Evidence for genetic control of adult weight plasticity in the snail Helix aspersa

Phenotypic plasticity and canalization are important topics in quantitative genetics and evolution. Both concepts are related to environmental sensitivity. The latter can be modeled using a model with genetically structured environmental variance. This work reports the results of a genetic analysis of adult weight in the snail Helix aspersa. Several models of heterogeneous variance are fitted using a Bayesian, MCMC approach. Exploratory analyses using posterior predictive model checking and model comparisons based on the deviance information criterion favor a model postulating a genetically structured heterogeneous environmental variance. Our analysis provides a strong indication of a positive genetic correlation between additive genetic values affecting the mean and those affecting environmental variation of adult body weight. The possibility of manipulating environmental variance by selection is illustrated numerically using estimates of parameters derived from the snail data set.     

A new procedure to produce lignocellulosic anion exchangers from agricultural waste materials

Two lignocellulosic agricultural waste materials (LCM), sugarcane bagasse (BG) and rice hull (RH), were converted into weak-base anion exchanger and evaluated for their exchanger capacity for nitrate. Pure cellulose (PC) and pure alkaline lignin (PL) were also used as reference materials to elucidate possible reactivity in LCM. Epoxy and amino groups were introduced into BG, RH, PC and PL substrates after the reaction with epichlorohydrin and dimethylamine in the presence of pyridine and an organic solvent N,N-dimethylformamide (DMF). Amino group incorporation into cellulose decreased with the presence of water in the reaction mixture and increased with the reaction time and presence of a catalyst (pyridine). The highest maximum nitrate exchange capacity (Qmax) and yields of the prepared exchangers was obtained from PL (1.8 mmol g(-1) and 412.5%), followed by BG (1.41 mmol g(-1) and 300%), PC (1.34 mmol g(-1) and 166%) and RH (1.32 mmol g(-1) and 180%). The proposed synthetic procedure was effective in modifying PL, PC and LCM chemically resulting in a higher yield and nitrate removal capacity.     

Normal adult climbing fiber monoinnervation of cerebellar Purkinje cells in mice lacking MHC class I molecules

Some immune system proteins have recently been implicated in the development and plasticity of neuronal connections. Notably, proteins of the major histocompatibility complex 1 (MHC class 1) have been shown to be involved in synaptic plasticity in the hippocampus and the development of projection patterns in the visual system. We examined the possible role for the MHC class 1 proteins in one well-characterized example of synaptic exuberance and subsequent refinement, the climbing fiber (CF) to Purkinje cell (PC) synapse. Cerebella from adult mice deficient for two MHC genes, H2-D1 and H2-K1, and for beta2-microglobulin gene were examined for evidence of deficient elimination of supernumerary CF synapses on their PCs. Electrophysiological and morphological evidence showed that, despite the absence of these MHC class 1 molecules, adult PCs in these transgenic mice are monoinnervated as in wild-type animals. These findings indicate that, at the level of restriction of afferent number at this synapse, functional MHC class 1 proteins are not required.     

The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACP_Pks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACP_Pks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce α-alkyl β-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.Mycolic acids, α-branched and β-hydroxylated fatty acids of unusual chain length (C30-C90), are the hallmark of the Corynebacterineae suborder that includes the causative agents of tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Members of each genus biosynthesize mycolic acids of specific chain lengths, a feature used in taxonomy. For example, Corynebacterium holds the simplest prototypes (C32-C36), called “corynomycolic acids,” which result from an enzymatic condensation between two regular size fatty acids (C16–C18). In contrast, the longest mycolates (C60-C90) are the products of condensation between a very long meromycolic chain (C40-C60) and a shorter α-chain (C22-C26) (1). These so-called “eumycolic acids” are found in mycobacteria and display various structural features present on the meromycolic chain. Eumycolic acids are major and essential components of the mycobacterial envelope where they contribute to the formation of the outer membrane (2, 3) that plays a crucial role in the permeability of the envelope. They also impact on the pathogenicity of some mycobacterial species (4).The first in vitro mycolate biosynthesis assays have been developed using Corynebacterium cell-wall extracts in the presence of a radioactive precursor (5, 6) and have brought key information about this pathway. Yet, any attempt to fractionate these extracts to identify the proteins involved has ended in failure. Later, enzymes catalyzing the formation of the meromycolic chain and the introduction of functions have been discovered with the help of novel molecular biology tools (for review, see Ref. 1), culminating with the identification of the putative operon fadD32-pks13-accD4 that encodes enzymes implicated in the mycolic condensation step in both corynebacteria and mycobacteria (see Fig. 1) (7–9). AccD4, a putative carboxyltransferase, associates at least with the AccA3 subunit to form an acyl-CoA carboxylase (ACC)³ complex that most likely activates, through a C₂-carboxylation step, the extender unit to be condensed with the meromycolic chain (see Fig. 1). In Corynebacterium glutamicum, the carboxylase would metabolize a C16 substrate (8, 10), whereas in M. tuberculosis the purified complex AccA3-AccD4 was shown to carboxylate C24-C26 acyl-CoAs (11). Furthermore, FadD32, predicted to belong to a new class of long-chain acyl-AMP ligases (FAAL) (12), is most likely required for the activation of the meromycolic chain prior to the condensation reaction. At last, the cmrA gene controls the reduction of the β-keto function to yield the final mycolic motif (13) (see Fig. 1).Open in a separate windowFIGURE 1.Proposed scheme for the biosynthesis of mycolic acids. The asymmetrical carbons of the mycolic motif have a R,R configuration. R₁-CO, meromycolic chain; R₂, branch chain. In mycobacteria, R₁-CO = C40-C60 and R₂ = C20-C24; in corynebacteria, R₁-CO = C16-C18 and R₂ = C14-C16; X₁, unknown acceptor of the mycolic α-alkyl β-ketoacyl chains; X₂, unknown acceptor of the mycolic acyl chains.Although the enzymatic properties of the ACC complex have been well characterized (9, 11), those of Pks13 and FadD32 are poorly or not described. Pks13 is a type I polyketide synthase (PKS) made of a minimal module holding ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, and additional N-terminal ACP and C-terminal thioesterase domains (Fig. 1). Its ACP domains are naturally activated by the 4′-phosphopantetheinyl (P-pant) transferase PptT (14). The P-pant arm has the general function of carrying the substrate acyl chain via a thioester bond involving its terminal thiol group. In the present article we report the purification of a soluble activated form of the large Pks13 protein. For the first time, the loading mechanisms of both types of substrates on specific domains of the PKS were investigated. We describe a unique catalytic mechanism of the Pks13-FadD32 enzymatic couple and the development of an in vitro condensation assay that generates the formation of α-alkyl β-ketoacids, the precursors of mycolic acids.     

A role for myelin-associated peroxisomes in maintaining paranodal loops and axonal integrity

Demyelinating diseases of the nervous system cause axon loss but the underlying mechanisms are not well understood. Here we show by confocal and electron microscopy that in myelin-forming glia peroxisomes are associated with myelin membranes. When peroxisome biogenesis is experimentally perturbed in Pex5 conditional mouse mutants, myelination by Schwann cells appears initially normal. However, in nerves of older mice paranodal loops become physically unstable and develop swellings filled with vesicles and electron-dense material. This novel model of a demyelinating neuropathy demonstrates that peroxisomes serve an important function in the peripheral myelin compartment, required for long-term axonal integrity.     

Skin cancer--primary and secondary prevention (information campaigns and screening)--with a focus on children & sunbeds

Solar and artificial (sunbed) UV-exposure is the main risk factor for the development of epithelial skin cancer (basal cell carcinoma, BCC, and squamous cell carcinoma, SCC) as well for malignant melanoma (MM). UV exposure in childhood and adolescence is especially important. Therefore, adequate methods of primary prevention have continuously to be used and to be developed further to target these age-groups in order to reduce the risks of intensive UV-exposure. Primary prevention can effectively be combined with secondary prevention (early detection, screening) to reduce the burden of skin cancer and to decrease incidence, morbidity and mortality.     

A complex of catalytically inactive protein phosphatase-1 sandwiched between Sds22 and inhibitor-3

Protein Ser/Thr phosphatase-1 (PP1) associates with a host of proteins to form substrate-specific holoenzymes. Sds22 and Inhibitor-3 (I3) are two independently described ancient interactors of PP1. We show here by various approaches that Sds22 and I3 form a heterotrimeric complex with PP1, both in cell lysates and after purification. The stability of the complex depended on functional PP1 interaction sites in Sds22 and I3, indicating that PP1 is sandwiched between Sds22 and I3. Intriguingly, I3 could not be replaced in this complex by another PP1 interactor with the same PP1 binding motif. In vitro, Sds22 and I3 were potent inhibitors of PP1, but with only some substrates. The inhibition by Sds22 could be reproduced with synthetic Sds22 fragments comprising leucine-rich repeats (LRR) 2 and 5. Sds22 and LRR5 also slowly converted PP1 into a conformation that was inactive with all tested substrates. Cell lysates that were prepared under conditions that prevented the Sds22-induced inactivation of PP1 contained a catalytically inactive complex of Sds22, PP1, and I3, indicating that this complex exists in vivo. Therefore, our studies show that a pool of PP1 is complexly controlled by both Sds22 and I3.     

Escherichia coli FolC structure reveals an unexpected dihydrofolate binding site providing an attractive target for anti-microbial therapy

In some bacteria, such as Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The crystal structure of E. coli FolC is described in this paper. It showed strong similarities to that of the FPGS enzyme of Lactobacillus casei within the ATP binding site and the catalytic site, as do all other members of the Mur synthethase superfamily. FolC structure revealed an unexpected dihydropteroate binding site very different from the folate site identified previously in the FPGS structure. The relevance of this site is exemplified by the presence of phosphorylated dihydropteroate, a reaction intermediate in the DHFS reaction. L. casei FPGS is considered a relevant model for human FPGS. As such, the presence of a folate binding site in E. coli FolC, which is different from the one seen in FPGS enzymes, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy.