N-(2-Aminoethyl)-5-isoquinolinesulfonamide, a newly synthesized protein kinase inhibitor, functions as a ligand in affinity chromatography. Purification of Ca2+-activated, phospholipid-dependent and other protein kinases

We designed a simple procedure for the purification of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) from rabbit brain, using affinity chromatography with a new affinity adsorbent. The adsorbent was synthesized by attaching the amino residue of N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) to cyanogen bromide-activated Sepharose. H-9 is a potent competitive inhibitor of protein kinase C, cGMP-, and cAMP-dependent protein kinase with respect to ATP and exhibits inhibition constants of 18, 0.87, and 1.9 microM, respectively (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry, 23, 5036). A 960-fold purification was achieved in the two-step procedure, which entailed DEAE-cellulose and the affinity chromatography. The resultant preparation was essentially homogeneous, as indicated by polyacrylamide gel electrophoresis under conditions of denaturation with sodium dodecyl sulfate. The affinity of protein kinase C for the H-9-Sepharose was high, and the enzyme could not be eluted either by a high concentration of sodium chloride or by 40% glycerol. The protein kinase C could be eluted from H-9-Sepharose by the buffer containing both 0.2 M NaCl and 20% glycerol, thereby suggesting that the binding between protein kinase C and H-9-Sepharose was due to both hydrophobic and electrostatic interactions. H-9 coupled to Sepharose retained both cyclic nucleotide-dependent protein kinases and protein kinase C, and these enzymes could be eluted separately by the buffer containing L-arginine, a potent inhibitor of these three kinases. The novel aspects of these three multifunctional protein kinases can thus be investigated using isoquinolinesulfonamide derivatives.     

Two frameshift mutations in the cystic fibrosis gene

Cystic fibrosis (CF) is a recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We have identified in exon 7 two frameshift mutations, one caused by a two-nucleotide insertion and the other caused by a one-nucleotide deletion; these mutations--CF1154insTC and CF1213delT, respectively, are predicted to shift the reading frame of the protein and to introduce UAA(ochre) termination codons at residues 369 and 368.     

Effect of natural zeolite on methane production for anaerobic digestion of ammonium rich organic sludge

The effect of an inorganic additive on the methane production from NH(4+)-rich organic sludge during anaerobic digestion was investigated using different kinds of inorganic adsorbent zeolites (mordenite, clinoptilolite, zeolite 3A, zeolite 4A), clay mineral (vermiculite), and manganese oxides (hollandite, birnessite). The additions of inorganic materials resulted in significant NH4+ removals from the natural organic sludge ([NH4+]=1, 150 mg N/l), except for the H-type zeolite 3A and birnessite. However, an enhanced methane production was only achieved using natural mordenite. Natural mordenite also enhanced the methane production from the sludge with a markedly high NH4+ concentration (4500 mg N/l) during anaerobic digestion. Chemical analyses of the sludge after the digestion showed considerable increases in the Ca2+ and Mg2+ concentrations in the presence of natural mordenite, but not with synthetic zeolite 3A. The effect of Ca2+ or Mg2+ addition on the methane production was studied using Na(+)-exchanges mordenite and Ca2+ or Mg(2+)-enriched sludge. The simultaneous addition of Ca2+ ions and Na(+)-exchanged mordenite enhanced the methane production; the amount of produced methane was about three times greater than that using only the Na(+)-exchanged mordenite. In addition, comparing the methane production by the addition of natural mordenite or Ca2+ ions, the methane production with natural mordenite was about 1.7 times higher than that with only Ca2+ ions. The addition of 5% and 10% natural mordenite were suitable condition for obtaining a high methane production. These results indicated that the Ca2+ ions, which are released from natural mordenite by a Ca2+/NH4+ exchange, enhanced the methane production of the organic waste at a high NH4+ concentration. Natural mordenite has a synergistic effect on the Ca2+ supply as well on the NH4+ removal during anaerobic digestion, which is effective for the mitigation of NH4+ inhibition against methane production.     

Biogeochemical Signals from Deep Microbial Life in Terrestrial Crust

In contrast to the deep subseafloor biosphere, a volumetrically vast and stable habitat for microbial life in the terrestrial crust remains poorly explored. For the long-term sustainability of a crustal biome, high-energy fluxes derived from hydrothermal circulation and water radiolysis in uranium-enriched rocks are seemingly essential. However, the crustal habitability depending on a low supply of energy is unknown. We present multi-isotopic evidence of microbially mediated sulfate reduction in a granitic aquifer, a representative of the terrestrial crust habitat. Deep meteoric groundwater was collected from underground boreholes drilled into Cretaceous Toki granite (central Japan). A large sulfur isotopic fractionation of 20–60‰ diagnostic to microbial sulfate reduction is associated with the investigated groundwater containing sulfate below 0.2 mM. In contrast, a small carbon isotopic fractionation (<30‰) is not indicative of methanogenesis. Except for 2011, the concentrations of H₂ ranged mostly from 1 to 5 nM, which is also consistent with an aquifer where a terminal electron accepting process is dominantly controlled by ongoing sulfate reduction. High isotopic ratios of mantle-derived ³He relative to radiogenic ⁴He in groundwater and the flux of H₂ along adjacent faults suggest that, in addition to low concentrations of organic matter (<70 µM), H₂ from deeper sources might partly fuel metabolic activities. Our results demonstrate that the deep biosphere in the terrestrial crust is metabolically active and playing a crucial role in the formation of reducing groundwater even under low-energy fluxes.     

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

Differential susceptibility to oxidative stress of two scleractinian corals: antioxidant functioning of mycosporine-glycine

This study examined the importance of mycosporine-glycine (Myc-Gly) as a functional antioxidant in the thermal-stress susceptibility of two scleractinian corals, Platygyra ryukyuensis and Stylophora pistillata. Photochemical efficiency of PSII (F_v/F_m), activity of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), and composition and abundance of mycosporine-like amino acids (MAAs) in the coral tissue and in symbiotic zooxanthellae were analyzed during 12-h exposure to high temperature (33 °C). After 6- and 12-h exposures at 33 °C, S. pistillata showed a significantly more pronounced decline in F_v/F_m compared to P. ryukyuensis. A 6-h exposure at 33 °C induced a significant increase in the activities of SOD and CAT in both host and zooxanthellae components of S. pistillata while in P. ryukyuensis a significant increase was observed only in the CAT activity of zooxanthellae. After 12-h exposure, the SOD activity of P. ryukyuensis was unaffected in the coral tissue but slightly increased in zooxanthellae, whereas the CAT activity in the coral tissue showed a 2.5-fold increase. The total activity of antioxidant enzymes was significantly higher in S. pistillata than in P. ryukyuensis, suggesting that P. ryukyuensis is less sensitive to oxidative stress than S. pistillata. This differential susceptibility of the corals is consistent with a 20-fold higher initial concentration of Myc-Gly in P. ryukyuensis compared to S. pistillata. In the coral tissue and zooxanthellae of both species investigated, the first 6 h of exposure to thermal stress induced a pronounced reduction in the abundance of Myc-Gly but not in other MAAs. When exposure was prolonged to 12 h, the Myc-Gly pool continued to decrease in P. ryukyuensis and was completely depleted in S. pistillata. The delay in the onset of oxidative stress in P. ryukyuensis and the dramatic increase in the activities of the antioxidant enzymes in S. pistillata, which contains low concentrations of Myc-Gly suggest that Myc-Gly provides rapid protection against oxidative stress before the antioxidant enzymes are induced. These findings strongly suggest that Myc-Gly is functioning as a biological antioxidant in the coral tissue and zooxanthellae and demonstrate its importance in the survival of reef-building corals under thermal stress.     

Pharmacological characterization of a novel muscarinic partial agonist, YM796, in transfected cells expressing the m1 or m2 muscarinic receptor gene.

To investigate the pharmacological effect of a novel compound YM796, we performed radioligand binding experiments and correlative biochemical experiments using the transfected murine fibroblast B82 cells which expressed the m1 and m2 muscarinic receptor genes (cloned cell lines designated as LK3-3 and M2LKB2-2, respectively). [3H](-)methyl-3-quinuclidinyl benzilate [( 3H](-)MQNB) binding in these transfected cell lines was inhibited by different optical isomers of YM796 and other muscarinic drugs, atropine, pirenzepine, AF-DX 116, as well as selected agonists. (-)YM796, (+)YM796 and (+/-)YM796 inhibited [3H](-)MQNB binding in LK3-3 cells with Ki values of 16.4 microM, 30.1 microM and 21.8 microM and in M2LKB2-2 cells with Ki values of 52.0 microM, 108 microM and 77.1 microM, respectively. From functional assays we found the two isomers, (-)YM796 and (+)YM796 had different intrinsic activities for the M1 and M2 muscarinic receptors. (-)YM796 revealed agonistic activity: stimulation of [3H]IP1 accumulation in LK3-3 cells with an EC50 value of 26.5 microM, which was less efficacious (the Emax value was 5.6 times basal) than carbachol, a full agonist (the Emax value was 17.2 times basal). Interestingly, (-)YM796 did not show significant inhibition of cAMP formation in M2LKB2-2 cells except at extremely high concentrations (greater than 1mM). (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. These results suggest that (-)YM796 represents a muscarinic partial agonist with functional selectivity for the M1 muscarinic receptors whereas (+)YM796 shows no efficacy for either M1 or M2 muscarinic receptors in these transfected cells.     

Discrimination between forms of vitamin E by humans with and without genetic abnormalities of lipoprotein metabolism.

To study the mechanisms of discrimination between various forms of vitamin E, four normal subjects, one patient with lipoprotein lipase deficiency, and three patients with abnormal apolipoprotein B-100 production were given an oral dose containing three tocopherols labeled with differing amounts of deuterium (2R,4'R,8'R-alpha-(5,7-(C2H3)2)tocopheryl acetate (d6-RRR-alpha-tocopheryl acetate), 2S,4'R,8'R-alpha-5-(C2H3)tocopheryl acetate (d3-SRR-alpha-tocopheryl acetate), and 2R,4'R,8'R-gamma-(3,4-2H)tocopherol (d2-RRR-gamma-tocopherol). The tocopherol contents of plasma, red cells, and lipoproteins were measured up to 76 h after the dose. In normal subjects all three tocopherols were absorbed and secreted in chylomicrons with equal efficiencies. Both d2-gamma- and d3-SRR-alpha-tocopherols peaked at similar concentrations in the other lipoprotein fractions, then decreased similarly, but 2-4 times more rapidly than did d6-RRR-alpha-tocopherol. A lipoprotein lipase-deficient patient and a patient with prolonged production of chylomicrons with absent apolipoprotein B-100 also demonstrated the lack of discrimination between tocopherols during absorption. Despite abnormal apolipoprotein B-100 production in two patients, the "VLDL" was preferentially enriched in d6-RRR-alpha-tocopherol. Our results show that there is no discrimination between the three tocopherols during absorption and secretion in chylomicrons, but subsequently there is a preferential enrichment of very low density lipoprotein (VLDL) with RRR-alpha-tocopherol. Catabolism of this VLDL results in the maintenance of plasma RRR-alpha-tocopherol concentrations.