Analysis of the ratio of alpha- to beta-globin and globin messenger ribonucleic acid content of fractionated rabbit erythroid bone-marrow cells.

The ratio of alpha- to beta-globin mRNA was measured by hybridization of a constant amount of highly purified alpha- or beta-globin cDNA (complementary DNA) with increasing amounts of RNA in the range up to 20% cDNA hybridization, where an essentially linear reaction is obtained. Statistical analysis indicates that the ratio of alpha- to beta-globin can be measured within a maximal error of +/- 0.3 and in most cases is better than +/- 0.15. Under these conditions there is no significant deviation from the ratio of 1.3 in the alpha- to beta-globin mRNA ratio of RNA isolated from erythroid cells rich in pronormoblasts through to reticulocytes. If the ratio of alpha- to beta-globin mRNA exceeded 1.7 or was less than 0.9 in pronormoblasts, it would be detected in these experiments. The overall globin mRNA content increases to a maximal value in the fractions rich in basophilic normoblasts of 30,000--50,000 molecules/cell. However, the accuracy of these determinations is not as great as for the ratio determinations, and no significant deviations were seen except in the cells rich in pronormoblasts, which contained less globin mRNA than the later stages.     

A change in membrane microviscosity of mouse neuroblastoma cells in association with morphological differentiation

Changes in membrane microviscosity as well as in membrane constituents of mouse neuroblastoma clone N-18 were studied in association with neurite formation. The membrane microviscosity studied by fluorescence technique increased with the formation of neurites. The concomitant increase increase in the ratio of cholesterol to phospholipids was also observed.     

Studies on a phospholipase B from Penicillium notatum. Purification, properties, and mode of action.

1. Phospholipase B which hydrolyzes both the acyl ester bonds of diacylphospholipids (diacyl-hydrolase) and the acyl ester bond of monoacylphospholipids or lysophospholipids, [monoacyl-hydrolase or lysophospholipase, EC 3.1.1.5] was purified from Penicillium notatum about 2000-fold over the crude extract. The final preparation was homogeneous on disc electrophoresis. The apparent molecular weight, determined by gel filtration on Sephadex G-200, was about 116,000. The isoelectric point was pH 4.0. 2. The purified enzyme was a glycoprotein. The carbohydrate content was approximately 30%, consisting of mannose, glucose, and glucosamine. The amino acid composition was also determined. 3. The ratio of monoacyl-hydrolase to diacyl-hydrolase activities was influenced by the physical state of the substrate in the assay system. It was about 1 : 1 or 100 : 1 in the presence of absence of Triton X-100, respectively, and the latter value remained constant throughout the purification procedures. 4. Both enzyme activities had the same pH optimum, 4.0, and were heat-labile. None of the metals tested had any effect on either activity except for Fe2+ and Fe3+. Diisopropyl fluorophosphate at relatively high concentrations completely inhibited both enzyme activities. 5. The Michaelis-Menten constants (Km) of the enzyme for egg lecithin were about 1.5 and 25 mM in the absence and presence of Triton X-100, respectively. The Km value for dicaproyllecithin was 9.8 mM in the absence of Triton X-100. 6. Using a mixture of 1-[14C]stearoyl-lecithin and 2-[14C]oleoyl-lecithin in the presence of Triton X-100 as a substrate, it was found that the P. notatum phospholipase B attacked the acyl ester bonds sequentially, first the 2-acyl and then 1-acyl groups.     

The synthesis and biological activity of certain pentaazaacenaphthylenes, hexaazaacenaphthylenes and their corresponding nucleosides

Synthesis of the tricyclic nucleoside 8-amino-6-N-methyl-2-(beta-D-ribo-furanosyl)-1,2,3,5,6,7-hexaazaacena phthylene (5) has been accomplished by the ring closure of an appropriately substituted pyrazolo[3,4-d]-pyrimidine nucleoside followed by the requisite chemical conversions. The formation, isolation and structural elucidation of two unexpected nucleosides formed by a reductive ring cleavage of the hexaazaacenaphthylene ring system is discussed. A comparison of the antitumor and biological activity of 5 with the structurally related tricyclic pentaazaacenaphthylene nucleoside which is currently in phase II clinical trials at the 5'-phosphate pro-drug is also presented.     

Protective effects of coenzyme Q10 and α-tocopherol against free radical-mediated liver cell injury

In an attempt to provide further confirmation of the antioxidant role of reduced form of coenzyme Q homologue (CoQ_nH₂) and α-tocopherol (α-Toc), we incubated isolated rat hepatocytes with a water-soluble radical initiator, 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) in the presence or absence of exogenously added coenzyme Q₁₀ (CoQ₁₀) or α-Toc for 3 h at 37°C under an atmosphere of 95% oxygen and 5% carbon dioxide. In the control experiment without adding AAPH it was confirmed that added CoQ₁₀ and α-Toc were incorporated into the cells and some CoQ₁₀ were converted to CoQ₁₀H₂. Incubation of hepatocytes with 50 mM AAPH resulted in the formation of thiobarbituric acid-reactive substances and the decrease in cell viability and both were inhibited by exogenously added CoQ₁₀ or α-Toc in a dose-dependent manner. The decrease in endogenous CoQ₉H₂ and α-Toc levels was observed by the addition of AAPH. Addition of CoQ₁₀ inhibited the oxidation of CoQ₉H₂ to CoQ₉ dose-dependently while the addition of α-Toc did not. These data suggest that both CoQ_nH₂ and α-Toc act as antioxidants and can inhibit free radical-mediated cell injury.     

Receptor-like cytoplasmic kinases are pivotal components in pattern recognition receptor-mediated signaling in plant immunity

Innate immunity is generally initiated with recognition of conserved pathogen-associated molecular patterns (PAMPs). PAMPs are perceived by pattern recognition receptors (PRRs), leading to activation of a series of immune responses, including the expression of defense genes, ROS production and activation of MAP kinase. Recent progress has indicated that receptor-like cytoplasmic kinases (RLCKs) are directly activated by ligand- activated PRRs and initiate pattern -triggered immunity (PTI) in both Arabidopsis and rice. To suppress PTI, pathogens inhibit the RLCKs by many types of effectors, including AvrAC, AvrPphB and Xoo1488. In this review, we summarize recent advances in RLCK-mediated PTI in plants.     

Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.     

Intracranial Somatosensory Responses with Direct Spinal Cord Stimulation in Anesthetized Sheep

The efficacy of spinal cord stimulators is dependent on the ability of the device to functionally activate targeted structures within the spinal cord, while avoiding activation of near-by non-targeted structures. In theory, these objectives can best be achieved by delivering electrical stimuli directly to the surface of the spinal cord. The current experiments were performed to study the influence of different stimulating electrode positions on patterns of spinal cord electrophysiological activation. A custom-designed spinal cord neurostimulator was used to investigate the effects of lead position and stimulus amplitude on cortical electrophysiological responses to spinal cord stimulation. Brain recordings were obtained from subdural grids placed in four adult sheep. We systematically varied the position of the stimulating lead relative to the spinal cord and the voltage delivered by the device at each position, and then examined how these variables influenced cortical responses. A clear relationship was observed between voltage and electrode position, and the magnitude of high gamma-band oscillations. Direct stimulation of the dorsal column contralateral to the grid required the lowest voltage to evoke brain responses to spinal cord stimulation. Given the lower voltage thresholds associated with direct stimulation of the dorsal column, and its possible impact on the therapeutic window, this intradural modality may have particular clinical advantages over standard epidural techniques now in routine use.