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PubMed2003

Inhibitory effects of rosmarinic acid on Lck SH2 domain binding to a synthetic phosphopeptide.

In the course of screening inhibitors from the methanol (MeOH) extracts of 168 medicinal plants against lymphocyte cell-specific kinase (Lck) Src -homology 2 (SH2) binding to a synthetic phosphotyrosine-containing peptide (phosphopeptide), we isolated rosmarinic acid from the MeOH extract of Prunella vulgaris, which showed specific inhibitory activity. The IC 50 value for Lck SH2 binding to phosphopeptide (SGSGEEPQpYEEIPI) of hamster polyomavirus middle-sized tumor (hmT pY324) was 7 microM. However, even at concentrations of 0.1 to 1000 microM, no significant inhibitions were observed against other SH2 domains binding such as the growth factor receptor binding protein 2 (Grb2) SH2 domain to phosphopeptide of Shc and phospholipase Cgamma1 (PLCgamma1) SH2 domain to translational elongation factor 1alpha (EF1alpha) C-terminal. Rosmarinic acid inhibited interleukin-2 (IL-2) gene expression by 50 % at a concentration of 8 microM in Jurkat cells stimulated with anti-CD3 and anti-CD4 antibodies. FK506 and cyclosporin A (CsA) employed as positive controls showed less than 30 % inhibition at the same concentration. In addition, rosmarinic acid inhibited the intracellular [Ca 2+] i increase in Jurkat cells after T cell activation in a dose-dependent manner at concentrations of 1.4 to 140 microM of rosmarinic acid, which is one of the earliest responses of antigen-specific T cell receptor (TCR) and of the upstream pathway of IL-2 expression. Taken together, these results suggest that rosmarinic acid has the potential to specifically inhibit Lck SH2 domain binding to its cognate ligand, including ZAP-70, Cbl, HS-1, and PLCgamma1, and Lck-dependent Ca 2+ signaling pathway of its downstream effector and finally to modulate IL-2 gene expression after T cell activation.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Application of capillary electrophoresis for organic acid analysis in herbal studies.

A capillary electrophoresis (CE) procedure has been developed for the separation of 25 inorganic and organic acid anions using a buffer system consisting of 15 mM tris(hydroxymethyl)aminomethane, 3 mM 1,2,4-benzenetricarboxylic acid, 1.5 mM tetraethylenepentaamine (TEPA) and 20% methanol with pH adjusted to 8.4. A good separation of organic acids extracted from a mixture of Chinese traditional medicine (TCM) containing three herbs, Flos chrysthemi, Spica prunellae, and Folium mori was obtained using the procedure developed with satisfactory working range (0.20-77 mg/g), low detection limit (90-190 microg/g), and good repeatability (relative standard deviation 4.47-6.99%, n = 4). A satisfactory extraction of organic acids was achieved within 20 min using 0.1 M NaOH. The addition of TEPA to provide a reduced electroosmotic flow (EOF) environment was shown to remove interfering organic compounds extracted from TCM. The applicability of using organic acids as markers for determining the mixing ratio of constituent herbs for a TCM mixture was investigated using a three-component mixture with a 1:1:1 mixing ratio. A satisfactory mixing ratio of 1.04:1.09:0.98 was obtained using the methodology developed based on organic acids as markers. The application of our method for determining more complicated TCM mixtures has been discussed.

Buffers↗
PubMed2003

Rosmarinic acid inhibits TCR-induced T cell activation and proliferation in an Lck-dependent manner.

Lck is a T cell-restricted Src family protein tyrosine kinase that plays pivotal roles in TCR-mediated signaling. We aimed to identify novel agents that could disrupt the molecular interaction of the Src homology 2-domain of Lck (Lck SH2) with its binding partners, with the expectation that this would impair TCR signaling and generate immunosuppression. Large-scale screening of plant extracts indicated that rosmarinic acid (RosA) in extracts of Prunella vulgaris consistently inhibits the interaction between Lck SH2 and a peptide containing its consensus binding sequence (pYEEI). The inhibitory effect of RosA was specific for SH2 domains of Src family protein tyrosine kinase. RosA inhibited TCR-induced-Ca(2+) mobilization and IL-2 promoter activation but not phorbol 12-myristate 13-acetate/ionomycin-induced IL-2 promoter activation, indicating its point of inhibition at the membrane proximal site of TCR signaling. Furthermore, RosA inhibited TCR-induced splenocyte proliferation as well as one-way MLR at an IC(50) of 25-50 microM and inhibited cytokine expression such as IL-2 and IFN-gamma. Here, we first report RosA as an inhibitor of TCR-signaling and subsequent T cell proliferation.

Calcium↗
PubMed1998

Multiple mating and cooperative breeding in polygynandrous alpine accentors. II. Male mating tactics

I studied the timing and frequency of male copulatory behaviour in alpine accentors, Prunella collaris, in Japan, with respect to dominance status, age and development of the cloacal protuberance. Males used one of three mating tactics depending on their dominance status. Alpha males guarded several females in succession, but only during the females' fertile period, while gamma males copulated rarely and only with unguarded females. The tactics of beta males were flexible: with unguarded females they usually adopted a frequent-copulation tactic, but when two females had synchronous egg-laying periods, beta males associated closely with whichever female the alpha male left unguarded. When alpha males closely guarded a female, beta males guarded this same female for short periods before and after the guarding period of the alpha male. Subordinate rank restricted a male's opportunities for mating but males of all dominance ranks were sexually active and had fully developed cloacal protuberances. Frequent copulation plus part-time mate guarding (as found in beta males) and opportunistic copulation (as found in gamma males) appears to be a conditional strategy whereby young, subordinate males are 'making the best of a bad job'. A long-term study over 10 years showed that males moved up in social rank with increasing age but 78.9% of males remained subordinate because alpha males occupied the top rank for much of their lifetime. Copyright 1998 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed1998

Multiple mating and cooperative breeding in polygynandrous alpine accentors. I. Competition among females

The reproductive strategy of female alpine accentors, Prunella collaris, was studied on the summit of Mt Norikura in Japan. Alpine accentors formed polygynandrous groups consisting of three to six males and three to five females; however, each female laid eggs in her own nest. Within each group, females were organized in stable, linear hierarchies based on age. All females were sexually active but high-ranking females copulated more frequently with several males from the group. In contrast, the degree of multiple mating and frequency of copulations of low-ranking females was lower because their sexual activity was interrupted by more dominant females. All copulations were preceded by female solicitation. When the young hatched, males helped to feed the chicks of several females with whom they had copulated. The main factor influencing reproductive success was the amount of parental care that was available for feeding the chicks. High-ranking females had paternal help from two or more males and secured a high provisioning rate for the brood, which increased fledging success and nestling weight. The reproductive success of low-ranking females was lower because the brood did not receive sufficient paternal investment and so often starved to death early in the nestling period. These results suggest that the greatest potential benefit females might obtain from multiple mating is the subsequent assistance of several males in caring for the offspring. I discuss the implications of these observations with the idea that competition among females for males brings about multiple mating as a female reproductive strategy. Copyright 1998 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed1999

Feather mites, pectoral muscle condition, wing length and plumage coloration of passerines.

I compared the feather mite (Acari, Proctophyllodidae) loads of moulting birds with features of the new plumage that they were growing. I examined 21 samples, each sample containing individuals of the same species, sex and age class (juvenile, yearling or adult). I used nine species: wren, Troglodytes troglodytes; dunnock, Prunella modularis; robin, Erithacus rubecula; blue tit, Parus caeruleus; great tit, P. major; chaffinch, Fringilla coelebs; greenfinch, Carduelis chloris; linnet, C. cannabina; and yellowhammer, Emberiza citrinella. As previously reported for the house finch, Carpodacus mexicanus, birds with more feather mites grew duller plumage and relatively shorter wings than less infested individuals of the same sex and age class. They also had lower protein reserves judged by the shape of their pectoral muscle. Mite load was usually a better predictor of coloration than pectoral muscle score, but the opposite was true for changes in wing length. In contrast to house finches, birds whose plumage was dull before moult had higher mite loads while moulting. Even if feather mites do not cause dull plumage or short wings, they appear to be a good indicator of birds in poor physiological condition. Copyright 1999 The Association for the Study of Animal Behaviour.

Journal Article↗
PubMed2006

Ticks (Ixodidae) from passerine birds in the Carpathian region.

Birds have been found to be a reservoir host of borrelia. In order to assess the situation in Slovakia ticks were collected from a total of 3057 mist-netted, ringed and released passerine birds in two locations at 500 m (in 2001) and 1000 m (in 2003) above sea level in the Bukovské Vrchy Hills, part of the Carpathian region in the north-east of Slovakia. A total of 75 birds of 16 species were infested with subadult ticks of Ixodes ricinus species (prevalence of parasitization 5%). Sixty-two larvae from 31 birds of 9 species and 80 nymphs from 52 birds of 15 species were found. The highest intensity of parasitization was observed on blackbirds Turdus merula, song thrushes T. philomelos and dunnocks Prunella modularis. Six Ixodes ricinus adult ticks were found on humans working with birds, and one I. ricinus female tick on their dog. In ticks, the presence of Rickettsia sp., Coxiella burnetii, Borrelia burgdorferi sensu lato and members of the Anaplasmataceae and Piroplasmidae, were investigated by polymerase chain reaction, followed by sequence analysis. Rickettsia sp. was found in 1 nymph from the European robin Erithacus rubecula, in 3 adult ticks (1 male, 2 females) from humans and in the tick from the dog. The closely related Ehrlichia- like species "Schotti variant" was detected in 1 nymph from the song thrush. Borrelia afzelii was identified in 1 male and B. garinii in 1 female tick collected on humans. Ixodes ricinus was found to be the vector of a wide spectrum of tick-borne pathogens in a mountainous area of the Carpathians. Because of the low yield of ticks and pathogens the importance of birds as reservoir hosts is still poorly understood.

Animals↗
PubMed2005

Clematis mandshurica protected to apoptosis of rat chondrocytes.

OBJECTIVE: To investigate the effect of SKI 306X, a purified extract from the mixture of three herbs, i.e. Clematis mandshurica, Trichosanthes kirilowii and Prunella vulgaris, on apoptosis in chondrocytes. DESIGN: Rat chondrocyte cell line RCJ3.1C.18 cells were incubated with 1 microM staurosporin and SKI 306X or each of its components. Cell viability was determined by trypan blue exclusion assay. Induction of apoptosis was determined by nuclear condensation or fragmentation after Hoechst staining. Amount of apoptosis was quantified both by nuclear morphology and flow cytometry. Expression level of Bcl-2, and caspase-3 and PARP activations were assayed by Western blot. RESULTS: SKI 306X significantly prevented staurosporin-induced apoptosis. Among its three components, only Clematis mandshurica significantly decreased the amount of staurosporin-induced apoptosis. Although the level of Bcl-2 expression was decreased after staurosporin treatment, it was sustained after the combination treatment with Clematis mandshurica. Whereas staurosporin induced the degradation of 32 kDa caspase-3 precursor and the production of 85-kDa cleavage products of PARP in a time-dependent fashion, Clematis mandshurica treatment prevented those manifestations. CONCLUSIONS: Pharmacological efficacy of SKI 306X protecting osteoarthritis in part may result from the inhibition of apoptosis in chondrocytes by Clematis mandshurica.

Animals↗
PubMed2000

A comparison of human immunodeficiency virus type-1 protease inhibition activities by the aqueous and methanol extracts of Chinese medicinal herbs.

The aqueous and methanol extracts of thirty-one herbs traditionally used as anti-fever remedies in China were screened for their in vitro inhibition on human immunodeficiency virus type-1 protease (HIV-1 PR). The activity of recombinant HIV-1 protease was determined by sequence-specific cleavage at the Tyr-Pro bond of the fluorogenic substrate (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)- Arg) or by HPLC anaylsis of the cleavage products after incubation of the enzyme with a synthetic peptide substrate (Acetyl-Ser-Gln-Asn-Tyr-Pro-Val-Val-amide). Among the herbal extracts examined, the aqueous extracts of Prunella vulgaris and Scutellaria baicalensis and the methanol extracts of Woodwardia unigemmata, Paeonica suffruticosa and Spatholobus suberectus elicited significant inhibition (>90%) at a concentration of 200 microg/ml.

HIV Protease↗
PubMed2001

A comparison of HIV-1 integrase inhibition by aqueous and methanol extracts of Chinese medicinal herbs.

The aqueous and methanol extracts of twenty herbs traditionally used in Chinese medicine were screened for anti-HIV-1 integrase activity in a non-radioactive ELISA-based HIV-1 integrase assay. The screening was performed at an herb extract concentration of 50 microg/ml. It was found that most of the aqueous and methanol herb extracts could elicit strong inhibition of HIV-I integrase activity. The inhibition was most likely due to tannins or polyphenolics in the herb extracts. In most of the herb extracts, 40-80% of the anti-HIV-1 integrase activity could be removed after passing through a minicolumn of polyamide resin. After removal of polyphenolic compounds, the methanol extract of Paeonia suffruticosa still exerted potent inhibition of HIV-1 integrase (EC50 = 15 microg/ml) and the aqueous extract of Prunella vulgaris caused moderate inhibition (EC50 = 45 microg/ml). The results support the view that herbs represent a rich source of anti-HIV compounds.

Drugs, Chinese Herbal↗
PubMed2000

Antioxidative and free radical scavenging activities of selected medicinal herbs.

The antioxidative and superoxide- and hydroxyl radical-scavenging activities and pro-oxidant effect of twelve selected medicinal herbs were studied. The aqueous extracts of Coptis chinensis, Paeonia suffruticosa, Prunella vulgaris and Senecio scandens exhibited the highest potency in inhibiting rat erythrocyte hemolysis and lipid peroxidation in rat kidney and brain homogenates. The aforementioned four herbs also demonstrated strong superoxide- and hydroxyl radical-scavenging activity, but exerted only a slight pro-oxidant effect.

Animals↗
PubMed2002

Prevalence of Borrelia burgdorferi sensu lato in the nightingale (Luscinia megarhynchos) and other passerine birds.

In order to explore the involvement of terrestrial birds in the ecology of Lyme disease spirochetes, we determined the presence of Borrelia burgdorferi s. I. DNA in tick larvae feeding on the hosts. Birds were caught at several study sites along the Rhine valley in SW Germany between August 1999 and March 2001. A total of 987 Ixodes ricinus larvae were collected from 225 birds belonging to 20 host species. The following four passerine species that have not yet been subject to detailed reservoir competence analyses were investigated: Nightingale (Luscinia megarhynchos), Dunnock (Prunella modularis), Chiffchaff (Phylloscopus collybita) and Reed warbler (Acrocephalus scirpaceus). Borrelia DNA was analysed in attached ticks and for one species, the Nightingale, blood samples were included. Borrelia DNA was amplified by means of the polymerase chain reaction (PCR) targeting the ospA gene of B. burgdorferi s. l. and 1-2 larvae per bird were analysed. On the basis of one larva per individual bird, Borrelia DNA could be detected in six out of nine larval ticks from the Nightingale, in one out of ten ticks from the Dunnock, in three of nine ticks from the Chiffchaff, and in two out of 21 larval ticks from Reed warblers. Five out of nine ticks removed from Robins Erithacus rubecula in winter were found to be Borrelia positive. Blood samples of Nightingales caught during the breeding period were ospA-gene-positive in 71 of 138 birds (51%).

Animals↗
PubMed2006

Where in Europe should we look for sources of the cutaneous trematode Collyriclum faba infections in migrating birds?

Cutaneous cysts with trematodes of Collyriclum faba have been found in birds during their spring and post-breeding migrations in the Czech Republic. During spring migrations, C. faba was found in one dunnock Prunella modularis, two European robins Erithacus rubecula, three common nightingales Luscinia megarhynchos, one song thrush Turdus philomelos and one great reed warbler Acrocephalus arundinaceus. During post-breeding migration, the same parasite was found in one garden warbler Sylvia borin, one whitehroat S. communis, three goldcrests Regulus regulus and one Eurasian treecreeper Certhia familiaris. The newly identified hosts of C. faba are dunnock, common nightingale, song thrush, great reed warbler and Eurasian treecreeper. Geographical areas of the birds' infection were identified from an analysis of reports on ringed birds of the same species, the time necessary for the development of cutaneous cysts with C. faba and the time of their survival, and hitherto known geographical areas of endemic occurrence of C. faba. It is presumed that birds trapped during spring migrations were infected in some montane and submontane regions in south-western Europe (the Alps, the Apennines). Birds infected during autumn migration or post-breeding vagrancy could have been infected in the Central European Carpathians, the region of C. faba endemic occurrence. For migrating birds, the impact of C. faba infections has not been hitherto assessed.

Animal Migration↗
PubMed1978

Salmonellosis in wild birds feeding at sewage treatment works.

Between June 1976 and August 1977 faeces were collected from 599 wild British birds caught during ringing operations at two sewage treatment works in south-east England. Samples were incubated with selenite-F broth to detect the presence of Salmonella. Salm. anatum was isolated from one bird, a Dunnock Prunella modularis an incidence of 0.17% of the total birds examined and 3.23% of the Dunnocks. Comparisons are drawn with previously reported studies and it is suggested that sewage treatment works play little part in the transmission of Salmonella infections to wild birds feeding there.

Animals↗
PubMed2002

Effects of SKI 306X, a new herbal agent, on proteoglycan degradation in cartilage explant culture and collagenase-induced rabbit osteoarthritis model.

OBJECTIVE: Protective effects of SKI 306X, a natural herbal product extracted from three herbs Clematis mandshurica, Trichosanthes kirilowii, and Prunella vulgaris, on articular cartilage was examined and compared with other osteoarthritis (OA) drugs using in vitro and in vivo models. METHODS: In vitro culture of rabbit articular cartilage explants was used as a model to measure the effects of drugs on the matrix degradation. The recombinant human interleukin-1alpha (rhIL-1alpha, 5 ng/ml) was added to induce proteoglycan (PG) degradation and the degree of PG degradation was assessed by measuring the amount of glycosaminoglycan (GAG) released into the culture medium. In in vivo experiment, collagenase was intraarticularly injected twice into the right knee joint of rabbits to induce OA-like change, and test agents were orally administered once a day for 28 days. The degrees of OA-like changes were evaluated through a histological examination. RESULTS: In vitro study revealed SKI 306X inhibited the degradation of PG in a concentration-dependent manner. Trichosanthes kirilowii, which is one of the major components of SKI 306X, also significantly inhibited the GAG release in cartilage explant culture at 0.3 and 0.1 mg/ml. Dexamethasone and NSAIDs, such as diclofenac and rofecoxib, had no significant effects on the suppression of PG degradation. In in vivo studies, OA-like degeneration of the articular cartilage and synovial tissue was induced by injecting collagenase into the right knee joint of mature rabbits. At a dose of 200 mg/kg, SKI 306X reduced the OA-like histological changes, whereas diclofenac had no effect at 10 mg/kg. CONCLUSION: These results indicate that SKI 306X inhibited PG degradation in cartilage explant culture, and its prophylactic administration significantly protected the knee joint of rabbit from OA-like change in collagenase-induced experimental OA model. This strongly suggests that SKI 306X can be a good OA agent with some cartilage protection activity.

Analysis of Variance↗
PubMed2004

Borrelia burgdorferi sensu lato infection in passerine birds from the Mazurian Lake region (Northeastern Poland).

The aim of the present study was to evaluate a potential role of different passerine birds species in Mazurian Lake region (northeast Poland) in the spread of Borrelia burgdorferi sensu lato, the spirochaete that causes Lyme disease. A total number of 1254 birds (representing 42 species) were captured during the 3-year study period. Blood samples were collected from birds and analyzed with a nested polymerase chain reaction technique in order to detect fragments of the pathogen DNA. Positive results were obtained in 4.2% of all blood samples. Specifically, B. burgdorferi s.l. were detected in tree pipit (Anthus Erivialis; 21.1% of 19 birds), dunnock (Prunella modularis; 15.8% of 19 birds), chaffinch (Fringilla coelebs; 12.7% of 166 birds), song thrush (Turdus philometos; 9.3% of 54 birds), nuthatch (Sitta euopea; 7.7% of 26 birds), hawfinch (Coccothraustes coccothroustes; 6.7% of 15 birds), robin (Erithacus rebecula; 5.1% of 256 birds), blackbird (Turdus merula; 4.2% of 71 birds) and wren (Troglodytes troglodytes; 3.7% of 27 birds). Additionally, the incidence of the infection was analyzed in relation to the habitat in which the birds resided (mixed coniferous forest or alder swamp forest), months of the study (from April to October), age and sex, but the differences were not statistically significant.

Age Factors↗
PubMed1994

Moderate inhibition of mutagenicity and carcinogenicity of benzo[a]pyrene, 1,6-dinitropyrene and 3,9-dinitrofluoranthene by Chinese medicinal herbs.

The activity of six Chinese medicinal herbs against the environmental mutagens and carcinogens benzo[a]pyrene (B[a]P), 1,6-dinitropyrene (1,6-diNP) and 3,9-dinitrofluoranthene (3,9-diNF) was determined. Samples of Prunella spica, Rheum palmatum, Polygonum multiflorum, Agrimonia pilosa, Ephedra sinica and Teitoutou were tested in an in vitro system. Antimutagenic activity against B[a]P was marked in the presence of extracts (boiled for 2 h in a water bath) whereas that against 1,6-diNP and 3,9-diNF varied from 20 to 86%. The differences in inhibition might be due to inactivation of metabolic enzymes. An extract of P. multiflorum was divided into ether, ethyl acetate and water soluble fractions, which were tested for antimutagenic activity against B[a]P. The antimutagenic action of the ethyl acetate soluble fraction was substantial and dose-dependent. Tannins and related compounds were the major components of the extract, of which epigallocatechin, epigallocatechin gallate, epicatechin gallate and tannic acid strongly inhibited the mutagenicity of B[a]P (2.5 micrograms/plate) in Salmonella typhimurium TA98 with S9 mix. To confirm the results of the in vitro test system, F344/DuCrj male rats were given a subcutaneous injection of B[a]P. Thereafter, they received water extracts of the six Chinese medicinal herbs for 50 weeks and were examined for tumors. The P. multiflorum extract significantly reduced the tumor incidence.

Animals↗
PubMed2004

Learning fine-tunes a specific response of nestlings to the parental alarm calls of their own species.

Parent birds often give alarm calls when a predator approaches their nest. However, it is not clear whether these alarms function to warn nestlings, nor is it known whether nestling responses are species-specific. The parental alarms of reed warblers, Acrocephalus scirpaceus ("churr"), dunnocks, Prunella modularis ("tseep"), and robins, Erithacus rubecula ("seee") are very different. Playback experiments revealed that nestlings of all three species ceased begging only in response to conspecific alarm calls. These differences between species in response are not simply a product of differences in raising environment, because when newly hatched dunnocks and robins were cross-fostered to nests of the other two species, they did not develop a response to their foster species' alarms. Instead, they still responded specifically to their own species' alarms. However, their response was less strong than that of nestlings raised normally by their own species. We suggest that, as in song development, a neural template enables nestlings to recognize features of their own species' signals from a background of irrelevant sounds, but learning then fine-tunes the response to reduce recognition errors.

Animals↗