Using green fluorescent protein to understand the mechanisms of G-protein-coupled receptor regulation

G protein-coupled receptor (GPCR) activation is followed rapidly by adaptive changes that serve to diminish the responsiveness of a cell to further stimulation. This process, termed desensitization, is the consequence of receptor phosphorylation, arrestin binding, sequestration and down-regulation. GPCR phosphorylation is initiated within seconds to minutes of receptor activation and is mediated by both second messenger-dependent protein kinases and receptor-specific G protein-coupled receptor kinases (GRKs). Desensitization in response to GRK-mediated phosphorylation involves the binding of arrestin proteins that serve to sterically uncouple the receptor from its G protein. GPCR sequestration, the endocytosis of receptors to endosomes, not only contributes to the temporal desensitization of GPCRs, but plays a critical role in GPCR resensitization. GPCR down-regulation, a loss of the total cellular complement of receptors, is the consequence of both increased lysosomal degradation and decreased mRNA synthesis of GPCRs. While each of these agonist-mediated desensitization processes are initiated within a temporally dissociable time frame, recent data suggest that they are intimately related to one another. The use of green fluorescent protein from the jellyfish Aqueora victoria as an epitope tag with intrinsic fluorescence has facilitated our understanding of the relative relationship between GRK phosphorylation, arrestin binding, receptor sequestration and down-regulation.

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide

We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins.

Auringon asemaa seuraavan aurinkopaneelijärjestelmän kehittäminen

Tässä diplomityössä käsitellään Aurinkoa seuraavan aurinkopaneelijärjestelmän kehittämistä Suomen olosuhteisiin. Työ on tehty osana Lappeenrannan teknillisen yliopiston Green Campus-projektia, jossa tarkoituksena on tuottaa yliopistolle energiaa uusiutuvilla menetelmillä ja käyttää niitä apuna tutkimuksessa sekä opetuksessa. Tavoitteena työssä on ymmärtää Auringon seuraamisen hyödyt sekä mahdolliset haitat aurinkopaneeli sovellutuksissa. Aikaisemman tutkimustiedon ollessa vähäistä, on työssä pyritty löytämään laskentamalli tuottavuuden laskentaan riippumatta siitä, missä päin maapalloa aurinkopaneelijärjestelmä sijaitsee. Työ alkaa kirjallisuustutkimuksella, jossa käydään läpi aurinkopaneelien toimintaperiaate, aurinkoenergian ja auringonpaistetuntien suuruusluokat Suomessa, sekä Suomen sääoloista johtuvat vaatimukset aurinkopaneelijärjestelmille. Tämän jälkeen on vertailtu kaupallisia järjestelmiä. Lopuksi järjestelmällistä tuotesuunnittelua hyväksikäyttäen suunnitellaan oma versio Aurinkoa seuraavasta aurinkopaneelijärjestelmästä. Oman suunnitelman järkevyyttä simuloidaan pienoismallilla Matlab-Arduino ympäristössä ja pyritään löytämään mahdollisia heikkouksia. Suomessa aurinkoenergiasta 90 % saadaan maalis – syyskuun aikana. Nykyisillä akkujärjestelmillä aurinkoenergia Suomessa ei sovellu kuin täydentäväksi energianlähteeksi. Aurinkoa seuraamalla voidaan saavuttaa 25-30 % tuottavuuden lisäys kesäaikana verrattuna staattiseen järjestelmään. Talvella hyöty tippuu 0-10 % luokkaan. Pienoismallilla simuloidut ohjaustavat osoittivat, että Aurinkoa on mahdollista seurata ilman sensoreita laskemalla Auringon paikka tähtitieteen kaavoista.

Nucleotide-sugar transporters: structure, function and roles in vivo

The glycosylation of glycoconjugates and the biosynthesis of polysaccharides depend on nucleotide-sugars which are the substrates for glycosyltransferases. A large proportion of these enzymes are located within the lumen of the Golgi apparatus as well as the endoplasmic reticulum, while many of the nucleotide-sugars are synthesized in the cytosol. Thus, nucleotide-sugars are translocated from the cytosol to the lumen of the Golgi apparatus and endoplasmic reticulum by multiple spanning domain proteins known as nucleotide-sugar transporters (NSTs). These proteins were first identified biochemically and some of them were cloned by complementation of mutants. Genome and expressed sequence tag sequencing allowed the identification of a number of sequences that may encode for NSTs in different organisms. The functional characterization of some of these genes has shown that some of them can be highly specific in their substrate specificity while others can utilize up to three different nucleotide-sugars containing the same nucleotide. Mutations in genes encoding for NSTs can lead to changes in development in Drosophila melanogaster or Caenorhabditis elegans, as well as alterations in the infectivity of Leishmania donovani. In humans, the mutation of a GDP-fucose transporter is responsible for an impaired immune response as well as retarded growth. These results suggest that, even though there appear to be a fair number of genes encoding for NSTs, they are not functionally redundant and seem to play specific roles in glycosylation.

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

Mouse PNAS-4 (mPNAS-4) has 96% identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Association of postalimentary lipemia with atherosclerotic manifestations

We identified different lipemic and metabolic responses after the ingestion of a standardized meal by healthy adults and related them to atherosclerotic markers. Samples from 60 normolipidemic adults were collected before and after a liquid meal (40 g fat/m² body surface) at 0, 2, 4, 6, and 8 h for measurements of lipids, free fatty acids (FFA), insulin, cholesteryl ester transfer protein (CETP), autoantibodies to epitopes of oxidized LDL (oxLDL Ab), lipolytic activities, and apolipoprotein E polymorphism. Mean carotid intima-media thickness (cIMT) was determined by Doppler ultrasound. The volunteers were classified into early (N = 39) and late (N = 31) triacylglycerol (TAG) responders to the test meal. Late responders showed lower HDL cholesterol concentration at fasting and in the TAG peak, lower insulin and higher FFA concentrations compared to early responders. Multivariate regression analyses showed that mean cIMT was associated with gender (male) and age in early responders and by cholesterol levels at the 6th hour in late responders. oxLDL Ab were explained by lipoprotein lipase and negatively by hepatic lipase and oxLDL Ab (fasting period) by CETP (negative) and FFA (positive). This study is the first to identify a postalimentary insulin resistance state, combined with a reduced CETP response exclusively among late responders, and the identification of the regulators of postalimentary atherogenicity. Further research is required to determine the metabolic mechanisms described in the different postalimentary phenotypes observed in this study, as well as in different pathological states, as currently investigated in our laboratory.

Obtenção de ácidos graxos por cristalização do óleo de pescado fracionado por hidrólise enzimática

Este trabalho teve por objetivo estabelecer um processo para obter e caracterizar os produtos da cristalização das frações do óleo de pescado hidrolisado com lipase pancreática. A hidrólise enzimática foi realizada em 60 minutos com uma concentração de substrato de 1262 mmols, 38 °C, pH 8 e com uma concentração de extrato enzimático de 7,647 mg.mL-1. Os produtos da hidrólise foram separados por cromatografia em coluna e caracterizados por cromatografia em camada delgada e cromatografia em fase gasosa. A temperatura de cristalização foi controlada entre 5 °C e -18 °C. A fração TAG foi melhor cristalizada a 0 °C enquanto que as frações DAG + AGL e MAG foram cristalizadas a 5 °C. Foi observado que o ácido docosahexaenoico (DHA) concentra-se na fase líquida da fração DAG + AGL representando 97,17% em relação ao óleo de pescado. O ácido palmítico foi cristalizado na fração DAG, perfazendo aproximadamente 50% do total de ácidos graxos. Foi observado que os ácidos graxos insaturados de cadeia longa, principalmente o EPA, concentram-se na fase sólida da fração TAG, esses resultados sugerem que o processo combinado de hidrólise enzimática seguido de cristalização possibilita a obtenção destes ácidos graxos do óleo de pescado.

Characterisation of Cladonia rangiferina transcriptome and genome, and the effects of dehydration and rehydration on its gene expression

Lichens are symbiotic organisms, which consist of the fungal partner and the photosynthetic partner, which can be either an alga or a cyanobacterium. In some lichen species the symbiosis is tripartite, where the relationship includes both an alga and a cyanobacterium alongside the primary symbiont, fungus. The lichen symbiosis is an evolutionarily old adaptation to life on land and many extant fungal species have evolved from lichenised ancestors. Lichens inhabit a wide range of habitats and are capable of living in harsh environments and on nutrient poor substrates, such as bare rocks, often enduring frequent cycles of drying and wetting. Most lichen species are desiccation tolerant, and they can survive long periods of dehydration, but can rapidly resume photosynthesis upon rehydration. The molecular mechanisms behind lichen desiccation tolerance are still largely uncharacterised and little information is available for any lichen species at the genomic or transcriptomic level. The emergence of the high-throughput next generation sequencing (NGS) technologies and the subsequent decrease in the cost of sequencing new genomes and transcriptomes has enabled non-model organism research on the whole genome level. In this doctoral work the transcriptome and genome of the grey reindeer lichen, Cladonia rangiferina, were sequenced, de novo assembled and characterised using NGS and traditional expressed sequence tag (EST) technologies. RNA extraction methods were optimised to improve the yield and quality of RNA extracted from lichen tissue. The effects of rehydration and desiccation on C. rangiferina gene expression on whole transcriptome level were studied and the most differentially expressed genes were identified. The secondary metabolites present in C. rangiferina decreased the quality – integrity, optical characteristics and utility for sensitive molecular biological applications – of the extracted RNA requiring an optimised RNA extraction method for isolating sufficient quantities of high-quality RNA from lichen tissue in a time- and cost-efficient manner. The de novo assembly of the transcriptome of C. rangiferina was used to produce a set of contiguous unigene sequences that were used to investigate the biological functions and pathways active in a hydrated lichen thallus. The de novo assembly of the genome yielded an assembly containing mostly genes derived from the fungal partner. The assembly was of sufficient quality, in size similar to other lichen-forming fungal genomes and included most of the core eukaryotic genes. Differences in gene expression were detected in all studied stages of desiccation and rehydration, but the largest changes occurred during the early stages of rehydration. The most differentially expressed genes did not have any annotations, making them potentially lichen-specific genes, but several genes known to participate in environmental stress tolerance in other organisms were also identified as differentially expressed.

Modulaarinen arkkitehtuuri tuoteperheen kehittämisessä

Modulaarisen tuotteen kehittäminen tehostaa yrityksen kilpailukykyä ja helpottaa asiakkaiden tarpeiden tyydyttämistä. Tuoteperheen edut verrattaessa massatuotantoon ovat laajempi tuotevalikoima sekä parempi tuotettavuus massaräätälöintiin verrattuna. Eri tuotevaihtoehtoja ja moduuleja on mahdollista kehittää rinnakkain. Moduloitava tuoteperhe helpottaa yrityksen eri vaiheita aina tuotteen suunnittelusta huoltotoimenpiteisiin ja lopulta tuotteen purkamiseen. Asiakkaille tärkeitä hyötyjä moduloinnin osalta ovat tuotteiden parempi laatu ja huollettavuus. Täysin uuden modulaarisen tuoteperheen kehittäminen vaatii runsaasti resursseja suunnitteluosastolla. Modulaarisessa tuotteessa suunnittelutyö voidaan kohdistaa vain tietyn moduulin kohdalle ja suunnitteluaikoja saadaan täten lyhennettyä. Tässä kandidaatintyössä tutkittiin, miten hitsausautomaatiosovelluksissa modulaarisuus on toteutettu sekä pohditaan kehityskohteita, koska hitsausautomaatiosovelluksia tuotetaan runsaasti asiakasräätälöintinä. Tarkasteltavana tuoteperheenä oli robottihitsausportaalit ja hitsaustornit.

Jätteiden kemiallisten ominaisuuksien spektrometriset online-mittausmenetelmät

Tässä diplomityössä käsiteltiin spektrometrisia online-mittausmenetelmiä jätteiden kemiallisten ja fysikaalisten ominaisuuksien määrittämiseksi. Tavoitteena oli selvittää, mitä ominaisuuksia menetelmillä voidaan mitata ja kuinka luotettavia tuloksia mittauksilla saadaan. Diplomityössä suoritettiin kirjallisuuskatsaus, jossa käsiteltiin kolmen spektrometrisen menetelmän soveltuvuutta reaaliaikaisiin jätemittauksiin. Työn empiirisessä osassa FPXRFanalysaattorilla mitattiin neljän eri jätenäytteen alkuainepitoisuuksia. Mittauksen tarkoituksena oli selvittää, mitä alkuaineita menetelmällä voidaan mitata. FPXRF-analysaattorilla saatuja tuloksia verrattiin ICP-MS-menetelmällä saatuihin tuloksiin regressioanalyysin avulla. Työssä todettiin, että FPXRF-analysaattori sopii parhaiten kaliumin, kalsiumin, ja raudan pitoisuuksien määrittämiseen. Lisäksi lyijyn, sinkin, kromin, kloorin, kuparin, kadmiumin, arseenin, fosforin, molybdeenin ja vanadiinin määrittäminen on mahdollista, mutta tarkan pitoisuuden saamiseksi laboratoriomenetelmien käyttö voi olla tarpeen. Tutkituista jätenäytteistä menetelmä soveltui parhaiten tuhkalle ja kompostille niiden fyysisten ominaisuuksien, kuten homogeenisuuden ja kosteuspitoisuuden takia. Biojätteelle menetelmä soveltui huonosti. FPXRF-analysaattorin luotettavuuteen vaikuttaa näytteen kosteuspitoisuus, homogeenisuus, partikkelikoko, mittaustapa ja laitteen kalibrointi. Työssä tarkastelluilla menetelmillä ei voida tällä hetkellä täysin korvata laboratorioanalyyseja. FPXRF-analysaattoria voidaan kuitenkin käyttää kvalitatiiviseen tai semikvantitatiiviseen haitta-aineiden analysointiin, millä voidaan vähentää kalliiden laboratorioanalyysien tarvetta.

Asiakasohjautuvan hitsaustuotannon kehittäminen PK-konepajassa

Tässä diplomityössä tarkasteltiin asiakasohjautuvan hitsaustuotannon kehittämistä PK-konepajassa. Työn tarkoituksena oli kerätä taustatietoa asiakasohjautuvan hitsaustuotannon erityispiirteistä ja tarkastella hitsaustuotannon kehittämistä. Työssä tutustuttiin hitsausprosesseihin ja niiden kehitysversioihin sekä hitsausaineisiin. Tärkeänä osa-alueena olivat hitsin ja hitsaustuotannon laadun ja laadunhallinnan sekä hitsauksen mekanisoinnin ja automatisoinnin tarkastelu hitsauksen työsuojelua ja hitsausergonomiaa unohtamatta. Tämän jälkeen kartoitettiin yrityksen nykytilanne ja laadittiin kehittämistoimenpiteet sekä tarvittavat laskelmat ja kehitystyön implementointi. Sen jälkeen vertailtiin tehostettua toimintaa vanhaan toimintaan sekä tehtiin johtopäätökset toiminnan kehittämisestä.