Occurrence of the medicinal leech (Hirudo medicinalis) in birds’ nests

Occurrence and breeding of Hirudo medicinalis were recorded in birds’ nests in the fishing ponds and water bodies used extensively by anglers in south-eastern Poland, in 4 of 11 studied bird species (Circus aeruginosus, Fulica atra, Cygnus olor, Chroicocephalus ridibundus). Factors important for the distribution and density of this leech were: nest height, pH of the water and oxygen concentration in the water inside the nests, the body-length of the nesting birds. Our data show that (1) H. medicinalis chooses nests as habitats that are safe in water bodies rich in predators (particularly fish) and provide stable feeding conditions; (2) fishing ponds and other water bodies used extensively by anglers in a landscape modified by human pressure can be an important secondary habitat for the medicinal leech. As the nesting activity of some aquatic birds apparently favors the occurrence of H. medicinalis, active protection of aquatic birds and creating sites appropriate for their breeding may be an indirect way for the conservation of the leech in such areas.     

The effects of trans linoleic acid on the concentration of serum prostaglandin f2α and platelet aggregation

In order to determine the effects of dietary trans linoleate on the concentration of PGF_2α and its precursor acid, and on platelet aggregation, four groups of rats were fed a diet containing hydrogenated coconut oil (HCO, treatment A); 9 trans, 12 trans linoleate (trans linoleate, treatment B); an equal mixture of 9 cis linoleate (cis linoleate) and trans linoleate (treatment C); and cis linoleate (treatment D); respectively for 12 weeks. The levels of arachidonic acid as a percent of total serum lipids were 5.8, 0.8, 25.2 and 30.0, respectively for treatments A, B, C, and D. The serum concentrations of PGF_2α were 3.2, 0.31, 14.1 and 27.4 ng/ml, respectively for treatments A, B, C, and D. Thus, trans linoleate caused decreased biosynthesis of PGF_2α probably resulting from decreased level of its precursor acid. The magnitude of the inhibition of the platelet aggregation by indomethacin was greater in treatment A than in treatment B. The difference in the magnitude of the inhibition between treatment C and D was not apparent at a high concentration of indomethacin, however, at a lower concentration (0.01 mM) the percent inhibition was 3 and 42 for treatments C and D, respectively. These results indicate that levels of dietary linoleate can affect physiological responses through an effect on the amount of PG synthesized in animal tissues.     

Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

Phenolic composition and medicinal usage of Psidium guajava Linn.: Antifungal activity or inhibition of virulence?

Psidium guajava is a Myrtaceae plant whose medicinal properties are recognized in several locations. The use of teas and tinctures prepared from their leaves has been used to combat infections caused by fungi of the genus Candida. In this study, aqueous extracts of leaves and hydroethanolic were tested to verify the antifungal potential and its chemical composition has been investigated. The microbiological assays were performed by broth microdilution to determine the minimum inhibitory concentration (MIC) and from these the minimum fungicidal concentration was performed (MFC) by subculturing on solid media. A cell viability curve was obtained for demonstration of inhibition of fungal growth of strains of Candida albicans and Candida tropicalis. Tests to check morphological changes by the action of the extracts were performed in microcultive cameras depleted environment at concentrations of MIC/2, MIC and MIC × 2. Extracts analyzed by high performance liquid chromatography demonstrated flavonoids and phenolic acids. The extracts showed fungistatic effect and no fungicide with MIC >8192 μg/mL, MFC above 8192 μg/mL. The IC₅₀ was calculated ranging from 1803.02 to 5623.41 μg/mL. It has been found that the extracts affect the morphological transition capability, preventing the formation of pseudohyphae and hyphae. Teas and tinctures, therefore, have the potential antifungal, by direct contact, causing inhibition of fungal multiplication and its virulence factor, the cell dimorphism, preventing tissue invasion. Further studies are needed to elucidate the biochemical pathways and genes assets involved in these processes.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Spontaneous activity of foregut and hindgut visceral muscle of the locust,Locusta migratoria—II. The effect of biogenic amines

1. At 10⁻⁸ M, 5-HT increased both the frequency and amplitude of contractions of isolated locust foregut. At 10⁻⁴ M the foregut general tonus was increased.2. Both spontaneously active and quiescent hindguts were less sensitive to 5-HT, showing only an increase in amplitude of contraction at 10⁻⁶−10⁻⁵ M.3. The Hill plot suggested that although the 5-HT receptor populations in these two gut divisions differed in affinities, they were essentially homogeneous.4. Octopamine (10⁻⁵−10⁻⁴ M) increased foregut contraction frequency but diminished amplitude.5. Octopamine action on the hindgut was varied. At 10⁻⁸ M it slightly increased tonus, while at 10⁻⁵ M it increased contraction amplitude without affecting frequency.6. At 10⁻⁴ M octopamine suppressed activity of spontaneously active preparations and lowered the tonus of quiescent preparations.7. Tyramine induced dose-dependent inhibition of foregut responses to 5-HT. The hindgut was exceptionally sensitive to tyramine, at only 10⁻⁸ M it suppressed 5-HT responses.8. Octopamine inhibited fore- and hindgut responses to 5-HT, but was less effective than tyramine.9. Locust fore- and hindgut have remarkably different pharmacological properties reflecting differences in innervation and in extrajunctional monoamine receptor affinities.     

Alterations in the extracellular catabolism of nucleotides and platelet aggregation induced by high-fat diet in rats: effects of α-tocopherol

The aim of this study was to assess whether α-tocopherol administration prevented alterations in the ectonucleotidase activities and platelet aggregation induced by high-fat diet in rats. Thus, we examined four groups of male rats which received standard diet, high-fat diet (HFD), α-tocopherol (α-Toc), and high-fat diet plus α-tocopherol. HFD was administered ad libitum and α-Toc by gavage using a dose of 50 mg/kg. After 3 months of treatment, animals were submitted to euthanasia, and blood samples were collected for biochemical assays. Results demonstrate that NTPDase, ectonucleotide pyrophosphatase/phosphodiesterase, and 5′-nucleotidase activities were significantly decreased in platelets of HFD group, while that adenosine deaminase (ADA) activity was significantly increased in this group in comparison to the other groups (P?<?0.05). When rats that received HFD were treated with α-Toc, the activities of these enzymes were similar to the control, but ADA activity was significantly increased in relation to the control and α-Toc group (P?<?0.05). HFD group showed an increased in platelet aggregation in comparison to the other groups, and treatment with α-Toc significantly reduced platelet aggregation in this group. These findings demonstrated that HFD alters platelet aggregation and purinergic signaling in the platelets and that treatment with α-Toc was capable of modulating the adenine nucleotide hydrolysis in this experimental condition.     

Adrenoceptor α2A signalling countervails the taming effects of synchronous cyclic nucleotide-elevation on thrombin-induced human platelet activation and aggregation

The healthy vascular endothelium constantly releases autacoids which cause an increase of intracellular cyclic nucleotides to tame platelets from inappropriate activation. Elevating cGMP and cAMP, in line with previous reports, cooperated in the inhibition of isolated human platelet intracellular calcium-mobilization, dense granules secretion, and aggregation provoked by thrombin. Further, platelet alpha granules secretion and, most relevant, integrin α_IIaβ₃ activation in response to thrombin are shown to be prominently affected by the combined elevation of cGMP and cAMP. Since stress-related sympathetic nervous activity is associated with an increase in thrombotic events, we investigated the impact of epinephrine in this setting. We found that the assessed signalling events and functional consequences were to various extents restored by epinephrine, resulting in full and sustained aggregation of isolated platelets. The restoring effects of epinephrine were abolished by either interfering with intracellular calcium-elevation or with PI3-K signalling. Finally, we show that in our experimental setting epinephrine likewise reconstitutes platelet aggregation in heparinized whole blood, which may indicate that this mechanism could also apply in vivo.     

Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca2+ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca2+/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca2+ titration of intact platelets using A23187 and Ca2+/HEDTA buffers, revealed half-maximal response at about 15 microM free Ca2+ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.     

Correlation between calpain-mediated cytoskeletal degradation and expression of platelet procoagulant activity. A role for the platelet membrane-skeleton in the regulation of membrane lipid asymmetry?

The relationship between platelet calpain-activity and platelet procoagulant-activity was investigated by comparison of the time course of their generation after platelet stimulation by calcium ionophore A23187, or by the combined action of collagen and thrombin, or during exposure of platelets to the local anesthetics dibucaine or tetracaine. In addition, the Ca²⁺ dose-response curves of both activities in intact platelets, obtained by stimulation with A23187 in the presence of Ca²⁺/HEDTA-buffers, were compared. Platelet procoagulant activity was determined by assaying for prothrombinase activity in the presence of saturating concentrations of factors Xa, Va, and prothrombin. Platelet calpain activity was monitored by the degradation of its major substrates (filamin, talin, myosin) and the formation of their fragments as judged from protein patterns after gel electrophoresis. Platelet stimulation by A23187 resulted in a fast increase in prothrombinase activity, reaching its maximum level after about 20 seconds. Filamin and talin were completely hydrolysed within 15 s, and myosin was partly degraded between 15 and 30 s after platelet activation. When platelets were activated by collagen plus thrombin, prothrombinase activity was generated with a sigmoid time course, the steepest increase being observed between 1 and 2 min after platelet activation. Proteolysis of filamin and talin occurred between 0.5 and 1.5 min after platelet activation, while degradation of myosin became visible after 2 to 2.5 min. Dibucaine and tetracaine were both found to be potent stimulators of prothrombinase activity, with half-maximal activities obtained at 0.7 and 2.8 mM, respectively. Using suboptimal concentrations of both local anesthetics, it was found that the generation of prothrombinase activity closely paralleled that of calpain activity over a time course of 1 hour. Ca²⁺ titration of intact platelets using A23187 and Ca²⁺/HEDTA buffers, revealed half-maximal response at about 15 μM free Ca²⁺ for both calpain and prothrombinase activity. These findings strongly suggest a causal relationship between generation of a procoagulant platelet surface and calpain-mediated degradation of filamin, talin, and myosin. Since an increased procoagulant activity reflects an increased exposure of phosphatidylserine at the platelet outer surface, the present findings suggest that platelet cytoskeletal proteins are involved in the regulation of membrane lipid asymmetry.     

Contributions to the mathematical theory of epidemics—II. The problem of endemicity

(1) A mathematical investigation has been made of the prevalence of a disease in a population from which certain individuals are being removed as the result of the disease, whilst fresh individuals are being introduced as the result of birth or immigration. Allowance is made for the effects of the immunity produced as the result of an attack of the disease, but the effect of deaths from other causes is not taken into account, and the action of the disease is supposed to be independent of the age of the individual. (2) As a special case of the above, results have been obtained for a closed population in which no deaths occur and to which no fresh individuals are added, but in which the individuals after being infected acquire immunity, and then may be again infected. A threshold density of population exists analogous to that described in the previous paper, which is such that no disease can exist in a population, the density of which is below the threshold. (3) In other special cases investigated when either immigration or birth is operative in the supply of fresh individuals, as well as in the general case, only one steady state of disease is possible. To reach this state the population must be of a certain density which will be determined by the functions characterizing the infectivity, morbidity, etc., of the disease. (4) Increase of the immigration rate or of the birth-rate results in an increase in the rate of infection of the healthy individuals and also in the percentage rate of infection, the percentage of sick, and in the percentage of mortality from the disease. This result is, of course, a necessary consequence of our assumption that the disease is the only cause of death. (5) More particular results have been obtained by substituting constants in the place of the undetermined functions assumed in the general theory. Further, under these conditions the nature of the steady states has been more fully investigated and it has been shown that in all cases, except one, the steady states are stable ones. In the exception, a disturbance would result in purely periodic oscillations about the steady state.     

The effects and mechanism of flavonoid–rePON1 interactions. Structure–activity relationship study

Flavonoids are plant phenolic secondary metabolites that are widely distributed in the human diet. These antioxidants have received much attention because of their neuroprotective, cardioprotective, and chemopreventive actions. While a major focus has been on the flavonoids’ antioxidant properties, there is an emerging view that many of the potential health benefits of flavonoids and their in vivo metabolites are due to modulatory actions in cells through direct interactions with proteins, and not necessarily due to their antioxidant function. This view relies on the observations that flavonoids are present in the circulation at very low concentrations, which are not sufficient to exert effective antioxidant effects. The enzyme paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL), and is responsible for many of HDLs’ antiatherogenic properties. We previously showed that the flavonoid glabridin binds to rePON1 and affects the enzyme’s 3D structure. This interaction protects the enzyme from inhibition by an atherogenic component of the human carotid plaque. Here, we broadened our study to an investigation of the structure–activity relationships (SARs) of 12 flavonoids from different subclasses with rePON1 using Trp-fluorescence quenching, modeling calculations and Cu²⁺-induced low-density lipoprotein (LDL) oxidation methods. Our findings emphasize the ‘protein-binding’ mechanism by which flavonoids exert their beneficial biological role toward rePON1. Flavonoids’ capacity to interact with the enzyme’s rePON1 hydrophobic groove mostly dictates their pro/antioxidant behavior.     

Energy and biological evolution—II. The mathematical structure of equilibrium states

We examine certain mathematical structures presented in Part I. The most important of these are the energy structures determined by the couple (ω×E, ψ) the space of causality defined by ψ^-1(0) and the notion of collapsibility, i.e., the descent of a species from a higher to a lower equilibrium configuration as a result of energy loss. This work was supported by an Associateship of The International Centre for Theoretical Physics, P.O. Box 586, Miramare, 34100 Trieste, Italy.