Site-directed mutagenesis establishes aspartic acids-227 and -342 as essential for enzyme activity in an isomalto-dextranase from Arthrobacter globiformis

Isomalto-dextranase, from Arthrobacter globiformis T6, is a member of the glycoside hydrolase family 27. However, the alignments of the whole amino acid sequence are distinct from other members of this family. The enzymes cleave the glycosidic bond of the substrate in two different manners: either retaining or inverting the anomeric configuration. We believe that a retaining enzyme is involved in a two-step, double-displacement mechanism utilizing active site carboxylic acids as the nucleophile and general acid/base catalysts in the hydrolytic reaction. The critical amino acid residues at the isomalto-dextranase active site that catalyzes the hydrolysis reaction of dextran have been identified and the roles of nine amino acid residues (D107, D163, D227, D295, D340, D342, D373, D396, and E420) in the isomalto-dextranase from A. globiformis analyzed by site-directed mutagenesis. Of 15 mutant enzymes that were prepared, eight had reduced activities for dextran hydrolysis. Aspartic acids-227 and -342, which are part of the apparent catalytic dyad, were essential for hydrolase activity toward dextran.     

Temporal and spatial patterns of mass flowerings on the Malay Peninsula

We propose a hypothesis to explain the temporal and spatial patterns of mass flowerings in dipterocarp tree species on the Malay Peninsula. The literature on these mass flowerings reveals that during 1980-2002 at least 11 flowerings occurred at irregular intervals of 1-6 yr in a lowland rain forest. Five of them were typical mass flowerings-a high density of flowering trees and the characteristic sequential flowering of Shorea species. The 11 flowerings were classified into two flowering times: spring and autumn. There is evidence that low temperature and drought triggered the flowerings. Therefore, the seasonality of mass flowerings is characterized by the annual patterns of rainfall and low temperature. In addition, changes in El Ni?o-Southern Oscillation (ENSO) may play important roles in determining the supra-annual occurrence of mass flowerings. Flowering surveys on the Malay Peninsula implied that regions with spring or autumn mass flowerings corresponded geographically to those regions that had one cool season (December-February) or two (December-February and June-August), respectively. This finding anticipates the seasonal pattern and geographical distribution of mass flowerings on the Malay Peninsula.     

Structure determination and conformational change induced by tyrosine phosphorylation of the N-terminal domain of the alpha-chain of pig gastric H+/K+-ATPase

It has been well established that phosphorylation is an important reaction for the regulation of protein functions. In the N-terminal domain of the alpha-chain of pig gastric H(+)/K(+)-ATPase, reversible sequential phosphorylation occurs at Tyr 10 and Tyr 7. In this study, we determined the structure of the peptide involving the residues from Gly 2 to Gly 34 of pig gastric H(+)/K(+)-ATPase and investigated the tyrosine phosphorylation-induced conformational change using CD and NMR experiments. The solution structure showed that the N-terminal fragment has a helical conformation, and the peptide adopted two alpha-helices in 50% trifluoroethanol (TFE) solvent, suggesting that the peptide has a high helical propensity under hydrophobic conditions. Furthermore, the CD and NMR data suggested that the structure of the N-terminal fragment becomes more disordered as a result of phosphorylation of Tyr 10. This conformational change induced by the phosphorylation of Tyr 10 might be an advantageous reaction for sequential phosphorylation and may be important for regulating the function of H(+)/K(+)-ATPase.     

Carboxyhemoglobin formation following smoke inhalation injury in sheep is interrelated with pulmonary shunt fraction

Carboxyhemoglobin (COHb) formation is triggered by the inducible isoform of heme oxygenase (HO-1) catalyzing carbon monoxide (CO) production through breakdown of heme molecules, exposure to CO or both. In the setting of CO poisoning, COHb is regarded as a reliable marker characterizing both severity of injury and efficacy of treatment strategies. This study was designed as a prospective laboratory experiment to elucidate potential interdependencies between COHb generation, oxygenation, and pulmonary shunt fraction (Qs/Qt) in an ovine model of smoke inhalation injury. Chronically instrumented ewes (n=15) were repeatedly subjected to cotton smoke (4 x 12 breaths) according to an established protocol. This approach resulted in a progressive increase in COHb formation that was interrelated with the degree of Qs/Qt (P<0.001) and inversely correlated with both arterial and mixed venous HbO(2) saturation (r=-0.96 and -0.93). Although the arteriovenous COHb gradient successively decreased over time, COHb determined in venous blood underestimated the arterial content.     

Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction

To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from beta-galactosidase transgenic and recombination activating gene 1 (RAG-1) deficient double-mutant mice (H-2b) were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice (H-2d). The human pancreatic cancer cell line Capan-1 was subcutaneously xenotransplanted into SCID recipients and stromal formation was analyzed on day 14 and on day 28. Immunohistochemical and immunofluorescence studies revealed that BM-derived endothelial cells (X-gal/CD31 or H-2b/CD31 double-positive cells) and myofibroblasts (X-gal/alpha-smooth muscle actin or H-2b/alpha-smooth muscle actin double-positive cells) were present within and around the cancer nests. On day 14, the frequencies of BM-derived endothelial cells and BM-derived myofibroblasts were 25.3+/-4.4% and 12.7+/-9.6%, respectively. On day 28, the frequency of BM-derived endothelial cells was 26.7+/-9.7%, which was similar to the value on day 14. However, the frequency of BM-derived myofibroblasts was significantly higher (39.8+/-17.1%) on day 28 than on day 14 (P<0.05). The topoisomerase IIalpha-positive ratio was 2.2+/-1.2% for the H-2b-positive myofibroblasts, as opposed to only 0.3+/-0.4% for the H-2b-negative myofibroblasts, significant proliferative activity was observed in the BM-derived myofibroblasts (P<0.05). Our results indicate that BM-derived myofibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development.     

Blocking of alpha 5 integrin stimulates production of TGF-beta and PAI-1 by human mesangial cells

Expression of integrin, which mediates cell-matrix interaction, is affected by several cytokines, in particular by transforming growth factor-beta (TGF-beta). However, it is unknown whether, in an opposite way, a specific integrin is involved in cytokine synthesis. We tested this hypothesis. Function-blocking anti-alpha 5 integrin (fibronectin receptor) antibody increased TGF-beta secretion in growth-arrested human mesangial cells (2.3-fold) compared with control IgG or anti-alpha v beta 3 integrin (receptor for several matrix proteins) antibody. It also increased the secretion of plasminogen activator inhibitor-1 (PAI-1), a protein associated with matrix increase, by 3.2-fold. The increase in PAI-1 secretion induced by anti-alpha 5 integrin antibody was not abrogated by anti-TGF-beta neutralizing antibody. These results indicate that function-blocking of anti-alpha 5 integrin stimulates TGF-beta as well as PAI-1 production, suggesting that alpha 5 integrin is involved in fibrotic process. Function-modulation of a specific integrin thus appears to play a role in glomerular remodeling.     

Factors that contribute to efficient catalytic activity of a small Ca2+-dependent deoxyribozyme in relation to its RNA cleavage function

Recently, we found a small Ca(2+)-dependent deoxyribozyme (unmodified), d(GCCTGGCAG(1)G(2)C(3)T(4)A(5)C(6)A(7)A(8)C(9)G(10)A(11)GTCCCT), with cleavage activity for its RNA substrate, r(AGGGACA downward arrow UGCCAGGC) ( downward arrow denotes the RNA cleavage site), in the presence of Ca(2+) and developed a functional SPR sensor chip with this deoxyribozyme [Okumoto, Y., Ohmichi, T., and Sugimoto, N. (2002) Biochemistry 41, 2769-2773]. In the study presented here, to clarify the factors contributing to the efficient catalytic activity of the unmodified deoxyribozyme, RNA cleavage reactions were carried out using 24 mutant deoxyribozymes containing one unnatural DNA nucleotide, such as dI (2'-deoxyinosine), 7-deaza-dG, 2-aminopurine, 7-deaza-dA, 2-amino-dA, dm(5)C (5-methyl-2'-deoxycytosine), or d(P)C (5-propynyl-2'-deoxycytosine). The K(m) values (Michaelis constants) with the mutants that lacked N7 and O6 of G(1) and O6 of G(2) were 4.5 and 6.6 times that of the unmodified one, respectively. The k(cat) value (cleavage rate constant) with the mutants that lacked O6 of G(10) was 0.025 times that of the unmodified one. The results of UV melting curves, SPR kinetics, and CD spectra supported the quantitative idea that the catalytic activity of the unmodified form was achieved using Ca(2+). On the basis of these results, a preliminary model for two G(1) x A(8) and G(2) x A(7) mismatched base pairs such as G(anti) x A(anti) formed in the catalytic loop is proposed. The factor of 10 increase in the k(cat)/K(m) value of the mutant deoxyribozyme, which has C(9) substituted with d(P)C, suggests that the base stacking interaction between the substituted propynyl group in dC and the nearest-neighbor base grew stronger. Thus, substituting d(P)C for dC in the catalytic loop would be one of the best ways to increase the catalytic activity of the deoxyribozyme.     

Changes of low-frequency vibrational modes induced by universal 15N- and 13C-isotope labeling in S2/S1 FTIR difference spectrum of oxygen-evolving complex

The effects of universal (15)N- and (13)C-isotope labeling on the low- (650-350 cm(-1)) and mid-frequency (1800-1200 cm(-1)) S(2)/S(1) Fourier transform infrared (FTIR) difference spectrum of the photosynthetic oxygen-evolving complex (OEC) were investigated in histidine-tagged photosystem (PS) II core particles from Synechocystis sp. PCC 6803. In the mid-frequency region, the amide II modes were predominantly affected by (15)N-labeling, whereas, in addition to the amide II, the amide I and carboxylate modes were markedly affected by (13)C-labeling. In the low-frequency region, by comparing a light-induced spectrum in the presence of ferricyanide as the electron acceptor, with the double difference S(2)/S(1) spectrum obtained by subtracting the Q(A)(-)/Q(A) from the S(2)Q(A)(-)/S(1)Q(A) spectrum, considerable numbers of bands found in the light-induced spectrum were assigned to the S(2)/S(1) vibrational modes in the unlabeled PS II core particles. Upon (13)C-labeling, changes were observed for most of the prominent bands in the S(2)/S(1) spectrum. Although (15)N-labeling also induced changes similar to those by (13)C-labeling, the bands at 616(-), 605(+), 561(+), 555(-), and 544(-) cm(-1) were scarcely affected by (15)N-labeling. These results indicated that most of the vibrational modes found in the low-frequency spectrum are derived from the coupling between the Mn-cluster and groups containing nitrogen and/or carbon atom(s) in a direct manner and/or through hydrogen bonding. Interestingly, an intensive band at 577(-) cm(-1) was not affected by (15)N- and (13)C-isotope labeling, indicating that this band arises from the mode that does not include either nitrogen or carbon atoms, such as the skeletal vibration of the Mn-cluster or stretching vibrational modes of the Mn-ligand.