Dissertationen zum Thema „Isoenzymes Analysis“

Lettuce, in its cultivated form (Lactuca sativa L.), is a crop of economic importance with several characteristics which make it well suited for biochemical and genetic studies. Biochemical traits such as proteins and enzymes have been studied extensively in many plant species and constitute an important experimental approach to physiological, evolutionary, taxonomic and breeding studies. Seed proteins were extracted from an array of lettuce cultivars. Polyacrylamide gel electrophoresis was employed in a comparative analysis of the soluble protein and isozyme characteristics of seed from each cultivar. Some fall-desert, winter-desert, and coastal cultivars were distinguishable based upon variation in soluble proteins or esterase isozymes. Multiple forms of the carboxylic ester hydrolases or esterases have been shown to occur in a wide variety of plants; their role in the plant cell is still however poorly understood. The inheritance of the esterase isozymes was analyzed by a microelectrophoresis technique which enabled the analysis of individual seeds in the progeny from a cross involving two winter-desert cultivars of contrasting banding phenotypes. Two distinct banding patterns were observed in single seeds of these cultivars; their F₁ hybrid showed a summation of parental patterns, the F₂ segregated in a 1:2:1 phenotypic ratio for these esterase patterns, and backcross segregation ratios were 1:1. Banding pattern differences could be accounted for by the segregation of a single gene with codominant action. Quantitative as well as qualitative differences in esterase activity were observed between the parental lines from two different years of production. Some esterase isozymes were developmentally regulated. Molecular weight determination experiments verified the presence of two gene products of 56 and 62.5 Kd.

Allozyme frequencies and general protein patterns were surveyed among selected Texas marine fishes and shellfishes to illustrate the application of biochemical genetic techniques to stock and species identification in fisheries management.

The Leishmania donovani complex comprises four described species: L. donovani, L. archibaldi, L. infantum and L. chagasi. L. chagasi is the only New World species and has been considered similar to L. infantum, although some authors insist on maintenance of its independent species status. L. donovani has at least two major epidemiological subgroups whose relationships are poorly understood. In this thesis, molecular biological techniques were used to investigate the taxonomy and phylogenetic relationships within the L. donovani complex, with isoenzyme analysis (lEA) as reference technique. Random amplification of polymorphic DNA (RAPD) was used to provide anonymous genetic markers which allowed overall comparisons of genomes. Selected target genes and intergenic regions were also amplified by the polymerase chain reaction (PCR), namely the major surface protease (msp or gp63), the mini-exon and the ribosomal internal transcribed spacer (ITS). PCR products of intergenic regions between msp genes (ITG/CS and ITG/L), mini-exon and ITS were analysed by restriction fragment length polymorphism (RFLP). Phylogenies generated from each of the methods were compared with that of IEA. L. infantum and L. chagasi were found to be synonymous, whilst L. donovani was found to be more polymorphic than L. infantum and a fourth possible species in the complex, L. archibaldi, was not supported. Six genetic groups of strains were identified in the L. donovani complex, based on all DNA based analyses, which agreed with IEA typing. Pooled data from RFLP and RAPD analyses generated robust phylogenies which were congruent with ITG/CS RFLP and msp DNA sequence based phylogenies, but not with lEA phylogenies. The evolutionary history of the L. donovani complex is analysed in the light of the present results. The diverse typing methods were also evaluated and genetic markers suggested, that are applicable to classification and typing of L. donovani species and strains.

Over the last decade, the innate immune system has been the subject of extensive research. Often overlooked by the robustness and specificity of the adaptive immune system, the innate immune system is proving to be just as complex. The identification of several families of pattern recognition receptors (PRRs) has revealed an ancient yet multifaceted system of proteins that are responsible for initiating host defense. A wide array of pathogens, from virus to bacteria, is detected using this assortment of receptors. One such family, the Toll-like receptors (TLRs), has been at the forefront of this research. To date, 10 TLRs have been described in the human genome. Activation of TLRs leads to the induction of immune-related genes that ultimately control the response of the host. However, the signaling pathways emanating from activated TLRs and other PRRs are not fully understood. In particular, the pathway leading to the activation of interferon regulatory factor 3 (IRF3), a transcription factor crucial for the induction of type I interferon, remains undefined. IRF3 activation occurs as the consequence of viral infection and through the activation of TLRs 3 and 4 by dsRNA and lipopolysaccharide (LPS), respectively. The focus of this research is to describe components of the IRF3 activation pathway, partly through the analysis of TLR signal transduction. IRF3 normally resides in the cytoplasm of cells. Upon infection with certain viruses and bacteria, IRF3 is activated though phosphorylation at its C-terminus. Phosphorylated IRF3 homodimerizes and associates with co-activators CBP-p300. After translocating to the nucleus, the activate IRF3 complex induces the activation of type 1 interferon and interferon related genes. Little is known about the pathways that lead to the activation of IRF3, especially the kinases involved. In this study we report that the non-canonical IкB kinase homologues, IкB kinase epsilon (IKKε) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-кB activation, are also essential components of the IRF3 signaling pathway. In particular, mouse embryonic fibroblasts from TBK1 deficient mice fail to activate IRF3 in response to both viral infection and stimulation with LPS or poly (IC), a dsRNA analog. Thus, both IKKε and TBK1 play a critical role in innate immunity and host defense. In addition to viral infection, IRF3 activation also occurs via the activation of TLR3 and 4. TLRs signal through a subfamily of Toll-IL-1-Resistance (TIR) domain containing adapter molecules. One such adapter, MyD88, is crucial for all TLRs, with the exception of TLR3. MyD88 participates in a signal transduction pathway culminating in the activation of the transcription factor NF-кB. Studies from MyD88-deficient mice reveal that both TLR3 and 4 still are capable of activating NF-кB, although with slightly delayed kinetics. Another aspect of the MyD88-independent signal transduction pathway is the activation of IRF3. A second TIR domain containing adapter molecule called Mal/Tirap was discovered and originally thought to mediate the MyD88-independent pathway. However, Mal-deficient mice were found to be defective in both TLR2 and 4 mediated NF-кB activation. We hypothesized that other TIR domain containing adapters could mediate this MyD88-independent pathway of TLR3 and 4 leading to the activation of IRF3. Two additional TIR adapters were discovered, TRIF and TRAM. TRIF was shown to mediate TLR3 signal transduction. In this study, we report that both TRIF and TRAM mediate the activation of the MyD88-independent pathway in response to LPS/TLR4 activation. Unlike any of the other known TIR domain containing adapters, TRAM appears to be restricted to the LPS/TLR4 activation pathway while TRIF plays a role in both TLR3 and TLR4 pathways leading to IRF3 target gene expression. Our studies revealed that TRAM could be acting upstream of TRIF in the LPS/TLR4 pathway. To this end, we sought to determine the localization of TRAM within the cell. We found that TRAM localizes to the plasma membrane. TRAM localization is the result of myristoylation since mutation of the predicted myristoylation site (G2A) resulted in the re-distribution of TRAM from the membrane into the cytoplasm. Reconstitution of TRAM-deficient macrophages with TRAM G2A is unable to rescue LPS/TLR4 signal transduction. Thus, myristoylation and membrane association of TRAM are critical for LPS/TLR4 signal transduction. The data generated in this dissertation extends our understanding of the signaling pathways of the innate immune system. Indeed, the molecules and pathways described herein could prove to be beneficial targets for ameliorating symptoms of disease, both autoimmune and pathogen-associated. Finally, the research described here will spur further insight into the complex signaling pathways of a once ignored arm of the immune system.

Giardia lamblia, an enteric protozoan parasite, infects a large number of individuals worldwide. In South Africa prevalences ranging between 4 and 63% are documented, however, the impact of giardiasis is underreseached in this country. Giardia infections vary from asymptomatic carriage or a self-limiting acute symptomatic illness to chronic, debilitating malabsorption syndrome. The factors responsible for development of symptomatic versus asymptomatic infection are poorly understood. It is believed by some that host factors determine the clinical outcome of infection. On the other hand, the possibility of the existence of pathogenic and non-pathogenic strains (a situation akin to Entamoeba spp.) remains to be explored. One requirement for investigation of the potential contribution of strain differences to pathogenecity of infection is establishment of laboratory cultures of different strains isolated from symptomatic and asymptomatic patients. The present study was undertaken to develop and modify existing methods for: (i) establishment of laboratory cultures of Giardia trophozoites from excystation of faecal cysts, (ii) long-term maintenance and cryopreservation of the cultures and (iii) preliminary characterisation methodology. One thousand and twenty-three stool specimens were collected from day care centres, hospital wards and Hlabisa hospital laboratory. A further 6246 were retrieved from the Microbiology Laboratory at King Edward VIII Hospital and screened by direct wet preparation. Giardia was detected by light microscopy following formol-ether concentration (127 of 1023 samples) or direct examination of wet preparations (78 of 6246 samples). Cysts were purified from the positive specimens by sucrose gradient separation. Viability was assessed by a dye-exclusion method (eosin). Three in vitro excystation techniques were employed in an attempt to obtain trophozoites for initiation and establishment of viable cultures thereof. Culture conditions were optimised using two reference strains of Giardia, WB & H7 (obtained from the National Institutes of Health, USA). The percentage excystation ranged between 0-42% with all the in vitro methods of excystment. Excysted trophozoites remained viable in TYI-S-33 culture medium for periods ranging between 12-72 hours or up to 9 days, and gradually died, hence viable trophozoite cultures could not be established. Some culture initiates (overall 65%) were lost through overwhelming bacterial and!or fungal contaminants. An animal model was subsequently set up in which C57BL/6 and Praomys (Mastomys) coucha mice were used for in vivo excystation experiments. 1-3 day old suckling mice were intragastrically injected with 10,5 -cysts/ ml in 0,1 ml distilled water. Trophozoites were retrieved from the stomachs of infected mice 7-10 days after inoculation and cultivated in TYI-S-33 medium. Six local isolates were axenised using the in vivo excystation method. They have been maintained for more than 15 months in culture after stabilates and Iysates of confluent growths had been cryopreserved in Liquid Nitrogen. Successful (100%) retrieval of the cryopreserved cultures has been achieved. Seven isoenzyme electrophoresis systems have been set up and optimised. Reproducible results were obtained in six of the enzymes. Some differences in banding patterns of the enzymes were demonstrated.
Thesis (M.Med.Sc.)-University of Natal, Durban, 1999.

Bibliography: leaves 143-150.
xxii, 150 leaves : col. ill. ; 30 cm.
Isozyme analysis was carried out on sweet cherry (Prunus avium) leaves. Cultivars were identified and compared. Progeny from controlled hybridisations were examined to determine inheritance patterns of isozymes. Isozymes were also used to determine gene flow in cherry orchards and to determine pollen donors of selected cultivars.
Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, (1996?)

Indiana University-Purdue University Indianapolis (IUPUI)
Seven human alcohol dehydrogenase genes (which encode the primary enzymes involved in alcohol metabolism) are grouped into classes based on function and sequence identity. While the Class I ADH isoenzymes contribute significantly to ethanol metabolism in the liver, Class IV ADH isoenzymes are involved in the first-pass metabolism of ethanol. It has been suggested that the ability to efficiently oxidize ethanol occurred late in primate evolution. Kinetic data obtained from the Class I ADH isoenzymes of marmoset and brown lemur, in addition to data from resurrected ancestral human Class IV ADH isoenzymes, supports this proposal--suggesting that two major events which occurred during primate evolution resulted in major adaptations toward ethanol metabolism. First, while human Class IV ADH first appeared 520 million years ago, a major adaptation to ethanol occurred very recently (approximately 15 million years ago); which was caused by a single amino acid change (A294V). This change increases the catalytic efficiency of the human Class IV enzymes toward ethanol by over 79-fold. Secondly, the Class I ADH form developed 80 million years ago--when angiosperms first began to produce fleshy fruits whose sugars are fermented to ethanol by yeasts. This was followed by the duplication and divergence of distinct Class I ADH isoforms--which occurred during mammalian radiation. This duplication event was followed by a second duplication/divergence event which occurred around or just before the emergence of prosimians (some 40 million years ago). We examined the multiple Class I isoforms from species with distinct dietary preferences (lemur and marmoset) in an effort to correlate diets rich in fermentable fruits with increased catalytic capacity toward ethanol oxidation. Our kinetic data support this hypothesis in that the species with a high content of fermentable fruit in its diet possess greater catalytic capacity toward ethanol.

Indiana University-Purdue University Indianapolis (IUPUI)
The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.

Indiana University-Purdue University Indianapolis (IUPUI)
ALDH isoenzymes are known to impact the sensitivity of certain neoplastic cells toward cyclophosphamides and its analogs. Despite its bone marrow toxicity, cyclophos-phamide is still used to treat various recalcitrant forms of cancer. When activated, cyclo-phosphamide forms aldophosphamide that can spontaneously form the toxic phospho-ramide mustard, an alkylating agent unless detoxified by ALDH isozymes to the carbox-yphosphamide metabolite. Prior work has demonstrated that the ALDH1A1 and ALDH3A1 isoenzymes can convert aldophosphamide to carboxyphosphamide. This has also been verified by over expression and siRNA knockdown studies. Selective small molecule inhibitors for these ALDH isoenzymes are not currently available. We hypothe-sized that novel and selective small molecule inhibitors of ALDH3A1 would enhance cancer cells’ sensitivity toward cyclophosphamide. If successful, this approach can widen the therapeutic treatment window for cyclophosphamides; permitting lower effective dos-ing regimens with reduced toxicity. An esterase based absorbance assay was optimized in a high throughput setting and 101, 000 compounds were screened and two new selective inhibitors for ALDH3A1, which have IC50 values of 0.2 µM (CB7) and 16 µM (CB29) were discovered. These two compounds compete for aldehyde binding, which was vali-dated both by kinetic and crystallographic studies. Structure activity relationship dataset has helped us determine the basis of potency and selectivity of these compounds towards ALDH3A1 activity. Our data is further supported by mafosfamide (an analog of cyclo-phosphamide) chemosensitivity data, performed on lung adenocarcinoma (A549) and gli-oblastoma (SF767) cell lines. Overall, I have identified two compounds, which inhibit ALDH3A1’s dehydrogenase activity selectively and increases sensitization of ALDH3A1 positive cells to aldophosphamide and its analogs. This may have the potential in improving chemotherapeutic efficacy of cyclophosphamide as well as to help us understand better the role of ALDH3A1 in cells. Future work will focus on testing these compounds on other cancer cell lines that involve ALDH3A1 expression as a mode of chemoresistance.

Face às dúvidas que persistem sobre a origem híbrida dos sobreiros de cortiça preguenta”, a qual é argumento incontornável na discussão recorrente sobre os povoamentos mistos de sobreiro e azinheira em Portugal, e para obter estimativas credíveis da taxa de hibridação nestes povoamentos, estabeleceram-se critérios genéticos de discriminação entre as duas espécies e os híbridos. O presente trabalho documenta os seguintes progressos nessa investigação: I. Descoberta e validação de marcadores isoenzimáticos para a distinção entre sobreiro e híbridos ou descendentes destes; II. Confirmação do estatuto híbrido de árvores conhecidas como carvalhos cerqueiros através de marcadores genéticos; III. Estimativa duma taxa de hibridação muito baixa (inferior a 0,1%), nas parcelas mistas estudadas; IV. Detecção em sobreiros como em azinheiras de marcadores de introgressão da outra espécie; V. Análise preliminar de sobreiros de cortiça preguenta; VI. Análise preliminar das descendências de híbridos, incluindo a demonstração de que intervêm grãos de pólen de qualquer uma das espécies parentais, assim como uma taxa relativamente elevada de aparentes autopolinizações; VII. Análise preliminar das frequências alélicas em dois loci marcadores, um de cada espécie, e dedução dum valor neutral de Ne relativamente baixo (entre 5 e 7); VIII. Demonstração da mesma metodologia noutras espécies do género Quercus e táxones aparentados. Embora ainda sem carácter definitivo, os valores estimados da taxa de hibridação entre sobreiro e azinheira indicam tratar-se dum evento geralmente excepcional, podendo admitir-se que localmente seja mais frequente. O método desenvolvido pode ser aplicado em larga escala para a detecção de híbridos em materiais de propagação de sobreiro, com custo relativamente baixo, e pode ainda contribuir para o esclarecimento de várias questões relacionadas com a biologia da reprodução das duas espécies e dos híbridos entre elas, na continuação do que o presente trabalho estudou preliminarmente.