The Time Required for Dormancy Release in Arabidopsis Is Determined by DELAY OF GERMINATION1 Protein Levels in Freshly Harvested Seeds

Seed dormancy controls the start of a plant's life cycle by preventing germination of a viable seed in an unfavorable season. Freshly harvested seeds usually show a high level of dormancy, which is gradually released during dry storage (after-ripening). Abscisic acid (ABA) has been identified as an essential factor for the induction of dormancy, whereas gibberellins (GAs) are required for germination. The molecular mechanisms controlling seed dormancy are not well understood. DELAY OF GERMINATION1 (DOG1) was recently identified as a major regulator of dormancy in Arabidopsis thaliana. Here, we show that the DOG1 protein accumulates during seed maturation and remains stable throughout seed storage and imbibition. The levels of DOG1 protein in freshly harvested seeds highly correlate with dormancy. The DOG1 protein becomes modified during after-ripening, and its levels in stored seeds do not correlate with germination potential. Although ABA levels in dog1 mutants are reduced and GA levels enhanced, we show that DOG1 does not regulate dormancy primarily via changes in hormone levels. We propose that DOG1 protein abundance in freshly harvested seeds acts as a timer for seed dormancy release, which functions largely independent from ABA.     

One-step purification of a functional, constitutively activated form of visual arrestin

Desensitization of agonist-activated G protein-coupled receptors (GPCRs) requires phosphorylation followed by the binding of arrestin, a ~48 kDa soluble protein. While crystal structures for the inactive, 'basal' state of various arrestins are available, the conformation of 'activated' arrestin adopted upon interaction with activated GPCRs remains unknown. As a first step towards applying high-resolution structural methods to study arrestin conformation and dynamics, we have utilized the subtilisin prodomain/Profinity eXact? fusion-tag system for the high-level bacterial expression and one-step purification of wild-type visual arrestin (arrestin 1) as well as a mutant form (R175E) of the protein that binds to non-phosphorylated, light-activated rhodopsin (Rho?). The results show that both prodomain/Profinity eXact? fusion-tagged wild-type and R175E arrestins can be expressed to levels approaching 2-3 mg/l in Luria-Bertani media, and that the processed, tag-free mature forms can be purified to near homogeneity using a Bio-Scale? Mini Profinity eXact? cartridge on the Profinia? purification system. Functional analysis of R175E arrestin generated using this approach shows that it binds to non-phosphorylated rhodopsin in a light-dependent manner. These findings should facilitate the structure determination of this 'constitutively activated' state of arrestin 1 as well as the monitoring of conformational changes upon interaction with Rho?.     

Existence of memory in membrane channels: analysis of ion current through a voltage‐dependent potassium single channel

Ion current fluctuation of voltage‐dependent potassium channel in LβT2 cells has been investigated by autocorrelation function and DFA (detrended fluctuation analysis) methods. The calculation of the autocorrelation function exponent and DFA exponent of the sample was based on the digital signals or the 0–1 series corresponding to closing and opening of channels after routine evolution, rather than the sequence of sojourn times. The persistent character of the correlation of the time series was evident from the slow decay of the autocorrelation function. DFA exponent α was significantly greater than 0.5. The main outcome has been the demonstration of the existence of memory in this ion channel. Thus, the ion channel current fluctuation provided information about the kinetics of the channel protein. The result suggests the correlation character of the ion channel protein non‐linear kinetics indicates whether the channel is open or not.     

Mechanisms of Prostate Permeability Triggered by Microbubble-Mediated Acoustic Cavitation

The objective of this research was to study the mechanisms of opening of the blood?Cprostate barrier and increased permeability of prostate tissue induced by microbubble cavitation. Thirty-five rabbits were randomly divided into four study groups: (1) control group and groups exposed to (2) microbubble alone, (3) ultrasound alone, or (4) combined intervention (ultrasound?+?microbubble group). Evans blue (EB) tracer was used to gauge the changes of permeability of prostate tissue. Furthermore, light and electron microscopy analyses were conducted, as well as the western blot analysis of expression of gap junction (Cx43) protein. We observed that EB concentration in prostate tissue was significantly greater in the ultrasound?+?microbubble group compared with either intervention alone (p?<?0.05, both comparisons). Furthermore, light microscopy of tissue samples from animals exposed to ultrasound?+?microbubble showed epithelial cell disarrangement, loss of interstitial structure, and thickness of fibrous stroma. In line with these findings, electron microscopy analysis demonstrated widening of cell gaps and broken cell connections, as well as more dense lysosomes and secretary granules, and mitochondrial swelling. These changes were absent in the animals exposed to microbubble or ultrasound alone. Finally, only combined treatment with microbubble or ultrasound significantly elevated expression of Cx43 (p?<?0.05 vs. control group). In conclusion, increases of permeability of prostate tissue by acoustic cavitation appear to involve opening of tight junctions, widening of intracellular spaces, changes in the structure of acinar cell membrane, enhancement of vesicular transport, and loosening of fibrous stroma. Increased expression of cell gap junction protein will help to restore normal connections between cells and the blood?Cprostate barrier after the treatment.     

Usefulness of Speckle Tracking Imaging to Assess Myocardial Contractility in Intra-Abdominal Hypertension: Study in a Mini-Pig Model

This study evaluated the usefulness of speckle tracking imaging (STI) in assessment of myocardial contractility in intra-abdominal hypertension experimentally induced in mini-pigs. To this effect, 12 mini-pigs were anesthetized with intravenous injection of 3?% sodium pentobarbital, hemorrhaged to reach the shock status, and resuscitated with excessive volume of lactated Ringer??s solution. The animals were either sham-operated (study group 1) or underwent treatment with intra-abdominal volume increment (study group 2). Observations were made prior to induction of shock, 1?h after shock, 2?h after induction of intra-abdominal hypertension, and 8 and 12?h after treatment. The heart rate and mean artery pressure were conventionally measured. STI was used to assess radial and circumferential strains of segmental ventricular wall. The results obtained demonstrated that myocardial contractility, as manifested by radial and circumferential strains of different ventricular wall segments, was decreased after induction of intra-abdominal hypertension. Treatment with intra-abdominal volume increment was able to decrease heart rate and intra-bladder pressure (indicator of effectiveness of treatment) and significantly improved myocardial contractility of involved ventricular wall segments. In conclusion, STI is a useful method to assess myocardial regional functions.     

Molecular characterization of the GHRH/GHRH-R and its effect on GH synthesis and release in orange-spotted grouper (Epinephelus coioides)

Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide that stimulates growth hormone (GH) synthesis and secretion in the pituitary gland. In this paper, the full-length cDNAs of orange-spotted grouper GHRH and its receptor (GHRH-R) were cloned. The grouper GHRH cDNA is 713 bp in length and encodes a 141-aa precursor that includes an 18-aa signal peptide, a 27-aa mature GHRH mature peptide and a 47-aa carboxyl terminus. The grouper GHRH-R cDNA sequence is 1495 bp in length, encoding a 422-aa receptor with seven transmembrane domains. Tissue distribution analyses showed that both GHRH and GHRH-R mRNAs were predominantly expressed in the brain, while the GHRH-R mRNA was also abundantly detected in the pituitary gland. Both GHRH and GHRH-R mRNAs were expressed throughout embryonic development from the multi-cell stage to the newly hatched larvae stage, and the highest GHRH and GHRH-R expressions appeared at the brain vesicle stage and the heart stage, respectively. In vitro studies performed on the grouper pituitary primary cells showed that a synthetic grouper GHRH-NH(2) increased both GH mRNA expression and GH protein release in a dose-dependent manner. Together, these results suggest that the newly obtained grouper GHRH was able to stimulate GH synthesis and release, similar to its mammalian counterparts.