Synthesis and SAR of arylaminoethyl amides as noncovalent inhibitors of cathepsin S: P3 cyclic ethers

The synthesis and structure-activity relationship of a series of arylaminoethyl amide cathepsin S inhibitors are reported. Optimization of P3 and P2 groups to improve overall physicochemical properties resulted in significant improvements in oral bioavailability over early lead compounds. An X-ray structure of compound 37 bound to the active site of cathepsin S is also reported.     

ECOLOGICAL CONDITIONS AFFECT EVOLUTIONARY TRAJECTORY IN A PREDATOR–PREY SYSTEM

The arms race of adaptation and counter adaptation in predator–prey interactions is a fascinating evolutionary dynamic with many consequences, including local adaptation and the promotion or maintenance of diversity. Although such antagonistic coevolution is suspected to be widespread in nature, experimental documentation of the process remains scant, and we have little understanding of the impact of ecological conditions. Here, we present evidence of predator–prey coevolution in a long-term experiment involving the predatory bacterium Bdellovibrio bacteriovorus and the prey Pseudomonas fluorescens , which has three morphs (SM, FS, and WS). Depending on experimentally applied disturbance regimes, the predator–prey system followed two distinct evolutionary trajectories, where the prey evolved to be either super-resistant to predation (SM morph) without counter-adaptation by the predator, or moderately resistant (FS morph), specialized to and coevolving with the predator. Although predation-resistant FS morphs suffer a cost of resistance, the evolution of extreme resistance to predation by the SM morph was apparently unconstrained by other traits (carrying capacity, growth rate). Thus we demonstrate empirically that ecological conditions can shape the evolutionary trajectory of a predator–prey system.     

An assay for social interaction in Drosophila fragile X mutants

We developed a novel assay to examine social interactions in Drosophila and, as a first attempt, apply it here at examining the behavior of Drosophila Fragile X Mental Retardation gene (dfmr1) mutants. Fragile X syndrome is the most common cause of single gene intellectual disability (ID) and is frequently associated with autism. Our results suggest that dfmr1 mutants are less active than wild-type flies and interact with each other less often. In addition, mutants for one allele of dfmr1, dfmr1^B55, are more likely to come in close contact with a wild-type fly than another dfmr1^B55 mutant. Our results raise the possibility of defective social expression with preserved receptive abilities. We further suggest that the assay may be applied in a general strategy of examining endophenoypes of complex human neurological disorders in Drosophila, and specifically in order to understand the genetic basis of social interaction defects linked with ID.Key words: Drosophila, Fragile X, autism, social behavior, novel assay     

Aging specifically impairs amnesiac-dependent memory in Drosophila

Age-related memory impairment (AMI) is observed in many species. However, it is uncertain whether AMI results from a specific or a nonspecific decay in memory processing. In Drosophila, memory acquired after a single olfactory conditioning paradigm has three distinct phases: short-term memory (STM), middle-term memory (MTM), and longer-lasting anesthesia-resistant memory (ARM). Here, we demonstrate that age-related defects in olfactory memory are identical to those of the MTM mutant amnesiac (amn). Furthermore, amn flies do not exhibit an age-dependent decrease in memory, in contrast to other memory mutants. The absence of AMI in amn flies is restored by expression of an amn transgene predominantly in DPM cells. Thus, we propose that AMI in flies results from a specific decrease in amn-dependent MTM.     

Characterization of Drosophila nitric oxide synthase: a biochemical study

The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW. The denatured molecular mass of the expressed protein was 152kDa along with a proteolytically cleaved product of 121kDa. The DNOS heme protein exhibited very low Ca(2+)/calmodulin-dependent NO synthase activity. The trypsin digestion patterns were different from nNOS. The full-length DNOS protein had high degree of stability against trypsin. The activity assay of trypsin-digested protein confirmed the same result. Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity. Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3-4M urea. Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN. The bacterial expression of DNOS full-length protein represents an important development in structure-function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant.     

Cloning and Mutagenesis of a Cytochrome P-450 Locus from Bradyrhizobium japonicum That Is Expressed Anaerobically and Symbiotically

Cytochromes P-450, which in many organisms participate in the metabolism of a variety of endobiotic and xenobiotic substances, are synthesized by symbiotic bacteroids of Bradyrhizobium japonicum. Polyclonal antibodies were raised against two cytochromes P-450 (CYP112 and CYP114) purified from bacteroids. A lambda gt11 expression clone of B. japonicum USDA 110 DNA that reacted with the anti-CYP112 antibody was obtained and was used to screen a library of USDA 110 genomic DNA in pLAFR1 for a clone of the P-450 locus. Forced expression of subclones of the P-450 locus in Escherichia coli produced polypeptides that reacted with either the anti-CYP112 antibody or the anti-CYP114 antibody; no cross-reactivity was evident. A Western blot (immunoblot) analysis showed that neither protein was present in free-living aerobically grown B. japonicum cells, but that both proteins were present in cells grown anaerobically, as well as in bacteroids. A mutant strain disrupted in the CYP112 locus produced neither CYP112 nor CYP114, indicating that the mutation was polar for CYP114. The mutant produced effective nodules on soybeans, even though the bacteroids contained no detectable P-450. This suggests that the cytochromes P-450 which we examined are not involved in an essential symbiotic function.     

Analysis of 6 VNTR loci by ‘multiplex’ PCR and automated fluorescent detection

The polymerase chain reaction was used to amplify six small variable number of tandem repeat loci in two reactions (D19S20 co-amplifying with D17S5 and D1S80; D17S766 co-amplifying with D16S83 and D17S24). When coupled with fluorescent detection of the products, this provides a rapid, highly discriminating automated test. Preferential amplification of small alleles, leading to allelic dropout was found to occur in D19S20 and D16S83. Population databases are presented for Caucasians and Afro-Caribbeans at loci D19S20, D16S83 and D17S24, and for Asians at D19S20.     

Amino Acids Translocated from Turgid and Water-stressed Barley Leaves : II. Studies with N and C

Movement of labeled amino acids from leaf blades to sheaths was followed after supplying ¹³NH₃ gas, ¹⁴CO₂, or ¹⁴C-amino-acids to attached blades of barley plants.