Reduced fertility of female mice lacking CD81

In somatic cells, the tetraspanins CD81 and CD9 associate with each other, with additional tetraspanins and with non-tetraspanin molecules to form proteolipidic complexes. Here we show that CD81 is expressed on the surface of oocytes where it associates with tetraspanin-enriched membrane structures. A major CD9 and CD81 partner, CD9P-1, is also expressed by oocytes. Deletion of CD81 gene in mice results in a 40% reduction of female fertility. In vitro insemination indicated that this infertility is due to a deficiency of oocytes to fuse with sperm. While the fertility of CD9-/- mice is severely but not completely impaired, double knock-out CD9-/- CD81-/- mice were completely infertile indicating that CD9 and CD81 play complementary roles in sperm-egg fusion. Finally, a fraction of CD9 was transferred from CD81-/- oocytes to sperm present in the perivitelline space indicating that the defect of fusion of CD81-/- oocytes does not result from an impaired initial gamete interaction.     

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.     

Promoter variant-dependent mRNA expression of the MEF2A in longissimus dorsi muscle in cattle

The myocyte enhancer factor 2A (MEF2A) gene encodes a member of the myocyte enhancer factor 2 (MEF2) protein family that is involved in vertebrate skeletal, cardiac, and smooth muscle development and differentiation during myogenesis. According to recent studies, MEF2 genes might be major regulators of postnatal skeletal muscle growth; thus, they are considered to be important, novel candidates for muscle development and body growth in farm animals. The aim of the present study was to search for polymorphisms in the bovine MEF2A gene and analyze their effect on the MEF2A mRNA expression level in the longissimus dorsi muscle of Polish Holstein-Fresian cattle. In total, 4094?bp of the whole coding sequence and the promoter region of MEF2A were re-sequenced in 30 animals, resulting in the detection of 6 novel variants as well as one previously reported SNP. Three linked mutations in the promoter region (-780T/G, g.-768T/G, and g.-222A/G) and only two genotypes were identified in two Polish breeds (TTA/TTA and TTA/GGG). Three SNPs in the coding region [g.1599G/A (421aa), g.1626G/A (429aa), and g.1641G/A (434aa)] appeared to be silent substitutions and segregated as two intragene haplotypes: GGG and AAA. Expression analysis showed that the mutations in the promoter region are highly associated with the MEF2A mRNA level in the longissimus dorsi muscle of bulls carrying two different genotypes. The higher MEF2A mRNA level was estimated in the muscle of bulls carrying the TTA/TTA (p<0.01) genotype as compared with those with TTA/GGG. The results obtained suggest that the nucleotide sequence mutation in MEF2A might be useful marker for body growth traits in cattle.     

Distinct patterns of MMP-9 and MMP-2 activity in slow and fast twitch skeletal muscle regeneration in vivo

Skeletal muscles exhibit great plasticity and an ability to reconstruct in response to injury. However, the repair process is often inefficient and hindered by the development of fibrosis. We explored the possibility that during muscle repair, the different regeneration ability of the fast (extensor digitorum longus; EDL) and slow twitch (Soleus) muscles depends on the differential expression of metalloproteinases (MMP-9 and MMP-2) involved in the remodeling of the extracellular matrix. Our results show that MMP-9 and MMP-2 are present in the intact muscle and are up-regulated after crush-induced muscle injury. The expression and the activity of these two enzymes depend on the type of muscle and the phase of muscle regeneration. In the regenerating Soleus muscle, elevated levels of MMP-9 occurred during the myolysis and reconstruction phase. In contrast, regenerating EDL muscles exhibited decreased MMP-9 levels during myolysis and increased MMP-2 activity at the reconstruction phase. Moreover, satellite cells (mononuclear myoblasts) derived from Soleus and EDL muscles showed no differences in localization or activity of MMP-9 and MMP-2 during proliferation and differentiation in vitro. MMP-9 activity was present during all stages of myoblast differentiation, whereas MMP-2 activity reached its highest level during myoblast fusion. We conclude that MMPs are involved in muscle repair, and that fast and slow twitch muscles exhibit different patterns of MMP-9 and MMP-2 activity.     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

Structural properties of bombesin-like peptides revealed by surface-enhanced Raman scattering on roughened silver electrodes

This work presents a Fourier-transform absorption infrared, Fourier-transform Raman, and surface-enhanced Raman scattering (SERS) study of the following peptides belonging to the bombesin-like family: phyllolitorin, [Leu(8)]phyllolitorin, NMB, NMC, and PG-L. The SERS study was undertaken to understand the adsorption mechanism of bombesin-like peptides on an electrochemically roughened silver electrode surface and to show changes in the adsorption mechanism with alterations in amino acids and small tertiary structures. The SERS spectra presented here shows bands mainly associated with the Trp(8) residue vibrations. The presence of mainly pyrrole coring vibrations for phyllolitorin and [Leu(8)]phyllolitorin and mainly benzene coring modes for NMB and NMC indicated that these groups interact with the roughened silver electrode surface. Furthermore, N(1)--C(8) and C(3)--C(9) bonds of the PG-L indole ring seemed to have nearly a vertical orientation on the electrode surface. In addition, distinct vibrations of the C--S fragment were observed in the SERS spectra of [Leu(8)]phyllolitorin and PG-L. The strong enhancement of the nu(C==O) vibration in the [Leu(8)]phyllolitorin SERS spectrum yielded evidence that the intact C==O bond(s) bind strongly to the silver electrode surface, whereas NMC, phyllolitorin, and NMB were located near the silver surface. This finding was supported by the presence of the nu(C--C(==O)) mode. The amide I band observed at 1642 and 1634 cm(-1) for NMB and NMC, respectively, and the Raman amide III band seen in the 1282-1249 cm(-1) range for all peptides except PG-L, indicate that the strongly hydrogen-bonded alpha-helical conformation and random-coil structure are favored for binding to the surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 980-992, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Lysophosphatidic acids, cyclic phosphatidic acids and autotaxin as promising targets in therapies of cancer and other diseases

Lysophospholipids have long been recognized as membrane phospholipid metabolites, but only recently lysophosphatidic acids (LPA) have been demonstrated to act on specific G protein-coupled receptors. The widespread expression of LPA receptors and coupling to several classes of G proteins allow LPA-dependent regulation of numerous processes, such as vascular development, neurogenesis, wound healing, immunity, and cancerogenesis. Lysophosphatidic acids have been found to induce many of the hallmarks of cancer including cellular processes such as proliferation, survival, migration, invasion, and neovascularization. Furthermore, autotaxin (ATX), the main enzyme converting lysophosphatidylcholine into LPA was identified as a tumor cell autocrine motility factor. On the other hand, cyclic phosphatidic acids (naturally occurring analogs of LPA generated by ATX) have anti-proliferative activity and inhibit tumor cell invasion and metastasis. Research achievements of the past decade suggest implementation of preclinical and clinical evaluation of LPA and its analogs, LPA receptors, as well as autotaxin as potential therapeutic targets.