Hydrolysis of thioester analogs by rat liver phospholipase A1

The hydrolysis of thioester containing phospholipids by rat liver plasmalemma phospholipase A1 was measured in a continuous spectrophotometric assay. In this assay thioester substrates were employed which, upon hydrolysis, liberated a free thiol which was reacted with 4,4'-dithiopyridine to yield the product 4-thiopyridone that absorbs at 324 nm. Thioester substrates, prepared by chemical synthesis, were used in phospholipid and Triton X-100 micelles for kinetic analysis carried out according to the method of Hendrickson and Dennis (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739). Vmax, Ks, and Km values obtained for various isomers and racemic mixtures of the synthetic thioester analogs are compared with corresponding oxyester substrates. Unnatural sn-1 isomers competitively inhibited the hydrolysis of natural sn-3 isomers of phosphatidylethanolamine and phosphatidic acid. Furthermore, the sn-1 isomer of phosphatidic acid was hydrolyzed by phospholipase A1, but with lower catalytic efficiency than the sn-3 isomer. The presence of a thioester at the sn-1 position did not change the Vmax significantly, as compared to the oxyester phospholipids. When two thioesters were present on the phospholipid molecule, the Vmax was decreased significantly. A convenient synthesis of 1-monothioester analogs of phospholipids is reported. The results presented show the usefulness of the spectrophotometric assay for measuring phospholipase A1 activity as well as the influence of racemic mixtures and thioesters on the hydrolytic rate.     

Use of nuclear morphometry characteristics to distinguish between normal and abnormal cervical glandular histologies.

This is a methodological study exploring the use of quantitative histopathology applied to the cervix to discriminate between normal and cancerous (consisting of adenocarcinoma and adenocarcinoma in situ) tissue samples. The goal is classifying tissue samples, which are populations of cells, from measurements on the cells. Our method uses one particular feature, the IODs-Index, to create a tissue level feature. The specific goal of this study is to find a threshold for the IODs-Index that is used to create the tissue level feature. The main statistical tool is Receiver Operating Characteristic (ROC) curve analysis. When applied to the data, our method achieved promising results with good estimated sensitivity and specificity for our data set. The optimal threshold for the IODs-Index was found to be 2.12.     

Biological soil crusts in ecological restoration: emerging research and perspectives

Drylands encompass over 40% of terrestrial ecosystems and face significant anthropogenic degradation causing a loss of ecosystem integrity, services, and deterioration of social‐ecological systems. To combat this degradation, some dryland restoration efforts have focused on the use of biological soil crusts (biocrusts): complex communities of cyanobacteria, algae, lichens, bryophytes, and other organisms living in association with the top millimeters of soil. Biocrusts are common in many ecosystems and especially drylands. They perform a suite of ecosystem functions: stabilizing soil surfaces to prevent erosion, contributing carbon through photosynthesis, fixing nitrogen, and mediating the hydrological cycle in drylands. Biocrusts have emerged as a potential tool in restoration; developing methods to implement effective biocrust restoration has the potential to return many ecosystem functions and services. Although culture‐based approaches have allowed researchers to learn about the biology, physiology, and cultivation of biocrusts, transferring this knowledge to field implementation has been more challenging. A large amount of research has amassed to improve our understanding of biocrust restoration, leaving us at an opportune time to learn from one another and to join approaches for maximum efficacy. The articles in this special issue improve the state of our current knowledge in biocrust restoration, highlighting efforts to effectively restore biocrusts through a variety of different ecosystems, across scales and utilizing a variety of lab and field methods. This collective work provides a useful resource for the scientific community as well as land managers.     

Postnatal gender-dependent maturation of cellular cysteine uptake

Background. In view of the functional capacity of glutathione synthesis in premature infants, and because the availability of cysteine is one the rate limiting steps in glutathione synthesis, we hypothesized that the low glutathione levels in premature infants may be due to immaturity of the active cellular uptake of cysteine.

Objective. To document in cells from newborn infants the effect of maturity and gender on cysteine uptake and consequently on glutathione levels.

Methods. Incorporation of L-[35S] cysteine was measured in leukocytes from cord blood and from tracheal aspirates (TAC) of newborn infants of varying (gestational as well as postnatal) ages and gender. Cysteine uptake was correlated with glutathione in TAC.

Results. The maturity of newborn girls positively influences cysteine uptake, which is responsible for 78% of the variation in their glutathione content. However, in newborn boys, gestational and postnatal ages did not influence the cysteine uptake.

Discussion. Cysteine uptake appears to be the limiting step explaining the reported gender-related differences in glutathione as well as the low levels of this central antioxidant found in premature infants. The immature cysteine uptake found in cells from premature infants raises questions about the bioavailability of this conditionally essential amino acid in regimens of parenteral nutrition for human neonates.     

Correction to: Genetic characterization of Addison’s disease in Bearded Collies

Diversity of the CRISPR locus of Mycobacterium tuberculosis complex has been studied since 1997 for molecular epidemiology purposes. By targeting solely the 43 spacers present in the two first sequenced genomes (H37Rv and BCG), it gave a biased idea of CRISPR diversity and ignored diversity in the neighbouring cas-genes. We set up tailored pipelines to explore the diversity of CRISPR-cas locus in Short Reads. We analyzed data from a representative set of 198 clinical isolates as evidenced by well-characterized SNPs. We found a relatively low diversity in terms of spacers: we recovered only the 68 spacers that had been described in 2000. We found no partial or global inversions in the sequences, letting always the Direct Variant Repeats (DVR) in the same order. In contrast, we found an unexpected diversity in the form of: SNPs in spacers and in Direct Repeats, duplications of various length, and insertions at various locations of the IS6110 insertion sequence, as well as blocks of DVR deletions. The diversity was in part specific to lineages. When reconstructing evolutionary steps of the locus, we found no evidence for SNP reversal. DVR deletions were linked to recombination between IS6110 insertions or between Direct Repeats. This work definitively shows that CRISPR locus of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages. The evolutionary mechanisms that we discovered could be involved in bacterial adaptation but in a way that remains to be identified.     

Altered infant feeding patterns in boys with acquired nonsyndromic cryptorchidism

BACKGROUND : Genetic and environmental factors likely influence susceptibility to nonsyndromic cryptorchidism, a common disease presenting at birth or in later childhood. We compared cases and controls to define differential risk factors for congenital versus acquired cryptorchidism. METHODS : We compared questionnaire and clinical data from cases of congenital cryptorchidism (n = 230), acquired cryptorchidism (n = 182) and hernia/hydrocele (n = 104) with a group of healthy male controls (n = 358). Potential predictor variables (p < 0.2 in univariable analysis) were included in stepwise multivariable logistic regression models. RESULTS : Temporary (odds ratio [OR], 0.5; 95% confidence interval [CI], 0.4–0.8) or exclusive (OR, 0.6; 95% CI, 0.4–0.9) breastfeeding was reduced and soy formula feeding increased (OR, 1.8; 95% CI, 1.2–2.9) in acquired but not congenital or hernia/hydrocele groups. The highest risk estimates were observed for primary soy formula feeding with limited or no breastfeeding (OR 2.5; 95% CI, 1.4–4.3; adjusted OR, 2.7; 95% CI, 1.4–5.4) in the acquired group. Primary feeding risk estimates were equivalent or strengthened when multivariable models were limited to age greater than 2 years, full‐term or not small for gestational age, or Caucasian subjects. Pregnancy complications and increased maternal exposure to cosmetic or household chemicals were not consistently associated with either form of cryptorchidism in these models. CONCLUSIONS : Our data support reduced breastfeeding and soy formula feeding as potential risk factors for acquired cryptorchidism. Although additional studies are needed, hormonally active components of breast milk and soy formula could influence the establishment of normal testis position in the first months of life, leading to apparent ascent of testes in childhood. Birth Defects Research (Part A), 2012. © 2012 Wiley Periodicals, Inc.     

The cyclic AMP cascade is altered in the fragile X nervous system

Fragile X syndrome (FX), the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP). Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP) cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.