Recognition of polyadenylation sites in yeast pre-mRNAs by cleavage and polyadenylation factor

Recognition of poly(A) sites in yeast pre-mRNAs is poorly understood. Employing an in vitro cleavage system with cleavage and polyadenylation factor (CPF) and cleavage factor IA we show that the efficiency and positioning elements are dispensable for poly(A)-site recognition within a short CYC1 substrate in vitro. Instead, U-rich elements immediately upstream and downstream of the poly(A) site mediate cleavage-site recognition within CYC1 and ADH1 pre-mRNAs. These elements act in concert with the poly(A) site to produce multiple recognition sites for the processing machinery, since combinations of mutations within these elements were most effective in cleavage inhibition. Intriguingly, introduction of a U-rich element downstream of the GAL7 poly(A) site strongly enhanced cleavage, underscoring the importance of downstream sequences in general. RNA- binding analyses demonstrate that cleavage depends on the recognition of the poly(A)-site region by CPF. Consistent with in vitro results, mutation of sequences upstream and downstream of the poly(A) site affected 3'-end formation in vivo. A model for yeast pre-mRNA cleavage-site recognition outlines an unanticipated high conservation of yeast and mammalian 3'-end processing mechanisms.

Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian influenza virus in chickens and ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.

Proteolytic processing of the P2/nucleocapsid cleavage site is critical for human immunodeficiency virus type 1 RNA dimer maturation

Differences in virion RNA dimer stability between mature and protease-defective (immature) forms of human immunodeficiency virus type 1 (HIV-1) suggest that maturation of the viral RNA dimer is regulated by the proteolytic processing of the HIV-1 Gag and Gag-Pol precursor proteins. However, the proteolytic processing of these proteins occurs in several steps denoted primary, secondary, and tertiary cleavage events and, to date, the processing step associated with formation of stable HIV-1 RNA dimers has not been identified. We show here that a mutation in the primary cleavage site (p2/nucleocapsid [NC]) hinders formation of stable virion RNA dimers, while dimer stability is unaffected by mutations in the secondary (matrix/capsid [CA], p1/p6) or a tertiary cleavage site (CA/p2). By introducing mutations in a shared cleavage site of either Gag or Gag-Pol, we also show that the cleavage of the p2/NC site in Gag is more important for dimer formation and stability than p2/NC cleavage in Gag-Pol. Electron microscopy analysis of viral particles shows that mutations in the primary cleavage site in Gag but not in Gag-Pol inhibit viral particle maturation. We conclude that virion RNA dimer maturation is dependent on proteolytic processing of the primary cleavage site and is associated with virion core formation.

Surface structure of SnOxNy thin films deposited using a filtered cathodic arc for novel energy storage systems

Tin oxide/nitride (SnOxNy) thin films were synthesised using a filtered cathodic vacuum arc deposition system. These films were deposited at room temperature with increasing amounts of reactive nitrogen gas to alter the nanostructure. To understand the surface structure of the coatings several techniques were used including scanning electron microscopy (SEM), atomic force microscopy (AFM), x-ray photoelectron spectroscopy (XPS), x-ray diffraction (XRD) and x-ray absorption spectroscopy (XAS). Preliminary results have shown that a cathodic arc can be used to deposit smooth films which exhibit a mixed tin oxide/nitride structure.

A truncated fragment of Src protein kinase generated by calpain-mediated cleavage is a mediator of neuronal death in excitotoxicity

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N-terminal unique domain. This generates a truncated Src fragment of ?52 kDa, which we localized predominantly to the cytosol. A cell membrane-permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke.

The effects of mechanical stress, environment and cathodic protection on the degradation and failure of coatings: An overview

Organic coatings have been used in conjunction with cathodic protection as the most economical method of corrosion protection by the oil and gas pipeline industry. In a bid to prolong the life of the pipelines, the degradation and failure of pipeline coatings under the effects of major influencing factors including mechanical stress, the environmental corrosivity and cathodic protection have been extensively investigated over the past decades. This paper provides an overview of recent research for understanding coating degradation under the effect of these factors, either individually or in combination. Electrochemical impedance spectroscopy remains the primary and the most commonly used technique of studying the degradation of organic coatings, although there have been attempts to use other techniques such as electrochemical polarization (both dynamic and static), electrochemical noise, Scanning Kelvin Probe, Fourier Transform Infrared Spectroscopy, Differential Scanning Calorimetry and Dynamic Mechanical Analyser. Major knowledge and technological gaps in the investigation of the combined effects of mechanical stress, environmental corrosivity and cathodic protection on coating degradation have been identified.

Studying the cathodic disbondment of coatings using electrochemical impedance spectroscopy

The disbondment of protective organic coatings is a widely reported pipeline coating failure mode in the oil and gas industry. Traditional methods of evaluating cathodic disbondment of pipeline coatings are based on visual inspection of pipeline conditions, and laboratory testing of cathodic disbondment resistance (CDR) using standard methods such as ASTM G8. Although some other laboratory-based techniques, such as scanning kelvin probe and scanning acoustic microscopy have been used to study the cathodic disbondment (CD) of coatings, these are often difficult to apply in practical testing. Over the past decade, electrochemical impedance spectroscopy (EIS) has been employed as a potential method for measuring CD. This paper reports preliminary results from an EIS study designed to characterise CD behaviour of epoxy coatings under excessive cathodic protection. EIS data correlated well with the area of disbonded coating. Analysis of EIS data can provide valuable information on the initiation and rates of CD.

Understanding the effects of electrical interference signals and the environment on the effectiveness of cathodic protection

Cathodic protection (CP) failure due to excursions from safe CP levels is a challenge for the protection and maintenance of buried energy pipelines. Although research shows that stray current is a major factor contributing to CP failure, there is little consensus on how 'big' the excursions (either in magnitude, length or frequency) need to be in order to cause pipeline corrosion problems. This uncertainty has caused difficulties in selecting suitable parameters in relevant industry standards. This paper provides a brief review of past research on different factors affecting CP efficiency. Preliminary results from new electrochemical cells designed to develop an understanding of how CP excursions away from the 'safe' level can lead to corrosion problems are also presented.

A review of techniques for the monitoring of cathodic shielding and corrosion under disbonded coatings

The mitigation of external corrosion of energy pipelines by a combination of barrier coatings and Cathodic Protection (CP) is not always effective. Even when design specifications are properly met, the shielding of cathodic protection current from reaching steel surface by disbonded barrier coatings, often referred to as cathodic shielding, may lead to severe corrosion problems such as deep pitting, high and near neutral pH Stress Corrosion Cracking (SCC) and Microbiologically Induced Corrosion (MIC). Unfortunately, current indirect assessment methods used in the pipeline industry have serious difficulties in detecting such corrosion problems. This paper provides a brief review of current techniques and their limitations when being applied under complex buried pipeline environmental conditions. The main purpose is to identify potential methods that could be utilised in the design of new monitoring probes specific for the monitoring of cathodic shielding and corrosion of disbonded coatings in the pipeline industry.