants. The specificity of the suppressors and the criteria for molecular classification of the mutants are discussed.

A genetic study of primary and secondary reversions of some tryptophanA auxotrophs of Salmonella typhimurium

1. The linkage order of four tryA mutants of S. typhimurium, and cysB-12, is:Attempts to plot the position of tryA-50 were unsuccessful.2. Some of the reversions of tryA-8, tryA-47, tryA-56 and tryA-50 were analysed genetically; tryA-52 does not revert. All four auxotrophs gave reversions that were phenotypically and genetically indistinguishable from that expected by back-mutation of the original mutant site.3. Both tryA-8 and tryA-50 produced reversions that grew as wild-type but were due to unlinked suppressor mutations. Some of these were super-suppressors in that they suppressed both tryA-8 and tryB-4; others suppressed many site mutants in the tryA gene but did not suppress tryB-4.4. All the slow-growing reversions of tryA-8, tryA-50 and tryA-56, and a minority of the semi-fast reversions of tryA-8, were due to unlinked suppressors.5. All the slow-growing reversions of tryA-47, the semi-fast reversions of tryA-56 and the majority of the semi-fast reversions of tryA-8 were due to genetic changes that were inseparable, in very extensive experiments, from their original mutant site.6. Slow-growing reversions of tryA-47 produced faster growing mutants. Some of these were due to mutation in unlinked modifying genes and in others the genetic change was within the tryA gene. Nine of the latter had the genetic change just to the left of the 47S site; in one the change was inseparable from the 47S site. None had this further change to the right of the 47S site. These further changes, in the absence of the 47S site, gave prototrophic phenotypes; they are inter-site suppressors.

Recombination within the am gene of Neurospora crassa

Maps have been made showing the order of am mutant sites using (a) frequencies of am+ recombinants from crosses between am mutants and (b) the modes of distribution of the am+ recombinants among the two parental and two recombinant classes with respect to the flanking markers inos and sp.It is possible to arrange the sites in an order such that, in almost all the crosses yielding useful numbers of am+ recombinants, the sp allele originally in coupling with the distal am mutant site occurs in the majority of the recombinants. No such consistent pattern was found with respect to the inos marker. The order obtained by reference to sp agreed with the best order deducible from recombination frequencies.The data are consistent with the hypothesis that am+ recombinants arise by a process of gene conversion, that there is a gradient of conversion frequencies from the right (inos or distal) end of the gene to the left (sp or proximal) end, and that conversion tends to be associated (though less than 50% of the time) with crossing-over, especially on the distal side.There is no obvious relationship between the map position of a given am mutant and the properties of the protein product of the mutant gene.