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.     

Regulation of rat ovarian cell growth and steroid secretion

A cultured rat ovarian cell line (31 A-F(2)) was used to study the effect of growth factors (epidermal growth factor [EGF] and fibroblast growth factor [FGF]), a survival factor (ovarian growth factor [OGF]), a hormone (insulin), and an iron-binding protein (transferring) on cell proliferation and steroid production under defined culture conditions. EGF and insulin were shown to be mitogenic (half-maximal response at 0.12 nM and 0.11 muM, respectively) for 31A-F(2) cells incubated in serum-free medium. EGF induced up to three doublings in the cell population, whereas insulin induced an average of one cell population doubling. FGF, OGF, and transferrin were found not to have any prominent effect on cell division when incubated individually with 31A-F(2) cells in serum-free medium. However, a combination of EGF, OGF, insulin, and transferrin stimulated cell division to the same approximate extent as cells incubated in the presence of 5 percent fetal calf serum. EGF or insulin did not significantly affect total cell cholesterol levels (relative to cells incubated in serum-free medium) when incubated individually with 31A-F(2) cells. However, cell cholesterol levels were increased by the addition of OGF (250 percent), FGF (370 percent), or a combination of insulin and EGF (320 percent). Progesterone secretion from 31A-F(2) cells was enhanced by EGF (25 percent), FGF (80 percent), and insulin (115 percent). However, the addition of a mitogenic mixture of EGF, OGF, insulin, and transferrin suppressed progesterone secretion 150 percent) below that of control cultures. These studies have permitted us to determine that EGF and insulin are mitogenic factors that are required for the growth of 31A-F(2) cells and that OGF and transferrin are positive cofactors that enhance growth. Also, additional data suggest that cholesterol and progesterone production in 31A-F(2) cells can be regulated by peptide growth factors and the hormone insulin.     

CONSORF: a consensus prediction system for prokaryotic coding sequences

CONSORF is a fully automatic high-accuracy identification system that provides consensus prokaryotic CDS information. It first predicts the CDSs supported by consensus alignments. The alignments are derived from multiple genome-to-proteome comparisons with other prokaryotes using the FASTX program. Then, it fills the empty genomic regions with the CDSs supported by consensus ab initio predictions. From those consensus results, CONSORF provides prediction reliability scores, predicted frame-shifts, alternative start sites and best pair-wise match information against other prokaryotes. These results are easily accessed from a website.     

The effects of acrylamide on brain creatine kinase: Inhibition kinetics and computational docking simulation

The occurrence of acrylamide is frequently observed in processed foods. Therefore, the harmful effects of acrylamide on metabolic enzymes are important to understand. We studied the inhibitory effects of acrylamide on the brain creatine kinase (CK-BB). We found that CK-BB was kinetically inactivated by acrylamide accompanied by the disruption of the hydrophobic surface. Acrylamide mainly interacted with the thiol (–SH) residue of CK-BB and resulted in alkylation. A computational docking simulation supported that acrylamide directly bound to the active site of CK-BB where cysteine and glycine residues interacted mainly. The inhibition kinetics combined with computational prediction can be useful in order to have insights into the mechanisms regarding environmentally hazardous factors at the molecular level.     

Neuromuscular organization and aminergic modulation of contractions in the Drosophila ovary

Background  

The processes by which eggs develop in the insect ovary are well characterized. Despite a large number of Drosophila mutants that cannot lay eggs, the way that the egg is moved along the reproductive tract from ovary to uterus is less well understood. We remedy this with an integrative study on the reproductive tract muscles (anatomy, innervation, contractions, aminergic modulation) in female flies.     

The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis.     

Different activation treatments for successful development of bovine oocytes following intracytoplasmic sperm injection

In this study, the developmental capacity and cytogenetic composition of different oocyte activation protocols was evaluated following intracytoplasmic sperm injection (ICSI) of in vitro matured bovine oocytes. Motile spermatozoa selected by Percoll density gradient were treated with 5 mM dithiothreitol (DTT) and analysed for ultrastructural changes of the head using transmission electron microscopy (TEM). The alterations in sperm morphology after DTT treatment for different times (15, 30 and 60 min) were 10%, 45-55% and 70-85%, respectively. Further, a partial decondensation of sperm heads was observed after DTT treatment for 30 min. Oocytes were injected with sperm treated with DTT for 30 min. In group 1, sperm injection was performed without any activation stimulus to the oocytes. In group 2, sham injection without sperm was performed without activating the oocytes. Oocytes injected with sperm exposed to 5 microM ionomycin for 5 min (group 3), 5 microM ionomycin + 1.9 mM dimethylaminopurine (DMAP) for 3 h (group 4) and 5 microM ionomycin + 3 h culture in M199 + 1.9 mM DMAP (group 5) were also evaluated for cleavage, development and chromosomal abnormality. Cleavage and development rates in groups 1, 2 and 3 were significantly (p < 0.05) lower than those in groups 4 and 5. The incidence of chromosomal abnormality in the embryos treated directly with DMAP after ionomycin (group 4) was higher than in group 5. We conclude that immediate DMAP treatment after ionomycin exposure of oocytes results in arrest of release of the second polar body, and thus leads to changes in chromosomal pattern. Therefore, the time interval between ionomycin and DMAP plays a crucial role in bovine ICSI.     

Nucleotide polymorphism at the alcohol dehydrogenase locus of pocket gophers, genus Geomys

Using the strictly neutral model as a null hypothesis, we tested for deviations from expected levels of nucleotide polymorphism at the alcohol dehydrogenase locus (Adh-1) within and among four species of pocket gophers (Geomys bursarius major, G. knoxjonesi, G. texensis llanensis, and G. attwateri). The complete protein-encoding region was examined, and 10 unique alleles, representing both electromorphic and cryptic alleles, were used to test hypotheses (e.g., the neutral model) concerning the maintenance of genetic variation. Nineteen variable sites were identified among the 10 alleles examined, including 9 segregating sites occurring in synonymous positions and 10 that were nonsynonymous. Several statistical methods, including those that test for within-species variation as well as those that examine variation within and among species, failed to reject the null hypothesis that variation (both within and between species of Geomys) at the Adh locus is consistent with the neutral theory. However, there was significant heterogeneity in the ratio of polymorphism to divergence across the gene, with polymorphisms clustered in the first half of the coding region and fixed differences clustered in the second half of the gene. Two alternative hypotheses are discussed as possible explanations for this heterogeneity: an old balanced polymorphism in the first half of the gene or a recent selective sweep in the second half of the gene.