The goal of this project was to determine if Glutathione S-Transferase Mu 1 (GSTM1), a key antioxidant enzyme, is altered in diabetes. We compared GSTM1 levels between hearts of type I diabetic Akita mice and wild-type mice using Western blotting. Western blotting for GSTM1 had previously been unsuccessful because of the low amounts of this enzyme in the heart. The Western blotting procedure was optimized through experiments to determine if treatment of the membrane prior to blocking and during primary and secondary antibody incubation increased the sensitivity of the blot. We found that treatment of the membrane in 0.01% glutaraldehyde for 20 minutes before blocking and incubating with the primary antibody in the presence of 75% TTBS (Tris-buffered saline with Tween 20) increased band intensity by 8.92-fold when compared to the control. In addition, the addition of 5% PEG-8000 and the use of 1% BSA in the primary and secondary antibody incubations increased Western blot sensitivity. The improved Western blotting procedure allowed for the detection and quantification of the GSTM1 in the Akita and control samples. After quantifying and normalizing the protein loaded, we found that GSTM1 levels were similar in Akita mice than in normal mice. Hence, the levels of GSTM1 do not seem to be affected in type 1 diabetic heart dysfunctions.
Plant innate immunology introduces a new plasmid containing a promoter, UBQ10pro, which does not react with 35Spro. Previously, two 35Spro promoters on different plasmids inside the plant arabidopsis co-silenced each other and prevented fluorescent proteins from being produced. This fluorescent nucleic membrane marker protein allows her to see how chloroplasts and stromule extensions interact with the nucleus in the immune responses. Majken transferred the gene tRFP-WIP1 from a vector containing 35Spro to a vector with UBQ10pro. This allows the cell to produce both proteins in large quantities that allow the organelles to be studied. In the future, this research and plasmid will help farmers and agricultural scientist protect and understand plant health leading to increased crop yield and quality.
Analyzing the Role of the Protein Kinase, STY46, in Regulating the Sugar Starvation Response in Arabidopsis thaliana
When plants undergo environmental stresses, they experience sugar starvation (SS), causing a shift in carbon fluxes and signal transduction cascades to alter the plant’s metabolism and growth processes. This series of events, called the sugar starvation response (SSR), is crucial in determining plants’ survival under diminished carbon levels. It is speculated that the protein kinase, STY46, is a potential master regulator of the sugar starvation response. Compared to wild type plants, Arabidopsis with genetically altered levels of STY46 are expected to have differences in carbon use and plant growth. To meet this objective, four transgenic Arabidopsis thaliana lines (knockout, complementation, inducible overexpression and constitutive overexpression) will be generated to examine carbon partitioning and allocation under sugar starvation. The specific aim of this paper was to generate construct for a STY46 complementation line and discuss future research to be performed using the constructs. Positive results were obtained in preliminary gel electrophoresis, since restriction enzyme digest of the transformed plasmid construct appeared accurate. However, when the STY46 gene was sequenced, the results were inconclusive due to the inability of the primers to bind onto the plasmid. This most likely occurred due to either incorrect primer design or untransformed plasmids sent to sequencing.
Greenhouse studies were performed to examine the effects of two carbon sources on Anaerobic Soil Disinfestation (ASD) of Agrobacterium tumefaciens and Pseudomonas synxantha inoculum. The first experiment was performed using Yolo silty clay loam soil from the UC Davis Armstrong Field Facility, which will be referred to as clay soil. The experimental design was composed of treatment pots in a controlled greenhouse environment, each with an irrigated soil amended with rice bran at a rate of 9, 7, or 5 tons/acre, or molasses at a rate of 4.5, 2.25, or 1.125 tons/acre. Inoculum bags containing the bacteria A. tumefaciens and P. synxantha were buried in these pots, which were then covered with an impermeable tarp. The second experiment only considered the treatment of 9 tons/acre of rice bran, but used soil with a Hanford fine sandy loam texture, which will be referred to as sandy soil. Untreated control pots were also present for comparison in both experiments. At three and seven days after initiating Anaerobic Soil Disinfestation, three pots from each treatment were taken to enumerate the bacteria through dilution plating on selective media. Populations of A. tumefaciens in the 1.125 tons/acre molasses treatment tended to decrease with time, but the populations at seven days were not significant different from time zero across treatments. Populations of P. synxantha in the 9 tons/acre rice bran treatment also tended to decrease with time, but the populations were also not significantly different from time zero across treatments. Anaerobic conditions were lost after five days for both the 1.125 tons/acre molasses 2.25 tons/acre molasses, with lower efficacy of ASD over time for molasses compared to the rice bran treatments. A. tumefaciens populations decreased to undetectable levels by seven days in the sandy soils exposed to ASD conditions. These data indicate that soil texture may play a major role in ASD efficacy which will affect where ASD will be used commercially in the state of California. Overall, the comparison of rice bran and molasses as carbon sources for ASD was inconclusive; however, it was clearly shown that rice bran at rates below commercial levels of 9 tons/acre were effective at inducing anaerobic conditions in the soil.
Biomarkers to Signify Early Detection of a Spontaneous Mouse Mutation In the Palmitoyl Transferase Zdhhc13 Gene
The Zdhhc13 gene in mice is partly responsible for the encoding of palmitoyl acyltransferase (PAT) enzymes in mice. A naturally occurring recessive mutation in the mice causes a nonsense base substitution, leading to a truncated form of the Zdhhc13 protein. Mutant mice experienced an increased susceptibility to tumor multiplicity and malignant progression of papillomas after chemically induced skin carcinogenesis. Our goal in this research is to identify potential biomarkers that signify early detection of mutation in the Zdhhc13 gene. This is mainly done through MetaboAnalyst, a metabolite analysis program used to identify possible biomarkers, achieve group separation, and analyze pathways of metabolites to see their role in a functional gene. After the metabolite concentrations from three groups of mice are normalized, statistical procedures are conducted, and significant compounds are identified. Our graphs showed fair separation between homozygous WT, heterozygous, and homozygous mutant mice, with heterozygous mice showing characteristics closer to homozygous mutant mice than homozygous WT mice. Significant biomarkers identified include cysteine, taurine, L-pyroglutamic acid, and L-glutamic acid, which all have a direct role or a connection to processes in the skin.
Proteins transport across the chloroplast membrane through the translocon of the outer and inner membranes (Toc and Tic, respectively). In previous studies, the translocon was successfully plugged with a biotinylated precursor, RSSU-HC, bound to a large avidin molecule. Recently, the precursor was systematically shortened to find the minimum number of amino acids for a protein to span both membranes. From this, researchers can determine the protein import pathway and whether they import in a linear or folded manner.
The primary function of proteasomes is to degrade mis-folded and unwanted proteins. Tripeptidyl peptidase II (TPPII) is a newly discovered protease that works downstream of the ubiquitin-proteasome system. It is required in eukaryotic cells for cleaving of larger protein degradation products. However, little is known about its function in other cellular processes. TPPII’s responses to stress and DNA damage could be areas of research for cancer therapy, if TPPII can be studied better. Homogenization is a process used to break down cells for subsequent analysis or purification of proteins. Accurate analysis of protein expression and protease activity in the cell can be studied by good homogenization procedures and sample preparation. This study investigated the effect of various homogenization and assay buffers to determine the optimal buffer for homogenization and activity assays of cardiac TPPII. The results demonstrated that a homogenization buffer containing polyvinylpyrrolidone and glycerol produced more desirable results compared to buffers currently used for TPPII analysis
Structural and kinetic analysis of hydrolase mutants and subsequent impact on predicting functionality with Michaelis-Menten mechanics
Understanding about enzymes has grown due to the use of computational algorithms in predicting the correlation between structure and function. These algorithms have had critical roles in the redesign and reengineering of native enzymes to enhance catalytic behavior. In this paper, front-end programs were used to model, interact with, and redesign a glycoside hydrolase enzyme. The structural model was coupled with Michaelis-Menten kinetics to yield the Kcat and Km kinetic constants for three mutants. The compiled dataset will be essential to tracking patterns and trends across enzyme families. It may also be useful in future efforts to redesign enzymes using predictive methods.
Kinetic Analysis of Three Glycoside Hydrolase Mutants to Discover Associations Between Michaelis-Menten Constants and Protein Structure
The need to process and analyze novel enzymes is exponentially growing; meanwhile the rate to do so has not kept up. Therefore, computational methods are necessary to continue to explore an ever increasing amount of new enzymes. This project intends to enhance computational methods through the collection of experimental data from mutant proteins to further determine how protein structure affects enzyme efficiency. To do so, the Michaelis-Menten kinetic constants, Kcat and KM values, will be gathered on three different glycoside hydrolase mutants; subsequently, the data will be contributed to an algorithm that will better predictions of kinetic constants based on protein structure through computational modeling.
"Understanding the molecular basis of Postharvest Chilling Injury in tomato fruit: Generation of RD29A::CBF1 transgenic construct "
For decades, consumers have been purchasing refrigerated produce and storing it in refrigerators at home. Although this approach helps to preserve the produce for a certain period of time after harvest, many fruits and vegetables are damaged by the cold in a disorder called Postharvest Chilling Injury (PCI). Although PCI has been extensively studies, the molecular basis of its early events still remain poorly understood. The general goal of this project was to study those processes in tomatoes. The C-binding factor 1, or CBF1 gene, regulates cold tolerance, and the objective is to introduce CBF1 from a cold tolerant species, Solanum habrochaites, into the cold-sensitive cultivated tomato, Solanum lycopersicum. The aim of this work was to synthesize RD29A::CBF1 gene constructs which will be transformed into S. lycopersicum to assess cold tolerance in fruit after harvest. The two constructs created were RD29A::ShCBF1 using CBF1 from S. habrochaites and RD29A::SlCBF1 using CBF1 from S. lycopersicum. RD29A, ShCBF1, and SlCBF1 were amplified using Assembly Polymerase Chain Reaction (PCR). After purification, RD29A::ShCBF1 and RD29A::SlCBF1 constructs were spliced into pCAMBIA1300 vector plasmids and transformed into competent E. coli cells which copied the plasmids after multiple rounds of replication. The plasmids were extracted and diagnostic restriction digest was conducted with the EcoRI enzyme which yielded positives in colony #1 of RD29A::ShCBF1 and colony #21 of RD29A::SlCBF1. These were then sent for sequencing. This portion of the project culminated without the corroboration of the identities of the two engineered gene constructs due to time constraints. Even so, this brings research one step closer to reducing PCI in produce and decreasing the overall losses in agriculture.
Enzymes play a fundamental role in catalytic functions integral to many biological processes. The use of computational design to engineer the structure and predict enzymatic function hold the potential to enhance catalytic efficiency. Here, 3-D protein-folding simulators were used to structurally mutate the BglB enzyme's primary structure for three mutants. The computationally designed mutants, then produced and purified, were analyzed according to Michaelis-Menten kinetics that provided further insight on the mutants' catalytic efficiency (Kcat/Km). The compiled data will be important for developing and refining computational algorithms that can successfully predict correlations between structure and function.
In today's fermented foods industry, a single harmful type of bacteria can destroy a whole crop yield. However, the presence of lactic acid bacteria can kill these detrimental types of bacteria because of its acidic nature. Nathan's experiment focuses on testing the different stressful environments LAB can endure so as to give more concrete data on the environments in which LAB can grow and be of service to farmers and agricultural companies.
Plants are subject to many stresses daily. In order to efficiently deal with all of these stresses, plants have devised a general stress response which allows them to immediately respond before executing a specialized response to deal with the stressor. Multiple biological pathways in cells have been observed to activate the Rapid Stress Response Element (RSRE), which is the cis-element that plays a part in activating the general stress response. Previous research conducted on the effects of various chemicals on RSRE activation indicated a possible relationship between Mitogen Activated Protein Kinase (MAPK) pathways’ and RSRE activation. The individual effects of MAPK inhibitors on wound response, pathogen response, and basal response in one week old Arabidopsis thaliana seedlings will give a clearer picture of how MAPK pathways are utilized in those responses. This study shows that MAPK modulating chemicals are able to enhance specific stress responses. Additionally, one MAPK modulator was able to delay all RSRE induction. Since MAPK pathways are conserved in animals, studying MAPK pathways’ role in plants could be applicable to human stress responses.
Nelson Chou conducts a research project in the Department of Plant Sciences. The mentors of this young scholar are Dr. Johan Six and Dr. Sandra Yanni of the Agroecology Lab group. The objective the research is to explore the effects of photodegradation on littler and soil organic matter and its magnitude and rate in a California grassland. Photodegradation is an abiotic process in which sunlight, composed of both ultraviolet and visible light, degrades organic materials and can therefore facilitate subsequent microbial decomposition of organic matter. The carbon and nitrogen amounts of soil, grass litter, light effect on decomposition will be strategically analyzed. These understandings will form a basis of accurate prediction of annual decomposition rates of highly sun-exposed ecosystems such as the California grasslands.
Synthesizing MUC1, an important protein in human epithelial cells that protects cells from external disease. This protein is overexpressed in the cancer cells of the colon, ovaries, lung, and pancreas. By synthesizing various forms of this protein, Nicholas and his co-workers hope to begin developing a vaccine that can target and help the immune system destroy cancer cells while leaving normal cells intact.