Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size

Autophagy is an intracellular bulk degradation system. We established mouse fibroblast lines coupling the Tet-off system with an Atg5-/- mouse embryonic fibroblast line to artificially regulate autophagic ability. In the presence of doxycycline (Dox), Atg5 expression was completely suppressed and these cells were autophagy-defective. After removal of Dox, autophagic ability was restored within 6 h. Very low levels of Atg5 could induce an autophagy competent state. We applied this novel system to examine the contribution of autophagy to controlling cell size. Cell size reduction in response to starvation was significantly inhibited in cells unable to undergo autophagy. The generated cell lines will be useful reagents for future mechanistic studies into the regulation and physiologic significance of autophagy.     

Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.     

Pancreatoblastoma. A case report with special emphasis on squamoid corpuscles with optically clear nuclei rich in biotin

BACKGROUND: Pancreatoblastoma (PBL) is a rare neoplasm that generally occurs in the pediatric age group and shows unique histopathology, including squamoid corpuscles that may contain tumor cells with optically clear nuclei (OCN) rich in biotin. In the English-language literature there have been two reports on the cytology of PBL, but neither of them refers to the cytologic features of squamoid corpuscles. CASE: A 3-year-old boy with nausea and general fatigue was referred to our center. Imaging studies showed an approximately 7.5-cm, left-sided abdominal mass and multiple metastases in the lung. The abdominal mass was biopsied, and its histology showed solid cellular nests with occasional acinar differentiation and squamoid corpuscles. Imprint cytology of the biopsied sample displayed cellular epithelial nests with focal acinar structures and foci composed of larger cells with a low nuclear/cytoplasmic ratio. These foci contained a few tumor cells with biotin-rich OCN and were determined to be squamoid corpuscles. CONCLUSION: Detection of occasional squamoid corpuscles with biotin-rich OCN can be useful in making a diagnosis of PBL on cytologic samples.     

Safety of Intracoronary Infusion of 20 Million C-Kit Positive Human Cardiac Stem Cells in Pigs

Background

There is mounting interest in using c-kit positive human cardiac stem cells (c-kit^pos hCSCs) to repair infarcted myocardium in patients with ischemic cardiomyopathy. A recent phase I clinical trial (SCIPIO) has shown that intracoronary infusion of 1 million hCSCs is safe. Higher doses of CSCs may provide superior reparative ability; however, it is unknown if doses >1 million cells are safe. To address this issue, we examined the effects of 20 million hCSCs in pigs.

Methods

Right atrial appendage samples were obtained from patients undergoing cardiac surgery. The tissue was processed by an established protocol with eventual immunomagnetic sorting to obtain in vitro expanded hCSCs. A cumulative dose of 20 million cells was given intracoronarily to pigs without stop flow. Safety was assessed by measurement of serial biomarkers (cardiac: troponin I and CK-MB, renal: creatinine and BUN, and hepatic: AST, ALT, and alkaline phosphatase) and echocardiography pre- and post-infusion. hCSC retention 30 days after infusion was quantified by PCR for human genomic DNA. All personnel were blinded as to group assignment.

Results

Compared with vehicle-treated controls (n=5), pigs that received 20 million hCSCs (n=9) showed no significant change in cardiac function or end organ damage (assessed by organ specific biomarkers) that could be attributed to hCSCs (P>0.05 in all cases). No hCSCs could be detected in left ventricular samples 30 days after infusion.

Conclusions

Intracoronary infusion of 20 million c-kit positive hCSCs in pigs (equivalent to ~40 million hCSCs in humans) does not cause acute cardiac injury, impairment of cardiac function, or liver and renal injury. These results have immediate translational value and lay the groundwork for using doses of CSCs >1 million in future clinical trials. Further studies are needed to ascertain whether administration of >1 million hCSCs is associated with greater efficacy in patients with ischemic cardiomyopathy.     

Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size

Autophagy is an intracellular bulk degradation system. We established mouse fibroblast lines coupling the Tet-off system with an Atg5(-/-) mouse embryonic fibroblast line to artificially regulate autophagic ability. In the presence of doxycycline (Dox), Atg5 expression was completely suppressed and these cells were autophagy-defective. After removal of Dox, autophagic ability was restored within 6h. Very low levels of Atg5 could induce an autophagy competent state. We applied this novel system to examine the contribution of autophagy to controlling cell size. Cell size reduction in response to starvation was significantly inhibited in cells unable to undergo autophagy. The generated cell lines will be useful reagents for future mechanistic studies into the regulation and physiologic significance of autophagy.     

Functional Consequences of Various Leucine Mutations in the M3/M4 Loop of the Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase α-Subunit

Leucines were mutated within the sequence L³¹¹ILGYTWLE³¹⁹ of the extracellular loop flanking the third (M3) and fourth (M4) transmembrane segments (M3/M4 loop) of the Torpedo Na⁺,K⁺-ATPase α-subunit. Replacement of Leu³¹¹ with Glu resulted in a considerable loss of Na⁺,K⁺-ATPase activity. Replacement of Leu³¹³ with Glu shifted the equilibrium of E₁P and E₂P toward E₁P and reduced the rate of the E₁P to E₂P transition. The reduction of the transition rate and stronger inhibition of Na⁺,K⁺-ATPase activity by Na⁺ at higher concentrations together suggest that there is interference of Na⁺ release on the extracellular side in the Leu³¹³ mutant. Thus, Leu³¹³ could be in the pathway of Na⁺ exit. Replacement of Leu³¹⁸ with Glu yielded an enzyme with significantly reduced apparent affinity for both vanadate and K⁺, with an equilibrium shifted toward E₂P and no alteration in the transition rate. The reduced vanadate affinity is due to the lower rate of production of vanadate-reactive [K⁺ ₂]E₂ caused by inhibition of dephosphorylation through reduction of the K⁺ affinity of E₂P. Thus, Leu³¹⁸ may be a critical position in guiding external K⁺ to its binding site.     

Steroid Series

3β-Acetoxy-B-nor-5β-cholestan-6-one (Ia) afforded only one isolatable oxime (IIa), while oximation of 3β, 17β-diacetoxy-B-nor-5β-androstan-6-one (Ib) yielded two isomeric oximes (IIb and IIIb). 7-Aza-5β-cholestan-3β-ol (VIa), 7-aza-5β-androstane-3β, 17β-diol (VIc), and 6-aza-5β-androstane-3β, 17β-diol (VIIc) were synthesized by Beckmann rearrangement of these oximes, followed by reduction with lithium aluminium hydride. The structure of the aza-steroids were established by conversion of the intermediate lactams (IVa, b) into the lactones (IXa, b), prepared from the 3β-acetoxy-B-nor-6-oxo-5β-steroids (Ia, b) by Baeyer Villiger reaction.