Isolation and Characterization of Ovomucin - a Bioactive Agent of Egg White

The hen’s egg is a source of new life. Therefore, it contains many biologically active compounds. In addition to being a very nutritious food and also commonly used in the food industry due to its many techno-functional properties, the egg can serve as a source of compounds used as nutra-, pharmaand cosmeceuticals. One such interesting compound is ovomucin, an egg white protein responsible for the gel-like properties of thick egg white. Previous studies have indicated that ovomucin and ovomucin-derived peptides have several different bioactive properties. The objectives of the present study were to develop isolation methods for ovomucin, to characterize the structure of ovomucin, to compare various egg fractions as sources of ovomucin, to study the effects of various dissolving methods for ovomucin, and to investigate the bioactive properties of ovomucin and ovomucin-derived peptides. A simple and rapid method for crude ovomucin separation was developed. By using this method crude ovomucin was isolated within hours, compared to the 1-2 days (including a dialysis step) needed when using several other methods. Structural characterization revealed that ovomucin is composed of two subunits, α- and β-ovomucin, as egg white protein formerly called α1-ovomucin seemed to be ovostatin. However, it might be possible that ovostatin is associated within β- and α-ovomucin. This interaction could even have some effect on the physical nature of various egg white layers. Although filtration by-product fraction was a very prominent source of both crude and β-ovomucin, process development has reduced its amount so significantly that it has no practical meaning anymore. Thus, the commercial liquid egg white is probably the best option, especially if it generally contains amounts of β-ovomucin as high as were found in these studies. Crude ovomucin was dissolved both by using physical and enzymic methods. Although sonication was the most effective physical method for ovomucin solubilisation, colloid milling seemed to be a very promising alternative. A milk-like, smooth and opaque crude ovomucin suspension was attained by using a colloid mill. The dissolved ovomucin fractions were further tested for bioactive properties, and it was found that three dissolving methods tested produced moderate antiviral activity against Newcastle disease virus, namely colloid milling, enzymatic hydrolysis and a combination of sonicaton and enzymatic hydrolysis. Moreover, trypsin-digested crude ovomucin was found to have moderate antiviral activity against avian influenza virus: both subtype H5 and H7.

Measles virus infection – Interplay between virus and host cells

Measles, caused by measles virus (MV), is a highly contagious viral disease causing severe respiratory infection and a typical rash. Despite the availability of a protective vaccine, measles is still the leading vaccine-preventable cause of childhood mortality worldwide. The high mortality associated with the disease is mainly due to an increased susceptibility to secondary infections during the period of immunosuppression that continues for several weeks after recovery. The present study was undertaken to elucidate the role of cytoskeletal components in the regulation of MV infection. The most interesting finding was that MV replication was activated in unstimulated peripheral blood mononuclear cells (PBMC) when globular actin was converted into the filamentous form with jasplakinolide. This provides a new aspect in our understanding of MV infection in PBMC. In the second part of the thesis we investigated MV-induced structural changes of cellular nuclear matrix, which is a proteinaceous framework of the nucleus similar to the cytoskeleton in the cytoplasm. We showed that cleavage of nuclear markers was virusspecific and a general caspase inhibitor rescued MV-infected cells from cell death. Furthermore, we studied MV-induced innate immune mechanisms in lung epithelial and endothelial cells. Our results showed that MV infection resulted in activation of the double stranded RNA (dsRNA) binding molecules melanoma differentiation-associated gene 5 (mda-5), retinoic acid inducible gene I (RIG-I), and toll-like receptor 3 (TLR3) gene expression, followed by high expression of antiviral cytokine mRNA.

Virus Infections in Early Childhood, Cow’s Milk Formula Exposure and Genetic Predisposition in the Development of Diabetes-Associated Autoimmunity

Varhaislapsuuden virusinfektioiden, lehmänmaitopohjaisen äidinmaitovastikeen ja geneettisen alttiuden merkitys diabetekseen liittyvän autoimmuniteetin kehittymisessä Tyypin 1 diabetes on autoimmuunisairaus, joka syntyy haiman insuliinia tuottavien beta-solujen tuhouduttua elimistön oman immuunipuolustusjärjestelmän hyökkäyksen seurauksena. Sekä perimän että ympäristötekijöiden arvellaan vaikuttavan tautiprosessiin, mutta taudin tarkkaa syntymekanismia ei tunneta. Tutkimuksen tarkoituksena oli selvittää varhaislapsuuden ympäristötekijöiden vaikutusta beta-soluautoimmuniteetin syntyyn, erityispaino tutkimuksessa oli ympäristötekijöiden yhteisvaikutuksessa sekä geneettisten riskitekijöiden ja ympäristötekijöiden vuorovaikutuksessa. Varhaislapsuudessa sairastettu sytomegalovirus- tai enterovirusinfektio ei lisännyt beta-soluautoimmuniteetin riskiä lapsilla, joilla on geneettisesti kohonnut riski sairastua tyypin 1 diabetekseen. Ennen puolen vuoden ikää sairastettu rotavirusinfektio lisäsi hieman tyypin 1 diabetekseen liittyvän autoimmuniteetin riskiä. Tarkemmassa analyysissa varhaislapsuuden enterovirusinfektio osoittautui kuitenkin autovasta-aineiden muodostumisen riskitekijäksi niiden lasten joukossa, jotka olivat saaneet lehmänmaitopohjaista äidinmaidon vastiketta ensimmäisten elinkuukausien aikana. Tämä löydös viittaa enterovirusinfektion ja lehmänmaitopohjaisen vastikkeen yhteisvaikutukseen tyypin 1 diabetekseen liittyvän autoimmuniteetin synnyssä. Löydösten mukaan PTPN22 geenin C1858T polymorfismi vaikuttaa CD4+ T solujen aktivaatioon ja proliferaatiovasteeseen, 1858T alleeliin liittyy alentunut T-soluresepto-rivälitteinen aktivaatio. 1858T alleelin kantajuuteen liittyy lisäksi lisääntynyt autovasta-aineiden ja kliinisen diabeteksen ilmaantuvuus. Tämä yhteys rajoittui yksilöihin, jotka olivat altistuneet lehmänmaitopohjaiselle vastikkeelle ennen kuuden kuukauden ikää. Tulosten mukaan sekä ympäristötekijöiden väliset yhteisvaikutukset että perimä vaikuttavat yksittäisen ympäristötekijän merkitykseen tyypin 1 diabetekseen liittyvän autoimmuniteetin synnyssä. Nämä yhteisvaikutukset ympäristötekijöiden kesken ja perimän ja ympäristötekijöiden välillä selittävät aiemmin julkaistujen tulosten ristiriittaisuutta tutkimuksissa, joissa on analysoitu vain yhden ympäristötekijän vaikutusta diabeteksen ilmaantuvuuteen.

Functional Genomic Approaches for Deciphering Plant-Virus Interactions

Plant-virus interactions are very complex in nature and lead to disease and symptom formation by causing various physiological, metabolic and developmental changes in the host plants. These interactions are mainly the outcomes of viral hijacking of host components to complete their infection cycles and of host defensive responses to restrict the viral infections. Viral genomes contain only a small number of genes often encoding for multifunctional proteins, and all are essential in establishing a viral infection. Thus, it is important to understand the specific roles of individual viral genes and their contribution to the viral life cycles. Among the most important viral proteins are the suppressors of RNA silencing (VSRs). These proteins function to suppress host defenses mediated by RNA silencing and can also serve in other functions, e.g. in viral movement, transactivation of host genes, virus replication and protein processing. Thus these proteins are likely to have a significant impact on host physiology and metabolism. In the present study, I have examined the plant-virus interactions and the effects of three different VSRs on host physiology and gene expression levels by microarray analysis of transgenic plants that express these VSR genes. I also studied the gene expression changes related to the expression of the whole genome of Tobacco mosaic virus (TMV) in transgenic tobacco plants. Expression of the VSR genes in the transgenic tobacco plants causes significant changes in the gene expression profiles. HC-Pro gene derived from the Potyvirus Y (PVY) causes alteration of 748 and 332 transcripts, AC2 gene derived from the African cassava mosaic virus (ACMV) causes alteration of 1118 and 251transcripts, and P25 gene derived from the Potyvirus X (PVX) causes alterations of 1355 and 64 transcripts in leaves and flowers, respectively. All three VSRs cause similar up-regulation in defense, hormonally regulated and different stress-related genes and down-regulation in the photosynthesis and starch metabolism related genes. They also induce alterations that are specific to each viral VSR. The phenotype and transcriptome alterations of the HC-Pro expressing transgenic plants are similar to those observed in some Potyvirus-infected plants. The plants show increased protein degradation, which may be due to the HC-Pro cysteine endopeptidase and thioredoxin activities. The AC2-expressing transgenic plants show a similar phenotype and gene expression pattern as HC-Pro-expressing plants, but also alter pathways related to jasmonic acid, ethylene and retrograde signaling. In the P25 expressing transgenic plants, high numbers of genes (total of 1355) were up-regulated in the leaves, compared to a very low number of down-regulated genes (total of 5). Despite of strong induction of the transcripts, only mild growth reduction and no other distinct phenotype was observed in these plants. As an example of whole virus interactions with its host, I also studied gene expression changes caused by Tobacco mosaic virus (TMV) in tobacco host in three different conditions, i.e. in transgenic plants that are first resistant to the virus, and then become susceptible to it and in wild type plants naturally infected with this virus. The microarray analysis revealed up and down-regulation of 1362 and 1422 transcripts in the TMV resistant young transgenic plants, and up and down-regulation of a total of 1150 and 1200 transcripts, respectively, in the older plants, after the resistance break. Natural TMV infections in wild type plants caused up-regulation of 550 transcripts and down-regulation of 480 transcripts. 124 up-regulated and 29 down-regulated transcripts were commonly altered between young and old TMV transgenic plants, and only 6 up-regulated and none of the down-regulated transcripts were commonly altered in all three plants. During the resistant stage, the strong down-regulation in translation-related transcripts (total of 750 genes) was observed. Additionally, transcripts related to the hormones, protein degradation and defense pathways, cell division and stress were distinctly altered. All these alterations may contribute to the TMV resistance in the young transgenic plants, and the resistance may also be related to RNA silencing, despite of the low viral abundance and lack of viral siRNAs or TMV methylation activity in the plants.