The stromal side of the cytochrome b6f complex regulates state transitions.

In oxygenic photosynthesis, state transitions distribute light energy between Photosystem I and Photosystem II. This regulation involves reduction of the plastoquinone pool, activation of the State Transitions 7 (STT7) protein kinase by the cytochrome b6f complex, and phosphorylation and migration of Light Harvesting Complex II (LHCII). Here, we show that in Chlamydomonas reinhardtii, the C-terminus of the cyt b6 subunit PetB acts on phosphorylation of STT7 and state transitions. We used site-directed mutagenesis of the chloroplast petB gene to truncate (remove L215b6) or elongate (add G216b6) the cyt b6 subunit. Modified complexes are devoid of heme ci and degraded by FTSH protease, revealing that salt bridge formation between cyt b6 (PetB) and subunit IV (PetD) is key to the assembly of the complex. In double mutants where FTSH is inactivated, modified cyt b6f accumulated but the phosphorylation cascade was blocked. We also replaced the arginine interacting with heme ci propionate (R207Kb6). In this modified complex, heme ci is present but the kinetics of phosphorylation are slower. We show that highly phosphorylated forms of STT7 accumulated transiently after reduction of the PQ pool and represent the active forms of the protein kinase. Phosphorylation of the LHCII targets is favored at the expense of the protein kinase, and the migration of LHCII towards PSI is the limiting step for state transitions.

Technical refinements of the scapular tip-free flap for mandibular reconstruction.

BACKGROUND: The use of scapular tip chimeric free flaps (STFFs) for reconstructing mandibular defects has recently become popular, but its utility relative to other bone-containing free flaps remains debatable. The aim of the report is to describe how technical modification of STFF impacted in its use for mandibular reconstruction also commenting results obtained in a unicentric series of patients. PATIENTS AND METHODS: Patients undergoing mandibular reconstruction using an STFF from January 1, 2014 to June 1, 2022 were retrospectively enrolled in this report. We collected data on chimeric flap type, bone management, vascular pedicles, and the final outcomes. In total, 31 patients (13 men and 18 women) with a mean age of 68 years were enrolled. According to the classification system of Urken, 15 patients had body defects, while 7 had ramus defects, another 7 had symphysis defects, and 2 had both ramus and bodily defects. STFF was always harvested working in two equips simultaneously, in supine position. Dissection included preparation of chimeric components of the flap as latissimus dorsi, serratus and scapular tip. After pedicle dissection scapular bone was cut basing on reconstructive needing with a rectangular (stick) shape including the border of the scapula. In cases of longer bone harvesting, circumflex pedicle was also included to perfuse the upper portion of the scapular border. In five cases, the STFF was harvested with only the scapular angle component, and was thus a composite osteomuscular flap; for the remaining 26 cases, a chimeric STFF was used. Circumflex pedicle was included for eight patients. Six of the seven patients with symphyseal defects underwent a single osteotomy. RESULTS: The average length of the harvested was 69.92 mm (maximum length = 104 mm). The average height of transplanted bone was 26.78 mm (maximum height = 44.2 mm). Mouth-opening was normal in 25 patients, limited in 6 patients, and severely impaired in no patients. The cosmetic results were rated as excellent by 20 patients, good by 8 patients, and poor by 3 patients. CONCLUSION: The STFF is an excellent option for mandibular reconstruction when other flaps are not available and for patients in poor general condition. Technical innovations here presented made possible to harvest long bone segments with accurate shape thanks to osteotomies if needed and with adequate soft tissues components of the chimeric flap, ensuring satisfactory functional and cosmetic results.

Inflammation and the Potential Implication of Macrophage-Microglia Polarization in Human ASD: An Overview.

Autism spectrum disorder (ASD) is a heterogeneous collection of neurodevelopmental disorders, difficult to diagnose and currently lacking treatment options. The possibility of finding reliable biomarkers useful for early identification would offer the opportunity to intervene with treatment strategies to improve the life quality of ASD patients. To date, there are many recognized risk factors for the development of ASD, both genetic and non-genetic. Although genetic and epigenetic factors may play a critical role, the extent of their contribution to ASD risk is still under study. On the other hand, non-genetic risk factors include pollution, nutrition, infection, psychological states, and lifestyle, all together known as the exposome, which impacts the mother's and fetus's life, especially during pregnancy. Pathogenic and non-pathogenic maternal immune activation (MIA) and autoimmune diseases can cause various alterations in the fetal environment, also contributing to the etiology of ASD in offspring. Activation of monocytes, macrophages, mast cells and microglia and high production of pro-inflammatory cytokines are indeed the cause of neuroinflammation, and the latter is involved in ASD's onset and development. In this review, we focused on non-genetic risk factors, especially on the connection between inflammation, macrophage polarization and ASD syndrome, MIA, and the involvement of microglia.

Maxillary reconstruction with scapular tip chimeric free flap.

PURPOSE: Purpose of the article is to discuss the use of the scapular tip free flap (STFF) for the reconstruction of maxillary defects. METHODS: A retrospective evaluation of patients who underwent maxillary reconstruction with STFF is presented. Patients were evaluated with respect to complications, function, and cosmesis. RESULTS: Study population consisted of 53 patients. All flaps survived and partial bone resorption only occurred in a young patient. Minor complications included two instances of partial muscular necrosis. The donor site was primarily closed in all patients. Mouth opening was assessed as good (>3 cm) in 41 patients, partially limited (2-3 cm) in 9 patients, and limited (<2 cm) in 3 patients. Dental rehabilitation was achieved in 35 patients; esthetic results were assessed by patient as excellent in 19 patients, good in 28 patients, and poor in 6 patients. CONCLUSIONS: The scapular tip chimeric free flap represents an indispensable tool for reconstructive head and neck microsurgery. The main advantages of this technique are very low donor site morbidity and a long pedicle, as well as the potential for harvesting multiple flaps in a chimeric design; STFF represents the first choice for treatment of small postero-lateral defects of the maxilla, and of wide and complex through-and-through defects involving all components of the midface.

Development and validation of new analytical methods using sea urchin embryo bioassay to evaluate dredged marine sediments.

Management of dredged materials disposal is regulated by several environmental normative requirements, and it is often supported by the integration of chemical data with ecotoxicological characterization. The reliability of a bioassay to assess the potential toxicity of dredged sediments requires the selection of quality criteria that should be based on simple analytical methods and easily understandable hazard for politicians and environmental managers. The sea urchin embryo-toxicity bioassay is considered an essential component for evaluating the quality of sediments in harbour areas but its use, when based exclusively on the observation of normal vs. abnormal embryos, may alter the interpretation of the results, overestimating the risk assessment. To improve the reliability of this assay in establishing a causative relationship between quality of sediments and sea urchin embryonic development, here we developed and validated three Integrative Toxicity Indexes (ITI 2.0, ITI 3.0, ITI 4.0), modifying the already-known ITI (here ITI 1.0). Based on this aim, we used Taranto harbour as a model pilot-study to compare results to those obtained from standard criteria. Among the tested indexes, the ITI 4.0, discriminating strictly developmental delay and morphological defects from fertilized egg to gastrula stage, resulted in the most promising.

A stromal region of cytochrome b6f subunit IV is involved in the activation of the Stt7 kinase in Chlamydomonas.

The cytochrome (cyt) b6f complex and Stt7 kinase regulate the antenna sizes of photosystems I and II through state transitions, which are mediated by a reversible phosphorylation of light harvesting complexes II, depending on the redox state of the plastoquinone pool. When the pool is reduced, the cyt b6f activates the Stt7 kinase through a mechanism that is still poorly understood. After random mutagenesis of the chloroplast petD gene, coding for subunit IV of the cyt b6f complex, and complementation of a ΔpetD host strain by chloroplast transformation, we screened for impaired state transitions in vivo by chlorophyll fluorescence imaging. We show that residues Asn122, Tyr124, and Arg125 in the stromal loop linking helices F and G of cyt b6f subunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cyt b6f and recombinant Stt7 kinase domain show that cyt b6f enhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cyt b6f complex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane.

Long-Lasting Activity of ERK Kinase Depends on NFATc1 Induction and Is Involved in Cell Migration-Fusion in Murine Macrophages RAW264.7.

Macrophages are mononuclear cells that become osteoclasts (OCs) in the presence of two cytokines, macrophage colony-stimulating factor (M-CSF), and receptor activator of NF-κB ligand (RANKL). RANKL binding to its specific receptor RANK leads to OCs differentiation mainly by nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). In our previous study, the analysis of the protein network in NFATc1-knockdown cells, using the Ingenuity Pathway Analysis (IPA), showed a link between NFATc1 and Mitogen-activated protein kinase kinase (MEK)-extracellular receptor kinase (ERK) signaling pathway. Therefore, this study aimed to extend our knowledge of the relationship between NFATc1 and the ERK. Here, we demonstrate that delayed ERK1/2 phosphorylation in pre-OC RANKL-induced depends on NFATc1. Indeed, the knockdown of NFATc1 reduced the phosphorylation of ERK1/2 (60%) and the pharmacological inhibition of the ERK1/2 kinase activity impairs the expression of NFATc1 without preventing its translocation into the nucleus. Furthermore, silencing of NFATc1 significantly reduced RANKL-induced migration (p < 0.01), and most pre-OCs are still mononuclear after 48 h (80 ± 5%), despite the presence of actin rings. On the other hand, the inhibitors FR180204 and PD98059 significantly reduced RANKL-induced cell migration (p < 0.01), leading to a reduction in the number of multinucleated cells. Finally, we suggest that long-lasting ERK activity depends on NFATc1 induction and is likely linked to cell migration, fusion, and OC differentiation.

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b6f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast.

MITF: an evolutionarily conserved transcription factor in the sea urchin Paracentrotus lividus.

Microphthalmia-associated transcription factor (MITF) is a member of MYC superfamily, associated with melanocyte cells, as it was discovered in depigmented mice. However, over the last years it was found to be involved in many cellular signaling pathways, among which oncogenesis, osteoclast differentiation, and stress response. In mammals, Mitf gene mutations can cause diverse syndromes affecting pigmentation of eyes or skin, bone defects and melanomas. As MITF protein homologs were also found in some invertebrates, we have isolated and characterized the MITF cDNAs from the sea urchin Paracentrotus lividus, referred to as Pl-Mitf. The in silico study of the secondary and tertiary structure of Pl-Mitf protein showed high conserved regions mostly lying in the DNA binding domain. To understand the degree of evolutionary conservation of MITF, a phylogenetic analysis was performed comparing the Pl-Mitf deduced protein with proteins from different animal species. Moreover, the analysis of temporal and spatial expression pattern of Pl-Mitf mRNA showed that it was expressed from the onset of gastrulation of the sea urchin embryo to the pluteus larva, specifically in primary mesenchymes cells (PMCs), the sea urchin skeletogenic cells, and in the forming archenteron, the larval gut precursor. In silico protein-protein interactions analysis was used to understand the association of MITF with other proteins. Our results put in evidence the conservation of the MITF protein among vertebrates and invertebrates and may provide new perspectives on the pathways underlying sea urchin development, even if further functional analyses are needed.

Identification of lipid and saccharide constituents of whole microalgal cells by ¹³C solid-state NMR.

Microalgae are unicellular organisms in which plasma membrane is protected by a complex cell wall. The chemical nature of this barrier is important not only for taxonomic identification, but also for interactions with exogenous molecules such as contaminants. In this work, we have studied freshwater (Chlamydomonas reinhardtii) and marine (Pavlova lutheri and Nannochloropsis oculata) microalgae with different cell wall characteristics. C. reinhardtii is covered by a network of fibrils and glycoproteins, while P. lutheri is protected by small cellulose scales, and the picoplankton N. oculata by a rigid cellulose wall. The objective of this work was to determine to what extent the different components of these microorganisms (proteins, carbohydrates, lipids) can be distinguished by ¹³C solid-state NMR with an emphasis on isolating the signature of their cell walls and membrane lipid constituents. By using NMR experiments which select rigid or mobile zones, as well as ¹³C-enriched microalgal cells, we improved the spectral resolution and simplified the highly crowded spectra. Interspecies differences in cell wall constituents, storage sugars and membrane lipid compositions were thus evidenced. Carbohydrates from the cell walls could be distinguished from those incorporated into sugar reserves or glycolipids. Lipids from the plasmalemma and organelle membranes and from storage vacuoles could also be identified. This work establishes a basis for a complete characterization of phytoplankton cells by solid-state NMR.

Carbonic anhydrase inhibition blocks skeletogenesis and echinochrome production in Paracentrotus lividus and Heliocidaris tuberculata embryos and larvae.

Carbonic anhydrases (CAs) are a family of widely distributed metalloenzymes, involved in diverse physiological processes. These enzymes catalyse the reversible conversion of carbon dioxide to protons and bicarbonate. At least 19 genes encoding for CAs have been identified in the sea urchin genome, with one of these localized to the skeletogenic mesoderm (primary mesenchyme cells, PMCs). We investigated the effects of a specific inhibitor of CA, acetazolamide (AZ), on development of two sea urchin species with contrasting investment in skeleton production, Paracentrotus lividus and Heliocidaris tuberculata, to determine the role of CA on PMC differentiation, skeletogenesis and on non-skeletogenic mesodermal (NSM) cells. Embryos were cultured in the presence of AZ from the blastula stage prior to skeleton formation and development to the larval stage was monitored. At the dose of 8 mmol/L AZ, 98% and 90% of P. lividus and H. tuberculata embryos lacked skeleton, respectively. Nevertheless, an almost normal PMC differentiation was indicated by the expression of msp130, a PMC-specific marker. Strikingly, the AZ-treated embryos also lacked the echinochrome pigment produced by the pigment cells, a subpopulation of NSM cells with immune activities within the larva. Conversely, all ectoderm and endoderm derivatives and other subpopulations of mesoderm developed normally. The inhibitory effects of AZ were completely reversed after removal of the inhibitor from the medium. Our data, together with new information concerning the involvement of CA on skeleton formation, provide evidence for the first time of a possible role of the CAs in larval immune pigment cells.

Combined Effects of Cadmium and UVB Radiation on Sea Urchin Embryos: Skeleton Impairment Parallels p38 MAPK Activation and Stress Genes Overexpression.

Human and natural activities release many pollutants in the marine environment. The mixture of pollutants can affect many organisms concurrently. We used Paracentrotus lividus as a model to analyze the effects on signal transduction pathways and stress gene expression in embryos exposed continuously to double stress, i.e., cadmium (Cd) from fertilization and UVB at cleavage (Cd/UVB-embryos). By microscopical inspection, we evaluated embryonic morphology after 72 h of development. Tissue-specific markers were used to assess mesoderm differentiation by immunofluorescence. We analyzed p38MAPK, ERK1/2, and JNK activation by Western blot and mRNA profiles of Pl-MT, Pl-14-3-3epsilon, and Pl-jun genes by real-time quantitative polymerase chain reaction (qPCR) and the localization of their transcripts by whole mount in situ hybridization (WMISH). We found that the Cd/UVB combined exposure induced morphological malformations in 76% of pluteus embryos, mainly affecting the development of the skeleton, including the normal branching of skeletal roads. In Cd/UVB-embryos, p38MAPK was activated 1 h after UVB exposure and a remarkable overexpression of the Pl-MT, Pl-14.3.3epsilon, and Pl-jun genes 24 h after UVB exposure. Pl-MT and Pl-14.3.3epsilon mRNAs were misexpressed as they were localized in a position different from that observed in wild-type embryos, i.e., the intestine. On the contrary, Pl-jun mRNA has remained localized in the skeletogenic cells despite their displacement in exposed embryos. In conclusion, Cd/UVB exposure affected skeletal patterning producing alternative morphologies in which p38MAPK activation and Pl-MT, Pl-14.3.3epsilon, and Pl-jun gene overexpression seem linked to a protective role against the stress response induced by Cd/UVB.

The requirement for carotenoids in the assembly and function of the photosynthetic complexes in Chlamydomonas reinhardtii.

We have investigated the importance of carotenoids on the accumulation and function of the photosynthetic apparatus using a mutant of the green alga Chlamydomonas reinhardtii lacking carotenoids. The FN68 mutant is deficient in phytoene synthase, the first enzyme of the carotenoid biosynthesis pathway, and therefore is unable to synthesize any carotenes and xanthophylls. We find that FN68 is unable to accumulate the light-harvesting complexes associated with both photosystems as well as the RC subunits of photosystem II. The accumulation of the cytochrome b6f complex is also strongly reduced to a level approximately 10% that of the wild type. However, the residual fraction of assembled cytochrome b6f complexes exhibits single-turnover electron transfer kinetics comparable to those observed in the wild-type strain. Surprisingly, photosystem I is assembled to significant levels in the absence of carotenoids in FN68 and possesses functional properties that are very similar to those of the wild-type complex.

Supramolecular assemblies based on complexes of nonionic amphiphilic cyclodextrins and a meso-tetra(4-sulfonatophenyl)porphine tributyltin(IV) derivative: potential nanotherapeutics against melanoma.

Amphiphilic cyclodextrin (ACyD) provides water-soluble and adaptable nanovectors by modulating the balance between the hydrophobic and hydrophilic chains at both CyD sides. This work aimed to design nanoassemblies based on nonionic and hydrophilic ACyD (SC6OH) for the delivery of a poor-water-soluble organotin(IV)-porphyrin derivative [(Bu3Sn)4TPPS] to melanoma cancer cells. To characterize the porphyrin derivatives under simulated physiological conditions, a speciation was performed using complementary techniques. In aqueous solution (≤ 20 µM), (Bu3Sn)4TPPS primarily exists as a monomer (2 in Figure 1), as suggested by the low static anisotropy (ρ ≈ 0.02) with a negligible formation of porphyrin supramolecular aggregates. MALDI-TOF spectra indicate the presence of moieties (i.e., [(Bu3Sn)3TPPS](-)) that are derivatives of the monomeric species. Spectrofluorimetry coupled with potentiometric measurements primarily assesses the presence of the hydrolytic [(Bu3Sn)4TPPS (OH)4](4-) species under physiological conditions. Nanoassemblies of (Bu3Sn)4TPPS/SC6OH were prepared by dispersion of organic films in PBS at pH 7.4 and were investigated using a combination of spectroscopic and morphological techniques. The UV-vis and emission fluorescence spectra of the (Bu3Sn)4TPPS/SC6OH reveal shifts in the peculiar bands of the organotin(IV)-porphyrin derivative due to its interaction with the ACyD supramolecular assemblies in aqueous solution. The mean size was within the range of 100-120 nm. The ξ-potential was negative (-16 mV) for the (Bu3Sn)4TPPS/SC6OH nanoassemblies, with an entrapment efficiency of approximately 67%. The intracellular delivery, cytotoxicity, nuclear morphology and cell growth kinetics were evaluated via fluorescence microscopy on A375 human melanoma cells. The delivery of (Bu3Sn)4TPPS by ACyD with respect to free (Bu3Sn)4TPPS increases the internalization efficiency and cytotoxicity to induce apoptotic cell death and, at lower concentrations, changes the cellular morphology and prevents cell proliferation.

Special Issue on "Insights on Ecotoxicological Effects of Anthropogenic Contaminants in Aquatic Organisms".

In human history, many key points have characterized technological progress, such as the use of metals, which began in prehistoric times and continues to the present day, with many industrial uses [...].

Carbonic anhydrases in development: morphological observations and gene expression profiling in sea urchin embryos exposed to acetazolamide.

Carbonic anhydrases (CANs) are conserved metalloenzymes catalysing the reversible hydration of carbon dioxide into protons and bicarbonate, with important roles in cells physiology. Some CAN-coding genes were found in sea urchin genome, although only one involved in embryonic skeletogenesis was described in Paracentrotus lividus. Here, we investigated gene expression patterns of P. lividus embryos cultured in the presence of acetazolamide (AZ), a CAN inhibitor, to combine morphological defects with their molecular underpinning. CAN inhibition blocked skeletogenesis, affected the spatial/temporal expression of some biomineralization-related genes, inhibited embryos swimming. A comparative analysis on the expression of 127 genes in control and 3 h/24 h AZ-treated embryos, using NanoString technology, showed the differential expression of genes encoding for structural/regulatory proteins, with different embryonic roles: biomineralization, transcriptional regulation, signalling, development and defence response. The study of the differentially expressed genes and the signalling pathways affected, besides in silico analyses and a speculative 'interactomic model', leads to predicting the presence of various CAN isoforms, possibly involved in different physiological processes/activities in sea urchin embryo, and their potential target genes/proteins. Our findings provide new valuable molecular data for further studies in several biological fields: developmental biology (biomineralization, axes patterning), cell differentiation (neural development) and drug toxicology (AZ effects on embryos/tissues).

Optimizing the results of facial animation surgery: Botulinum toxin injection into free functional gracilis flap transfer.

Although neuromuscular gracilis transplantation is the best choice for facial reanimation in patients with congenital or inveterate palsy, the results are not completely satisfactory. Ancillary procedures developed to achieve better symmetry of the smile and reduce the hypercontractility of the transplanted muscle have been reported. However, the intramuscular injection of botulinum toxin has not been described for this purpose. Patients undergoing gracilis injections of botulinum toxin after facial reanimation surgery between September 1, 2020, and June 1, 2022, were retrospectively enrolled in this study. We collected photographs taken before and 20-30 days after injection and compared the symmetry of the face using software. Nine patients with a mean age of 23.56 years (range, 7-56 years) were enrolled. Reinnervation of the muscle was provided by the contralateral healthy facial nerve via a sural cross-graft (four patients), by the ipsilateral masseteric nerve (three cases), and by the contralateral masseteric and facial nerve (two). Using Emotrics software, we identified differences in the commissure excursion discrepancy of 3.82 mm, the smile angle discrepancy of 0.084°, and the dental show discrepancy of 1.49 mm; the average difference in the commissure height deviation was 2.26 mm (P = 0.02), and those in the upper- and lower-lip height deviation were 1.05 mm and 1.49 mm, respectively. Gracilis injection of botulinum toxin after gracilis transplantation is a safe and feasible procedure that could be applicable to all patients with asymmetric smiles related to excessive transplant contraction. It yields good esthetic results with little to no related morbidity.

Analysis of the P. lividus sea urchin genome highlights contrasting trends of genomic and regulatory evolution in deuterostomes.

Sea urchins are emblematic models in developmental biology and display several characteristics that set them apart from other deuterostomes. To uncover the genomic cues that may underlie these specificities, we generated a chromosome-scale genome assembly for the sea urchin Paracentrotus lividus and an extensive gene expression and epigenetic profiles of its embryonic development. We found that, unlike vertebrates, sea urchins retained ancestral chromosomal linkages but underwent very fast intrachromosomal gene order mixing. We identified a burst of gene duplication in the echinoid lineage and showed that some of these expanded genes have been recruited in novel structures (water vascular system, Aristotle's lantern, and skeletogenic micromere lineage). Finally, we identified gene-regulatory modules conserved between sea urchins and chordates. Our results suggest that gene-regulatory networks controlling development can be conserved despite extensive gene order rearrangement.

Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance.

Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.

Phylogenetic analysis and expression patterns of p16 and p19 in Paracentrotus lividus embryos.

P16 and P19 are two small acidic proteins involved in the formation of the biomineralized skeleton of sea urchin embryos and adults. Here, we describe the cloning and the embryonic temporal and spatial expression profiles of p16 and p19 mRNAs, identified for the first time in Paracentrotus lividus. Phylogenetic analysis showed a high degree of similarity of the deduced Pl-P16 and Pl-P19 sequences with the Lytechinus variegatus and Strongylocentrotus purpuratus orthologs. While only a reduced similarity with other phyla, including mammals, was detected, their implication in biomineralized tissues calls for their conservation in evolution. By comparative quantitative PCR and in situ hybridization, we found that Pl-p16 and Pl-p19 expression was restricted to skeletogenic cells throughout embryogenesis, with transcript levels peaking at the late gastrula stage. Dissimilar Pl-p16 and Pl-p19 spatial expression within the primary mesenchyme cell syncytium at the gastrula and pluteus stages suggests the occurrence of a different regulation of gene transcription.