Search

If not fertilized, synchronous apoptosis is induced in starfish eggs at approximately 11h after stimulation with the hormone, 1-methyladenine. In this study, a membrane-impermeant substrate of caspase-3, acetyl-Asp-Glu-Val-Asp-coumarylamido-4-methanesulfonic acid (Ac-DEVD-CAMS), was synthesized and microinjected into a starfish egg. Caspase-3 activity in unfertilized egg was detected approximately 30min before blebbing by quantifying the accumulation rate of a membrane-impermeant, fluorogenic product, 7-aminocoumarin-4-methanesulfonic acid (ACMS), using a photomultiplier mounted on a fluorescence microscope. When active recombinant human caspase-3 was microinjected into an egg at 3h after 1-methyladenine treatment, the injected caspase-3 activity decreased and disappeared within 2h. This decrease is probably due to proteasome-dependent degradation of the enzyme, since the injected caspase-3 was degraded and a proteasome inhibitor blocked its degradation. In contrast, in aged eggs at approximately 10h after 1-methyladenine treatment, no degradation of the injected caspase-3 was observed, suggesting that endogenous caspase-3 may stabilize at this point, therefore, inducing apoptosis.

The syntheses of novel rigid dendritic ligands and their ruthenium complexes, in which 1,3,5-phenylene-based hydrocarbon dendron units were directly connected to a phenanthroline ligand at the 3 and 8 positions, are described. Preliminary absorption and fluorescence results are indicative of highly efficient energy transfer within the rigid metallodendrimers.

The phosphoenolpyruvate carboxylase (PEPC) isozyme involved in C4 photosynthesis is known to undergo reversible regulatory phosphorylation under illuminated conditions, thereby decreasing the enzyme's sensitivity to its feedback inhibitor, L-malate. For the direct assay of this phosphorylation in intact maize leaves, phosphorylation state-specific antibodies to the C4-form PEPC were prepared. The antibodies were raised in rabbits against a synthetic phosphorylated 15-mer peptide with a sequence corresponding to that flanking the specific site of regulatory phosphorylation (Ser15) and subsequently purified by affinity-chromatography. Specificity of the resulting antibodies to the C4-form PEPC phosphorylated at Ser15 was established on the basis of several criteria. The antibodies did not react with the recombinant root-form of maize PEPC phosphorylated in vitro. By the use of these antibodies, the changes in PEPC phosphorylation state were semi-quantitatively monitored under several physiological conditions. When the changes in PEPC phosphorylation were monitored during the entire day with mature (13-week-old) maize plants grown in the field, phosphorylation started before dawn, reached a maximum by mid-morning, and then decreased before sunset. At midnight dephosphorylation was almost complete. The results suggest that the regulatory phosphorylation of C4-form PEPC in mature maize plants is controlled not only by a light signal but also by some other metabolic signal(s). Under nitrogen-limited conditions the phosphorylation was enhanced even though the level of PEPC protein was decreased. Thus there seems to be some compensatory regulatory mechanism for the phosphorylation.

The syntheses of aureobasidin A (AbA) derivatives with alkyl chains and their in vitro structure-biological activity relationships are discussed. The analogs replaced at positions 6, 7, or 8 of AbA with either L-glutamic acid, delta-hydroxy-L-norvaline, or delta-hydroxy-N-methyl-L-norvaline are prepared. The gamma-carboxyl or delta-hydroxyl group of these new amino acids was coupled with acids, alcohols, or amines with alkyl chains. While the analogs having L-glutamic acid residue at positions 6 or 8 showed weak activity, esterification of the gamma-carboxyl group with benzyl or shorter alkyl (C4 or C6) alcohols, significantly enhanced the activities. Introduction of longer alkyl (C14) chain to the same amino acids residues at positions 6, 7, or 8 resulted in total loss of antifungal activity. Among the lipophilic analogs in [L-Glu6] derivatives, the C6 alcohol ester showed the strongest antifungal activity against Candida spp. so far tested. None of the derivatives showed activity against Cryptococcus neoformans.

The first total synthesis of antifungal cyclic depsipeptide aureobasidin A is described. The synthesis was achieved mainly using bromotris(pyrrolidino)phosphonium hexafluorophosphate (PyBroP) as a coupling reagent. Peptide cyclization was carried out between L- allo -isoleucine (L- a lle 1 ) and L- Pro 9 residues in the linear nonapeptide at the final step of the synthesis. Synthesized aureobasidin A was completely identical with the natural antibiotic with respect to antifungal activity and physicochemical properties. Unusual reactions due to N -methylamino acid, an oxazoline-mediated reaction and an N , O -acyl migration, are also described.

A monooleoyl glycerophospho heptapeptide was synthesized as a candidate of the endogenous pathogen of essential hypertension. Synthesis of the target compound was achieved by the fragment condensation of the phosphorylated tetrapeptide with a His-containing tripeptide.

A chemical synthesis was performed of an octapeptide with a phosphonic ester linkage, which corresponds to a variation of the partial sequence of a gene product of erb B-2. The phosphonic ester linkage was successfully prepared through a coupling reaction of magnesium salt of the hydroxypeptide with phosphonochloridate, without damage of the peptide bond. The free aminophosphonic peptide as the final product gave a single peak on the ODS column, and a reasonable ratio of the component amino acids. The reaction series exploited here might be useful as a general procedure for preparation of the phosphonic peptide.

A main component of ganglioside M5 from egg of the sea urchin, Anthocidaris crassispina, Neu5Gcα2 → 6Glcβ1 → 1Cer (1), which has N-glycolyl group in neuraminic acid part, as well as phytosphingosine and saturated nonhydroxy fatty acid in ceramide part was first synthesized starting from N-acetylneuraminic acid.

A reactive ion etching (RIE) technique using a C 2 H 6 , H 2 , and O 2 mixture was applied to the fabrication of InGaAsP/InP P-substrate partially inverted buried heterostructure laser diodes, which have been commercially produced for their superior characteristics. The addition of O 2 suppressed side etching and made it possible to fabricate ridge mesa structures for the laser diodes with a height of 4 μm and a width of 1μm with superior controllability. The effects of O 2 addition were investigated by Auger electron spectroscopy and a mechanism suppressing side etching was examined

A spider toxin NSTX-3 obtained from the venom of Papua New Guinean spider, Nephila maculata was synthesized in order to confirm its proposed structure and to supply the sample for the biological tests. The structure of NSTX-3 is now established as 2,4-dihydroxyphenylacetyl-L-Asn→Cad←Pua←L-Arg, where Cad stands for cadaverine and Pua for putreanine.

The isomers of sialic acid with 6-acetylamino group, 1a and 1b were synthesized from furanose 3a and 3b respectively obtained by aldol condensation of D -glucose with oxalacetic acid.

A main component of ganglioside M5 from egg of the sea urchin, Anthocidaris crassispina, Neu5Gcα2 → 6Glcβ1 → 1Cer (1), which has N-glycolyl group in neuraminic acid part, as well as phytosphingosine and saturated nonhydroxy fatty acid in ceramide part was first synthesized starting from N-acetylneuraminic acid.

Chemistry-based investigation is reviewed which led to identification of the active entities responsible for the immunostimulating potencies of peptidoglycan and lipopolysaccharide. Though these glycoconjugates which ubiquitously occur in wide range of bacteria as the essential components of their cell envelopes have long been known to enhance the immunological responses of higher animals, neither the precise chemical structures required nor the mechanism of their action had been [corrected] elucidated until early 1970s. Chemical synthesis of partial structures of peptidoglycan proved N-acetylmuramyl-L-alanyl-D-isoglutamine to be the minimum structure responsible for the activity and led to later identification of its receptor protein Nod2 present in animal cells. Another active partial structure of peptidoglycan, gamma-D-glutamyl-meso-diaminopimelic acid, and its receptor Nod1 were also identified as well. With regard to lipopolysaccharide, its glycolipid part named lipid A was purified and the structure studied. Chemically synthesized lipid A according to the newly elucidated structure exhibited full activity described for lipopolysaccharide known as endotoxin. Synthetic homogeneous lipid A and its structural analogues and labeled derivatives enabled precise studies of their interaction with receptor proteins and the mechanism of their action. Chemical synthesis of homogeneous partial structures of peptidoglycan and lipopolysaccharide gave unequivocal evidences for the concept that definite small molecular parts of these complex macromolecular bacterial glycoconjugates are specifically recognized by their respective receptors and trigger our defense system now widely recognized as innate immunity.

Reactive ion etching of InP using a C2H6/H2/O2 mixture was investigated. The effects of oxygen gas added to a C2H6/H2 gas were systematically investigated. A very smooth etched surface was obtained by the addition of O2, even when the etching depth was 3.5 μm. Mass spectrum of the gases in the etching chamber was also investigated during etching. It was found that the O2 gas reduced the formation of hydrocarbon polymers in the etching process.

The isomers of sialic acid with 6-acetylamino group, 1a and 1b were synthesized from furanose 3a and 3b respectively obtained by aldol condensation of D -glucose with oxalacetic acid.

Total synthesis of a lanthionine peptide nisin was achieved by the successive condensations of four segments including cyclic lanthionine peptide parts and the C-terminal linear segment including dehydroalanine, followed by the deprotection procedure with anhydrous HF. Modified reaction conditions for the Hofmann degradation were applied to prepare the dehydroalanine residue from the 2,3-diaminopropionic acid residue. The final deprotection with anhydrous HF proceeded without decomposition of the dehydroamino acid residues which are generally labile in acidic media. The synthetic nisin was completely identical with the natural one in all respects, resulting in a confirmation of the proposed structure.

N-Acetylneuramimic acid (sialic acid) 1 was synthesized through relatively short steps from d -glucose via a key intermediate 1,5-lactone derivative obtained from the pyranose isomer of the aldol condensation products of d -glucose with oxalacetic acid.

In this paper, we compared the chemical and biological activities of natural and synthetic clavamines, N-[2, 4-dihydroxyphenylacetyl-4-L-asparaginyl)-N'-[N-(L-arginyl-glycyl-L-alanyl)-8-amino-4-azaoctanoyl]-1, 5-pentane-diamine (DPA→Asn→Cad←Ptr←Ala←Gly←Arg)[DPA, 2, 4-dihydroxyphenylacetic acid; Cad, 1.5-pentanediamine (cadaverine); Ptr, 8-amino-4-azaoctanoic acid (putreanine, or carboxyethylputrescine)], and found that both were identical. Both compounds showed the same Rf value in thin-layer chromatography and the same retention time in high performance liquid chromatography (HPLC). Both hydrolyzates consisted of the same components, such as Gly, DPA, Ala, Asp, Arg and Ptr in gas chromatography. The Edman degradation of both compounds also gave exactly the same sequence. The 1H-NMR spectrum and fast atom bombardment (FAB) mass spectrum of the synthetic compound coincided with those of the natural one. Both were active on the insects used. Clavamine was the main component of the insecticidal activity in the crude venom. By this synthetic study, the structure of a new venom insecticide was thus established. The synthetic compound in abundant quantity will be advantageous for practical use as an insecticide over natural ones, because the latter is available only in small amounts. Also, it will be useful for the exploitation of a new field of biochemistry for polyamines.

Nephritogenoside (1), proposed as a nephritogenic triglycosylhenicosapeptide with asparagine N-α-glycoside linkage, was first synthesized by the coupling of triglycosyldipeptide active ester with the nonadecapeptide (3–21) using allyloxycarbonyl group as the final protecting group. A β-anomeric glycopeptide was also prepared.

A spider toxin NSTX-3 obtained from the venom of Papua New Guinean spider, Nephila maculata was synthesized in order to confirm its proposed structure and to supply the sample for the biological tests. The structure of NSTX-3 is now established as 2,4-dihydroxyphenylacetyl-L-Asn→Cad←Pua←L-Arg, where Cad stands for cadaverine and Pua for putreanine.

The structure of ancovenin, a new peptide inhibitor of angiotensin I converting enzyme was determined chemically. Ancovenin is composed of 16 amino acid residues including three lanthionine residues which form a complex tricyclic structure bridged by sulfide bonds. Moreover, this peptide includes dehydroalanine as one of its unusual amino acids. In the present study, plausible mechanisms of the Edman degradation of dehydroalanine and lanthionine-containing peptides were also elucidated.

N-Acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]) injected intraperitoneally significantly increased the number of cells entering the peritoneal cavity of guinea pigs primed with liquid paraffin or thioglycollate. There was a close relationship between peritoneal polymorphonuclear leukocyte (PMN) accumulation and the uptake of glucosamine by macrophages in guinea pigs treated with a variety of bacterial cell surface components such as cell wall peptidoglycan subunits and bacterial or synthetic lipid A. The PMN accumulation was also facilitated by the intraperitoneal transfer of the peritoneal macrophages that had been stimulated by MDP in vitro. Furthermore, cell-free lavage fluids taken from the peritoneum of MDP-treated guinea pigs also initiated the influx of PMNs when introduced into the peritoneal cavities of liquid paraffin-pretreated guinea pigs. These results suggest that a soluble factor which attracts neutrophils is produced by MDP-treated macrophages. Partial characterization of the factor is described.

A lysophosphatidylserine with 19:4 acyl group proposed as a candidate of DLIS-2, an endogenous digoxin-like substance was chemically synthesized. The synthetic compound showed a significant activity of Na + ,K + -ATPase inhibition, which has been accepted as a major indicator activity for the essential hypertension.

The ability of synthetic lipid A analogs to induce prostaglandin synthesis in macrophages was compared with that of native lipopolysaccharide. The synthetic preparations comprised monomeric or dimeric derivatives of D-glucosamine with different patterns of substitution by phosphate and tetradecanoic, (R)-3-hydroxytetradecanoic, and (R)-3-tetradecanoyloxytetradecanoic acid. All of these preparations are structurally distinct from native lipid A (principally regarding the position of fatty acid substitution) and hence have been previously shown to be endotoxically inactive in many biological tests. It was found that many of these synthetic samples exhibit strong activity in inducing prostaglandin E2 and prostaglandin F2 alpha, with some of them having activity comparable to that of lipopolysaccharide. Experiments with macrophages of C3H/HeJ mice enabled us to differentiate between endotoxin-specific (in the case of dimeric preparations) and endotoxin-nonspecific (for monomeric preparations) mechanisms for the induction of prostaglandins. These results indicate that there is a difference in the mechanism of induction of prostaglandin synthesis between monomeric lipid A's and dimeric or native lipid A structures.

In a study on structure-activity relationship of spider toxin NSTX-3 obtained from venom of Nephila maculata, nine analogs or fragments of natural toxin were synthesized. Among them, des-Arg- NSTX-3 was found to be the minimum structure for blocking the excitatory transmission in the crustacean neuromuscular synapse. A positive charge of the arginine part was concluded to play the most important role for exhibition of the activity.

Clavamine, a new insecticidal toxin isolated from the venom of Joro spider (Nephila clavata) was totally synthesized in order to confirm the proposed structure. In clavamine, a fragment of glycylalanine is inserted in the structure of NSTX-3, a New Guinean spider toxin. The synthesis determined the chemical structure of clavamine unambiguously.

A series of 1,3,5-phenylene-based rigid dendritic porphyrins were synthesized by Suzuki coupling between a porphyrin core and dendron units. The intramolecular energy transfer was studied by absorption and fluorescence spectroscopies. The encapsulation of the porphyrin core within the 1,3,5-phenylene dendron units was found to provide highly efficient energy transfer from the dendron units to the porphyrin core. The dendritic wedge structure affected the energy transfer efficiency. The 1,3,5-phenylene-based rigid dendron units act as highly efficient light-harvesting antennae. These dendritic porphyrins have also been examined as C(60) hosts and substrate-selective oxidation catalysts. The attachment of the second generation of 1,3,5-phenylene-based dendron units with the porphyrin core enabled a stable inclusion of C(60) in toluene. Furthermore, the size and shape of the nanospace in the rigid dendritic porphyrins were found to affect the selectivity of substrates in the catalytic olefin oxidations.

Ganglioside M5 (NeuGcalpha2-6Glcbeta1-1'Cer), the main ganglioside in sea urchin and sand dollar eggs, exists mainly in the endoplasmic reticulum and yolk granules in unfertilized eggs. To study the localization of ganglioside M5 after fertilization, early embryos were stained with an anti-ganglioside M5 monoclonal antibody. Using immunofluorescent and immunoelectron microscopy, intense label was observed in the outer surface and cytoplasm of embryos. These results indicate that ganglioside M5 was secreted during embryogenesis and localized in the extracellular matrix (ECM). When living embryos were incubated in sea water containing 7-nitrobenz-2-oxa-1,3-diazole labeled-ganglioside M5 (NBD-M5), the ECM and plasma membrane were strongly stained. Since the localization of NBD-M5 in the ECM was similar to that of extracellular M5, NBD-M5 was likely to be useful to examine the fate of extracellular ganglioside M5. Interestingly, NBD-M5 was incorporated in subcortical vesicles during embryogenesis, suggesting that the extracellular ganglioside M5 is transported into the cytoplasm. When fertilized eggs were incubated with NBD-M5 and tetramethylrhodamine dextran (a marker dye for endocytotic vesicles), colocalization of the dyes was observed in the vesicles. Thus, it was concluded that NBD-M5 in the ECM and/or plasma membrane was internalized in the cells by endocytosis, suggesting that extracellular M5 is transported from the ECM to endocytotic vesicles.

Activity of phosphoeno/pyruvate carboxylase (PEPC) for C4 photosynthesis is upregulated by light (reviewed in [1, 2]). The regulation is mediated by a specific protein kinase (PEPC-PK) that phosphorylates Ser near N-terminus (Ser-15 in the maize Ca-form PEPC). This phosphorylation is dependent on light intensity in Ca-plants [3]. Hartwell e al [4] reported that PEPC-PK was regulated by the level of translatable mRNA. No evidence, however, has been obtained to indicate how PEPC-PKs or its mRNAs are regulated and there has been no report about the gene for PEPC-PK.

GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha1--6fucosyltransferase (alpha1-6FucT; EC 2.4.1.68), which catalyzes the transfer of fucose from GDP-Fuc to N-linked type complex glycopeptides, was purified from a Triton X-100 extract of porcine brain microsomes. The purification procedures included sequential affinity chromatographies on GlcNAcbeta1-2Manalpha1-6(GlcNAcbeta1-2Manalpha1- 2)Manbeta1-4GlcNAcbet a1-4GlcNAc-Asn-Sepharose 4B and synthetic GDP-hexanolamine-Sepharose 4B columns. The enzyme was recovered in a 12% final yield with a 440, 000-fold increase in specific activity. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a major band corresponding to an apparent molecular mass of 58 kDa. The alpha1-6FucT has 575 amino acids and no putative N-glycosylation sites. The cDNA was cloned in to pSVK3 and was then transiently transfected into COS-1 cells. alpha1-6FucT activity was found to be high in the transfected cells, as compared with non- or mock-transfected cells. Northern blotting analyses of rat adult tissues showed that alpha1-6FucT was highly expressed in brain. No sequence homology was found with other previously cloned fucosyltransferases, but the enzyme appears to be a type II transmembrane protein like the other glycosyltransferases.

An assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase (alpha 1-6FucT; EC 2.4.1.68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine gamma-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. [equation: see text] The substrate and guanosine diphospho-fucopyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HPLC. Quantitation of the product involved measurement of the fluorescence intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGnF-bi-Asn-PABA (product) were analyzed by 1H NMR. The enzymatic product was also analyzed by 1H NMR and was found to have alpha 1-6fucose at the reducing end GlcNAc. This method is highly specific for alpha 1-6FucT and is applicable for various experiments, including purification and cell culture ones.

A protein having a high-affinity binding site for [3H]mepyramine (MBP) was purified to homogeneity from rat liver membranes. The purified MBP has a single type of binding site for [3H]mepyramine with Kd value of 18.5 nM, and its molecular weight was determined to be 56,000 by SDS polyacrylamide gel electrophoresis. Amino acid sequences of twelve tryptic peptides derived from MBP are highly homologous with those of rat debrisoquine 4-hydroxylase (cytochrome P450 2D1) and other rat P450 2D subfamily members. In immunoblotting analysis, an antibody against rat P450 2D1 stained a band corresponding to MBP with Mr of 56,000; its migration position was clearly different from that of rat P450 2D1. Substrates and inhibitors of debrisoquine 4-hydroxylase potently displace [3H]-mepyramine binding to MBP. Quinine and quinidine showed 400 and 80 times, respectively, higher affinity for MBP than for debrisoquine 4-hydroxylase. These results suggest that MBP is a novel P450 2D family member.