Higher‐order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm‐to‐vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi‐scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X‐ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo‐EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher‐order chain structures that are broken upon interaction with the receptor Atg19 in vitro. The stoichiometry of these cargo–receptor complexes is key to maintaining the size of the Cvt aggregate in vivo. Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.     

Endothelial cell junctions

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.     

Genomic organization and chromosomal localization of three members of the UDP-N-acetylgalactosamine: polypeptide N- acetylgalactosaminyltransferase family

A homologous family of UDP- N -acetylgalactosamine: polypeptide N - acetylgalactosaminyltransferases (GalNAc-transferases) initiate O- glycosylation. These transferases share overall amino acid sequence similarities of approximately 45-50%, but segments with higher similarities of approximately 80% are found in the putative catalytic domain. Here we have characterized the genomic organization of the coding regions of three GalNAc-transferase genes and determined their chromosomal localization. The coding regions of GALNT1 , -T2 , and -T3 were found to span 11, 16, and 10 exons, respectively. Several intron/exon boundaries were conserved within the three genes. One conserved boundary was shared in a homologous C. elegans GalNAc- transferase gene. Fluorescence in situ hybridization showed that GALNT1 , -T2 , and -T3 are localized at chromosomes 18q12-q21, 1q41-q42, and 2q24-q31, respectively. These results suggest that the members of the polypeptide GalNAc-transferase family diverged early in evolution from a common ancestral gene through gene duplication.      

Oxidative stress in skin fibroblasts cultures from patients with Parkinson's disease

Background  

In the substantia nigra of Parkinson's disease (PD) patients, increased lipid peroxidation, decreased activities of the mitochondrial complex I of the respiratory chain, catalase and glutathione-peroxidase, and decreased levels of reduced glutathione have been reported. These observations suggest that oxidative stress and mitochondrial dysfunction play a role in the neurodegeneration in PD. We assessed enzymatic activities of respiratory chain and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with PD.     

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte

By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.     

Prospects for estimating nucleotide divergence with RAPDs

The technique of random amplification of polymorphic DNA (RAPD), which is simply polymerase chain reaction (PCR) amplification of genomic DNA by a single short oligonucleotide primer, produces complex patterns of anonymous polymorphic DNA fragments. The information provided by these banding patterns has proved to be of great utility for mapping and for verification of identity of bacterial strains. Here we consider whether the degree of similarity of the banding patterns can be used to estimate nucleotide diversity and nucleotide divergence. With haploid data, fragments generated by RAPD-PCR can be treated in a fashion very similar to that for restriction-fragment data. Amplification of diploid samples, on the other hand, requires consideration of the fact that presence of a band is dominant to absence of the band. After describing a method for estimating nucleotide divergence on the basis of diploid samples, we summarize the restrictions and criteria that must be met when RAPD data are used for estimating population genetic parameters.      

Intra-articular CD1c-expressing myeloid dendritic cells from rheumatoid arthritis patients express a unique set of T cell-attracting chemokines and spontaneously induce Th1, Th17 and Th2 cell activity

Introduction

Myeloid dendritic cells (mDCs) are potent T cell-activating antigen-presenting cells that have been suggested to play a crucial role in the regulation of immune responses in many disease states, including rheumatoid arthritis (RA). Despite this, studies that have reported on the capacity of naturally occurring circulating mDCs to regulate T cell activation in RA are still lacking. This study aimed to evaluate the phenotypic and functional properties of naturally occurring CD1c (BDCA-1)⁺ mDCs from synovial fluid (SF) compared to those from peripheral blood (PB) of RA patients.

Methods

CD1c⁺ mDC numbers and expression of costimulatory molecules were assessed by fluorescence-activated cell sorting (FACS) analysis in SF and PB from RA patients. Ex vivo secretion of 45 inflammatory mediators by mDCs from SF and PB of RA patients was determined by multiplex immunoassay. The capacity of mDCs from SF to activate autologous CD4⁺ T cells was measured.

Results

CD1c⁺ mDC numbers were significantly increased in SF versus PB of RA patients (mean 4.7% vs. 0.6%). mDCs from SF showed increased expression of antigen-presenting (human leukocyte antigen (HLA) class II, CD1c) and costimulatory molecules (CD80, CD86 and CD40). Numerous cytokines were equally abundantly produced by mDCs from both PB and SF (including IL-12, IL-23, IL-13, IL-21). SF mDCs secreted higher levels of interferon γ-inducible protein-10 (IP-10), monokine induced by interferon γ (MIG) and, thymus and activation-regulated chemokine (TARC), but lower macrophage-derived chemokine (MDC) levels compared to mDCs from PB. mDCs from SF displayed a strongly increased capacity to induce proliferation of CD4⁺ T cells associated with a strongly augmented IFNγ, IL-17, and IL-4 production.

Conclusions

This study suggests that increased numbers of CD1c⁺ mDCs in SF are involved in the inflammatory cascade intra-articularly by the secretion of specific T cell-attracting chemokines and the activation of self-reactive T cells.     

Induction/synchronization of oestrus and ovulation in dairy goats with different short term treatments and fixed time intrauterine or exocervical insemination system

Two experiments were carried out on Ionica dairy goats in order to test the efficiency of: (1) short term-5-day combined progestogen-PGF2α-GnRH treatments on induction/synchronization of oestrus and fertility after natural mating in lactating goats and during the transition period (Experiment 1); (2) short term-9-day FGA-PGF2α-eCG treatments on synchronizing oestrus and ovulation (Experiment 2.1) and artificial insemination (AI) fixed time system in synchronized does (Experiment 2.2), during the breeding season. In Experiment 1, four treatment groups (N=24) were considered: (1) FPe-11d - control, FGA intravaginal sponges (11 days)+PGF2α (9th d)+eCG (11th d); (2) FPe-5d, FGA (5 days)+PGF2α (5th d)+eCG (5th d); (3) PFe-5d, PGF2α (D0)+FGA (5 days)+eCG (5th d); (4) GPe-5d, GnRH (D0)+PGF2α (5th d)+eCG (5th d). Goats were checked for oestrus and naturally mated. The occurrence of oestrus was 75.0, 78.3, 86.4, and 58.3% for groups 1-4, respectively, with significant differences (P<0.05) between groups 3 and 4. Interval to oestrus was earlier (P<0.05) in GPE-5d than in FPe-11d control group. There were no differences between the groups (P>0.05) in fertility or in prolificacy. In Experiment 2.1, 22 goats were subdivided into two treatment groups (N=11): (T1) FPe-11d (control), FGA (11 days)+PGF2α (9th d)+eCG (11th d); (T2) FPe-9d, FGA (9 days)+PGF2α (7th d)+eCG (9th d). Oestrus and ovulation times were monitored every 4h; ovulation rate was also determined. The induction of oestrus ranged from 91 to 100% and all goats ovulated. Intervals to oestrus, from the onset of oestrus to ovulation, from sponge removal to ovulation, and ovulation rates were 28.2±4.9 and 26.0±4.0h, 25.3±9.2 and 28.9±7.4h, 53.5±7.6 and 54.9±7.1h, 3.7±1.6 and 2.4±1.4 corpora lutea (P<0.05) for T1 and T2, respectively. In T2 a great abnormal ovulatory response was observed. In Experiment 2.2, 48 goats were synchronized with FPe-9d treatment and subjected to AI, performed 50h after s.r. with frozen semen, and subdivided into 2 AI system groups (N=24): T3, exocervical AI (100×10(6)Spz/doe); T4, intrauterine AI (20×10(6)Spz/doe). Fertility rate was higher (P<0.05) in T4. It seems that short term-5-day combined progestogen-PGF2α-GnRH-eCG treatments need to be investigated for AI fixed time.