Comparative Analysis of kdp and ktr Mutants Reveals Distinct Roles of the Potassium Transporters in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803

Photoautotrophic bacteria have developed mechanisms to maintain K⁺ homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K⁺ uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K⁺ (Kdp). The contributions of each of these K⁺ transport systems to cellular K⁺ homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli, where Kdp conferred K⁺ uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K⁺ uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K⁺ depletion. Correspondingly, Kdp-mediated K⁺ uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19, encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K⁺ required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K⁺ concentration under conditions of limited K⁺ in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K⁺ availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K⁺ levels under fluctuating environmental conditions.     

Effects of gas pressurization with ethylene on the ultrastructure of the yeast Saccharomyces cerevisiae

We investigated ultrastructural changes in the yeast Saccharomyces cerevisiae when exposed to compressed ethylene gas. Transmission electron microscopy (TEM) revealed that intracellular organelles in yeast cells treated with compressed ethylene at up to 0.640 MPa (6.4 atm), especially the nuclear and plasma membranes, were seriously damaged.     

Characterization of the Rv3378c gene product, a new diterpene synthase for producing tuberculosinol and (13R, S)-isotuberculosinol (nosyberkol), from the Mycobacterium tuberculosis H37Rv genome

The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of 1H- and 13C-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.     

6,7-Dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives as novel corticotropin-releasing factor 1 receptor antagonists

To identify an orally active corticotropin-releasing factor 1 receptor antagonist, a series of 6,7-dihydro-5H-cyclopenta[d]pyrazolo[1,5-a]pyrimidines and their derivatives were designed, synthesized and evaluated. An in vitro study followed by in vivo and pharmacokinetic studies of these heterotricyclic compounds led us to the discovery of an orally active CRF1 receptor antagonist. The results of a structure-activity relationship study are presented.     

Quantum chemical modeling of perovskite: An investigation of piezoelectricity in ferrite of yttrium

In a previous article, we used Hartree-Fock (HF) theory to study the piezoelectricity in BaTiO₃. In this paper, we applied the Douglas-Kroll-Hess second order scalar relativistic method to investigate the possible piezoelectric properties in the perovskite YFeO₃ structure, which has not yet been studied experimentally. The 30s20p13d and 31s21p17d Gaussian basis sets for the Fe (⁵D) and Y (²D) atoms, respectively, were built with the Generator Coordinate HF method. After contraction to [13s7p5d] and [13s8p7d], in combination with the 20s14p/6s4p basis set for the O (³P) atom from literature, they had their quality evaluated using calculations of the total and the orbital energies for the ²FeO⁺¹ and ¹YO⁺¹ fragments. The dipole moment, the total energy, and the total atomic charges in YFeO₃ in C_s space group were calculated. The results and the analysis lead us to believe that the perovskite YFeO₃ does not present piezoelectric properties.     

In vivo tracking of transplanted mononuclear cells using manganese-enhanced magnetic resonance imaging (MEMRI)

Background

Transplantation of mononuclear cells (MNCs) has previously been tested as a method to induce therapeutic angiogenesis to treat limb ischemia in clinical trials. Non-invasive high resolution imaging is required to track the cells and evaluate clinical relevance after cell transplantation. The hypothesis that MRI can provide in vivo detection and long-term observation of MNCs labeled with manganese contrast-agent was investigated in ischemic rat legs.

Methods and Findings

The Mn-labeled MNCs were evaluated using 7-tesla high-field magnetic resonance imaging (MRI). Intramuscular transplanted Mn-labeled MNCs were visualized with MRI for at least 7 and up to 21 days after transplantation in the ischemic leg. The distribution of Mn-labeled MNCs was similar to that of ¹¹¹In-labeled MNCs measured with single-photon emission computed tomography (SPECT) and DiI-dyed MNCs with fluorescence microscopy. In addition, at 1–2 days after transplantation the volume of the site injected with intact Mn-labeled MNCs was significantly larger than that injected with dead MNCs, although the dead Mn-labeled MNCs were also found for approximately 2 weeks in the ischemic legs. The area covered by CD31-positive cells (as a marker of capillary endothelial cells) in the intact Mn-MNCs implanted site at 43 days was significantly larger than that at a site implanted with dead Mn-MNCs.

Conclusions

The present Mn-enhanced MRI method enabled visualization of the transplanted area with a 150–175 µm in-plane spatial resolution and allowed the migration of labeled-MNCs to be observed for long periods in the same subject. After further optimization, MRI-based Mn-enhanced cell-tracking could be a useful technique for evaluation of cell therapy both in research and clinical applications.     

Where Do Neurologists Look When Viewing Brain CT Images? An Eye-Tracking Study Involving Stroke Cases

The aim of this study was to investigate where neurologists look when they view brain computed tomography (CT) images and to evaluate how they deploy their visual attention by comparing their gaze distribution with saliency maps. Brain CT images showing cerebrovascular accidents were presented to 12 neurologists and 12 control subjects. The subjects' ocular fixation positions were recorded using an eye-tracking device (Eyelink 1000). Heat maps were created based on the eye-fixation patterns of each group and compared between the two groups. The heat maps revealed that the areas on which control subjects frequently fixated often coincided with areas identified as outstanding in saliency maps, while the areas on which neurologists frequently fixated often did not. Dwell time in regions of interest (ROI) was likewise compared between the two groups, revealing that, although dwell time on large lesions was not different between the two groups, dwell time in clinically important areas with low salience was longer in neurologists than in controls. Therefore it appears that neurologists intentionally scan clinically important areas when reading brain CT images showing cerebrovascular accidents. Both neurologists and control subjects used the "bottom-up salience" form of visual attention, although the neurologists more effectively used the "top-down instruction" form.