Self-Assembly of Star Micelle into Vesicle in Solvents of Variable Quality: The Star Micelle Retains Its Core–Shell Nanostructure in the Vesicle

Intra- and intermolecular interactions of star polymers in dilute solutions are of fundamental importance for both theoretical interest and hierarchical self-assembly into functional nanostructures. Here, star micelles with a polystyrene corona and a small ionic core bearing platinum­(II) complexes have been regarded as a model of star polymers to mimic their intra- and interstar interactions and self-assembled behaviors in solvents of weakening quality. In the chloroform/methanol mixture solvents, the star micelles can self-assemble to form vesicles, in which the star micelles shrink significantly and are homogeneously distributed on the vesicle surface. Unlike the morphological evolution of conventional amphiphiles from micellar to vesicular, during which the amphiphilic molecules are commonly reorganized, the star micelles still retain their core–shell nanostructures in the vesicles and the coronal chains of the star micelle between the ionic cores are fully interpenetrated

CAND1 is not required for regulation of the SCF^FWD-1 ubiquitin ligase.

<p>Western blot analyses showing degradation of FRQ (A) and FWD-1 (C) in wild-type and <i>cand1^KO</i> strains after addition of cycloheximide (10 mg/mL). Densitometric analyses from four independent experiments showing the degradation of FRQ (B) and FWD-1 (D). Arrows point out FWD-1 protein bands. Asterisks indicate nonspecific bands detected by FRQ antibody (A) or FWD-1 antibody (C).</p

CSN containing mutant CSN-5 efficiently prevents degradation of substrate receptors of CRLs.

<p>Western blot analyses with labeled antibody showing degradation of Myc-SCON-2 (A), Myc-FBP94 (C), Myc-BTB1 (E), and Myc-DCAF11 (G) after addition of cycloheximide (10 mg/mL) in the wild-type, <i>csn-5^KO</i>, <i>csn-5^KO</i> complementation with CSN-5 and CSN-5H127A strains. Densitometric analyses from four independent experiments showing the degradation of Myc-SCON-2 (B), Myc-FBP94 (D), Myc-BTB1 (F), and Myc-DCAF11 (H).</p

Mutations in the JAMM motif of CSN-5 partially restore SCF-mediated FRQ degradation in the <i>csn-5^KO</i> strain.

<p>(A) Western blot analyses showing degradation of FRQ protein in <i>csn-5</i> mutant and the different CSN-5 complementation strains after addition of cycloheximide (10 mg/mL). Asterisks indicate nonspecific bands detected by FRQ antibody. (B) Densitometric analyses from four independent experiments showing the degradation of FRQ in different strains.</p

CSN-5–mutated CSN efficiently prevents degradation of components of the SCF^FWD-1 complex.

<p>(A–C) Western blot analyses with labeled antibodies showing degradation of Myc-Cul1 (A), Myc-SKP-1 (B), and FWD-1 (C) after addition of cycloheximide (10 mg/mL) in the wild-type, <i>csn-5^KO</i>, and different CSN-5 complementation strains. Arrows point out FWD-1 protein bands. Asterisks indicate nonspecific bands detected by FWD-1 antibody. (D–F) Densitometric analyses from four independent experiments showing the degradation of Myc-Cul1 (D), Myc-SKP-1 (E), and FWD-1 (F).</p

CAND1 is not required for regulation of the circadian rhythm.

<p>(A–C) Normal conidiation rhythms of <i>cand1</i> mutants in dark–dark (A), light–dark (B), and temperature cycles (C) measured by race tube assays. At least four replicates were tested under each condition. Black lines indicate the growth fronts every 24 h.</p

Mutations within the JAMM motif of CSN-5 abolish CSN–mediated deneddylation activity for Cul1, Cul3, and Cul4.

<p>(A) Amino acid alignment of conserved JAMM motifs of CSN-5 homologs from <i>Neurospora crassa</i> (Ncr), <i>Homo sapiens</i> (Hsa), <i>Arabidopsis thaliana</i> (Ath), <i>Drosophila melanogaster</i> (Dme), <i>Caenorhabditis elegans</i> (Cel), and <i>Schizosaccharomyces pombe</i> (Spb). (B) Predicted structure of the <i>N. crassa</i> CSN-5 JAMM domain. The structure was generated by the SWISS-MODEL using the structure of the pre-mRNA splicing factor Prp8 as template (PDB accession number 2P8R), and the functional sites (His127, His129, and Asp140) were mapped according to the structure alignment with the AfJAMM structure (PDB accession number 1R5X). (C–E) Western blot analyses with c-Myc antibody of expression profiles of Myc-Cul1 (C), Myc-Cul3 (D), and Myc-Cul4 (E) in the wild-type strain, <i>csn-5^KO</i>, and CSN-5 complementation strains. (F) Western blot analysis showing the expression of Cul4 in the wild-type, <i>cul4^KO</i>, and <i>csn-5^KO</i> strains. (G) Western blot analyses with c-Myc antibody of expression profiles of Myc-Cul1 in the wild-type strain, <i>csn-5^KO</i>, and <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5 or <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5tri complementation strains. (H) Western blot analyses showing the expression of endogenous Cul4 in the wild-type strain, <i>csn-5^KO</i>, <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5 or <i>csn-5^KO</i>, pcsn-5-Myc-CSN-5tri complementation strains. The asterisk indicates a nonspecific cross-reacted protein band recognized by our Cul4 antiserum (in F and H).</p

Point mutations do not disrupt integrity of the CSN or interactions of the CSN with Cul1 and Cul4.

<p>(A) Immunoprecipitation assays confirming interactions between different versions of Flag-CSN-5 and Myc-CSN-6. Wild-type strain and wild-type strain expressing Myc-CSN-6 were used as negative controls. (B) The Myc-CSN-5, Myc-CSN-5H127A, Myc-CSN-5H129A, or Myc-CSN-5D140N in <i>csn-5^KO</i> properly incorporates into CSN complex. (C) Silver-stained SDS-PAGE showing the two-step purification of Myc-His-CSN-5, Myc-His-CSN-5H127A, Myc-His-CSN-5H129A, or Myc-His-CSN-5D140N in the <i>csn-5^KO</i> strains. A wild-type strain was used as the negative control. CSN subunits identified by mass spectrometry analysis in products of Myc-His-CSN-5 or Myc- His-CSN-5H127A are indicated. Asterisks indicate the two IgG bands. (D) Immunoprecipitation assays confirming the interaction between different versions of Flag-CSN-5 and Myc-Cul1. (E) Immunoprecipitation assays showing the interaction between different versions of Myc-CSN-5 and endogenous Cul4. The asterisk indicates a nonspecific cross-reacted protein band recognized by our Cul4 antiserum.</p

Rescue of growth and developmental defects in the <i>csn-5^KO</i> strain by the expression of JAMM-motif mutant CSN-5.

<p>(A) Wild-type, <i>csn-5^KO</i>, and the different CSN-5 complementation strains growing on slants containing QA. <i>csn-5^KO</i> strains produced significantly less conidia and aerial hyphae than wild-type or CSN-5 complementation strains. (B) Growth rates of the wild-type, <i>csn-5^KO</i>, and CSN-5 complementation strains, measured at 25°C by race tube assays in constant darkness after 1 d of light treatment. (C–E) Rescue of conidiation rhythms in the different CSN-5 complementation strains, measured by race tube assay in dark–dark (C), light–dark (D), and temperature cycles (E). At least four replicates were tested under each condition. Black lines indicate the growth fronts every 24 h.</p

<i>ARMC5</i> mutations in familial and sporadic primary bilateral macronodular adrenal hyperplasia

<div><p>To investigate <i>Armadillo repeat-containing 5</i> (<i>ARMC5</i>) mutations in Chinese patients with familial and sporadic primary bilateral macronodular adrenal hyperplasia (PBMAH), we performed clinical data collection and <i>ARMC5</i> sequencing for three PBMAH families and 23 sporadic PBMAH patients. <i>ARMC5</i> pathogenic germline mutations were identified in all 3 PBMAH families. Secondary <i>ARMC5</i> somatic mutations were found in two adrenal nodules from two PBMAH family members with <i>ARMC5</i> germline mutations. PBMAH family members with <i>ARMC5</i> pathogenic germline mutations displayed various clinical manifestations. <i>ARMC5</i> pathogenic germline mutations were identified in 5 sporadic PBMAH patients among whom one patient displayed both hypercortisolism and primary aldosteronism. We detected a total of 10 <i>ARMC5</i> pathogenic mutations, of which 8 had not been previously reported. Our results suggest that <i>ARMC5</i> pathogenic germline mutations are common in familial and sporadic Chinese PBMAH patients, and demonstrate the importance of <i>ARMC5</i> screening in PBMAH family members to detect patients with insidious PBMAH.</p></div