Photolysis quantum yield measurements in the near-UV; a critical analysis of 1-(2-nitrophenyl)ethyl photochemistry.

The photolysis quantum yield, Qp, of 1-(2-nitrophenyl)ethyl phosphate (caged Pi) measured in the near-UV (342 nm peak with 60 nm half-bandwidth) is 0.53 and is based on results reported in 1978 (Biochemistry, 17, 1929-1935). This article amplifies methodology for determining that Qp in view of different recent estimates.

Agonist-induced Ca2+ sensitization in smooth muscle: redundancy of Rho guanine nucleotide exchange factors (RhoGEFs) and response kinetics, a caged compound study.

Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca(2+)]i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca(2+) sensitization. Gα12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca(2+)-sensitized force are not well understood.

Orientation of the N-terminal lobe of the myosin regulatory light chain in skeletal muscle fibers.

The orientation of the N-terminal lobe of the myosin regulatory light chain (RLC) in demembranated fibers of rabbit psoas muscle was determined by polarized fluorescence. The native RLC was replaced by a smooth muscle RLC with a bifunctional rhodamine probe attached to its A, B, C, or D helix. Fiber fluorescence data were interpreted using the crystal structure of the head domain of chicken skeletal myosin in the nucleotide-free state.

Pulmonary manifestations of relapsing polychondritis.

Relapsing polychondritis (RP) is a chronic multisystemic disease characterized by recurrent episodes of cartilage inflammation throughout the body. The lower respiratory tract is involved in 20% to 50% of patients and results in significant morbidity. Effective medical therapies and airway interventions are available in experienced centers; however, no single treatment is curative, and the prognosis of RP with airway disease remains overall guarded.

Relapsing polychondritis and airway involvement.

Objective: To assess the prevalence and characteristics of airway involvement in relapsing polychondritis (RP).

Methods: Retrospective chart review and data analysis of RP patients seen in the Rheumatology Clinic and the Complex Airway Center at Beth Israel Deaconess Medical Center from January 2004 through February 2008.

Results: RP was diagnosed in 145 patients.

Orientation of the essential light chain region of myosin in relaxed, active, and rigor muscle.

The orientation of the ELC region of myosin in skeletal muscle was determined by polarized fluorescence from ELC mutants in which pairs of introduced cysteines were cross-linked by BSR. The purified ELC-BSRs were exchanged for native ELC in demembranated fibers from rabbit psoas muscle using a trifluoperazine-based protocol that preserved fiber function. In the absence of MgATP (in rigor) the ELC orientation distribution was narrow; in terms of crystallographic structures of the myosin head, the LCD long axis linking heavy-chain residues 707 and 843 makes an angle (beta) of 120-125 degrees with the filament axis.

Studies of decarboxylation in photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds.

Photolysis of alpha-carboxy-2-nitrobenzyl (CNB) caged compounds, studied here by time-resolved IR and UV spectroscopy, involves at least two pathways. In one, a conventional 2-nitrobenzyl type rearrangement takes place to release the photoprotected species via rapid decay of an aci-nitro intermediate. The alpha-carboxylate moiety of the CNB group is retained and the final by-product from this pathway is 2-nitrosophenylglyoxylate.

Toward protein structure in situ: comparison of two bifunctional rhodamine adducts of troponin C.

As part of a program to develop methods for determining protein structure in situ, sTnC was labeled with a bifunctional rhodamine (BR or BSR), cross-linking residues 56 and 63 of its C-helix. NMR spectroscopy of the N-terminal domain of BSR-labeled sTnC in complex with Ca(2+) and the troponin I switch peptide (residues 115-131) showed that BSR labeling does not significantly affect the secondary structure of the protein or its dynamics in solution. BR-labeling was previously shown to have no effect on the solution structure of this complex.

Relapsing polychondritis: prevalence of expiratory CT airway abnormalities.

Purpose: To retrospectively determine the prevalence of expiratory computed tomographic (CT) abnormalities, including malacia and air trapping, in patients with relapsing polychondritis and to retrospectively determine the frequency with which expiratory abnormalities are accompanied by inspiratory abnormalities on CT scans.

Materials And Methods: Institutional review board approval was obtained, and informed consent was not required for this retrospective HIPAA-compliant study. A computerized hospital information system was used to identify all patients with clinically diagnosed or biopsy-proved relapsing polychondritis who were referred for CT airway imaging during a 17-month period.

Smooth muscle myosin: regulation and properties.

The relationship of the biochemical states to the mechanical events in contraction of smooth muscle cross-bridges is reviewed. These studies use direct measurements of the kinetics of Pi and ADP release. The rate of release of Pi from thiophosphorylated cycling cross-bridges held isometric was biphasic with turnovers of 1.

The fast tumble signal in bacterial chemotaxis.

We have analyzed repellent signal processing in Escherichia coli by flash photorelease of leucine from photolabile precursors. We found that 1). response amplitudes of free-swimming cell populations increased with leucine jump concentration, with an apparent Hill coefficient of 1.

Myosin regulatory light chain phosphorylation and strain modulate adenosine diphosphate release from smooth muscle Myosin.

The effects of myosin regulatory light chain (RLC) phosphorylation and strain on adenosine diphosphate (ADP) release from cross-bridges in phasic (rabbit bladder (Rbl)) and tonic (femoral artery (Rfa)) smooth muscle were determined by monitoring fluorescence transients of the novel ADP analog, 3'-deac-eda-ADP (deac-edaADP). Fluorescence transients reporting release of 3'-deac-eda-ADP were significantly faster in phasic (0.57 +/- 0.

Biphasic excitation by leucine in Escherichia coli chemotaxis.

Leucine concentration jumps (applied by photolysis of inert derivatives) triggered swim or tumble responses in Escherichia coli mutants lacking Tsr or Tar, respectively. Wild-type E. coli bacteria were attracted in spatial assays when the initial leucine concentration difference was 5 to 120 micro M but were repulsed when it was over 0.

Photolytic cleavage of 1-(2-nitrophenyl)ethyl ethers involves two parallel pathways and product release is rate-limited by decomposition of a common hemiacetal intermediate.

Time-resolved FTIR spectroscopic studies of the flash photolysis of several 1-(2-nitrophenyl)ethyl ethers derived from aliphatic alcohols showed that a long-lived hemiacetal intermediate was formed during the reaction. Breakdown of this intermediate was rate-limiting for product release. One of these compounds (methyl 2-[1-(2-nitrophenyl)ethoxy]ethyl phosphate, 9) was studied in detail by a combination of time-resolved FTIR and UV-vis spectroscopy.

Combined single-molecule force and fluorescence measurements for biology.

Recent advances in single-molecule techniques allow the application of force to an individual biomolecule whilst simultaneously monitoring its response using fluorescent probes. The effects of applied mechanical load on single-enzyme turnovers, biomolecular interactions and conformational changes can now be studied with nanometer precision and millisecond time resolution.

In situ orientations of protein domains: troponin C in skeletal muscle fibers.

A recently developed approach for mapping protein-domain orientations in the cellular environment was used to investigate the Ca(2+)-dependent structural changes in the tropomyosin/troponin complex on the actin filament that regulate muscle contraction. Polarized fluorescence from bifunctional rhodamine probes attached along four alpha helices of troponin C (TnC) was measured in permeabilized skeletal muscle fibers. In relaxed muscle, the N-terminal lobe of TnC is less closed than in crystal structures of the Ca(2+)-free domain, and its D helix is approximately perpendicular to the actin filament.

NMR structure of a bifunctional rhodamine labeled N-domain of troponin C complexed with the regulatory "switch" peptide from troponin I: implications for in situ fluorescence studies in muscle fibers.

The structure of the calcium-saturated regulatory domain of skeletal troponin C (sNTnC) complexed with the switch peptide comprising residues 115-131 of troponin I (TnI), and with a bifunctional rhodamine fluorescent label attached to residues 56 (E56C) and 63 (E63C) on the C helix of sNTnC, has been determined using nuclear magnetic resonance (NMR) spectroscopy. The structure shows that the integrity of the C helix is not altered by the E(56,63)C mutations or by the presence of the bifunctional rhodamine and that the label does not interact with the hydrophobic cleft of sNTnC. Moreover, the overall fold of the protein and the position of the TnI peptide are similar to those observed previously with related cardiac NTnC complexes with residues 147-163 of cardiac TnI [Li et al.

Orientation changes of the myosin light chain domain during filament sliding in active and rigor muscle.

Structural changes in myosin power many types of cell motility including muscle contraction. Tilting of the myosin light chain domain (LCD) seems to be the final step in transducing the energy of ATP hydrolysis, amplifying small structural changes near the ATP binding site into nanometer-scale motions of the filaments. Here we used polarized fluorescence measurements from bifunctional rhodamine probes attached at known orientations in the LCD to describe the distribution of orientations of the LCD in active contraction and rigor.

Altered immune response in adult women exposed to diethylstilbestrol in utero.

Objective: Between 1940 and 1970, 1.5 million female fetuses were exposed to diethylstilbestrol in utero. Numerous deleterious effects on reproductive anatomic and physiologic characteristics have been documented in these women.

Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.

Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.