No description available.

Thesis (doctoral)--Rijksuniversiteit Groningen, 1996. / Includes bibliographical references (p. [109]-119).

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Abstract Secretory carbonic anhydrase isoenzyme VI (CA VI) catalyses the reversible hydration of carbon dioxide (CO2 + H2O ↔ HCO3- + H+). Low concentrations of salivary CA VI are associated with high decayed, missing or filled teeth (DMFT) index scores and a high incidence of acid injury in the upper gastrointestinal tract plus lowered taste and smell perception. Two mechanisms of action for CA VI have been proposed: acid neutralisation and growth factor function. In the present study the distribution and catalytic properties of CA VI have been examined in order to further clarify its mechanisms of action and biological significance. CA VI was found to be present and secreted by the alveolar epithelium of the mammary gland, serous acinar cells of lingual von Ebner’s glands, serous demilune cells of posterior lingual mucous glands and serous cells of submucosal tracheobronchial glands. CA VI was also found in the serous cells in the tracheobronchial mucosal epithelium, taste pore, taste bud, base of the tracheobronchial cilia, bronchiolar Clara cells and enamel pellicle. An immunofluorometric assay showed that the mean concentration of CA VI in colostral milk was eight times higher than that in mature milk (35 mg/l vs. 4.5 mg/l). Stopped-flow spectroscopy measurements revealed that the dehydration activity of CA VI is moderate (maximum kcat = 3.0 × 105 · s-1). The finding that CA VI is a potent catalyst of acid neutralisation emphasizes the possible role of the pellicle bound CA VI in local neutralisation of the acidic metabolic products of dental biofilm. The function of CA VI in von Ebner’s glands’ saliva is likely taste stimuli modification via CA activity although other functions may exist. Its role in milk or respiratory tract mucus remains open, however, as these secretions do not have significant acid predispositions that would need enzymatic catalysis for removal.

M.Sc. / To successfully intervene in the HIV/AIDS pandemic, knowledge of the pathogenesis of the disease and factors that stimulate or inhibit viral replication are crucial. Plants are expected to produce antiviral compounds since viruses form one of the major groups of plant pathogens. The objective in this project was to investigate the effects of 6 plant extracts on immune responses as well as evaluate their potential anti-HIV activity. Plant species tested were: Hypoxis hemerocal/idea, Elephantorrhiza elephantina, Spirulina platensis, Echinacea purpurea, Echinacea pal/ida and Cannabis sativa. Extracts were prepared via 24 hour extraction or 12 hour reflux in H20, methanol and ethanol in a 1:5 ratio. The crude extracts were analysed by TLC and HPLC and shown to consist of complex related mixtures of compounds. Using LC-MS, partial identification of methanol extracts revealed the following expected compounds: 9- octadecenoic acid (E)- in Hypoxis hemerocallidea, 4H-1-benzopyran-4-one,2-(3,4- dihydroxyphenyl)-7 -(13-D-glucopyranosyloxy)-5-hydroxy- in E/ephantorrhiza e/ephantina, ethanol,2-butoxy-,phosphate in Spirulina platensis, 2-propenoic acid,3-(3,4-dihydroxyphenyl)- in Echinacea purpurea, 2-propenoic acid,3-(3,4-dihydroxyphenyl)- in Echinacea pal/ida and ~-9- tetrahydrocannabivarin in Cannabis sativa tincture. Viability assays using tetrazolium salts (XTT) gave a qualitative picture of events allowing us to assess host cell responses and extract toxicity. Extracts exhibited intrinsic absorbances at some visible wavelengths but did not interfere at the wavelengths used in this viability assay. Having analysed cell viability it was thought prudent to report on the type of cell death induced by either HIV or the extracts, so Annexin-V (indicator of apoptosis) and PI (indicator of necrosis) detected by flow cytometry was employed. Results obtained revealed that cells were driven towards necrosis rather than apoptosis. None of the extracts showed significant in vitro toxicity in CEMss, CEMNKR. U937, Jurkat and PM1 cells or ex vivo in PBMC at a concentration range of 1000f..lg/ml-4f.!g/ml. Viability assays were also an indirect indication of HIV's effect on the cells. As for the effect of extracts on the immune system, IL-2 secretion was stimulated by most of the extracts. The effect of plant extracts on HIV activity was also investigated by looking at core protein levels (p24 was generally decreased by methanol extracts), reverse transcriptase activity (no detectable influence) and envelope glycoprotein levels (gp120 levels were only marginally reduced). It appears that Echinacea purpurea, Echinacea pal/ida and Spirulina platensis have immune enhancing abilities, while Hypoxis hemerocallidea, Elephantorrhiza e/ephantina and Cannabis sativa have dual purposes by enhancing both immunity and inhibiting HIV activity.

The acquired immunodeficiency syndrome (AIDS) in humans, which is caused by the human immunodeficiency virus type 1 (HIV-1) remains among the leading causes of death worldwide. Although HAART has reduced HIV mortality significantly, adhering to the recommended drug schemes, significant toxicities experienced by treated patients, and the high mutation rate of the virus that seem to easily circumvent the action of these drugs emphasize the need for alternative treatment strategies. Medicinal plants are a good source for the discovery of novel antimicrobial chemotherapeutic agents. Reverse transcription is the most essential step for viral replication to succeed successfully. This makes reverse transcriptase the prime target for antiviral therapy against HIV. Emphasis was placed on the discovery of plants with inhibitory activity against HIV-1 reverse transcriptase. Crude extracts from the active plant(s) was screened in vitro for their ability to suppress HIV replication in suitable cell systems. The potential of isolating and identifying the active principle(s) was also investigated. Crude extracts from different parts of Gunnera perpensa showed similar amounts of inhibition: aqueous extracts (97% „b 0.110%SD), methanol/chloroform extracts (94% „b 2.374%SD), rhizome extracts (96% „b 0.475%SD), stem extracts (94% „b 3.723%SD), leaf extracts (96% „b 1.097% SD). Crude extracts were found to be significantly (P„T0.027) non-toxic to CEM.NKR.CCR5 cells and PBMCs at 5 ƒÝg/ml. In acutely infected CEM.NKR.CCR5 cells, acutely infected PBMCs, and chronically infected PBMCs Gunnera perpensa extracts did not significantly (P>0.05) increase cell viability or reduced HIV core protein content, over 4 days. The in vitro test did therefore not reflect the findings with the reverse transcriptase assay. Activity-guided fractionations of Gunnera perpensa rhizome extract lead to the collection of a significantly active fraction. NMR studies revealed the presence of an epimeric mixture of glucose ¡§contaminants¡¨ in this active fraction. The presence of these ¡§contaminants¡¨ concealed the true structure of the active principle. Gunnera perpensa was identified as containing a potential active principle that significantly inhibits recombinant HIV reverse transcriptase. Unfortunately, in vitro experiments could not confirm this finding. The identity and structure of the active principle remains unidentified. Future studies will be concerned with in vitro antiviral studies of the pure active principle. Furthermore, preliminary tests indicated that some of the original collection of crude extracts had anti-bacterial and anti-malarial activities. These findings can be investigated in future. / Dr. D. Meyer

No description available.

A detailed study was made in this doctoral thesis on the development and differentiation of the vertebrate pituitary gland, with the aim to establish a fate map in Rathke's pouch of the origin of different hormone producing cells present in the adult pituitary gland, that explain if the differences observed in the distribution pattern of different hormone producing cells in the adult is the consecuence of differences in their development. For this reason, the study was made in two vertebrate groups, Mammals and Avian, that present notable differences in their hormone producing cell distribution patterns. The results allowed us to conclude that the origin of different hormone producing cells in Rathke’s pouch determine their definitive distribution in the adult gland. At the same time, the relationship between proliferation and differentiation was studied, showing us that after differentiation, hormone producing cells continue proliferating with a low rate, contributing to the establishment of differentiated populations. Using immunochemicals and in situ hidridization techniques, the expression of different molecules such as hypothalamic releasing factors; different peptides, whose role as modulators in different pituitary axis have been proposed in the adult animal; different calcium binding proteins and transcription factors in relation to the differentiation of different hormone producing cells, was also studied in this work, allowing us to establish different relationships between some of these factors and specific aspects of the development and differentiatin of the pituitary gland.

Historically, the causative agent of infectious coryza has been identified as the NAD requiring bacterium Haemophilus paragallinarum and the implementation of an intensive vaccination program led to the effective control of this contagious upper respiratory infection. More recently, however, a decline in the protective capacity of a vaccine conditioned immune response was noted, with a number of contributing factors, including the emergence of a fast-growing NAD-independent bacterium, which has largely replaced the traditional NAD-dependent variety. As such, accurate, reproducible methods for determining and continually monitoring the type of infecting bacteria was necessitated. To address this need, strains of H. paragallinarum were evaluated according to both their phenotypic and their genotypic properties, in a combination serodiagnostic approach. A data bank of NAD-dependent H. paragallinarum reference strain and field isolate serovar-specific fingerprints was established on both a whole cell and outer membrane protein level. Visual comparative analysis of the qualitatively and quantitatively similar outer membrane protein patterns of all strains of NAD independency studied with the formulated data bank, indicate that the NAD-independent strains displayed profiles typical of serovar C-3. The outer membrane proteins have been identified as putative virulence determinants and, as such, were characterised according to their surface location, susceptibility to heat modification, functional role as endotoxins, sequence homology to structural membrane counterparts, and finally, their ability to induce an immune response. These studies represent novel efforts and form the foundation for identifying those antigens responsible for maintaining an infection in the host milieu. Ribotype analysis served as an adjunct to phenotypic observations, with the local NAD-independent field isolates being identified as serotype A. These contradictory outcomes call for the creation of a set of reference strains specific for NAD-independent isolates. The identification of restriction fragment length polymorphisms in the conserved 16S rRNA gene sequences indicate the potential application of this method for type assignment, requiring the recognition of a battery of versatile restriction enzymes to generate serovar-specific polymorphic profiles. The complexity of serotype allocation demands that a combination approach in which genotypic analyses complement phenotypic-based methods of haemagglutination inhibition and outer membrane protein profiling. The groundwork for implementation of such a system has been accomplished. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

The complement system is an important immune system mechanism involved in both the recognition and elimination of invading pathogens. It is activated by three different pathways: The classical pathway, which relies on binding of C1, and results in the cleavage of C4 and C2 through activation of C1r and C1s; the alternative pathway that relies on the spontaneous hydrolysis of C3 and the lectin pathway. The lectin pathway is activated by binding of Mannan-binding lectin (MBL) or the ficolins (L-ficolin, H-ficolin and M-ficolin) to microbial binding motifs, and the subsequent activation of the MBL-associated serine proteases (MASP) 1/ 2/ 3. Of these MASP2 has been identified as the enzyme responsible for the activation of complement by C4 and C2 cleavage. The work presented here will focus on four different aspects of the lectin pathway: specificity and stoichiometry of the L-ficolin protein complex, expression of H-ficolin, substrate characterization for MASP1 and investigation of the prothrombin activation potential of MASP2. L-ficolin binding specificity was investigated using glycan array technology, and it was found that L-ficolin, instead of recognizing single monosaccharides like MBL, instead binds to extended oligosaccharide structures. The binding to these was dependent not only on the presence of acetyl groups, but also on their orientation in space. It was also found that L-ficolin in serum is found as a multimeric protein complex composed of 18 polypeptide chains and associated with one MASP dimer. The expression of H-ficolin resulted in the generation of a stable mammalian cell line producing oligomerized and biologically functional H-ficolin. MASP1 substrate specificity was investigated by two different procedures. Firstly fractionated plasma was subjected to MASP1 treatment in an attempt to identify a plasma protein substrate. This did not yield any substrate candidates, since only cleavage of the protease inhibitor α-2-macroglobulin could be detected. Additionally the thrombin-like activity of MASP1 was investigated through cleavage experiments done with factor XIII and fibrinogen. These experiments showed that the factor XIII cleavage site for MASP1 and thrombin is identical. This was also found for the fibrinogen β-chain but not for the α-chain showing that MASP1 interaction with fibrinogen is distinct from that of thrombin. An earlier observation that MASP2 was capable of activating prothrombin and generating thrombin was further characterized. Here it was shown that the activation of prothrombin by MASP2 is identical to that by factor Xa, which is the enzyme undertaking this role in the coagulation system, and that the activation can result in deposition of fibrin on the surface upon which MASP2 is bound. The prothrombin activation potential of MASP2 was also utilized to develop a new MASP2 activity assay, which was shown to be capable of measuring MASP2 activity, when MASP2 is bound, via MBL (or L-ficolin) to appropriate surfaces.