Fluorescence-based imaging of cellular defect in lysinuric protein intolerance (LPI)

Fluoresenssiperusteiset kuvantamismenetelmät lysinurisen proteiini-intoleranssin (LPI) soluhäiriön tutkimuksessa Lysinurinen proteiini-intoleranssi on suomalaiseen tautiperintöön kuuluva autosomaalisesti peit¬tyvästi periytyvä sairaus, jonka aiheuttaa kationisten aminohappojen kuljetushäiriö munuaisten ja ohutsuolen epiteelisolujen basolateraalikalvolla. Aminohappojen kuljetushäiriö johtaa moniin oirei¬siin, kuten kasvuhäiriöön, osteoporoosiin, immuunijärjestelmän häiriöihin, oksenteluun ja runsaspro¬teiinisen ravinnon nauttimisen jälkeiseen hyperammonemiaan. LPI-geeni SLC7A7 (solute carrier family 7 member 7) koodaa y+LAT1 proteiinia, joka on basolateraali¬nen kationisten ja neutraalien aminohappojen kuljettimen kevyt ketju, joka muodostaa heterodimee¬rin raskaan alayksikön 4F2hc:n kanssa. Tällä hetkellä SLC7A7-geenistä tunnetaan yli 50 LPI:n aiheut¬tavaa mutaatiota. Tässä tutkimuksessa erityyppisiä y+LAT1:n LPI-mutaatiota sekä yhdeksän C-terminaalista polypep¬tidiä lyhentävää deleetiota kuvannettiin nisäkässoluissa y+LAT1:n GFP (green fluorescent protein) -fuusioproteiineina. Tulokset vahvistivat muissa soluissa tehdyt havainnot siitä, että 4F2hc on edel¬lytyksenä y+LAT1:n solukalvokuljetukselle, G54V-pistemutantti sijaitsee solukalvolla samoin kuin vil¬lityyppinen proteiini, mutta lukukehystä muuttavia ja proteiinia lyhentäviä mutantteja ei kuljeteta solukalvoon. Lisäksi havaittiin, että poikkeuksena tästä säännöstä ovat y+LAT1-deleetioproteiinit, joista puuttui korkeintaan 50 C-terminaalista aminohappoa. Nämä lyhentyneet kuljettimet sijaitsevat solukalvolla kuten villityyppiset ja LPI-pistemutanttiproteiinit. Dimerisaation osuutta kuljetushäiriön synnyssä tutkittiin käyttämällä fluorescence resonance energy transfer (FRET) menetelmää. Heterodimeerin alayksiköistä kloonattiin ECFP (cyan) ja EYFP (yellow) fuusioproteiinit, joita ilmennettiin nisäkässoluissa, ja FRET mitattiin virtaussytometri-FRET -menetel¬mällä (FACS-FRET). Tutkimuksissa kaikkien mutanttien havaittiin dimerisoituvan yhtä tehokkaasti. Kul¬jetushäiriön syynä ei siten ole alayksiköiden dimerisaation estyminen mutaation seurauksena. Tutkimuksessa havaittiin, että kaikki mutantti-y+LAT1-transfektiot tuottavat vähemmän transfektoi¬tuneita soluja kuin villityyppisen y+LAT1:n transfektiot. Solupopulaatioissa, joihin oli tranfektoitu lu¬kukehystä muuttava tai stop-kodonin tuottava mutaatio havaittiin suurempi kuolleisuus kuin saman näytteen transfektoitumattomissa soluissa, kun taas villityyppistä tai G54V-pistemutanttia tuottavas¬sa solupopulaatiossa oli pienempi kuolleisuus kuin saman näytteen fuusioproteiinia ilmentämättö¬missä soluissa. Tulos osoittaa mutanttiproteiinien erilaiset vaikutukset niitä ilmentäviin soluihin, joko suoraan y+LAT1:n tai 4F2hc:n kautta aiheutuneina. LPIFin SLC7A7 lähetti-RNA:n määrä ei merkittävästi poikennut villityyppisen määrästä fibroblasteissa ja lymfoblasteissa. SLC7A7:n promoottorianalyysissä oli osoitettavissa säätelyalueita geenin 5’ ei-koo¬daavalla alueella sekä ensimmäisten kahden intronin alueella. LPI-taudin tautimekanismin kannalta keskeisin tekijä on kuitenkin aminohappokuljetuksen häiriö, jonka vaikutuksesta näistä aminohapoista riippuvaiset prosessit elimistössä eivät toimi normaalisti. Havaittu virheellinen y+LAT1/4F2hc kuljetuskompleksin sijainti edellyttää lisätutkimuksia sen mahdol¬lisen kliinisen merkityksen selvittämiseksi.

Improvements in the Assessment of Bacterial Viability and Killing

It is axiomatic that our planet is extensively inhabited by diverse micro-organisms such as bacteria, yet the absolute diversity of different bacterial species is widely held to be unknown. Different bacteria can be found from the depths of the oceans to the top of the mountains; even the air is more or less colonized by bacteria. Most bacteria are either harmless or even advantageous to human beings but there are also bacteria, which can cause severe infectious diseases or spoil the supplies intended for human consumption. Therefore, it is vitally important not only to be able to detect and enumerate bacteria but also to assess their viability and possible harmfulness. Whilst the growth of bacteria is remarkably fast under optimum conditions and easy to detect by cultural methods, most bacteria are believed to lie in stationary phase of growth in which the actual growth is ceased and thus bacteria may simply be undetectable by cultural techniques. Additionally, several injurious factors such as low and high temperature or deficiency of nutrients can turn bacteria into a viable but non-culturable state (VBNC) that cannot be detected by cultural methods. Thereby, various noncultural techniques developed for the assessment of bacterial viability and killing have widely been exploited in modern microbiology. However, only a few methods are suitable for kinetic measurements, which enable the real-time detection of bacterial growth and viability. The present study describes alternative methods for measuring bacterial viability and killing as well as detecting the effects of various antimicrobial agents on bacteria on a real-time basis. The suitability of bacterial (lux) and beetle (luc) luciferases as well as green fluorescent protein (GFP) to act as a marker of bacterial viability and cell growth was tested. In particular, a multiparameter microplate assay based on GFP-luciferase combination as well as a flow cytometric measurement based on GFP-PI combination were developed to perform divergent viability analyses. The results obtained suggest that the antimicrobial activities of various drugs against bacteria could be successfully measured using both of these methods. Specifically, the data reliability of flow cytometric viability analysis was notably improved as GFP was utilized in the assay. A fluoro-luminometric microplate assay enabled kinetic measurements, which significantly improved and accelerated the assessment of bacterial viability compared to more conventional viability assays such as plate counting. Moreover, the multiparameter assay made simultaneous detection of GFP fluorescence and luciferase bioluminescence possible and provided extensive information about multiple cellular parameters in single assay, thereby increasing the accuracy of the assessment of the kinetics of antimicrobial activities on target bacteria.

Inactive endosialidase-based detection of bacterial and oncofetal polysialic acid

Polysialic acid is a carbohydrate polymer which consist of N-acetylneuraminic acid units joined by alpha2,8-linkages. It is developmentally regulated and has an important role during normal neuronal development. In adults, it participates in complex neurological processes, such as memory, neural plasticity, tumor cell growth and metastasis. Polysialic acid also constitutes the capsule of some meningitis and sepsis-causing bacteria, such as Escherichia coli K1, group B meningococci, Mannheimia haemolytica A2 and Moraxella nonliquefaciens. Polysialic acid is poorly immunogenic; therefore high affinity antibodies against it are difficult to prepare, thus specific and fast detection methods are needed. Endosialidase is an enzyme derived from the E. coli K1 bacteriophage, which specifically recognizes and degrades polysialic acid. In this study, a novel detection method for polysialic acid was developed based on a fusion protein of inactive endosialidase and the green fluorescent protein. It utilizes the ability of the mutant, inactive endosialidase to bind but not cleave polysialic acid. Sequencing of the endosialidase gene revealed that amino acid substitutions near the active site of the enzyme differentiate the active and inactive forms of the enzyme. The fusion protein was applied for the detection of polysialic acid in bacteria and neuroblastoma. The results indicate that the fusion protein is a fast, sensitive and specific reagent for the detection of polysialic acid. The use of an inactive enzyme as a specific molecular tool for the detection of its substrate represents an approach which could potentially find wide applicability in the specific detection of diverse macromolecules.

Luminescent Lanthanide Reporters: New Concepts for Use in Bioanalytical Applications

Lanthanides represent the chemical elements from lanthanum to lutetium. They intrinsically exhibit some very exciting photophysical properties, which can be further enhanced by incorporating the lanthanide ion into organic or inorganic sensitizing structures. A very popular approach is to conjugate the lanthanide ion to an organic chromophore structure forming lanthanide chelates. Another approach, which has quickly gained interest, is to incorporate the lanthanide ions into nanoparticle structures, thus attaining improved specific activity and binding capacity. The lanthanide-based reporters usually express strong luminescence emission, multiple narrow emission lines covering a wide wavelength range, and exceptionally long excited state lifetimes enabling timeresolved detection. Because of these properties, the lanthanide-based reporters have found widespread applications in various fields of life. This study focuses on the field of bioanalytical applications. The aim of the study was to demonstrate the utility of different lanthanide-based reporters in homogeneous Förster resonance energy transfer (FRET)-based bioaffinity assays. Several different model assays were constructed. One was a competitive bioaffinity assay that utilized energy transfer from lanthanide chelate donors to fluorescent protein acceptors. In addition to the conventional FRET phenomenon, a recently discovered non-overlapping FRET (nFRET) phenomenon was demonstrated for the first time for fluorescent proteins. The lack of spectral overlap in the nFRET mechanism provides sensitivity and versatility to energy transfer-based assays. The distance and temperature dependence of these phenomena were further studied in a DNA-hybridization assay. The distance dependence of nFRET deviated from that of FRET, and unlike FRET, nFRET demonstrated clear temperature dependence. Based on these results, a possible excitation mechanism operating in nFRET was proposed. In the study, two enzyme activity assays for caspase-3 were also constructed. One of these was a fluorescence quenching-based enzyme activity assay that utilized novel inorganic particulate reporters called upconverting phosphors (UCPs) as donors. The use of UCPs enabled the construction of a simple, rather inexpensive, and easily automated assay format that had a high throughput rate. The other enzyme activity assay took advantage of another novel reporter class, the lanthanidebinding peptides (LBPs). In this assay, energy was transferred from a LBP to a green fluorescent protein (GFP). Using the LBPs it was possible to avoid the rather laborious, often poorly repeatable, and randomly positioned chemical labeling. In most of the constructed assays, time-resolved detection was used to eliminate the interfering background signal caused by autofluorescence. The improved signal-to-background ratios resulted in increased assay sensitivity, often unobtainable in homogeneous assay formats using conventional organic fluorophores. The anti-Stokes luminescence of the UCPs, however, enabled the elimination of autofluorescence even without time-gating, thus simplifying the instrument setup. Together, the studied reporters and assay formats pave the way for increasingly sensitive, simple, and easily automated bioanalytical applications.

Switchable Lanthanide Luminescence for Detection of Biomolecules

Binary probes are oligonucleotide probe pairs that hybridize adjacently to a complementary target nucleic acid. In order to detect this hybridization, the two probes can be modified with, for example, fluorescent molecules, chemically reactive groups or nucleic acid enzymes. The benefit of this kind of binary probe based approach is that the hybridization elicits a detectable signal which is distinguishable from background noise even though unbound probes are not removed by washing before measurement. In addition, the requirement of two simultaneous binding events increases specificity. Similarly to binary oligonucleotide probes, also certain enzymes and fluorescent proteins can be divided into two parts and used in separation-free assays. Split enzyme and fluorescent protein reporters have practical applications among others as tools to investigate protein-protein interactions within living cells. In this study, a novel label technology, switchable lanthanide luminescence, was introduced and used successfully in model assays for nucleic acid and protein detection. This label technology is based on a luminescent lanthanide chelate divided into two inherently non-luminescent moieties, an ion carrier chelate and a light harvesting antenna ligand. These form a highly luminescent complex when brought into close proximity; i.e., the label moieties switch from a dark state to a luminescent state. This kind of mixed lanthanide complex has the same beneficial photophysical properties as the more typical lanthanide chelates and cryptates - sharp emission peaks, long emission lifetime enabling time-resolved measurement, and large Stokes’ shift, which minimize the background signal. Furthermore, the switchable lanthanide luminescence technique enables a homogeneous assay set-up. Here, switchable lanthanide luminescence label technology was first applied to sensitive, homogeneous, single-target nucleic acid and protein assays with picomolar detection limits and high signal to background ratios. Thereafter, a homogeneous four-plex nucleic acid array-based assay was developed. Finally, the label technology was shown to be effective in discrimination of single nucleotide mismatched targets from fully matched targets and the luminescent complex formation was analyzed more thoroughly. In conclusion, this study demonstrates that the switchable lanthanide luminescencebased label technology can be used in various homogeneous bioanalytical assays.