Deletions in the acidic lipid-binding region of the plasma membrane Ca2+ pump. A mutant with high affinity for Ca2+ resembling the acidic lipid-activated enzyme

The C-terminal segment of the loop between transmembrane helices 2 and 3 (A(L) region) of the plasma membrane Ca(2+) pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca(2+) pump isoform h4xb with deletions in the A(L) region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296-349 had full Ca(2+) transport activity, but deletions involving the segment of amino acids 350-356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the V(max) of mutant d296-349 was similar to that of the recombinant wild type pump, but its K(0.5) for Ca(2+) was about 5-fold lower. The addition of calmodulin increased the V(max) and the apparent Ca(2+) affinity of both the wild type and d296-349 enzymes indicating that the activating effects of calmodulin were not affected by the deletion. At low concentrations of Ca(2+) and in the presence of saturating amounts of calmodulin, the addition of phosphatidic acid increased about 2-fold the activity of the recombinant wild type pump. In contrast, under these conditions phosphatidic acid did not significantly change the activity of mutant d296-349. Taken together these results suggest that (a) deletion of residues 296-349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the A(L) region; (b) the A(L) region acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca(2+) affinity of the PMCA to the lipid composition of the membrane; and (c) the function of the A(L) region is independent of the autoinhibition by the C-terminal calmodulin-binding region.     

Charity or empowerment? The role of COVAX for low and middle-income countries

What has the past reaction to the COVID-19 pandemic taught us? We have seen that many low and middle-income countries (LMICs) still lack access to vaccines, and it seems little progress has been made in the last few months and year. This article discusses whether the current strategies, most notably, vaccine donations by the international community and the COVID-19 global access facility COVAX, offer meaningful solutions to tackle the problem. At the centre of our analysis, we compare the concepts of “donations” and “charity” with “vaccine equity” and the “empowerment” of poorer countries. We suggest that the achievement of fair global vaccine production requires that our global approach is supportive of the idea of empowerment. We, therefore, need structural reforms, which would most importantly include capacity building, to positively impact this goal and to take the interests of the global poor seriously.     

TRAF6 regulates proliferation and differentiation of skeletal myoblasts

We could recently demonstrate an important role of receptor interacting protein-2 (RIP2), an activator of nuclear factor kappa B (NF-κB) and a target of activated receptors of the tumor necrosis factor receptor (TNFR) type, in myogenic differentiation and regeneration. Here, we analyze a potential role of TNFR associated factor 6 (TRAF6), which also associates with the cytoplasmic domain of TNFR type, but also IL-1-R and TLR type receptors, and activates NF-κB, in these processes. Specifically, we show that during myogenic differentiation in vitro, traf6 gene expression is downregulated in normal myoblasts, but not in rhabdomyosarcoma cells, suggesting a role of the TRAF6 protein in this process. Inhibition of traf6 expression using specific siRNAs led to an inhibition of both myoblast proliferation and differentiation, whereas inhibition of the TRAF6 effector NF-κB alone in our system only blocked proliferation. Finally, we demonstrate that the traf6 gene is downregulated in skeletal muscle tissue of the dystrophic mdx mouse. Taken together, these data argue for a role of TRAF6 in the regulation of skeletal muscle differentiation and regeneration.     

In vivo studies on putative Shine-Dalgarno sequences of the halophilic archaeon Halobacterium salinarum

The involvement of Shine-Dalgarno sequences in the translation of mRNA in halophilic archaea was investigated for two gvp genes involved in gas vesicle formation in Halobacterium salinarum PHH1. With the exception of gvpA and gvpO, all reading frames of the p-gvpDEFGHIJKLM and p-gvpACNO mRNAs contained upstream of the AUG start codon a putative Shine-Dalgarno (SD) sequence that is complementary to the 3'-end of the small ribosomal subunit RNA. The importance of the SD sequences of gvpG and gvpH was investigated in Haloferax volcanii transformants, and an alteration of the SD sequence resulted in a reduction of the amount of the GvpG or GvpH protein. For a more quantitative analysis the region upstream of gvpH was fused to the bgaH reading frame encoding an enzyme with beta-galactosidase activity as reporter. Scanning mutagenesis within the mRNA leader demonstrated that mutations adjacent to the putative SD sequence GGAGGUCA did not influence the efficiency of translation, whereas constructs harbouring an altered SD sequence yielded only 5-50% of the beta-galactosidase activities obtained with the wild-type SD element. A complete mutation of the SD sequence still yielded 20% of the wild-type activity. Alterations in the spacing of the SD sequence and the translation initiation codon of gvpH indicated that a distance of 4 or 10 nucleotides yielded a similar beta-galactosidase activity as found with the 7 nt spacing of the SD element in wild type, whereas a distance of 1 nt resulted in the loss of translation. A complete deletion of the 5'-UTR resulting in a leaderless mRNA yielded an enhanced beta-galactosidase activity in transformants implying that the initiation of translation involved a mechanism other than a specific mRNA-rRNA interaction.     

The Absoluteness of Semantic Processing: Lessons from the Analysis of Temporal Clusters in Phonemic Verbal Fluency

Background

For word production, we may consciously pursue semantic or phonological search strategies, but it is uncertain whether we can retrieve the different aspects of lexical information independently from each other. We therefore studied the spread of semantic information into words produced under exclusively phonemic task demands.

Methods

42 subjects participated in a letter verbal fluency task, demanding the production of as many s-words as possible in two minutes. Based on curve fittings for the time courses of word production, output spurts (temporal clusters) considered to reflect rapid lexical retrieval based on automatic activation spread, were identified. Semantic and phonemic word relatedness within versus between these clusters was assessed by respective scores (0 meaning no relation, 4 maximum relation).

Results

Subjects produced 27.5 (±9.4) words belonging to 6.7 (±2.4) clusters. Both phonemically and semantically words were more related within clusters than between clusters (phon: 0.33±0.22 vs. 0.19±0.17, p<.01; sem: 0.65±0.29 vs. 0.37±0.29, p<.01). Whereas the extent of phonemic relatedness correlated with high task performance, the contrary was the case for the extent of semantic relatedness.

Conclusion

The results indicate that semantic information spread occurs, even if the consciously pursued word search strategy is purely phonological. This, together with the negative correlation between semantic relatedness and verbal output suits the idea of a semantic default mode of lexical search, acting against rapid task performance in the given scenario of phonemic verbal fluency. The simultaneity of enhanced semantic and phonemic word relatedness within the same temporal cluster boundaries suggests an interaction between content and sound-related information whenever a new semantic field has been opened.     

Distribution, formation and regulation of gas vesicles

A range of bacteria and archaea produce intracellular gas-filled proteinaceous structures that function as flotation devices in order to maintain a suitable depth in the aqueous environment. The wall of these gas vesicles is freely permeable to gas molecules and is composed of a small hydrophobic protein, GvpA, which forms a single-layer wall. In addition, several minor structural, accessory or regulatory proteins are required for gas vesicle formation. In different organisms, 8-14 genes encoding gas vesicle proteins have been identified, and their expression has been shown to be regulated by environmental factors. In this Review, I describe the basic properties of gas vesicles, the genes that encode them and how their production is regulated. I also discuss the function of these vesicles and the initial attempts to exploit them for biotechnological purposes.     

Reduced NODAL signaling strength via mutation of several pathway members including FOXH1 is linked to human heart defects and holoprosencephaly

Abnormalities of embryonic patterning are hypothesized to underlie many common congenital malformations in humans including congenital heart defects (CHDs), left-right disturbances (L-R) or laterality, and holoprosencephaly (HPE). Studies in model organisms suggest that Nodal-like factors provide instructions for key aspects of body axis and germ layer patterning; however, the complex genetics of pathogenic gene variant(s) in humans are poorly understood. Here we report our studies of FOXH1, CFC1, and SMAD2 and summarize our mutational analysis of three additional components in the human NODAL-signaling pathway: NODAL, GDF1, and TDGF1. We identify functionally abnormal gene products throughout the pathway that are clearly associated with CHD, laterality, and HPE. Abnormal gene products are most commonly detected in patients within a narrow spectrum of isolated conotruncal heart defects (minimum 5%-10% of subjects), and far less commonly in isolated laterality or HPE patients (approximately 1% for each). The difference in the mutation incidence between these groups is highly significant. We show that apparent gene dosage discrepancies between humans and model organisms can be reconciled by considering a broader combination of sequence variants. Our studies confirm that (1) the genetic vulnerabilities inferred from model organisms with defects in Nodal signaling are indeed analogous to humans; (2) the molecular analysis of an entire signaling pathway is more complete and robust than that of individual genes and presages future studies by whole-genome analysis; and (3) a functional genomics approach is essential to fully appreciate the complex genetic interactions necessary to produce these effects in humans.     

Calorimetric Study of Cowpea Protein Isolates. Effect of Calcium and High Hydrostatic Pressure

The thermal properties of cowpea protein isolates (CPI) were studied by differential scanning calorimetry under the influence of various conditions. An increase in the pH of protein extraction, from 8.0 to 10.0, during CPI preparation promoted a partial denaturation of cowpea proteins. Increases in enthalpy change of denaturation (ΔH) and temperature of denaturation (Td) were detected with increasing protein concentration from 7.5 to 10.5% (w/w). This behavior suggests that denaturation involves a first step of dissociation of protein aggregates. Calcium induced thermal stabilization in cowpea proteins, the increase in Td was ca. 0.3 °C/mM for protein dispersions of 7.5% (w/w) for 0 to 40 mM CaCl₂. High hydrostatic pressure (HHP) induced denaturation in CPI in a pressure level dependent manner. The presence of calcium protected cowpea proteins towards HHP-induced denaturation when pressure level was 400 MPa, but not when it was 600 MPa. Thermal properties of cowpea protein isolates were very sensitive to processing conditions, these behaviors would have implications in processing of CPI-containing foodstuff.