Regulation
of 11-b HSD1 gene expression in the brain by DHEA.
1*
and TOM GEOGHEGAN2, 1Department of Biology, Bellarmine
University, Louisville, KY, 40205, and 2Department of Biochemistry
and Molecular Biology, University of Louisville, KY, 40202.
DHEA (dehydropiandrosterone) is a C-19 adrenal steroid precursor for androgens and estrogens. It is a sterol that circulates in highest concentration in humans (micromolar levels). The DHEA levels are highest between the ages of 20 to 30 years and decrease gradually with age. DHEA is important because it has shown to have many unique functions. It acts as a neurosteroid; improve cognitive function; enhances immune function. It also has anti-diabetic and anti-obesity effects. We hypothesize that some of those function are achieved by changing the biochemistry in the brain. One very important finding is DHEA’s ability to down regulate the expression of the 11b-hydroxysteroid dehydrogenases type 1 (11b-HSD1) gene in the liver as well as some other glucocorticoids targeted tissues. The product of this gene is mostly a reductase, which will regenerate active glucocorticoids from their inactive form in targeted tissues. Since 11b-HSD1 is also expressed in brain, we hypothesize that DHEA also regulates its expression in the brain. In order to address this question, we used semi-quantitative RT-PCR to measure the mRNA level of 11b-HSD1 in brain tissues from either untreated rats or DHEA treated rats. This publication was made possible by NIH Grant Number P20 RR16481 from the BRIN Program of the National Center for Research Resources.
Diminished
skeletal arteriolar constrictor response as a function of age.
REBECCA
DENNY1*, KATHLEEN
HAMILTON2, CANDICE THOMAS2, and JEFF C. FALCONE2.
1Department of Biology, Bellarmine University, Louisville, KY, 40205,
and 2Department of Physiology and Biophysics,
University of Louisville, KY,
40202.
Older humans have a number of vascular problems, which suggest a decreased ability to vasoconstrict, so we have examined first-order arterioles in young (n=8; 3-7 months) and old (n=5; 20-24 months) rats. The SD, WKY, SHR, and F344BNF1 strains of rats have been used. We hypothesized that the skeletal muscle arteriolar constriction response is diminished in the aged rats as compared to the young. The arterioles we used were isolated from the rat cremaster, a skeletal muscle. The arterioles were cannulated between two glass pipettes and were kept at a constant pressure of 90 cmH2O. Different drug concentrations of norepinephrine, phenylephrine, and prostaglandins were added to the vessel bath and diameter was measured to produce concentration-response curves. It appears that the response in the aged arterioles was diminished with each drug added. The response even seemed to be diminished with the dilators added, adenosine and acetylcholine. This study suggests that the arteriolar responses to constrictor agents in aged animals are less than that of young animals. This publication was made possible by NIH Grant Number P20 RR16481 from the BRIN Program of the National Center for Research Resources.
Isolating
the ephrin A5 signal pathway in chick embryo. SEAN
HOBAN1*
and ERIC WONG2, 1Department of Biology, Bellarmine
University, Louisville, KY, 40205, and 2Eric Wong, Department of
Biology, University of Louisville, 40292.
The millions of neurons of the developing central nervous system must grow on very specific paths for long distances in order to make complex connections with their targets. During growth, a neuron extends in a single, random direction and, depending on the conditions it encounters, will advance in that direction or withdraw and reform in another direction. One of these regulatory molecules is Ephrin A5, an inhibitory molecule that induces a transient collapse of the growth cone, causing the neuron to seek another direction in which to grow. Currently the proteins that make up the signal transduction pathway of this process are unknown, and finding them is difficult. Interestingly, Ephrin A5 is important in the development of the chick visual system, a classic and highly accessible system for the study of axon guidance. We decided the best way to isolate the components of the pathway would be to isolate the Ephrin A5 receptors from the cells of this system and attempt to catch any proteins that are bound to them, using an affinity column coated with Ephrin A5. Then we would attempt to identify any proteins that showed up, and finally, try to define its role in Ephrin signaling. This publication was made possible by NIH Grant Number P20 RR16481 from the BRIN Program of the National Center for Research Resources.
Metabolism
of base propenals.
CLINT MOREHEAD1*,
MATTHEW WEST2, SANJAY SRIVASTAVA2 and AHRUNI BHATNAGAR2,
1Department of Biology, Bellarmine University, Louisville, KY 40205,
and 2Division of Cardiology, University of Louisville, KY 40202.
Reactive oxygen species (ROS) are the major source of DNA damage. Although most of the damage is repaired, cumulative DNA injury due to ROS has been suggested to be responsible for spontaneous carcinogenesis and aging. Unquenched ROS induce a variety of modifications in DNA such as the formation of highly cytotoxic base propenals. Background levels of these compounds have been detected in healthy human cells. The base propenals also represent the major products of antieoplastic agents such as bleomycin. Once the base propenals are released, due to their a,b-unsaturation, they readily react with existing DNA to form pyrimido[1,2-a]purin-10(3H)-one (M1G) adducts. The objective of this study was to: (i) Identify the biochemical processes involved in the metabolism and detoxification of base propenals; (ii) Test the catalytic efficiency of aldose reductase (AR) in reducing base propenals and their glutathione and N-acetyl cysteine conjugates; (iii) Assess the contribution of AR-mediated reduction to the overall cellular metabolism of base propenals. It was determined that (i) glutathiolation and reduction are the main metabolic transformations of base propenals, that (ii) the reduction of glutathional base propenals is catalyzed by AR, and that (iii) because it prevents spontaneous dissociation of the conjugate and diminishes its electrophilicity, AR catalyzed reduction may be a critical detoxification route of DNA-derived aldehydes. This publication was made possible by NIH Grant Number P20 RR16481 from the BRIN Program of the National Center for Research Resources.
Mobilization
of P element transposons to create mutations in novel genes in Drosophila
melanogaster. ANGELA THACKER, SHINGIRIRAI NYAMWANZA
and JOHN RAWLS, Kentucky Biomedical Research Infrastructure Network Summer
Research Program, Department of Biology, University of Kentucky, Lexington, KY
40506.
With completion of the fruitfly genome project, the powerful genetic analysis system of Drosophila melanogaster may be applied to an even broader range of developmental and physiological issues. We have carried out genetic screens to isolate mutations affecting two very different cell processes: 1) control of pyrimidine degradation in animal cells, and 2) mRNA sorting and translation control during spermatogenesis. First, the gene for the second enzyme in pyrimidine degradation, dihydropyrimidinase, has been localized by the genome project to a site in chromosome 3 (the CRMP gene). In an attempt to create knockout mutations in this gene, we have mobilized a P element transposon near CRMP’s 5’ end. Second, genetic studies and analysis of transgenic animals have shown that mutation of the ms(3)127-10 gene causes precocious translation of mRNAs in pre-meiotic spermatocytes, RNAs that are normally stored and translated in post-meiotic spermatids. The ms(3)127-10 mutation has been localized to a small region in chromosome 3 within which the Drosophila genome project database identifies about twelve genes. To determine which of these controls translation during spermatogenesis, we have mobilized two P element transposons near the ms(3)127-10 gene, to create mutations in and near it. In each of these three screens, we made use of the transposon w+ eye color marker to identify and isolate animals in which the transposon was lost or modified. We will report on these genetic screens, the spectrum of transposon mobilization events created, and the properties of some of the mutations that we have isolated.
A
screening strategy to identify protective properties of genes regulated by
ischemic preconditioning. BARBRA
PEPER1,
JASON WILLIAMS2, YIRO GUO2, ROBERTO BOLLI2 and
GREGG ROKOSH2, 1Department of Biology, Bellarmine
University, Louisville, KY, 40205, and 2Division of Cardiology,
University of Louisville, KY, 40202.
Short cycles of ischemia can prepare myocardium to withstand longer periods of ischemia resulting in decreased myocardial damage. Known as ischemic preconditioning (IPC), this phenomenon can be witnessed in tow phases: early (within 1 hour) and late (after 12 hours). The changes occurring during early phase do not last long; however, changes occurring during the late phase last for days, as they incorporate increased protein synthesis. Research has begun to examine the changes associated with late phase IPC in order to determine what protection these changes in protein synthesis offer. Novel gene candidates involved in ischemic preconditioning, HAX-1 and ARF2, were isolated form murine mRNA and amplified by PCR. These cDNA segments were cloned and run through Real Time PCR (RT) for Microarray analysis validation. RT revealed minimal amounts of both gene products expressed ischemic preconditioned murine DNA samples. An in vitro model to determine the protective effects of Phsophatidylinositol-4-phosphate-5-kinase (PIPK), a novel candidate gene revealed through Microarray analysis of IPC mice, involved exposed transfected Cos7 cells to various concentrations of hydrogen peroxide. Cells were run through Fluorescence Activated Cell Sorting (FACS) to determine any protective benefits of PIPK against apoptosis. Results demonstrated little protection at all concentrations. This publication was made possible by NIH Grant Number P20 RR16481 from the BRIN Program of the National Center for Research Resources.
Design
of PCR primers based on DNA and protein sequence comparisons of allergenic
pollen proteins in ragweed. TADD
N. ROBERTS*, and
DAVE L. ROBINSON, Department of Biology, Bellarmine University, Louisville, KY,
40205.
It has been
estimated that 20% of the U.S. population has allergies, most commonly hayfever
caused by exposure to ragweed (Ambrosia spp.) pollen. There are five
major classes of ragweed pollen allergens that have been sequenced and submitted
to the NCBI GenBank. These have been found in four different ragweed species.
The known protein and DNA sequences of different ragweed pollen allergens were
obtained from the GenBank and aligned using a multiple sequence alignment tool (AlignX;
Vector NTI, Informax, Inc.). We focused on the two ragweed species that cause
most hayfever: Giant Ragweed (A. trifida L.) and Common Ragweed (A.
artemisiifolia L.). In comparing these two species, the DNA sequence
of pollen allergen Amb-5 was 78% identical, while the protein sequence (45 amino
acids) was 100% identical. A protein sequence for pollen allergen Amb-3 from A.elatior
L. (now classified as A. artemisiifolia L.) has been
published, but no protein or DNA sequences are available for the same allergen
in Giant Ragweed. In order to attempt PCR amplification of a Amb-3 gene in Giant
Ragweed we designed degenerate primers using the BackTranslate program (Vector
NTI). Since there is some level of evolutionary conservation observed in the
Amb-5 sequences, there may also be similarity in Amb-3. DNA was extracted from
Common Ragweed leaves and used, with these primers, for PCR amplification of a
putative Amb-3 gene.
This publication was made possible by NIH Grant Number P20 RR16481 from the BRIN
Program of the National Center for Research Resources.
The
Isolation and Characterization of a Possible
The amidohydrolase superfamily is
comprised of metalloenzymes that share a common overall structural fold.
Dihydroorotase, phosphotriesterase and urease are examples of three
members of this superfamily that share the five conserved amino acid residues as
well as the common ab
barrel core structure commonly found in all members of the family.
Primary sequence alignments performed through database searches can aide
in identifying possible new members of this superfamily.