Inhibitory effect of α-hydrazinoornithine on egg cleavage in sea urchin eggs

In fertilized sea urchin eggs which are kept in sea water containing α-hydrazinoornithine (αHO) at a concentration above 1 mM from the time of fertilization, cleavage is delayed markedly. The third cleavage is almost completely blocked by 3 mM αHO. Hydrazine, as well as ornithine, exerts no harmful effect on egg cleavage. αHO causes competitive inhibition of ornithine decarboxylase (ODC) in the egg homogenate. Polyamine levels decrease in fertilized eggs treated with αHO. The addition of ornithine (above 3 mM) to an egg culture containing αHO prevents the αHO-induced delay of cleavage. Putrescine (0.2–0.5 mM), which is the product of the reaction catalyzed by ODC, also relieves egg cleavage from the inhibited state. The same effect occurs in the presence of spermidine (0.2–0.5 mM) or spermine (0.1–0.8 mM). Especially, spermine (0.5 mM) completely cancels the inhibitory effect of αHO on egg cleavage. Egg cleavage is delayed only slightly in the presence of each polyamine (above 2 mM).     

Inhibition of glutamate racemase by substrate–product analogues

d-Glutamate is an essential biosynthetic building block of the peptidoglycans that encapsulate the bacterial cell wall. Glutamate racemase catalyzes the reversible formation of d-glutamate from l-glutamate and, hence, the enzyme is a potential therapeutic target. We show that the novel cyclic substrate–product analogue (R,S)-1-hydroxy-1-oxo-4-amino-4-carboxyphosphorinane is a modest, partial noncompetitive inhibitor of glutamate racemase from Fusobacterium nucleatum (FnGR), a pathogen responsible, in part, for periodontal disease and colorectal cancer (K_i = 3.1 ± 0.6 mM, cf. K_m = 1.41 ± 0.06 mM). The cyclic substrate–product analogue (R,S)-4-amino-4-carboxy-1,1-dioxotetrahydro-thiopyran was a weak inhibitor, giving only ∼30% inhibition at a concentration of 40 mM. The related cyclic substrate–product analogue 1,1-dioxo-tetrahydrothiopyran-4-one was a cooperative mixed-type inhibitor of FnGR (K_i = 18.4 ± 1.2 mM), while linear analogues were only weak inhibitors of the enzyme. For glutamate racemase, mimicking the structure of both enantiomeric substrates (substrate–product analogues) serves as a useful design strategy for developing inhibitors. The new cyclic compounds developed in the present study may serve as potential lead compounds for further development.     

Site-specific cleavage of DNA–RNA hybrids by zinc finger/FokI cleavage domain fusions

Zinc-finger proteins of the Cys₂His₂ type bind DNA–RNA hybrids with affinities comparable to those for DNA duplexes. Such zinc-finger proteins were converted into site-specific cleaving enzymes by fusing them to the FokI cleavage domain. The fusion proteins are active and under optimal conditions cleave DNA duplexes in a sequence-specific manner. These fusions also exhibit site-specific cleavage of the DNA strand within DNA–RNA hybrids albeit at a lower efficiency (≃50-fold) compared to the cleavage of the DNA duplexes. These engineered endonucleases represent the first of their kind in terms of their DNA–RNA cleavage properties, and they may have important biological applications.     

Study of trans–trans farnesol effect on hyphae formation by Yarrowia lipolytica

Dimorphism is an ability of certain fungi related to its adaptation to the environment and provides a selective advantage under stress conditions and is associated to the development of human diseases. Hyphae inducing- and inhibitory-effect of farnesol on hyphae formation by the dimorphic yeast Yarrowia lipolytica was evaluated through digital image analysis. The agitation speed of the culture was the most effective hyphae inducer in comparison to bovine calf serum and N-acetylglucosamine. In low agitation system, bovine calf serum was more effective for hyphae formation inducing 57 % of hyphae transition. Farnesol inhibited hyphae formation even in low concentration (300 μM) and this effect increased with increasing concentrations. In the presence of N-acetylglucosamine, this effect was more evident in comparison to the presence of bovine calf serum, which might have protected the cells from farnesol. Digital image analysis was an important tool to evaluate this phenomenon.     

Inhibition of ovulation and fertilization by indomethacin and effect of prostaglandin-f2α on early pregnancy in the rabbit

Ovulation induced by HCG in rabbits was blocked by a single subcutaneous injection of 6 mg/kg indomethacin given 6 h after the insemination and HCG treatment. In addition, a time-dependent inhibition in the fertilization rate after indomethacin treatment was also recorded. This suggests that indomethacin, when given at a critical time and at an appropriate dose level, not only blocks ovulation but also interferes with fertilization.Treatment with graded amounts of prostaglandin-F_2α incorporated in a Silastic-polyvinylpyrrolidone-gel (PVP) had marginal to no effect after intravaginal placement on 4- or 6-day pregnancy. However, 5 mg PGF_2α Silastic-PVP tube when placed intravaginally on Day-7 of pregnancy resulted in termination of pregnancy in 66% of the treated does. This implies that young corpora lutea are resistant to PGF_2α treatment and that pregnancy at the time of ovo-implantation can be terminated by PGF_2α incorporated in a Silastic-PVP tube.     

Inhibition of methane production from whey by heavy metals – protective effect of sulfide

A whey solution was used as a substrate for methane production in an anaerobic fixed-bed reactor. At a hydraulic retention time of 10 days, equivalent to a space loading of 3.3 kg (m³day)⁻¹, 90% of the chemical oxygen demand was converted to biogas. Only a little propionate remained in the effluent. Toxicity tests with either copper chloride, zinc chloride or nickel chloride were performed on effluent from the reactor. Fifty per cent inhibition of methanogenesis was observed in the presence of ≥10 mg CuCl₂ l⁻¹≥40 mg ZnCl₂ l⁻¹ and ≥60 mg NiCl₂ l⁻¹, respectively. After exposure to Cu²⁺, Zn²⁺ or Ni²⁺ ions for 12 days, complete recovery of methanogenesis by equimolar sulfide addition was possible upon prolonged incubation. Recovery failed, however, for copper chloride concentrations ≥40 mg l⁻¹. If the sulfide was added simultaneously with the three heavy metal salts, methanogenesis was only slightly retarded and the same amount of methane as in non-inhibited controls was reached either 1 day (40 mg ZnCl₂ l⁻¹) or 2 days later (10 mg CuCl₂ l⁻¹). Up to 60 mg NiCl₂ l⁻¹ had no effect if sulfide was present. Sulfide presumably precipitated the heavy metals as metal sulfides and by this means prevented heavy metal toxicity. Received: 8 October 1999 / Received revision: 3 January 2000 / Accepted: 4 January 2000     

The effect of nutrients on carbon and nitrogen fixation by the UCYN-A–haptophyte symbiosis

Symbiotic relationships between phytoplankton and N₂-fixing microorganisms play a crucial role in marine ecosystems. The abundant and widespread unicellular cyanobacteria group A (UCYN-A) has recently been found to live symbiotically with a haptophyte. Here, we investigated the effect of nitrogen (N), phosphorus (P), iron (Fe) and Saharan dust additions on nitrogen (N₂) fixation and primary production by the UCYN-A–haptophyte association in the subtropical eastern North Atlantic Ocean using nifH expression analysis and stable isotope incubations combined with single-cell measurements. N₂ fixation by UCYN-A was stimulated by the addition of Fe and Saharan dust, although this was not reflected in the nifH expression. CO₂ fixation by the haptophyte was stimulated by the addition of ammonium nitrate as well as Fe and Saharan dust. Intriguingly, the single-cell analysis using nanometer scale secondary ion mass spectrometry indicates that the increased CO₂ fixation by the haptophyte in treatments without added fixed N is likely an indirect result of the positive effect of Fe and/or P on UCYN-A N₂ fixation and the transfer of N₂-derived N to the haptophyte. Our results reveal a direct linkage between the marine carbon and nitrogen cycles that is fuelled by the atmospheric deposition of dust. The comparison of single-cell rates suggests a tight coupling of nitrogen and carbon transfer that stays balanced even under changing nutrient regimes. However, it appears that the transfer of carbon from the haptophyte to UCYN-A requires a transfer of nitrogen from UCYN-A. This tight coupling indicates an obligate symbiosis of this globally important diazotrophic association.     

Inhibition of staphylococcal α-toxin. The effect of aromatic polysulphonic acids on the lethal effect of α-toxin in mice

1. Certain aromatic polysulphonic acids, previously tested for inhibition of the haemolytic activity of staphylococcal α-toxin, together with some additional related compounds, were tested as possible inhibitors of α-toxin in mice. 2. Compounds that inhibited the haemolytic activity of α-toxin at concentrations of 0·16mm or less [compounds (I), (II), (IV), (V), (VII) and (VIII)] were found to inhibit the lethal effect of α-toxin. 3. With the exception of compound (VIII), amounts of 1mg. were required to inhibit 4 LD₅₀ of toxin when the test compounds were premixed with α-toxin before injection; comparable inhibition with 0·3mg. of compound (VIII) was achieved without prolonged premixing. 4. Mixtures of α-toxin and compounds (I) and (II) containing an excess of test compound showed markedly diminished inhibitory activities. 5. The `half-molecule' analogues of group 1 [compounds (III) and (XVIII)] were non-inhibitory. 6. Compounds (I)–(V), when administered separately from α-toxin by the same route (intraperitoneal), were active only when injected almost simultaneously with toxin, whereas compounds (VII) and (VIII) were strikingly inhibitory when injected 15min. before or after the toxin. 7. Compound (VIII) failed to inhibit the lethal effect of α-toxin when injected by a different route (intravenous).     

Inhibition of cholinesterase and monoamine oxidase-B activity by Tacrine–Homoisoflavonoid hybrids

A series of Tacrine–Homoisoflavonoid hybrids were designed, synthesised and evaluated as inhibitors of cholinesterases (ChEs) and human monoamine oxidases (MAOs). Most of the compounds were found to be potent against both ChEs and MAO-B. Among these hybrids, compound 8b, with a 6 carbon linker between tacrine and (E)-7-hydroxy-3-(4-methoxybenzylidene)chroman-4-one, proved to be the most potent against AChE and MAO-B with IC₅₀ values of 67.9 nM and 0.401 μM, respectively. This compound was observed to cross the blood–brain barrier (BBB) in a parallel artificial membrane permeation assay for the BBB (PAMPA-BBB). The results indicated that compound 8b is an excellent multifunctional promising compound for development of novel drugs for Alzheimer’s disease (AD).     

Inhibition of β-amyloid–induced neurotoxicity by planar analogues of procyanidin B3

Reactive oxygen species (ROS) are known to be produced during the amyloid beta (Aβ) aggregation process. Both ROS production and Aβ fibril formation can result in nerve cell injury. Proanthocyanidins are oligomers of catechin that can act as inhibitors of Aβ aggregation. Procyanidin B3 (Cat-Cat), the dimer of (+)-catechin, can easily cross the blood-brain barrier. Previously, we synthesized two derivatives of Cat-Cat, namely Cat-PCat and PCat-PCat, in which the geometry of one or both catechin molecules in Cat-Cat was constrained to be planar. The antioxidative activities of Cat-PCat and PCat-PCat were found to be stronger than that of Cat-Cat, with PCat-PC at exhibiting the most potent activity. These compounds are predicted to protect against Aβ-induced neurotoxicity via inhibition of Aβ aggregation as well as by antioxidative effects toward Aβ-induced intracellular ROS generation. PCat-PCat exhibited the most potent neuroprotective effects against Aβ-induced cytotoxicity, which resulted from inhibition of β-sheet structure formation during the Aβ aggregation process. PCat-PCat may be a promising lead compound for the treatment of Alzheimer’s disease.     

The effect of disturbance on an ant–plant mutualism

Protective ant–plant mutualisms—where plants provide food or shelter to ants and ants protect the plants from herbivores—are a common feature in many ecological communities, but few studies have examined the effect of disturbance on these interactions. Disturbance may affect the relationship between plants and their associated ant mutualists by increasing the plants’ susceptibility to herbivores, changing the amount of reward provided for the ants, and altering the abundance of ants and other predators. Pruning was used to simulate the damage to buttonwood mangrove (Conocarpus erectus) caused by hurricanes. Pruned plants grew faster than unpruned plants, produced lower levels of physical anti-herbivore defenses (trichomes, toughness), and higher levels of chemical defenses (tannins) and extrafloral nectaries. Thus, simulated hurricane damage increased plant growth and the amount of reward provided to ant mutualists, but did not have consistent effects on other anti-herbivore defenses. Both herbivores and ants increased in abundance on pruned plants, indicating that the effects of simulated hurricane damage on plant traits were propagated to higher trophic levels. Ant-exclusion led to higher leaf damage on both pruned and upruned plants. The effect of ant-exclusion did not differ between pruned and unpruned plants, despite the fact that pruned plants had higher ant and herbivore densities, produced more extrafloral nectaries, and had fewer physical defenses. Another common predator, clubionid spiders, increased in abundance on pruned plants from which ants had been excluded. I suggest that compensatory predation by these spiders diminished the effect of ant-exclusion on pruned plants.     

The effect of landscape heterogeneity on host–parasite dynamics

Environmental heterogeneity has been shown to have a profound effect on population dynamics and biological invasions, yet the effect of its spatial structure on the dynamics of disease invasion in a spatial host–parasite system has received little attention. Here we explore the effect of environment heterogeneity using the pair approximation and the stochastic spatially explicit simulation in which the lost patches are clustered in a fragmented landscape. The intensity of fragmentation is defined by the amount and spatial autocorrelation of the lost habitat. More fragmented landscape (high amount of habitat loss, low clustering of lost patches) was shown to be detrimental to the parasitic disease invasion and transmission, which implies that the potential of using artificial disturbances as a disease-control agency in biological conservation and management. Two components of the spatial heterogeneity (the amount and spatial autocorrelation of the lost habitat) formed a trade-off in determining the host–parasite dynamics. An extremely high degree of habitat loss was, counter-intuitively, harmful to the host. These results enrich our understanding of eco-epidemiological, host–parasite systems, and suggest the possibility of using the spatial arrangement of habitat patches as a conservation tool for guarding focal species against parasitic infection and transmission.     

Inhibition of DNA polymerase α by gossypol

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase α (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase α with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase α does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase β in a dose-dependent manner, but had no effect on DNA polymerase γ. These results suggest that inhibition of DNA polymerase α may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.