PSF1 is essential for early embryogenesis in mice

Psf1 (partner of sld five 1) forms a novel heterotetramer complex, GINS (Go, Ichi, Nii, and San; five, one, two, and three, respectively, in Japanese), with Sld5, Psf2, and Psf3. The formation of this complex is essential for the initiation of DNA replication in yeast and Xenopus laevis egg extracts. Although all of the components are well conserved in higher eukaryotes, the biological function in vivo is largely unknown. We originally cloned the mouse ortholog of PSF1 from a hematopoietic stem cell cDNA library and found that PSF1 is expressed in blastocysts, adult bone marrow, and testis, in which the stem cell system is active. Here we used the gene-targeting technique to determine the physiological function of PSF1 in vivo. Mice homozygous for a nonfunctional mutant of PSF1 died in utero around the time of implantation. PSF1-/- blastocysts failed to show outgrowth in culture and exhibited a cell proliferation defect. Our data clearly indicate that PSF1 is required for early embryogenesis.     

Role of pressor mechanisms from the NTS and CVLM in control of arterial pressure

In the present study, we investigated the effects of inhibition of the caudal ventrolateral medulla (CVLM) with the GABA(A) agonist muscimol combined with the blockade of glutamatergic mechanism in the nucleus of the solitary tract (NTS) with kynurenic acid (kyn) on mean arterial pressure (MAP), heart rate (HR), and regional vascular resistances. In male Holtzman rats anesthetized intravenously with urethane/chloralose, bilateral injections of muscimol (120 pmol) into the CVLM or bilateral injections of kyn (2.7 nmol) into the NTS alone increased MAP to 186 +/- 11 and to 142 +/- 6 mmHg, respectively, vs. control: 105 +/- 4 mmHg; HR to 407 +/- 15 and to 412 +/- 18 beats per minute (bpm), respectively, vs. control: 352 +/- 12 bpm; and renal, mesenteric and hindquarter vascular resistances. However, in rats with the CVLM bilaterally blocked by muscimol, additional injections of kyn into the NTS reduced MAP to 88 +/- 5 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Moreover, in rats with the glutamatergic mechanisms of the NTS blocked by bilateral injections of kyn, additional injections of muscimol into the CVLM also reduced MAP to 92 +/- 2 mmHg and mesenteric and hindquarter vascular resistances below control baseline levels. Simultaneous blockade of NTS and CVLM did not modify the increase in HR but also abolished the increase in renal vascular resistance produced by each treatment alone. The results suggest that important pressor mechanisms arise from the NTS and CVLM to control vascular resistance and arterial pressure under the conditions of the present study.     

The formation of Zn-Chl a in Chlorella heterotrophically grown in the dark with an excessive amount of Zn2+

Chlorella, when heterotrophically cultivated in the dark, is able to grow with Zn2+ at 10-40 mM, which is 10 times the concentration lethal to autotrophically grown cells. However, the lag phase is prolonged with increasing concentrations of Zn2+; for example, in this study, 1 d of the control lag phase was prolonged to about 16 d with Zn2+ at 16.7 mM (x2,000 of the control). Once the cells started to grow, the log phase was finished within 4-6 d regardless of Zn concentration, which was almost the same as that of the control. The photosysystem I reaction center chlorophyll, P700, and the far-red fluorescence were detected only after the late log phase of the growth curve, suggesting that chlorophyll-protein complexes can be organized after cell division has ceased. Interestingly, at more than 16.7 mM of Zn2+, Zn-chlorophyll a was accumulated and finally accounted for about 25% of the total chlorophyll a in the late stationary phase. We found that the Zn-chlorophyll a was present in the thylakoid membranes and not in the soluble fractions of the cells. The rather low fluorescence yield at around 680 nm in the stationary phase suggests that Zn-chlorophyll a can transfer its excitation energy to other chlorophylls. Before accumulation of Zn-chlorophyll a, a marked amount of pheophytin a was temporally accumulated, suggesting that Zn-chlorophyll a could be chemically synthesized via pheophytin a.     

Cardiac progenitor cells in brown adipose tissue repaired damaged myocardium

Cardiomyocyte (CM) regeneration is limited in adult life and is not sufficient to prevent myocardial infarction. Hence, the identification of a useful source of CM progenitors is of great interest for possible use in regenerative therapy. Mesenchymal stem cells in bone marrow, embryonic stem cells, and skeletal myoblasts are known sources of CM repletion; however, there are a number of critical problems for clinical application. In this study, we succeeded to identify CM progenitor cells in brown adipose tissue (BAT). Moreover, we showed that CM progenitor cells in BAT that existed in CD29-positive population could differentiate into CM with high efficiency. To confirm the in vivo effect of CD29(+)BAT-derived cells (BATDCs), we transplanted these cells into infarct border zone of an acute myocardial infarction model in rat. Results clearly indicated that implantation of CD29(+) BATDCs led to the reduction of the infarction area and improvement of left ventricular function by replacing newly developed CMs in comparison with that by CD29(+) white adipose tissue-derived cells or control saline. These findings suggest that BATDCs are one of the useful sources for a new strategy in CM regeneration.     

In vivo kinematics and articular surface congruency of total ankle arthroplasty during gait

Relatively high rates of loosening and implant failure have been reported after total ankle arthroplasty. Abnormal kinematics and incongruency of the articular surface may cause increased contact pressure and rotational torque applied to the implant, leading to loosening and implant failure. We measured in vivo kinematics of two-component total ankle arthroplasty (TNK ankle), and assessed congruency of the articular surface during the stance phase of gait. Eighteen ankles of 15 patients with a mean age of 75±6 years (mean±standard deviation) and follow-up of 44±38 months were enrolled. Lateral fluoroscopic images were taken during the stance phase of gait. 3D-2D model-image registration was performed using the fluoroscopic image and the implant models, and three-dimensional kinematics of the implant and incongruency of the articular surface were determined. The mean ranges of motion were 11.1±4.6°, 0.8±0.4°, and 2.6±1.5° for dorsi-/plantarflexion, inversion/eversion, and internal/external rotation, respectively. At least one type of incongruency of the articular surface occurred in eight of 18 ankles, including anterior hinging in one ankle, medial or lateral lift off in four ankles, and excessive axial rotation in five ankles. Among the four ankles in which lift off occurred during gait, only one ankle showed lift off in the static weightbearing radiograph. Our observations will provide useful data against which kinematics of other implant designs, such as three-component total ankle arthroplasty, can be compared. Our results also showed that evaluation of lift off in the standard weightbearing radiograph may not predict its occurrence during gait.     

Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels

Vasculogenesis, the in-situ assembly of angioblast or endothelial progenitor cells (EPCs), may persist into adult life, contributing to new blood vessel formation. However, EPCs are scattered throughout newly developed blood vessels and cannot be solely responsible for vascularization. Here, we identify an endothelial progenitor/stem-like population located at the inner surface of preexisting blood vessels using the Hoechst method in which stem cell populations are identified as side populations. This population is dormant in the steady state but possesses colony-forming ability, produces large numbers of endothelial cells (ECs) and when transplanted into ischaemic lesions, restores blood flow completely and reconstitutes de-novo long-term surviving blood vessels. Moreover, although surface markers of this population are very similar to conventional ECs, and they reside in the capillary endothelium sub-population, the gene expression profile is completely different. Our results suggest that this heterogeneity of stem-like ECs will lead to the identification of new targets for vascular regeneration therapy.     

Autophagy machinery controls nitrogen remobilization at the whole-plant level under both limiting and ample nitrate conditions in Arabidopsis

? Processes allowing the recycling of organic nitrogen and export to young leaves and seeds are important determinants of plant yield, especially when plants are nitrate-limited. Because autophagy is induced during leaf ageing and in response to nitrogen starvation, its role in nitrogen remobilization was suspected. It was recently shown that autophagy participates in the trafficking of Rubisco-containing bodies to the vacuole. ? To investigate the role of autophagy in nitrogen remobilization, several autophagy-defective (atg) Arabidopsis mutants were grown under low and high nitrate supplies and labeled with at the vegetative stage in order to determine (15) N partitioning in seeds at harvest. Because atg mutants displayed earlier and more rapid leaf senescence than wild type, we investigated whether their defects in nitrogen remobilization were related to premature leaf cell death by studying the stay-green atg5.sid2 and atg5.NahG mutants. ? Results showed that nitrogen remobilization efficiency was significantly lower in all the atg mutants irrespective of biomass defects, harvest index reduction, leaf senescence phenotypes and nitrogen conditions. ? We conclude that autophagy core machinery is needed for nitrogen remobilization and seed filling.