Chemical Research with Undergraduate Collaborators: Building Cyclic Polymers

Undergraduate research in chemistry provides not only a meaningful experience for the students, but is essential in getting research done. This talk will focus on an ongoing project in my lab: designing large molecules of specific shapes by studying the fundamental reactions. While results will be discussed, the talk will be tailored towards a general audience. I will attempt to highlight the outstanding contributions made by Bucknell students that have worked in my lab.

Quantum Mechnical Investion of Cyclic 3',5'- Adenosine Monophosphate, the Second Hormonal Messenger

Full geometry optimizations using the PM3, AM1, 3-21G∗/HF and 6-31G∗/HF levels of theory were conducted on the syn and anti conformations of cyclic3′,5′-adenosine monophosphate (cAMP). Comparison of the anti crystal structures with the semiempirical and ab initio results revealed that the ab initio results agree well with the experimental results. The results of semiempirical calculations are in qualitative agreement with experimental and ab initio values, with the exception of the glycosyl torsion angle for the anti conformer. Sugar puckering, which is not handled properly by semiempirical methods for unconstrained sugars, nucleosides, nucleotides and nucleotide base pairs, is modeled reasonably well by the semiempirical methods for cAMP. This improvement results from the constraints introduced by the cyclization of AMP to form the phosphodiester.

Use of the Supermolecule Approach To Model the Syn and Anti Conformations of Solvated Cyclic 3‘,5‘-Adenosine Monophosphate

The supermolecule approach has been used to model the hydration of cyclic 3‘,5‘-adenosine monophosphate, cAMP. Model building combined with PM3 optimizations predict that the anti conformer of cAMP is capable of hydrogen bonding to an additional solvent water molecule compared to the syn conformer. The addition of one water to the syn superstructure with concurrent rotation of the base about the glycosyl bond to form the anti superstructure leads to an additional enthalpy of stabilization of approximately −6 kcal/mol at the PM3 level. This specific solute−solvent interaction is an example of a large solvent effect, as the method predicts that cAMP has a conformational preference for the anti isomer in solution. This conformational preference results from a change in the number of specific solute−solvent interactions in this system. This prediction could be tested by NMR techniques. The number of waters predicted to be in the first hydration sphere around cAMP is in agreement with the results of hydration studies of nucleotides in DNA. In addition, the detailed picture of solvation about this cyclic nucleotide is in agreement with infrared experimental results.

Cyclic alternating pattern in narcolepsy patients and healthy controls after partial and total sleep deprivation

To investigate the regulation NREM sleep at baseline and in morning recovery sleep after partial and total sleep deprivation (SD) in narcolepsy-cataplexy (NC) using cyclic alternating pattern (CAP).

Stochastic amplitude-modulated stretching of Rabbit flexor digitorum profundus tendons reduces stiffness compared to Cyclic Loading but does not affect Tenocyte Metabolism

Background It has been demonstrated that frequency modulation of loading influences cellular response and metabolism in 3D tissues such as cartilage, bone and intervertebral disc. However, the mechano-sensitivity of cells in linear tissues such as tendons or ligaments might be more sensitive to changes in strain amplitude than frequency. Here, we hypothesized that tenocytes in situ are mechano-responsive to random amplitude modulation of strain. Methods We compared stochastic amplitude-modulated versus sinusoidal cyclic stretching. Rabbit tendon were kept in tissue-culture medium for twelve days and were loaded for 1h/day for six of the total twelve culture days. The tendons were randomly subjected to one of three different loading regimes: i) stochastic (2 – 7% random strain amplitudes), ii) cyclic_RMS (2–4.42% strain) and iii) cyclic_high (2 - 7% strain), all at 1 Hz and for 3,600 cycles, and one unloaded control. Results At the end of the culture period, the stiffness of the “stochastic” group was significantly lower than that of the cyclic_RMS and cyclic_high groups (both, p < 0.0001). Gene expression of eleven anabolic, catabolic and inflammatory genes revealed no significant differences between the loading groups. Conclusions We conclude that, despite an equivalent metabolic response, stochastically stretched tendons suffer most likely from increased mechanical microdamage, relative to cyclically loaded ones, which is relevant for tendon regeneration therapies in clinical practice.

An essential bacterial-type cardiolipin synthase mediates cardiolipin formation in a eukaryote

Cardiolipin is important for bacterial and mitochondrial stability and function. The final step in cardiolipin biosynthesis is catalyzed by cardiolipin synthase and differs mechanistically between prokaryotes and eukaryotes. To study the importance of cardiolipin synthesis for mitochondrial integrity, membrane protein complex formation, and cell proliferation in the human and animal pathogenic protozoan parasite, Trypanosoma brucei, we generated conditional cardiolipin synthase-knockout parasites. We found that cardiolipin formation in T. brucei procyclic forms is catalyzed by a bacterial-type cardiolipin synthase, providing experimental evidence for a prokaryotic-type cardiolipin synthase in a eukaryotic organism. Ablation of enzyme expression resulted in inhibition of de novo cardiolipin synthesis, reduction in cellular cardiolipin levels, alterations in mitochondrial morphology and function, and parasite death in culture. By using immunofluorescence microscopy and blue-native gel electrophoresis, cardiolipin synthase was shown to colocalize with inner mitochondrial membrane proteins and to be part of a large protein complex. During depletion of cardiolipin synthase, the levels of cytochrome oxidase subunit IV and cytochrome c1, reflecting mitochondrial respiratory complexes IV and III, respectively, decreased progressively.

Hydraulic sustainability of soil-bentonite cutoff walls subjected to cyclic wetting and drying

This study investigated the effect of cyclic wetting and drying on the backfill used in soil-bentonite (SB) cutoff walls. For this purpose, model SB vertical cutoff wall backfills were prepared comprising of a fine grained mortar sand and 2% bentonite (by total weight) and 4% bentonite (by total weight). Results of the study indicate that the volume change is influenced by the bentonite content, that is, the increase in volume change increased with increasing bentonite content.

Topical application of 17β-estradiol (E2) improves skin flap survival through activation of endothelial nitric oxide synthase in rats

This study investigates the influence of 17β-estradiol (E2) on nitric oxide (NO) production in endothelial cell cultures and the effect of topical E2 on the survival of skin flap transplants in a rat model. Human umbilical vein endothelial cells were treated with three different E2 concentrations and nitrite (NO2) concentrations, as well as endothelial nitric oxide synthase (eNOS) protein expressions were analyzed. In vivo, random-pattern skin flaps were raised in female Wistar rats 14 days following ovariectomy and treated with placebo ointment (group 1), E2 as gel (group 2), and E2 via plaster (group 3). Flap perfusion, survival, and NO2 levels were measured on postoperative day 7. In vitro, E2 treatment increased NO2 concentration in cell supernatant and eNOS expression in cell lysates (p < 0.05). In vivo, E2 treated (gel and plaster groups) demonstrated significantly increased skin flap survival compared to the placebo group (p < 0.05). E2 plaster-treated animals exhibited higher NO2 blood levels than placebo (p < 0.05) paralleling the in vitro observations. E2 increases NO production in endothelial cells via eNOS activation. Topical E2 application can significantly increase survival of ischemically challenged skin flaps in a rat model and may augment wound healing in other ischemic situations via activation of NO production.

Role of RhoA/ROCK-dependent actin contractility in the induction of tenascin-C by cyclic tensile strain

In chick embryo fibroblasts, the mRNA for extracellular matrix protein tenascin-C is induced 2-fold by cyclic strain (10%, 0.3 Hz, 6 h). This response is attenuated by inhibiting Rho-dependent kinase (ROCK). The RhoA/ROCK signaling pathway is primarily involved in actin dynamics. Here, we demonstrate its crucial importance in regulating tenascin-C expression. Cyclic strain stimulated RhoA activation and induced fibroblast contraction. Chemical activators of RhoA synergistically enhanced the effects of cyclic strain on cell contractility. Interestingly, tenascin-C mRNA levels perfectly matched the extent of RhoA/ROCK-mediated actin contraction. First, RhoA activation by thrombin, lysophosphatidic acid, or colchicine induced tenascin-C mRNA to a similar extent as strain. Second, RhoA activating drugs in combination with cyclic strain caused a super-induction (4- to 5-fold) of tenascin-C mRNA, which was again suppressed by ROCK inhibition. Third, disruption of the actin cytoskeleton with latrunculin A abolished induction of tenascin-C mRNA by chemical RhoA activators in combination with cyclic strain. Lastly, we found that myosin II activity is required for tenascin-C induction by cyclic strain. We conclude that RhoA/ROCK-controlled actin contractility has a mechanosensory function in fibroblasts that correlates directly with tenascin-C gene expression. Previous RhoA/ROCK activation, either by chemical or mechanical signals, might render fibroblasts more sensitive to external tensile stress, e.g., during wound healing.

Sensitivity to endothelin-1 is decreased in isolated livers of endothelial constitutive nitric oxide synthase knockout mice

ABSTRACT: BACKGROUND: Hepatic sinusoidal resistance is regulated by vasoactive factors including endothelin-1 (ET-1) and nitric oxide (NO). In the absence of NO, vasoconstrictor response to endothelin is expected to predominate. Therefore, we hypothesized sensitivity to endothelin to be increased in mice lacking the endothelial cell NO synthase gene. Response of vascular resistance to endothelin was assessed in the in situ perfused liver of endothelial constitutive nitric oxide synthase (ecNOS) knockout and wild type mice. Livers were also harvested for RNA and protein isolation for quantitative PCR and Western blotting, respectively. The expression of endothelin receptors, isoenzymes of NO synthase, heme-oxygenase and adrenomedullin was quantified. RESULTS: Endothelin increased hepatic vascular resistance in a dose-dependent manner in both strains; however, this increase was significantly less in ecNOS knockout mice at physiologic concentrations. Expression of heme-oxygenases and adrenomedullin was similar in both groups, whereas inducible nitric oxide synthase (iNOS) protein was not detectable in either strain. mRNA levels of pre-pro-endothelin-1 and ETB receptor were comparable in both strains, while mRNA for ETA receptor was decreased in ecNOS knockouts. CONCLUSION: Livers of ecNOS knockout mice have a decreased sensitivity to endothelin at physiologic concentrations; this is associated with a decreased expression of ETA receptors, but not with other factors, such as iNOS, ETB receptors, adrenomedullin or heme-oxygenase. Further studies targeting adaptive changes in ETA receptor distribution and/or intracellular signaling downstream of the receptor are indicated.

Single-cell analysis divides bovine monocyte-derived dendritic cells into subsets expressing either high or low levels of inducible nitric oxide synthase

Dendritic cells (DC) are important cells at the interface between innate and adaptive immunity. DC have a key role in antigen processing and presentation to T cells. Effector functions of DC related to innate immunity have not been explored extensively. We show that bovine monocyte-derived DC (mDC) express inducible nitric oxide synthase (iNOS) mRNA and protein and produce NO upon triggering with interferon-gamma (IFN-gamma) and heat-killed Listeria monocytogenes (HKLM). An immunocytochemical analysis revealed that a sizeable subset (20-60%) copiously expresses iNOS (iNOShi) upon IFN-gamma/HKLM triggering, whereas the other subset expressed low levels of iNOS (iNOSlo). Monocyte-derived macrophages (mMphi) are more homogeneous with regard to iNOS expression. The number of cells within the iNOSlo mDC subset is considerably larger than the number of dead cells or cells unresponsive to IFN-gamma/HKLM. The large majority of cells translocated p65 to the nucleus upon triggering by IFN-gamma/HKLM. A contamination of mDC with iNOS-expressing mMphi was excluded as follows. (i) Cell surface marker analysis suggested that mDC were relatively homogeneous, and no evidence for a contaminating subset expressing macrophage markers (e.g. high levels of CD14) was obtained. (ii) iNOS expression was stronger in iNOShi mDC than in mMphi. The use of maturation-promoting stimuli revealed only subtle phenotypic differences between immature and mature DC in cattle. Nevertheless, these stimuli promoted development of considerably fewer iNOShi mDC upon triggering with IFN-gamma/HKLM. Immunocytochemical results showed that although a significant proportion of cells expressed iNOS only or TNF only upon triggering with IFN-gamma/HKLM, a significant number of cells expressed both iNOS and TNF, suggesting that TNF and iNOS producing (TIP) DC are present within bovine mDC populations obtained in vitro.