A possible mechanism for enzymic depilation of skins
- Authors: Brady, Dean
- Date: 1989
- Subjects: Chemistry, Technical , Tanning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3877 , http://hdl.handle.net/10962/d1001611
- Description: Streptomyces fradiae is a bacterium which has been previously found to produce extracellular enzymes which are capable of wool degradation and skin depilation. Streptomyces fradiae 3739 and other strains of Streptomyces were found in this study to be able to degrade a keratin source (wool) to a considerable degree. However according to the evidence of SEM micrographs presented here the highly keratinised spindle cells of the paracortex are fairly resistant to protease attack, and it is the cementation material which binds these cells together which is initially degraded by the proteases. A large degree of correlation was found with the strains of Streptomyces studied, between the ability of the individual strains to degrade wool and the ability of their extracellular proteases to reduce the depilation load of sheepskins. With further analysis S. fradiae 3739 was found to produce at least one amylase and four or more proteases. The proteases as a group had maximal proteolytic activity in the 8.0-9.0 pH unit range, and were considerably thermostabilised by the inclusion of calcium ions into the reaction solution. The protease group was found to cause depilation of merino sheepskins. For comparative purposes a protease produced by a strain of Proteus vulgaris isolated from a staling hide with hair slip (natural depilation) was studied. The protease activity was maximal in the alkaline region between 8.0-9.0 pH units. Tbe protease appeared to be a single enzyme with a molecular mass of approximately 44 000 daltons. The protease was maximally active at 40°C, although it was only thermostable to 30°C. The enzyme was ineffectual as a depilant except when the skin was pre-treated with a strong alkali, preferably including sodium sulphite in the protease preparation. One of the most important differences between the extracellular proteases of S. fradiae and P. vulgaris was that the former were greater in variety and caused a greater decrease in the depilation load of sheepskins than the latter. Further research with mixtures of commercial proteases provided evidence that a synergistic depilatory effect occurs when proteases of complementary bond specificities are used in conjunction in enzymic depilatory preparations. Some form of strong alkali treatment of skins was found to be necessary to produce leather of the prerequisite quality when the skin was depilated by proteases, otherwise the skin was found to be depleted and stiff. Calcium hydroxide alone was found to be inadequate for this task, probably owing to the fact that it is less alkaline than the lime-sulphide mixture. The calcium hydroxide (lime) must therefore be used in conjunction with sodium hydroxide (which makes the solution as alkaline as that of the lime-sulphide solution) to produce leather comparable to that produced by the lime sulphide treatment. A combination of the information provided by the present research and that gleaned from the relevent literature allows for the construction of a model to represent the possible mechanism of enzymic depilation of skins, in which depilation is caused by the disruption of the basement membrane at the dermal-epidermal junction by the degradation of its constituent molecular components by general proteases, resulting in the removal of the epidermis and its associated wool or hair
- Full Text:
- Authors: Brady, Dean
- Date: 1989
- Subjects: Chemistry, Technical , Tanning
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3877 , http://hdl.handle.net/10962/d1001611
- Description: Streptomyces fradiae is a bacterium which has been previously found to produce extracellular enzymes which are capable of wool degradation and skin depilation. Streptomyces fradiae 3739 and other strains of Streptomyces were found in this study to be able to degrade a keratin source (wool) to a considerable degree. However according to the evidence of SEM micrographs presented here the highly keratinised spindle cells of the paracortex are fairly resistant to protease attack, and it is the cementation material which binds these cells together which is initially degraded by the proteases. A large degree of correlation was found with the strains of Streptomyces studied, between the ability of the individual strains to degrade wool and the ability of their extracellular proteases to reduce the depilation load of sheepskins. With further analysis S. fradiae 3739 was found to produce at least one amylase and four or more proteases. The proteases as a group had maximal proteolytic activity in the 8.0-9.0 pH unit range, and were considerably thermostabilised by the inclusion of calcium ions into the reaction solution. The protease group was found to cause depilation of merino sheepskins. For comparative purposes a protease produced by a strain of Proteus vulgaris isolated from a staling hide with hair slip (natural depilation) was studied. The protease activity was maximal in the alkaline region between 8.0-9.0 pH units. Tbe protease appeared to be a single enzyme with a molecular mass of approximately 44 000 daltons. The protease was maximally active at 40°C, although it was only thermostable to 30°C. The enzyme was ineffectual as a depilant except when the skin was pre-treated with a strong alkali, preferably including sodium sulphite in the protease preparation. One of the most important differences between the extracellular proteases of S. fradiae and P. vulgaris was that the former were greater in variety and caused a greater decrease in the depilation load of sheepskins than the latter. Further research with mixtures of commercial proteases provided evidence that a synergistic depilatory effect occurs when proteases of complementary bond specificities are used in conjunction in enzymic depilatory preparations. Some form of strong alkali treatment of skins was found to be necessary to produce leather of the prerequisite quality when the skin was depilated by proteases, otherwise the skin was found to be depleted and stiff. Calcium hydroxide alone was found to be inadequate for this task, probably owing to the fact that it is less alkaline than the lime-sulphide mixture. The calcium hydroxide (lime) must therefore be used in conjunction with sodium hydroxide (which makes the solution as alkaline as that of the lime-sulphide solution) to produce leather comparable to that produced by the lime sulphide treatment. A combination of the information provided by the present research and that gleaned from the relevent literature allows for the construction of a model to represent the possible mechanism of enzymic depilation of skins, in which depilation is caused by the disruption of the basement membrane at the dermal-epidermal junction by the degradation of its constituent molecular components by general proteases, resulting in the removal of the epidermis and its associated wool or hair
- Full Text:
Isolation, purification and effect of ligands on the nicotinic cholinergic receptor
- Authors: Kapp, Eugene Anthony
- Date: 1989
- Subjects: Ligands (Biochemistry) , Nicotinic receptors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4550 , http://hdl.handle.net/10962/d1018235
- Description: The nicotinic cholinergic receptor protein of the fish electric organ, Torpedo fuscomaculata, has been isolated, purified and shown to represent a true model for the nAChR from other species and higher vertebrates. It is an integral membrane protein composed of four different subunits, tightly associated with other functional, but non-specific proteins. Purification of the nicotinic cholinergic receptor by chromatofocusing demonstrates an improved method over that of affinity and ion-exchange chromatography. Gel chromatography and SDS-polyacrylamide gel electrophoresis show evidence of four subunits; a(40-44 kDa), 6(53 kDa ),'Y(63 kDa) and 6(66 kDa) despite some degradation of receptor molecules by intracellular proteases. Spectrophotometric and fluorimetric studies of receptor-ligand interactions, show the functional and chemical integrity of the receptor to remain intact after solubilisation. The effect of cholinergic ligands on purified receptor preparations indicate quenching of the intrinsic fluorescence of the receptor. Agonists, like acetylcholine, bind and cause local conformational transitions, changing the active region from a hydrophobic to a hydrophilic environment. This phenomenon is illustrated by the 10-fold increase in fluorescence when the receptor is in a desensitised state. Antagonists, such as d-Tubocurarine, block this conformational transition. In vitro rectus abdominis muscle preparations . show the nitrosamines, dimethylnitrosamine and diphenylnitrosamine, to be true agonists of the nAChR. However their low affinity and specificity for the receptor precludes them as photoaffmity labelling agents. Photoactivation of dimethylnitrosamine occurs when associated with an acidic hydrogen at the active site of the receptor, suggesting energy-transfer labelling to be more facile than photoaffmity labelling. The membrane-bound receptor, in the presence of these nitrosamines, undergoes conformational transitions regulating the opening and closing of the ion-channel. Desensitisation and receptor activation are shown to involve one and the same molecular transition.
- Full Text:
- Authors: Kapp, Eugene Anthony
- Date: 1989
- Subjects: Ligands (Biochemistry) , Nicotinic receptors
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4550 , http://hdl.handle.net/10962/d1018235
- Description: The nicotinic cholinergic receptor protein of the fish electric organ, Torpedo fuscomaculata, has been isolated, purified and shown to represent a true model for the nAChR from other species and higher vertebrates. It is an integral membrane protein composed of four different subunits, tightly associated with other functional, but non-specific proteins. Purification of the nicotinic cholinergic receptor by chromatofocusing demonstrates an improved method over that of affinity and ion-exchange chromatography. Gel chromatography and SDS-polyacrylamide gel electrophoresis show evidence of four subunits; a(40-44 kDa), 6(53 kDa ),'Y(63 kDa) and 6(66 kDa) despite some degradation of receptor molecules by intracellular proteases. Spectrophotometric and fluorimetric studies of receptor-ligand interactions, show the functional and chemical integrity of the receptor to remain intact after solubilisation. The effect of cholinergic ligands on purified receptor preparations indicate quenching of the intrinsic fluorescence of the receptor. Agonists, like acetylcholine, bind and cause local conformational transitions, changing the active region from a hydrophobic to a hydrophilic environment. This phenomenon is illustrated by the 10-fold increase in fluorescence when the receptor is in a desensitised state. Antagonists, such as d-Tubocurarine, block this conformational transition. In vitro rectus abdominis muscle preparations . show the nitrosamines, dimethylnitrosamine and diphenylnitrosamine, to be true agonists of the nAChR. However their low affinity and specificity for the receptor precludes them as photoaffmity labelling agents. Photoactivation of dimethylnitrosamine occurs when associated with an acidic hydrogen at the active site of the receptor, suggesting energy-transfer labelling to be more facile than photoaffmity labelling. The membrane-bound receptor, in the presence of these nitrosamines, undergoes conformational transitions regulating the opening and closing of the ion-channel. Desensitisation and receptor activation are shown to involve one and the same molecular transition.
- Full Text:
Effects of vitamin A on tumour and untransformed cells
- Authors: De Villiers, Diane Lynette
- Date: 1988
- Subjects: Vitamin A , Vitamin A in the body , Cancer -- Prevention
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3881 , http://hdl.handle.net/10962/d1001615
- Description: Vitamin A and its chemical analogues (retinoids) are known to play a role in the maintenance and differentiation of epithelial tissue. Retinoids have been shown to inhibit carcinogenesis in a number of tissues in experimental animals and to inhibit the growth of various untransformed and cancer cell lines in vitro. This study investigated the effect of retinyl acetate supplemented at concentrations of 1 μM, 5 μM, 10 μM and 100 μM to in vitro cultured untransformed LLCMK cells, and transformed BL-6 melanoma and human hepatoma cell lines. A small but non-significant effect of vitamin A addition on the growth of the untransformed cells was observed, while substantial inhibition of proliferation of the two tumour cell lines was found. At the cytotoxic level of 100 μM supplemented vitamin A, all three cell lines showed marked inhibition of growth. This led to an electron microscopy study to examine the ultrastructural effect of the vitamin A addition. At the low non-toxic levels of vitamin A addition (1 - 10 μM), no ultrastructural changes were observed in the untransformed cells. However, at a level of 5 μM and 10 μM vitamin A addition in the tumour cells, an increase in the size of suspected lipid droplets was observed. At the cytotoxic level of 100 μM supplemented vitamin A, large lipid droplets were very apparent, as was much cellular degeneration. This effect was more marked in the tumour cells than in the untransformed cells. The lipid nature of the droplets was confirmed by using the lipid stain, Sudan IV. In order to investigate the effect of added vitamin A at the cell surface level, an ELISA system was used to quantify the level of the cell surface glycoprotein, fibronectin, in the culture media. Vitamin A plays an important role in the production of mature fibronectin by participating in the glycosylation of the molecule. This study showed no major effect of added vitamin A on the release of fibronectin into the culture media. This did not, however, exclude the possibility that the vitamin A was involved in the production and enhanced binding of fibronectin to the cell surface, and was possibly also exerting an effect on the availability of fibronectin receptors. Further studies would, however, be required to substantiate such effects of vitamin A supplementation. No single mechanism of action of vitamin A on tumour cell growth inhibition was identified, but the possibility that at least two mechanisms exist, was suggested
- Full Text:
- Authors: De Villiers, Diane Lynette
- Date: 1988
- Subjects: Vitamin A , Vitamin A in the body , Cancer -- Prevention
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3881 , http://hdl.handle.net/10962/d1001615
- Description: Vitamin A and its chemical analogues (retinoids) are known to play a role in the maintenance and differentiation of epithelial tissue. Retinoids have been shown to inhibit carcinogenesis in a number of tissues in experimental animals and to inhibit the growth of various untransformed and cancer cell lines in vitro. This study investigated the effect of retinyl acetate supplemented at concentrations of 1 μM, 5 μM, 10 μM and 100 μM to in vitro cultured untransformed LLCMK cells, and transformed BL-6 melanoma and human hepatoma cell lines. A small but non-significant effect of vitamin A addition on the growth of the untransformed cells was observed, while substantial inhibition of proliferation of the two tumour cell lines was found. At the cytotoxic level of 100 μM supplemented vitamin A, all three cell lines showed marked inhibition of growth. This led to an electron microscopy study to examine the ultrastructural effect of the vitamin A addition. At the low non-toxic levels of vitamin A addition (1 - 10 μM), no ultrastructural changes were observed in the untransformed cells. However, at a level of 5 μM and 10 μM vitamin A addition in the tumour cells, an increase in the size of suspected lipid droplets was observed. At the cytotoxic level of 100 μM supplemented vitamin A, large lipid droplets were very apparent, as was much cellular degeneration. This effect was more marked in the tumour cells than in the untransformed cells. The lipid nature of the droplets was confirmed by using the lipid stain, Sudan IV. In order to investigate the effect of added vitamin A at the cell surface level, an ELISA system was used to quantify the level of the cell surface glycoprotein, fibronectin, in the culture media. Vitamin A plays an important role in the production of mature fibronectin by participating in the glycosylation of the molecule. This study showed no major effect of added vitamin A on the release of fibronectin into the culture media. This did not, however, exclude the possibility that the vitamin A was involved in the production and enhanced binding of fibronectin to the cell surface, and was possibly also exerting an effect on the availability of fibronectin receptors. Further studies would, however, be required to substantiate such effects of vitamin A supplementation. No single mechanism of action of vitamin A on tumour cell growth inhibition was identified, but the possibility that at least two mechanisms exist, was suggested
- Full Text:
Isolation and identification of possible analgesics and antihypertensive agents from antidesma venosum
- Authors: Mashimbye, Mahlori Jeffrey
- Date: 1986
- Subjects: Antidesma , Analgesics , Hypotensive agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3882 , http://hdl.handle.net/10962/d1001616
- Description: This investigation originated from a suggestion by Noristan Laboratories, Pretoria, that because Black people were using the roots of A. venosum E. MEY. ex. TUL for treating headache, the plant might contain analgesics. No previous chemical investigation has been carried out on this plant but from previous work done on other species antihypertensive agents were expected to be present
- Full Text:
- Authors: Mashimbye, Mahlori Jeffrey
- Date: 1986
- Subjects: Antidesma , Analgesics , Hypotensive agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3882 , http://hdl.handle.net/10962/d1001616
- Description: This investigation originated from a suggestion by Noristan Laboratories, Pretoria, that because Black people were using the roots of A. venosum E. MEY. ex. TUL for treating headache, the plant might contain analgesics. No previous chemical investigation has been carried out on this plant but from previous work done on other species antihypertensive agents were expected to be present
- Full Text:
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