Nanovesicles Are Secreted during Pollen Germination and Pollen Tube Growth： A Possible Role in Fertilization

Dear Editor, During recent decades, a novel mechanism of secretion has been identified in a wide range of mammalian cells. It involves the release of bioactive membrane nanovesicles （30-100 nm）, termed exosomes, upon the fusion of multivesicular bodies with the plasma membrane （Thery et al., 2009）. Exosomes are implicated in diverse functions, such as scavenging of archaic proteins, intercellular messengers delivering cell-specific signals, and vehicles for transmissible pathogens. Exosomes have also been described in other organisms such as bacte- ria, Drosophila, and fungi.     

DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters

The spatial and temporal organization of DNA replication was investigated in living cells with a green fluorescent protein fusion to the DNA polymerase clamp PCNA. In situ extractions and photobleaching experiments revealed that PCNA, unlike RPA34, shows little if any turnover at replication sites, suggesting that it remains associated with the replication machinery through multiple rounds of Okazaki fragment synthesis. Photobleaching analyses further showed that the transition from earlier to later replicons occurs by disassembly into a nucleoplasmic pool of rapidly diffusing subcomponents and reassembly at newly activated sites. The fact that these replication sites were de novo assembled in close proximity to earlier ones suggests that activation of neighboring origins may occur by a domino effect possibly involving local changes in chromatin structure and accessibility.     

The folding of the hepatitis C virus internal ribosome entry site depends on the 3′-end of the viral genome

Hepatitis C virus (HCV) translation initiation is directed by an internal ribosome entry site (IRES) and regulated by distant regions at the 3′-end of the viral genome. Through a combination of improved RNA chemical probing methods, SHAPE structural analysis and screening of RNA accessibility using antisense oligonucleotide microarrays, here, we show that HCV IRES folding is fine-tuned by the genomic 3′-end. The essential IRES subdomains IIIb and IIId, and domain IV, adopted a different conformation in the presence of the cis-acting replication element and/or the 3′-untranslatable region compared to that taken up in their absence. Importantly, many of the observed changes involved significant decreases in the dimethyl sulfate or N-methyl-isatoic anhydride reactivity profiles at subdomains IIIb and IIId, while domain IV appeared as a more flexible element. These observations were additionally confirmed in a replication-competent RNA molecule. Significantly, protein factors are not required for these conformational differences to be made manifest. Our results suggest that a complex, direct and long-distance RNA–RNA interaction network plays an important role in the regulation of HCV translation and replication, as well as in the switching between different steps of the viral cycle.     

Effects of supplementation with plant extract product containing carvacrol,cinnamaldehyde and capsaicin on serum metabolites and enzymes during the finishing phase of feedlot-fed bull calves

This study investigated the in vivo effects of a commercial blend of plant extracts (carvacrol, cinnamaldehyde and capsaicin) on serum metabolic parameters closely connected with energy and protein metabolism (glucose; l-lactate; non-esterified fatty acids, NEFA; urea nitrogen, SUN; creatinine; total protein, TSP) and enzymes associated with hepatic function (aspartate-aminotransferase, AST and gamma-glutamyl transferase, GGT) in finishing-stage Belgian Blue bull calves maintained in a commercial feedlot. Monitoring was performed over 86 days in 24 animals randomly allotted to two groups: (1) a control group (CTR, no supplementation; n = 10), and (2) a group receiving dietary supplementation with a commercial blend of plant extracts (PEX, 100 mg/kg DM of concentrate; n = 14). Under the conditions of our study, supplementation with the commercial blend did not give detrimental effects, but the opposite: the decrease in serum l-lactate, NEFA and creatinine levels and the increase in SUN concentrations; suggests an improvement in the energy status and protein turnover of the supplemented animals.     

Fatty acyl-AMP as an intermediate in fatty acid reduction to aldehyde in luminescent bacteria

The acyl protein synthetase component (50K) of the fatty acid reductase complex from the luminescent system of Photobacterium phosphoreum has been found to catalyze the activation of fatty acid via formation of an enzyme bound acyl-AMP (carboxyphosphate mixed anhydride) immediately prior to the acylation of the enzyme. PPi-ATP exchange and nucleotide binding experiments are dependent on fatty acid and indicate that the fatty acyl-AMP is directly formed and that an adenylated enzyme intermediate is not part of the mechanism. The formation of acyl-AMP from fatty acid and ATP is reversible with a standard free energy of -2 kcal/mol, and is dependent on Mg2+. The fatty acyl-AMP intermediate has been isolated and shown to be part of the pathway of fatty acid reduction. The 34K component of the complex, which strongly stimulates the acylation of the 50K protein by fatty acyl-AMP or fatty acid and ATP, is not required for the formation of acyl-AMP showing that it differentially affects the fatty acid activation and acylation steps catalyzed by the 50K protein.     

SP-A permeabilizes lipopolysaccharide membranes by forming protein aggregates that extract lipids from the membrane

Surfactant protein A (SP-A) is known to cause bacterial permeabilization. The aim of this work was to gain insight into the mechanism by which SP-A induces permeabilization of rough lipopolysaccharide (Re-LPS) membranes. In the presence of calcium, large interconnected aggregates of fluorescently labeled TR-SP-A were observed on the surface of Re-LPS films by epifluorescence microscopy. Using Re-LPS monolayer relaxation experiments at constant surface pressure, we demonstrated that SP-A induced Re-LPS molecular loss by promoting the formation of three-dimensional lipid-protein aggregates in Re-LPS membranes. This resulted in decreased van der Waals interactions between Re-LPS acyl chains, as determined by differential scanning calorimetry, which rendered the membrane leaky. We also showed that the coexistence of gel and fluid lipid phases within the Re-LPS membrane conferred susceptibility to SP-A-mediated permeabilization. Taken together, our results seem to indicate that the calcium-dependent permeabilization of Re-LPS membranes by SP-A is related to the extraction of LPS molecules from the membrane due to the formation of calcium-mediated protein aggregates that contain LPS.     

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.     

Antimicrobial resistance and class I integrons in Salmonella enterica isolates from wild boars and Bísaro pigs

The antibiotic resistance phenotype and genotype and the integron type were characterized in 58 Salmonella enterica isolates recovered from Bísaro pigs and wild boars (20 S. Typhimurium, 17 S. Rissen, 14 S. Enteritidis and 7 S. Havana). Most S. Typhimurium isolates (15/20 of Bísaro pigs and wild boars) showed ampicillin, chloramphenicol, streptomycin, tetracycline, sulfonamide, and amoxicillin-clavulanic acid resistances. Of the 17 S. Rissen isolates of both origins, 13 were resistant to ampicillin, tetracycline and trimethoprim-sulfamethoxazole. Among the S. Enteritidis isolates of Bísaro pigs, eight were nalidixic acid-resistant and three were sulfonamide-resistant. The tet(A) or tet(G) genes were detected in most tetracycline-resistant isolates. The intI1 gene was identified in 72.5% of S. enterica isolates in which the conserved region 3' of class 1 integrons (qacEΔ1+sul1) was also amplified, whereas none had the intI2 gene. The dfrA12+orfF+aadA2 gene cassette arrangement was found in the variable region of class 1 integrons in 14 S. Rissen isolates. Fifteen S. Typhimurium isolates had two integrons with variable regions of 1000 and 1200 bp that harbored the aadA2 and blaPSE-1 gene cassettes, respectively. In these isolates the floR and tet(G) genes were also amplified, indicative of the genomic island 1 (SGI1). Salmonella Typhimurium and S. Rissen of animal origin frequently show a multi-antimicrobial resistant phenotype, which may have implications in public health.     

Sterculic acid antagonizes 7-ketocholesterol-mediated inflammation and inhibits choroidal neovascularization

Sterculic acid is a cyclopropene fatty acid with numerous biological activities. In this study we demonstrate that sterculic acid is a potent inhibitor of endoplasmic reticulum (ER) stress and related inflammation caused by 7-ketocholesterol (7KCh). 7KCh is a highly toxic oxysterol suspected in the pathogenesis of various age-related diseases such as atherosclerosis, Alzheimer's disease and age-related macular degeneration. Sterculic acid demonstrated to be 5-10 times more effective than other anti-inflammatory fatty acids at inhibiting 7KCh-mediated inflammatory responses in cultured cells. In vivo, sterculic acid was effective at inhibiting the formation of choroidal neovascularization (CNV) in the laser-injury rat model. Our data suggests that sterculic acid may be useful in treating CNV in certain forms of age-related macular degeneration.