Third Annual Research Showcase, April 11, 2007
“The Effect of Mercuric Ion APTT on PT Clotting Tests”
Advisor Professor Charles Spillert, New Jersey Medical School
Mercuric ion (Hg2+) has been found to increase coagulant activity in whole blood. We conducted an in vitro study to determine whether mercuric chloride (HgCl2) has an effect on blood coagulation when added to whole blood solutions containing either PT or APTT reagent. We used 10 µl of 10% PT reagent and 10 µl of 10% APTT reagent and added them to separate solutions of 990 µl of human citrated whole blood (CWB). Mercuric chloride was added to each reagent sample and compared to the samples containing only reagent. The incubation period was 5 minutes at 37ºC. The blood clotting time was measured in seconds using a Sonoclot Coagulation Analyzer. PT and HgCl2 resulted in no change in clotting time when compared to that of just PT. APTT, in combination with HgCl2, resulted in a significant reduction in clotting time when compared to that of just APTT (p<0.01). The data in this study suggests that HgCl2 and APTT concurrently stimulate coagulation.
Krystian F. Jarosz
“Assessing Genetic Susceptibility to Early Onset Periodontitis”
Advisor Professor S. Diehl, New Jersey Dental School
Aggressive periodontitis is a rare destructive disease of the periodontal tissue that occurs in young individuals. Recently there has been an increased amassing of evidence for a genetic susceptibility to this classification of periodontal disease. This study aims to explore such a hypothesis of genetically programmed disease-genes via genotyping with SNP genetic markers. The study incorporates reaction plate preparation, assay operation, reading of the genotype, and lastly statistical analysis of obtained findings. Studies such as this are performed with the broad intention of future genetic therapy applications such as the preparation of personalized drugs that may ameliorate disease symptoms, or prevent disease altogether.
Jasneet Kaur and Tao Lin ( Math)
“Impact of Constitutively Active RhoA and Change in Cell Shape on Mitotic Spindle Orientation”
Advisors Professor Amitabha Bose and Professor Edward Bonder
The orientation of the mitotic spindle in a cell determines the cleavage plane for cytokinesis. Expression of constitutively active RhoA, a member of Rho GTPase family that is involved in signaling the rearrangement of actin cytoskeleton, has been observed to lead to a rounded cell shape as well as the misorientation of the mitotic spindle. In this report, we have examined the relationship between the change in cell shape caused by RhoA and the misorientation of the mitotic spindle. Normal IAR-2 rat liver epithelial cells, RhoA activated IAR-2 cells, and RhoA activated IAR-2 cells treated with several different concentrations of Y-27632, a highly potent, cell-permeable, selective inhibitor of Rho-associated protein kinase, were observed by confocal microscopy. This inhibitory action of Y-27632 relaxes actin-myosin contraction and allows the cell shape of the RhoA activated cells to return to normal. As a result, the relationship between cell shape and spindle orientation can be studied by analyzing the shapes and spindle angles of the different cells. From the data, it was concluded that the change in cell shape caused by the effect of constitutively active RhoA did not directly lead to a misoriented spindle. We propose that the reduction in amount of cortical flow as a result of over expression of RhoA has a more significant role in regulating the mitotic spindle orientation. Through mathematical modeling we show that the effect of cell shape is minimal compared to the effect of cortical flow in determining the angle of the mitotic spindle.
Olga Khorkova, PhD Student in Biology
“Long-Term Effects of Neuromodulatory Input on Ionic Current Interactions”
Advisor Jorge Golowasch
Reliability of respiration, heartbeat and digestion depends on the stability of output (bursting activity) of the central pattern generators (CPGs) controlling these functions. The bursting activity in turn depends on the characteristics of the ionic currents expressed by CPG neurons. Here we show that CPG neurons with different functions (e.g. pacemaker vs follower neurons) have distinct regulation of their currents levels and of their correlations. We further demonstrate that neurons with different functions respond differently during adaptation to changes in environmental inputs, such as the loss of neuromodulator supply after decentralization of crab stomatogastric ganglion (STG). Such differences could be essential for the simultaneous maintenance of stable output of single neurons and CPG networks under constantly changing environmental conditions.
“Structural Insights into Hydrolytic Mechanism of Antibiofilm Agent Dispersin B”
Advisor Dr. N. Ramasubbu, New Jersey Dental School
Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix (PGA) which is a linear polymer of N-acetylglucosamine residues in Î²(1,6)-linkage. Dispersin B (DspB), a soluble glycoside hydrolase produced by Actinobacillus actinomycetemcomitans (Aa) degrades PGA. DspB, is an (Î²/Î±) TIM-barrel protein and belongs to family 20 glycosyl hydrolases in which a conserved amino acid pair, aspartate-glutamate, is present (Asp183-Glu184, DspB numbering). In addition, the active site of DspB contains another acidic residue Glu332 at about 5Ã… away from Glu184. Objective: To understand the role of each of these acidic residues in the hydrolytic mechanism.
Methods: Using site-directed mutagenesis, biological and biochemical characterization, we investigated the role of Asp183, Glu184 and Glu332. Results: We found that Glu184 and Glu332 residues are essential for DspB activity. Mutation of each of these causes a significant reduction in the enzymatic activity. The variant Glu184Gln requires a 10-fold increase in enzyme concentration (>1000 nM) for measurable activity in kinetic as well as biofilm assay whereas Glu332Gln is inactive even at 1000 nM. In contrast, both DspB and Asp183Ala exhibited similar kinetics at 100 nM concentration; however, Asp183Ala showed a 12-fold loss in activity compared to DspB. Similar results were obtained in a 96-well biofilm detachment assay as well.
Conclusion: The loss of activity in the Glu184 and Glu332 variants suggests that DspB might hydrolyze PGA through a mechanism similar to the substrate-assisted mechanism proposed earlier. Based on our results, it appears that Asp183, Glu184 and Glu332 play a significant role in the hydrolysis of PGA.
“Temperature Dependency of Circadian Clocks in Drosophila”
Advisor Professor Issac Edery, Rutgers-New Brunswick
The circadian clock, an internal biological clock, allows, among other things, an organism to adjust its daily activity patterns by sensing changes in environmental queues such as light/dark (LD) cycles and temperature. Splicing of the Drosophila melanogaster period (per) intron 8 (dmpi8) in the 3’UTR has been associated with temperature sensitivity of circadian clocks in Drosophila. High splicing efficiency is linked with early evening activity peak and low splicing efficiency with later mainly nocturnal evening activity peaks in D. melanogaster (Canton-S; a strain originating from N. America). Cold temperatures are associated with high splicing efficiency and warm temperatures with low splicing efficiency. This makes biological sense by providing a mechanism whereby flies avoid the hot midday hours during warm days. The SR proteins sc35, srp54, nop5, xl6, rbp1, sf2 may play a role in splicing regulation, however, silencing expression of a single protein at a time showed no affect. Silencing multiple related proteins at a time may be the key to determining the role of these SR proteins in splicing regulation of dmpi8.