title: Understanding Allosteric Arginine Mutations Using Macromolecular Rate Theory

authors: Hamill, Carlin James

subjects: macromolecular rate theory

abstract: Enzyme catalysed reaction rates have been traditionally modelled with the Arrhenius and Eyring-Polanyi equations. These models assume that the reaction rate is exponential with temperature, and thus the natural log of the reaction rate versus 1/T is linear. Significant deviations from these models at high temperatures has traditionally been attributed to thermal denaturation. An increasing body of evidence has shown that denaturation alone is insufficient to account for these deviations from Arrhenius behaviour. Macromolecular rate theory (MMRT) accounts for these deviations with the introduction of the activation heat capacity (Δ𝐶𝑃‡) to the rate equation. The activation heat capacity is a consequence of the restriction in conformational freedom along the reaction coordinate, as an enzyme moves from the enzyme-substrate complex to the transition state complex – thus for an enzymatic reaction the activation heat capacity is negative. A non-zero activation heat capacity imparts temperature dependence to the activation entropy and activation enthalpy, introducing curvature to the rate equation independent of thermal denaturation. The activation heat capacity may itself be temperature dependent. MMRT equations of increasing complexity have been developed to reflect this and are suitable for different applications.
This thesis explores the effects of allosteric arginine mutations on the temperature dependence of enzyme rates through the lens of MMRT and evolution using the model enzyme MalL. An in-depth analysis of a previously characterised arginine mutant is described along with four additional arginine mutants. The arginine mutants were designed to mimic urea ligand binding across the enzyme surface. These mutants were characterised kinetically and with biophysical methods. Two were further characterised structurally, with high resolution structures being produced. These mutant enzymes showed significant rate improvements at low temperatures, suggesting two possible mechanisms for evolution towards psychrophily. These arginine mutants showed significant improvement in crystallographic resolution, indicating surface arginine mutations may be a general route for crystallographic improvement.

text: Understanding Allosteric Arginine Mutations Using Macromolecular Rate Theory Enzyme catalysed reaction rates have been traditionally modelled with the Arrhenius and Eyring-Polanyi equations. These models assume that the reaction rate is exponential with temperature, and thus the natural log of the reaction rate versus 1/T is linear. Significant deviations from these models at high temperatures has traditionally been attributed to thermal denaturation. An increasing body of evidence has shown that denaturation alone is insufficient to account for these deviations from Arrhenius behaviour. Macromolecular rate theory (MMRT) accounts for these deviations with the introduction of the activation heat capacity (Δ𝐶𝑃‡) to the rate equation. The activation heat capacity is a consequence of the restriction in conformational freedom along the reaction coordinate, as an enzyme moves from the enzyme-substrate complex to the transition state complex – thus for an enzymatic reaction the activation heat capacity is negative. A non-zero activation heat capacity imparts temperature dependence to the activation entropy and activation enthalpy, introducing curvature to the rate equation independent of thermal denaturation. The activation heat capacity may itself be temperature dependent. MMRT equations of increasing complexity have been developed to reflect this and are suitable for different applications.
This thesis explores the effects of allosteric arginine mutations on the temperature dependence of enzyme rates through the lens of MMRT and evolution using the model enzyme MalL. An in-depth analysis of a previously characterised arginine mutant is described along with four additional arginine mutants. The arginine mutants were designed to mimic urea ligand binding across the enzyme surface. These mutants were characterised kinetically and with biophysical methods. Two were further characterised structurally, with high resolution structures being produced. These mutant enzymes showed significant rate improvements at low temperatures, suggesting two possible mechanisms for evolution towards psychrophily. These arginine mutants showed significant improvement in crystallographic resolution, indicating surface arginine mutations may be a general route for crystallographic improvement.

year: 2024

title: Digital Smarts: Enhancing learning and teaching ﻿[Introduction]

authors: Wright, Noeline

abstract: This book is a partnership on many levels—between co-editors, with and among the other chapter authors, external, international reviewers, and eventually with you, the book’s readership. Our colleagues have also had to trust us in the mentoring, leadership and fruition of this project. We also hope that the work is trusted in the sense of having a quality assurance process that stands up as rigorous and befitting an academic text. We will address that aspect in more detail later in this introduction.
Partnership, trust and integrity are implicit in any edited book development that grows from within a shared context such as ours, the University of Waikato’s Faculty of Education.

text: Digital Smarts: Enhancing learning and teaching ﻿[Introduction] This book is a partnership on many levels—between co-editors, with and among the other chapter authors, external, international reviewers, and eventually with you, the book’s readership. Our colleagues have also had to trust us in the mentoring, leadership and fruition of this project. We also hope that the work is trusted in the sense of having a quality assurance process that stands up as rigorous and befitting an academic text. We will address that aspect in more detail later in this introduction.
Partnership, trust and integrity are implicit in any edited book development that grows from within a shared context such as ours, the University of Waikato’s Faculty of Education.

year: 2015

title: Investigation of in vitro diabetic inflammation and  treatment with Piper excelsum tea extract

authors: Harvey, Savannah

subjects: Piper excelsum

abstract: Induction of inflammation through hyperglycaemia and adiposity has been shown to contribute to the pathogenicity of type 2 diabetes (T2D). The pro-inflammatory cytokine, tumour necrosis factor α (TNF-α) was the first cytokine found to bridge the connection between the immune system and T2D. This was found due to its increased production in the disease state. TNF-α has specifically been linked to the onset of insulin resistance in T2D models. Evidence has shown that heat shock protein 60 (HSP60) may also play a role as a danger signal for the immune system as it can result in the induction of inflammation. Currently, there are no pharmaceutical options for T2D patients which target inflammation. Tea made from the leaves of kawakawa (Piper excelsum) has been used for many generations by Māori as a traditional rākau rongoā (traditional medicine) to treat a range of ailments including inflammation. The aim of this project was to investigate the modulation of inflammation using a commercially available kawakawa tea in human cancer cells in vitro. 

This project involved measuring the growth rate, cytotoxicity, TNF-α production and HSP60 expression upon exposure to three treatments: high glucose, lipopolysaccharide (LPS) or kawakawa tea extract (KTE).

This study showed that a high concentration of KTE (1000 µg/mL) inhibited cell growth in both HEPG2 and HeLa cell lines. Cells treated with this concentration also showed typical morphological signs of apoptosis. Cytotoxicity testing confirmed that there was a significant (p <0.05) decrease in mitochondrial dehydrogenase activity for this treatment which confirmed the cells were likely dying through apoptosis. Over a course of 24-hours three LPS treatments were found to be cytotoxic. These were the 10 ng/mL, 10 µg/mL, and 100 µg/mL concentrations of LPS. After 48-hours all treatments apart from 100 µg/mL KTE treatment were also found to be cytotoxic. The cytotoxicity for these treatments was either due to a reduction in mitochondrial dehydrogenase activity (infers mitochondrial stress) or increased lactate dehydrogenase (LDH) release from cells (infers necrotic cell death). These cytotoxicity experiments allowed for identification of which concentrations should be selected or removed for future experiments. 

Cytotoxicity levels were taken into consideration and experiments were run investigating the induction of TNF-α using enzyme-linked immunosorbent assays (ELISA). Issues regarding negative readings from samples arose which resulted in protocol changes and extensive trouble shooting. It was found that extended exposure times were required to attenuate a positive result from the positive control, LPS. 

HSP60 expression was also measured for human cells exposed to high glucose and KTE treatments. No change in HSP60 expression was detected. However, 24-hour LPS exposure resulted in a significant increase in HSP60 expression compared to the control for the 100 ng/mL LPS treatment. Two concentrations were investigated; 100 ng/mL LPS and 100 µg/mL LPS which resulted in 1.4- and 2.1-fold increases compared to the control, respectively.

Overall, no conclusion could be drawn regarding the modulation of inflammation by KTE. However, this study was able to identify the effect the tea has on human cancer cell growth and the nature of cytotoxicity the tea poses to the cells. It is recommended to continue this research exploring different kawakawa tea blends and using alternative in vitro cell lines and whole animal models.

text: Investigation of in vitro diabetic inflammation and  treatment with Piper excelsum tea extract Induction of inflammation through hyperglycaemia and adiposity has been shown to contribute to the pathogenicity of type 2 diabetes (T2D). The pro-inflammatory cytokine, tumour necrosis factor α (TNF-α) was the first cytokine found to bridge the connection between the immune system and T2D. This was found due to its increased production in the disease state. TNF-α has specifically been linked to the onset of insulin resistance in T2D models. Evidence has shown that heat shock protein 60 (HSP60) may also play a role as a danger signal for the immune system as it can result in the induction of inflammation. Currently, there are no pharmaceutical options for T2D patients which target inflammation. Tea made from the leaves of kawakawa (Piper excelsum) has been used for many generations by Māori as a traditional rākau rongoā (traditional medicine) to treat a range of ailments including inflammation. The aim of this project was to investigate the modulation of inflammation using a commercially available kawakawa tea in human cancer cells in vitro. 

This project involved measuring the growth rate, cytotoxicity, TNF-α production and HSP60 expression upon exposure to three treatments: high glucose, lipopolysaccharide (LPS) or kawakawa tea extract (KTE).

This study showed that a high concentration of KTE (1000 µg/mL) inhibited cell growth in both HEPG2 and HeLa cell lines. Cells treated with this concentration also showed typical morphological signs of apoptosis. Cytotoxicity testing confirmed that there was a significant (p <0.05) decrease in mitochondrial dehydrogenase activity for this treatment which confirmed the cells were likely dying through apoptosis. Over a course of 24-hours three LPS treatments were found to be cytotoxic. These were the 10 ng/mL, 10 µg/mL, and 100 µg/mL concentrations of LPS. After 48-hours all treatments apart from 100 µg/mL KTE treatment were also found to be cytotoxic. The cytotoxicity for these treatments was either due to a reduction in mitochondrial dehydrogenase activity (infers mitochondrial stress) or increased lactate dehydrogenase (LDH) release from cells (infers necrotic cell death). These cytotoxicity experiments allowed for identification of which concentrations should be selected or removed for future experiments. 

Cytotoxicity levels were taken into consideration and experiments were run investigating the induction of TNF-α using enzyme-linked immunosorbent assays (ELISA). Issues regarding negative readings from samples arose which resulted in protocol changes and extensive trouble shooting. It was found that extended exposure times were required to attenuate a positive result from the positive control, LPS. 

HSP60 expression was also measured for human cells exposed to high glucose and KTE treatments. No change in HSP60 expression was detected. However, 24-hour LPS exposure resulted in a significant increase in HSP60 expression compared to the control for the 100 ng/mL LPS treatment. Two concentrations were investigated; 100 ng/mL LPS and 100 µg/mL LPS which resulted in 1.4- and 2.1-fold increases compared to the control, respectively.

Overall, no conclusion could be drawn regarding the modulation of inflammation by KTE. However, this study was able to identify the effect the tea has on human cancer cell growth and the nature of cytotoxicity the tea poses to the cells. It is recommended to continue this research exploring different kawakawa tea blends and using alternative in vitro cell lines and whole animal models.

year: 2021