The Investigation of Shrinkage in Apoptotic Bodies

By Ashley Ilinkoski and Michael Model

Objectives

Apoptosis is programmed cell death where the cell shrinks then eventually fragments and dies, also considered the point of no return. This method can possibly be controlled by putting cells in different media to ensure ions pouring into the cell rather than out. Shrinkage is one of the most universal and specific signs of apoptotic cell death. (Some data suggest that shrinkage due to water loss is the requirement for apoptosis). However, in some apoptotic systems shrinkage occurs late, after irreversible cell damage has occurred. Such systems provide a good model to investigate the exact role of shrinkage in apoptotic development. One specific hypothesis we wish to address is that shrinkage is necessary for cell fragmentation into “apoptotic bodies”.

Methods

Apoptosis is induced in HeLa cells with camptothecin or RNA polymerase actinomycin D. After the onset of irreversible changes (i.e., morphological blebbing, mitochondrial depolarization or cytochrome c release) treatments designed to inhibit cell water loss are applied. Aqua porin inhibitors are also being introduced to the cells to see if the porins close to sustain cell life by cutting off water transport. The results include formation of apoptotic bodies and DNA fragmentation. The loss of cell material is monitored by the TIE-TTD microscopy and DNA fragmentation by the TUNEL assay.

Results

Preliminary data indicates that high-potassium medium that opposes water loss prevents formation of apoptotic bodies. Conversely, high-sodium medium that activates water loss results in active cell fragmentation. Future work will clarify these results.

Oxytocin (Oxt) is a nine amino acid neuropeptide that was thought to be invariant in its sequence across species. However, recent work in primates has found that in some New World Monkeys there can be one or two amino acid substitutions in the protein. So, to assess whether or not these alternative sequences are functional in mice, we set out to perform a grooming bioassay. We hypothesized that these alternative forms of Oxt would have differential effects on grooming compared to the native protein. To test this hypothesis we performed stereotaxic surgery on mice and implanted guide cannulae aimed at the third ventricle. Following recovery from surgery, mice were injected with two µl of each of four treatments over the course of four days: Saline, Oxytocin, Peptide 1, and Peptide 2. Following each microinjection subjects were videotaped for 30 minutes and the amount of grooming scored by an observer blind to each treatment. At the completion of the study site checks were performed to verify the location of the microinjections. While we are still in the process of analyzing our data- all of our treatments were successfully delivered to the lateral ventricle; thus, no animals will need to be excluded from the study. We predict that treatment with Peptide 1 and 2 will result in less grooming compared to treatment with Oxt.

Vernal pools fill during the spring when snowmelt and rainwater gather in depressions in the ground. Undecomposed leaf litter from previous years will fall into these depressions and become both a shelter and a food source for many invertebrates. Leaf litter from different tree species were tested to compare the invertebrate communities that colonized in each and, ultimately, to determine whether one leaf species was colonized quicker or more abundantly than another. Invertebrates were identified to the family level. The family Asellidae was the most abundant invertebrate type in all leaf species litters, accounting for 56% of the total invertebrate count, followed by the family Chironomidae with 26% of the total. A multivariate analysis showed that there was no significant difference between invertebrate communities among leaf litter types. Samples were heavily dominated by a few families of invertebrates and were colonized by very few other families. The similarity between communities showed that the invertebrates likely perform many of the same ecological functions on different leaf types. As a whole community, this ecological function is primarily to breakdown larger leaf litter so that smaller organisms can continue the decomposition process.

My abstract talks about obesity as a problem and how triiodothyronine (T3) is changed due to calorie restriction in rat plasma.

Problem: Roots are the major type of plant tissue that contributes to soil organic carbon. Our study was designed to test whether variation in root chemical and morphological traits change decomposition and soil carbon sequestration rates. Compared to tulip roots (Liriodendron tulipifera), elm roots (Ulmus americana) have higher lignin:Nitrogen ratio, but finer diameter, as well as greater root tip abundance. Based on morphological traits, we expect elm roots to decompose faster because of their higher surface area and fine morphology causing them to easily break into the soil. Based on chemical properties, we expect that tulip roots will decompose faster because they have lower lignin:nitrogen ratio. Since microbial communities can adapt to the quality of locally available nutrients, it is expected that decay rates will be accelerated for tissues that have a ‘home field advantage,’ being more similar to neighboring tree species.

Methods: Litterbags filled with soil and elm or tulip roots, including treatment groups of either 1st and 2nd order roots, 3rd and 4th order roots, or entire root systems, were left to decompose for 42 weeks in under trees of both species in riparian forest at Jennings Woods.

Results: Tulip roots decomposed faster than elm roots, implying that root tissue chemistry has a greater effect on decomposition than morphological characteristics. The strength of our predicted relationships varied between treatment groups. Decomposition occurred faster under tulip trees regardless of the identity of the roots, giving evidence against ‘home field advantage’ theory.