Limitation of the Ellman method: cholinesterase activity measurement in the presence of oximes

The Ellman method for assaying thiols is widely used for cholinesterase activity measurement. Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine [ATCh]) hydrolysis by the cholinesterase. Oximes, reactivators of inhibited cholinesterase, are nucleophiles that also react with ATCh (oximolysis), producing thiocholine and (indirectly) TNB ion. The aim of this study was to characterize ATCh oximolysis. Therefore, we measured the oximolysis between oximes (K027 and HI-6) and ATCh in the presence of DTNB at different pH values, taking into account the final concentration of a product that is thiocholine. To confirm oximate ion involvement in the nucleophilic attack, we also determined the reaction rate between the oximes and ATCh, without DTNB, at different pH values by measuring the decrease in oximate ion absorption over time. The oximate ion of K027 reacted 14 times faster with ATCh (306M(-1)min(-1)) than the oximate ion of HI-6 (22M(-1)min(-1)). However, the rate constants obtained with the Ellman method were 84M(-1)min(-1) for K027 and 22M(-1)min(-1) for HI-6. Our results confirmed that the rate obtained with K027 using the Ellman method is actually the rate of the Ellman reaction itself. This suggests that the Ellman method cannot be used uncritically to evaluate oxime reaction with choline esters, in particular when oximolysis is faster than the Ellman reaction itself at a given pH.     

Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1

Crisponi syndrome is a severe autosomal recessive condition that is phenotypically characterized by abnormal, paroxysmal muscular contractions resembling neonatal tetanus, large face, broad nose, anteverted nares, camptodactyly, hyperthermia, and sudden death in most cases. We performed homozygosity mapping in five Sardinian and three Turkish families with Crisponi syndrome, using high-density single-nucleotide polymorphism arrays, and identified a critical region on chromosome 19p12-13.1. The most prominent candidate gene was CRLF1, recently found to be involved in the pathogenesis of cold-induced sweating syndrome type 1 (CISS1). CISS1 belongs to a group of conditions with overlapping phenotypes, also including cold-induced sweating syndrome type 2 and Stuve-Wiedemann syndrome. All these syndromes are caused by mutations of genes of the ciliary neurotrophic factor (CNTF)-receptor pathway. Here, we describe the identification of four different CRLF1 mutations in eight different Crisponi-affected families, including a missense mutation, a single-nucleotide insertion, and a nonsense and an insertion/deletion (indel) mutation, all segregating with the disease trait in the families. Comparison of the mutation spectra of Crisponi syndrome and CISS1 suggests that neither the type nor the location of the CRLF1 mutations points to a phenotype/genotype correlation that would account for the most severe phenotype in Crisponi syndrome. Other, still-unknown molecular factors may be responsible for the variable phenotypic expression of the CRLF1 mutations. We suggest that the syndromes can comprise a family of "CNTF-receptor-related disorders," of which Crisponi syndrome would be the newest member and allelic to CISS1.     

Sperm DNA adducts impair fertilization during ICSI but not during IVF

Many studies emphasize the influence of the status of spermatozoal nucleus on fertilization, mainly with regard to DNA fragmentation. This study was undertaken to analyze the influence of DNA adducts content in spermatozoa on fertilization during assisted reproduction. Ovarian hyperstimulation, oocyte retrieval and laboratory work-up in 61 IVF (in vitro fertilization) and 118 ICSI (intracytoplasmic sperm injection) first cycles were performed according to the same protocol. Semen analysis was made according to WHO Manual (1999). DNA adducts assay in spermatozoa was performed by 32Ppostlabeling method. In total 331 fertilizable oocytes were obtained during IVF and 659 during ICSI. Both groups differed significantly by sperm count, motility and morphology but not by the concentration of DNA adducts in spermatozoa (0.0306 +/- 0.0217 in IVF versus 0.0373 +/- 0.0321 in ICSI). The fertilization rate during IVF was significantly influenced by sperm count (p=0.0002) and motility (p=0.0037) but not by DNA adducts concentration (p=0.30528), whereas during ICSI was positively influenced by sperm motility (p=0.04669) and negatively by DNA adducts concentration (p=0.00796). DNA adducts concentration in spermatozoa significantly negatively influences fertilization rate during ICSI, but not during IVF.     

Age and seasonal effects on predator-sensitive foraging in squirrel monkeys (Saimiri sciureus): a field experiment

A field experiment was conducted to examine the effect of perceived predation risk on the use of foraging areas by juvenile and adult primates under different conditions of local food abundance. Wild squirrel monkeys, Saimiri sciureus, were observed in an experiment conducted during the dry and the wet seasons at a site in Eastern Amazonia, Brazil. Animals were presented with feeding platforms that differed in food quantity and exposure to aerial predators through varying vegetative cover. In the dry season, juveniles and adults chose platforms based solely on food quantity. However, in the wet season, juveniles foraged preferentially on high-reward platforms only if cover level also was high (i.e., potentially offered greater concealment from predators). In contrast, adults showed the same pattern of platform use regardless of season. These results indicate that age and local resource availability based on seasonality affect whether primates forage in a predator-sensitive manner. Juveniles may be more sensitive to predation risk when foraging, and individuals may take fewer risks when resource abundance is high in their environment.     

Responses of squirrel monkeys to seasonal changes in food availability in an eastern Amazonian forest

Tropical forests are characterized by marked temporal and spatial variation in productivity, and many primates face foraging problems associated with seasonal shifts in fruit availability. In this study, I examined seasonal changes in diet and foraging behaviors of two groups of squirrel monkeys (Saimiri sciureus), studied for 12 months in Eastern Brazilian Amazonia, an area characterized by seasonal rainfall. Squirrel monkeys were primarily insectivorous (79% of feeding and foraging time), with fruit consumption highest during the rainy season. Although monkeys fed from 68 plant species, fruit of Attalea maripa palms accounted for 28% of annual fruit-feeding records. Dietary shifts in the dry season were correlated with a decline in ripe A. maripa fruits. Despite pronounced seasonal variation in rainfall and fruit abundance, foraging efficiency, travel time, and distance traveled remained stable between seasons. Instead, squirrel monkeys at this Eastern Amazonian site primarily dealt with the seasonal decline in fruit by showing dietary flexibility. Consumption of insects, flowers, and exudates increased during the dry season. In particular, their foraging behavior at this time strongly resembled that of tamarins (Saguinus sp.) and consisted of heavy use of seed-pod exudates and specialized foraging on large-bodied orthopterans near the forest floor. Comparisons with squirrel monkeys at other locations indicate that, across their geographic range, Saimiri use a variety of behavioral tactics during reduced periods of fruit availability.     

All-trans retinoic acid suppresses exocrine differentiation and branching morphogenesis in the embryonic pancreas

Recent evidence has shown that retinoic acid (RA) signalling is required for early pancreatic development in zebrafish and frog but its role in later development in mammals is less clear cut. In the present study, we determined the effects of RA on the differentiation of the mouse embryonic pancreas. Addition of all-trans retinoic acid (atRA) to embryonic pancreatic cultures induced a number of changes. Branching morphogenesis and exocrine differentiation were suppressed and there was premature formation of endocrine cell clusters (although the total area of beta cells was not different in control and atRA-treated buds). We investigated the mechanism of these changes and found that the premature formation of beta cells was associated with the early expression of high-level Pdx1 in the endocrine cell clusters. In contrast, the suppressive effect of RA on exocrine differentiation may be due to a combination of two mechanisms (i) up-regulation of the extracellular matrix component laminin and (ii) enhancement of apoptosis. We also demonstrate that addition of fibroblast growth factor (FGF)-10 is able to partially prevent apoptosis and rescue exocrine differentiation and branching morphogenesis in atRA-treated cultures but not in mice lacking the FGF receptor 2-IIIb, suggesting the effects of FGF-10 are mediated through this receptor.     

Effect of T3 treatment on glutathione redox pool and its metabolizing enzymes in mitochondrial and post-mitochondrial fractions of adult rat testes

T3 (3,3', 5-triiodo-L-thyronine; 20 microg/100 g body weight/day in 0.01 N NaOH, i.p for 1, 3 and 5 days) treatment modulated reduced (GSH) and oxidized (GSSG) glutathione contents along with the activities of its metabolizing enzymes (such as glutathione peroxidase, glutathione reductase and glutathione S-transferase) in the testis of Wistar rats. However, the magnitude and nature of changes in the above biochemical parameters in response to T3 treatment were noticed to be different between mitochondrial and post-mitochondrial fractions. This was accompanied with elevated levels of lipid hydroperoxide and ascorbic acid in the crude homogenate of testis. The level of hydrogen peroxide in the post-mitochondrial fractions of testes did not change on first day, decreased on 3rd day and increased on 5th day of the hormone treatment when compared to respective controls. Nevertheless, its content in mitochondria was significantly elevated in response to all the three durations of the hormone treatment having the highest induction on 3rd day. The changes observed in the levels of GSH and GSSG and its metabolizing enzymes in response to T3 treatment reflect an alteration in the redox state of testis, which may be a causative factor for the impairment of testicular physiology as a consequence of oxidative stress.