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 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.

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.

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.

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.

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.

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.

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.

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.

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.

A chromogenic substrate 1, 5-bromo-4-chloroindol-3-yl 5-acetamido-3,5-dideoxy-alpha-D-glycero-D-galacto-2-nonulopyranosidon ic acid (X-Neu5Ac), has been synthesized to facilitate the screening of bacterial colonies or plaques for the detection of either natural or mutant neuraminidase activity. Substrate 1 was hydrolyzed by neuraminidase isolated from Clostridium perfringens to release a halogenated indol-3-ol 2 that undergoes rapid aerobic oxidation to form the dark blue pigment, 5,5'-dibromo-4,-4'-dichloroindigo 3. Preliminary kinetic studies indicate that this compound is a good substrate (Km 0.89 x 10(-3) M) for neuraminidase and is quite stable under identical conditions in the absence of enzyme. These results suggest that X-Neu5Ac 1 can be useful to screen for bacterially-encoded enzyme production directly on agar plates.

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.

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.

A group of spider toxins (JSTX, NSTX, argiopin, argiotoxin etc.) share a basic common structure and have been reported to block strongly quisqualate- and kainate-sensitive glutamate responses in vertebrate and invertebrate nervous systems. They are presumed to be potent antagonists of both quisqualate and kainate receptors and may serve as useful tools for characterizing these receptors. We report here the synthesis of tritium-labeled NSTX-3 and the characterization of its binding sites in the rat brain. We found that high- and low-affinity binding sites exist in the cerebellum (Kd = 7.75 and 202 nM, Bmax = 0.37 and 5.54 pmol/mg protein, respectively). Synthetic NSTX analogs strongly inhibited [3H]NSTX-3 binding in the cerebellum (IC50 = 10(-7)-10(-6) M), whereas competitive agonists of glutamate receptors (AMPA, quisqualate, NMDA, kainate, glutamate and aspartate) exhibited weak or no inhibitory effects.

N-Acetylmuramyl-L-alanyl-D-isoglutamine corresponding to the minimum structure responsible for the immunoadjuvant activity of bacterial cell walls was acylated at the 6-hydroxyl group with various mycolic acids of long carbon chains in order to test their possible antitumor effects. Mycolic acids used were extracted from cell walls of Mycobacterium, Nocardia, and Corynebacterium. All the mycoloyl derivatives synthesized show antitumor activity based on a principle of immunotherapy.

A synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), failed to produce polyarthritis with a wide dose range in a water-in-oil emulsion of mineral oil such as liquid paraffin, Drakeol, or heavy mineral oil. MDP, however, produced moderate to severe arthritis with almost 100% incidence in a water-in-oil emulsion made up of Difco incomplete adjuvant, which consists of Bavol F as an oil vehicle and Arlacel A as an emulsifier. N-acetylmuramyl-L-alanyl-L-isoglutamine did not produce arthritis, whereas 4,6-diacetyl-MDP produced the disease. Bacterial peptidoglycans, such as disaccharide peptides which were N-acetylglucosaminyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine and N-acetylglucosaminyl-6,o-acetyl-N-acetylmuramyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelyl-D-alanine, also produced polyarthritis with low incidence in Difco oil but not in the other mineral oils described above. MDP and bacterial disaccharide peptides were able to produce the disease even in the latter mineral oil only when the concentration of Arlacel A was increased from 15% to 20 to 30% in the oil. We concluded that one of the minimal essential structures responsible for development of this disease is MDP, although the role of the oil vehicle remained uncertain, and there is no direct correlation between granulona formation and arthritogenicity of MDP.

To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-(2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O [(R)3-tetradecanoyloxytetradecanoyl]-beta-D-glucopyranosyl]-3-O-[(R) -3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-alpha -D-glucopyranose 1,4'-bis(phosphate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.

The effects of polyclonal B cell activation (PBA) of cell walls and their cell wall fractions obtained from several kinds of gram-positive bacteria were studied using the anti-sheep red blood cell (SRBC) or anti-trinitrophenylated (TNP) SRBC plaque forming cell (PFC) responses of cultured spleen cells from Balb/c, athymic nu/nu, their littermates (nu/+), C3H/He (LPS-responder), C3H/HeJ (LPS-non-responder), (CBA/N × Balb/c) F1 male with an X-linked defect in B cell function and the F1 female mice. The cell walls of Staphylococcus epidermidis (ATCC 155), Lactobacillus plantarum (ATCC 8014), Micrococcus lysodeikticus (NCTC 2665), Mycobacterium rhodochrous (ATCC 184), Streptomyces gardneri (ATCC 23911) and Nocardia corynebacteriodes (ATCC 14898) had the ability to induce polyclonal B cell responses in the spleen cells of Balb/c, nu/nu, nu/+, C3H/He and C3H/HeJ mice. The cell wall fractions prepared by enzymatic digestion from the cell walls of S. epidermidis, S. gardneri or N. corynebacteriodes were also capable of inducing polyclonal B cell responses. The responses of spleen cells from (CBA/N × Balb/c) F1 male mice to these active preparations, except the cell walls of M. rhodochrous, were much lower than those of the F1 female mice. These findings indicate that the majority of the cell wall preparations lacks PBA ability for spleen cells with the CBA/N defect, except for the cell walls of M. rhodochrous which possess this ability. The PBA-ability of synthetic peptidoglycan, muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine, MDP), was also examined, and a similar activity was observed in MDP.

Thirteen 6-O-acyl-N-acetylmuramyl-L-alanyl-D-isoglutamines (6-O-acyl-MDPs), including four inactive D-isoasparagine and L-isoglutamine analogs, were tested for their pyrogenicity and immunopotentiating activity to stimulate primary humoral and cellular immune responses in guinea pigs to a model protein antigen, ovalbumin, when administered in various vehicles. Among them, derivatives whose muramic acid residue was substituted by alpha-branched (and beta-hydroxylated) higher fatty acids at the carbon-6 position, especially 6-O-(2-tetradecylhexadecanoyl)-MDP (B3O-MDP) and, to a lesser extent, 6-O-(3-hydroxy-2-docosylhexacosanoyl)-MDP (BH48-MDP) and its L-serine analog [BH48-MDP(L-Ser)], were found to exert strong adjuvant activity in both the induction of delayed-type hypersensitivity and the stimulation of circulating precipitating antibody levels when combined with nonirritating vehicles (liposomes, squalene-in-water emulsion, and phosphate-buffered saline). These vehicles did not efficiently support the adjuvant activity of MDP, the parent molecule of the above lipophilic derivatives. Pyrogenicity tests showed that introduction of alpha-branched higher fatty acid groups but not of straight, long-chain fatty acids at the 6-position of the muramic acid residue resulted in marked decrease of the pyrogenicity inherent to MDP via intravenous administration.

Re-mutant lipopolysaccharide (Re LPS) of Salmonella minnesota profoundly inhibited the growth of murine B-cell lines, designated CYG34 and CYG101, established from long term bone-marrow culture. When CYG101 cells were cultured together with Re LPS at concentrations of 2-100μg/ml, the cell proliferation was dose-dependently suppressed. Re LPS hardly or slightly affected the growth of a murine T-cell line, BW5147, pre-B cell lines, 70Z/3 and CYG 8, and a myeloma cell line, X63Ag8. S-form LPS of Escherichia coli and S. typhimurium, Re-mutant LPS of S. minnesota and natural lipid A of E. coil, S. typhimurium and S. minnesota also inhibited the growth of CYG101 cells. However, degraded polysaccharide of S-form LPS of S. typhimurium did not inhibit the cell growth. Synthetic compounds 406, corresponding to a biosynthetic disaccharide lipid A precursor, and 506, corresponding to lipid A of the E. coli type, also inhibited the growth of CYG101 cells. These results suggest that the growth inhibition of cell lines by LPS is selective or specific to the cell line and that the lipid A portion of LPS is involved in this growth inhibition. Double acyl groups of lipid A may not play any role in the inhibition of cell growth.

Silkworm, Bombyx mori , produces self-denfense substance lepidopteran in a haemolymph when it is vaccinated with killed Escherichia coli . Lepidopteran family contains at least three congeners A, B and C, all of which were isolated and determined in amino acid sequences. Furthermore, total synthesis of lepidopteran A was achieved. For elucidation of structure-activity relationship of lepidopteran, twenty one different kinds of peptide fragments were then synthesized. From results of antibacterial tests for those peptides, it is concluded that N-terminal region particularly the Arg -Trp2 sequence is very important for exhibition of the biological activity. The minimal sequence of lepidopteran providing significant bioactivity is the peptide fragment (1–18). α-Helix content in the peptide chain does not necessarily guarantee a manifestation of the activity. An important factor to strengthen the activity seems to be a hydrophobicity in the molecule in addition to the N-terminal region. The assumption was confirmed by preparation of long aliphatic amide of the fragment (1–18) which showed almost comparable activity to that of the original lepidopteran.

Chemical syntheses are described of polyacylated β(1→6) glucosamine disaccharide 1,4′-bis(phosphate), which corresponds to the proposed structure of E. coli lipid A, and of its dephospho derivatives. The synthetic bisphosphate proved to be identical with the corresponding natural specimen. The chemical structure of lipid A was thus established.

Two antigens, cholera toxin (CT) and a synthetic albumin-conjugated 16-residue peptide derived from the circumsporozoite (CS) protein of Plasmodium falciparum sporozoites, were tested as immunogens in rabbits. The malaria peptide-albumin conjugate by itself was completely nonimmunogenic, and although cholera toxin was immunogenic it also expressed considerable native toxicity. After attachment of CT to liposomes containing ganglioside GM1, toxicity of CT was completely eliminated and antigenicity was enhanced. Therefore liposomes may be capable of reducing toxicity of certain potentially dangerous antigens such as toxins. After incorporation of the malaria peptide-albumin conjugate into liposomes a high titre of specific antibodies was induced against the malaria peptide but not against albumin. These antibodies also reacted with native CS protein. Three adjuvants, including lipid A and two types of lipophilic muramyl dipeptide, were compared and found to be effective in liposomes. Based on the conversion of synthetic P. falciparum CS peptide from a nonimmunogenic to an immunogenic form and on the 'toxoiding' effect of liposomes for CT, it is concluded that liposomes should be considered as being a useful carrier for antigens and adjuvants for vaccines for poorly antigenic or toxic substances.

The biological activities of synthetic glycolipids, which were chemically synthesized and based on the structure of lipid A of the lipopolysaccharide (LPS) from Escherichia coli, were examined with special reference to their adjuvant activity on the induction of delayed-type hypersensitivity, activity on the induction of tumour necrotic factor (TNF) and tumour regressive activity on line 10 hepatoma in strain 2 guinea pigs. Among them, a compound structurally corresponding to free E. coli lipid A (compound 506) as well as LPS exhibited potent adjuvant activity in the induction of delayed-type hypersensitivity in guinea pigs and TNF inducing activity in the sera of mice which were presensitized with Propionibacterium acnes. Compound 506 showed potent lethal toxicity in the intravenous administration of BALB/c mice presensitized with P. acnes. The regressive activity on line 10 hepatoma was observed by the multiple intralesional injection of squalane-treated compounds 504 and 505 in strain 2 guinea pigs.

A pentaacyl precursor of lipid A biosynthesis, termed precursor Ib, and a structural isomer have been chemically synthesized. These compounds were, in comparison to synthetic Escherichia-coli type lipid A or lipopolysaccharide, analyzed for their activity in typical endotoxin test systems. It was found that both precursor Ib and the isomer exhibited similar or only slightly lower pyrogenic, lethal and Shwartzman-phenomenon-inducing activity than lipid A. All preparations were comparable in their B-lymphocyte mitogenicity, macrophage-activating capacity and immunoreactivity towards lipid A antisera. The proton nuclear magnetic resonance spectra of the 1-dephospho derivative of synthetic and bacterial precursor Ib were indistinguishable proving that the previously proposed structure for precursor Ib is correct.

Two new ureido amino acids, lividine ( 1 ) and grateloupine ( 2 ), were isolated from Grateloupia livida and Grateloupia filicina respectively. The structures of both amino acids were assumed mainly by NMR spectra and determined as N w -carbamoyl-L-citrulline for lividine and as N -carbamoyl- Y -aminobutyric acid for grateloupine by comparison with the synthetic compounds respectively.

The peptide N -acetylmuramyl- l -alanyl- d -isoglutamine (MDP), which has adjuvant activities, and 17 of its derivatives and analogs were synthesized and assayed to elucidate the structure necessary for adjuvant activity in induction of experimental allergic encephalomyelitis (EAE) in guinea pigs. The results revealed the importance of the d configuration and the α-carboxamide group of the isoglutaminyl residue of MDP for adjuvant activity. Replacement of the l -alanyl residue of MDP by d -alanine, but not by l -serine or glycine, resulted in a marked decrease in the activity. The β-methyl glycoside of MDP was found to be more active than the α-methyl derivative. 6- O -Stearoyl- N -acetylmuramyl- l -alanyl- d -isoglutamme showed activity.

Synthetic and biological studies continued in our laboratories con- tributed to elucidate the chemical structure and biological functions of en- dotoxin, which is lipopolysaccharide (LPS) located at the cell surface of gram-negative bacteria. In this paper the effect of structural variation on the biological activities is discussed of lipophilic part of LPS. The recent result of our synthetic approach toward the most simple LPS isolated and characterized from Escherichia goJ Re mutant cells is also described. ~~~~~~ O-hOH)r

Thirty two semisynthetic tuberactinomycins were prepared by introduction of various amino acids to amino group of tuberactinamine N, a cyclic peptide moiety of tuberactinomycin N and O, which was isolated from natural tuberactinomycin N by acid treatment with liberation of γ-hydroxy-β-lysine of the branched part. Among introduced amino acids, β-amino acids were synthesized from corresponding α-amino acids by modified Arndt-Eistert reaction. After couplings of N-protected amino acids with tuberactinamine N, deprotections were carried out to give semisynthetic tuberactinomycins. From their minimum inhibitory concentrations against many bacteria, it was suggested that a branched part effects significantly to the strength of antimicrobial activities though most important active site must locate in the cyclic peptide moiety, and basicity and/or hydrophobicity of the branched part seemed to strengthen the antibacterial activity especially.

A new inhibitor of angiotensin I converting enzyme, I5B2, was isolated from the culture broth of Actinomadura sp. No. 937ZE-1. This compound contains N-methylvaline, tyrosine and 1-amino-2-(4-hydroxyphenyl)ethylphosphonic acid. The microorganism also produced another inhibitor, I5B1, which is identical with K-4 isolated from Actinomadura sp. as an antihypertensive agent.

Dityromycin is a peptide antibiotic isolated from the culture broth of the soil microorganism, Streptomyces sp. strain No. AM-2504, showing activities against Bacillus, Corynebacterium, and Clostridium. Dityromycin was clarified to be composed of N-MeVal, Pro, Val, Phenylglycine, CH3NH2, N,N-Me2Thr and di-α-amino acid containing diphenylether moiety. The peptide sequence except one labile amino acid residue was determined by partial hydrolysis, chemical cleavage, and the Edman degradation. The unknown amino acid was finally deduced to be the dehydroamino acid containing epoxide, resulting in an elucidation of whole structure of the antibiotic.

The binding of 14C-labelled tuberactinomycin O was analysed in equilibrium dialysis cells. The ionic conditions and the concentration of the labelled drug used in the binding assays allowed the binding of just one drug molecule per non-programmed ribosome. Under these conditions, the occupation of the ribosomal P-site by deacylated tRNAPhe in the presence of poly(U) increased the amount of [14C]tuberactinomycin O bound by a factor of two. Kanamycin, gentamicin and neomycin reduced the binding of tuberactinomycin O, whereas chloramphenicol, tetracycline, streptomycin and puromycin had no effect. A stimulation of the binding of tuberactinomycin O was found upon addition of erythromycin.

The structure of the oligosaccharide portion of an E.coli Re lipopolysaccharide was determined as α-KDO-(2→4)-α-KDO-(2→6)-β-GlcN-α(1→6)-α-GlcN, bisphosphorylated at positions 1 and 4′. Taking into account the previous determination of the acylation pattern of the GlcN disaccharide, the total structure of E.coli Re LPS was thus established.

A synthetic lipid A (preparation 516), containing seven acyl groups and representing one component of natural free lipid A of Salmonella minnesota R595, has been investigated for biological activity in a number of endotoxin test systems. It was found that the synthetic preparation was, in typical in vivo endotoxin tests (lethality, pyrogenicity, Shwartzman reactivity) as well as in its antigenicity and macrophage activation capacity, significantly less active than natural Salmonella lipid A. However, in other in vitro assay systems (B-cell mitogenicity, complement activation, Limulus amoebocyte lysate gelation) it expressed similar activity as Salmonella lipid A.

A synthetic muramyl dipeptide (MDP), which has the minimal adjuvant-active structure that can substitute for Mycobacterium in complete Freund's adjuvant, could stimulate peritoneal macrophages of the guinea pig to produce T-cell activating soluble factors. The culture supernatant of MDP-stimulated macrophages could help the proliferative response of the T-lymphocyte-enriched fraction of lymph nodes to PHA. It also helped the antigenic activation of immune T lymphocytes deprived of macrophages to produce macrophage migration inhibitory factor (MIF). Gel filtration of the culture supernatant showed that both of the helping activities were found mainly in the fraction emerging at the 53,000- to 85,000-MW range. A series of MDP analogs which are known to be adjuvant active or inactive was tested for their ability to stimulate macrophages to produce these factors. The adjuvant activity of these synthetic compounds was found to be parallel to the ability to stimulate the production of these T-cell activating monokines. Since both of these activities are highly dependent on the precise stereochemical structures of the MDP analogs, the parallelism of these activities would suggest the possibility that the immunopotentiating effect of the MDP and its analogs is related at least partially to their activity to stimulate macrophages to produce T-cell activating monokines.

Interferon-inducing, pyrogenic and proclotting enzyme of horseshoe crab activation activities of chemically synthesized lipid A analogues were investigated and compared with the same activities of a natural lipid A. These analogues are nonphosphorylated, C-1 or C-4′ monophosphorylated and C-1,4′ bisphosphorylated derivatives of β-1,6-linked d-glucosamine disaccharide possessing both ester-bound and amide-bound fatty acid substituents. Fatty acid substituents of the analogues are tetradecanoyl (C14), (R)-3-hydroxytetradecanoyl (C14-OH) or (R)-3-tetradecanoyloxytetradecanoyl [C14-O-(C14)] groups. The biological activities of the samples were assayed after solubilization with triethylamine and complexing with bovine serum albumin. Interferon-inducing activity was exhibited by both the C-1 monophosphorylated compounds examined. Ester-bound and amide-bound fatty acid substituents of these compounds are both C14 or C14 and C14-OH, respectively. Nonphosphorylated, C-4′ monophosphorylated and C-1,4′ bisphosphorylated compounds possessing the same fatty acid substituents as those of the C-1 monophosphorylated compounds showed no detectable interferon-inducing activity. C-4′ monophosphorylated compounds possessing C14-OH as ester-bound and C14-OH or C14-O-(C14) as amide-bound fatty acid substituents exhibited interferon-inducing activity, but nonphosphorylated compounds possessing the same fatty acid substituents did not. None of the analogues exhibited significant pyrogenicity nor proclotting enzyme of horseshoe crab activation activity under the conditions employed in this study.

Synthetic lipid A part structures corresponding structurally to a biosynthetic lipid A disaccharide precursor have been analyzed for endotoxic activity in several systems in vivo and in vitro. It was found that a synthetic beta-1,6-linked D-glucosamine disaccharide, which carries four molar equivalents of (R)-3-hydroxytetradecanoyl residues in positions 2, 3, 2' and 3' and phosphoryl groups in positions 1 and 4' (preparation 406), exhibited lethal toxicity, B lymphocyte mitogenicity, the capacity to engender prostaglandin formation in macrophages and to induce endotoxic tolerance, as well as serological lipid A antigenicity. On a weight basis, preparation 406 was of comparable activity to lipid A precursor and bacterial free lipid A. Preparation 406, like lipid A precursor, lacked, however, the ability to induce the local Shwartzman phenomenon and both preparations were of moderate pyrogenicity. Two further synthetic analogues which contained only one phosphoryl group (preparation 404 at C-4', preparation 405 at C-1) showed comparable or diminished activity depending on the test system employed, except in the capacity to inactivate complement where they exhibited, in contrast to preparation 406, significant activity. The results show that the endotoxic principle of lipopolysaccharides, as postulated previously is embedded in the lipid A component. Our results also suggest initial conclusions on the structural requirements for the expression of endotoxin activities.

Total syntheses of [Ala3, Ala4]-, [Ala3]-, and [Ala4]-tuberactinomycin O corresponding to deoxy analogs of natural antibiotic tuberactinomycin O were achieved for the purpose of elucidation of relationship between chemical structure and biological activity. Cyclization of the pentapeptide intermediates at two different positions was investigated. Minimum inhibitory concentrations of the synthetic analogs against Mycobacterium or Corynebacterium indicated that both hydroxy groups of two serine residues of tuberactinomycins are not required for antibacterial activity, nor participated to drug-resistancy of bacilli. These analogs were found to have the same conformations in solution as those of natural antibiotic from the results of NMR, ORD, and CD measurements.

Synthetic lipid A analogs (compounds 404 through 406) were examined for their immunopharmacological activities. These compounds had two amide-bound and two ester-bound (R)-3-hydroxytetradecanoyl groups at the C-2 and C-2' and the C-3 and C-3' positions, respectively, of beta (1-3)glucosamine disaccharide. In all of the in vitro assays, these synthetic compounds exhibited high activities comparable to those of a reference lipid A prepared from Escherichia coli O8:K27 Re-mutant strain F515. The compounds activated the clotting enzyme cascade of the horseshoe crab, activated the human complement via the classical pathway, caused polyclonal B-cell activation, stimulated the phagocytosis of sheep erythrocytes by murine peritoneal macrophages, and enhanced the migration of human polymorphonuclear leukocytes. They also increased the thymidine uptake of splenocytes of BALB/c nu/nu and C3H/HeN mice but not those of C3H/HeJ (a nonresponder to lipopolysaccharide). A dephosphorylated derivative, compound 403, was barely active in all of the above assays except for the enhancement of polymorphonuclear leukocyte migration. However, compounds 404 through 406 were feeble in pyrogenicity and could not prepare the local Shwartzman reaction, although they were very lethal to galactosamine-loaded mice. Therefore, synthetic lipid A analogs described here were fully immunopharmacologically active in in vitro assays, but all of them were far less active than natural E. coli F515 lipid A regarding the biological activities characteristic of endotoxic lipopolysaccharides and lipid A's. The high lethal toxicity of compound 406 (1,4'-bisphosphate) to the galactosamine-loaded mice may not reflect its real toxicity to normal mice. In all activities examined, compound 406 was quite comparable to a biosynthetic lipid A precursor, a natural counterpart of compound 406. The immunopharmacological activities of these newly synthesized lipid A analogs, especially compound 406, were much stronger than those of compounds that had been synthesized earlier by using the originally proposed model of the lipid A structure. The findings described in this report justify the acylation pattern of a disaccharide backbone of lipid A, revised on the basis of recent analytical studies. The low in vivo endotoxic activities of the present lipid A analogs are most probably due to the fact that the kinds of acyl groups were different from those of the complete lipid A from E. coli, although there were no differences in the acylation positions on the disaccharide backbone.

Various structural analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine, which had been shown to be the minimum structure required for the immunoadjuvant activity of bacterial cell walls, were synthesized in order to investigate in more detail the relationship between the activity and chemical structures. Particularly, the compounds of the structures corresponding to the cell walls of special bacteria without immunoadjuvant activity were prepared in this study. Several novel procedures including the preparation of a branched glutamyl peptide were newly exploited to accomplish the syntheses of muramyl peptides with complicated structures. As a result of this synthetic study, new informations were obtained on the structural requirement for the immunoadjucant activity.