Structure of the heterodimeric complex between CAD domains of CAD and ICAD

We present here the structure of the complex between the CAD domain of caspase activated deoxyribonuclease (CAD) and the CAD domain of its inhibitor (ICAD), determined by nuclear magnetic resonance spectroscopy. The two domains adopt a very similar fold, which consists of an alpha-helix and a beta-sheet, and are aligned side by side in the complex. Notably, the positive charges on the strand beta2 at one end of the beta-sheet of CAD and negative charges around the opposite end of the beta-sheet of ICAD are paired in the complex. Point mutations of the charged amino acids at this interface, on either CAD or ICAD, prevented formation of the functional CAD-ICAD complex. This implies that the interaction between the CAD domains of CAD and ICAD is an essential step in the correct folding of CAD in the complex.     

Troglitazone has a reducing effect on thromboxane production

Effects of troglitazone, an antidiabetic thiazolidinedione that enhances insulin sensitivity, on thromboxane (TX) production were assessed in human erythroleukemia (HEL) cells and human platelets. Measurement of TX was performed by using the gas chromatography/selected ion monitoring (GC/SIM) method. We found that troglitazone reduced the TX production from HEL cells and human platelets. Furthermore, troglitazone also reduced arachidonic acid (AA)-induced TX production from HEL cell and thrombin-induced TX release from platelets. In addition, we compared the effect of troglitazone with that of alpha-tocopherol and BRL 49653. Other thiazolidinedione compound BRL 49653 had effects similar to troglitazone, but alpha-tocopherol had no effect on TX production. Our findings suggest that the thiazolidinedione group had an antithrombotic effect and was beneficial in preventing vascular complications often observed in diabetes mellitus.     

The role of silk threads as lifelines for caterpillars: pattern and significance of lifeline-climbing behaviour

Abstract.  1. Most lepidopteran larvae use all of their legs (thoracic legs and abdominal prolegs) when walking on solid substrates. When caterpillars involuntarily or intentionally drop from the tree canopy, they can regain their original position by climbing silk lifelines spun out from the head spinnerets. However, the taxonomic distribution of this climbing behaviour in the Lepidoptera is unknown.
2. Here, lifeline-climbing behaviour is reported in 13 lepidopteran species belonging to different taxa (five superfamilies and six families: Zygaenidae, Drepanidae, Geometridae, Lymantriidae, Noctuidae, and Nymphalidae). Caterpillars usually used only the three pairs of thoracic legs to climb lifelines, although they use different methods to walk on solid substrates, according to their taxonomic grouping and number of prolegs.
3. Results suggest that lifeline-climbing behaviour using only the thoracic legs is common among various lepidopteran taxa. The majority of species (12 of 13) climbed lifelines by alternating movement of the left and right set of thoracic legs, aided by side-to-side body movements. Only one of the 13 species, the geometrid Naxa seriaria (Motschulsky), climbed lifelines by drawing them down with its thoracic legs, aided by abdominal looping movements. While side-to-side movement of the abdomen was previously reported in lifeline climbing, this is the first report of the use of looping movements.     

Pineal circadian clocks gate arylalkylamine N-acetyltransferase gene expression in the mouse pineal gland

In mammals it has been thought that the circadian clock localizes only in the suprachiasmatic nucleus of the hypothalamus. Recent studies have revealed that certain brain regions and peripheral tissues may also have intrinsic circadian clocks. However, the roles played by 'peripheral circadian clocks' have not been fully elucidated. In this study, we investigated their function using mouse pineal glands, and found that expression of the arylalkylamine N-acetyltransferase (Aa-Nat, EC 2.3.1.87, the rate-limiting enzyme of melatonin synthesis) gene after adrenergic receptor stimulation depended on the time of day even in vitro (gating). Phase-dependent Aa-Nat responses were observed in both melatonin-proficient and melatonin-deficient mouse pineal glands. Phosphodiesterases are unlikely to suppress Aa-Nat induction because a phosphodiesterase inhibitor itself had no effect on the mRNA levels. Puromycin was ineffective in inducing Aa-Nat mRNA levels in either the presence or absence of isoproterenol, suggesting that newly synthesized proteins may not be necessary to gate the Aa-Nat responses. We also discovered circadian dependence of the expression of Period1-luminescence in Period1-luciferase transgenic mouse pineal glands: circadian clocks may be functional in culture. Aa-Nat mRNA levels showed no significant circadian rhythms in the absence of isoproterenol, thus suggesting that Aa-Nat mRNA levels are induced by adrenergic mechanisms, not by a pineal circadian clock. Our results suggest that the pineal circadian clock may determine timing when Aa-Nat gene expression can respond to inputs from the master circadian clock in the suprachiasmatic nucleus, e.g. adrenergic stimulation.     

Differentiation, distribution, and chemical state of intracellular trace elements in LAD2 mast cell line

Oxidation state changes of metallic ions are involved in the generation and biological defense against reactive oxygen species. The relationship between allergy and oxidative damage by metallic elements was studied by X-ray fluorescence analysis using a mast cell line. The distribution of metallic elements is changed by the induction of reactive oxygen species. In mast cells, the degranulation leading to antigen or calcium ionophore stimulation is related to excessive accumulation of iron and to its chemical state. X-ray absorption near-edge structure spectroscopy showed that the oxidation state of iron in the cells shifted from Fe(II) to Fe(III) in degranulation. This finding might have implications for understanding the mechanisms involved in IgE-mediated cell responses as seen in allergic reaction.     

Conservation of the E-function for Floral Organ Identity in Rice Revealed by the Analysis of Tissue Culture-induced Loss-of-Function Mutants of the <Emphasis Type="Italic">OsMADS1</Emphasis> Gene

Rapid progress in studies on flower development has resulted in refining the classical ‘ABC model’ into a new ‘ABCDE model’ to explain properly the regulation of floral organ identity. Conservation of E-function for flower organ identity among the dicotyledonous (dicot) plants has been revealed. However, its conservation in monocotyledonous (monocot) plants remains largely unknown. Here, we show the conservation of E-function in rice (Oryza sativaL.) by characterizing tissue culture-induced mutants of two MADS-box genes, OsMADS1and OsMADS5, which form a subclade within the well-supported clade of SEP-genes (E-function) phylogeny. Severe loss-of-function mutations of OsMADS1cause complete homeotic conversion of organs (lodicules, stamens, and carpels) of three inner whorls into lemma- and palea-like structures. Such basic deformed structure is reiterated along with the pedicel at the center of the same floret, indicating the loss of determinacy of the flower meristem. These phenotypes resemble the phenotypes caused by mutations of the dicot E-class genes, such as the Arabidopsis SEP123(SEPALLATA1/2/3) and the petunia FBP2(Floral Binding Protein 2), suggesting that OsMADS1play a very similar role in rice to that of defined E-class genes in dicot plants. In case of the loss-of-function mutation of OsMADS5, no defect in either panicles or vegetative organs was observed. These results demonstrate that OsMADS1clearly possesses E-function, and so, E-function is fundamentally conserved between dicot plants and rice, a monocot model plant.     

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.     

Identification of the heparin-binding region of snake venom vascular endothelial growth factor (VEGF-F) and its blocking of VEGF-A165

VEGF-A165 displays multiple effects through binding to KDR (VEGFR-2). Heparin/heparan sulfate-like molecules are known to greatly modulate their interaction. In fact, VEGF-A lacking a C-terminal heparin-binding region exhibits significantly reduced mitogenic activity. We recently found novel heparin-binding VEGFs in snake venom, designated VEGF-Fs, which specifically recognize KDR, rather than other VEGF receptors. VEGF-Fs virtually lack the C-terminal heparin-binding region when compared with other heparin-binding VEGF subtypes, despite their heparin-binding potential. The C-terminal region does not exhibit any significant homology with other known proteins or domains. In this study, we attempted to identify the heparin-binding region of VEGF-F using synthetic peptides. The C-terminal peptide of vammin (one of the VEGF-Fs, 19 residues) bound to heparin with similar affinity as native vammin. We then evaluated the effects of this peptide on the biological activity of VEGF-A165. The C-terminal peptide of VEGF-F exhibited specific blockage of VEGF-A165 activity both in vitro and in vivo. These observations demonstrate that the short C-terminal region of VEGF-F functions fully as the active heparin-binding domain and the corresponding peptide specifically blocks VEGF-A165, thus suggesting that the C-terminal heparin-binding region of VEGF-F recognizes similar heparin/heparan sulfate molecules as VEGF-A165. The present results will provide novel insight into VEGF-heparin interaction and may facilitate the design of new anti-VEGF drugs based on novel strategies.     

Synthesis of potent and selective inhibitors of Candida albicans N-myristoyltransferase based on the benzothiazole structure

Two parallel synthetic methods using solid-supported reagents were established to examine the rapid optimization of weak hit compound 1. Several compounds showed high potency in the low nanomolar range against N-myristoyltransferase. The structure-activity relationship (SAR) and antifungal activities of a series of novel 2-aminobenzothiazole N-myristoyltransferase inhibitors are presented.