Effects of nitrogen source on the synthesis of the UV-screening compound, scytonemin, in the cyanobacterium Nostoc punctiforme PCC 73102

The effects of nitrogen source (N(2), NO(3)(-) and NH(4)(+)) on scytonemin synthesis were investigated in the heterocystous cyanobacterium Nostoc punctiforme PCC 73102. With the required UVA radiation included, Nostoc synthesized three to seven times more scytonemin while fixing nitrogen than when utilizing nitrate or ammonium. A similar increase in scytonemin synthesis occurred when nitrate or ammonium became depleted by growth and Nostoc switched to diazotrophic metabolism with the differentiation of heterocysts. In addition, UVA-exposed cultures grown in medium with both NO(3)(-) and NH(4)(+) synthesized some scytonemin but synthesis increased when NH(4)(+) was depleted and growth had become dependent on NO(3)(-) reduction. Although the mechanism is unclear, these results suggest that the greater the restriction in nitrogen accessibility, the greater the production of scytonemin. Perhaps the entire response may be an interaction between this restriction and a resultant sensitivity to UV radiation that acts as a cue for determining the level of scytonemin synthesis. Scytonemin is a stable UVR screening compound and appears to be synthesized by cyanobacteria as a long-term solution for reducing UVR exposure and damage, but mainly or solely, when metabolic activity is absent. It is likely that during metabolic resurgence, the presence of a dense scytonemin sheath would facilitate the recovery process without the need for active defenses against UV radiation.     

The cyanobacterial UV-absorbing pigment scytonemin displays radical-scavenging activity

Scytonemin is a 544-Da hydrophobic pigment that can absorb UV-A radiation. It is present in cyanobacterial sheaths and is thought to function as a UV protectant. In this study, scytonemin was purified from the terrestrial cyanobacterium Nostoc commune, and its radical-scavenging activity was characterized. The purified scytonemin quenched an organic radical in vitro and accounted for up to 10% of the total activity of an ethanol extract of N. commune. These results suggest that the extracellular UV-absorbing pigment scytonemin has multiple roles, functioning as a UV sunscreen and an antioxidant relevant to anhydrobiosis in N. commune.     

Conformational and biological characterization of human parathyroid hormone hPTH(1-34) analogues containing beta-amino acid residues in positions 17-19

The N-terminal 1-34 fragment of parathyroid hormone (PTH) elicits the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(18) upon biological function, we synthesized and characterized the following human (h) PTH(1-34) analogues containing beta-amino acid residues: [analogues: see text]. Biological activity and binding affinity of analogue I are one order of magnitude lower than those of the parent compound. In analogue II, both binding affinity and biological activity are partially recovered. Analogues III and V have no binding affinity and very low biological activity. Both bioactivity and binding affinity are partially recovered in analogue IV. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, 2D-nuclear magnetic resonance and molecular dynamics calculations. The results confirmed the presence in all analogues of two helical segments located at the N-terminal and C-terminal sequences. The insertion of beta-amino acid residues around position 18 does not cause appreciable conformational differences in the five analogues. The differences in biological activity and binding affinity among the five analogues cannot be related to structural differences in the membrane mimetic environment reported in this study. Our results stress the importance of the side-chain functionalities in the sequence 17-19 for biological function.     

Improved synthesis of unique estradiol-linked platinum(II) complexes showing potent cytocidal activity and affinity for the estrogen receptor alpha and beta

We have recently reported the synthesis of a platinum(II) complex, made of estradiol, the female sex hormone, and a cisplatin analog, an anticancer drug, linked together by an eleven carbon atoms chain. The novel estradiol-Pt(II) hybrid molecule was synthesized in nine chemical steps with 10% overall yield. This new compound has been tested in vitro on estrogen-dependent (MCF-7) and -independent (MDA-MD-231) (ER(+) and ER(-)) cell lines. Interestingly, the biological activity was quite significant, more potent than that of cisplatin, the compound currently used in chemotherapy. The estrogen receptor binding affinity (ERBA) of this compound was very similar to that of 17beta-estradiol (E(2)) on both estrogen receptors (ERs), alpha and beta. In order to further study this type of molecule, we have decided to synthesize several analogs with the same estrogenic scaffold but with various chain lengths separating the estradiol from the toxic part of the molecule. This was planned in order to study the effect of the length of the linking chain on the biological activity of the hybrids. Four E(2)-Pt(II) hybrid molecules having 6-14 carbon atoms linking chain have been synthesized using a new synthetic methodology. They are synthesized in only eight chemical steps with 21% overall yield. The 17beta-estradiol-linked platinum(II) complexes have been tested for their receptor binding affinity as well as for their cytocidal activity on several breast cancer cell lines. The synthesis and biological results are reported herein.     

Assessing the ability of a short fluorinated antifreeze glycopeptide and a fluorinated carbohydrate derivative to inhibit ice recrystallization

A short fluorinated antifreeze glycopeptide (2) was synthesized and evaluated for ice recrystallization inhibition (IRI) activity. The activity of 2 was compared to native biological antifreeze AFGP 8 and a rationally designed C-linked AFGP analogue (OGG-Gal, 1). In addition, a simple fluorinated galactose derivative was prepared and its IRI activity was compared to non-fluorinated compounds. The results from this study suggest that the stereochemistry at the anomeric position in the carbohydrate plays a role in imparting ice recrystallization inhibition activity and that incorporation of hydrophobic groups such as fluorine atoms cause a decrease in IRI activity. These observations are consistent with the theory that fluorine atoms increase ordering of bulk water resulting in a decrease of IRI activity, supporting our previously proposed mechanism of ice recrystallization inhibition.     

Effect of environmental factors on the synthesis of scytonemin, a UV-screening pigment, in a cyanobacterium (Chroococcidiopsis sp.)

Abstract.The UV-screening pigment scytonemin is found in many species of ensheathed cyanobacteria. Past work has shown that the pigment is synthesized in response to exposure to UV-A irradiance. This study investigated the effect of other correlated stress factors including heat, osmotic and oxidative stress on the synthesis of scytonemin in a clonal cyanobacterial isolate ( Chroococcidiopsis sp.) from an epilithic desert crust. Stress experiments were carried out both in conjunction with UV-A irradiance and in isolation. Increases in both temperature and photooxidative conditions in conjunction with UV-A caused a synergistic increase in the rate of scytonemin production. In contrast, increased salt concentration under UV-A irradiance inhibited scytonemin synthesis. However, unlike the responses to temperature and oxidative stress, cells synthesized low levels of scytonemin under osmotic stress in the absence of scytonemin-inducing irradiance. These results suggest that scytonemin induction may be regulated as a part of a complex stress response pathway in which multiple environmental signals affect its synthesis.     

One-electron oxidation of "photo-Fenton" reagents and inhibition of lipid peroxidation

The "photo-Fenton" reagent, 2-mercaptopyridine N-oxide (MPO), which releases a hydroxyl radical on ultraviolet irradiation, has been found to act as an antioxidant. In the peroxidation of linoleate initiated by a water-soluble azo-initiator, MPO has about one-third the activity of the water-soluble vitamin E analogue Trolox C. In contrast, the oxygen-containing analogue, 2-hydroxypyridine N-oxide (HPO), does not have measurable antioxidant activity in this system. Both reagents react with hydroxyl radical with second order rate constants very close to the diffusion-controlled limit. With the less oxidising dithiocyanate radical anion, MPO reacts approximately 50 times more rapidly than HPO at pH>7. The more reducing properties of MPO result in its activity as an antioxidant and make it less suitable than HPO as a source of hydroxyl radicals for investigation of oxidative stress in biological systems.     

Dynamic function of the alkyl spacer of acetogenins in their inhibitory action with mitochondrial complex I (NADH-ubiquinone oxidoreductase)

Studies on the inhibitory mechanism of acetogenins, the most potent inhibitors of mitochondrial complex I (NADH-ubiquinone oxidoreductase), are useful for elucidating the structural and functional features of the terminal electron transfer step of this enzyme. Previous studies of the structure-activity relationship revealed that except for the alkyl spacer linking the two toxophores (i.e., the hydroxylated THF and the gamma-lactone rings), none of the multiple functional groups of these inhibitors is essential for potent inhibition. To elucidate the function of the alkyl spacer, two sets of systematically selected analogues were synthesized. First, the length of the spacer was varied widely. Second, the local flexibility of the spacer was specifically reduced by introducing multiple bond(s) into different regions of the spacer. The optimal length of the spacer for inhibition was approximately 13 carbon atoms. The decrease in the strength of the inhibitory effect caused by elongating the spacer from 13 carbons was much more drastic than that caused by shortening. Local flexibility in a specific region of the spacer was not important for the inhibition. These observations indicate that the active conformation of the spacer is not an extended form, and is not necessarily restricted to a certain rigid shape. Moreover, an analogue in which a spacer covering 10 carbon atoms was hardened into a rodlike shape still maintained a potent inhibitory effect. Our results strongly suggest that the spacer portion is free from steric congestion arising from the putative binding site probably because there is no cavity-like binding site for the spacer portion. The manner of acetogenin binding to the enzyme may not be explained by a simple "key and keyhole" analogy.     

Efficiency of transfer of essential polyunsaturated fatty acids versus organic carbon from producers to consumers in a eutrophic reservoir

One of the central paradigms of ecology is that only about 10% of organic carbon production of one trophic level is incorporated into new biomass of organisms of the next trophic level. Many of energy-yielding compounds of carbon are designated as ‘essential’, because they cannot be synthesized de novo by consumers and must be obtained with food, while they play important structural and regulatory functions. The question arises: are the essential compounds transferred through trophic chains with the same efficiency as bulk carbon? To answer this question, we measured gross primary production of phytoplankton and secondary production of zooplankton and content of organic carbon and essential polyunsaturated fatty acids of ω-3 family with 18–22 carbon atoms (PUFA) in the biomass of phytoplankton and zooplankton in a small eutrophic reservoir during two summers. Transfer efficiency between the two trophic levels, phytoplankton (producers) and zooplankton (consumers), was calculated as ratio of the primary production versus the secondary (zooplankton) production for both carbon and PUFA. We found that the essential PUFA were transferred from the producers to the primary consumers with about twice higher efficiency than bulk carbon. In contrast, polyunsaturated fatty acids with 16 carbon atoms, which are synthesized exclusively by phytoplankton, but are not essential for animals, had significantly lower transfer efficiency than both bulk carbon, and essential PUFA. Thus, the trophic pyramid concept, which implicitly implies that all the energy-yielding compounds of carbon are transferred from one trophic level to the next with the same efficiency of about on average 10%, should be specified for different carbon compounds.     

Antioxidative activity and chemical constituents of edible terrestrial alga Nostoc commune Vauch

The extract of terrestrial alga Nostoc commune Vauch. has high antioxidative activity. Our study on N. commune Vauch. resulted in the isolation of two β-ionone derivatives, nostocionone and 3-oxo-β-ionone, together with four indole alkaloids, scytonemin, reduced scytonemin, N-(p-coumaroyl)tryptamine, and N-acetyltryptamine. The structures of the isolated compounds were determined on the basis of 1D and 2D NMR and MS analyses. Among these isolates, nostocionone and reduced scytonemin demonstrated strong antioxidative activities which were assessed by using a β-carotene oxidation assay.     

Piperazine skeleton in the structural modification of natural products: a review

Piperazine moiety is a cyclic molecule containing two nitrogen atoms in positions 1 and 4, as well as four carbon atoms. Piperazine is one of the most sought heterocyclics for the development of new drug candidates with a wide range of applications. Over 100 molecules with a broad range of bioactivities, including antitumor, antibacterial, anti-inflammatory, antioxidant, and other activities, were reviewed. This article reviewed investigations regarding piperazine groups for the modification of natural product derivatives in the last decade, highlighting parameters that affect their biological activity.     

The synthesis of the UV-screening pigment,scytonemin, and photosynthetic performance in isolates from closely related natural populations of cyanobacteria (Calothrix sp.)

Two populations of the cyanobacterium Calothrix sp. found in Yellowstone thermal spring outflows differ greatly in their contents of scytonemin, a UV-screening pigment, and in their photosynthetic carbon assimilation rates. Clonal isolates from both populations were used to investigate these phenotypic differences. Identical partial 16S rDNA sequences ( approximately 900 bp) suggest a very close relationship between the two Calothrix populations and indicate that environmental differences may, in part, explain the field observations. The effects of native spring water on scytonemin synthesis and photosynthesis were tested during experiments using plated cells. Results show differences in the spring water environment were at least partly responsible for the differences in scytonemin content observed in the field. Furthermore, spring water effects on photosynthetic performance suggest adaptation in these strains to their spring of origin. Controlled experiments performed using cultures grown in artificial liquid medium showed no significant difference in photosynthetic carbon uptake between strains. However, significant differences were detected in their ability to synthesize scytonemin indicating genetic differences between populations. These findings suggest that both genetic and environmental differences are responsible for the naturally occurring variation in scytonemin content and photosynthetic ability in these two closely related populations.     

Structure and function of bovine pancreatic deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (bpDNase), the first DNase discovered, is the best characterized among various types of DNase. A catalytic mechanism has been suggested based on the X-ray structure of the bpDNase-octamer complex. In this review, we will focus on three aspects: 1) the distinctive functions of the two structural calcium atoms; 2) the biological functions of the two disulfides; and 3) the involvement of the N- and C-terminal fragments in the enzyme folding for activity.     

Different modulation of phospholipase A2 activity by saturated and monounsaturated N-acylethanolamines

The physiological functions of N-acylethanolamines (NAEs) are poorly understood, although many functions were suggested for these naturally occurring membrane components of plants and animals. The binding with cannabinoid receptors CB1 and CB2 was demonstrated for some NAEs, such as anandamide. However, the chemical nature of these molecules suggests that some of their biological effects on biomembranes could be related, at least partially, to physical interactions with the lipid bilayer. The present work studies the effect of saturated and monounsaturated NAEs on phospholipase A2 (PLA2) activity, which is dependent on lipid bilayer features. The present study, performed by 2-dimethylamino-(6-lauroyl)-naphthalene (Laurdan) fluorescence, demonstrates that the acyl chain length and the presence of a single double bond are crucial for the enzymatic activity modulation by NAEs. In fact, saturated NAEs with 10 carbon atoms don't affect the PLA2 activity, while NAEs with 12 and 16 carbon atoms largely activate the enzyme. On the other hand, an acyl chain length of 18 carbon atoms, with or without the presence of a double bond, only slightly affects the enzymatic activity. A structural model for NAE-lipid interactions is proposed in order to explain the differences in PLA2 activity modulation by these fatty acid derivatives.     

Methyl 5'-(6-aminopurin-9-yl)-5'-deoxy-beta-D-ribofuranoside 2',3'-cyclic monophosphate: a novel, biologically active, structural analogue of cyclic AMP

A novel structural analogue of cyclic AMP has been synthesized. This compound has been found to activate protein kinase from skeletal muscle (Ka 5.0 microM). It is virtually resistant to degradation by beef heart cAMP phosphodiesterase. It is an inhibitor of this enzyme with an [I]50 of 47.0 microM. The proliferation of cancer cells (HT-29) is inhibited by this compound. It represents the first example of a 2',3'-cyclic nucleotide with marked biological activity.     

Solid-phase synthesis of quinol fatty alcohols, design of N/O-substituted quinol fatty alcohols and comparative activities on axonal growth

Following the promising activity of Q2FA15 on axonal growth, two new series of N/O-substituted QFAs were synthesized, based on a SN2-type reaction. O-alkylated QFA bearing 14 carbon atoms on the side chain (n=14) shows a very potent activity on axonal growth though lowered when compared to Q2FA15. While O-alkylation allows good retention of the biological activity, N-alkylation abolishes it nonetheless. A solid-phase-supported synthesis of Q2FA15 allowing the conception of new hybrid compounds is also described.     

Structure-function relationship for saponin effects on cell cycle arrest and apoptosis in the human 1547 osteosarcoma cells: a molecular modelling approach of natural molecules structurally close to diosgenin

In this paper, eight natural molecules structurally close to diosgenin (five saponins: diosgenin, hecogenin, tigogenin, sarsasapogenin, smilagenin; two steroidal alkaloids: solasodine, solanidine; one sterol: stigmasterol) have been tested for their biological activities on human 1547 osteosarcoma cells. Differences in activity were studied in term of proliferation rate, cell cycle distribution and apoptosis induction. By using molecular modelling, two structural characteristics were calculated: spatial conformation and electron transfer capacity. The second property has been investigated by the HOMO repartition and the corresponding energy. Correlation between the experimental and the theoretical data permit us to highlight the importance of the hetero-sugar moiety and the 5,6-double bond in the biological activity (apoptosis and cell cycle arrest) on the human 1547 cell line. The importance of conformation at C-5 and C-25 carbon atoms was also discussed.     

Systematic peptide engineering and structural characterization to search for the shortest antimicrobial peptide analogue of gaegurin 5

As part of an effort to develop new, low molecular mass peptide antibiotics, we searched for the shortest bioactive analogue of gaegurin 5 (GGN5), a 24-residue antimicrobial peptide. Thirty-one kinds of GGN5 analogues were synthesized, and their biological activities were analyzed against diverse microorganisms and human erythrocytes. The structural properties of the peptides in various solutions were characterized by spectroscopic methods. The N-terminal 13 residues of GGN5 were identified as the minimal requirement for biological activity. The helical stability, the amphipathic property, and the hydrophobic N terminus were characterized as the important structural factors driving the activity. To develop shorter antibiotic peptides, amino acid substitutions in an inactive 11-residue analogue were examined. Single tryptophanyl substitutions at certain positions yielded some active 11-residue analogues. The most effective site for the substitution was the hydrophobic-hydrophilic interface in the amphipathic helical structure. At this position, tryptophan was the most useful amino acid conferring favorable activity to the peptide. The introduced tryptophan played an important anchoring role for the membrane interaction of the peptides. Finally, two 11-residue analogues of GGN5, which exhibited strong bactericidal activity with little hemolytic activity, were obtained as property-optimized candidates for new peptide antibiotic development. Altogether, the present approach not only characterized some important factors for the antimicrobial activity but also provided useful information about peptide engineering to search for potent lead molecules for new peptide antibiotic development.     

Inhibition of free radical-induced peroxidation of rat liver microsomes by resveratrol and its analogues

Resveratrol (3,5,4'-trans-trihydroxystilbene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidative activity. To find more efficient antioxidants by structural modification, resveratrol analogues, that is, 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), 4-hydroxy-trans-stilbene (4-HS) and 3,5-dihydroxy-trans-stilbene (3,5-DHS), were synthesized and their antioxidant activity studied for the free radical-induced peroxidation of rat liver microsomes in vitro. The peroxidation was initiated by either a water-soluble azo compound 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH) or Fe(2+)/ascorbate, and monitored by oxygen uptake and formation of thiobarbituric acid reactive substances (TBARS). It was found that all of these trans-stilbene derivatives are effective antioxidants against both AAPH- and iron-induced peroxidation of rat liver microsomes with an activity sequence of 3,4-DHS>4,4'-DHS>resveratrol>4-HS>3,5-DHS. The remarkably higher antioxidant activity of 3,4-DHS is discussed.     

Anti-tumor and anti-invasive effects of diverse delta-alkyllactones: dependence on molecular side-chain length, action period and intracellular uptake

New delta-alkyllactones (DALs) with diverse side-chain lengths (184-254 Da), which are structurally different from the widespread, naturally occurring delta-lactones of higher molecular weight (348-439 Da), such as camptothecin and sultriecin, were chemically synthesized and analyzed for their carcinostatic activity. Of the DALs with 11, 12, 13, 14, or 16 carbon atoms, delta-hexadecalactone (DH16:0) was the most carcinostatic when administered to Ehrlich ascites tumor (EAT) cells at 37 degrees C for 20 h, and measured by the mitochondrial dehydrogenase-based WST-1 assay. Prolongation of the administration period to 72 h enhanced the carcinostatic activity more markedly for DH16:0 than for other DALs. The carcinostatic activity of DALs was unexpectedly augmented by increasing the number of carbon atoms, in contrast to the conventional view that carcinostatic activity is attenuated by the addition of carbon atoms to fatty acids. Intracellular accumulation of DH16:0, as analyzed by gas chromatography, was detected (1.5 Pg/cell), whereas other DALs studied were rarely found. The results indicate a close relationship between carcinostatic activity and intracellular accumulation. Invasion of human fibrosarcoma HT-1080 cells through the reconstituted basement membrane was inhibited by several DALs, even at doses as low as 5-10% of those necessary for carcinostatic activity, suggesting an invasive mechanism different from carcinostasis. The invasion-inhibitory activity was intensified by increasing the number of carbon atoms, in a manner similar to that for the carcinostatic activity. The lifespan of EAT-cell-transplanted mice was markedly prolonged with DH16:0, presumably due to excellent distribution throughout the body and tumor cells. Thus DH16:0 may be a potent anticancer agent, in term of its carcinostatic, anti-invasive, and lifespan-prolonging activities.