S-nitrosylation of cytoskeletal proteins.

Nitric oxide has pronounced effects on cellular functions normally associated with the cytoskeleton, including cell motility, shape, contraction, and mitosis. Protein S-nitrosylation, the covalent addition of a NO group to a cysteine sulfur, is a signaling pathway for nitric oxide that acts in parallel to cyclic guanosine monophosphate (cGMP), but is poorly studied compared to the latter. There is growing evidence that S-nitrosylation of cytoskeletal proteins selectively alters their function.

Design and implementation of a student-taught course on research in regenerative medicine.

In the Undergraduate School of Engineering and Applied Sciences (SEAS) at the University of Virginia (UVa), there are few opportunities for undergraduate students to teach, let alone develop, an introductory course for their major. As two undergraduate engineering students (D. N.

Spread from the Sink to the Patient: Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs.

There have been an increasing number of reports implicating as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing from sink wastewater to the surrounding environment.

Direct measurement of cortical force generation and polarization in a living parasite.

Apicomplexa is a large phylum of intracellular parasites that are notable for the diseases they cause, including toxoplasmosis, malaria, and cryptosporidiosis. A conserved motile system is critical to their life cycles and drives directional gliding motility between cells, as well as invasion of and egress from host cells. However, our understanding of this system is limited by a lack of measurements of the forces driving parasite motion.

Loop 2 of myosin is a force-dependent inhibitor of the rigor bond.

Myosin's actin-binding loop (loop 2) carries a charge opposite to that of its binding site on actin and is thought to play an important role in ionic interactions between the two molecules during the initial binding step. However, no subsequent role has been identified for loop 2 in actin-myosin binding. We used an optical trap to measure bond formation and bond rupture between actin and rigor heavy meromyosin when loaded perpendicular to the filament axis.

Design, development, and evaluation of a novel retraction device for gallbladder extraction during laparoscopic cholecystectomy.

Background: A source of frustration during laparoscopic cholecystectomy involves extraction of the gallbladder through port sites smaller than the gallbladder itself. We describe the development and testing of a novel device for the safe, minimal enlargement of laparoscopic port sites to extract large, stone-filled gallbladders from the abdomen.

Methods: The study device consists of a handle with a retraction tongue to shield the specimen and a guide for a scalpel to incise the fascia within the incision.

Laser trap measurements of flagellar membrane motility.

In addition to swimming motility, which is driven by propagation of bends along the flagellum, the unicellular green alga Chlamydomonas exhibits an unusual and alternative form of whole cell locomotion, called gliding motility. In gliding motility, a large flagellar membrane glycoprotein mediates flagellar membrane adhesion to solid substrates. This in turn activates a transmembrane signaling system that initiates the movement of a cross-linked cluster of glycoproteins within the plane of the flagellar membrane by activating and/or recruiting isoforms of the motor proteins kinesin and dynein.

A High-Throughput Technique Reveals the Load- and Site Density-Dependent Kinetics of E-Selectin.

The kinetics of bond rupture between receptors and ligand are critically dependent on applied mechanical force. Force spectroscopy of single receptor-ligand pairs to measure kinetics is a laborious and time-consuming process that is generally performed using individual force probes and making one measurement at a time when typically hundreds of measurements are needed. A high-throughput approach is thus desirable.

Thermal denaturation of tilapia myosin and its subunits as affected by constantly increasing temperature.

Purified tilapia myosin was digested with α-chymotrypsin and purified to obtain heavy meromyosin (HMM) and light meromyosin (LMM). Biochemical properties of tilapia myosin, HMM, and LMM were characterized. Surface hydrophobicity (S(o) ) showed an increase for myosin and HMM between 30 and 40 °C and reached a plateau at 70 °C.

Force spectroscopy reveals multiple "closed states" of the muscle thin filament.

Tropomyosin (Tm) plays a critical role in regulating the contraction of striated muscle. The three-state model of activation posits that Tm exists in three positions on the thin filament: "blocked" in the absence of calcium when myosin cannot bind, "closed" when calcium binds troponin and Tm partially covers the myosin binding site, and "open" after myosin binding forces Tm completely off neighboring sites. However, we recently showed that actin filaments decorated with phosphorylated Tm are driven by myosin with greater force than bare actin filaments.

4-Azetidinyl-1-heteroatom linked cyclohexane antagonists of CCR2: patent evaluation.

This application discloses a series of di- and tri-substituted cyclohexanes as CCR2 receptor antagonists which are stated to be useful in treating inflammation and autoimmune diseases, such as type 2 diabetes and asthma. Although receptor binding of the compounds to CCR2 is demonstrated, there are no data to support the idea that these molecules are functional antagonists.

Direct regulation of striated muscle myosins by nitric oxide and endogenous nitrosothiols.

Background: Nitric oxide (NO) has long been recognized to affect muscle contraction, both through activation of guanylyl cyclase and through modification of cysteines in proteins to yield S-nitrosothiols. While NO affects the contractile apparatus directly, the identities of the target myofibrillar proteins remain unknown. Here we report that nitrogen oxides directly regulate striated muscle myosins.

The viscoelastic properties of microvilli are dependent upon the cell-surface molecule.

We studied at nanometer resolution the viscoelastic properties of microvilli and tethers pulled from myelogenous cells via P-selectin glycoprotein ligand 1 (PSGL-1) and found that in contrast to pure membrane tethers, the viscoelastic properties of microvillus deformations are dependent upon the cell-surface molecule through which load is applied. A laser trap and polymer bead coated with anti-PSGL-1 (KPL-1) were used to apply step loads to microvilli. The lengthening of the microvillus in response to the induced step loads was fitted with a viscoelastic model.

The Effects of Load on E-Selectin Bond Rupture and Bond Formation.

Molecular dissociation rates have long been known to be sensitive to applied force. We use a laser trap to provide evidence that rates of association may also be force-dependent. We use the thermal fluctuation assay to study single bonds between E-selectin and sialyl Lewis(a) (sLe(a)), the sugar on PSGL-1 to which the three selectins bind.

The reciprocal coordination and mechanics of molecular motors in living cells.

Molecular motors in living cells are involved in whole-cell locomotion, contractility, developmental shape changes, and organelle movement and positioning. Whether motors of different directionality are functionally coordinated in cells or operate in a semirandom "tug of war" is unclear. We show here that anterograde and retrograde microtubule-based motors in the flagella of Chlamydomonas are regulated such that only motors of a common directionality are engaged at any single time.

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit.

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding.