Photochemical transformation of 20-hydroxyecdysone: production of monomeric and dimeric ecdysteroid analogues.

Structural modification of 20-hydroxyecdysone (20E) based on photochemical transformation yielded dimeric ecdysteroid 7alphaH,7'alphaH-bis-[(20R,22R)-2beta,3beta,20,22,25-pentahydroxy-5beta-cholest-8(14)-en-6-one-7-yl] as a main product. Its structure was determined by detailed NMR analysis. Furthermore, two new monomeric analogues: 14-epi-20-hydroxyecdysone and 14-deoxy-14,18-cyclo-20-hydroxyecdysone were identified in addition to the earlier described 14-deoxy and 14-hydroperoxy derivatives of 20E. Formation of the specific and so far unique ecdysteroid dimer has not been observed in earlier photo-transformation studies. The transformed dimeric analogue of 20-hydroxyecdysone retained the high agonistic activity on the ecdysone receptor in the B(II)-bioassay compared with the original 20E.     

Synthesis of OSW-1 analogues and a dimer and their antitumor activities

Five analogues, including a 16-epi-isomer (6), and a 3-terephthalic acid linked dimer (8) of OSW-1 were synthesized. Their inhibitory activities on P388 and A-549 cells were detected.     

Different specific activities of the monomeric and oligomeric forms of plasmid DNA in transformation of B. subtilis and E. coli.

Summary (1) The low residual transforming activity in preparations of monomeric, supercoiled, circular (CCC) forms of the plasmids pC194 and pHV14 could be attributed to the presence in such isolates of a small number of contaminating multimeric molecules. (2) E. coli derived preparations of pHV14, an in vitro recombinant plasmid capable of replication in both E. coli and B. subtilis, contain oligomeric forms of plasmid DNA in addition to the prevalent monomeric CCC form. The specific transforming activity of pHV14 DNA for E. coli is independent of the degree of oligomerization, whereas in transformation of B. subtilis the specific activity of the purified monomeric CCC molecules is at least four orders of magnitude less than that of the unfractionated preparation. (3) Oligomerization of linearized pHV14 DNA by T4 ligase results in a substantial increase of specific transforming activity when assayed with B. subtilis and causes a decrease when used to transform E. coli.     

Biological activities of analogues of lipid A based chemically on the revised structural model. Comparison of mediator-inducing, immunomodulating and endotoxic activities

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (beta-1,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amide-bound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.     

Regulation of the actin-activated ATPase and in vitro motility activities of monomeric and filamentous Acanthamoeba myosin II.

The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba myosin II is inhibited by phosphorylation of 3 serine residues at the tip of the tail of each heavy chain. From previous studies, it had been concluded that the activity of each molecule in the filament was regulated by the global state of phosphorylation of the filament and was independent of its own phosphorylation state. The actin-activated Mg(2+)-ATPase activity of monomeric phosphorylated myosin II was not known because it polymerizes under the ionic conditions necessary for the expression of this activity. We have now found conditions to maintain myosin II monomeric and active during the enzyme assay. The actin-activated Mg(2+)-ATPase activities of monomeric dephosphorylated and phosphorylated myosin II were found to be the same as the activity of filamentous dephosphorylated myosin II. These results support the conclusion that phosphorylation regulates filamentous myosin II by affecting filament conformation. Consistent with their equivalent enzymatic activities, monomeric and filamentous dephosphorylated myosin II were equally active in an in vitro motility assay in which myosin adsorbed to a surface drives the movement of F-actin. In contrast to their very different enzymatic activities, however, filamentous and monomeric phosphorylated myosin II had similar activities in the in vitro motility assay; both were much less active than monomeric and filamentous dephosphorylated myosin II. One interpretation of these results is that the rate-limiting steps in the two assays are different and that, while the rate-limiting step for actin-activated Mg(2+)-ATPase activity is regulated only at the level of the filament, the rate-limiting step for motility can also be regulated at the level of the monomer.     

Biological activities of Synanceja horrida (stonefish) venom.

Some biological and neurochemical properties of the venom of stonefish (Syanceja horrida) were investigated. The venom exhibited oedema-inducing, haemolytic, hyaluronidase, thrombin-like, alkaline phosphomonoesterase, 5' nucleotidase, acetylcholinesterase, phosphodiesterase, arginine esterase, and arginine amidase activities. Recalcification clotting time, prothrombin, and kaolin-cephalin clotting times were increased 1.7-2.3- and 2.4-fold respectively. The LD50 (i.v. mouse) was 300 micrograms/Kg. Its effects on uptake and stimulation of neurotransmitter synthesis and release were observed in rat brain synaptosomes. In the presence of 100 micrograms venom, uptake of [methyl-3H] choline in rat brain synaptosomes was inhibited 70%, while that of 4-amino-n-[U-14C] butyric acid was inhibited 20%. The toxin also stimulated the release of [3H]-acetylcholine from the synaptosomes.     

Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo

Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala(2), its clinical application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogues. In this study, we investigated GLP-1 analogues including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala(2)]GLP-1 and [Gly(2)]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biological effectiveness of [D-Ala(2)]GLP-1 was also observed in diabetic db/db mice. Similarly, improved biological activity of acetyl- and hexenoic-His(1)-GLP-1, glucagon((1-5)-, glucagon((1-10))-, PACAP(1-5)-, VIP(1-5)-, and secretin((1-10))-GLP-1 was observed, despite normal or lower receptor binding and activation in vitro. [Ala(8/11/12/16)] substitutions also increased biological activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biological activity. All other modified peptides examined showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the alpha-amino group of His(1) and replacement of Ala(2) result in significantly increased biological effects of GLP-1 in vivo, likely due to decreased degradation rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biological activity.     

Biological activities of new monohydroxylated brassinosteroid analogues with a carboxylic group in the side chain

Thirteen monohydroxylated brassinosteroids analogues were synthesized and tested for their biological activity in plant and animal systems. The cytotoxic activity of the products was studied using human normal and cancer cell lines with 28-homocastasterone as positive control, their brassinolide type activity was established using the bean second-internode test with 24-epibrassinolide as standard.     

Synthetic peptides related to the dermorphins. II. Synthesis and biological activities of new analogues

A new series of analogues of the potent opiate-like peptides dermorphins (mainly tetra- and pentapeptides) were synthesized in order to better evaluate the structure-activity relationships. Relative potencies were referred to dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂), the prototype of this class of frog skin peptides. Peripheral opioid activity (guinea pig ileum and mouse vas deferens) was determined for all the dermorphin analogues. For a selected number of them also central analgesic (hot plate and tail-flick tests) and cataleptic activities were assayed in the rat by intracerebroventricular administration.     

Short-chain oligopeptides with copper(II) binding properties: The impact of specific structural modifications on the copper(II) coordination abilities

A series of linear tetrapeptides containing two histidyl residues in position 2 and 4, namely DHGH, DHGdH, KHGH, KHGdH, Ac-DHGH-NH₂, Ac-DHGdH-NH₂, Ac-KHGH-NH₂, and Ac-KHGdH-NH₂, were synthesized and characterised. Their copper(II) binding properties were investigated in depth through a variety of physicochemical methods. Potentiometric titrations were first carried out to establish the stoichiometry and the stability of the resulting copper(II)-peptide complexes. The copper(II) chromophores that are formed in the various cases in dependence of pH were subsequently characterised by extensive spectroscopic analysis (UV-Vis, EPR, CD) in strict correlation with potentiometric data. The effects of the nature of the first amino acid (Lys versus Asp) and of N-terminal amino group protection on copper(II) binding were specifically addressed. On turn, the careful comparison of the copper(II) coordination abilities of the linear peptides with those of their cyclic analogs provided insight into the effects of cyclization on the overall metal binding properties.     

Biological and enzymatic activities of Micrurus sp. (Coral) snake venoms

The venoms of Micrurus lemniscatus carvalhoi, Micrurus frontalis frontalis, Micrurus surinamensis surinamensis and Micrurus nigrocinctus nigrocinctus were assayed for biological activities. Although showing similar liposome disrupting and myotoxic activities, M. frontalis frontalis and M. nigrocinctus nigrocinctus displayed higher anticoagulant and phospholipase A2 (PLA2) activities. The latter induced a higher edema response within 30 min. Both venoms were the most toxic as well. In the isolated chick biventer cervicis preparation, M. lemniscatus carvalhoi venom blocked the indirectly elicited twitch-tension response (85+/-0.6% inhibition after a 15 min incubation at 5 microg of venom/mL) and the response to acetylcholine (ACh; 55 or 110 microM), without affecting the response to KCl (13.4 mM). In mouse phrenic nerve-diaphragm preparation, the venom (5 microg/mL) produced a complete inhibition of the indirectly elicited contractile response after 50 min incubation and did not affect the contractions elicited by direct stimulation. M. lemniscatus carvalhoi inhibited 3H-L-glutamate uptake in brain synaptosomes in a Ca2+-, but not time, dependent manner. The replacement of Ca2+ by Sr2+ and ethylene glycol-bis(beta-aminoethyl ether) (EGTA), or alkylation of the venom with p-bromophenacyl bromide (BPB), inhibited 3H-L-glutamate uptake. M. lemniscatus carvalhoi venom cross-reacted with postsynaptic alpha-neurotoxins short-chain (antineurotoxin-II) and long-chain (antibungarotoxin) antibodies. It also cross-reacted with antimyotoxic PLA2 antibodies from M. nigrocinctus nigrocinctus (antinigroxin). Our results point to the need of catalytic activity for these venoms to exert their neurotoxic activity efficiently and to their components as attractive tools for the study of molecular targets on cell membranes.     

Biological activities of synthetic analogues of Alternaria alternata toxin (AAL-toxin) and fumonisin in plant and mammalian cell cultures

In a search for an analogue of AAL-toxin with high phytotoxicity and low mammalian toxicity, aminopentols [(AP₁), hexacetyl AP₁ and N-acetyl AP₁], and nine analogues (1–9), were tested for toxicity to duckweed (Lemna pausicostata), susceptible tomato (asc/asc) leaf discs, black nightshade leaf discs and mammalian cell lines, including dog kidney (MDCK), rat liver hepatoma (H4TG) and mouse fibroblasts (NIH3T3). These were compared with AAL-toxin and fumonisin B₁ (FB₁). Analogue 9 at 10 μM increased cellular leakage and chlorophyll loss from both tomato and black nightshade leaf discs. The diester 9 was the most active in the duckweed bioassay, but it was much less toxic to MDCK and H4TG cells with an IC₅₀ of 200 μM compared to 10 μM for FB₁. Analogue 9 and FB₁ showed similar low toxicities (IC₅₀ = 150 μM) to NIH3T3 cells. Among the substances tested, only analogue 9 had significant phytotoxicity and low mammalian toxicity, indicating some potential for development of safe and effective natural herbicides.     

Trinuclear copper(II) complexes of bis(acylhydrazone) ligands. Structural analysis and magnetic properties of a sulfato-bridged hexanuclear dimer

The synthesis and spectroscopic characterization of the new symmetric, ligand bis(salicylaldehyde)methyliminodiacetylhydrazone (H₄MeImds) is reported. The reactions of H₄ MeImds with copper(II) chloride, nitrate and sulfate give trinuclear metal complexes of formula Cu₃(MeImds)Cl₂ · CHCl₃ · H₂O, Cu₃(MeImds)(NO₃)₂ · 2H₂O and Cu₃(MeImds)(SO₄) · 8H₂O; the trinuclear species are obtained in spite of the used metal:ligand molar ratio. The X-ray crystal structure of [Cu₃(MeImds)(SO₄)(H₂O)₃] · 5H₂O is determined; it consists of linear units, with the nonadentate ligand bound to three copper ions. The trimetallic units are bridged via sulfate anions to give hexanuclear dimers with an imposed centre of symmetry. The magnetic properties of [Cu₃(MeImds)(SO₄)(H₂O)₃] · 5H₂O are characterized by a significant antiferromagnetic coupling within the trimetallic units and by a weak antiferromagnetic coupling between them. The analogous trinuclear copper(II) complexes Cu₃(HImds)Cl₂ · H₂O and Cu₃(HImds)(SO₄) · 4H₂O (H₅Imds, bis(salicylaldehyde)iminodiacetylhydrazone) were also prepared and spectroscopically characterized.     

Biological activity of trisporoids and trisporoid analogues in Mucor mucedo (-)

In the course of their sexual interactions, zygomycete fungi communicate via an elaborate series of carotene-derived compounds, namely trisporic acid and its biosynthetic progenitors. A novel building-block strategy allowed the systematic generation of structurally modified trisporoids along with putative early biosynthetic precursors for physiological tests. The impact of discrete structural elements was documented by the ability of individual compounds to induce sexually committed hyphae in Mucor mucedo. The activity screening contributed to establish general structure-function relationships for trisporoid action. Most crucial for activity were the dimension of the longer side chain, the polarity of functional groups at C(4) and C(13), and the number of conjugated double bonds in the side chain. The presence of an oxygen substituent at the cyclohexene ring is not essential for function. The overall biological activity apparently results from the combination of the various structural elements.     

The monomeric arrangement in the dimer of Escherichia coli RNA polymerase holoenzyme studied with (scanning) transmission electron microscopy

A model is presented for the arrangement of the monomers in the dimer of Escherichia coli RNA polymerase holoenzyme. The dimers, formed at low salt concentration in the presence of magnesium ions, were negatively stained and photographed in TEM and STEM. The profiles of the monomers forming the dimers were classified. In 61% (134) of the dimers, both monomeric profiles could be classified. We determined the regions on the monomer which interact in the dimer, and the frequencies with which the various possible contacts between regions occur. We conclude that the binding site on the monomer is constituted by one of the α subunits and the β subunit, and that the association between the monomers is isologous.In the absence of Mg²⁺, we found that the holoenzyme exists mainly as a monomer at low salt concentration. The differences in the degree of oligomerization of RNA polymerase at low salt concentrations are discussed in view of our model.