N-Bridged bicyclic sulfonamides as inhibitors of γ-secretase

The structural modification of a series of [3.3.1] bicyclic sulfonamide based γ-secretase inhibitors is described. Appropriate substitution on the bicyclic scaffold provides a significant increase in the metabolic stability of the compounds resulting in an improved in vivo metabolic profile.     

Cycloheximide genotoxicity in in vitro and in vivo test systems

The aim of this study was to investigate if there was any genotoxic effect produced by the antibiotic cycloheximide, widely used as a fungicide in agriculture as well as in everyday laboratory practice. The battery of test systems included the bacterium Salmonella typhimurium (strains TA98 and TA100), the yeast Saccharomyces cerevisiae (D7), Allium cepa somatic cells and mouse bone marrow cells. This combination of test systems enabled us to establish possible effects caused by cycloheximide at different levels of the genome and to indicate a possible mechanism of action. The results obtained in experiments showed that cycloheximide did not induce frameshift or base-pair substitution mutations in S. typhimurium regardless of metabolic activation. In S. cerevisiae cycloheximide had only toxic effects but no increase of mitotic gene conversion was noticed under the conditions of the experiment. However, in A. cepa somatic cells as well as in mouse bone marrow cells cycloheximide showed its activity causing different genetic damages, e.g., chromosome breaks, mitotic disturbances and nuclear abnormalities.     

Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

A novel S phase inhibitor in fission yeast.

We have cloned a novel fission yeast gene, spd1, which causes G1 arrest when overexpressed. Deleting the gene results in cells being accelerated through G1 into S phase in certain circumstances when the G1-->S phase control is compromised. We have found that the encoded 14 kDa protein is cell cycle regulated, declining in level during S phase, and that p14spd1 physically associates with p34cdc2 in vivo when overexpressed, suggesting that p14spd1 may regulate S phase progression via an interaction with p34cdc2. We conclude that p14spd1 is a negative regulator of S phase, and that it may be part of the control ensuring an orderly onset of S phase or part of a G1-->S phase checkpoint control.     

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey- lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.      

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.