One of the major limitations on research into the control of eukaryotic DNA replication has been the lack of any cell-free system that initiates DNA replication in vitro. The first part of the disseration describes the establishment of a eukaryotic system, derived from the activated eggs of the South African clawed toad, Xenopus laevis, that efficiently initiates and completes DNA replication in vitro. Using a variety of biochemical techniques I show that DNA added to the extract in the form of sperm nuclei is efficiently replicated over a period of 4 - 6 hours. Replication of nuclear DNA represents a single round of semiconservative, semidiscon-tinuous replication. The extract will also replicate naked DNA incubated in it, regardless of sequence, though less efficiently than nuclear templates. This is probably related to the unusual ability of the egg extract to assemble apparently normal interphase nuclei from any DNA molecule incubated in it Evidence is presented that initiation, rather than chain elongation, is the rate-limiting step for replication in vitro. In this and in other ways the cell-free system behaves as though it were an early embryo blocked in a single cell cycle. The second part of the dissertation describes experiments that examine the control of DNA replication in the extract The first set of experiments suggest that on replication, DNA is marked in some way so that it can no longer act as a substrate for further initiation. This provides a mechanism by which the template DNA is replicated precisely once per incubation in vitro (or per cell cycle in vivo). The second set of experiments investigate the relationship between nuclear assembly and the initiation of DNA replication in vitro. A novel method for quantifying DNA replication in intact nuclei using the nucleotide analogue biotin-11-dUTP is described. This technique reveals that although they are in the common cytoplasm of the egg extract, different nuclei start to replicate at different times. Entry into S-phase is characterised by a burst of many synchronous or near-synchronous initiations within individual nuclei. This means that nuclei act as independent and integrated units of replication in the cell-free system, and suggests a fundamental role for nuclear assembly in controlling DNA replication in vitro.

Yucca (Yucca schidigera) and Soapbark (Quillaja saponaria), both native desert plants, are major commercial sources of saponin extracts. Yucca schidigera is native to the southwestern United States and to the arid Mexican desert, and Quillaja saponaria is found in arid areas of Chile. Saponins have detergent or surfactant properties with both water-soluble and fat-soluble components.The use of natural saponins from yucca and soapbark as an additive in the diet, has improved production in aquatic organisms. The purpose of this study was to evaluate the effect of inclusion of Nutrafito plus (NTF+), which is a mixture of Yucca and Soapbark extracts, on growth, survival, and development of Nile tilapia Oreochromis niloticus and the effect on growth, survival, development, and digestive enzyme activities for juvenile Pacific white shrimp Litopenaeus vannamei.The extracts were included at different levels in the diets of tilapia and shrimp in four experiments. In experiment 1, at the end of 6 weeks there was no significant difference (p > 0.05) between growth rates of tilapia fed 6 different diets, with no mortality nor abnormal behavior in any of the treatments. Water quality parameters were determined weekly and remained within recommended limits for Nile tilapia culture.In the second trial, at the end of 40 days, there was no significant difference (p > 0.05) between growth rates of fish fed 7 different diets with various levels of Nutrafito. There were no mortalities during the experiment.In the third trial, 20 tanks (140 L each) were stocked with 10 shrimp each. Tanks were divided into 5 treatments with 4 replicates each. There was significant difference (p < 0.05) between growth rates of shrimp fed 5 different diets, with higher growth rate at higher levels of inclusion.In the fourth trial, 15 tanks (140 L each) were stocked with 10 shrimp each. Tanks were divided into 5 treatments with 3 replicates each. There was significant difference between growth rates and feed conversion ratio of shrimp fed 5 different diets. In addition, an analysis for digestive enzymes activity was done and no significant difference (p > 0.05) was found between treatments.

by Henry, Kam-yin Cheung. / Thesis (M.Ph.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 119-126.

by Lee Heung Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1988. / Bibliography: leaves 189-203.

Pregnane X receptor (PXR) is a ligand-activated transcription factor that plays a role in a broad array of biological processes, including drug metabolism and transport. Ginkgo biloba is an herb commonly used to improve cognitive function. In primary cultures of rat hepatocytes, Ginkgo biloba induces the mRNA expression of CYP3A23, a target gene for rat PXR. The present study tested the hypothesis that Ginkgo biloba activates PXR. Cultured HepG2 human hepatoma cells were transfected with the full-length human PXR (pCR3-hPXR), the full-length mouse PXR (pCR3-mPXR), or an empty vector (pCR3) in addition to a reporter plasmid (XREM-CYP3A4-LUC; firefly luciferase) and an internal control plasmid (phRL-TK; Renilla luciferase). At 24 h after transfection, cells were treated for 24 h with Ginkgo biloba extract and luciferase activity was measured. The extract at 200 µg/ml increased mouse and human PXR activity by 3.0-fold and 9.5-fold, respectively, indicating that Ginkgo biloba more effectively activates human PXR. Dose-response experiments showed that the extract produced a log-linear increase over the range of 200–800 µg/ml. To determine whether Ginkgo biloba extract induces human PXR target gene expression, cultured LS180 human colon adenocarcinoma cells were treated for 72 h with the extract. Total cellular RNA was isolated and reverse transcribed. CYP3A4, CYP3A5, and ABCB1 cDNAs were amplified by real-time PCR. Ginkgo biloba extract at 200, 400, and 800 µg/ml increased CYP3A4 mRNA expression by 1.7-, 2.4-, and 2.5-fold, respectively. The extract at the same concentrations increased the mRNA expression of CYP3A5 (1.3 to 3.6-fold) and ABCB1 (2.7 to 6.4-fold). To determine whether the increased expression involved PXR activation, cells were treated with a PXR antagonist, L-sulforaphane, and Ginkgo biloba extract. L-sulforaphane at 5, 10, and 20 µM decreased CYP3A4 mRNA expression by 54%, 78%, and 93%, respectively, in cells co-treated with the extract. A similar pattern of response was obtained with CYP3A5 and ABCB1. In cells co-treated with the extract, L-sulforaphane (5 and 10 µM) was not cytotoxic and did not decrease PXR mRNA expression. Our data from cell culture experiments indicate that Ginkgo biloba activates PXR and increases CYP3A4, CYP3A5, and ABCB1 mRNA expression.

Pregnane X receptor (PXR) is a ligand-activated transcription factor that plays a role in a broad array of biological processes, including drug metabolism and transport. Ginkgo biloba is an herb commonly used to improve cognitive function. In primary cultures of rat hepatocytes, Ginkgo biloba induces the mRNA expression of CYP3A23, a target gene for rat PXR. The present study tested the hypothesis that Ginkgo biloba activates PXR. Cultured HepG2 human hepatoma cells were transfected with the full-length human PXR (pCR3-hPXR), the full-length mouse PXR (pCR3-mPXR), or an empty vector (pCR3) in addition to a reporter plasmid (XREM-CYP3A4-LUC; firefly luciferase) and an internal control plasmid (phRL-TK; Renilla luciferase). At 24 h after transfection, cells were treated for 24 h with Ginkgo biloba extract and luciferase activity was measured. The extract at 200 µg/ml increased mouse and human PXR activity by 3.0-fold and 9.5-fold, respectively, indicating that Ginkgo biloba more effectively activates human PXR. Dose-response experiments showed that the extract produced a log-linear increase over the range of 200–800 µg/ml. To determine whether Ginkgo biloba extract induces human PXR target gene expression, cultured LS180 human colon adenocarcinoma cells were treated for 72 h with the extract. Total cellular RNA was isolated and reverse transcribed. CYP3A4, CYP3A5, and ABCB1 cDNAs were amplified by real-time PCR. Ginkgo biloba extract at 200, 400, and 800 µg/ml increased CYP3A4 mRNA expression by 1.7-, 2.4-, and 2.5-fold, respectively. The extract at the same concentrations increased the mRNA expression of CYP3A5 (1.3 to 3.6-fold) and ABCB1 (2.7 to 6.4-fold). To determine whether the increased expression involved PXR activation, cells were treated with a PXR antagonist, L-sulforaphane, and Ginkgo biloba extract. L-sulforaphane at 5, 10, and 20 µM decreased CYP3A4 mRNA expression by 54%, 78%, and 93%, respectively, in cells co-treated with the extract. A similar pattern of response was obtained with CYP3A5 and ABCB1. In cells co-treated with the extract, L-sulforaphane (5 and 10 µM) was not cytotoxic and did not decrease PXR mRNA expression. Our data from cell culture experiments indicate that Ginkgo biloba activates PXR and increases CYP3A4, CYP3A5, and ABCB1 mRNA expression.

1. Relevance of the work First medicine used by man was of herbal origin, i.e. prepared from plants, their parts and mixtures. After thousands of years, these preparations have not lost their relevance. Increasingly more and more clinical research is done on verification of efficiency and safety of herbal medicine, whereas methods of modern analysis help to ensure their quality and stability. This allows to objectively determine the potential of herbal medicine use, to foresee their possible side effects and to warn against their interaction with other drugs. It is essential that medicinal plants and their parts should be treated as a whole of biologically active substances. A lot of resources are allocated annually to the development of new herbal preparations and research of new plants, analysis of their chemical composition, pharmacological toxicological action, and interaction of plants with other medicinal drugs. By interacting with regulatory systems of the organism, herbal drugs inhibit or stimulate them and primarily affect subjective symptoms. Herbal medicine must be individually prescribed according to the peculiarities of the patient’s ailment and body. Herbal medicine can evoke reactions of the body, which depend on individual characteristics of the patient and on effect of the herbal drugs. There must be no doubts, though, that this medicine is better tolerated and its side effects are considerably milder that those of synthetic preparations. This can be... [to full text]

This work studies the ability to produce effective surfactant and adhesive formulations using surface active biological material extracted from different biomass sources using alkaline extraction methods. Two urban waste biomass sources were used to produce surfactants, Return Activated Sludge (RAS), and solid Urban Refuse (UR). The third biomass source investigated was isolated mustard protein (MP). RAS and MP extracts were investigated for adhesive production. The results indicate that extracts from the waste biomass sources, RAS and UR, can be combined with a commercial surfactant, sodium dioctyl sulfosuccinate (AOT), to produce surfactants with low interfacial tensions against various oils. These highly surface-active formulations were shown to be useful in the removal of bitumen from contaminated sand. RAS and MP showed potential as protein-based wood adhesives. These sources were used in adhesive formulations to produce a strong bond strength under low-pressure, ambient pressing conditions.

This work studies the ability to produce effective surfactant and adhesive formulations using surface active biological material extracted from different biomass sources using alkaline extraction methods. Two urban waste biomass sources were used to produce surfactants, Return Activated Sludge (RAS), and solid Urban Refuse (UR). The third biomass source investigated was isolated mustard protein (MP). RAS and MP extracts were investigated for adhesive production. The results indicate that extracts from the waste biomass sources, RAS and UR, can be combined with a commercial surfactant, sodium dioctyl sulfosuccinate (AOT), to produce surfactants with low interfacial tensions against various oils. These highly surface-active formulations were shown to be useful in the removal of bitumen from contaminated sand. RAS and MP showed potential as protein-based wood adhesives. These sources were used in adhesive formulations to produce a strong bond strength under low-pressure, ambient pressing conditions.

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