A mutant Escherichia coli tyrosyl-tRNA synthetase utilizes the unnatural amino acid azatyrosine more efficiently than tyrosine

Alloproteins, proteins that contain unnatural amino acids, have immense potential in biotechnology and medicine. Although various approaches for alloprotein production exist, there is no satisfactory method to produce large quantities of alloproteins containing unnatural amino acids in specific positions. The tyrosine analogue azatyrosine, l-beta-(5-hydroxy-2-pyridyl)-alanine, can convert the ras-transformed phenotype to normal phenotype, presumably by its incorporation into cellular proteins. This provided the stimulus for isolation of a mutant tyrosyl-tRNA synthetase (TyrRS) capable of charging azatyrosine to tRNA. A plasmid library of randomly mutated Escherichia coli tyrS (encoding TyrRS) was made by polymerase chain reaction techniques. The desired TyrRS mutants were selected by screening for in vivo azatyrosine incorporation of E. coli cells transformed with the mutant tyrS plasmids. One of the clones thus isolated, R-6-A-7, showed a 17-fold higher in vivo activity for azatyrosine incorporation than wild-type TyrRS. The mutant tyrS gene contained a single point mutation resulting in replacement of phenylalanine by serine at position 130 in the protein. Structural modeling revealed that position 130 is located close to Asp(182), which directly interacts with tyrosyladenylate. Kinetic analysis of aminoacyl-tRNA formation by the wild-type and mutated F130S TyrRS enzymes showed that the specificity for azatyrosine, measured by the ratios of k(cat)/K(m) for tyrosine and the analogue, increased from 17 to 36 as a result of the F130S mutation. Thus, the high discrimination against azatyrosine is significantly reduced in the mutant enzyme. These results suggest that utilization of F130S TyrRS for in vivo protein biosynthesis may lead to efficient production of azatyrosine-containing alloproteins.     

Dynamic function of the alkyl spacer of acetogenins in their inhibitory action with mitochondrial complex I (NADH-ubiquinone oxidoreductase)

Studies on the inhibitory mechanism of acetogenins, the most potent inhibitors of mitochondrial complex I (NADH-ubiquinone oxidoreductase), are useful for elucidating the structural and functional features of the terminal electron transfer step of this enzyme. Previous studies of the structure-activity relationship revealed that except for the alkyl spacer linking the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings), none of the multiple functional groups of these inhibitors is essential for potent inhibition. To elucidate the function of the alkyl spacer, two sets of systematically selected analogues were synthesized. First, the length of the spacer was varied widely. Second, the local flexibility of the spacer was specifically reduced by introducing multiple bond(s) into different regions of the spacer. The optimal length of the spacer for inhibition was approximately 13 carbon atoms. The decrease in the strength of the inhibitory effect caused by elongating the spacer from 13 carbons was much more drastic than that caused by shortening. Local flexibility in a specific region of the spacer was not important for the inhibition. These observations indicate that the active conformation of the spacer is not an extended form, and is not necessarily restricted to a certain rigid shape. Moreover, an analogue in which a spacer covering 10 carbon atoms was hardened into a rodlike shape still maintained a potent inhibitory effect. Our results strongly suggest that the spacer portion is free from steric congestion arising from the putative binding site probably because there is no cavity-like binding site for the spacer portion. The manner of acetogenin binding to the enzyme may not be explained by a simple "key and keyhole" analogy.     

Temporal and Motor Representation of Rhythm in Fronto-Parietal Cortical Areas: An fMRI Study

When sounds occur with temporally structured patterns, we can feel a rhythm. To memorize a rhythm, perception of its temporal patterns and organization of them into a hierarchically structured sequence are necessary. On the other hand, rhythm perception can often cause unintentional body movements. Thus, we hypothesized that rhythm information can be manifested in two different ways; temporal and motor representations. The motor representation depends on effectors, such as the finger or foot, whereas the temporal representation is effector-independent. We tested our hypothesis with a working memory paradigm to elucidate neuronal correlates of temporal or motor representation of rhythm and to reveal the neural networks associated with these representations. We measured brain activity by fMRI while participants memorized rhythms and reproduced them by tapping with the right finger, left finger, or foot, or by articulation. The right inferior frontal gyrus and the inferior parietal lobule exhibited significant effector-independent activations during encoding and retrieval of rhythm information, whereas the left inferior parietal lobule and supplementary motor area (SMA) showed effector-dependent activations during retrieval. These results suggest that temporal sequences of rhythm are probably represented in the right fronto-parietal network, whereas motor sequences of rhythm can be represented in the SMA-parietal network.     

Mannose-specific lectin from the mushroom Hygrophorus russula

A lectin was purified from the mushroom Hygrophorus russula by affinity chromatography on a Sephadex G-50 column and BioAssist S cation exchange chromatography and designated H. russula lectin (HRL). The results of sodium dodecyl sulfate-polyaclylamidegel electrophoresis, gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry of HRL indicated that it was composed of four identical 18.5?kDa subunits with no S-S linkage. Isoelectric focusing of the lectin showed bands near pI 6.40. The complete sequence of 175 amino acid residues was determined by amino acid sequencing of intact or enzyme-digested HRL. The sequence showed homology with Grifola frondosa lectin. The cDNA of HRL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA consisted of 528?bp encoding 176 amino acids. In hemagglutination inhibition assay, α1-6 mannobiose was the strongest inhibitor and isomaltose, Glcα1-6Glc, was the second strongest one, among mono- and oligosaccharides tested. Frontal affinity chromatography indicated that HRL had the highest affinity for Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc, and non-reducing terminal Manα1-6 was essential for the binding of HRL to carbohydrate chains. The sugar-binding specificity of HRL was also analyzed by using BIAcore. The result from the analysis exhibited positive correlations with that of the hemagglutination inhibition assay. All the results suggested that HRL recognized the α1-6 linkage of mannose and glucose, especially the Manα1-6 bond. HRL showed a mitogenic activity against spleen lymph cells of an F344 rat. Furthermore, an enzyme-linked immunosorbent assay showed strong binding of HRL to human immunodeficiency virus type-1 gp120.     

Ubiquitin ligase CHIP suppresses cancer stem cell properties in a population of breast cancer cells

Cancer stem cells (CSCs) have several distinctive characteristics, including high metastatic potential, tumor-initiating potential, and properties that resemble normal stem cells such as self-renewal, differentiation, and drug efflux. Because of these characteristics, CSC is regarded to be responsible for cancer progression and patient prognosis. In our previous study, we showed that a ubiquitin E3 ligase carboxyl terminus of Hsc70-interacting protein (CHIP) suppressed breast cancer malignancy. Moreover, a recent clinical study reported that CHIP expression levels were associated with favorable prognostic parameters of patients with breast cancer. Here we show that CHIP suppresses CSC properties in a population of breast cancer cells. CHIP depletion resulted in an increased proportion of CSCs among breast cancers when using several assays to assess CSC properties. From our results, we propose that inhibition of CSC properties may be one of the functions of CHIP as a suppressor of cancer progression.     

Requirement of the activity of hepatocyte growth factor activator inhibitor type 1 for the extracellular appearance of a transmembrane serine protease matriptase in monkey kidney COS-1 cells

Hepatocyte growth factor activator inhibitor type I (HAI-1) is a membrane-bound, serine protease inhibitor with two protease-inhibitory domains (Kunitz domain I and II). HAI-1 is known as a physiological inhibitor of a membrane-bound serine protease, matriptase. Paradoxically, however, HAI-1 has been found to be required for the extracellular appearance of the protease in an expression system using a monkey kidney COS-1 cell line. In the present study, we show using COS-1 cells that co-expression of recombinant variants of HAI-1 with the inhibition activity toward matriptase, including a variant consisting only of Kunitz domain I (the domain responsible for inhibition of matriptase), allowed for the appearance of this protease in the conditioned medium, whereas that of the variants without the activity did not. These findings suggest that the inhibition activity toward matriptase is critical for the extracellular appearance of protease in COS-1 cells.     

Variation in the herd composition counts of sika deer

Herd composition counts (HCC) are commonly used to assess population status in deer. We evaluated the reliability of HCC of sika deer (Cervus nippon Temminck) using repeated counts and by comparing estimated sex ratios and calf-to-female ratios of marked deer on Nakanoshima Island, Hokkaido, Japan between April 1999 and October 2000. Although both total counts and sex and age ratios fluctuated greatly by month, seasonal changes showed a relatively small variance. This suggested that seasonal changes in behavior within sex and age classes contributed to biased ratio estimates obtained from HCC. Route counts should be used as a relative population abundance index with estimates of detection probability, especially for females. Adult sex ratios and age ratios from HCC were unbiased during the rutting season (October–November), and age ratios in spring could be used if yearlings are counted as adults.     

Enzymatic characterization of peroxisomal and cytosolic betaine aldehyde dehydrogenases in barley

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is an important enzyme that catalyzes the last step in the synthesis of glycine betaine, a compatible solute accumulated by many plants under various abiotic stresses. In barley ( Hordeum vulgare L.), we reported previously the existence of two BADH genes ( BBD1 and BBD2 ) and their corresponding proteins, peroxisomal BADH (BBD1) and cytosolic BADH (BBD2). To investigate their enzymatic properties, we expressed them in Escherichia coli and purified both proteins. Enzymatic analysis indicated that the affinity of BBD2 for betaine aldehyde was reasonable as other plant BADHs, but BBD1 showed extremely low affinity for betaine aldehyde with apparent K_m of 18.9 μ M and 19.9 m M , respectively. In addition, V_max/K_m with betaine aldehyde of BBD2 was about 2000-fold higher than that of BBD1, suggesting that BBD2 plays a main role in glycine betaine synthesis in barley plants. However, BBD1 catalyzed the oxidation of ω-aminoaldehydes such as 4-aminobutyraldehyde and 3-aminopropionaldehyde as efficiently as BBD2. We also found that both BBDs oxidized 4- N -trimethylaminobutyraldehyde and 3- N -trimethylaminopropionaldehyde.     

Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen–specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54^high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54^high or B2^high leukemia cells preferentially survived in vivo in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis in vitro and in vivo. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy.