Pigment cell mechanism of postembryonic stripe pattern formation in the Japanese four‐lined snake

Postembryonic changes in the dermal and epidermal pigment cell architecture of the striped and nonstriped morph of the Japanese four‐lined snake Elaphe quadrivirgata were examined to reveal stripe pattern formation after hatching. The striped and nonstriped morphs were distinguishable at the hatching, suggesting that the basis of stripe pattern was formed during embryonic development. In the striped morph, the color of stripes changed from red‐brown in juveniles to vivid dark‐brown in adults, and density of dermal melanophore increased much more in the stripe than background dorsal scales with growth. This increase in density of dermal melanophore was accompanied not only by the increased epidermal melanophore density but also by the change in vertical structures of dermal melanophore. By contrast, the density of epidermal and dermal melanophore evenly increased over the dorsal scales in the nonstriped morph. Thus, the increased vividness of the stripe pattern after hatching is achieved through localized increase of melanophore density particularly in the stripe region but not over the whole dorsal scales. J. Morphol. 277:196–203, 2016. © 2015 Wiley Periodicals, Inc.     

Loss‐of‐function mutations in the APX1 gene result in enhanced selenium tolerance in Arabidopsis thaliana

It is generally recognized that excess selenium (Se) has a negative effect on the growth and development of plants. Numerous studies have identified key genes involved in selenium tolerance in plants; however, our understanding of its molecular mechanisms is far from complete. In this study, we isolated an Arabidopsis selenium‐resistant mutant from the mutant XVE pool lines because of its increased root growth and fresh weight in Se stress, and cloned the gene, which encodes the cytosolic ascorbate peroxidase (APX1). Two other APX1 gene knockout allelic lines were also selenium resistant, and the APX1‐complementary COM1 restored the growth state of wild type under Se stress. In addition, these APX1 allelic lines accumulated more Se than did wild‐type plants when subjected to Se stress. Further analysis revealed that the APX1‐mediated Se tolerance was associated, at least in part, with the enhanced activities of antioxidant enzymes catalase, glutathione peroxidase and glutathione reductase. Moreover, enhanced Se resistance of the mutants was associated with glutathione (GSH), which had the higher expression level of GSH1 gene involved in GSH synthesis and consequently increased GSH content. Our results provide genetic evidence indicating that loss‐of‐function of APX1 results in tolerance to Se stress.     

Identification of two TYRP1 loss‐of‐function alleles in Valais Red sheep

The Valais Red sheep breed is a local breed of the Swiss canton Valais. Although the breed is characterised by its brown colour, black animals occasionally occur and the objective of this study was to identify the causative genetic variants responsible for the obvious difference. A GWAS using high‐density SNP data to compare 51 brown and 38 black sheep showed a strong signal on chromosome 2 at the TYRP1 locus. Haplotype analyses revealed three different brown‐associated alleles. The WGS of three sheep revealed four protein‐changing variants within the TYRP1 gene. Three of these variants were associated with the recessively inherited brown coat colour. This includes the known missense variant TYRP1:c.869G>T designated as b^Soay and two novel loss‐of‐function variants. We propose to designate the frame‐shift variant TYRP1:c.86_87delGA as b^VS1 and the nonsense variant TYRP1:c.1066C>T as b^VS2. Interestingly, the b^VS1 allele occurs only in local breeds of Switzerland whereas the b^VS2 allele seems to be more widespread across Europe.     

Born blonde: a recessive loss‐of‐function mutation in the melanocortin 1 receptor is associated with cream coat coloration in Antarctic fur seals

Although the genetic basis of color variation has been extensively studied in humans and domestic animals, the genetic polymorphisms responsible for different color morphs remain to be elucidated in many wild vertebrate species. For example, hypopigmentation has been observed in numerous marine mammal species but the underlying mutations have not been identified. A particularly compelling candidate gene for explaining color polymorphism is the melanocortin 1 receptor (MC1R), which plays a key role in the regulation of pigment production. We therefore used Antarctic fur seals (Arctocephalus gazella) as a highly tractable marine mammal system with which to test for an association between nucleotide variation at the MC1R and melanin‐based coat color phenotypes. By sequencing 70 wild‐type individuals with dark‐colored coats and 26 hypopigmented individuals with cream‐colored coats, we identified a nonsynonymous mutation that results in the substitution of serine with phenylalanine at an evolutionarily highly conserved structural domain. All of the hypopigmented individuals were homozygous for the allele coding for phenylalanine, consistent with a recessive loss‐of‐function allele. In order to test for cryptic population structure, which can generate artefactual associations, and to evaluate whether homozygosity at the MC1R could be indicative of low genome‐wide heterozygosity, we also genotyped all of the individuals at 50 polymorphic microsatellite loci. We were unable to detect any population structure and also found that wild‐type and hypopigmented individuals did not differ significantly in their standardized multilocus heterozygosity. Such a lack of association implies that hypopigmented individuals are unlikely to suffer disproportionately from inbreeding depression, and hence, we have no reason to believe that they are at a selective disadvantage in the wider population.     

Two novel ANK1 loss‐of‐function mutations in Chinese families with hereditary spherocytosis

Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia disorder. ANK1 mutations account for most HS cases, but pathogenicity analysis and functional research have not been widely performed for these mutations. In this study, in order to confirm diagnosis, gene mutation was screened in two unrelated Chinese families with HS by a next‐generation sequencing (NGS) panel and then confirmed by Sanger sequencing. Two novel heterozygous mutations (c.C841T, p.R281X and c.T290G, p.L97R) of the ANK1 gene were identified in the two families respectively. Then, the pathogenicity of the two new mutations and two previously reported ANK1 mutations (c.C648G, p.Y216X and c.G424T, p.E142X) were studied by in vitro experiments. The four mutations increased the osmotic fragility of cells, reduced the stabilities of ANK1 proteins and prevented the protein from localizing to the plasma membrane and interacting with SPTB and SLC4A1. We classified these four mutations into disease‐causing mutations for HS. Thus, conducting the same mutation test and providing genetic counselling for the two families were meaningful and significant. Moreover, the identification of two novel mutations enriches the ANK1 mutation database, especially in China.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

Loss‐of‐function of OsSTN8 suppresses the photosystem II core protein phosphorylation and interferes with the photosystem II repair mechanism in rice (Oryza sativa)

STN8 kinase is involved in photosystem II (PSII) core protein phosphorylation (PCPP). To examine the role of PCPP in PSII repair during high light (HL) illumination, we characterized a T–DNA insertional knockout mutant of the rice (Oryza sativa) STN8 gene. In this osstn8 mutant, PCPP was significantly suppressed, and the grana were thin and elongated. Upon HL illumination, PSII was strongly inactivated in the mutants, but the D1 protein was degraded more slowly than in wild‐type, and mobilization of the PSII supercomplexes from the grana to the stromal lamellae for repair was also suppressed. In addition, higher accumulation of reactive oxygen species and preferential oxidation of PSII reaction center core proteins in thylakoid membranes were observed in the mutants during HL illumination. Taken together, our current data show that the absence of STN8 is sufficient to abolish PCPP in osstn8 mutants and to produce all of the phenotypes observed in the double mutant of Arabidopsis, indicating the essential role of STN8‐mediated PCPP in PSII repair.     

The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest‐derived melanophores

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest‐derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell‐type‐specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin‐synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores in zebrafish.     

α‐MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV‐induced DNA damage in human melanocytes

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of α‐melanocortin (α‐MSH) that were more potent and stable than the physiological α‐MSH, and mimicked its photoprotective effects against UV‐induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified α‐MSH core His⁶‐d ‐Phe⁷‐Arg⁸, which contained different N‐capping groups, C‐terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C‐terminal modifications. The most effective C‐terminal tripeptide mimicked α‐MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non‐functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.     

Gain‐of‐function mutations cluster in distinct regions associated with the signalling pathway in the PAS domain of the aerotaxis receptor,Aer

The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD‐containing PAS domain. Here, we randomly mutagenized the region encoding residues 14–119 of the PAS domain and found 72 aerotaxis‐defective mutants, 24 of which were gain‐of‐function, signal‐on mutants. The mutations were mapped onto an Aer homology model based on the structure of the PAS–FAD domain in NifL from Azotobacter vinlandii. Signal‐on lesions clustered in the FAD binding pocket, the β‐scaffolding and in the N‐cap loop. We suggest that the signal‐on lesions mimic the ‘signal‐on’ state of the PAS domain, and therefore may be markers for the signal‐in and signal‐out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the β‐scaffolding and the N‐cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS β‐scaffold and residue Q248C in the HAMP AS‐2 helix.     

Negative modulation of the GABAAρ1 receptor function by l‐cysteine

l ‐Cysteine is an endogenous sulfur‐containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson′s and Alzheimer′s disease. l ‐Cysteine can modulate the activity of ionic channels, including voltage‐gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l ‐cysteine on responses mediated by homomeric GABA_Aρ1 receptors, which are known for mediating tonic γ‐aminobutyric acid (GABA) responses in retinal neurons. GABA_Aρ1 receptors were expressed in Xenopus laevis oocytes and GABA‐evoked chloride currents recorded by two‐electrode voltage‐clamp in the presence or absence of l ‐cysteine. l ‐Cysteine antagonized GABA_Aρ1 receptor‐mediated responses; inhibition was dose‐dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration‐response curves for GABA were shifted to the right in the presence of l ‐cysteine without a substantial change in the maximal response. l ‐Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N‐ethyl maleimide. Our results suggest that redox modulation is not involved during l ‐cysteine actions and that l ‐cysteine might be acting as a competitive antagonist of the GABA_Aρ1 receptors.

     

Natural loss‐of‐function mutation of EDR1 conferring resistance to tomato powdery mildew in Arabidopsis thaliana accession C24

To screen for potentially novel types of resistance to tomato powdery mildew Oidium neolycopersici, a disease assay was performed on 123 Arabidopsis thaliana accessions. Forty accessions were fully resistant, and one, C24, was analysed in detail. By quantitative trait locus (QTL) analysis of an F₂ population derived from C24 × Sha (susceptible accession), two QTLs associated with resistance were identified in C24. Fine mapping of QTL‐1 on chromosome 1 delimited the region to an interval of 58 kb encompassing 15 candidate genes. One of these was Enhanced Disease Resistance 1 (EDR1). Evaluation of the previously obtained edr1 mutant of Arabidopsis accession Col‐0, which was identified because of its resistance to powdery mildew Golovinomyces cichoracearum, showed that it also displayed resistance to O. neolycopersici. Sequencing of EDR1 in our C24 germplasm (referred to as C24‐W) revealed two missing nucleotides in the second exon of EDR1 resulting in a premature stop codon. Remarkably, C24 obtained from other laboratories does not contain the EDR1 mutation. To verify the identity of C24‐W, a DNA region containing a single nucleotide polymorphism (SNP) unique to C24 was sequenced showing that C24‐W contains the C24‐specific nucleotide. C24‐W showed enhanced resistance to O. neolycopersici compared with C24 not containing the edr1 mutation. Furthermore, C24‐W displayed a dwarf phenotype, which was not associated with the mutation in EDR1 and was not caused by the differential accumulation of pathogenesis‐related genes. In conclusion, we identified a natural edr1 mutant in the background of C24.     

New insights into the half‐of‐the‐sites reactivity of human aldehyde dehydrogenase 1A1

Aldehyde dehydrogenases (ALDHs) couple the oxidation of aldehydes to the reduction of NAD(P)⁺. These enzymes have gained importance as they have been related to the detoxification of aldehydes generated in several diseases involving oxidative stress. It has been determined that tetrameric ALDHs work only with two of their four active sites (half‐of‐the‐sites reactivity), but the mechanistic reason for this feature remains unknown. In this study, tetrameric human aldehyde dehydrogenase class 1A1 (ALDH1A1) was dimerized to study the correlation of the oligomeric structure with the presence of half‐of‐the‐sites reactivity. Stable dimers from ALDH1A1 were generated by combining the mutation of two residues of the dimer–dimer interface in the tetramer (previously shown to render a low‐active and unstable enzyme) and the fusion of green fluorescent protein (GFP) in the C‐terminus of the mutant. Some kinetic properties of the GFP‐fusion mutant resembled those of human aldehyde dehydrogenase class 3A1, a native dimer, in that the fusion dimer did not show burst in the generation of nicotinamide adenine dinucleotide (NADH) and was less sensitive to the action of specific modulators. The presence of primary isotope effect indicated that the rate‐limiting step changed from NADH release to hydride transfer. The mutant showed higher activity with malondialdehyde and acrolein and was more resistant to inactivation by acrolein compared with the wild type. The mutant kinetic profile showed two hyperbolic components when the substrates were varied, suggesting the presence of two active sites with different affinities and catalytic capacities. In conclusion, the ALDH1A1–GFP dimeric mutant exhibits full site reactivity, suggesting that only the tetrameric structure induces the half‐of‐the‐sites reactivity. Proteins 2013; 81:1330–1339. © 2013 Wiley Periodicals, Inc.     

Anti‐HIV‐1 potency of the CRISPR/Cas9 system insufficient to fully inhibit viral replication

The range of genome‐editing tools has recently been expanded. In particular, an RNA‐guided genome‐editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)‐associated 9 (Cas9) system, has many applications for human diseases. In this study, guide RNA (gRNA) to target gag, pol and a long terminal repeat of HIV‐1 was designed and used to generate gRNA‐expressing lentiviral vectors. An HIV‐1‐specific gRNA and Cas9 were stably dually transduced into a highly HIV‐1‐susceptible human T‐cell line and the inhibitory ability of the anti‐HIV‐1 CRISPR/Cas9 lentiviral vector assessed. Although clear inhibition of the early phase of HIV‐1 infection was observed, as evaluated by a VSV‐G‐pseudotyped HIV‐1 reporter system, the anti‐HIV‐1 potency in multiple rounds of wild type (WT) viral replication was insufficient, either because of generation of resistant viruses or overcoming of the activity of the WT virus. Thus, there are potential difficulties that must be addressed when considering anti‐HIV‐1 treatment with the CRISPR/Cas9 system alone.     

Activation mechanisms of the first sphingosine‐1‐phosphate receptor

Activation of the first sphingosine‐1‐phosphate receptor (S1PR₁) promotes permeability of the blood brain barrier, astrocyte and neuronal protection, and lymphocyte egress from secondary lymphoid tissues. Although an agonist often activates the S1PR₁, the receptor exhibits high levels of basal activity. In this study, we performed long‐timescale molecular dynamics and accelerated molecular dynamics (aMD) simulations to investigate activation mechanisms of the ligand‐free (apo) S1PR₁. In the aMD enhanced sampling simulations, we observed four independent events of activation, which is characterized by close interaction between Y311^7.53 and Y221^5.58 and increased distance between the intracellular ends of transmembrane (TM) helices 3 and 6. Although TM helices TM3, TM6, TM5 and, TM7 are associated with GPCR activation, we discovered that their movements are not necessarily correlated during activation. Instead, TM5 showed a decreased correlation with each of these regions during activation. During activation of the apo receptor, Y221^5.58 and Y311^7.53 became more solvated, because a water channel formed in the intracellular pocket. Additionally, a lipid molecule repeatedly entered the receptor between the extracellular ends of TM1 and TM7, providing important insights into the pathway of ligand entry into the S1PR₁.     

Differential regulation of NMDA receptor function by DJ‐1 and PINK1

Dysfunction of PTEN‐induced kinase 1 (PINK1) or DJ‐1 promotes neuronal death and is implicated in the pathogenesis of Parkinson’s disease, but the underlying mechanisms remain unclear. Given the roles of N‐methyl‐d‐ aspartate receptor (NMDAr)‐mediated neurotoxicity in various brain disorders including cerebral ischemia and neurodegenerative diseases, we investigated the effects of PINK1 and DJ‐1 on NMDAr function. Using protein overexpression and knockdown approaches, we showed that PINK1 increased NMDAr‐mediated whole‐cell currents by enhancing the function of NR2A‐containing NMDAr subtype (NR2ACNR). However, DJ‐1 decreased NMDAr‐mediated currents, which was mediated through the inhibition of both NR2ACNR and NR2B‐containing NMDAr subtype (NR2BCNR). We revealed that the knockdown of DJ‐1 enhanced PTEN expression, which not only potentiated NR2BCNR function but also increased PINK1 expression that led to NR2ACNR potentiation. These results indicate that NMDAr function is differentially regulated by DJ‐1‐dependent signal pathways DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR. Our results further showed that the suppression of DJ‐1, while promoted NMDA‐induced neuronal death through the overactivation of PTEN/NR2BCNR‐dependent cell death pathway, induced a neuroprotective effect to counteract DJ‐1 dysfunction‐mediated neuronal death signaling through activating PTEN/PINK1/NR2ACNR cell survival–promoting pathway. Thus, PINK1 acts with DJ‐1 in a common pathway to regulate NMDAr‐mediated neuronal death. This study suggests that the DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR pathways may represent potential therapeutic targets for the development of neuroprotection strategy in the treatment of brain injuries and neurodegenerative diseases such as Parkinson’s disease.     

Identification and characterization of the zebrafish glutathione S‐transferase Pi‐1

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S‐transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi‐1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3‐month‐old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione‐conjugating activity toward 1‐chloro‐2,4‐dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH‐dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.