Mutational analysis and modeling reveal functionally critical residues in transmembrane segments 1 and 3 of the UapA transporter

Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA.     

Effects of Ce doping on the luminescent property of Ca3SiO4Cl2:Eu phosphor for green lighting

White light‐emitting diodes (LEDs) for green lighting are new solutions for energy saving and environmental protection. Ca₃SiO₄Cl₂:Ce,Eu is an efficient phosphor for white LEDs. Effective energy transfer from Ce³⁺ to Eu²⁺ occurs in Ca₃SiO₄Cl₂:Ce,Eu due to good spectrum overlap between the emission band of Ca₃SiO₄Cl₂:Ce and the excitation band of Ca₃SiO₄Cl₂:Eu, and hues vary systematically from blue to green at different Ce concentrations. A great improvement in the luminescent property of Ca₃SiO₄Cl₂:Eu has been observed on Ce³⁺ doping, which is attributed to energy transfer from Ce³⁺ to Eu²⁺ and an increase in the number of luminescent centers (Eu²⁺) on Ce doping. The optimal sample has a quantum efficiency of up to 75%, and can be an efficient green phosphor for white LEDs. Copyright © 2014 John Wiley & Sons, Ltd.     

Dynamic expression of N‐myc in mouse embryonic development using an enhanced green fluorescent protein reporter gene in the N‐myc locus

N‐myc belongs to the Myc oncogene family and plays an essential role in mammalian embryonic development. The expression of N‐myc is dynamically regulated during embryonic development; however, its expression pattern has not been well characterized due to the lack of a suitable animal model. In this paper, a genetically modified mouse model was generated in which the enhanced green fluorescent protein (EGFP) coding sequence was inserted into the N‐myc locus, so that endogenous N‐myc expression could be traced by the signal of EGFP. The EGFP signal in the transgenic mouse was confirmed to be consistent with the expression pattern of endogenous N‐myc by fluorescence microscopy and immunohistochemical staining. Furthermore, the spatial and temporal expression of EGFP was observed in the central and peripheral nervous system, heart, lung and kidney, given the known indispensable role of N‐myc in their formation. EGFP was also strongly detected in the liver, paranephros and the epithelium of the intestine. The EGFP signal can be used to trace N‐myc expression in this transgenic mouse model. N‐myc expression was observed in specific locations and cell lineages, and dynamically changed during embryonic development. The changing N‐myc expression pattern seen in mouse embryonic development and the animal model described in this paper provide important insights and a new tool to research N‐myc function.     

Oral mucosal lesions in denture wearers

doi:10.1111/j.1741‐2358.2009.00289.x
Oral mucosal lesions in denture wearers Objective: To determine the prevalence of oral mucosal lesions (OMLs) and denture‐related mucosal lesions (DMLs) in denture wearers and to co‐relate the prevalence with age, gender, type of denture and any systemic conditions. Materials and methods: Dental records of 380 denture wearers were retrospectively reviewed for OMLs and DMLs. Results: We found 45% of the denture wearers had DMLs and 60.8% had OMLs not related to denture wearing. Although the prevalence of DMLs was higher in complete denture wearers than in partial denture wearers (49% vs. 42.2%), this difference was not significant. The most common DMLs were traumatic ulcer (19.5%) and denture‐induced stomatitis (18.1%). When analysed by type, traumatic ulcer, denture hyperplasia, frictional keratosis and candidiasis were more common in complete denture wearers, whereas denture‐induced stomatitis was more common in partial denture wearers. Frictional keratosis was more common in men than in women. The prevalence of OMLs not related to denture wearing was higher in complete denture wearers than in partial denture wearers, and the most common OML was fissured tongue (27.6%). No association between DMLs and systemic conditions or xerostomic drugs was noted. Conclusion: No differences in the prevalence of DMLs in association with denture type were found. The prevalence of OMLs not related to denture wearing was higher in complete denture wearers than in partial denture wearers. This difference was affected by age, and the data were similar to the findings observed in the elderly.     

Cotranslational Folding Increases GFP Folding Yield

Protein sequences evolved to fold in cells, including cotranslational folding of nascent polypeptide chains during their synthesis by the ribosome. The vectorial (N- to C-terminal) nature of cotranslational folding constrains the conformations of the nascent polypeptide chain in a manner not experienced by full-length chains diluted out of denaturant. We are still discovering to what extent these constraints affect later, posttranslational folding events. Here we directly address whether conformational constraints imposed by cotranslational folding affect the partitioning between productive folding to the native structure versus aggregation. We isolated polyribosomes from Escherichia coli cells expressing GFP, analyzed the nascent chain length distribution to determine the number of nascent chains that were long enough to fold to the native fluorescent structure, and calculated the folding yield for these nascent chains upon ribosome release versus the folding yield of an equivalent concentration of full-length, chemically denatured GFP polypeptide chains. We find that the yield of native fluorescent GFP is dramatically higher upon ribosome release of nascent chains versus dilution of full-length chains from denaturant. For kinetically trapped native structures such as GFP, folding correctly the first time, immediately after release from the ribosome, can lead to lifelong population of the native structure, as opposed to aggregation.     

Ultrastructural changes at gap junctions between lesioned crayfish axons

Summary In crayfish, the severed distal segment of single lateral giant axon (SLGA) often survives for at least 10 months after lesioning if this segment retains a septal region of apposition with an adjacent, intact SLGA. In control (unsevered) SLGAs, this septal region usually contains gap junctions and 50–60 nm vesicles near the axolemma of both SLGAs. From 1–14 days after lesioning, the distal segment of a severed SLGA undergoes obvious ultrastructural changes in mitochondria and neurotubular organization compared to control SLGAs or to adjacent, intact SLGAs in the same animal. Gap junctions are very difficult to locate in severed SLGAs within 24 h after lesioning. From two weeks to ten months after lesioning, the surviving stumps of severed SLGAs often appear remarkably normal except that structures normally associated with the presence of gap junctions remain very difficult to find.These and other data suggest that SLGA distal segments receive trophic support from adjacent, intact SLGAs. The mechanism of this support probably could not be via diffusion across gap junctions between intact and severed SLGAs since gap junctions largely disappear after lesioning. However, trophic maintenance could occur via the exocytotic — pinocytotic action of 50–60 nm vesicles which are always present on both sides of the septum between an intact SLGA and a severed SLGA distal segment.This work was supported by NIH research grant NS-14412 and and RCDA 00070 to G.D.B.     

How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach

Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.     

Small GTPases in vesicle trafficking

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.