All-trans-retinal is a closed-state inhibitor of rod cyclic nucleotide-gated ion channels

Rod vision begins when 11-cis-retinal absorbs a photon and isomerizes to all-trans-retinal (ATR) within the photopigment, rhodopsin. Photoactivated rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP, thereby closing cyclic nucleotide-gated (CNG) ion channels. After isomerization, ATR dissociates from rhodopsin, and after a bright light, this release is expected to produce a large surge of ATR near the CNG channels. Using excised patches from Xenopus oocytes, we recently showed that ATR shuts down cloned rod CNG channels, and that this inhibition occurs in the nanomolar range (aqueous concentration) at near-physiological concentrations of cGMP. Here we further characterize the ATR effect and present mechanistic information. ATR was found to decrease the apparent cGMP affinity, as well as the maximum current at saturating cGMP. When ATR was applied to outside-out patches, inhibition was much slower and less effective than when it was applied to inside-out patches, suggesting that ATR requires access to the intracellular surface of the channel or membrane. The apparent ATR affinity and maximal inhibition of heteromeric (CNGA1/CNGB1) channels was similar to that of homomeric (CNGA1) channels. Single-channel and multichannel data suggest that channel inhibition by ATR is reversible. Inhibition by ATR was not voltage dependent, and the form of its dose-response relation suggested multiple ATR molecules interacting per channel. Modeling of the data obtained with cAMP and cGMP suggests that ATR acts by interfering with the allosteric opening transition of the channel and that it prefers closed, unliganded channels. It remains to be determined whether ATR acts directly on the channel protein or instead alters channel-bilayer interactions.     

A matrix metalloproteinase gene is expressed at the boundary of senescence and programmed cell death in cucumber

Cell-cell and extracellular cell matrix (ECM) interactions provide cells with information essential for controlling morphogenesis, cell-fate specification, and cell death. In animals, one of the major groups of enzymes that degrade the ECM is the matrix metalloproteinases (MMPs). Here, we report the characterization of the cucumber (Cucumis sativus L. cv Marketmore) Cs1-MMP gene encoding such an enzyme likely to play a role in plant ECM degradation. Cs1-MMP has all the hallmark motif characteristics of animal MMPs and is a pre-pro-enzyme having a signal peptide, propeptide, and zinc-binding catalytic domains. Cs1-MMP also displays functional similarities with animal MMPs. For example, it has a collagenase-like activity that can cleave synthetic peptides and type-I collagen, a major component of animal ECM. Cs1-MMP activity is completely inhibited by a hydroxamate-based inhibitor that binds at the active site of MMPs in a stereospecific manner. The Cs1-MMP gene is expressed de novo at the end stage of developmental senescence, prior to the appearance of DNA laddering in cucumber cotyledons leaf discs and male flowers. As the steady-state level of Cs1-MMP mRNA peaks late in senescence and the pro-enzyme must undergo maturation and activation, the protease is probably not involved in nutrient remobilization during senescence but may have another function. The physiological substrates for Cs1-MMP remain to be determined, but the enzyme represents a good candidate for plant ECM degradation and may be involved in programmed cell death (PCD). Our results suggest that PCD occurs only at the culmination of the senescence program or that the processes are distinct with PCD being triggered at the end of senescence.     

Transient and cyclic responses of strain-generated potential in rabbit patellar tendon are frequency and pH dependent

The goal of this study was to expand understanding of strain-generated potential (SGP) in ligamentous or tendinous tissues. Most SGP studies in the past have focused on cartilage or bone. Herein, rabbit patellar tendon (PT) was used as a model. Each patellar tendon had two Ag/AgCl electrodes inserted at axial positions of 1/4 and 1/2 from patellar to tibial insertions. Each specimen was electrically isolated, gripped in a servohydraulic test system, and then subjected to a short session of uniaxial haversine tension (2.5 percent maximum strain) at a frequency of 0.5, 1.0, 2.0, or 5.0 Hz. A cyclic (sinusoidal) electrical potential superimposed upon a larger transient (exponentially asymptotic) potential was consistently observed. Upon termination of loading, the cyclic SGP ended, and the shifted baseline of the SGP exponentially decayed and asymptotically returned to a residual potential which over all specimens was not different than the original potential. The transient and cyclic SGPs were frequency dependent (P < 0.001, P = 0.06, respectively). To our knowledge, this transient portion of the SGP, although theoretically predicted by Suh (1996, Biorheology, 33, pp. 289-304) and Chen (1996, Ph.D. thesis, University of Wisconsin-Madison) has not been observed in other experiments using different protocols. Additional PTs were dehydrated and the rehydrated in solution at different pH levels. The magnitude of SGPs increased in basic solution (pH 9.5) but diminished in pH 4.7 buffer. This pH dependency suggests that electrokinetics is the dominant mechanism for the transient and cyclic responses of the SGPs, although this study does not provide direct evidence.     

Programmed cell death in cell cultures

In plants most instances of programmed cell death (PCD) occur in a number of related, or neighbouring, cells in specific tissues. However, recent research with plant cell cultures has demonstrated that PCD can be induced in single cells. The uniformity, accessibility and reduced complexity of cell cultures make them ideal research tools to investigate the regulation of PCD in plants. PCD has now been induced in cell cultures from a wide range of species including many of the so-called model species. We will discuss the establishment of cell cultures, the fractionation of single cells and isolation of protoplasts, and consider the characteristic features of PCD in cultured cells. We will review the wide range of methods to induce cell death in cell cultures ranging from abiotic stress, absence of survival signals, manipulation of signal pathway intermediates, through the induction of defence-related PCD and developmentally induced cell death.     

Abnormalities in uridine homeostatic regulation and pyrimidine nucleotide metabolism as a consequence of the deletion of the uridine phosphorylase gene

We report in the present study the critical role of uridine phosphorylase (UPase) in uridine homeostatic regulation and pyrimidine nucleotide metabolism, employing newly developed UPase-/- mice. Our data demonstrate that the abrogation of UPase activity led to greater than a 6-fold increase in uridine concentrations in plasma, a 5-6-fold increase in lung and gut, and a 2-3-fold increase in liver and kidney, as compared with wild type mice. Urine uridine levels increased 24-fold normal in UPase-/- mice. Uridine half-life and the plasma retention of pharmacological doses of uridine were significantly prolonged. Further, in these UPase-/- mice, abnormal uridine metabolism led to disorders of various nucleotide metabolisms. In the liver, gut, kidney, and lung of UPase-/- mice, total uridine ribonucleotide concentrations increased 2-3 times as compared with control mice. Cytidine ribonucleotides and adenosine and guanosine ribonucleotides also increased, although to a lesser extent, in these organs. Most significant deoxyribonucleotide changes were present in the gut and lung of UPase-/- mice. In these tissues, dTTP concentration increased more than 4-fold normal, and dCTP, dGTP, and dATP concentrations rose 1-2 times normal. In kidney, dTTP concentration increased 2-fold normal, and dCTP and dGTP concentrations rose less than 1-fold normal. In addition, the accumulated uridine in plasma and tissues efficiently reduced 5-fluorouracil host toxicity and altered the anesthetic effect of pentobarbital. These data indicate that UPase is a critical enzyme in the regulation of uridine homeostasis and pyrimidine nucleotide metabolism, and 5-fluorouracil activity.     

Drosophila Nedd4, a ubiquitin ligase,is recruited by Commissureless to control cell surface levels of the roundabout receptor

Crossing the midline produces changes in axons such that they are no longer attracted to the midline. In Drosophila, Roundabout reaches high levels on axons once they have crossed the midline, and this prohibits recrossing. Roundabout protein levels are regulated by Commissureless. We show that Commissureless binds to and is regulated by the ubiquitin ligase DNedd4. We further show that the ability of Commissureless to regulate Roundabout protein levels requires an intact DNedd4 binding site and ubiquitin acceptor sites within the Commissureless protein. The ability of Commissureless to regulate Robo in the embryo also requires a Commissureless/DNedd4 interaction. Our results show that changes in axonal sensitivity to external cues during pathfinding across the midline makes use of ubiquitin-dependent mechanisms to regulate transmembrane protein levels.     

Function-based selection and characterization of base-pair polymorphisms in a promoter of Escherichia coli RNA polymerase-sigma(70)

We performed two sets of in vitro selections to dissect the role of the -10 base sequence in determining the rate and efficiency with which Escherichia coli RNA polymerase-sigma(70) forms stable complexes with a promoter. We identified sequences that (i) rapidly form heparin-resistant complexes with RNA polymerase or (ii) form heparin-resistant complexes at very low RNA polymerase concentrations. The sequences selected under the two conditions differ from each other and from the consensus -10 sequence. The selected promoters have the expected enhanced binding and kinetic properties and are functionally better than the consensus promoter sequence in directing RNA synthesis in vitro. Detailed analysis of the selected promoter functions shows that each step in this multistep pathway may have different sequence requirements, meaning that the sequence of a strong promoter does not contain the optimal sequence for each step but instead is a compromise sequence that allows all steps to proceed with minimal constraint.