VIAF, a conserved inhibitor of apoptosis (IAP)-interacting factor that modulates caspase activation

Inhibitor of apoptosis (IAP) proteins are involved in the suppression of apoptosis, signal transduction, cell cycle control and gene regulation. Here we describe the cloning and characterization of viral IAP-associated factor (VIAF), a highly conserved, ubiquitously expressed phosphoprotein with limited homology to members of the phosducin family that associates with baculovirus Op-IAP. VIAF bound Op-IAP both in vitro and in intact cells, with each protein displaying a predominantly cytoplasmic localization. VIAF lacks a consensus IAP binding motif, and overexpression of VIAF failed to prevent Op-IAP from protecting human cells from a variety of apoptotic stimuli, suggesting that VIAF does not function as an IAP antagonist. VIAF was unable to directly inhibit caspase activation in vitro and a reduction of VIAF protein levels by RNA interference led to a decrease in Bax-mediated caspase activation, suggesting that VIAF functions to co-regulate the apoptotic cascade. Finally, VIAF is a substrate for ubiquitination mediated by Op-IAP. Thus, VIAF is a novel IAP-interacting factor that functions in caspase activation during apoptosis.     

The influence of meal size on prey DNA detectability in piscivorous birds

Molecular methods allow noninvasive assessment of vertebrate predator–prey systems at high taxonomic resolution by examining dietary samples such as faeces and pellets. To facilitate the interpretation of field‐derived data, feeding trials, investigating the impacts of biological, methodological and environmental factors on prey DNA detection, have been conducted. The effect of meal size, however, has not yet been explicitly considered for vertebrate consumers. Moreover, different noninvasively obtained sample types remain to be compared in such experiments. Here, we present a feeding trial on abundant piscivorous birds, Great Cormorants (Phalacrocorax carbo), to assess meal size effects on postfeeding prey DNA detection success. Faeces and pellets were sampled twice a day after the feed of large (350–540 g), medium (190–345 g) and small (15–170 g) fish meals contributing either a large (>79%) or small (<38%) share to the daily consumption. Samples were examined for prey DNA and fish hard parts. Molecular analysis of faeces revealed that both large meal size and share had a significantly positive effect on prey DNA detection rate postfeeding. Furthermore, large meals were detectable for a significantly longer time span with a detection limit at ~76 hr and a 50% detection probability at ~32 hr postfeeding. In pellets, molecular methods reliably identified the meal consumed the previous day, which was not possible via morphological analysis or when examining individual faeces. The less reliable prey DNA detection of small meals or meal shares in faeces signifies the importance of large numbers of dietary samples to obtain reliable trophic data.     

Subtidal and intertidal mussel beds (Mytilus edulis L.) in the Wadden Sea: diversity differences of associated epifauna

In 1997 and 1998, surveys were performed to compare species composition, abundance and diversity of non-attached epifauna (>1 mm) in low intertidal and adjacent shallow subtidal zones of three mussel beds (Mytilus edulis L.) near the island of Sylt in the North Sea. The community structure was similar when compared within tidal zones: no significant differences in species numbers and abundances were recorded between locations and between years. A comparison between tidal zones, however, revealed higher diversity, species densities and total species numbers in the subtidal (per 1,000 cm²: H ′=2.0±0.16; 12 ±1 species density; 22 species) than the intertidal zone (per 1,000 cm²: H ′=0.7±0.27; 6±2 species density; 19 species). Abundances significantly dropped with increasing submergence from 2,052 (±468) m^–2 to 1,184 (±475) m^–2. This was mainly due to significantly higher densities of both juvenile periwinkles, Littorina littorea, and crabs, Carcinus maenas, in intertidal mussel beds. However, many less dominant species were significantly more abundant in subtidal mussel beds. This study revealed that in the non-attached epifaunal community of mussel beds the tidal level effect within metres was strong, whilst the spatial variability in a much wider (kilometre) range but the same tidal level was negligible. The high epifaunal diversity in the subtidal zone suggests that the protective measures for mussel beds against the effects of mussel fishery should be extended from the intertidal to the subtidal zone, if the integrity of the mussel bed community in the Wadden Sea National Park is to be maintained. Electronic Publication     

Ferritin—a mediator of apoptosis?

Previously we have demonstrated an apoptosis inducing activity for a rat hepatocyte conditioned medium (CM) presumably mediated by acidic isoferritins. Here, we present support for this assumption since isoferritins purified from different rat hepatocyte CM significantly enhanced the frequency of apoptotic cells in primary rat hepatocytes, an effect completely inhibited by a neutralizing anti-H-ferritin antibody. The apoptosis induction appears to be related to a 43 kDa ferritin subunit contained in the isoferritins released from primary hepatocytes, presumably representing a ferritin heavy/light chain heterodimer. In addition, these isoferritins immunologically crossreact with antibodies raised against placental isoferritin p43-PLF (which also contains a 43 kDa ferritin subunit) and melanoma-derived H-chain ferritin, representing ferritin isoforms which reveal immunomodulatory properties. Furthermore, p53 and FasL are upregulated upon isoferritin treatment in a time dependent mode, and apoptosis induction can be suppressed by neutralizing anti-FasL antibodies. Proapoptotic Bid is upregulated too and translocated into mitochondria in primary hepatocytes exposed to the isoferritins purified from the CM. Finally, epidermal growth factor (EGF) and dexamethasone (DEX), which counteract proapoptotic mitochondrial signalling, almost completely abolished the proapoptotic effect of the hepatocyte derived isoferritins. In conclusion, our findings demonstrate that acidic isoferritins with homology to immunomodulatory ferritin isoforms (p43-PLF, melanoma-derived-H-chain ferritin) are released from hepatocytes in vitro, and are able to stimulate upregulation of p53 and mediate apoptosis involving Fas (CD95) signalling as well as addressing the intrinsic mitochondrial proapoptotic pathway.     

Generation and selection of ribozyme variants with potential application in protein engineering and synthetic biology

Over the past two decades, RNA catalysis has become a major topic of research. On the one hand, naturally occurring ribozymes have been extensively investigated concerning their structure and functional mechanisms. On the other hand, the knowledge gained from these studies has been used to engineer ribozyme variants with novel properties. In addition to RNA engineering by means of rational design, powerful techniques for selection of ribozymes from large pools of random sequences were developed and have been widely used for the generation of functional nucleic acids. RNA as catalyst has been accompanied by DNA, and nowadays a large number of ribozymes and deoxyribozymes are available. The field of ribozyme generation and selection has been extensively reviewed. With respect to the field of biotechnology, RNA and DNA catalysts working on peptides or proteins, or which are designed to control protein synthesis, are of utmost importance and interest. Therefore, in this review, we will focus on engineered nucleic acid catalysts for peptide synthesis and modification as well as for intracellular control of gene expression.     

Molecular markers in breeding for virus resistance in barley

The soil-borne barley yellow mosaic virus disease (BaMMV, BaYMV, BaYMV-2) and the aphid-transmitted barley yellow dwarf virus (BYDV) are serious threats to winter barley cultivation. Resistance to barley yellow mosaic virus disease has been identified in extensive screening programmes and several recessive resistance genes have been mapped, e.g. rym4, rym5, rym9, rym11, rym13. In contrast to barley yellow mosaic virus disease, no complete resistance to BYDV is known in the barley gene pool, but tolerant accessions have been identified and QTL for BYDV-tolerance have been detected on chromosomes 2HL and 3HL. The use of resistance and tolerance in barley breeding can be considerably improved today by molecular markers (RFLPs, RAPDs, AFLPs, SSRs, STSs, SNPs), as they facilitate (i) efficient genotyping and estimation of genetic diversity; (ii) reliable selection on a single plant level independent of symptom expression in the field (iii) acceleration of back crossing procedures; (iv) pyramiding of resistance genes; (v) detection of QTL and marker-based combination of positive alleles; and (vi) isolation of resistance genes via map-based cloning.     

Mechanisms Regulating Insulin Response to Intragastric Glucose in Lean and Non-Diabetic Obese Subjects: A Randomized,Double-Blind,Parallel-Group Trial

Background/Objectives

The changes in blood glucose concentrations that result from an oral glucose challenge are dependent on the rate of gastric emptying, the rate of glucose absorption and the rate of insulin-driven metabolism that include the incretins, glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1). The rate of insulin-driven metabolism is clearly altered in obese subjects, but it is controversial which of these factors is predominant. We aimed to quantify gastric emptying, plasma insulin, C-peptide, glucagon and glucose responses, as well as incretin hormone secretions in obese subjects and healthy controls during increasing glucose loads.

Subjects/Methods

The study was conducted as a randomized, double-blind, parallel-group trial in a hospital research unit. A total of 12 normal weight (6 men and 6 women) and 12 non-diabetic obese (BMI > 30, 6 men and 6 women) participants took part in the study. Subjects received intragastric loads of 10 g, 25 g and 75 g glucose dissolved in 300 ml tap water.

Results

Main outcome measures were plasma GLP-1 and GIP, plasma glucagon, glucose, insulin, C-peptide and gastric emptying. The primary findings are: i) insulin resistance (P < 0.001) and hyperinsulinemia (P < 0.001); ii) decreased insulin disposal (P < 0.001); iii) trend for reduced GLP-1 responses at 75 g glucose; and iv) increased fasting glucagon levels (P < 0.001) in obese subjects.

Conclusions

It seems that, rather than changes in incretin secretion, fasting hyperglucagonemia and consequent hyperglycemia play a role in reduced disposal of insulin, contributing to hyperinsulinemia and insulin resistance.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01875575","term_id":"NCT01875575"}}NCT01875575     

Preclinical Evaluation of a Replication-Deficient Intranasal ΔNS1 H5N1 Influenza Vaccine

Background

We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (ΔNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a “self-adjuvant effect”, in turn increasing vaccine immunogenicity. As a result, ΔNS1 viruses elicit strong B- and T- cell mediated immune responses.

Methodology/Principal Findings

We applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5∶3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log₁₀ TCID_50/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains.

Conclusion/Significance

Our findings show that intranasal immunization with the replication deficient H5N1 ΔNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of ΔNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.     

Requirement of the SecB chaperone for export of a non-secretory polypeptide in Escherichia coli

Summary The SecB protein of Escherichia coli is a cytosolic component of the export machinery which can prevent some precursors from prematurely folding into export-incompatible conformations by binding to the newly synthesised polypeptide. The feature(s) of target proteins recognised by SecB, however, are unclear and have been a matter of controversy. Also, it has not been asked if binding of SecB is specific for secretory proteins. We demonstrate here that a non-secretory polypeptide, a fragment of a tail fiber protein of phage T4, fused to the signal peptide of the outer membrane protein OmpA has a very strong SecB requirement for export and that the signal peptide itself cannot, at least not alone, be responsible for this action of SecB. The data reported, together with those of the literature, suggest that SecB recognizes the polypeptide backbone of the target protein.