Role of ErbB2 and ErbB4 in Cancer Growth, Prognosis and as Targets for Immunotherapy

ErbB receptors (EGFR, ErbB2, ErbB3 and ErbB4) are growth factor receptors that regulate signals of cell differentiation, proliferation, migration and survival. Inappropriate activation of these receptors is associated with the development and severity of many cancers and has prognostic and predictive value in cancer therapy. Drugs, such as therapeutic antibodies, targeted against EGFR and ErbB2, are currently used in therapy of breast, colorectal and head and neck cancers. The role of ErbB4 in tumorigenesis has remained relatively poorly understood. Alternative splicing produces four different isoforms of one ErbB4 gene. These isoforms (JM-a, JM-b, CYT-1 and CYT-2) are functionally dissimilar and proposed to have different roles in carcinogenesis. The juxtamembrane form JM-a undergoes regulated intramembrane proteolysis producing a soluble receptor ectodomain and an intracellular domain that translocates into the nucleus and regulates transcription. Nuclear signaling via JM-a isoform stimulates cancer cell proliferation. This study aimed to develop antibodies targeting the proposed oncogenic ErbB4 JM-a isoform that show potential in inhibiting ErbB4 dependent tumorigenesis. Also, the clinical relevance of ErbB4 shedding in cancer was studied. The currently used monoclonal antibody trastuzumab, targeting ErbB2, has shown efficacy in breast cancer therapy. In this study novel tissues with ErbB2 amplification and trastuzumab sensitivity were analyzed. The results of this study indicated that a subpopulation of breast cancer patients demonstrate increased shedding and cleavage of ErbB4. A JM-a isoform-specific antibody that inhibited ErbB4 shedding and consequent activation of ErbB4 had anti-tumor activity both in vitro and in vivo. Thus, ErbB4 shedding associates with tumor growth and specific targeting of the cleavable JM-a isoform could be considered as a strategy for developing novel ErbB-based cancer drugs. In addition, it was demonstrated that ErbB2 amplification is common in intestinal type gastric cancers with poor clinical outcome. Trastuzumab inhibited growth of gastric and breast cancer cells with equal efficacy. Thus, ErbB2 may be a useful target in gastric cancer.

PCR amplification and sequence analyses of reverse transcriptase-like genes in Crinipellis perniciosa isolates

Reverse transcriptase (RT) sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative RT sequences were analyzed in the genome of the pathogenic fungus C. perniciosa, the causal agent of witches' broom disease of cocoa. A 394 bp fragment was amplified from genomic DNA of different isolates of C. perniciosa belonging to C-, L-, and S-biotypes and collected from various geographical areas. The cleavage of PCR products with restriction enzymes and the sequencing of various RT fragments indicated the presence of several sequences showing transition events (G:C to A:T). Southern blot analysis revealed high copy numbers of RT signals, forming different patterns among C-, S-, and L-biotype isolates. Sequence comparisons of the predicted RT peptide indicate a close relationship with the RT protein from thegypsy family of LTR-retrotransposons. The possible role of these retrotransposons in generating genetic variability in the homothallic C. perniciosa is discussed.

The Integrin Tail: A Tale of Cell Motility and Division

Integrin transmembrane receptor functions are regulated by adaptor molecules binding to their alpha and beta subunit intracellular domains, or tails, thus affecting integrin traffic and adhesion during e.g. cell motility. Interestingly, many cellular proteins function in both cell motility and cell division, thus raising the possibility that integrins might be involved in regulating the cell cycle. A thorough understanding of cell division is essential in cell biology and in human malignancies. It is well established that failures to complete cell cycle can give rise to genetically unstable cells with tumorigenic properties. Transformed cells promote the disruption of intercellular adhesions such as tight junctions, and this correlates with the onset of cell motility, invasion and unfavorable prognosis in cancer. In this study, we analyzed integrin regulation, mediated by adaptor binding to the  subunit tail, during cell motility and cell division. We revealed a novel molecular mechanism by which Rab21, through association with the integrin alpha subunits, drives integrin endosomal traffic during mitotic phases. In addition, we found indications for this finding in vivo, as RAB21 gene deletions were mapped in ovarian and prostate cancer samples. Importantly, the multinucleated phenotype of cultured ovarian cancer cells could be reverted by Rab21 overexpression. In this thesis work, we also show how the tight junction protein ZO-1 unexpectedly interacts with the 5 integrin cytoplasmic domain in the lamellipodia to promote cell motility and at the cleavage furrow to support separation of the daughter cells. The alpha5-ZO-1 complex formation was dependent on PKC which regulates ZO-1 phosphorylation and its subcellular localization. In addition, by an in situ detection method, we showed that a subset of metastatic human lung cancers expressed the alpha5beta-ZO-1 complex. Taken together, we were able to identify new molecular pathways that regulate integrin functions in an alpha tail-mediated fashion. These findings firmly suggest that genetic alterations in integrin traffic may lead to progression of tumorigenesis as a result of failed cell division. Also, the interplay of integrins and ZO-1 in forming spatially regulated adhesive structures broadens our view of crosstalk between pathways and distinct adhesive structures that can be involved in cancer cell biology.

Novel Players in the Integrin Signaling Orchestra: TCPTP and MDGI

Metastases are the major cause of cancer deaths. Tumor cell dissemination from the primary tumor utilizes dysregulated cellular adhesion and upregulated proteolytic degradation of the extracellular matrix for progeny formation in distant organs. Integrins are transmembrane adhesive receptors mediating cellcell and cellmatrix interactions that are crucial for regulating cell migration, invasion, proliferation, and survival. Consequently, increased integrin activity is associated with augmented migration and invasion capacity in several cancer types. Heterodimeric integrins consist of an alpha - and beta-subunit that are held together in a bent conformation when the receptor is inactive, but extension and separation of subdomains is observed during receptor activation. Either inside-out or outside-in activation of receptors is possible through the intracellular molecule binding to an integrin cytoplasmic domain or extracellular ligand association with an integrin ectodomain, respectively. Several regulatory binding partners have been characterized for integrin cytoplasmic beta-domains, but the regulators interacting with the cytoplasmic alpha-domains have remained elusive. In this study, we performed yeast two-hybrid screens to identify novel binding partners for the cytoplasmic integrin alpha-domains. Further examination of two plausible candidates revealed a significant coregulatory role of an integrin alpha-subunit for cellular signaling processes. T-cell protein tyrosine phosphatase (TCPTP) showed a specific interaction with the cytoplasmic tail of integrin alpha1. This association stimulated TCPTP phosphatase activity, leading to negative regulation of epidermal growth factor receptor (EGFR) signaling and diminished anchorage-independent growth. Another candidate, mammary-derived growth inhibitor (MDGI), exhibited binding to several different integrin cytoplasmic alpha-tails through a conserved GFFKR sequence. MDGI overexpression in breast cancer cells altered EGFR trafficking and caused a remarkable accumulation of EGFR in the cytoplasm. We further demonstrated in vivo that MDGI expression induced a novel form of anti-EGFR therapy resistance. Moreover, MDGI binding to α-tails retained integrin in an inactive conformation attenuating integrin-mediated adhesion, migration, and invasion. In agreement with these results, sustained MDGI expression in breast cancer patients correlated with an increased 10-year distant disease-free survival. Taken together, the integrin signaling network is far from a complete view and future work will doubtless broaden our understanding further.

Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

Characterisation of Human Neutral and Lysosomal alpha-Mannosidases

Neutral alpha-mannosidase and lysosomal MAN2B1 alpha-mannosidase belong to glycoside hydrolase family 38, which contains essential enzymes required for the modification and catabolism of asparagine-linked glycans on proteins. MAN2B1 catalyses lysosomal glycan degradation, while neutral α-mannosidase is most likely involved in the catabolism of cytosolic free oligosaccharides. These mannose containing saccharides are generated during glycosylation or released from misfolded glycoproteins, which are detected by quality control in the endoplasmic reticulum. To characterise the biological function of human neutral α-mannosidase, I cloned the alpha-mannosidase cDNA and recombinantly expressed the enzyme. The purified enzyme trimmed the putative natural substrate Man9GlcNAc to Man5GlcNAc, whereas the reducing end GlcNAc2 limited trimming to Man8GlcNAc2. Neutral α-mannosidase showed highest enzyme activity at neutral pH and was activated by the cations Fe₂+, Co2+ and Mn₂+, Cu2+ in turn had a strong inhibitory effect on alpha-mannosidase activity. Analysis of its intracellular localisation revealed that neutral alpha-mannosidase is cytosolic and colocalises with proteasomes. Further work showed that the overexpression of neutral alpha-mannosidase affected the cytosolic free oligosaccharide content and led to enhanced endoplasmic reticulum associated degradation and underglycosylation of secreted proteins. The second part of the study focused on MAN2B1 and the inherited lysosomal storage disorder α-mannosidosis. In this disorder, deficient MAN2B1 activity is associated with mutations in the MAN2B1 gene. The thesis reports the molecular consequences of 35 alpha-mannosidosis associated mutations, including 29 novel missense mutations. According to experimental analyses, the mutations fall into four groups: Mutations, which prevent transport to lysosomes are accompanied with a lack of proteolytic processing of the enzyme (groups 1 and 3). Although the rest of the mutations (groups 2 and 4) allow transport to lysosomes, the mutated proteins are less efficiently processed to their mature form than is wild type MAN2B1. Analysis of the effect of the mutations on the model structure of human lysosomal alpha-mannosidase provides insights on their structural consequences. Mutations, which affect amino acids important for folding (prolines, glycines, cysteines) or domain interface interactions (arginines), arrest the enzyme in the endoplasmic reticulum. Surface mutations and changes, which do not drastically alter residue volume, are tolerated better. Descriptions of the mutations and clinical data are compiled in an α-mannosidosis database, which will be available for the scientific community. This thesis provides a detailed insight into two ubiquitous human alpha-mannosidases. It demonstrates that neutral alpha-mannosidase is involved in the degradation of cytosolic oligosaccharides and suggests that the regulation of this α-mannosidase is important for maintaining the cellular homeostasis of N-glycosylation and glycan degradation. The study on alpha-mannosidosis associated mutations identifies multiple mechanisms for how these mutations are detrimental for MAN2B1 activity. The α-mannosidosis database will benefit both clinicians and scientific research on lysosomal alpha‑mannosidosis.

The regulation and function of collagenase-3 (MMP-13) in cutaneous wound healing and squamous cell carcinoma

Matrix metalloproteinase-13 (MMP-13) is a potent proteolytic enzyme, whose expression has been previously associated with fetal bone development and postnatal bone remodeling and with adult gingival wound healing. MMP-13 is also known to be involved in the growth and invasion of various cancers including squamous cell carcinoma (SCC) of the skin. The aim of this study was to further elucidate the function and regulation of MMP-13 in wound repair and cancer. In this study, it was shown that fetal skin fibroblasts express MMP-13 in response to transforming growth factor-β in a p38 MAP kinase dependent manner. In addition, MMP-13 was found to be expressed in vivo by wound fibroblasts in human fetal skin grafted on SCID mice. Adenovirally delivered expression of MMP-13 enhanced collagen matrix contraction by fibroblasts in vitro in association with altered cytoskeletal structure, enhanced proliferation and survival. These results indicate that MMP-13 is involved in cell-mediated collagen matrix remodeling and suggest a role for MMP-13 in superior matrix remodeling and scarless healing of fetal skin wounds. Using an MMP-13 deficient mouse strain, it was shown that MMP-13 is essential for the normal development of experimental granulation tissue in mice. MMP-13 was implicated in the regulation of myofibroblast function and angiogenesis and the expression of genes involved in cellular proliferation and movement, immune response, angiogenesis and proteolysis. Finally, epidermal mitogen, keratinocyte growth factor (KGF) was shown to suppress the malignant properties of skin SCC cells by downregulating the expression of several target genes with potential cancer promoting properties, including MMP-13, and by reducing SCC cell invasion. These results provide evidence that MMP-13 potently regulates cell viability, myofibroblast function and angiogenesis associated with wound healing and cancer. In addition, fibroblasts expressing MMP-13 show high collagen reorganization capacity. Moreover, the results suggest that KGF mediates the anti-cancer effects on skin SCC

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

From the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo production. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53'09" S and 48°26'42" W) - non breeding season (NBS), comprehending January to March; and breeding season (BS), August to October. Thirty queens were neutered. Ovaries were classified according to their status and were sliced in PBS for cumulus oocyte complex (COC) releasing. Grade I COC were washed three times in H-MEM supplemented with BSA, glutamine, sodium pyruvate, cysteine, streptomycin and penicillin. Oocytes were incubated in groups of 20-30 in 400µL of DMEM supplemented with FSH, LH, estradiol, IGF-I and basic fibroblast growth factor under mineral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. COC were fertilized in Ham's F-10 medium supplemented with BSA, cysteine, pyruvate and streptomycin/penicillin (culture medium) with fresh semen selected through swim up technique. Eighteen hours later, the presumptive zygotes were denuded, the percentage of cleavage was determined and every 10 zygotes were transferred to 100mL drops of culture medium for culture during three days. After 72 hours of culture the percentage of morulae formation was evaluated and these structures were transferred to drops of the same culture medium. At the eighth day of culture blastocyst formation was analyzed. During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries, they were 63.95, 39.54 and 24.41. The results of this experiment demonstrate that no statistically significant difference (P<0.05) was verified in the frequency of cleaved embryos and morulae and blastocyst formation when comparing the three ovarian conditions in the same season. However the breeding season presented better results considering cleavage and morulae and blastocyst formation.

Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Fracture modes and acoustic emission characteristics of hydrogen-assisted cracking in high-strength low-alloy steel weldment

The aim of the present paper is to study the relationship between the fracture modes in hydrogen-assisted cracking (HAC) in microalloied steel and the emission of acoustic signals during the fracturing process. For this reason, a flux-cored arc weld (FCAW) was used in a high-strength low-alloy steel. The consumable used were the commercially available AWS E120T5-K4 and had a diameter of 1.6 mm. Two different shielding gases were used (CO2 and CO2+5% H2) to obtain complete phenomenon characterization. The implant test was applied with three levels of restriction stresses. An acoustic emission measurement system (AEMS) was coupled to the implant test apparatus. The output signal from the acoustic emission sensor was passed through an electronic amplifier and processed by a root mean square (RMS) voltage converter. Fracture surfaces were examined by scanning electron microscopy (SEM) and image analysis. Fracture modes were related with the intensity, the energy and the number of the peaks of the acoustic emission signal. The shielding gas CO2+5% H2 proved to be very useful in the experiments. Basically, three different fracture modes were identified in terms of fracture appearance: microvoid coalescence (MVC), intergranular (IG) and quasi-cleavage (QC). The results show that each mode of fracture presents a characteristic acoustic signal.

Seulamurskaimen murskausominaisuuksien tutkiminen ja kehittäminen

Työn tavoitteena oli kehittää seulamurskaimen murskausprosessia selvittämällä murskaukseen vaikuttavia tekijöitä. Murskausprosessin sisäistämisen myötä voidaan saavuttaa parempi jaekoon hallinta, parantaa kapasiteettia sekä luoda tehokkaampia murskaussovelluksia. Murskausprosessia tutkittiin aluksi murskaukseen vaikuttavien teorioiden kautta. Teoriapohjaista tietoa ja murskaustapahtumaa tutkittiin käytännön testeillä, joissa testattiin seulamurskainta useilla eri materiaaleilla ja erilaisilla tuotevariaatioilla. Testeissä kerättiin näytteitä jaekoon jatkotutkimusta varten sekä saatujen tulosten pohjalta vertailtiin eri tuotevariaatioiden keskeisiä eroja. Saavutettuja tuloksia analysoitiin ja kehitettiin uusia potentiaalisia ratkaisuja seulamurskaimiin. Seulamurskaimen nykyinen rakenne on kehitetty ennen kaikkea seulontasovelluksiin ja sen toiminta perustuu rungon sisällä materiaalin pyörivään liikkeeseen. Murskausprosessissa on kuitenkin oleellista pyrkiä vähentämään sisäistä pyörintäliikettä, joka on hidasteena kappaleiden murskaukselle. Lisäksi seulamurskaimessa on useita rakenteita, jotka on kehitetty erityisesti seulontaa varten ja niiden uudenlaisella rakennesuunnittelulla voitaisiin saavuttaa huomattavasti parempia tuloksia murskauksen kannalta. Nämä tekijät kuitenkin vaativat huomattavan paljon tehoa, mikä puolestaan saattaa asettaa uusia haasteita suunnitteluun, sillä seulamurskaimessa käytettävissä oleva teho on varsin rajallinen.

Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS) located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST)-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

A liver metalloendopeptidase which degrades the circulating hypotensive peptide hormones bradykinin and atrial natriuretic peptide

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human liver using successive steps of chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-200. The purified enzyme hydrolyzed the Pro7-Phe8 bond of bradykinin and the Ser25-Tyr26 bond of atrial natriuretic peptide. No cleavage was produced in other peptide hormones such as vasopressin, oxytocin or Met- and Leu-enkephalin. This enzyme activity was inhibited by 1 mM divalent cation chelators such as EDTA, EGTA and o-phenanthroline and was insensitive to 1 µM phosphoramidon and captopril, specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. With Mr 85 kDa, the enzyme exhibits optimal activity at pH 7.5. The high affinity of this endopeptidase for bradykinin (Km = 10 µM) and for atrial natriuretic peptide (Km = 5 µM) suggests that it may play a physiological role in the inactivation of these circulating hypotensive peptide hormones.

The photodynamic and non-photodynamic actions of porphyrins

Porphyrias are a family of inherited diseases, each associated with a partial defect in one of the enzymes of the heme biosynthetic pathway. In six of the eight porphyrias described, the main clinical manifestation is skin photosensitivity brought about by the action of light on porphyrins, which are deposited in the upper epidermal layer of the skin. Porphyrins absorb light energy intensively in the UV region, and to a lesser extent in the long visible bands, resulting in transitions to excited electronic states. The excited porphyrin may react directly with biological structures (type I reactions) or with molecular oxygen, generating excited singlet oxygen (type II reactions). Besides this well-known photodynamic action of porphyrins, a novel light-independent effect of porphyrins has been described. Irradiation of enzymes in the presence of porphyrins mainly induces type I reactions, although type II reactions could also occur, further increasing the direct non-photodynamic effect of porphyrins on proteins and macromolecules. Conformational changes of protein structure are induced by porphyrins in the dark or under UV light, resulting in reduced enzyme activity and increased proteolytic susceptibility. The effect of porphyrins depends not only on their physico-chemical properties but also on the specific site on the protein on which they act. Porphyrin action alters the functionality of the enzymes of the heme biosynthetic pathway exacerbating the metabolic deficiencies in porphyrias. Light energy absorption by porphyrins results in the generation of oxygen reactive species, overcoming the protective cellular mechanisms and leading to molecular, cell and tissue damage, thus amplifying the porphyric picture.

Characterization of six rat strains (Rattus norvegicus) by mitochondrial DNA restriction fragment length polymorphism

Restriction fragment length polymorphism (RFLP) was used to examine the extent of mtDNA polymorphism among six strains of rats (Rattus norvegicus) - Wistar, Wistar Munich, Brown Norway, Wistar Kyoto, SHR and SHR-SP. A survey of 26 restriction enzymes has revealed a low level of genetic divergence among strains. The sites of cleavage by EcoRI, NcoI and XmnI were shown to be polymorphic. The use of these three enzymes allows the 6 strains to be classified into 4 haplotypes and identifies specific markers for each one. The percentage of sequence divergence among all pairs of haplotypes ranged from 0.035 to 0.33%, which is the result of a severe population constriction undergone by the strains. These haplotypes are easily demonstrable and therefore RFLP analysis can be employed for genetic monitoring of rats within animal facilities or among different laboratories.