Improvement of red color development on the surface of kuruma prawn Marsupenaeus japonicus under various conditions.

The degree of red color development on the surface of prawns by cooking is an important index for food quality. In this study, we tested several factors that are thought to influence the red color development to identify possible correlations with various conditions. Live kuruma prawns, Marsupenaeus japonicus, (15.4 cm, 25.2 g on average) were used in this study. In case of cooking at 100 °C for 1 min after 24 h of storage at 0 °C, 5 °C, and 20 °C, the red color development rate of prawns stored at 5 °C and 20 °C was significantly lower than that of prawns cooked just after killing. In case of cooking at 100 °C, 80 °C, and 60 °C after storage for 24 h at 0 °C, there was no color development at 60 °C and significantly less color development at 80 °C compared to cooking just after killing. Preparation using 1% sodium carbonate before cooking at 80 °C could compensate for the lack of red color development. Short exposure of live kuruma prawns to low-oxygen conditions had no influence on the color development, but putting the prawns in freshwater for 3 h significantly reduced the red color development rate. In conclusion, the storage time has little influence on the red color development when the cooking temperature is sufficiently high. However, in case a large amount of prawns is cooked followed by lowering the cooking temperature and/or prawns are exposed to serious stresses before cooking, an alkaline preparation could compensate for the lack of red color development.

Design and synthesis of 2-amino-6-(1H,3H-benzo[de]isochromen-6-yl)-1,3,5-triazines as novel Hsp90 inhibitors.

A novel series of 2-amino-1,3,5-triazines bearing a tricyclic moiety as heat shock protein 90 (Hsp90) inhibitors is described. Molecular design was performed using X-ray cocrystal structures of the lead compound CH5015765 and natural Hsp90 inhibitor geldanamycin with Hsp90. We optimized affinity to Hsp90, in vitro cell growth inhibitory activity, water solubility, and liver microsomal stability of inhibitors and identified CH5138303. This compound showed high binding affinity for N-terminal Hsp90α (Kd=0.52nM) and strong in vitro cell growth inhibition against human cancer cell lines (HCT116 IC50=0.098µM, NCI-N87 IC50=0.066µM) and also displayed high oral bioavailability in mice (F=44.0%) and potent antitumor efficacy in a human NCI-N87 gastric cancer xenograft model (tumor growth inhibition=136%).

Enantioselective synthesis of derivatives and structure-activity relationship study in the development of NA255 as a novel host-targeting anti-HCV agent.

Hepatitis C virus (HCV) infection represents a serious health-care problem. Previously we reported the identification of NA255 from our natural products library using a HCV sub-genomic replicon cell culture system. Herein, we report how the absolute stereochemistry of NA255 was determined and an enantioselective synthetic method for NA255 derivatives was developed. The structure-activity relationship of the NA255 derivatives and rat pharmacokinetic profiles of the representative compounds are disclosed.

Design and synthesis of novel macrocyclic 2-amino-6-arylpyrimidine Hsp90 inhibitors.

Macrocyclic compounds bearing a 2-amino-6-arylpyrimidine moiety were identified as potent heat shock protein 90 (Hsp90) inhibitors by modification of 2-amino-6-aryltriazine derivative (CH5015765). We employed a macrocyclic structure as a skeleton of new inhibitors to mimic the geldanamycin-Hsp90 interactions. Among the identified inhibitors, CH5164840 showed high binding affinity for N-terminal Hsp90α (K(d)=0.52nM) and strong anti-proliferative activity against human cancer cell lines (HCT116 IC(50)=0.15µM, NCI-N87 IC(50)=0.066µM). CH5164840 displayed high oral bioavailability in mice (F=70.8%) and potent antitumor efficacy in a HCT116 human colorectal cancer xenograft model (tumor growth inhibition=83%).

Trial for quality control in mercury contents by using tail muscle of full-cycle cultured bluefin tuna (Thunnus orientalis).

Substantial amounts of mercury are usually present in tuna muscle, with levels in excess of 10 times the standard safety value present in some individuals. Inspection of individual fish for mercury content would be desirable but may not be cost-effective. In this study, we tried to establish a low-cost system for checking the mercury content of tuna by using a tail muscle that is usually discarded. The samples used in this experiment were bluefin tuna, cultured in the Fisheries Laboratory of Kinki University (Oshima Experimental Station, Wakayama, Japan). They were raised from eggs spawned in 2002. Ninety-eight individuals, weighing 22.3 to 61.6 kg, were selected between December 2004 and November 2005. In nine individuals, the mercury content of the tail was compared with that of the whole body. The total mercury level was measured using the reduction vaporizing atomic absorption method after acid digestion. Except for the front of the abdomen, where the mercury content was lower (0.490 ppm), the mercury content of other parts of the fish did not differ from that of the tail muscle (0.631 ppm). Therefore, the overall mercury concentration in bluefin tuna could be estimated to be almost the same and/or lower than that of the tail muscle. On the basis of these results, for 1 year we investigated the quantity of mercury in full-cycle cultured bluefin tuna that were shipped. The mercury concentration showed no increase irrespective of increases of body weight.

Molecular properties of the two-component cell lysis system encoded by prophage phigaY of Lactobacillus gasseri JCM 1131T: cloning, sequencing, and expression in Escherichia coli.

Shotgun cloning of the Lactobacillus gasseri JCM 1131T whole DNA yielded two recombinant plasmids, p118gaY1 and p118gaY2, which directed cell lysis activity. Sequencing analysis revealed that the two plasmids carried almost identical inserted genes in following orders (truncated genes, in parentheses): in p118gaY1, (orf149)-orf92-holgaY-lysgaY-orf35-attL-(mnaAgaY1); in p118gaY2, (orfXgaY1)-orf169-orf149-orf92-holgaY-lysgaY-orf35-attP-(intgaY). The lysgaY-encoded protein (designated as LysgaY, 33.7 kDa) showed significant homology with putative muramidases (peptidoglycan-degrading enzyme) of the Lactobacillus phage phiadh, Lj965, Lj928, LL-H, mv4, and mv1. By zymogram analysis, LysgaY overproduced in Escherichia coli exhibited lytic activity towards 17 Gram-positive bacterial strains, including lactobacilli, lactococci, and staphylococci. The holgaY-encoded protein (15.7 kDa) contained three potential transmembrane helices, resembling putative holins (cytoplasmic membrane-disrupting protein) of Lj928 and Lj965. On the other hand, another clone p118gaYR obtained by EcoRI-shotgun cloning carried the (ptsCgaY1)-attR-(intgaY) genes. Three sequences, attL, attP, and attR, had a 47-bp common (core) sequence, and the core of attR was located in 3' region of a potential tRNA(Arg) gene. These results suggested that (i) attL and attR are phage-host junctions, left- and right-arms, respectively, (ii) attP is a phage attachment site, and (iii) intgaY is an integrase gene for phage integration and/or excision. After mitomycin C-induction, phage particles were demonstrated by electron microscopy. The prophage (phigaY) is somewhat leaky in the host, and has the two-component lysis system (HolgaY-LysgaY), closely resembling that of Lj928 as well as Lj965.

The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage varphiWMY: cloning, sequencing, expression, and mutational analysis in Escherichia coli.

From the genome library of Staphylococcus warneri M, the two successive cell-lysis genes (holWMY and lytWMY) were cloned and characterized. The lytWMY gene encoded a protein (LysWMY), whose calculated molecular mass and pI were 54 kDa and 8.95, respectively. When overproduced in Escherichia coli, lysWMY directed a protein of 45 kDa (smaller than the predicted molecular mass), having N-terminal 13 residues identical with those predicted from DNA. Comparative analysis revealed that LysWMY significantly resembles the putative N-acetylmuramoyl-L-alanine amidases encoded by the staphylococcal phages phi11, 80 alpha, and Twort. Examination of modular organization of LysWMY identified three putative domains CHAP (for D-alanyl-glycyl endopeptidase), amidase (L-muramoyl-L-alanine amidase), and SH3 (cell wall recognition). Gene knockout analysis revealed that each of the two domains of CHAP and amidase was responsible for cell-lytic activity on a zymogram gel. Site-directed mutation of Cys29Ala, His92Ala, or Asn114Ala in the CHAP domain substantially reduced cell-lytic activity, suggesting that this Cys-His-Asn triad is crucial for the enzymatic function. On the other hand, the holWMY gene encoded a protein (HolWMY) with molecular mass and pI of 16 kDa and 4.36; this protein contained two potential transmembrane helices, resembling other predicted holins (a cytoplasmic membrane-disrupting protein) encoded by the S. aureus phage, phi11, 80 alpha, and Twort. Upon mitomycin C exposure of S. warneri M, a prophage (phiWMY) was induced and the virion was examined under electron microscopy. PCR amplification and sequencing revealed the presence of the holWMY-lysWMY genes in the phage genome.

Characterization of lytic enzyme activities of Lactobacillus gasseri with special reference to autolysis.

Lactobacillus gasseri JCM 1130 and JCM 1131(T) exhibited autolytic activity in agar containing autoclaved cells of each strain as substrate. By zymogram analysis of JCM 1131(T), two lytic bands with apparent molecular masses of 54.5 and 35 kDa, were detected. Similarly, JCM 1130 yielded two lytic bands with apparent molecular masses of 35 and 33.5 kDa. In simple buffers as well, JCM 1131(T) suffered a drastic decrease in cell turbidity, but JCM 1130 did not undergo the decrease. The optimal pH for autolysis of JCM 1131(T) was in the range of 6.0-7.0, and the lysis was completely inhibited at pH 4-5. The lysis of JCM 1131(T) was suppressed by NaCl, in a concentration-dependent way. When subjected to UV irradiation or mitomycin C (MMC) treatment, cultures of both strains elicited conspicuous turbidity decrease after 2-4 h of growth, suggesting the occurrence of prophage induction. The 35-kDa lytic band of JCM 1131(T) and the 33.5-kDa protein of JCM 1130 were considerably increased by UV irradiation.

Synthesis and biological activities of benzofuran antifungal agents targeting fungal N-myristoyltransferase.

The C-4 side chain modification of lead compound 1 has resulted in the identification of a potent and selective Candida albicans N-myristoyltransferase (CaNmt) inhibitor RO-09-4609, which exhibits antifungal activity against C. albicans in vitro. Further modification of its C-2 substituent has led to the discovery of RO-09-4879, which exhibits antifungal activity in vivo. The drug design is based on X-ray crystal analysis of a CaNmt complex with benzofuran derivative 4a. The optimization incorporates various biological investigations including a quasi in vivo assay and pharmacokinetic study. The computer aided drug design, synthesis, structure-activity relationships, and biological properties of RO-09-4879 are described in detail.

Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 3.

A new series of acid-stable antifungal agents having strong inhibitory activity against Candida albicans N-myristoyltransferase (CaNmt) has been developed starting from acid-unstable benzofuranylmethyl aryl ether 2. The inhibitor design is based on X-ray crystallographic analysis of a CaNmt complex with aryl ether 3. Among the new inhibitors, pyridine derivative 8b and benzimidazole derivative 8k showed clear antifungal activity in a murine systemic candidiasis model.

Molecular analysis of the lysis protein Lys encoded by Lactobacillus plantarum phage phig1e.

The putative cell-lysis gene lys of Lactobacillus plantarum G1e phage phig1e encodes for a 442 amino-acids protein Lys. The N-terminal region (about 80 amino acids) of Lys consists of two discrete regions (the signal-peptide-like domain and the DE domain containing putative active sites of endolysin). To elucidate functions of the regions of Lys, mutational (random, site-directed, and/or fusion) analysis was performed. The plasmid pNdEHL, expressing the wild type Lys protein under promoter of lacZ' gene in Escherichia coli, was constructed. Two molecular species (44 kDa; referred to as pre-Lys, and 42 kDa; mature-Lys) from the protein extract of XL1-Blue/pNdEHL were detected on a sodium dodecyl sulfate gel and zymogram with L. plantarum G1e cells. Based on the N-terminal amino acid sequences, the two molecules were determined as; pre-Lys (the amino acid position deduced from lys gene, 1-7) MKLKNKL, mature-Lys (27-33) QTLSSQS. The mature Lys was hardly detected in the cells treated with sodium azide. These results suggested that the N-terminal 26 amino acids region of Lys precursor form is possibly processed posttranslationally, by a SecA-dependent manner at least in E. coli. Analysis of the point mutants (pLD36A, pLE39A, pLE55A, pLE67A and pLD71A), indicated that the acidic residues (aspartic acids at position 36, 71 and glutamic acids at position 39, 55) of N-terminal region and the serine at the position 48 of phig1e Lys are essential for the lytic activity.

Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 2.

Modification of the C-2 position of a benzofuran derivative 6 (RO-09-4609), an N-myristoyltransferase (Nmt) inhibitor, has led us to discover antifungal agents that are active in a murine systemic candidiasis model. The drug design is based on the analysis of a crystal structure of a Candida Nmt complex with 2. The optimization has been guided by various biological evaluations including a quasi in vivo assay and pharmacokinetic analysis.