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Objective To investigate the basal amino acid metabolism and impact of growth hormone (GH) therapy in short-stature children born small for gestational age (short SGA children). Methods In this age-matched case-control study, the basal blood levels of amino acids, asymmetric dimethylarginine (ADMA), and nitrite/nitrate (NOx) were compared between 24 short SGA children and 25 age-matched normal children. Changes in these parameters were assessed for 12 months in 12 short SGA children initiating GH therapy (Group A) and 12 age-matched short SGA children without GH therapy (Group B). Results The arginine levels were significantly lower in the short SGA than in normal children. The ADMA levels were significantly higher and NOx levels were significantly lower in the short SGA than normal children. In Group A, the ADMA level was significantly lower and NOx level was significantly higher at 6 months than at baseline. At 12 months, the ADMA level in Group A began to increase, but the NOx level remained the same. Group B showed no significant changes. Conclusions This study is the first to show that ADMA is promoted and nitric oxide is suppressed in short SGA children and that GH therapy affects the production of ADMA and nitric oxide.

Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) is synthesized from tyramine (TA) by the catalysis of tyramine-β-hydroxylase (TβH) originated in the insect nervous system. Both TA and OA act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. Herein, the inhibitory activity of 1-arylimidazole-2(3 H )-thiones (AITs) was tested on cloned Drosophila tyramine-β-hydroxylase (DmTβH) expressed in Bombyx mori strain. Radiolabelled 3 H-TA was used to analyze the activity of AITs exhibited inhibitory effects on DmTβH, whose ID 50 values range from 0.02 to 2511 nM where DmTβH was inhibited in a dose-dependent manner at pH 7.6 and 25 °C during a 30 min of incubation. To understand the catalytic role of the TβH, a three dimensional structure of the TβH from Drosophila melanogaster was constructed by homology modeling using the Phyre2 web server with 100% confidence. The modeled three-dimensional structure of TβH was used to perform the docking study with AITs. This may give more insights to precise design of inhibitors for TβH to control insect’s population.

The insect nervous system contains biogenic amines such octopamine (OA), which is synthesized from tyramine (TA) by catalysis of tyramine-β-hydroxylase (TβH). In this study, the Drosophila 70 kDa tyramine-β-hydroxylase (DmTβH) protein was purified after the recombinant nucleopolyhedrovirus isolated from Bombyx mori (BmNPV) containing the TβH gene was injected into the hemocoel of the fifth instar larvae from the d17 B. mori strain. Western blot analysis revealed an immunoreactive band with a molecular mass of 70 kDa. The products formed by incubating the enzyme reaction mixture were separated and detected by reverse phase high-performance liquid chromatography. The optimum pH, temperature, and incubation time for the conversion of TA to OA were 7.6, 25 °C, and 30 min, respectively. The inhibitory experiments using various concentrations of 1-(2-methoxy-5-methylphenyl) imidazole-2(3 H )-thione (MMIT) showed that MMIT inhibited DmTβH dose-dependently and that this method can be applied for screening DmTβH inhibitors.

EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine), a nucleoside reverse transcriptase inhibitor with extremely potent anti-HIV activity, was concisely synthesized from (R)-glyceraldehyde acetonide in an 18% overall yield by a 12-step sequence involving highly diastereoselective ethynylation of an α-alkoxy ketone intermediate. The present synthesis is superior, both in overall yield and in the number of steps, to the previous one which required 18 steps from an expensive starting material and resulted in a modest overall yield of 2.5%.

The storage glucans of Cyanidioschyzon merolae [clade L-1 (cyanidian algae), order Porphyridiales, subclass Bangiophycidae], which is considered to be one of the most primitive rhodophytes, were analyzed to understand the early evolution of the glucan structure in the Rhodophyta. Chain-length distribution analysis of the glucans of cyanidian algae demonstrated that while the glucans of Cyanidium caldarium and Galdieria sulphuraria are of the glycogen type, those of C. merolae are of the semiamylopectin type, as in other lineages of the Rhodophyta. Gel permeation chromatography, however, showed that the glucans of C. merolae do not include amylose, being different from those of other Bangiophycidae species. Identifi cation by MALDI–TOF-MS and enzyme assaying of glucan granulebound proteins indicated that phosphorylase, but not starch synthase, is included. Thus, C. merolae has an unusual glucan and bound-protein composition for the Bangiophycidae, appearing to be a member of the Florideophycidae. The fi nding that the alga does not contain amylose or the related enzyme, granule-bound starch synthase, is, however, consistent with previously reported results of molecular phylogenetic analysis of starch synthases. Our results support an evolutionary scenario defi ned by the loss of starch and reversion to glycogen synthesis during the evolution of cyanidian algae, and suggest the possibility that a C. merolae like primitive rhodophyte might have evolved into the Florideophycidae.

Rice (Oryza sativa) allelic sugary1 (sug1) mutants defective in isoamylase 1 (ISA1) accumulate varying levels of starch and phytoglycogen in their endosperm, and the activity of a pullulanase-type of a debranching enzyme (PUL) was found to correlate closely with the severity of the sug1 phenotype. Thus, three PUL-deficient mutants were generated to investigate the function of PUL in starch biosynthesis. The reduction of PUL activity had no pleiotropic effects on the other enzymes involved in starch biosynthesis. The short chains (DP < or = 13) of amylopectin in PUL mutants were increased compared with that of the wild type, but the extent of the changes was much smaller than that of sug1 mutants. The alpha-glucan composition [amylose, amylopectin, water-soluble polysaccharide (WSP)] and the structure of the starch components (amylose and amylopectin) of the PUL mutants were essentially the same, although the average chain length of the B(2-3) chains of amylopectin in the PUL mutant was approximately 3 residues longer than that of the wild type. The double mutants between the PUL-null and mild sug1 mutants still retained starch in the outer layer of endosperm tissue, while the amounts of WSP and short chains (DP < or = 7) of amylopectin were higher than those of the sug1 mutant; this indicates that the PUL function partially overlaps with that of ISA1 and its deficiency has a much smaller effect on the synthesis of amylopectin than ISA1 deficiency and the variation of the sug1 phenotype is not significantly dependent on the PUL activities.

A reliable quantitative assay method for starch branching enzyme (BE) remains to be established whereas it has been required to characterize BE towards understanding the regulatory mechanism for the synthesis of starch in plant tissues. We describe a new quantitative assay method for BE activities with malto-oligosaccharides, amylose and amylopectin as substrates by using bicinchoninic acid (BCA) for measuring directly the reducing terminals of linear glucans formed after debranching the reaction products. The BCA method not only can be performed by simple procedures and easy handling with the use of cheap toxic-free reagents, but also shows highly color-stable properties after the treatment of the reaction mixture with the color-yielding reagents compared with the modified Park-Johnson method (Carbohydr. Res., 94, 205-213(1981)). The intensity and the spectrum of the purple color generated in the treated assay mixture are maintained in sugars and glucans in the range of glucose and amylose with degree of polymerization up to at least 1658. The BCA method can be also applied for characterization of BE when amylopectin is used as glucan substrate. In this paper we report this efficient, reproducible and quantitative BCA method by showing some experimental results obtained in an attempt to determine the kinetic parameters of BEIIb from rice endosperm.

SSI accounts for 60% of the total SS activity in the soluble fraction in the developing rice en- dosperm. Rice SSI-deficient mutants were identified by using reverse genetics, and the chain-length analysis of the endosperm starch showed that SSI distinctly synthesizes DP 8―12 chains from short DP 6―7 chains emerg- ing from the A chains and the branch points in the B1 chains of amylopectin. 1) In this study, to evaluate by in vitro study the function of recombinant SSI of rice (rSSI), the change in the chain-length distribution of glyco- gen or amylopectin in the activity band after rSSI enzymatic reactions in native-PAGE gel was examined. When glycogen was used as the substrate, the -glucan produced in the rSSI activity band on the native- PAGE gel had specifically fewer DP 6 chains and more DP 8 chains than unmodified glycogen did. When rice amylopectin was used as the substrate, the -glucan produced in the rSSI activity band showed the oscillation of a short turn in the range of DP 20 compared with the unmodified amylopectin; the chains with DP 6, 7, 10, 11, 13 and 17 decreased, while the extent of decrease was reduced in the chains with DP 8, 12 and 15. These results suggest that rSSI preferentially elongates the chains up to DP 8 from the short and long B1 and B2 chains with DP 6 or 7 from branch points to the non-reduced end as well as the A chains with DP 6 or 7 by adding one or two glucose moieties.

The general transcription factor TFII-I, with the corresponding gene name GTF2I, is an unusual transcriptional regulator that associates with both basal and signal-induced transcription factors. TFII-I consists of six GTF2I repeat domains, called I-repeats R1-R6. The structure and function of the GTF2I domain are not clearly understood, even though it contains a helix-loop-helix motif, which is considered to be the protein-protein interaction area, based on biochemical analyses. Here, we report the solution structure of the fifth repeat of the six GTF2I repeat domains from murine TFII-I, which was determined by heteronuclear multidimensional NMR spectroscopy (PDB code 1Q60). The three-dimensional structure of the GTF2I domain is classified as a new fold, consisting of four helices (residues 8-24, 34-39, 63-71, and 83-91), two antiparallel beta strands (residues 44-47 and 77-80), and a well-defined loop containing two beta-turns between sheet 1 and helix 3. All of the repeats probably have similar folds to that of repeat 5, because the conserved residues in the GTF2I repeat domains are assembled on the hydrophobic core, turns, and secondary structure elements, as revealed by a comparison of the sequences of the first through the sixth GTF2I repeats in TFII-I.

Starch synthase IIIa (SSIIIa)-deficient rice (Oryza sativa) mutants were generated using retrotransposon insertion and chemical mutagenesis. The lowest migrating SS activity bands on glycogen-containing native polyacrylamide gel, which were identified to be those for SSIIIa, were completely absent in these mutants, indicating that they are SSIIIa null mutants. The amylopectin B2 to B4 chains with degree of polymerization (DP) ≥ 30 and the M r of amylopectin in the mutant were reduced to about 60% and 70% of the wild-type values, respectively, suggesting that SSIIIa plays an important part in the elongation of amylopectin B2 to B4 chains. Chains with DP 6 to 9 and DP 16 to 19 decreased while chains with DP 10 to 15 and DP 20 to 25 increased in the mutants amylopectin. These changes in the SSIIIa mutants are almost opposite images of those of SSI-deficient rice mutant and were caused by 1.3- to 1.7-fold increase of the amount of SSI in the mutants endosperm. Furthermore, the amylose content and the extralong chains (DP ≥ 500) of amylopectin were increased by 1.3- and 12-fold, respectively. These changes in the composition in the mutants starch were caused by 1.4- to 1.7-fold increase in amounts of granules-bound starch synthase (GBSSI). The starch granules of the mutants were smaller with round shape, and were less crystalline. Thus, deficiency in SSIIIa, the second major SS isozyme in developing rice endosperm affected the structure of amylopectin, amylase content, and physicochemical properties of starch granules in two ways: directly by the SSIIIa deficiency itself and indirectly by the enhancement of both SSI and GBSSI gene transcripts.

Four starch synthase I (SSI)-deficient rice (Oryza sativa) mutant lines were generated using retrotransposon Tos17 insertion. The mutants exhibited different levels of SSI activities and produced significantly lower amounts of SSI protein ranging from 0% to 20% of the wild type. The mutant endosperm amylopectin showed a decrease in chains with degree of polymerization (DP) 8 to 12 and an increase in chains with DP 6 to 7 and DP 16 to 19. The degree of change in amylopectin chain-length distribution was positively correlated with the extent of decrease in SSI activity in the mutants. The structural changes in the amylopectin increased the gelatinization temperature of endosperm starch. Chain-length analysis of amylopectin in the SSI band excised from native-polyacrylamide gel electrophoresis/SS activity staining gel showed that SSI preferentially synthesized DP 7 to 11 chains by elongating DP 4 to 7 short chains of glycogen or amylopectin. These results show that SSI distinctly generates DP 8 to 12 chains from short DP 6 to 7 chains emerging from the branch point in the A or B1 chain of amylopectin. SSI seemingly functions from the very early through the late stage of endosperm development. Yet, the complete absence of SSI, despite being a major SS isozyme in the developing endosperm, had no effect on the size and shape of seeds and starch granules and the crystallinity of endosperm starch, suggesting that other SS enzymes are probably capable of partly compensating SSI function. In summary, this study strongly suggested that amylopectin chains are synthesized by the coordinated actions of SSI, SSIIa, and SSIIIa isoforms.

In mammals, the circadian and stress systems (both centers of which are located in the hypothalamus) are involved in adaptation to predictable and unpredictable environmental stimuli, respectively. Although the interaction and relationship between these two systems are intriguing and have been studied in different ways since the "pre-clock gene" era, the molecular interaction between them remains largely unknown. Here, we show by systematic molecular biological analysis that acute physical stress elevated only Period1 (Per1) mRNA expression in mouse peripheral organs. Although behavioral rhythms in vivo and peripheral molecular clocks are rather stable against acute restraint stress, the results of a series of promoter analyses, including chromatin immunoprecipitation assays, indicate that a glucocorticoid-responsive element in the Per1 promoter is indispensable for induction of this mRNA both in vitro and in vivo. These results suggest that Per1 can be a potential stress marker and that a third pathway of Per1 transcriptional control may exist in addition to the clock-regulated CLOCK-BMAL1/E-box and light-responsive cAMP-responsive element-binding protein/cAMP-responsive element pathways.

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.

EI-1941-1 ( 1a ) and EI-1941-2 ( 2a ) accompanied by EI-1941-3 ( 3 ) have been isolated from culture broth of Farrowia sp. E-1941 as the inhibitors of interleukin-1β converting enzyme. The structures of 1a , 2a , and 3 were elucidated by the analysis of NMR and MS data, and finally the absolute stereochemistries of 1a and 2a were confirmed by optical rotation data, or X-ray crystallographic analysis of p -bromobenzoate, 2b , respectively.

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique alpha/beta sandwich fold composed of two alpha helices and four antiparallel beta strands and has a characteristic turn named the TY-turn between alpha1 and alpha2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the beta sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the beta2 and beta3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting beta2 and beta3.

The BolA-like proteins are widely conserved from prokaryotes to eukaryotes. The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA-like protein. The overall topology is alphabetabetaalphaalphabetaalpha, in which beta(1) and beta(2) are antiparallel, and beta(3) is parallel to beta(2). This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA-like proteins are assembled on the one side of the protein.

Ascorbic acid (vitamin C) is a cofactor required in catecholamine synthesis for conversion of dopamine to norepinephrine by dopamine beta-hydroxylase. Mutant mice lacking the plasma membrane ascorbic acid transporter (SVCT2) have severely reduced tissue levels of ascorbic acid and die after birth. We therefore investigated whether these mice might have impaired synthesis of catecholamines. Levels of catecholamines in brain were unaffected by SVCT2 deficiency. In heart, the only evidence for impaired dopamine beta-hydroxylase activity was a twofold increase in tissue dopamine. An influence of the deficiency on tissue catecholamines was most prominent in the adrenals where norepinephrine was decreased by 50% and epinephrine, by 81%. On the ultrastructural level, adrenal chromaffin cells in SVCT2 null mice showed depletion of catecholamine storage vesicles, increased amounts of rough endoplasmic reticulum, signs of apoptosis, and increased glycogen storage. Decreased plasma levels of corticosterone indicated additional effects of the deficiency on adrenal cortical function. These data show that deranged catecholamine system function in SVCT2 null mice is largely restricted to the adrenal medulla and cannot account for the lethality in these animals. The data, however, establish a crucial role for ascorbic acid in adrenal chromaffin cell function.

The in vitro metabolism of fenthion and its sulfoxide (fenthion sulfoxide) in sea bream (Pagrus major) and goldfish (Carassius auratus) was investigated and compared with that in rats. Fenthion was oxidized to fenthion sulfoxide and the oxon derivative, but not to its sulfone, in the presence of NADPH by liver microsomes of sea bream, goldfish, and rats. These liver microsomal activities of the fish were lower than those of rats but were of the same order of magnitude. The NADPH-linked oxon- and sulfoxide-forming activities of liver microsomes of the fish and rats were inhibited by SKF 525-A, metyrapone, alpha-naphthoflavone, and carbon monoxide. The oxidizing activity to fenthion sulfoxide was also inhibited by alpha-naphthylthiourea. Several cytochrome P450 isoforms and flavin-containing monooxygenase 1 exhibited these oxidase activities. Fenthion sulfoxide was reduced to fenthion with liver cytosol of the fish and rats upon addition of 2-hydroxypyrimidine, N(1)-methylnicotinamide, or butyraldehyde, each of which is an electron donor of aldehyde oxidase, under anaerobic conditions. The activity was inhibited by menadione, beta-estradiol, and chlorpromazine, which are inhibitors of aldehyde oxidase. The activities in the fish livers were similar to those of rat liver. Aldehyde oxidase purified from the livers of sea bream and rats exhibited the reducing activity. Thus, fenthion and fenthion sulfoxide are interconvertible in fish and rats through the activities of cytochrome P450, flavin-containing monooxygenase, and aldehyde oxidase.

Cellular RNA in Schizosaccharomyces pombe cells drastically decreases in amount during nitrogen starvation. Previously, we found and purified a soluble RNA-degrading enzyme whose activity drastically increased in the cells of S. pombe undergoing nitrogen starvation. The enzyme was a nuclease encoded by pnu1(+). In this study, the increase in the RNA-degrading activity and the decrease in cellular RNA level are examined in a null-mutant of pnu1(+) (pnu1Delta). During nitrogen starvation, wild-type cells show an apparent increase in RNA-degrading activity, whereas the pnu1Delta cells do not. The wild-type cells show a drastic decrease in cellular RNA amount, whereas the pnu1Delta cells show only a slight decrease. These results suggest that Pnu1 nuclease is implicated in the decrease in cellular RNA amount during nitrogen starvation, probably via the RNA-degrading activity. The increase in the RNA-degrading activity is independent of both the Wis1 stress-activated MAP kinase cascade and Tor1 signaling pathway, but it is strongly dependent on isp6(+), a gene for a possible protease, whose expression is induced during nitrogen starvation. A disruption mutant for isp6(+) (isp6Delta) is deficient in both the increase in the RNA-degrading activity and the drastic decrease in the cellular RNA amount during nitrogen starvation, which suggests that isp6(+) is involved in the RNA degradation via regulating the RNA-degrading activity of Pnu1.

Soybean extracts (SBE) containing isoflavone glycosides were cultured with Ganoderma lucidum mycelia producing beta-glucosidase. The anti-angiogenic effects of the cultivated product, containing rich in genistein, named GCP (genistein combined polysaccharide), were assessed with chick chorioallantoic membranes (CAM) and a mouse dorsal air-sac model. Beta-glucosidase produced by the mycelia converted the isoflavone glycosides into aglycons. A test of volunteers showed that serum concentrations of genistein in the subjects treated with GCP (n = 4) at 3 h after administration were significantly higher than those in the subjects treated with SBE (n = 4). GCP inhibited angiogenesis in CAM, and the activity of GCP was greater than that of SBE. GCP inhibited the formation of new vessels induced by colon carcinoma cells in vivo.

A concise enantioselective total synthesis of 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), an extremely potent anti-HIV agent, has been accomplished from (R)-glyceraldehyde acetonide in 18% overall yield by a 12-step sequence involving a highly diastereoselective ethynylation of an α-alkoxy ketone intermediate.

Substrate selectivities of microbial proline 4-hydroxylase and proline 3-hydroxylases, all of which were purified from recombinant Escherichia coli , were investigated. l 2-Azetidine carboxylate, 3,4-dehydro- l -proline and l -pipecolinic acid were hydroxylated by those enzymes in regio- and stereospecific manner.

The in vitro metabolism of p, p'-DDT (p, p'-dichlorodiphenyltrichloroethane), an important environmental pollutant, was examined in fish, focusing on reductive dechlorination. When p, p'-DDT was incubated with hepatopancreas microsomes or the blood of goldfish, Carassius auratus, in the presence of both a reduced pyridine nucleotide and FMN, a dechlorinated metabolite, p, p'-DDD (p, p'-dichlorodiphenyldichloroethane) was formed under anaerobic conditions. These reductase activities were inhibited by carbon monoxide. Although the microsomes or blood was boiled, the dechlorinating activity was not abolished. Hemoglobin and hematin exhibited the reductase activity toward p, p'-DDT with NADH and FMN. The activity of hematin was also exhibited with FMNH2. The reductive dechlorination appears to proceed nonenzymatically by the reduced flavin, catalyzed by the heme group of hemoproteins.

The in vitro metabolism of fenthion, an insecticide, was examined in fish, focusing on the interconversion between fenthion and its sulfoxide (fenthion sulfoxide). When fenthion was incubated with hepatopancreas microsomes of goldfish, Carassius auratus, in the presence of NADPH, the oxidized metabolite, fenthion sulfoxide was formed, but not fenthion sulfone. The oxidase activity was inhibited by SKF 525-A, and partly by α-naphthylthiourea. In contrast, fenthion sulfoxide was reduced back to fenthion by the hepatopancreas cytosol of goldfish in the presence of 2-hydroxypyrimidine, an electron donor for aldehyde oxidase. The activity was markedly inhibited by menadione, an inhibitor for aldehyde oxidase. The interconversion appears to be mediated mainly by the cytochrome P450 system and aldehyde oxidase.

From the ether extract of the liverwort Odontoschisma denudatum, three novel ent-bibsane- and a novel dolabellane-type diterpenoids have been isolated. Their absolute structures were determined by a combination of 2D-NMR, X-ray crystallographic analysis, modified Mosher method, and chemical degradation.

In the course of screening for inhibitors of the lymphocyte kinase, Lck (p56lck), aiming at novel immunosuppressants, we isolated a novel alkaloid, lymphostin (LK6-A), from the culture broth of Streptomyces sp. KY11783. Lymphostin was produced in a fermentation medium supplemented with a highly porous polymer resin, which prevented the degradation of this compound in the culture broth. Lymphostin inhibited the kinase activity of Lck with an IC50 value of 0.05 microM, and exhibited potent inhibitory activity against the mixed lymphocyte reaction (MLR) with an IC50 value of 0.009 microM.

Novel compounds MS-681a, b, c and d were isolated from the culture broth of a fungal strain KY6568. The strain was identified as Myrothecium sp. from its morphological characteristics. MS-681a, b, c and d inhibited the activity of purified smooth muscle myosin light chain kinase with IC50 values of 0.11, 0.29, 0.095 and 0.26 microM, respectively. Cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase and protein kinase C were not inhibited at 100 microM by MS-681 compounds.

EI-1507-1 and -2, novel interleukin-1 beta converting enzyme (ICE) inhibitors, were isolated from the culture broths of Streptomyces sp. E-1507. EI-1507-1 and EI-1507-2 selectively inhibited the recombinant human ICE activity with IC50 values of 0.23 and 0.42 microM, respectively. EI-1507-1 and EI-1507-2 also inhibited mature interleukin-1 beta secretion from THP-1 cells with IC50 values of 1.1 and 1.4 microM, respectively.

A novel cyclic peptide, MS-271, was isolated from the culture broth of an actinomycete, Streptomyces sp. M-271 as an inhibitor of smooth muscle myosin light chain kinase (MLCK). MS-271 inhibited the MLCK from chicken gizzard with an IC50 value of 8 microM. MS-271 did not inhibit cyclic AMP-dependent protein kinase, protein kinase C or calcium/calmodulin-dependent cyclic nucleotide phosphodiesterase at concentrations up to 400 microM. The primary structure of MS-271 was identical to that of siamycin I, an anti-HIV peptide isolated from a microbial source.

The three-dimensional structure of the endothelin B receptor (ETB) selective antagonist RES-701-1 has been determined by 1H NMR in deuterated dimethyl sulphoxide. RES-701-1 consists of 16 amino acid residues with a novel internal linkage between the beta-carboxyl group of Asp9 and the alpha-amino group of Gly1. The structural calculations were carried out with the combined use of distance geometry and simulated annealing. The result indicates that RES-701-1 adopts an extraordinary folding; the 'tail' (Trp10-Trp16) passes through the 'ring' region (Gly1-Asp9). Several critical NOEs directly support this extraordinary folding. The folding of RES-701-1 turned out to be the same as that Frèchet et al. calculated for RP 71955 which possesses the same internal linkage as RES-701-1. The obtained structure suggested that the region consisting of Thr6, Ala7, Tyr14 and Tyr15 and/or, the region consisting of Asn2, Tyr14 and Tyr15 are involved in a binding with ETB.

RES-701-1 is an ET B receptor selective antagonist obtained from Streptmyces sp. (IC 50 = 10 nM). The chemically synthesized RES-701-1 which exhibits only micromolar affinity for ET B receptor was examined by 1 H NMR and FAB-MS. The results indicated that the primary structures of the synthetic and the authentic RES-701-1 are identical but the 3D structure is completely different between them.

The structure of RES-701-1, a novel microbial endothelin type B receptor antagonist, was determined. Protein chemical data and FAB-MS have identified RES-701-1 to be a cyclic polypeptide consisting of 16 common L-amino acids. This compound is so stable against proteolysis that its enzymatic digestion under standard conditions proved to be very difficult. Therefore, the primary sequence of RES-701-1 was determined by the application of advanced protein chemical methods to be Gly1-Asn2-Trp3-His4-Gly5-Thr6-Ala7-Pro8-++ +Asp9-Trp10-Phe11-Phe12-Asn13- Tyr14-Tyr15-Trp16. The compound is cyclized between the beta-carboxyl group of Asp9 and the alpha-amino group of Gly1.

MS-282a and MS-282b were isolated from the culture broth of Streptomyces tauricus ATCC 27470 as inhibitors of smooth muscle myosin light chain kinase (MLCK). MS-282a and MS-282b inhibited the activity of chicken gizzard MLCK with IC50 values of 3.8 microM and 5.2 microM, respectively. Cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase and protein kinase C were not inhibited by 150 microM MS-282a at all. It is likely that MS-282a blocks MLCK activity by antagonizing calmodulin since 1) the compound inhibited calmodulin-dependent but not calmodulin-independent activity of MLCK; 2) the inhibition of MLCK was antagonized by increasing concentrations of calmodulin, and 3) the compound inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase.

Firefly luciferase genes have been isolated from approximately 20 species of Lampyrinae, Luciolinae, and Photurinae. These are mostly nocturnal luminescent species that use light signals for sexual communication. In this study, we isolated three cDNAs for firefly luciferase from Psilocladinae ( Cyphonocerus ruficollis ) and Ototretinae ( Drilaster axillaris and Stenocladius azumai ), which are diurnal non-luminescent or weakly luminescent species that may use pheromones for communication. The amino acid sequences deduced from the three cDNAs showed 81–89% identities to each other and 60–81% identities with known firefly luciferases. The three purified recombinant proteins showed luminescence and fatty acyl-CoA synthetic activities, as observed in other firefly luciferases. The emission maxima by the three firefly luciferases (λmax, 545–546 nm) were shorter than those by known luciferases from the nocturnal fireflies (λmax, 550–568 nm). These results suggest that the primary structures and enzymatic properties of luciferases are conserved in Lampyridae, but the luminescence colors were red-shifted in nocturnal species compared to diurnal species.

Assignments of 1H, 15N, and 13C magnetic resonances for ribonuclease H from Escherichia coli have been completed using double- and triple-resonance 2D and 3D NMR experiments. These assignments include all types of 1H, 15N, and 13C nuclei detectable by NMR. The enzyme used, which cleaves the RNA moiety of an RNA-DNA duplex, consists of 155 amino acid residues and has 1962 nuclei (227 nitrogen, 762 carbons, and 973 protons) observable independently by NMR. Among those, 1868 nuclei (95%) have been assigned. Two methods, 3D HCH and 13C-13C-1H heteroSQC/homoSQC, were newly devised to complete the side chain assignments. These methods were used to elucidate the -CH2- and -C-CH-substructures. Triple-resonance experiments to detect other types of substructures, (e.g., -N-CH- and -C-NH-) were also applied. In total, 10 kinds of 3D NMR experiments were used to complete the assignments. The chemical shifts obtained through the assignments were analyzed in terms of the tertiary structure of the protein molecule. Among the 13C chemical shifts, larger secondary shifts (deviations from shifts at the random coil state) were observed for the C alpha, C beta, and C' nuclei, which reflect the local structures on the backbone, that is, the alpha-helix, beta-sheet, and left-handed helix, respectively.

We isolated the cDNA of a luciferase isotype (LcLuc2) from the Japanese firefly, Luciola cruciata (Lampyridae, Coleoptera). The gene product of LcLuc2 (LcLuc2) showed 59% amino acid identity with firefly luciferase LcLuc1, which was previously identified in L. cruciata. The recombinant LcLuc2 showed both luminescence activity and fatty acyl-CoA synthetic activity comparable to those of LcLuc1. The spectral maxima of the luminescence by LcLuc1 and LcLuc2 were 554 and 543 nm, respectively. Reverse transcription-PCR analysis showed that the transcripts of LcLuc1 were abundant in the lanterns of larva, adult male, and adult female, whereas both LcLuc1 and LcLuc2 were expressed in eggs. The luminescence spectra of the lantern extracts from larva, adult male, and adult female were in good agreement with that of recombinant LcLuc1. On the other hand, the emission maximum of the extract from eggs was between those of LcLuc1 and LcLuc2. These results suggest that L. cruciata possesses two luciferases: LcLuc1 is responsible for the major luminescence in larva and adult, whereas LcLuc1 and LcLuc2 are responsible for the dim glow in firefly eggs.

International audience; Lowering of the CO(2) concentration in the environment induces development of a pyrenoidal starch sheath, as well as that of pyrenoid and CO(2)-concentrating mechanisms, in many microalgae. In the green algae Chlamydomonas and Chlorella, activity of granule-bound starch synthase (GBSS) concomitantly increases under these conditions. In this study, effects of the GBSS-defective mutation (sta2) on the development of pyrenoidal starch were investigated in Chlamydomonas. Stroma starch- and pyrenoid starch-enriched samples were obtained from log-phase cells grown with air containing 5% CO(2) (high-CO(2) conditions favouring stromal starch synthesis) and from those transferred to low-CO(2) conditions (air level, 0.04% CO(2), favouring pyrenoidal starch synthesis) for 6h, respectively. In the wild type, total starch content per culture volume did not increase during the low-CO(2) conditions, in spite of the development of pyrenoidal starch, suggesting that degradation of some part of stroma starch and synthesis of pyrenoid starch simultaneously occur under these conditions. Even in the GBSS-deficient mutants, pyrenoid and pyrenoid starch enlarged after lowering of the CO(2) concentration. However, the morphology of the pyrenoid starch was thinner and more fragile than the wild type, suggesting that GBSS does affect the morphology of pyrenoidal starch. Surprisingly normal GBSS activity is shown to be required to obtain the high A-type crystallinity levels that we now report for pyrenoidal starch. A model is presented explaining how GBSS-induced starch granule fusion may facilitate the formation of the pyrenoidal starch sheath.

In monocots, starch branching enzyme II (BEII) was functionally differentiated into BEIIa and BEIIb after separation from the dicots, and in cereals BEIIb plays a distinct role in amylopectin biosynthesis in the endosperm. The present study was conducted to examine to what extent a green algal BEII has an overlapping function with BEIIb in starch biosynthesis by introducing the Chlorella BEII gene into an amylose-extender ( ae ) mutant of rice. Chlorella BEII was found to complement the contribution of the rice endosperm BEIIb to the structures of amylopectin and starch granules because these mutated phenotypes were recovered almost completely to those of the wild type by the expression of Chlorella BEII. When the recombinant BE enzymes were incubated with the rice ae amylopectin, the branching pattern of Chlorella BEII was much more similar to that of rice BEIIb rather than rice BEIIa. Detailed analyses of BE reaction products suggests that BEIIb and Chlorella BEII only transfer chains with a degree of polymerization (DP) of 6 and 7, whereas BEIIa preferably transfers short chains with a DP of about 6–11. These results show that the Chlorella BEII is functionally similar to rice BEIIb rather than BEIIa.

The assignments of individual magnetic resonances of backbone nuclei of a larger protein, ribonuclease H from Escherichia coli, which consists of 155 amino acid residues and has a molecular mass of 17.6 kDa are presented. To remove the problem of degenerate chemical shifts, which is inevitable in proteins of this size, three-dimensional NMR was applied. The strategy for the sequential assignment was, first, resonance peaks of amides were classified into 15 amino acid types by 1H-15N HMQC experiments with samples in which specific amino acids were labeled with 15N. Second, the amide 1H-15N peaks were connected along the amino acid sequence by tracing intraresidue and sequential NOE cross peaks. In order to obtain unambiguous NOE connectivities, four types of heteronuclear 3D NMR techniques, 1H-15N-1H 3D NOESY-HMQC, 1H-15N-1H 3D TOCSY-HMQC, 13C-1H-1H 3D HMQC-NOESY, and 13C-1H-1H 3D HMQC-TOCSY, were applied to proteins uniformly labeled either with 15N or with 13C. This method gave a systematic way to assign backbone nuclei (N, NH, C alpha H, and C alpha) of larger proteins. Results of the sequential assignments and identification of secondary structure elements that were revealed by NOE cross peaks among backbone protons are reported.

Plastidial phosphorylase (Pho1) accounts for ∼96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30°C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20°C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures.

Kapurimycin A3 is a new antitumor antibiotic isolated from a Streptomyces. It contains the anthrapyrone skeleton and a beta,gamma-unsaturated delta-keto carboxylic acid moiety in the structure. In vitro, kapurimycin causes single-strand cleavage of supercoiled pBR322 DNA. The diminished cytotoxicity and DNA cleaving activity for 13-decarboxykapurimycin A3 indicates that the beta, gamma-unsaturated delta-keto carboxylic acid moiety is important for the activity of kapurimycin. Kapurimycin A3 binds to calf thymus DNA at 4 degrees C, and the thermal treatment of this adduct results in release of a guanine covalently attached to C-16 of kapurimycin via one of its nitrogen atoms. Thus, the epoxide is the alkylating functional group of kapurimycin, and this is consistent with the lack of DNA cleaving and cytotoxic activities for 14,16-deoxy-14,16-dihydroxykapurimycin. These findings have revealed that DNA strand scission by kapurimycin is due to the alkylation of guanine by ring opening of the epoxide group of kapurimycin, depurination of modified guanine, and presumably subsequent hydrolysis of the phosphate ester backbone at the resultant apurinic sites.

A new strategy for the sequential assignment of backbone proton resonances in larger proteins involving a unique combination of four types of heteronuclear three-dimensional (3D) NMR spectroscopies is reported. This method relies on the uniform labeling of amide nitrogens with 15N and of alpha-carbons with 13C. Heteronuclear 1H-15N TOCSY-HMQC and NOESY-HMQC experiments can reveal connections between cross-peaks arising from the NHi-C alpha Hi-1 and NHi-C alpha Hi connectivities in the finger-print region in in general. They also specifically reveal the sequential amide-amide connectivities among the amide cross-peaks for the alpha-helices. Heteronuclear 1H-13C HMQC-TOCSY and HMQC-NOESY experiments can reveal connections between cross-peaks arising from the NHi-C alpha Hi and NHi+1-C alpha Hi connectivities in the finger-print region in general. The combination of the two sets of results reveals the complete unambiguous sequential connection of cross-peaks for the proton resonances in the peptide backbone. The application of the new strategy is reported for a protein, ribonuclease H, with a molecular weight of 17.6 kDa.

The three-dimensional structure of an alpha-amylase inhibitor, HAIM, composed of 78 amino acids, was analyzed by two-dimensional NMR techniques. Sequence-specific assignments were made for the amino acid residues from Ile-6 to Cys-72. Distance geometry analysis of the interresidue NOEs revealed that the HAIM molecule consists of two beta-sheets, as is the case in a homologous alpha-amylase inhibitor, Tendamistat, though one of its beta-strands is much shorter than that of Tendamistat. The combination of molecular modeling from Tendamistat and distance geometry analysis was confirmed to be useful for our purpose.

This study aimed to determine whether a shark muscle oil-olive oil mixture influences activators of human angiogenesis. The mixture completely abolished the stimulation induced by vascular endothelial growth factor (VEGF), fibroblast growth factor-2, transforming growth factor-beta, and platelet-derived growth factor. This suggests that it may compete with these growth factors for their binding sites on the endothelial cell surface either by binding to the growth factor or by blocking the actual receptor. The possibility of the oil binding to the VEGF receptor was studied through the use of soluble forms of the receptors (VEGF-R1 and VEGF-R2). It was found that the shark oil-olive oil inhibited the formation of the complexes of VEGF with both of the receptors. This could have been because the oil bound to either the VEGF or the receptor or both. To determine which is possible, the shark oil-olive oil was mixed with the receptors. The molecular size of the receptors increased, and these larger forms of the receptor had reduced capacity for complexing with VEGF. Therefore, one mode of potential anti-angiogenic action of the shark muscle oil-olive oil is the inhibition of the activity of a number of stimulatory molecules, including VEGF. This study demonstrates that the blend of shark and olive oils antagonizes VEGF activity by binding to at least two receptors for the factor, thereby inhibiting the activation by the growth factor.

The structure of the C-terminal antifreeze-like (AFL) domain of human sialic acid synthase was determined by NMR spectroscopy. The structure comprises one alpha- and two single-turn 3(10)-helices and two beta-strands, and is similar to those of the type III antifreeze proteins. Evolutionary trace analyses of the type III antifreeze protein family suggested that the class-specific residues in the human and bacterial AFL domains are important for their substrate binding, while the class-specific residues of the fish antifreeze proteins are gathered on the ice-binding surface.

An array of 50 MHz quartz microbalances (QMBs) coated with a dendronizedpolymer was used to detect small amounts of volatile organic compounds (VOCs) in the gasphase. The results were compared to those obtained with the commonly used 10 MHzQMBs. The 50 MHz QMBs proved to be a powerful tool for the detection of VOCs in thegas phase, therefore, they represent a promising alternative to the much more delicatesurface acoustic wave devices (SAWs).

Lymphostin (LK6-A), a novel immunosuppressant, was isolated from the culture broth of Streptomyces sp. KYI 1783. Lymphostin showed potent inhibitory activity against the lymphocyte kinase, Lck (p56lck). The structure of lymphostin was determined by spectroscopic methods to be a novel tricyclic aromatic alkaloid. A key feature of the structure is the pyrrolo[4, 3, 2-de]quinoline moiety which is rare among natural products.

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.