Schistosoma mansoni: differential expression of cathepsins L1 and L2 suggests discrete biological functions for each enzyme

Schistosoma mansoni cathepsins L1 (SmCL1) and L2 (SmCL2) were expressed as active recombinant proteinases in Saccharomyces cerevisiae. The recombinant enzymes exhibited substrate preferences characteristic of cathepsin-L-like cysteine proteinases. However, the enzymes differed in their substrate specificities; SmCL1 cleaved Boc-Val-Leu-Lys-NHMec with a higher efficiency than it cleaved Z-Phe-Arg-NHMec, whereas the opposite was true for SmCL2. The enzymes also differed in their pH profiles of activity; SmCL1 exhibited a broad pH profile with an optimum of pH 6. 5, while SmCL2 was active only in the acidic pH range with an optimum of 5.35. Immunoblot and RT-PCR analyses revealed that the native forms of both SmCL1 and SmCL2 are expressed in male and female worms, but at higher levels in adult female compared to male schistosomes. Additionally, both enzymes were observed in the excretory/secretory products of adult worms. The RT-PCR analysis indicated that neither enzyme is expressed in S. mansoni eggs or in miracidia, suggesting that the cathepsin-L-like activity that has been previously reported to be expressed in these stages may be the product of another gene(s). Cercariae do not express SmCL2, but appear to express SmCL1 in its inactive precursor form. Together with the findings of previous immunolocalization and phylogenetic analyses, the results reported here demonstrate that SmCL1 and SmCL2 are distinct cathepsin cysteine proteinases and strongly suggest that they play discrete biological roles.     

The structure of Antirrhinum centroradialis protein (CEN) suggests a role as a kinase regulator

Expression of the plant protein centroradialis (CEN) leads to a morphological switch between shoot growth and the development of flower structures (inflorescence). We have determined the crystal structure of Antirrhinum CEN to 1.9 A resolution. This structure confirms the CEN proteins as a subset of the family of phosphatidylethanolamine-binding proteins (PEBP), as predicted from sequence homology. Mammalian forms of PEBP have been found to act as inhibitors of MAP kinase signalling, a central signalling cascade regulating cell differentiation. CEN and PEBP proteins share a similar topology dominated by a large central beta-sheet. The strong conservation of a binding pocket at one end of this sheet which is capable of binding phosphoryl ligands, suggests the biological effects of CEN, like PEBP, arise from the ability of this region to form complexes with phosphorylated ligands, hence interfering with kinases and their effectors.     

Characterisation of nitrilase and nitrile hydratase biocatalytic systems

Biocatalytic transformations converting aromatic and arylaliphatic nitriles into the analogous related amide or acid were investigated. These studies included synthesis of the -substituted nitrile 3-hydroxy-3-phenylpropionitrile, subsequent enrichment and isolation on this substrate of nitrile-degrading microorganisms from the environment, and a comparative study of enzymatic reactions of nitriles by resting cell cultures and enzymes. Each biocatalyst exhibited a distinctive substrate selectivity profile, generally related to the length of the aliphatic chain of the arylaliphatic nitrile and the position of substituents on the aromatic ring or aliphatic chain. Cell-free nitrilases generally exhibited a narrower substrate range than resting whole cells of Rhodococcus strains. The Rhodococcus strains all exhibited nitrile hydratase activity and converted -hydroxy nitriles (but did not demonstrate enantioselectivity on this substrate). The biocatalysts also mediated the synthesis of a range of -hydroxy carboxylic acids or amides from aldehydes in the presence of cyanide. The use of an amidase inhibitor permits halting the nitrile hydratase/amidase reaction at the amide intermediate.     

ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway

The ARF tumor suppressor is widely regarded as an upstream activator of p53-dependent growth arrest and apoptosis. However, recent findings indicate that ARF can also regulate the cell cycle in the absence of p53. In search of p53-independent ARF targets, we isolated nucleophosmin (NPM/B23), a protein we show is required for proliferation, as a novel ARF binding protein. In response to hyperproliferative signals, ARF is upregulated, resulting in the nucleolar retention of NPM and concomitant cell cycle arrest. The Mdm2 oncogene outcompetes NPM/B23 for ARF binding, and introduction of Mdm2 reverses ARF's p53-independent properties: in vitro, NPM is released from ARF-containing protein complexes, and in vivo S phase progression ensues. ARF induction by oncogenes or replicative senescence does not alter NPM/B23 protein levels but rather prevents its nucleocytoplasmic shuttling without inhibiting rRNA processing. By actively sequestering NPM in the nucleolus, ARF utilizes an additional mechanism of tumor suppression, one that is readily antagonized by Mdm2.     

DnaG interacts with a linker region that joins the N- and C-domains of DnaB and induces the formation of 3-fold symmetric rings

Loading of the replicative ring helicase onto the origin of replication (oriC) is the final outcome of a well coordinated series of events that collectively constitute a primosomal cascade. Once the ring helicase is loaded, it recruits the primase and signals the switch to the polymerization mode. The transient nature of the helicase–primase (DnaB–DnaG) interaction in the Escherichia coli system has hindered our efforts to elucidate its structure and function. Taking advantage of the stable DnaB–DnaG complex in Bacillus stearothermophilus, we have reviewed conflicting mutagenic data from other bacterial systems and shown that DnaG interacts with the flexible linker that connects the N- and C-terminal domains of DnaB. Furthermore, atomic force microscopy (AFM) imaging experiments show that binding of the primase to the helicase induces predominantly a 3-fold symmetric morphology to the hexameric ring. Overall, three DnaG molecules appear to interact with the hexameric ring helicase but a small number of complexes with two and even one DnaG molecule bound to DnaB were also detected. The structural/functional significance of these data is discussed and a speculative structural model for this complex is suggested.     

Delay discounting and probability discounting as related to cigarette smoking status in adults

This study examined relations between adult smokers and non-smokers and the devaluation of monetary rewards as a function of delay (delay discounting, DD) or probability (probability discounting, PD). The extent to which individuals discount value, either as a function of a reward being delayed or probabilistic, has been taken to reflect individual differences in impulsivity. Those who discount most are considered most impulsive. Previous research has shown that adult smokers discount the value of delayed rewards more than adult non-smokers. However, in the one published study that examined probability discounting in adult smokers and non-smokers, the smokers did not discount the value of probabilistic rewards more than the non-smoker controls. From this past research, it was hypothesized that measures of delay discounting would differentiate between smokers and non-smokers but that probability discounting would not. Participants were 54 (25 female) adult smokers (n = 25) and non-smokers (n = 29). The smokers all reported smoking at least 20 cigarettes per day, and the non-smokers reported having never smoked. The results indicated that the smokers discounted significantly more than the non-smokers by both delay and probability. Unlike past findings, these results suggest that both delay and probability discounting are related to adult cigarette smoking; however, it also was determined that DD was a significantly stronger predictor of smoking than PD.     

Measuring state changes in human delay discounting: an experiential discounting task

A new experiential discounting task (EDT) is presented. Unlike existing question-based measures of delay discounting, which rely on imagined consequences during task completion, this EDT requires that participants experience choice consequences (i.e. delays and pseudo consumatory responses) during the measurement period. As a preliminary examination of this task's sensitivity to variability in discounting, 12 participants (six females) completed a timing test (production and reproduction), a question-based measure of delay discounting, and the EDT during non-sleep-deprived (awake 7 h) and sleep-deprived (awake 21 h) conditions. Based on evidence that sleep deprivation increases impulsive behavior, it was hypothesized that participants would underrepresent time intervals in both production and reproduction procedures and discount significantly more with the discounting procedures while sleep deprived. Unfortunately, data from the question-based discounting procedure could not be reported due to invalid task completion. However, as hypothesized, certain production and reproduction intervals were underrepresented on the timing test, and discounting was significantly steeper on the EDT when participants were sleep deprived. Also, rate of discounting on the EDT was better characterized by a hyperbolic function than by exponential function, which is consistent with previous delay-discounting research. These preliminary results suggest this EDT may be a useful measure for assessing state changes in discounting processes.     

Lead identification of a potent benzopyranone selective estrogen receptor modulator

Starting from a phenol screening hit (6), three series of benzopyranone selective estrogen receptor modulators (SERMs) have been designed, synthesized, and analyzed for both estrogen receptor alpha binding affinity and in vitro activity in two cell assays. The lead compound identified, SP500263 (13), was more potent than raloxifene and tamoxifen in a cell-based assay measuring inhibition of interleukin-6 release.