Mechanism of activation for Zap-70 catalytic activity.

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a zeta-associated protein of M(r) 70,000 (Zap-70) as a critical protein tyrosine kinase in T-cell activation and development. During T-cell activation, Zap-70 becomes associated via its src homology type 2 (SH2) domains with tyrosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) sequences in the cytoplasmic zeta chain of the T-cell receptor. An intriguing conundrum is how Zap-70 is catalytically activated for downstream phosphorylation events. To address this question, we have used purified Zap-70, tyrosine phosphorylated glutathione S-transferase (GST)-Zeta, and GST-Zeta-1 cytoplasmic domains, and various forms of ITAM-containing peptides to see what effect binding of zeta had upon Zap-70 tyrosine kinase activity. The catalytic activity of Zap-70 with respect to autophosphorylation increased approximately 5-fold in the presence of 125 nM phosphorylated GST-Zeta or GST-Zeta-1 cytoplasmic domain. A 20-fold activity increase was observed for phosphorylation of an exogenous substrate. Both activity increases showed a GST-Zeta concentration dependence. The increase in activity was not produced with nonphosphorylated GST-Zeta, phosphorylated zeta, or phosphorylated ITAM-containing peptides. The increase in Zap-70 activity was SH2 mediated and was inhibited by phenylphosphate, Zap-70 SH2, and an antibody specific for Zap-70 SH2 domains. Since GST-Zeta and GST-Zeta-1 exist as dimers, the data suggest Zap-70 is activated upon binding a dimeric form of phosphorylated zeta and not by peptide fragments containing a single phosphorylated ITAM. Taken together, these data indicate that the catalytic activity of Zap-70 is most likely activated by a trans-phosphorylation mechanism.

Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase.

The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines. To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P. aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker. The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain. It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase. The dodecameric organization of catabolic P. aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

delta-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNA(Glu) mediated pathway. The enzyme glutamyl tRNA(Glu) reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNA(Glu) reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione S-transferase (GST). GST-glutamyl tRNA(Glu) reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNA(Glu) to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol.micrograms-1.min-1. The fusion protein used tRNA(Glu) from barley chloroplasts preferentially to E. coli tRNA(Glu) and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an approximately equal to 60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

Imaging ROMK1 inwardly rectifying ATP-sensitive K+ channel protein using atomic force microscopy.

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1. We have used atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica. ROMK1-GST appears in images as a particle composed of two units of similar size. Analyses of images indicate that the two units have volumes of approximately 118 nm3, which is close to the theoretical volume of a globular protein of approximately 65 kDa (the molecular mass of ROMK1-GST). Native GST exists as a dimer, and the images obtained here are consistent with the ROMK1-GST fusion protein's existence as a heterodimer. In experiments on ROMK1 in aqueous solution, single molecules appear to aggregate, but contact to the mica was maintained. Addition of ATP to the solution produced a change in height of the aggregates. This change (which was reversible) suggests that ATP induces a structural change in the ROMK1 protein. The data show that atomic force microscopy is a useful tool for examination of purified protein molecules under near-physiological conditions, and furthermore, that structural alterations in the proteins may be continuously investigated.

Functional interaction and colocalization of the herpes simplex virus 1 major regulatory protein ICP4 with EAP, a nucleolar-ribosomal protein.

The herpes simplex virus 1 infected cell protein 4 (ICP4) binds to DNA and regulates gene expression both positively and negatively. EAP (Epstein-Barr virus-encoded small nuclear RNA-associated protein) binds to small nonpolyadenylylated nuclear RNAs and is found in nucleoli and in ribosomes, where it is also known as L22. We report that EAP interacts with a domain of ICP4 that is known to bind viral DNA response elements and transcriptional factors. In a gel-shift assay, a glutathione S-transferase (GST)-EAP fusion protein disrupted the binding of ICP4 to its cognate site on DNA in a dose-dependent manner. This effect appeared to be specifically due to EAP binding to ICP4 because (i) GST alone did not alter the binding of ICP4 to DNA, (ii) GST-EAP did not bind to the probe DNA, and (iii) GST-EAP did not influence the binding of the alpha gene trans-inducing factor (alphaTIF or VP16) to its DNA cognate site. Early in infection, ICP4 was dispersed throughout the nucleoplasm, whereas EAP was localized to the nucleoli. Late in infection, EAP was translocated from nucleoli and colocalized with ICP4 in small, dense nuclear structures. The formation of dense structures and the colocalization of EAP and ICP4 did not occur if virus DNA synthesis and late gene expression were prevented by the infection of cells at the nonpermissive temperature with a mutant virus defective in DNA synthesis, or in cells infected and maintained in the presence of phosphonoacetate, which is an inhibitor of viral DNA synthesis. These results suggest that the translocation of EAP from the nucleolus to the nucleoplasm is a viral function and that EAP plays a role in the regulatory functions expressed by ICP4.

Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma.

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.

Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal.

Squid synaptotagmin (Syt) cDNA, including its open reading frame, was cloned and polyclonal antibodies were obtained in rabbits immunized with glutathione S-transferase (GST)-Syt-C2A. Binding assays indicated that the antibody, anti-Syt-C2A, recognized squid Syt and inhibited the Ca(2+)-dependent phospholipid binding to the C2A domain. This antibody, when injected into the preterminal at the squid giant synapse, blocked transmitter release in a manner similar to that previously reported for the presynaptic injection of members of the inositol high-polyphosphate series. The block was not accompanied by any change in the presynaptic action potential or the amplitude or voltage dependence of the presynaptic Ca2+ current. The postsynaptic potential was rather insensitive to repetitive presynaptic stimulation, indicating a direct effect of the antibody on the transmitter release system. Following block of transmitter release, confocal microscopical analysis of the preterminal junction injected with rhodamine-conjugated anti-Syt-C2A demonstrated fluorescent spots at the inner surface of the presynaptic plasmalemma next to the active zones. Structural analysis of the same preparations demonstrated an accumulation of synaptic vesicles corresponding in size and distribution to the fluorescent spots demonstrated confocally. Together with the finding that such antibody prevents Ca2+ binding to a specific receptor in the C2A domain, these results indicate that Ca2+ triggers transmitter release by activating the C2A domain of Syt. We conclude that the C2A domain is directly related to the fusion of synaptic vesicles that results in transmitter release.

Geographical structuring in the mtDNA of Italians.

Geographical patterns of mtDNA variation were studied in 12 Italian samples (1072 individuals) by two different spatial autocorrelation methods. Separate analyses of the frequencies of 12 restriction morphs show North-South clines, differences between Sardinia and the mainland populations, and the effects of isolation by distance. A recently developed autocorrelation statistic summarizing molecular similarity at all sites (AIDA; autocorrelation index for DNA analysis) confirms the presence of a clinical pattern; differences between random pairs of haplotypes tend to increase with their geographical distance. The partition of gene diversity, however, reveals that most variability occurs within populations, whereas differences between populations are minor (GST = 0.057). When the data from the 12 samples are pooled, two descriptors of genetic variability (number of polymorphic sites and average sequence difference between pairs of individuals) do not behave as expected under neutrality. The presence of clinal patterns, Tajima's tests, and a simulation experiment agree in suggesting that population sizes increased rapidly in Italy and Sicily but not necessarily so in Sardinia. The distribution of pairwise sequence differences in the Italian peninsula (excluding Sardinia) permits a tentative location of the demographic increase between 8000 and 20,500 years ago. These dates are consistent with archaeological estimates of two distinct expansion processes, occurring, respectively, in the Neolithic and after the last glacial maximum in the Paleolithic. Conversely, there is no genetic evidence that such processes have had a major impact on the Sardinian population.

Electrophile and antioxidant regulation of enzymes that detoxify carcinogens.

Detoxication (phase 2) enzymes, such as glutathione S-transferases (GSTs), NAD(P)H:(quinone-acceptor) oxidoreductase (QR), and UDP-glucuronsyltransferase, are induced in animal cells exposed to a variety of electrophilic compounds and phenolic antioxidants. Induction protects against the toxic and neoplastic effects of carcinogens and is mediated by activation of upstream electrophile-responsive/antioxidant-responsive elements (EpRE/ARE). The mechanism of activation of these enhancers was analyzed by transient gene expression of growth hormone reporter constructs containing a 41-bp region derived from the mouse GST Ya gene 5'-upstream region that contains the EpRE/ARE element and of constructs in which this element was replaced with either one or two consensus phorbol 12-tetradecanoate 13-acetate (TPA)-responsive elements (TREs). When these three constructs were compared in Hep G2 (human) and Hepa 1c1c7 (murine) hepatoma cells, the wild-type sequence was highly activated by diverse inducers, including tert-butylhydroquinone, Michael reaction acceptors, 1,2-dithiole-3-thione, sulforaphane,2,3-dimercapto-1-propanol, HgCl2, sodium arsenite, and phenylarsine oxide. In contrast, constructs with consensus TRE sites were not induced significantly. TPA in combination with these compounds led to additive or synergistic inductions of the EpRE/ARE construct, but induction of the TRE construct was similar to that induced by TPA alone. Transfection of the EpRE/ARE reporter construct into F9 cells, which lack endogenous TRE-binding proteins, produced large inductions by the same compounds, which also induced QR activity in these cells. We conclude that activation of the EpRE/ARE by electrophile and antioxidant inducers is mediated by EpRE/ARE-specific proteins.

Polymorphic simple sequence repeat regions in chloroplast genomes: applications to the population genetics of pines.

Simple sequence repeats (SSRs), consisting of tandemly repeated multiple copies of mono-, di-, tri-, or tetranucleotide motifs, are ubiquitous in eukaryotic genomes and are frequently used as genetic markers, taking advantage of their length polymorphism. We have examined the polymorphism of such sequences in the chloroplast genomes of plants, by using a PCR-based assay. GenBank searches identified the presence of several (dA)n.(dT)n mononucleotide stretches in chloroplast genomes. A chloroplast (cp) SSR was identified in three pine species (Pinus contorta, Pinus sylvestris, and Pinus thunbergii) 312 bp upstream of the psbA gene. DNA amplification of this repeated region from 11 pine species identified nine length variants. The polymorphic amplified fragments were isolated and the DNA sequence was determined, confirming that the length polymorphism was caused by variation in the length of the repeated region. In the pines, the chloroplast genome is transmitted through pollen and this PCR assay may be used to monitor gene flow in this genus. Analysis of 305 individuals from seven populations of Pinus leucodermis Ant. revealed the presence of four variants with intrapopulational diversities ranging from 0.000 to 0.629 and an average of 0.320. Restriction fragment length polymorphism analysis of cpDNA on the same populations previously failed to detect any variation. Population subdivision based on cpSSR was higher (Gst = 0.22, where Gst is coefficient of gene differentiation) than that revealed in a previous isozyme study (Gst = 0.05). We anticipate that SSR loci within the chloroplast genome should provide a highly informative assay for the analysis of the genetic structure of plant populations.

Molecular cloning and characterization of a conserved nuclear serine(threonine) protein kinase.

Human, Drosophila melanogaster, and Caenorhabditis elegans cDNA clones encoding homologues of a serine(threonine) protein kinase (EC 2.7.1.37) (designated Ndr protein kinase) have been isolated and sequenced. The human and Drosophila cDNAs predict polypeptides of 54 kDa and 52 kDa, respectively, which share approximately 80% amino acid similarity. Northern analysis of human tissues revealed a ubiquitously expressed 3.9-kb transcript. Recombinant GST-Ndr underwent intramolecular autophosphorylation on serine and threonine residues in vitro but failed to transphosphorylate several standard protein kinase substrates. Transfection of the human cDNA into COS-1 cells resulted in the appearance of an intense nuclear staining in cells analyzed by indirect immunofluorescence; deletion mutagenesis identified a short basic peptide, KRKAETWKRNRR, responsible for the nuclear accumulation of Ndr. Thus, Ndr is a conserved and widely expressed nuclear protein kinase. The closest known relative of this previously uncharacterized kinase is Dbf2, a budding yeast protein kinase required for the completion of nuclear division.

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2.

EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general transcription factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted transcription factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

Variação sazonal do metabolismo energético no primeiro ciclo anual de lagartos teiú Tupinambis merianae: correlatos com atividades diárias, adiposidade e proteção antioxidante

Lagartos teiú eclodem no verão e enfrentam o desafio de crescer e armazenar substratos em um curto período de tempo, antes do início do período de jejum e depressão metabólica (≈80%) a temperaturas amenas durante o inverno (≈17 °C). No despertar, o aumento do metabolismo e a reperfusão de órgãos favoreceriam a ocorrência de estresse oxidativo. Na primeira parte do presente estudo investigou−se os ajustes que compatibilizam as demandas em teiús neonatos, especialmente na pré-hibernação, por meio da gravação do comportamento em vídeo e da análise da massa dos corpos gordurosos abdominais e do nível plasmático de corticosterona (CORT) durante o primeiro ciclo anual. No início do outono a massa corpórea dos teiús foi 27 g e o comprimento rostro−cloacal 9,3 cm e aumentaram 40% e 20%, respectivamente, ao longo do outono, enquanto que as taxas diminuíram progressivamente até atingirem o valor zero no início do inverno. Na primavera, a massa corpórea dos teiús aumentou 80% em relação ao despertar e dobrou em relação ao final do verão; o comprimento acumulou um aumento de 27% em relação ao final do verão. A massa relativa dos corpos gordurosos foi 3,7% no início do outono e diminuiu nos meses subsequentes; no despertar, este estoque acumulou uma perda de 63% da sua massa. No início do outono 74% dos teiús estavam ativos por 4,7 h e permaneceram 2 h assoalhando diariamente; ao longo do outono o número de animais ativos e o tempo em atividade diminuíram até que todos se tornaram inativos. Na primavera 83% dos teiús estavam ativos por 7 h e permaneceram 4 h assoalhando. Um padrão sazonal similar foi observado na atividade locomotora e na alimentação. No outono, a alimentação cessou antes da atividade diária e os teiús tornaram−se afágicos algumas semanas antes da entrada em hibernação. Os maiores níveis de CORT foram observados no início do outono, reduzindo progressivamente até valores 75 e 86% menores na dormência e despertar, respectivamente; na primavera os níveis de CORT foram 32% menores em comparação com o início do outono. Este padrão sugere um papel da CORT nos ajustes que promovem a ingestão de alimento e a deposição de substratos energéticos no outono. A redução da atividade geral no final do outono contribuiria para a economia energética e manutenção da massa corpórea, apesar da redução da ingestão de alimento. O curso temporal das alterações fisiológicas e comportamentais em neonatos reforça a ideia de que a dormência sazonal nos teiús é o resultado da expressão de um ritmo endógeno. Na segunda parte do estudo foi investigada a hipótese de que ocorreriam ajustes das defesas antioxidantes durante a hibernação, em antecipação ao despertar. Foram analisados marcadores de estresse oxidativo e antioxidantes em vários órgãos de teiús em diferentes fases do primeiro ciclo anual. A CS, um indicador do potencial oxidante, não variou no fígado e foi menor no rim e no pulmão na hibernação. As enzimas antioxidantes revelaram (1) um efeito abrangente de redução das taxas na hibernação e despertar; por exemplo, GR e CAT foram menores em todos órgãos analisados e a GST tendeu a diminuir no fígado e no rim, embora constante no coração e no pulmão. A G6PDH no fígado e no rim não variou. (2) No fígado, a GST, a Se−GPX e o teor de TBARS foram maiores na atividade de outono em relação à primavera e a Se−GPX permaneceu elevada na hibernação. (3) No fígado, a SOD foi maior na hibernação e despertar em relação ao outono e a Mn−SOD seguiu este padrão. Em contraste, no rim, coração e pulmão a SOD foi menor na hibernação e as taxas se recuperaram no coração e pulmão no despertar. A Mn−SOD seguiu este padrão no pulmão. A concentração e o estado redox da glutationa não variaram no fígado, rim e coração; no pulmão o teor de Eq−GSH e GSH foi menor na hibernação, com tendência à recuperação no despertar. O teor de PC no rim foi maior na hibernação e diminuiu no despertar. No fígado, as alterações no jejum se assemelham às sazonais, como sugerem a inibição da CAT e GR e aumento da Se−GPX. Os efeitos do jejum na primavera no rim diferem dos efeitos sazonais, como sugerem a redução do teor de Eq−GSH e GSH e o aumento da razão GSSG:GSH, a redução da G6PDH e o aumento de PC. No conjunto, houve um efeito predominante de redução das taxas enzimáticas na hibernação e no despertar, exceto pelas taxas aumentadas da SOD e Se−GPX no fígado e pela recuperação da SOD no coração e da GR, SOD e Mn−SOD no pulmão no despertar. As elevadas taxas das enzimas antioxidantes no teiú em comparação a outros ectotermos e a ausência de evidências de estresse oxidativo no despertar sugerem que a atividade enzimática remanescente é suficiente para prevenir danos aos tecidos face às flutuações do metabolismo

Identification of YAP as sperm-PAWP’s predominant binding partner in MII-arrested oocytes and the relevance of this WWI domain modular interaction to oocyte activation

PAWP, a candidate sperm-borne oocyte activating factor, induces oocyte activation and acts upstream of the calcium signalling pathway, however, PAWP’s downstream signalling pathway in oocyte cytoplasm remains to be uncovered. Data from our lab suggested that the interacting partner of PAWP, at least in the frog (Xenopus laevis) model may be YAP, a highly expressed protein in amphibian and mammalian oocytes. Therefore, the objectives of this study were to confirm that PAWP’s predominant binding partner in Xenopus laevis oocyte is YAP; to determine if mammalian oocyte activation is also dependent on PAWP-YAP interaction; and to verify that the PAWP-YAP interaction during oocyte activation is dependent on the WWI domain module. By immunohistochemistry, YAP was localized predominantly in the cytosol of metaphase II-arrested Xenopus laevis oocytes, where presumably the PAWP-YAP interaction occurs. Utilizing Far Western blotting, YAP was identified as the predominant binding partner of PAWP, in metaphase II-arrested frog (Xenopus laevis), swine (Sus scrofa) and mouse (mus musculus) oocytes. The specificity of this interaction was then tested on Far Western blotting of mouse ovarian and oocyte cytosolic extracts, by competition with both wild-type and point-mutated recombinant WWI domains derived from YAP. The removal of GST from the wild-type WWI-GST fusion protein was a requirement for effective blockage of WWI module interaction between PAWP and YAP. As expected, the mutated WWI domain was ineffective in inhibiting the PAWP-YAP interaction. To conclude, this study identified YAP as the predominant binding partner of PAWP in both amphibian and mammalian oocytes, and showed this interaction is dependent on the WWI modular interaction. The results allow us to test the functional relevance of this WWI modular interaction during oocyte activation in vivo, in the future.

Personligt varumärke med Spotify : En kvantitativ studie om identitetsbyggande genom digital musikanvändning.

I denna studie har det framkommit ett genomgående mönster som visar att de personer som framhäver sin musiklyssning på Spotify gör det för att de vill att någon skall se att de har lyssnat på en specifik musik. Dessutom anser dessa personer att musik är jätteviktigt och döljer aktivt sina musiklistor och gör dem privata på Spotify. Däremot finns det personer som inte aktivt gör sina spellistor osynliga och dessa är sådana som inte anser att bilden av en person ändras om de ser att denne lyssnar på oväntad musik. Det framkom även att personer som aktivt döljer vad de lyssnar på gör det då de anser att musiken de ibland lyssnar på är pinsam. Dessa personer anser att musik säger mycket om en person, varför de aktivt döljer sina lyssningar på Spotify. De personer som även döljer sina spellistor gör det på grund av att de följs av andra Spotify-användare. Någonting som var relativt överraskande i denna studie var att Justin Bieber fortfarande ses som någonting pinsamt att lyssna på, vilket även bevittnas i inledningen till denna studie. Vi hade ändå trott att han hade blivit mer socialt accepterad. Dessutom kom vi fram till att de som nyttjar musikanvändningen i syfte att skapa ett personligt varumärke gör det då de anser att musik säger mycket om en person, vilka även ändrar bilden av en person när man får se vad den personen egentligen lyssnar på. Dessutom anser även de som har ändrat sin bild av en annan person på grund av musikval att det finns musik som är pinsam, vilket även är de personer som värderar musik allra högst. De skapar sig ett personligt varumärke genom att dölja och framhäva vad de lyssnar på på Spotify. Sammanfattningsvis har vi kunnat konstatera att det finns en viss skillnad mellan olika typer av människor; de som värdesätter musik högre bryr sig mer om sitt personliga varumärke och sin digitala identitet utåt när det gäller musik, varför de manipulerar sin Spotify-användning mer än den grupp personer som inte värdesätter musik lika mycket. Med dessa resultat kan vi konstatera att de musikintresserade svenska Spotify-användarna (15-35) döljer och framhäver vad de lyssnar på och på så vis använder musikstreamingtjänsten för att lyfta fram sin image och därmed det personliga varumärket.