NMR investigation of the alkaline-like conformational transition of horse heart cytochrome c in the presence of exogenous thiazole

The conformational transition of horse heart cyt c in the presence of exogenous thiazole is investigated by NMR spectroscopy. Surprisingly, besides the native form and the ligand-bound form, another species (species A) exists at neutral pH. Titration of thiazole at alkaline condition indicates that this form has Lys as the axial ligands and in fact is one of alkaline cyt c conformers. At a high concentration of thiazole, species A has a dominant population at a pH of approximately 7. It is the first time that a pure alkaline conformer of cyt c is obtained at neutral pH. The observed NOEs show that species A maintains the overall protein fold although large structural rearrangements are expected in the distal pocket. Furthermore, the different effect of imidazole, pyridine and thiazole on the alkaline transformation of cyt c is discussed.     

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.     

Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis

Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.     

Counterion-induced actin ring formation

Actin filaments form rings and loops when > 20 mM divalent cations are added to very dilute solutions of phalloidin-stabilized filamentous actin (F-actin). Some rings consist of very long single actin filaments partially overlapping at their ends, and others are formed by small numbers of filaments associated laterally. In some cases, undulations of the rings are observed with amplitudes and dynamics similar to those of the thermal motions of single actin filaments. Lariat-shaped aggregates also co-exist with rings and rodlike bundles. These polyvalent cation-induced actin rings are analogous to the toroids of DNA formed by addition of polyvalent cations, but the much larger diameter of actin rings reflects the greater bending stiffness of F-actin. Actin rings can also be formed by addition of streptavidin to crosslink sparsely biotinylated F-actin at very low concentrations. The energy of bending in a ring, calculated from the persistence length of F-actin and the ring diameter, provides an estimate for the adhesion energy mediated by the multivalent counterions, or due to the streptavidin-biotin bonds, required to keep the ring closed.     

Contribution of ΔpH and ΔE to Photosynthesis of Chlamydomonas Reinhardtii

The contribution of two components (pH and E) of the proton motive force to photosynthesis of C. reinhardtii was studied. Valinomycin, a photophosphorylation uncoupler, decreased significantly the fast phase (related mainly to the membrane electric potential) of millisecond delayed light emission (ms-DLE) of C. reinhardtii. Nigericin, another photophosphorylation uncoupler, decreased the slow phase (related mainly to the proton gradient) and partly also the fast phase of ms-DLE. Both valinomycin and nigericin decreased the net ATP content and photosynthetic rate of C. reinhardtii, but the inhibition by nigericin was stronger than that by valinomycin. Hence both components of the proton motive force contribute to photosynthesis and although the contribution of pH is larger than that of E, the latter is not negligible in photosynthesis of C. reinhardtii.     

A novel human hydroxysteroid dehydrogenase like 1 gene (HSDL1) is highly expressed in reproductive tissuesa

We report the cloning and characterization of a novel human hydroxysteroid dehydrogenase like gene (HSDL1) located on human chromosome 16q24.2. The HSDL1 cDNA is 3407 base pair in length, encoding a 309 amino acid polypeptide related to human 17-HSD3. Northern blot reveals that the HSDL1 is highly expressed in testis and ovary. In situ hybridization indicates that the expression of HSDL1 is predominantly increased in the prostate cancer tissue compared with the normal prostate tissue, which suggests that the gene expression is important to the arising of prostate cancer.     

缺血引起沙土鼠纹状体DARPP—32磷酸化状态的变化

采用蒙古沙土双侧颈总动脉阻断前脑缺血模型,以放射自显影（反向磷酸化,back-phosphorylation)及免疫印迹（Western blotting)法体外测定缺血时纹状体DARPP－32磷酸化水平和蛋白含量的变化,结果表明,短暂性缺血纹状体DARPP－32的免疫学活性和蛋白含量地明显改变。在缺血10min内,随缺血时间的延长,体外DARPP－32的[^32P]的掺入量在缺血5min时升高,在缺血2,7,10min时均降低,而反向磷酸化的测定结果表明体内DARPP－32磷酸化水平增高,说明缺血可诱导DARPP－32磷酸化水平变化。     

New chiral catalysts for reduction of ketones

The condensation of o-(diphenylphosphino)benzaldehyde and various chiral diamine gives a series of diimino-diphosphine tetradentate ligands, which are reduced with excess NaBH4 in refluxing ethanol to afford the corresponding diaminodiphosphine ligands in good yield. The reactivity of these ligands toward trans-RuCl2(DMSO)4 and [Rh(COD)Cl]2 had been investigated and a number of chiral Ru(II) and Rh(I) complexes with the PNNP-type ligands were synthesized and characterized by microanalysis and IR, NMR spectroscopic methods. The chiral Ru(II) and Rh(I) complexes have proved to be excellent catalyst precursors for the asymmetric transfer hydrogenation of aromatic ketones, leading to optically active alcohols in up to 97% ee.