Periodontitis and peri-implantitis biomarkers in human oral fluids and the null-allele mouse model

Tissue destruction associated with the periodontal disease progression is caused by a cascade of host and microbial factors and proteolytic enzymes. Aberrant laminin-332 (Ln-332), human beta defensin (hBD), and matrix metalloproteinase (MMP) functions have been found in oral inflammatory diseases. The null-allele mouse model appears as the next step in oral disease research. The MMP-8 knock-out mouse model allowed us to clarify the involvement of MMP-8 in vivo in oral and related inflammatory diseases where MMP-8 is suggested to play a key role in tissue destruction. The cleaved Ln-332 γ2-chain species has been implicated in the apical migration of sulcular epithelial cells during the formation of periodontal pockets. We demonstrated that increased Ln-332 fragment levels in gingival crevicular fluid (GCF) are strongly associated with the severity of inflammation in periodontitis. Porphyromonas gingivalis trypsin-like proteinase can cleave an intact Ln-332 γ2-chain into smaller fragments and eventually promote the formation of periodontal pockets. hBDs are components of an innate mucosal defense against pathogenic microbes. Our results suggest that P. gingivalis trypsin-like proteinase can degrade hBD and thus reduce the innate immune response. Elevated levels and the increased activity of MMPs have been detected in several pathological tissue-destructive conditions where MMPs are shown to cleave extracellular matrix (ECM) and basement membrane (BM) molecules and to facilitate tissue destruction. Elevated levels of MMP-8 have been reported in many inflammatory diseases. In periodontitis, MMP-8 levels in gingival crevicular fluid (GCF) and in peri-implant sulcular fluid (PISF) are elevated at sites of active inflammation, and the increased levels of MMP-8 are mainly responsible for collagenase activity, which leads to tissue destruction. MMP-25, expressed by neutrophils, is involved in inflammatory diseases and in ECM turnover. MMP-26 can degrade ECM components and serve as an activator of other MMP enzymes. We further confirmed that increased levels and activation of MMP-8, -25, and -26 in GCF, PISF, and inflamed gingival tissue are associated with the severity of periodontal/peri-implant inflammation. We evaluated the role of MMP-8 in P. gingivalis-induced periodontitis by comparing MMP-8 knock-out (MMP8-/-) and wild-type mice. Surprisingly, MMP-8 significantly attenuated P. gingivalis-induced site-specific alveolar bone loss. We also evaluated systemic changes in serum immunoglobulin and lipoprotein profiles among these mouse groups. P. gingivalis infection increased HDL/VLDL particle size in the MMP-8-/- mice, which is an indicator of lipoprotein responses during systemic inflammation. Serum total LPS and IgG antibody levels were enhanced in both mice groups. P. gingivalis-induced periodontitis, especially in MMP-8-/- mice, is associated with severe alveolar bone loss and with systemic inflammatory and lipoprotein changes that are likely to be involved in early atherosclerosis.

Studies on Matrix Metalloproteinase (MMP-2, -9 and -14) and β2 Integrin Targeting as Potential Anticancer Therapeutics

Proteolytic enzymes, such as matrix metalloproteinases (MMP), are associated to the progression of several cancers. They degrade extracellular components, which helps tumors to expand and cancer cells to escape from the primary site. Of all MMPs, gelatinases (MMP-2 and -9) and membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), in particular, are often associated to more aggressive types of head and neck carcinomas as well as to a poorer outcome in patient survival. Although therapies during the last decades have advanced, the mortality of the disease is still rather high and adjuvant therapies are searched for continuously. MMP-9 and MT1-MMP are also involved in neo-angiogenesis, which is necessary for tumor expansion. For this reason, we have identified synthetic peptides-targeting gelatinases and MT1-MMP, and have also evaluated their anticancer effects in vitro and in vivo. Antigelatinolytic peptides effectively inhibited tongue-carcinoma cell invasion and reduced the growth of xenografted tumors. In tumor samples of mice that were treated with antigelatinolytic peptides, the micro-vessel density was significantly reduced. We also identified a novel MT1-MMP targeting peptide and demonstrated that it exerted anticancer effects against several malignant cell lines in vitro. The effects of MT1-MMP inhibition on tongue-squamous cell carcinomas were evaluated by using xenograft tumors, which it effectively inhibited. Tranexamic acid was also demonstrated to inhibit tongue-squamous cell carcinoma invasion, most probably due to its ability to prevent the plasmin-mediated activation of proMMP-9. Leukocyte β2 integrins are another interesting option when evaluating targets for the therapeutic intervention of inflammatory conditions or malignancies of hematopoietic origin, since β2 integrins are expressed mainly by leukocytes. We identified a novel technique for screening small-molecule libraries against β2 integrins, and by using this technique we identified a novel αMβ2 integrin-binding chemical (IMB-10). IMB-10 significantly enhances leukocyte adhesion and inhibits their motility. We also demonstrated that IMB-10 can be used to inhibit inflammation and lymphoma growth in vivo. Interestingly, IMB-10 also reduced leukocyte tumor infiltration and inhibited tumor invasion.

Fibroblast nemosis in malignant progression of epithelial cells

Paracrine regulation between the components of the tumour microenvironment cancer cells, activated fibroblasts, immune and endothelial cells is under intense investigation. The signals between the different cell types are mediated by soluble factors, such as growth factors, proinflammatory cytokines and proteolytic enzymes. Nemosis is an experimental in vitro model of fibroblast activation, leading to increased production of such mediators. Nemotic activation of fibroblasts occurs as they are forced to cluster thereby forming a multicellular spheroid. The aim of the present studies was to elucidate the mechanisms underlying the nemotic response of cancer-associated fibroblasts (CAF) and the role of nemosis in paracrine regulation between activated fibroblasts and benign and malignant epithelial cells. The results presented in this thesis demonstrate that the nemotic response of CAFs and normal fibroblasts differs, and inter-individual variations exist between fibroblast populations. In co-culture experiments, fibroblasts increased colony formation of squamous cell carcinoma (SCC) cells, and CAFs further augmented this, highlighting the tumour-evolving properties of CAFs. Furthermore, fibroblast monolayers in those co-cultures started to cluster spontaneously. This kind of spontaneous nemosis response might take place also in vivo, although more direct evidence of this still needs to be obtained. The HaCaT skin carcinoma progression model was used to study the effects of benign and malignant keratinocytes on fibroblast nemosis. Benign HaCaT cells inhibited fibroblast nemosis, observed as inhibition of cyclooxygenase 2 (COX-2) induction in nemotic spheroids. In contrast, malignant HaCaTs further augmented the nemotic response by increasing expression of COX-2 and the growth factors hepatocyte growth factor / scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF), as well as causing a myofibroblastic differentiation of nemotic fibroblasts into fibroblasts resembling CAFs. On the other side of this reciprocal signalling, factors secreted into conditioned medium by the nemotic fibroblasts promoted proliferation and motility of the HaCaT cell lines. Notably, the nemotic fibroblast medium increased the expression of p63, a transcription factor linked to carcinogenesis, also in the highly metastatic HaCaT cells. These results emphasize the paracrine role of factors secreted by activated fibroblasts in driving tumour progression. We also investigated the epithelial-mesenchymal transition (EMT) of the HaCaT clones in response to transforming growth factor β (TGF-β), which is a well-characterized inducer of EMT. TGF-β caused growth arrest and loss of epithelial cell junctions in the HaCaT derivatives, but mesenchymal markers were not induced, suggesting a partial, but not complete EMT response. Inflammation induced by COX-2 has been proposed to be a key mechanism in EMT of benign cells. Corroborating this notion, COX-2 was induced only in benign, not in malignant HaCaT derivatives. Furthermore, in cells in which TGF-β caused COX-2 induction, migration was clearly augmented. The concept of treating cancer is changing from targeting solely the cancer cells to targeting the whole microenvironment. The results of this work emphasise the role of activated fibroblasts in cancer progression and that CAFs should also be taken into consideration in the treatment of cancer. The results from these studies suggests that nemosis could be used as a diagnostic tool to distinguish in vitro activated fibroblasts from tumour stroma and also in studying the paracrine signalling that is mediated to other cell types via soluble factors.

Regional differentiation in bull sperm plasma-membranes

Bull sperm heads and tails have been separated by proteolytic digestion (trypsin) and plasma membranes have been isolated, using discontinuous sucrose density gradient centrifugation. Plasma membrane bound Ca2+-ATPase is shown to be associated mostly with the tail membranes. Pyrene excimer fluorescence and diphenylhexatriene fluorescence polarization experiments indicate a more fluid lipid phase in the tail region. Differences in surface charge distribution have been found, using 1-anilinonaphthalene-8-sulfonate and Tb3+ as fluorescent probes.

Latent TGF-beta Binding Proteins : Adhesive functions and matrix association of LTBP-2 and potential functions of LTBP-1 and LTBP-3 in mesothelioma

Latent transforming growth factor-beta (TGF-beta) binding proteins (LTBPs) -1, -3 and -4 are ECM components whose major function is to augment the secretion and matrix targeting of TGF-beta, a multipotent cytokine. LTBP-2 does not bind small latent TGF-beta but has suggested functions as a structural protein in ECM microfibrils. In the current work we focused on analyzing possible adhesive functions of LTBP-2 as well as on characterizing the kinetics and regulation of LTBP-2 secretion and ECM deposition. We also explored the role of TGF-beta binding LTBPs in endothelial cells activated to mimic angiogenesis as well as in malignant mesothelioma. We found that, unlike most adherent cells, several melanoma cell lines efficiently adhered to purified recombinant LTBP-2. Further characterization revealed that the adhesion was mediated by alpha3beta1 and alpha6beta1 integrins. Heparin also inhibited the melanoma cell adhesion suggesting a role for heparan sulphate proteoglycans. LTBP-2 was also identified as a haptotactic substrate for melanoma cell migration. We used cultured human embryonic lung fibroblasts to analyze the temporal and spatial association of LTBP-2 into ECM. By We found that LTBP-2 was efficiently assembled to the ECM only in confluent cultures following the deposition of fibronectin (FN) and fibrillin-1. In early, subconfluent cultures it remained primarily in soluble form after secretion. LTBP-2 colocalized transiently with FN and fibrillin-1. Silencing of fibrillin-1 expression by lentiviral shRNAs profoundly disrupted the deposition of LTBP-2 indicating that the ECM association of LTBP-2 depends on a pre-formed fibrillin-1 network. Considering the established role of TGF-beta as a regulator of angiogenesis we induced morphological activation of endothelial cells by phorbol 12-myristate 13-acetate (PMA) and followed the fate of LTBP-1 in the endothelial ECM. This resulted in profound proteolytic processing of LTBP-1 and release of latent TGF-beta complexes from the ECM. The processing was coupled with increased activation of MT-MMPs and specific upregulation of MT1-MMP. The major role of MT1-MMP in the proteolysis of LTBP-1 was confirmed by suppressing the expression with lentivirally induced short-hairpin RNAs as well as by various metalloproteinases inhibitors. TGF-beta can promote tumorigenesis of malignant mesothelioma (MM), which is an aggressive tumor of the pleura with poor prognosis. TGF-beta activity was analyzed in a panel of MM tumors by immunohistochemical staining of phosphorylated Smad-2 (P-Smad2). The tumor cells were strongly positive for P-Smad2 whereas LTBP-1 immunoreactivity was abundant in the stroma, and there was a negative correlation between LTBP-1 and P-Smad2 staining. In addition, the high P-Smad2 immunoreactivity correlated with shorter survival of patients. mRNA analysis revealed that TGF-beta1 was the most highly expressed isoform in both normal human pleura and MM tissue. LTBP-1 and LTBP-3 were both abundantly expressed. LTBP-1 was the predominant isoform in established MM cell lines whereas the expression of LTBP-3 was high in control cells. Suppression of LTBP-3 expression by siRNAs resulted in increased TGF-beta activity in MM cell lines accompanied by decreased proliferation. Our results suggest that decreased expression of LTBP-3 in MM could alter the targeting of TGF-beta to the ECM and lead to its increased activation. The current work emphasizes the coordinated process of the assembly and appropriate targeting of LTBPs with distinct adhesive or cytokine harboring properties into the ECM. The hierarchical assembly may have implications in the modulation of signaling events during morphogenesis and tissue remodeling.

Factor VIII-hydrolyzing IgG in acquired and congenital hemophilia

Anti-factor VIII (FVIII) inhibitory IgG may arise as alloantibodies to therapeutic FVIII in patients with congenital hemophilia A, or as autoantibodies to endogenous FVIII in individuals with acquired hemophilia. We have described FVIII-hydrolyzing IgG both in hemophilia A patients with anti-FVIII IgG and in acquired hemophilia patients. Here, we compared the properties of proteolytic auto- and allo-antibodies. Rates of FVIII hydrolysis differed significantly between the two groups of antibodies. Proline-phenylalanine-arginine-methylcoumarinamide was a surrogate substrate for FVIII-hydrolyzing autoantibodies. Our data suggest that populations of proteolytic anti-FVIII IgG in acquired hemophilia patients are different from that of inhibitor-positive hemophilia A patients.

Potato virus A as a heterologous protein expression tool in plants

Viruksien käyttö tuotekehityksen ja tutkimuksen vaatimien proteiinien tuottamiseen, syötävien rokotteiden kehittämiseen ja geeniterapiaan edustavat kasvavia biotekniikan sovellusalueita. Perunan A-virus (PVA) kuuluu potyviruksiin, joiden proteiinit tuotetaan aluksi yhtenä suurena molekyylinä, joka pilkotaan yksittäisiksi proteiineiksi viruksen itsensä tuottamilla entsyymeillä. Siten virusgenomiin lisätty vieras geeni käännetään proteiiniksi virusproteiinien mukana. Lopputuloksena kaikkia proteiineja tuotetaan kasvisoluissa samansuuruinen määrä. Lisäksi, viruksen proteiinikuoren koontimekanismi sallii perintöaineksen merkittävän lisäyksen ilman että viruksen tartutuskyky merkittävästi heikkenee. Koska virus monistuu ja leviää koko kasviin, jo melko pieni määrä kasveja riittää huomattavan proteiinimäärän tuottamiseen esimerkiksi säännösten mukaisessa kasvihuoneessa. Tämän työn tarkoituksena oli muuntaa PVA:n genomia siten, että virus soveltuisi yhden vieraan proteiinin tai useiden erilaisten proteiinien samanaikaiseen tuottamiseen kasveissa. Aluksi kokeiltiin viruksen replikaasia ja kuoriproteiinia koodaavien genomialueiden välistä kohtaa ja ihmisestä peräisi olevaa geeniä, joka tuotti S-COMT-entsyymiä (katekoli-O-metyylitransferaasi). Sen aktiivisuuden rajoittaminen auttaa Parkinsonintaudin hoidossa. Kasvissa tuotettua S-COMT:ia voitaisiin käyttää lääkekehityksessä estolääkkeiden testaukseen. Kahden viikon kuluttua tartutuksesta tupakan lehdissä oli entsymaattisesti aktiivista S-COMT:ia n. 1 % lehden liukoisista proteiineista. PVA:n P1-proteiinia koodaavalta alueelta oli paikannettu kohta, johon ehkä voitaisiin siirtää vieras geeni. Asia varmistettiin siirtämällä tähän kohtaan meduusan geeni, joka tuottaa UV-valossa vihreänä fluoresoivaa proteiinia (GFP). GFP-geeniä kantava PVA levisi kasvissa ja lisääntyi n. 30-50 %:iin viruksen normaalista pitoisuudesta. Koko kasvi fluoresoi vihreänä UV-valossa. Vieras geeni voidaan sijoittaa myös potyviruksen P1- ja HCpro-proteiineja koodaavien alueiden väliin. Samaan PVA-genomiin siirrettiin kolme geeniä, yksi kuhunkin kolmesta kloonauskohdasta: GFP-geeni P1:n sisälle, merivuokon lusiferaasigeeni P1/HCpro-kohtaan ja bakteerin beta-glukuronidaasigeeni (GUS) replikaasi/kuoriproteiini-kohtaan. Virusgenomin ja itse viruksen pituudet kasvoivat 38 %, mutta virus säilytti tartutuskykynsä. Se levisi kasveissa saavuttaen n. 15 % viruksen normaalista pitoisuudesta. Kaikki kolme vierasta proteiinia esiintyivät lehdissä aktiivisina.

Biochemical and structural properties of Potato virus A VPg

The potato virus A (PVA) genome linked protein (VPg) is a multifunctional protein that takes part in vital infection cycle events such as replication and movement of the virus from cell to cell. VPg is attached to the 5´ end of the genome and is carried in the tip structure of the filamentous virus particle. VPg is also the last protein to be cleaved from the polyprotein. VPg interacts with several viral and host proteins and is phosphorylated at several positions. These features indicate a central role in virus epidemiology and a requirement for an efficient but flexible mechanism for switching between different functions. -- This study examines some of the key VPg functions in more detail. Mutations in the positively charged region from Ala38 to Lys44 affected the NTP binding, uridylylation, and in vitro translation inhibition activities of VPg, whereas in vivo translation inhibition was not affected. Some of the data generated in this study implicated the structural flexibility of the protein in functional activities. VPg lacks a rigid structure, which could allow it to adapt conformationally to different functions as needed. A major finding of this study is that PVA VPg belongs to the class of ´intrinsically disordered proteins´ (IDPs). IDPs are a novel protein class that has helped to explain the observed lack of structure. The existence of IDPs clearly shows that proteins can be functional and adapt a native fold without a rigid structure. Evidence for the intrinsic disorder of VPg was provided by CD spectroscopy, NMR, fluorescence spectroscopy, bioinformatic analysis, and limited proteolytic digestion. The structure of VPg resembles that of a molten globule-type protein and has a hydrophobic core domain. Approximately 50% of the protein is disordered and an α-helical stabilization of these regions has been hypothesized. Surprisingly, VPg structure was stabilized in the presence of anionic lipid vesicles. The stabilization was accompanied by a change in VPg structure and major morphological modifications of the vesicles, including a pronounced increase in the size and appearance of pore or plaque like formations on the vesicle surface. The most likely scenario seems to be an α-helical stabilization of VPg which induces formation of a pore or channel-like structure on the vesicle surface. The size increase is probably due to fusion or swelling of the vesicles. The latter hypothesis is supported by the evident disruption of the vesicles after prolonged incubation with VPg. A model describing the results is presented and discussed in relation to other known properties of the protein.

Developments and Microbiological applications in African foods: Emphasis on Nigerian Wara cheese

African indigenous foods have received limited research. Most of these indigenous foods are fermented and they form part of the rich nutritional culture of many groups in African countries. The industrialization and commercialisation of these indigenous African fermented foods should be preceded by a thorough scientific knowledge of their processing which can be vital in the elimination of hunger and poverty. This study highlighted emerging developments and the microbiology of cereal-based and cassava-based food products that constitute a major part of the human diet in most African countries. In addition, investigations were also carried out on the coagulant of the Calotropis procera plant used in traditional production of Nigerian Wara cheese and on the effects of adding a nisin producing Lactococcus lactis strain originating from human milk to Nigerian Wara cheese. Fermented cereal-based food such as ogi utilize popular African and readily available grains maize, millet or sorghum as substrates and is popular as a weaning diet in infants. In this study, the bulkiness caused by starch gelatinization was solved by amylase treatments in the investigation on cooked and fermented oat bran porridge. A similar treatment could reduce the viscosity of any cereal porridge. The properties of the Sodom apple leaves (Calotropis procera) extract in cheesemaking were studied. C. procera was affected by monovalent (K+ and Na+) and divalent (Mg2+ and Ca2+) cations during coagulation. The rennet strength of this coagulant was found to be 7 % compared to animal rennet at 35 °C. Increasing the incubation temperature to 70 °C increased the rennet strength 28-fold. The molecular weight of the partially purified protease was determined by SDS-PAGE and was confirmed by Zymography to be approximately 60 kilodaltons. The high proteolytic activity at 70 °C supported the suitability of the protease enzyme as a coagulant in future commercial production of Nigerian Wara cheese. It was also possible to extend the shelf life of Wara cheese by a nisin producing lactic acid bacteria Lactococcus lactis LAC309. The levels of nisin in both whey and curd fractions of Wara were investigated, results showed a 3 log reduction of toxicogenic Bacillus licheniformis spiked on Wara after 3 days. These studies are the first in Finland to promote the advancement of scientific knowledge in African foods. Recognizing these indigenous food products and an efficient transfer of technology from the developed countries to industrialize them are necessary towards a successful realization of the United Nations Millenium Development Program.

solation and characterization of monodeamidated derivatives of bovine pancreatic Ribonuclease A

The isolation and characterization of the initial intermediates formed during the irreversible acid denaturation of enzyme Ribonuclease A are described. The products obtained when RNase A is maintained in 0.5 M HCl at 30° for periods up to 20 h have been analyzed by ion-exchange chromatography on Amberlite XE-64. Four distinct components were found to elute earlier to RNase A; these have been designated RNase Aa2, Aa1c, Aa1b, and Aa1a in order of their elution. With the exception of RNase Aa2, the other components are nearly as active as RNase A. Polyacrylamide gel electrophoresis at near-neutral pH indicated that RNase Aa1a, Aa1b, and Aa1c are monodeamidated derivatives of RNase A; RNase Aa2 contains, in addition, a small amount of a dideamidated component. RNase Aa2, which has 75% enzymic activity as compared to RNase A, consists of dideamidated and higher deamidated derivatives of RNase A. Except for differences in the proteolytic susceptibilities at an elevated temperature or acidic pH, the monodeamidated derivatives were found to have very nearly the same enzymic activity and the compact folded structure as the native enzyme. Fingerprint analyses of the tryptic peptides of monodeamidated derivatives have shown that the deamidations are restricted to an amide cluster in the region 67–74 of the polypeptide chain. The initial acid-catalyzed deamidation occurs in and around the 65–72 disulfide loop giving rise to at least three distinct monodeamidated derivatives of RNase A without an appreciable change in the catalytic activity and conformation of the ribonuclease molecule. Significance of this specific deamidation occurring in highly acidic conditions, and the biological implications of the physiological deamidation reactions of proteins are discussed.

Prolamin degradation in sourdoughs

This thesis examines protein behaviours that occur during cereal fermentations. The focus is on the prolamin degradation in sourdoughs. The thesis also looks at what happens to the oat globulins during an oat bran acidification process. The cereal prolamins are unique proteins in many respects. The wheat prolamins (glutenins and gliadins) are responsible for the formation of the gluten that provides the viscoelastic properties to wheat doughs whereas the rye prolamins (secalins) are unable to develop gluten-like structures. In addition, many baking technological features, such as flavour, shelf-life and dough properties are affected by the protein degradation that might occur during processing. On the other hand, the prolamins contain protein structures that are harmful to gluten sensitive people. It is thus evident that the degradation of the prolamins in sourdough processes may be approached from various aspects. This thesis describes some of these approaches. Four different cereal fermentations were carried out. Wheat sourdough (WSD) and rye sourdough (RSD) fermentations represented traditional sourdoughs. A germinated-wheat sourdough (GWSD) was a novel sourdough type that was prepared using germinated wheat grains that had high and diverse proteolytic activities. The oat bran fermentation (OBF) represented a fermentation system that lacked functional cereal proteases. The high molecular weight glutenins and rye secalins were degraded during the WSD and RSD fermentations, respectively. It was noteworthy that in WSD only a very limited degradation of the gliadins occurred. The gliadins were, however, hydrolysed very extensively during the GWSD fermentation. No protein degradation was observable in the OBF system. Instead the acidification altered the solubility of the oat globulins and this finally led to their aggregation. This thesis confirms that the endogenous proteases of cereals hydrolyse cereal prolamins in sourdoughs. The thesis also shows that the proteolytic activity of the used cereal raw material determines the extent of proteolysis that occurs in sourdough. This means that bakers may adjust the protein degradation in their sourdoughs by selecting the raw material based on its proteolytic activity. The thesis also demonstrates that by using germinated grains, with high and diverse proteolytic activity in sourdough preparations, the prolamins can be extensively degraded. Whether such highly proteolytic food technology could be used to manufacture new gluten-free cereal-based products for gluten sensitive people remains to be solved.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Mechanisms for alphavirus nonstructural polyprotein processing

Replication and transcription of the RNA genome of alphaviruses relies on a set of virus-encoded nonstructural proteins. They are synthesized as a long polyprotein precursor, P1234, which is cleaved at three processing sites to yield nonstructural proteins nsP1, nsP2, nsP3 and nsP4. All the four proteins function as constitutive components of the membrane-associated viral replicase. Proteolytic processing of P1234 polyprotein is precisely orchestrated and coordinates the replicase assembly and maturation. The specificity of the replicase is also controlled by proteolytic cleavages. The early replicase is composed of P123 polyprotein intermediate and nsP4. It copies the positive sense RNA genome to complementary minus-strand. Production of new plus-strands requires complete processing of the replicase. The papain-like protease residing in nsP2 is responsible for all three cleavages in P1234. This study addressed the mechanisms of proteolytic processing of the replicase polyprotein in two alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SIN) representing different branches of the genus. The survey highlighted the functional relation of the alphavirus nsP2 protease to the papain-like enzymes. A new structural motif the Cys-His catalytic dyad accompanied with an aromatic residue following the catalytic His was described for nsP2 and a subset of other thiol proteases. Such an architecture of the catalytic center was named the glycine specificity motif since it was implicated in recognition of a specific Gly residue in the substrate. In particular, the presence of the motif in nsP2 makes the appearance of this amino acid at the second position upstream of the scissile bond a necessary condition for the cleavage. On top of that, there were four distinct mechanisms identified, which provide affinity for the protease and specifically direct the enzyme to different sites in the P1234 polyprotein. Three factors RNA, the central domain of nsP3 and the N-terminus of nsP2 were demonstrated to be external modulators of the nsP2 protease. Here I suggest that the basal nsP2 protease specificity is inherited from the ancestral papain-like enzyme and employs the recognition of the upstream amino acid signature in the immediate vicinity of the scissile bond. This mechanism is responsible for the efficient processing of the SFV nsP3/nsP4 junction. I propose that the same mechanism is involved in the cleavage of the nsP1/nsP2 junction of both viruses as well. However, in this case it rather serves to position the substrate, whereas the efficiency of the processing is ensured by the capability of nsP2 to cut its own N-terminus in cis. Both types of cleavages are demonstrated here to be inhibited by RNA, which is interpreted as impairing the basal papain-like recognition of the substrate. In contrast, processing of the SIN nsP3/nsP4 junction was found to be activated by RNA and additionally potentiated by the presence of the central region of nsP3 in the protease. The processing of the nsP2/nsP3 junction in both viruses occurred via another mechanism, requiring the exactly processed N-terminus of nsP2 in the protease and insensitive to RNA addition. Therefore, the three processing events in the replicase polyprotein maturation are performed via three distinct mechanisms in each of two studied alphaviruses. Distinct sets of conditions required for each cleavage ensure sequential maturation of P1234 polyprotein: nsP4 is released first, then the nsP1/nsP2 site is cut in cis, and liberation of the nsP2 N-terminus activates the cleavage of the nsP2/nsP3 junction at last. The first processing event occurs differently in SFV and SIN, whereas the subsequent cleavages are found to be similar in the two viruses and therefore, their mechanisms are suggested to be conserved in the genus. The RNA modulation of the alphavirus nonstructural protease activity, discovered here, implies bidirectional functional interplay between the alphavirus RNA metabolism and protease regulation. The nsP2 protease emerges as a signal transmitting moiety, which senses the replication stage and responds with proteolytic cleavages. A detailed hypothetical model of the alphavirus replicase core was inferred from the data obtained in the study. Similar principles in replicase organization and protease functioning are expected to be employed by other RNA viruses.

Studies on the variants of the protein toxins ricin and abrin

his study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101–109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.

Use of protein A gene fusions for the analysis of structure-function relationship of the transactivator protein C of bacteriophage Mu

A sensitive dimerization assay for DNA binding proteins has been developed using gene fusion technology. For this purpose, we have engineered a gene fusion using protein A gene of Staphylococcus aureus and C gene, the late gene transactivator of bacteriophage Mu. The C gene was fused to the 3' end of the gene for protein A to generate an A- C fusion. The overexpressed fusion protein was purified in a single step using immunoglobulin affinity chromatography. Purified fusion protein exhibits DNA binding activity as demonstrated by electrophoretic mobility shift assays. When the fusion protein A-C was mixed with C and analyzed for DNA binding, in addition to C and A-C specific complexes, a single intermediate complex comprising of a heterodimer of C and A-C fusion proteins was observed. Further, the protein A moiety in the fusion protein A-C does not contribute to DNA binding as demonstrated by proteolytic cleavage and circular dichroism (CD) analysis. The assay has also been applied to analyze the DNA binding domain of C protein by generating fusions between protein A and N- and C-terminal deletion mutants of C. The results indicate a role for the region towards the carboxy terminal of the protein in DNA binding. The general applicability of this method is discussed.