Synthesis and biological activity of adipokinetic hormone analogues with modifications in the 4-8 region

Several structural characteristics in the molecule of the locust adipokinetic hormone, AKH-I, have been investigated in terms of their importance in determining biologic activity. All modifications tested in this study resulted in analogues with decreased potency in comparison with the parent molecule. However, all analogues that were found to be active gave a full response, although often only at very high doses of peptide. This study has highlighted for the locust receptor(s) the vital role of the side chain of Thr(5), and the importance of positions 4 and 8. For example, when Trp(8) and Phe(4) were exchanged, the resulting analogue (Trp(4),Phe(8)-AKH-I) was one of the least active analogues tested in this study. Although Trp is tolerated quite well as a substitute for Phe(4), with only a 10-fold loss of potency, Phe is not favored as a substitute for Trp(8) (>300 times decrease in potency). On the other hand, 3-[2-napthyl] alanine (Nal) is a better substitute for Trp(8) (only a 100-fold loss in potency). We conclude that position 4 requires a phenyl ring in the side chain, and position 8 an indole ring.     

Molecular requirements for abscisic acid activity in two bioassay systems

Summary Twenty-two analogues of ABA have been tested in the Avena coleoptile and lettuce germination bioassays. Ten of these analogues were considerably more active than ABA itself as inhibitors of lettuce germination, but in the Avena coleoptile assay their activity never significantly exceeded that of ABA. The molecular requirements for activity also differ in the two assay systems, although the presence of the ring double bond is a requirement of both.     

Role of spore coats in the germinative response of Bacillus cereus to adenosine and its analogues.

Spores of the strain NCIB 8122 of Bacillus cereus have been depleted of coats by treatment with 0.1% sodium dodecyl sulfate--200 mM 2-mercaptoethanol--0.5 M NaCl (pH 9.6). The coat-depleted spores did not show any decrease in viability, heat resistance, refractility, dipicolinic acid content, or specific activities of several protoplastic enzymes. The germinative response of the coat-depleted spores to adenosine and several analogues thereof was found qualitatively similar to that obtained with intact spores. However, germination kinetics appeared to be affected by coat removal, since germination rate measured as loss of refractility was eight times slower even at inducer concentrations 10-fold higher than those required to promote optimal germination response of intact spores. Loss of heat resistance, on the other hand, was hardly affected by coat removal. These results suggest that, even though spore coats are not essential for the triggering reaction, they are required for a rapid evolution of the later events in the germination process.     

Germination of Bacillus cereus spores induced by purine ribosides and their analogs: effects of modification of base and sugar moieties of purine nucleosides on germination-inducing activity

Purine riboside and some of its analogs were tested for their ability to induce germination of Bacillus cereus T spores. Hypoxanthine and adenine showed no germination-inducing activity either in the present or absence of D-ribose or its phosphorylated derivatives. Purine riboside and 18 analogs with modified purine base were all able to induce germination of the spores to various extents. In contrast to this, the requirement for the sugar moiety in the purine riboside appeared to be more stringent. Only those nucleosides that contained either D-ribose or deoxy-D-ribose, and certain species of azole derivatives such as 5-aminoimidazole-4-carboxamide covalently linked to the C(1') of the sugar actively induced germination.     

Heat resistance, spore germination, and enterotoxigenicity of Clostridium perfringens

Heat resistance at 95 C, heat activation at 75 C, and germination response were determined for spores of 10 serotype strains of Clostridium perfringens type A, including five heat-resistant and five heat-sensitive strains. The D95-values ranged from 17.6 to 63.0 and from 1.3 to 2.8 for the heat-resistant and the heat-sensitive strains, respectively. The heat-activation values, the ratios between the heated and unheated viable counts of spore suspensions, ranged from 0.0035 to 0.65 and from 6.5 to 60.0 for the heat-sensitive and the heat-resistant strains, respectively. Spores of these strains were divided into two distinct germination types on the basis of their germination response; spores of the heat-resistant strains germinated in KC1 medium after heat activation (K-type), and spores of the heat-sensitive strains germinated in a mixture of L-alanine, inosine, and CaCl2 in the presence of CO2 without heat activation (A-type). The strains were tested for enterotoxigenicity by a reversed passive latex-agglutination (RPLA) test. All the heat-resistant strains were RPLA-positive, whereas the heat-sensitive strains were all RPLA-negative. A total of 37 strains of the organism isolated from food-poisoning outbreaks were tested for spore germination and enterotoxin formation. All of the 20 heat-resistant strains showed K-type spore germination and, except for three strains, were RPLA-positive, whereas all of the 17 heat-sensitive strains showed A-type spore germination and, except for only one strain, were RPLA-negative.     

Investigations on fungicides

A series of pyrazole derivatives, which are structural analogues of the systemic fungicide, carboxin (5,6-dihydro-2-methyl-i,4-oxathiin-3-carboxani-lide), have been synthesized and their antifungal properties investigated. 3,5-dimethylpyrazole-i-carboxanilides, although active in vitro and in leaf disk tests, showed no systemic antifungal activity. Certain 3,5-dimethylpyra-zole-4-carboxanilides, however, and their corresponding 1 -methyl derivatives, showed good activity in spore germination tests and high activity against wheat and broad bean rusts in vivo. In several instances, systemic antifungal activity was of the same order as that of carboxin, although generally accompanied by higher levels of phytotoxicity. 1 -Phenyl derivatives were essentially inactive. Substitution in the anilide ring by 3-methyl, 2-methyl or 3-chloro groups resulted in enhanced systemic activity, while 4-chloro, 4-ethoxy, 2-nitro and 3,4-dichloro substituents reduced activity.     

Mutagenicity of plant flavonoids: structural requirements for mutagenic activity in Salmonella typhimurium.

40 compounds structurally related to the plant flavonol quercetin were tested for mutagenic activity in Salmonella typhimurium strain TA98. 10 flavonols, quercetin, myricetin, rhamnetin, galangin, kaempferol, tamarixetin, morin, 3'-O-methylquercetin, 7,4'-di-O-methylquercetin and 5,7-di-O-methyl-quercetin, exhibited unequivocal mutagenic activity. 4 compounds, quercetin, myricetin, rhamnetin and 5,7-di-O-methylquercetin, were active without metabolic activation, although metabolic activation markedly enhanced their activity. All 4 have free hydroxyl groups at the 3' and 4' positions of the B ring. The other active compounds required an in vitro rat-liver metabolizing system for significant activity. Structural features which appear essential for mutagenic activity in this strain are a basic flavanoid ring structure with (1) a free hydroxyl group at the 3 position, (2) a double bond at the 2, 3 position, (3) a keto group at the 4 position, and (4) a structure which permits the proton of the 3-hydroxyl group to tautomerise to a 3-keto compound. The data are consistent with the requirement for a B ring structure that permits oxidation to quininoid intermediates. Free hydroxyl groups in the B ring are not essential for activity if a rat-liver metabolic activating system is employed. Data from 12 compounds which differ only at the essential sites described above indicate that the structural requirements for mutagenicity in strain TA100 are the same as those for activity in strain TA98. Based on the above structural requirements, a metabolic pathway for flavonol activation to DNA-reactive species is proposed.     

A transition state in pieces: major contributions of entropic effects to ligand binding by adenosine deaminase.

Nebularine undergoes hydration at the active site of adenosine deaminase, in a reaction analogous to a partial reaction in the displacement of ammonia from adenosine by water, to generate an inhibitory complex that captures much of the binding affinity expected of an ideal transition-state analogue. Enzyme affinities of several compounds related to nebularine 1,6-hydrate, and to its stable analog 2'-deoxycoformycin, were compared in an effort to identify the structural origins of strong binding. Binding of the stable transition-state analog inhibitor 2'-deoxycoformycin was rendered 9.8 kcal/mol less favorable by removal of substituent ribose, 9.7 kcal/mol less favorable by inversion of the 8-hydroxyl substituent of the diazepine ring, and 10.0 kcal/mol less favorable by removal of atoms 4-6 of the diazepine ring. Binding of the unstable transition-state analog nebularine hydrate was rendered at least 9.9 kcal/mol less favorable by removal of the 6-hydroxyl group and 10.2 kcal/mol less favorable by removal of atoms 1-3 of the pyrimidine ring. In each case, the enzyme exhibited only modest affinity (Kd greater than or equal to 10(-2) M) for the "missing piece", indicating that incorporation of 2 binding determinants within a single molecule permits an additional 7-12 kcal/mol of intrinsic binding energy to be manifested as observed binding energy. These results are consistent with earlier indications that adenosine deaminase may use 10.5 kcal/mol of the intrinsic free energy of binding of the two substrates to place them in positions appropriate for reaction at the active site, overcoming the unfavorable entropy change of -35 eu for the equilibrium of 1,6-hydration of purine ribonucleoside and reducing the equilibrium constant for attainment of the transition state in deamination of adenosine. Thus, adenosine deaminase may achieve up to 8 orders of magnitude of its catalytic power by converting the nonenzymatic, bimolecular, hydration reaction to a monomolecular reaction at its active site. Several new 6-substituted 1,6-dihydropurine ribonucleosides, prepared by photoaddition of formate and by low-temperature addition of organolithium reagents to a derivative of purine ribonucleoside, exhibited Ki values of 9-1400 microM against adenosine deaminase, in accord with the active site's considerable tolerance of bulky leaving groups in substrates. Inhibition by one diastereomer of 6-carboxy-1,6-dihydropurine ribonucleoside was found to be time-dependent, progressing from a weakly bound to a more strongly bound complex.     

Antimycobacterial and H1-antihistaminic activity of 2-substituted piperidine derivatives

2-Substituted derivatives of the antihistaminic agents Bamipine, Diphenylpyraline and of their 1-phenyl analogues were tested for their antimycobacterial and H(1)-antagonistic activities. They are strong H1-receptor antagonists and also inhibit the growth of mycobacterials with a maximum MIC of 6.25 microg/mL against Mycobacterium tuberculosis H(37)Rv. H1-receptor antagonistic potency was slightly decreased by substitution in ring position 2 and distinctly diminished by N-aryl substitution. The antimycobacterial potency of Diphenylpyraline was in general increased by substitution in ring position 2, whereas only a few Bamipine derivatives showed markedly improved activity. A correlation between the two activities was not detected for those compounds.     

The Presence of Low Molecular Weight Germinative Substances in Bacillus cereus T Spore Extract

An extract from intact spores of Bacillus cereus T having a germination-inducing activity was studied. Two distinct germinative principles were found through dialysis of the extract. One was diffusible through the dialysis membrane and the other was non-diffusible. The activity of the former fraction was inhibited by the addition of 1 mM glycoletherdiamine-N, N, N', N'-tetraacetic acid (GEDTA), whereas the latter fraction was inactive unless GEDTA was added to the assay system. The diffusible principle maintained the major portion of the activity found in the crude spore extract. By means of high-performance liquid chromatography (HPLC) using a gel permeation chromatography column, 9 fractions were obtained from the deproteinized diffusible fraction. Of those fractions, two fractions (No. 1 and No. 8) were responsible for the germination-inducing activity, but no reconstituted activity was observed unless both fractions No. 1 and No. 8 were added to the assay system. Amino acid analysis of fraction No. 1 revealed that the fraction was rich in free amino acids, especially in alanine. On the other hand, by the use of reverse-phase HPLC and fast atom bombardment mass spectrometry, it was concluded that the effective substance in fraction No. 8 was inosine. Based on these findings, it was suggested that the active substances in fraction No. 1 might be a free amino acid such as L-alanine and/or Ca²⁺ and a Ca²⁺-binding substance.     

Yeast spore germination: a requirement for Ras protein activity during re-entry into the cell cycle.

Saccharomyces cerevisiae spore germination is a process in which quiescent, non-dividing spores become competent for mitotic cell division. Using a novel assay for spore uncoating, we found that spore germination was a multi-step process whose nutritional requirements differed from those for mitotic division. Although both processes were controlled by nutrient availability, efficient spore germination occurred in conditions that did not support cell division. In addition, germination did not require many key regulators of cell cycle progression including the cyclin-dependent kinase, Cdc28p. However, two processes essential for cell growth, protein synthesis and signaling through the Ras protein pathway, were required for spore germination. Moreover, increasing Ras protein activity in spores resulted in an accelerated rate of germination and suggested that activation of the Ras pathway was rate-limiting for entry into the germination program. An early step in germination, commitment, was identified as the point at which spores became irreversibly destined to complete the uncoating process even if the original stimulus for germination was removed. Spore commitment to germination required protein synthesis and Ras protein activity; in contrast, post-commitment events did not require ongoing protein synthesis. Altogether, these data suggested a model for Ras function during transitions between periods of quiescence and cell cycle progression.     

Quantitative assessment of mRNA cap analogues as inhibitors of in vitro translation.

Fifty-eight analogues of the 5'-terminal 7-methylguanosine-containing cap of eukaryotic messenger RNA were synthesized and tested for their ability to inhibit in vitro protein synthesis. A new algorithm was developed for extracting KI, the dissociation constant for the cap analogue.eIF4E complex, from protein synthesis data. The results indicated that addition of a methyl group to the N2 of guanine produced more inhibitory compounds, but addition of a second methyl group to N2 decreased the level of inhibition dramatically. Aryl substitution at N7 improved the efficacy of guanine nucleoside monophosphate analogues. Substitution of the aromatic ring at the para position with methyl or NO2 groups abolished this effect, but substitution with Cl or F enhanced it. By contrast, aryl substitution at N7 in nucleoside di- or triphosphate analogues produced only minor effects, both positive and negative. By far the strongest determinants of inhibitory activity for cap analogues were phosphate residues. The beneficial effect of more phosphate residues was related more to anionic charge than to the number of phosphate groups per se. The second nucleotide residue in analogues of the form m7GpppN affected inhibitory activity in the order G > C > U > A, but there was no effect of 2'-O-modification. Opening the first ribose ring of m7GpppG analogues dramatically decreased activity, but alterations at the 2'-position of this ribose had no effect. Non-nucleotide-based cap analogues containing benzimidazole derivatives were inhibitory, though less so than those containing 7-methylguanine.     

Purification, properties, and function of flavokinase from rat intestinal mucosa

Flavokinase (ATP:riboflavin 5'-phosphotransferase) [EC 2.7.1.26] was purified to apparent homogeneity from rat intestinal mucosa by fractionation with ammonium sulfate, gel filtration, and flavin affinity chromatography. The addition of ATP to the enzyme solution was necessary for its binding to the affinity gel. The apparent molecular weight of the enzyme was estimated to be 13,500 by gel filtration on Sephadex G-100 and by SDS-PAGE. The properties of the enzyme, including its flavin specificity, were studied. Three types of riboflavin analogues were used for the flavin specificity study; namely, ones modified at the ribityl group, and at positions 3 and 8 of the isoalloxazine ring. Of the analogues modified at the ribityl group or position 3 of the isoalloxazine ring, only 2'-deoxyriboflavin was phosphorylated and then only weakly. On the other hand, most analogues modified at position 8 of the isoalloxazine ring were good substrates for the kinase, an appropriate increase in the substituent volume at position 8 of the isoalloxazine ring resulting in an increase in the Vmax value. In a previous paper on the mechanism of intestinal absorption of riboflavin, we proposed that one of the specific processes for the absorption of riboflavin is phosphorylation by flavokinase [Kasai, S. et al. (1988) J. Nutr. Sci. Vitaminol. 34, 265-280]. The present results support this conclusion because analogues that were absorbed at low concentrations through a process specific for riboflavin in our previous study were phosphorylated effectively by the enzyme, whereas those that were absorbed solely through simple diffusion at all concentrations were not phosphorylated or only phosphorylated weakly. The properties of the flavokinases from intestinal mucosa and liver were compared.     

Spore fitness components do not differ between diploid and allotetraploid species of Dryopteris (Dryopteridaceae)

BACKGROUND AND AIMS: Although allopolyploidy is a prevalent speciation mechanism in plants, its adaptive consequences are poorly understood. In addition, the effects of allopolyploidy per se (i.e. hybridization and chromosome doubling) can be confounded with those of subsequent evolutionary divergence between allopolyploids and related diploids. This report assesses whether fern species with the same ploidy level or the same altitudinal distribution have similar germination responses to temperature. The effects of polyploidy on spore abortion and spore size are also investigated, since both traits may have adaptive consequences. METHODS: Three allotetraploid (Dryopteris corleyi, D. filix-mas and D. guanchica) and three related diploid taxa (D. aemula, D. affinis ssp. affinis and D. oreades) were studied. Spores were collected from 24 populations in northern Spain. Four spore traits were determined: abortion percentage, size, germination time and germination percentage. Six incubation temperatures were tested: 8, 15, 20, 25 and 32 degrees C, and alternating 8/15 degrees C. KEY RESULTS: Allotetraploids had bigger spores than diploid progenitors, whereas spore abortion percentages were generally similar. Germination times decreased with increasing temperatures in a wide range of temperatures (8-25 degrees C), although final germination percentages were similar among species irrespective of their ploidy level. Only at low temperature (8 degrees C) did two allotetraploid species reach higher germination percentages than diploid parents. Allotetraploids showed faster germination rates, which would probably give them a competitive advantage over diploid parents. Germination behaviour was not correlated with altitudinal distribution of species. CONCLUSIONS: The results of this study suggest that (i) relative fitness of allopolyploids at sporogenesis does not differ from that of diploid parents and (ii) neither does allopolyploidization involve a change in the success of spore germination.     

Properties of substituted 2-trifluoromethylbenzimidazoles as uncouplers of oxidative phosphorylation

1. The activity of 25 substituted 2-trifluoromethylbenzimidazoles in uncoupling oxidative phosphorylation by rat-liver mitochondria has been compared. 2. For halogen- or mixed-halogen- and alkyl-substituted analogues, uncoupling activity was proportional to the acidity of the imidazole -NH group. Tetrachloro-2-trifluoromethylbenzimidazole was the most active (50% uncoupling of oxidative phosphorylation at 7.9x10(-8)m, pK5.04). Nitro-substituted analogues were less active than predicted from pK considerations or from partition-coefficient measurements. 3. Introduction of an -NH(2) or -CO(2)H substitutent caused a loss of uncoupling activity, as did alkylation at position 1 of the imidazole ring. 4. Benzimidazoles active as uncouplers stimulated mitochondrial adenosine triphosphatase but not all stimulated the oxidation of succinate in the absence of a phosphate acceptor. 5. 4,5-Dichloro-2-trifluoromethylbenzimidazole inhibited the succinate-oxidase system at about the same concentration required for uncoupling (0.52mum for 50% inhibition of both activities) and the site of this inhibition appears to lie between succinate dehydrogenase and cytochrome b.     

The rate constant describing slow-onset inhibition of yeast AMP deaminase by coformycin analogues is independent of inhibitor structure

(R)- and (S)-2'-deoxycoformycin, (R)-coformycin, and the corresponding 5'-monophosphates were compared as inhibitors of yeast AMP deaminase. The overall inhibition constants ranged from 4.2 mM for (S)-2'-deoxycoformycin to 10 pM for (R)-coformycin 5'-monophosphate, a difference of 3.8 x 10(8) in affinities. (R)-Coformycin, (R)-2'-deoxycoformycin 5'-monophosphate, and (R)-coformycin 5'-monophosphate exhibited both rapid and slow-onset inhibition. The S inhibitors and (R)-2'-deoxycoformycin exhibited classical competitive inhibition but no time-dependent onset of inhibition. The results indicate that the presence of the 2'-hydroxyl and 5'-phosphate and the R stereochemistry at the C-8 position of the diazepine ring are necessary for the optimum interaction of inhibitors with yeast AMP deaminase. This differs from the results for rabbit muscle AMP deaminase [Frieden C., Kurz, L. C., & Gilbert, H. R. (1980) Biochemistry 19, 5303-5309] and calf intestinal adenosine deaminase [Schramm, V. L., & Baker, D. C. (1985) Biochemistry 24, 641-646], in which a tetrahedral hydroxyl at C-8 in the R stereochemistry is sufficient for slow-onset inhibition with the coformycins. The results suggest that the transition state contains a tetrahedral carbon with the R configuration as a result of the direct attack of an oxygen nucleophile at C-6 of AMP. Slow-onset inhibition of yeast AMP deaminase is consistent with the mechanism [formula: see text] in which the combination of E and I is rapidly reversible. For these inhibitors, Ki varied by a factor of 3 x 10(3), and the overall inhibition constant (Ki*) varied by a factor of 2 x 10(5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of C-5'-nor-dideoxycarbanucleosides structurally related to neplanocin C

Purine carbanucleosides built on a 6-oxabicyclo[3.1.0]hexane template were synthesized from readily available 2-cyclopentenone employing a Mitsunobu reaction to incorporate the base onto the carbocyclic ring. Both adenosine and guanosine analogues exhibited moderate antiviral activity.     

Activation and germination of Bacillus thuringiensis spores in Manduca sexta larval gut fluid

Incubation of Bacillus thuringiensis HD-1 spores in the larval gut fluid of Manduca sexta (tobacco hornworm) resulted in increased viable counts, conversion to phase-dark spores, and a loss of absorbance in spore suspensions, indicative of spore germination. Heat-activated and untreated spores incubated in water did not exhibit these changes. Only when spores were heat activated and incubated in germinants L-alanine and adenosine did changes in the spores approximate those observed in gut fluid. These data suggest that M. sexta larval gut fluid induces the activation and germination of B. thuringiensis spores.     

Inhibition by adenosine of reactive oxygen metabolite production by human polymorphonuclear leucocytes.

The stimulation of reactive oxygen metabolite production from human polymorphonuclear leucocytes by chemotactic peptide (fMet-Leu-Phe) was inhibited by adenosine with a K0.5 of 0.6 microM. Dipyridamole (0.1 microM), an inhibitor of adenosine uptake, did not prevent the effect of adenosine. Non-metabolizable analogues could substitute for adenosine in the potency order N-ethoxycarboxamideadenosine greater than 2-chloroadenosine greater than adenosine greater than L-N6-(phenylisopropyl)adenosine = D-N6-(phenylisopropyl)adenosine, which is characteristic of an A2 adenosine receptor. The effects of adenosine, 2-chloroadenosine and N-ethoxycarboxamideadenosine were reversed by 8-phenyltheophylline. When endocytosis was inhibited with cytochalasin B, cells were still susceptible to adenosine receptor agonists. 2-Chloroadenosine (10 microM) reduced the activation of respiration in response to chemotactic peptide from 3.3-fold to 1.4-fold. Activation of reactive oxygen metabolite production in response to latex beads was not reversed by adenosine or its analogues. It was concluded that adenosine acts at an A2 adenosine receptor to antagonize the activation of polymorphonuclear leucocytes by those stimuli, such as chemotactic peptide, which cause an increase in the intracellular free Ca2+ concentration.     

Trans-N-deoxyribosylase: substrate specificity studies. Purine bases as acceptors.

A series of purine bases and analogues were tested as substrates for trans-N-deoxyribosylase (EC 2.4.2.6). It was observed that the pyrimidine ring and its substituents on positions 1, 2 and 6, are of minor importance. On the other hand only a few modifications are tolerated on the imidazole moiety, as follows. 1. A tautomeric proton must be present on the imidazole ring. The "usual" shift is between position 9 and 7. 2. The position of the tautomeric proton governs the site of substitution. 3. For steric reasons no substituent is allowed on position 8.