Identification of essential residues for the C-terminal cleavage of the mammalian LC3: a lesson from yeast Atg8

Atg8, a member of an evolutionarily conserved ubiquitin-like protein family, is involved in multiple membrane trafficking pathways including autophagy. In a recent study, we have identified two functional sites in the yeast Saccharomyces cerevisiae Atg8, one involving residues Tyr49 and Leu50, and the other--located on the opposite side of the molecule--residues Phe77 and Phe79. Here we extended our studies to the mammalian system and report that in LC3 residues Phe80 and Leu82, the equivalents of Phe77 and Phe79 in Atg8, are essential for its C-terminal cleavage. We propose that these residues are part of the Atg4 recognition site.     

Lying patterns of high producing healthy dairy cows after calving in commercial herds as affected by age,environmental conditions and production

An animal expresses its physiological and well-being status by its behaviour. Changes in behaviour can be associated with health, production or well-being problems and therefore with the profitability of the farm. The objectives of the present study were to analyse lying patterns of healthy cows, collected with a commercial behaviour sensor, in early lactation in relation to environmental conditions, age of the cow and production performance. In future, these results may be used as a ‘baseline’ for detection of alterations in behaviour that indicate health problems. The study involved 210 healthy multiparous Israeli Holstein cows in three commercial dairy farms. Only healthy cows during the first 28 days after calving were included in this study. Data were analysed in relation to calving season, age of cows and correlation between milk production and lying time.The results show that lying time increased significantly with age and is significantly (P < 0.05) higher in winter than in summer (summer lactation 2: 491 ± 17 min/day (mean ± SD), summer lactation 3 and more: 520 ± 25 min/day, winter lactation 2: 531 ± 25 min/day, winter lactation 3 and more: 579 ± 38 min/day).The proportion of positively and negatively milk production and lying time correlated cows is affected by calving season.This study indicates that behaviour variables in early lactation are affected by calving season, lactation number and type of correlation between milk production and lying time.     

Host-derived Langerhans cells persist after MHC-matched allografting independent of donor T cells and critically influence the alloresponses mediated by donor lymphocyte infusions

Mouse models of minor histocompatibility Ag-mismatched bone marrow transplantation were used to study donor dendritic cell (DC) reconstitution after conditioning, variables influencing the persistence of residual host DCs in different compartments, their phenotype, and their role in governing donor lymphocyte infusion (DLI)-mediated alloresponses. Reconstitution of all splenic DC subsets occurred rapidly after bone marrow transplantation and before T cell reconstitution. However, in contrast to MHC-mismatched chimeras, residual host-derived DCs persisted in the cutaneous lymph nodes (CLNs) of MHC-matched chimeras despite the presence or addition of donor T cells to the graft. The phenotype of these residual host-derived DCs in CLNs was consistent with Langerhans' cells (LCs). We confirmed their skin origin and found near-complete preservation of host-derived LCs in the skin. Host-derived LCs retained their ability to continuously traffic to the CLNs, expressed homogeneously increased levels of costimulatory molecules, and could capture and carry epicutaneously applied Ags. To determine the role of residual host LCs in governing DLI-mediated alloresponses, we administered DLI alone or after topical application of the TLR7 ligand imiquimod, which is known to enhance the LC emigration from the skin. DLI administration resulted in a decrease in host-derived DCs in the CLNs and increased recruitment of donor-derived DCs to the skin, whereas imiquimod augmented their alloreactivity. These results suggest uniqueness of the MHC-matched setting in relation to the persistence of host-derived DCs in the skin and points to a previously unrecognized role of host-derived LCs in the induction of DLI-mediated graft-vs-host alloresponses.     

NHEJ-deficient DT40 cells have increased levels of immunoglobulin gene conversion: evidence for a double strand break intermediate

Activation-induced cytidine deaminase (AID) likely initiates immunoglobulin gene-conversion (GC) by deaminating cytidines within the V-region of chicken B-cells. However, the intervening DNA lesion required to initiate GC remains elusive. GC could be initiated by a single strand break or a double strand break (DSB). To distinguish between these possibilities, we examined GC in the chicken DT40 B cell line deficient in non-homologous end joining (NHEJ). It is known that the NHEJ and homologous recombination DNA repair pathways compete for DSBs. In light of this, if a DSB is the major intermediate, deficiency in NHEJ should result in increased levels of GC. Here we show that DNA–PKcs^−/−/− and Ku70^−/− DT40 cells had 5- to 10-fold higher levels of GC relative to wildtype DT40 as measured by surface IgM reversion and sequencing of the V-region. These data suggest that a DSB is the major DNA lesion that initiates GC.     

Versatile roles of docosahexaenoic acid in the prenatal brain: from pro- and anti-oxidant features to regulation of gene expression

Docosahexaenoic acid (DHA) is the most ubiquitous polyunsaturated fatty acid (FA) in brain tissue. It is selectively esterified to amino phospholipids (PL) and therefore it is highly prevalent at the cytofacial site of the plasma membrane where it may specifically participate in intracellular events. A highly selective DHA accumulation prior to birth is the result of maternal supply via the placenta through a bio-magnification process. Supplements of DHA via the intra-amniotic route to the fetal rat increase brain DHA levels and also confer neuroprotection to fetuses subjected to global ischemic stress. The protective effect has been attributed to an enhanced free radical scavenging capacity of DHA. Dietary deprivation of linolenic acid (LNA) during the perinatal life on the other hand, resulted in losses of DHA from cerebral PLs [M. Schiefermeier, E. Yavin, n-3 deficient and DHA-enriched diets during critical periods of the developing prenatal rat brain, J. Lipid Res. 43 (2002) 124-131]. LNA deprivation also caused changes in a number of gene markers the identification of which was attained by a labor-intensive suppression subtractive hybridization protocol using mRNA from 2-week-old postnatal brains [E. Yakubov, P. Dinerman, F. Kuperstein, S. Saban, E. Yavin, Improved representation of gene markers on microarray by PCR-select subtracted cDNA targets, Mol. Brain Res. 137 (2005) 110-118]. Most notable was a remarkable elevation of dopamine (DA) receptor (D1 and D2) genes as evaluated by quantitative RT-PCR, SDS-PAGE gel electrophoresis and immunochemical staining [F. Kuperstein, E. Yakubov, P. Dinerman, S. Gil, R. Eylam, N. Salem Jr., E. Yavin, Overexpression of dopamine receptor genes and their products in the postnatal rat brain following maternal n-3 FA dietary deficiency, J. Neurochem. 95 (2005) 1550-1562]. Over-expression of DA receptors has been attributed to a compensatory mechanism resulting from impairment in DA neurotransmitter production, storage and processing. In conclusion, DHA is a versatile molecule with a wide range of actions spanning from participation in cellular oxidative processes and intracellular signaling to modulatory roles in gene expression and growth regulation.     

Widespread compensatory evolution conserves DNA-encoded nucleosome organization in yeast

Evolution maintains organismal fitness by preserving genomic information. This is widely assumed to involve conservation of specific genomic loci among species. Many genomic encodings are now recognized to integrate small contributions from multiple genomic positions into quantitative dispersed codes, but the evolutionary dynamics of such codes are still poorly understood. Here we show that in yeast, sequences that quantitatively affect nucleosome occupancy evolve under compensatory dynamics that maintain heterogeneous levels of A+T content through spatially coupled A/T-losing and A/T-gaining substitutions. Evolutionary modeling combined with data on yeast polymorphisms supports the idea that these substitution dynamics are a consequence of weak selection. This shows that compensatory evolution, so far believed to affect specific groups of epistatically linked loci like paired RNA bases, is a widespread phenomenon in the yeast genome, affecting the majority of intergenic sequences in it. The model thus derived suggests that compensation is inevitable when evolution conserves quantitative and dispersed genomic functions.     

Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind alpha-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the alpha-BTX/AChR system yielded 13-mer peptides that bind to alpha-BTX with high affinity and are potent inhibitors of alpha-BTX binding to AChR (IC(50) of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel beta-hairpin structure when bound to alpha-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.     

Localization of the PE methylation pathway and SR-BI to the canalicular membrane: evidence for apical PC biosynthesis that may promote biliary excretion of phospholipid and cholesterol

To better understand the regulation of biliary phospholipid and cholesterol excretion, canalicular membranes were isolated from the livers of C57BL/6J mice and abundant proteins separated by SDS-PAGE and identified by matrix-assisted laser desorption/ionization mass spectrometry. A prominent protein revealed by this analysis was betaine homocysteine methyltransferase (BHMT). This enzyme catalyzes the first step in a three-enzyme pathway that promotes the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Immunoblotting confirmed the presence of BHMT on the canalicular membrane, failed to reveal the presence of the second enzyme in this pathway, methionine adenosyltransferase, and localized the third enzyme of the pathway, PE N-methyltransferase (PEMT). Furthermore, immunfluorescence microscopy unambiguously confirmed the localization of PEMT to the canalicular membrane. These findings indicate that a local mechanism exists in or around hepatocyte canalicular membranes to promote phosphatidylethnolamine methylation and PC biosynthesis. Finally, immunoblotting revealed the presence and immunofluorescence microscopy unambiguously localized the scavenger receptor class B type I (SR-BI) to the canalicular membrane. Therefore, SR-BI, which is known to play a role in cholesterol uptake at the hepatocyte basolateral membrane, may also be involved in biliary cholesterol excretion. Based on these findings, a model is proposed in which local canalicular membrane PC biosynthesis in concert with the phospholipid transporter mdr2 and SR-BI, promotes the excretion of phospholipid and cholesterol into the bile.