Role of the gonad cytoplasmic core during oogenesis of the nematode Caenorhabditis elegans

In order to elucidate the function of the cytoplasmic core (or rachis: a structure specific of the nematode gonads), we have carried out a cytological study of this structure in the free-living nematode Caenorhabditis elegans, in wild-type and in several mutant strains showing an abnormal gametogenesis. We also performed an ultrastructural radioautographic study of RNA synthesis during oogenesis in order to examine the part played by the rachis in the transport of nutritive substances. Our results evidence for the first time a metabolite transfer from the germ cells to the cytoplasmic core and lead us to assign to the core a trophic role linked to oogenesis. A statistical analysis of silver grain distribution has led us to conclude that there is no accumulation of RNA labelling in any part of the cytoplasmic core. In addition, our studies performed on sterile mutant strains suggest that the cytoplasmic core may have a specific function in oogenesis determination.     

A distinct translation initiation mechanism generates cryptic peptides for immune surveillance

MHC class I molecules present a comprehensive mixture of peptides on the cell surface for immune surveillance. The peptides represent the intracellular protein milieu produced by translation of endogenous mRNAs. Unexpectedly, the peptides are encoded not only in conventional AUG initiated translational reading frames but also in alternative cryptic reading frames. Here, we analyzed how ribosomes recognize and use cryptic initiation codons in the mRNA. We find that translation initiation complexes assemble at non-AUG codons but differ from canonical AUG initiation in response to specific inhibitors acting within the peptidyl transferase and decoding centers of the ribosome. Thus, cryptic translation at non-AUG start codons can utilize a distinct initiation mechanism which could be differentially regulated to provide peptides for immune surveillance.     

Puromycin oligonucleotides reveal steric restrictions for ribosome entry and multiple modes of translation inhibition

Peptidyl transferase inhibitors have generally been studied using simple systems and remain largely unexamined In in vitro translation extracts. Here, we investigate the potency, product distribution, and mechanism of various puromycin-oligonucleotide conjugates (1 to 44 nt with 3'-puromycin) In a reticulocyte lysate cell-free translation system. Surprisingly, the potency decreases as the chain length of the oligonucleotide is increased in this series, and only very short puromycin conjugates function efficiently (IC50 < 50 microM). This observation stands in contrast with work on isolated large ribosomal subunits, which Indicates that many of the puromycin-oligonucleotide conjugates we studied should have higher affinity for the peptidyl transferase center than puromycin itself. Two tRNA(Al)-derived minihelices containing puromycin provide an exception to the size trend, and are the only constructs longer than 4 nt with any appreciable potency (IC50 = 40-56 microM). However, the puromycin minihelices inhibit translation by sequestering one or more soluble translation factors, and do not appear to participate in detectable peptide bond formation with the nascent chain. In contrast, puromycin and other short derivatives act in a factor-independent fashion at the peptidyl transferase center and readily become conjugated to the nascent protein chain. However, even for the short derivatives, much of the translation inhibition occurs without peptide bond formation between puromycin and the nascent chain, a revision of the classical model for puromycin function. This peptide bond-independent mode is likely a combination of multiple effects including inhibition of initiation and failure to properly recycle translation complexes that have reacted with puromycin.     

Pancreatic Stone Protein Predicts Postoperative Infection in Cardiac Surgery Patients Irrespective of Cardiopulmonary Bypass or Surgical Technique

Introduction

We investigated the role of pancreatic stone protein (PSP) in predicting the occurrence of infection in the postoperative course of cardiac surgery patients. Several biomarkers indicating the presence of inflammation and infection are available in the clinical routine; yet, their utility in the postoperative course of patients following cardiac surgery remains uncertain. Moreover, cardiopulmonary bypass, also referred to as “on-pump surgery”, increases the susceptibility to an exaggerated inflammatory state. However, the impact of such extracorporeal circulation on circulating PSP levels remains poorly understood.

Methods

In a prospective cohort of unselected patients undergoing cardiac surgery, we set out to elucidate the diagnostic accuracy of serum PSP levels as opposed to canonical biomarkers (CRP, WBC) of inflammation to discriminate between the presence of infection and surgical trauma,. In addition, we investigated whether the biomarkers were influenced by the surgical technique employed, i.e. on-pump vs. off-pump and minimally invasive surgery vs. sternotomy. Levels of circulating PSP and routine inflammatory biomarkers (CRP, WBC) were measured in samples taken from 120 patients at baseline as well as at postoperative day 1–3.

Results

Univariate analysis showed that among the biomarkers investigated, only PSP levels had discriminatory power to differentiate infection from surgical trauma in the postoperative course of the entire cohort of patients following cardiac surgery. With regard to cardiac surgical interventions, there was no significant association between the absence or presence of extracorporeal circulation and PSP levels. However, there was a significant difference in the slope of the rise of postoperative PSP between minimally invasive surgery as opposed to patients subjected to sternotomy.

Conclusion

In an unselected population of cardiac surgery patients, post-operative serum PSP levels were significantly associated with the presence of infection in both the on-pump and off-pump setting. Of note, the surgical technique employed (sternotomy vs. minimally invasive approach) had a significant impact on postoperative PSP levels.     

Cytotoxic Aggregation and Amyloid Formation by the Myostatin Precursor Protein

Myostatin, a negative regulator of muscle growth, has been implicated in sporadic inclusion body myositis (sIBM). sIBM is the most common age-related muscle-wastage disease with a pathogenesis similar to that of amyloid disorders such as Alzheimer''s and Parkinson''s diseases. Myostatin precursor protein (MstnPP) has been shown to associate with large molecular weight filamentous inclusions containing the Alzheimer''s amyloid beta peptide in sIBM tissue, and MstnPP is upregulated following ER stress. The mechanism for how MstnPP contributes to disease pathogenesis is unknown. Here, we show for the first time that MstnPP is capable of forming amyloid fibrils in vitro. When MstnPP-containing Escherichia coli inclusion bodies are refolded and purified, a proportion of MstnPP spontaneously misfolds into amyloid-like aggregates as characterised by electron microscopy and binding of the amyloid-specific dye thioflavin T. When subjected to a slightly acidic pH and elevated temperature, the aggregates form straight and unbranched amyloid fibrils 15 nm in diameter and also exhibit higher order amyloid structures. Circular dichroism spectroscopy reveals that the amyloid fibrils are dominated by β-sheet and that their formation occurs via a conformational change that occurs at a physiologically relevant temperature. Importantly, MstnPP aggregates and protofibrils have a negative effect on the viability of myoblasts. These novel results show that the myostatin precursor protein is capable of forming amyloid structures in vitro with implications for a role in sIBM pathogenesis.     

Potent anti-muscarinic activity in a novel series of quinuclidine derivatives

The synthesis and biological evaluation of a novel family of M(3) muscarinic antagonists are described. A systematic modification of the substituents to a novel alkyne-quinuclidine scaffold yielded original compounds displaying potent in vitro anticholinergic properties.