Beta-cyclodextrin inhibits the sweet taste suppressing activity of gurmarin by the formation of an inclusion complex with aromatic residues in gurmarin.

Our recent study in mice revealed that the inhibitory activity of gurmarin on the sweet taste responses was reduced significantly by the presence of beta-cyclodextrin (beta-CD). To investigate the mechanism involved in the action of beta-CD, physicochemical experiments were performed on the interaction of CDs with gurmarin examining the effect of CDs on the UV absorption spectrum of gurmarin and on the elution behavior in gel filtration (or exclusion) chromatography. Among the three kinds of cyclodextrins tested, beta-CD induced significant changes in the UV absorption spectrum of gurmarin that were characteristic of those found in the inclusion complex formation of tyrosine and tryptophan with beta-CD. The abnormal retention behavior of gurmarin in gel filtration resulting from hydrophobic interaction with the gel matrix reverted to normal in the presence of beta-CD in the elution buffer. These results suggest that the unique domain of gurmarin, in which five aromatic amino acid residues are all directed outwardly and form a hydrophobic cluster, is a possible site of interaction with the gurmarin-sensitive sweet taste receptor molecules in rodents.     

Topographic pattern of the rearrangement of the dystrophin gene in Japanese Duchenne muscular dystrophy

To compare the frequency and distribution of rearrangements in the dystrophin gene in Duchenne muscular dystrophy (DMD) between Japanese DMD patients and those in North America and Europe, Southern blot analyses of the dystrophin gene were carried out in 88 probands classified as DMD. Gene rearrangements were found in 61 (69%) subjects, and they were composed of partial gene deletions in 53 (60%) probands and partial duplications in 7 (8%) probands. A total deletion of the gene was found in 1 (1%) patient. Among 53 patients with deletions, 34 (64%) had breakpoints between introns 44 and 52 and 7 (13%) had breakpoints between introns 2 and 11. Both the frequency and the distribution of gene rearrangements found in this study were similar to those reported in North America and Europe. These data suggest that there are no ethnic or racial differences in the frequency and distribution of rearrangements thought to be caused by similar mechanisms in the dystrophin gene in all human racial groupings.     

A study of the native-denatured (N in equilibrium with D) transition in lysozyme. III. Effect of alteration of net charge by acetylation.

Measurement of the enzymic activity and fluorescence properties showed that the gross conformation of acetylated lysozyme [EC 3.2.1.17] is very similar to that of the native enzyme. On the other hand, protease digestion, t-butyl hypochloride modification and thermal denaturation experiments performed on native, acetylated, and guanidinated lysozymes showed that acetylation caused a small but significant shift of the N in equilibrium with D transition to the right. Thus it can be concluded that charge balance in a protein plays an important role in maintaining its conformation. The difference between equilibrium and kinetic methods of monitoring protein denaturation was also clarified.     

ASK1 and ASK2 differentially regulate the counteracting roles of apoptosis and inflammation in tumorigenesis

Apoptosis and inflammation generally exert opposite effects on tumorigenesis: apoptosis serves as a barrier to tumour initiation, whereas inflammation promotes tumorigenesis. Although both events are induced by various common stressors, relatively little is known about the stress‐induced signalling pathways regulating these events in tumorigenesis. Here, we show that stress‐activated MAP3Ks, ASK1 and ASK2, which are involved in cellular responses to various stressors such as reactive oxygen species, differentially regulate the initiation and promotion of tumorigenesis. ASK2 in cooperation with ASK1 functioned as a tumour suppressor by exerting proapoptotic activity in epithelial cells, which was consistent with the reduction in ASK2 expression in human cancer cells and tissues. In contrast, ASK1‐dependent cytokine production in inflammatory cells promoted tumorigenesis. Our findings suggest that ASK1 and ASK2 are critically involved in tumorigenesis by differentially regulating apoptosis and inflammation.     

New DNA binding ligands as a model of chromomycin A3

Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this study, new DNA-binding ligands were designed to mimic Chromomycin A(3) (CRA(3)), which contains a hydroxylated tetrahydroanthracene chromophore substituted with di and trisaccharides. The trisaccharide part of CRA(3) that is supposed to contribute to form the Mg(2+)-coordinated dimer was expected to be mimicked by a simple alkyl group attached to the chromophore part as new model compounds. The present study has successfully demonstrated that the new ligands form Mg(2+)-coordinated dimer complexes to exhibit DNA-binding affinity.     

Dynamic Bayesian network and nonparametric regression for nonlinear modeling of gene networks from time series gene expression data

We propose a dynamic Bayesian network and nonparametric regression model for constructing a gene network from time series microarray gene expression data. The proposed method can overcome a shortcoming of the Bayesian network model in the sense of the construction of cyclic regulations. The proposed method can analyze the microarray data as a continuous data and can capture even nonlinear relations among genes. It can be expected that this model will give a deeper insight into complicated biological systems. We also derive a new criterion for evaluating an estimated network from Bayes approach. We conduct Monte Carlo experiments to examine the effectiveness of the proposed method. We also demonstrate the proposed method through the analysis of the Saccharomyces cerevisiae gene expression data.     

Angiotensin-converting enzyme inhibitor inhibits dehydration-enhanced fever induced by endotoxin in rats

It has been reported that a host develops a marked fever under dehydrated conditions compared with normally hydrated conditions (11). The present study was carried out to investigate whether ANG II is involved in the enhancement seen in dehydrated rats of the fever induced by bacterial endotoxin. The results showed that intravenous injection of bacterial endotoxin produced a fever in dehydrated rats (rats deprived of water for 24 h) that was significantly greater than that seen in normally hydrated rats. In contrast, dehydration had no effect on the fever induced by intravenous interleukin-1beta (IL-1beta). Under dehydrated conditions, the enhanced endotoxin-induced fever was significantly inhibited by the angiotensin-converting enzyme inhibitor lisinopril, but the IL-1beta fever was not. These results suggest that the dehydration-induced enhancement of endotoxin fever is due, at least in part, to the action of ANG II, which elicits an increased production of pyrogenic cytokines such as IL-1.     

Characteristics of the Inhibitory Effect of Calmodulin on Specific [125I]Omega-Conotoxin GVIA Binding to Crude Membranes from Chick Brain

The characteristics of the inhibitory effect of calcium ion (Ca²⁺)/calmodulin (CaM) on specific [¹²⁵I]-omega-conotoxin GVIA (¹²⁵I--CTX) binding and on the labeling of ¹²⁵I--CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca²⁺/CaM depended on the concentrations of free Ca²⁺ and CaM. The IC₅₀ values for free Ca²⁺ and CaM were about 2.0 × 10^–8 M and 3.0 g protein/ml, respectively. The inhibitory effect of Ca²⁺/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290–309), but not by the calcineurin inhibitor FK506. Ca²⁺/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca²⁺ channel ₁ subunits) with ¹²⁵I--CTX using a cross-linker. These results suggest that Ca²⁺/CaM affects specific ¹²⁵I--CTX binding sites, probably N-type Ca²⁺ channel ₁ subunits, in crude membranes from chick whole brain.     

Analysis of the internal motion of free and ligand-bound human lysozyme by use of 15N NMR relaxation measurement: a comparison with those of hen lysozyme

Human lysozyme has a structure similar to that of hen lysozyme and differs in amino acid sequence by 51 out of 129 residues with one insertion at the position between 47 and 48 in hen lysozyme. The backbone dynamics of free or (NAG)3-bound human lysozyme has been determined by measurements of 15N nuclear relaxation. The relaxation data were analyzed using the Lipari-Szabo formalism and were compared with those of hen lysozyme, which was already reported (Mine S et al.. 1999, J Mol Biol 286:1547-1565). In this paper, it was found that the backbone dynamics of free human and hen lysozymes showed very similar behavior except for some residues, indicating that the difference in amino acid sequence did not affect the behavior of entire backbone dynamics, but the folded pattern was the major determinant of the internal motion of lysozymes. On the other hand, it was also found that the number of residues in (NAG)3-bound human and hen lysozymes showed an increase or decrease in the order parameters at or near active sites on the binding of (NAG)3, indicating the increase in picosecond to nanosecond. These results suggested that the immobilization of residues upon binding (NAG)3 resulted in an entropy penalty and that this penalty was compensated by mobilizing other residues. However, compared with the internal motions between both ligand-bound human and hen lysozymes, differences in dynamic behavior between them were found at substrate binding sites, reflecting a subtle difference in the substrate-binding mode or efficiency of activity between them.     

Comprehensive approach to genes involved in cell wall modifications in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The plant cell wall is of supermolecular architecture, and is composed of various types of heterogeneous polymers. A few thousand enzymes and structural proteins are directly involved in the construction processes, and in the functional aspects of the dynamic architecture in Arabidopsis thaliana. Most of these proteins are encoded by multigene families, and most members within each family share significant similarities in structural features, but often exhibit differing expression profiles and physiological functions. Thus, for the molecular dissection of cell wall dynamics, it is necessary to distinguish individual members within a family of proteins. As a first step towards characterizing the processes involved in cell wall dynamics, we have manufactured a gene-specific 70-mer oligo microarray that consists of 765 genes classified into 30 putative families of proteins that are implicated in the cell wall dynamics of Arabidopsis. By using this array system, we identified several sets of genes that exhibit organ preferential expression profiles. We also identified gene sets that are expressed differentially at certain specific growth stages of the Arabidopsis inflorescence stem. Our results indicate that there is a division of roles among family members within each of the putative cell wall-related gene families.