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1.
Background: Zoonotic cutaneous leishmaniasis (ZCL) due to Leishmania major is increasing in many parts of Iran. This disease originally is a disease found in gerbils. Leishmania parasites are transmitted by sandflies that live and breed in gerbil burrows. Nested PCR amplified Leishmania ITS1-5.8S rRNA gene in both main reservoir host “Rhombomys opimus” and in the “Phlebotomus papatasi” main vector of ZCL, in Iran. Population differentiation and seasonal variation of sandflies were analyzed at a microgeographical level in order to identify any isolation by distance, habitat or seasons. Methods: Populations of sandflies were sampled from the edges of villages in Natanz, Isfahan province, Iran, using the Centers for Disease Control traps and sticky papers. Individual sandflies were identified based on external and internal morphological characters. Nested PCR protocols were used to amplify Leishmania ITS1-5.8S rRNA gene, which were shown to be species-specific via DNA sequence. Results: A total of 4500 sandflies were collected and identified. P. papatasi, Phlebotomus sergenti and Phlebotomus jacusieli from genus Phlebotomus and Sergentomyia sintoni and Sergentomyia clydei from genus Sergentomyia were identified in this region. P. papatasi was the most abundant sandfly in the collections. Ten out of 549 female P. papatasi and four out of 19 R. opimus were found to be infected with L. major. Conclusion: Seasonal activity of sandflies starts in June and ends in November. Abundance of P. papatasi was in September. Finding and molecular typing of L. major in P. papatasi and R. opimus confirmed the main vector and reservoir in this region.Key Words: Leishmania major, Sandflies, Leishmaniasis, Iran  相似文献   

2.

Background:

Cutaneous leishmaniasis is one of the most important parasitic diseases in humans. In this disease, one of the responsible organisms is Leishmania major, which is transmitted by sandfly vector. There are specific differences in biochemical profiles and metabolite pathways in logarithmic and stationary phases of Leishmania parasites. In the present study, 1H NMR spectroscopy was used to examine the metabolites outliers in the logarithmic and stationary phases of promastigotes in L. major to enlighten more about the transmission mechanism in metacyclogenesis of L. major.

Methods:

Promastigote was cultured, logarithmic and stationary phases were separated by the peanut agglutinin, and cell metabolites were extracted. 1H NMR spectroscopy was applied, and outliers were analyzed using principal component analysis.

Results:

The most altered metabolites in stationary and logarithmic phases were limited to citraconic acid, isopropylmalic acid, L-leucine, ornithine, caprylic acid, capric acid, and acetic acid.

Conclusion:

1H NMR spectroscopy could play an important role in the characterization of metabolites in biochemical pathways during a metacyclogenesis process. These metabolites and their pathways can help in exploiting a transmission mechanism in metacyclogenesis, and outcoming data might be used in the metabolic network reconstruction of L. major modeling. Key Words: Leishmania major, Metabolomics, Principal component analysis  相似文献   

3.
Background: Currently, there are no effective vaccines against leishmaniasis, and treatment using pentavalent antimonial drugs is occasionally effective and often toxic for patients. The PTR1 enzyme, which causes antifolate drug resistance in Leishmania parasites encoded by gene pteridine reductase 1 (ptr1). Since Leishmania lacks pteridine and folate metabolism, it cannot synthesize the pteridine moiety from guanine triphosphate. Therefore, it must produce pteridine using PTR1, an essential part of the salvage pathway that reduces oxidized pteridines. Thus, PTR1 is a good drug-target candidate for anti-Leishmania chemotherapy. The aim of this study was the cloning, expression, and enzymatic assay of the ptr1 gene from Iranian lizard Leishmania as a model for further studies on Leishmania. Methods: Promastigote DNA was extracted from the Iranian lizard Leishmania, and the ptr1 gene was amplified using specific primers. The PCR product was cloned, transformed into Escherichia coli strain JM109, and expressed. The recombinant protein (PTR1 enzyme) was then purified and assayed. Results: ptr1 gene was successfully amplified and cloned into expression vector. Recombinant protein (PTR1 enzyme) was purified using affinity chromatography and confirmed by Western-blot and dot blot using anti-Leishmania major PTR1 antibody and anti-T7 tag monoclonal antibody, respectively. The enzymatic assay was confirmed as PTR1 witch performed using 6-biopterin as a substrate and nicotinamide adenine dinucleotide phosphate as a coenzyme. Conclusion: Iranian lizard Leishmania ptr1 was expressed and enzymatic assay was performed successfully. Key Words: Pteridine reductase 1 (PTR1), Leishmania, Gene expression  相似文献   

4.

Background:

Molecular diversity of Leishmania major and its morphological changes have become a controversial issue among researchers. Some aspects of polymorphic shapes of amastigotes in clinical manifestations along with molecular variation were evaluated among suspected patients of some exceptional zoonotic cutaneous leishmaniasis locations in Northern Khuzestan, Southwestern Iran.

Methods:

Suspected patients (n = 165) were sampled in zoonotic cutaneous leishmaniasis foci over two consecutive years during 2012-2014. Prepared smears were stained, scaled and measured by ocular micrometer. DNA was extracted from smears; ITS-rDNA and Cytochrome b (Cyt b) markers were amplified, and PCR products were digested by BsuR1 restriction enzyme. Then the RFLP and sequencing were employed.

Results:

Only L. major was identified in patients containing regular amastigotes'' shapes (oval or round) with a size of 2-4 µm in each of classical wet, dry, mixed lesions. Meanwhile, irregular shapes (spindle, pear, or cigarette) were observed separately in non-classical wet lesions with more than 4 µm. Interestingly, a few amastigotes with an external flagellum were observed in some lesions. All sequenced ITS-rDNA and Cyt b genes of L. major did not show any molecular variation (χ 2 P > 0.05), including only one common haplotype (GenBank access no. EF413075).

Conclusion:

Findings proved that unlike other endemic foci, there is not a meaningful correlation between phenotypic and genotypic features of L. major isolates. This study is considered as the first comprehensive report to incriminate morphometric shapes of L. major amastigotes, which enhances our knowledge concerning their relevance with various clinical appearances and genotypic traits. Key Words: Leishmania major, Nuclear gene, Mitochondrial gene, Amastigote shapes, Iran  相似文献   

5.
High resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy were employed as complementary metabolomic tools to dereplicate the chemical profile of the new and antitrypanosomally active sponge-associated bacterium Actinokineospora sp. EG49 extract. Principal Component (PCA), hierarchical clustering (HCA), and orthogonal partial least square-discriminant analysis (OPLS-DA) were used to evaluate the HRFTMS and NMR data of crude extracts from four different fermentation approaches. Statistical analysis identified the best culture one-strain-many-compounds (OSMAC) condition and extraction procedure, which was used for the isolation of novel bioactive metabolites. As a result, two new O-glycosylated angucyclines, named actinosporins A (1) and B (2), were isolated from the broth culture of Actinokineospora sp. strain EG49, which was cultivated from the Red Sea sponge Spheciospongia vagabunda. The structures of actinosporins A and B were determined by 1D- and 2D-NMR techniques, as well as high resolution tandem mass spectrometry. Testing for antiparasitic properties showed that actinosporin A exhibited activity against Trypanosoma brucei brucei with an IC50 value of 15 µM; however no activity was detected against Leishmania major and Plasmodium falciparum, therefore suggesting its selectivity against the parasite Trypanosoma brucei brucei; the causative agent of sleeping sickness.  相似文献   

6.

Background

Heat shock proteins (HSP) are highly conserved molecules with many immunological functions. They are highly immunogenic with important role in cancer immunotherapy and in vaccine development against infectious diseases. As adjuvant, HSP can augment the immunogenicity of weak antigens and can stimulate antigen presenting cells. Although vaccines have been successful for many infectious diseases, progress in leishmaniasis has not been achieved. In this report, the protective effect of HSP-enriched soluble leishmania antigen (SLA) was determined.

Methods

BALB/c mice were immunized 3× with HSP-enriched SLA and SLA alone and 10 days after final boost. They were infected with 106 stationary phase promastigote of Leishmania major and immunological responses were followed until nine weeks.

Results

No significant differences were observed in lymphocyte proliferation, footpad swelling, parasite burden, nitric oxide or IL-12 cytokine between HSP-enriched or SLA groups. Although the levels of IFN-γ, IL-4, TGF-β, IgG1 and IgG2b were increased in both groups, IFN-γ was significantly higher in SLA group and IgG2a in HSP-enriched SLA.

Conclusion

These results indicate that HSP direct the immune system towards Th2 pattern and does not have protective role in L. major infection. Key Words: Leishmaniasis, Heat shock proteins (HSP), Adjuvant  相似文献   

7.
Background:To study the anticancer activity of Plantago major, we assessed the effect of ethanolic, methanolic and acetonic extracts of this plant on HCT-116, SW-480, and HEK-293 cell lines as control. Methods:The cytotoxic activity, biocompatibility, and toxicity were evaluated by MTT assay, hemolysis, and Artemia salina-LD50 (on mice) tests, respectively. The analysis of the extracts was performed by GC-MS analysis. Results:The results showed that all the extracts had the most antiproliferative properties on the HCT-116 cell line. The P. major root extract was more effective than the aerial parts, and IC50 values for ethanolic, methanolic and acetonic root extracts were 405.59, 470.16, and 82.26 µg/mL, respectively on HCT-116 cell line at 72 h. Hemolysis degree of the ethanolic extract of aerial and root parts were approximately 1% at 400 μg/mL.. Using the ethanolic extracts, the Artemia survived every concentration, and no toxicity was observed. One week after the oral administration of different parts of P. major extracts, none of the mice died, even those were administered 2000 mg/kg. The results of GC/MS analysis showed that P. major extracts contain potential anticancer compounds, such as stearic acid (8.61%) in aerial parts of methanolic extract and 1,2- Benzenedicarboxylic acid, mono(2-ethylhexyl)ester (88.07% and 40.63%) in aerial and root parts of acetonic extract of P. major. Conclusions:Our findings suggest that the P. major is a source of potential compounds with antiproliferative properties. Key Words: Gas chromatography-mass spectrometry, HCT-116 cells, Hemolysis, Lethal dose 50  相似文献   

8.
Background: The cultured mesenchymal stem cells (MSC) have been used in many clinical trials; however, there are still some concerns about the cultural conditions. One concern is related to the use of FBS as a widely used xenogeneic supplement in the culture system. Human platelet-rich plasma (hPRP) is a candidate replacement for FBS. In this study, the effect of hPRP on MSC proliferation and osteogenic differentiation has been evaluated. Methods: Human adipose-derived stem cells (hADSC) were expanded. Cells from the third passage were characterized by flow cytometric analysis and used for in vitro experiments. Resazurin and alizarin red stains were used for cell proliferation and osteogenic differentiation assays, respectively. Results: Treatment with hPRP resulted in a statistically significant increase in cell proliferation compare to the negative control group (P<0.001). Cell proliferation in the 15% hPRP group was also significantly higher than that in the 10% hPRP group (P<0.05). Additionally, it caused less osteogenic differentiation of the hADSC compared to the FBS (P<0.001), but in comparison to negative control, it caused acceptable mineralization (P<0.001). Conclusion: These findings indicate that hPRP not only improves the proliferation but also it can be a suitable substitution in osteogenic differentiation for clinical purposes. However, the clinical application value of hPRP still needs more investigation. Key Words: Platelet-Rich Plasma, Adipose tissue, Stem Cells, Cell differentiation, Cell proliferation  相似文献   

9.
This study evaluated the anti-Leishmania amazonensis activity of a lipophilic extract from the brown alga Stypopodium zonale and atomaric acid, its major compound. Our initial results revealed high inhibitory activity for intracellular amastigotes in a dose-dependent manner and an IC50 of 0.27 μg/mL. Due to its high anti-Leishmania activity and low toxicity toward host cells, we fractionated the lipophilic extract. A major meroditerpene in this extract, atomaric acid, and its methyl ester derivative, which was obtained by a methylation procedure, were identified by nuclear magnetic resonance (NMR) spectroscopy. Both compounds inhibited intracellular amastigotes, with IC50 values of 20.2 μM (9 μg/mL) and 22.9 μM (10 μg/mL), and selectivity indexes of 8.4 μM and 11.5 μM. The leishmanicidal activity of both meroditerpenes was independent of nitric oxide (NO) production, but the generation of reactive oxygen species (ROS) may be at least partially responsible for the amastigote killing. Our results suggest that the lipophilic extract of S. zonale may represent an important source of compounds for the development of anti-Leishmania drugs.  相似文献   

10.
Organic extracts of 20 species of French seaweed have been screened against Trypanosoma brucei rhodesiense trypomastigotes, the parasite responsible for sleeping sickness. These extracts have previously shown potent antiprotozoal activities in vitro against Plasmodium falciparum and Leishmania donovani. The selectivity of the extracts was also evaluated by testing cytotoxicity on a mammalian L6 cell line. The ethyl acetate extract of the brown seaweed, Bifurcaria bifurcata, showed strong trypanocidal activity with a mild selectivity index (IC50 = 0.53 µg/mL; selectivity index (SI) = 11.6). Bio-guided fractionation led to the isolation of eleganolone, the main diterpenoid isolated from this species. Eleganolone contributes only mildly to the trypanocidal activity of the ethyl acetate extract (IC50 = 45.0 µM, SI = 4.0). However, a selective activity against P. falciparum erythrocytic stages in vitro has been highlighted (IC50 = 7.9 µM, SI = 21.6).  相似文献   

11.
《Plant Production Science》2013,16(3):216-222
Abstract

The present study was conducted to establish an efficient protocol of plantlet regeneration through somatic embryogenesis in garlic (Allium sativum L.). Root tips measuring 2 to 3 mm were excised and cultured on agar-solidified MS medium containing various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) for callus and embryo formation. The optimum concentration of 2,4-D was 0.5 μM. At a concentration higher than 1.0 μM, 2,4-D had an inhibitory effect on callus and embryo formation. Embryos germinated and formed rooted plantlets on MS solid medium containing 5.0 μM kinetin. The number of plantlets regenerated per root tip expiant depended on the concentration of 2,4-D in the callus initiation medium. The plantlets were established in the soil after acclimatization in a growth cabinet. Somatic embryos were morphologically characterized by scanning electron microscopy (SEM).  相似文献   

12.
《Plant Production Science》2013,16(4):392-397
abstract

The effects of various carbon sources, sucrose, glucose and fructose alone or in combination on the in vitro growth of banana plantlets were studied. Banana plants were cultured on the media supplemented with these carbon sources at 0.08 M for 13 weeks. The water potential of the medium was the highest in the medium supplemented with sucrose + glucose (-0.3 MPa), and was significantly lower in the medium supplemented with fructose alone or in combination with other carbon sources (-0.7 to -1.0 MPa) than in the other media. The leaf water potential was also the highest in the plants cultured on the medium supplemented with sucrose + glucose, and lowest in the plants cultured on that with fructose. The leaf water potential of plants cultured on sucrose + glucose, sucrose and glucose correlated well with their growth and photosynthetic activity, but the correlation was not observed in the plants cultured on fructose alone or in combination with other carbon sources. Plants cultured on fructose had a lower chlorophyll content (400 ptg dm-2) and lower photosynthetic rate (3 μmol02 m-2 s-1) than those cultured on sucrose + glucose (15,950 μgdm-2 for chlorophyll and 8.5 μmol02 m-2 s-1 for photosynthesis), and these differences were statistically significant. Both chlorophyll content and photosynthetic oxygen evolution were the highest in the plants cultured on sucrose + glucose, and the superior growth of plants on this medium was attributed to their high photosynthetic efficiency.  相似文献   

13.
The sulfated polysaccharides from Solieria filiformis (Sf), Botryocladia occidentalis (Bo), Caulerpa racemosa (Cr) and Gracilaria caudata (Gc) were extracted and extensively purified. These compounds were then subjected to in vitro assays to evaluate the inhibition of these polysaccharides on the growth of Leishmania (L.) amazonensis promastigotes. Under the same assay conditions, only three of the four sulfated polysaccharides were active against L. amazonensis, and the polysaccharide purified from Cr was the most potent (EC50 value: 34.5 μg/mL). The polysaccharides derived from Bo and Sf demonstrated moderate anti-leishmanial activity (EC50 values of 63.7 μg/mL and 137.4 μg/mL). In addition, we also performed in vitro cytotoxic assays toward peritoneal macrophages and J774 macrophages. For the in vitro cytotoxicity assay employing J774 cells, all of the sulfated polysaccharides decreased cell survival, with CC50 values of 27.3 μg/mL, 49.3 μg/mL, 73.2 μg/mL, and 99.8 μg/mL for Bo, Cr, Gc, and Sf, respectively. However, none of the sulfated polysaccharides reduced the cell growth rate of the peritoneal macrophages. These results suggest that macroalgae contain compounds with various chemical properties that can control specific pathogens. According to our results, the assayed sulfated polysaccharides were able to modulate the growth rate and cell survival of Leishmania (L.) amazonensis promastigotes in in vitro assays, and these effects involved the interaction of the sulfated polysaccharides on the cell membrane of the parasites.  相似文献   

14.
《Plant Production Science》2013,16(3):207-210
Abstract

This paper describes the effects of auxin added to the culture medium on main and branch root formation of banana (Musa spp.) shoots and growth characters of the plantlet rooted on the medium with and without auxin. Banana shoots cultured in vitro on Murashige and Skoog medium supplemented with 2 μM 1-naphthylacetic acid (NAA), rooted earlier and also had more adventitious roots than those cultured on the medium without NAA. However, the adventitious roots formed on the medium without NAA showed more lateral branching. Plant height and number of leaves per plantlet in in vitro culture were not influenced by the addition of NAA but under nursery conditions, plantlets rooted without NAA showed better growth in terms of days to the appearance of new leaf, plant height and number of leaves per plant. This might be due to the presence of abundant lateral roots. Even though auxins are generally known to promote rooting, NAA inhibited the formation of lateral roots in Banana plants.  相似文献   

15.
Background:CE is a zoonotic parasitic infection caused by Echinococcus granulosus worldwide and is associated with economic losses among livestock animals. EG95 is an immunogenic antigen from the E. granulosus. Lactococcus lactis has been prested as a safe vehicle for antigen delivery. The goal of this study was to design a novel L. lactis strain displaying EG95 as a vaccine delivery system. Methods:The eg95 encoding gene fragment fused to the M6 anchoring protein was cloned into the pNZ7021 vector, and L. lactis NZ9000 displaying recombinant EG95 was constructed. The expression of an approximately 32-kDa EG95 protein was confirmed by Western blotting and immunofluorescence analysis. The immune responses were evaluated in BALB/c mice immunized orally and subcutaneously with the live and killed recombinant L. lactis, respectively. Results:Total IgG level in mice immunized with heat-killed recombinant L. lactis (pNZ7021-eg95) significantly increased compared to the control group. sIgA was significantly higher in mice received live recombinant L. lactis (pNZ7021-eg95) compared to the control mice. Splenic lymphocytes from immunized mice represented the high levels of IFN-γ and the low-levels of IL-4 and IL-10. Conclusion:Our results indicate that immunization with EG95-expressing L. lactis can induce both specific humoral and cellular immune responses in mice. Key Words: Echinococcus granulosus, Lactococcus lactis, Immunization, Vaccines  相似文献   

16.
The culture supernatant of Paenibacillus sp. TKU036, a bacterium isolated from Taiwanese soils, showed high antioxidant activity (85%) when cultured in a squid pen powder (SPP)-containing medium at 37 °C for three days. Homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) was isolated and found to be the major antioxidant in the culture supernatant of the SPP-containing medium fermented by Paenibacillus sp. TKU036. Tryptophan was also present in the culture supernatant. The results of high-performance liquid chromatography (HPLC) fingerprinting showed that HGA and tryptophan were produced via fermentation but did not pre-exist in the unfermented SPP-containing medium. Neither HGA nor tryptophan was found in the culture supernatants obtained from the fermentation of nutrient broth or other chitinous material, i.e., medium containing shrimp head powder, by Paenibacillus sp. TKU036. The production of HGA via microorganisms has rarely been reported. In this study, we found that squid pen was a potential carbon and nitrogen source for Paenibacillus sp. Tryptophan (105 mg/L) and HGA (60 mg/L) were recovered from the culture supernatant. The isolated HGA was found to have higher antioxidant activity (IC50 = 6.9 μg/mL) than α-tocopherol (IC50 = 17.6 μg/mL). The anti-inflammatory activity of the isolated HGA (IC50 = 10.14 μg/mL) was lower than that of quercetin (IC50 = 1.14 μg/mL). As a result, squid pen, a fishery processing byproduct, is a valuable material for the production of tryptophan and the antioxidant and anti-inflammatory HGA via microbial conversion.  相似文献   

17.
《Plant Production Science》2013,16(4):301-304
Abstract

In Crotalaria juncea L., adventitious buds were formed in cotyledonary expiants cultured on 0.8% agar-solidified 1/2 MS basal medium containing B5 vitamins, 1.0 or 0.5 mg L–1 NAA, 5.0 or 10.0 mg L–1 BA and 3% sucrose. The frequency of adventitious bud formation was 30-45% in all combinations of NAA and BA. In histological observations, prominent mitotic figures were observed in several cells of the subepidermal palisade layers on the adaxial side of the expiants in contact with medium after 3 days of culture. Calli were formed within 6 days of culture. After 10 days of culture, numerous mature tracheary elements were produced at random in the proliferated regions, and cell divisions at the superficial region led to the formation of the meristematic structure. The shoot apex of the seedling produced numerous trichomes from superficial cells, but the adventitious bud formed on the cotyledon produced no trichomes. Initiation of the meristematic region in the expiant could be used as a target site for gene transfer experiments.  相似文献   

18.
Crude extracts and column fractions from the red algae Asparagopsis taxiformis and A. armata from the Strait of Messina (Italy) were screened for the production of antimicrobial compounds. Extracts from both species revealed remarkable antiprotozoal activity against Leishmania, revealing such algae as a great source of natural antiprotozoal products.  相似文献   

19.
The present study report a protocol for the efficient in vitro propagation of kenaf (Hibiscus cannabinus L., an industrial crop having high cellulosic fiber content) on hormone free MS medium using the shoot apex and nodal explants. Shoot tips and nodes were isolated from 15 days old seedlings cultivated on MS medium. Different combinations and concentrations of auxin/cytokinin were used and added to the MS medium to assess the shoot and root induction of theses explants. Several subcultures were drived in order to enhance the multiplication rate. Healthy and well developed in vitro propagated shoots were transferred for acclimatization under greenhouse conditions in pots filled with different substrates (sand + compost or perlite). Our results showed that shoots could elongate and root within 4-6 weeks on MS basal medium without any callus formation. However, addition of growth regulators to the MS medium leaded to a decrease in shoot and root induction rates. Indeed, the highest shoot regeneration frequency (90.5%) was obtained on MS control medium. Elongated shoots were transferred onto the same hormone free MS medium using five subcultures where the multiplication rate reached the highest value (3.66) at the fifth and last step. The in vitro rooted plantlets were acclimatized in greenhouse and successfully transplanted to natural conditions with 70% survival.  相似文献   

20.
To develop a protocol of Eleutherococcus koreanum Nakai adventitious root culture for production of biomass and bioactive compounds through bioreactors, different strengths of Murashige and Skoog (MS) medium were tested. After 5 weeks of culture, root growth at low salt strengths (1/4, 1/2, and 3/4 MS) was better than that at high salt strengths (1 and 2 MS), and the highest fresh and dry weight was achieved at 1/2 MS. The roots cultured at strengths exceeding 1 MS showed physiological abnormalities such as shorter, thicker, and less numerous roots compared to other treatments. Strengths over 1 MS caused physical dehydration that stimulated proline accumulation in the roots and decreased water potential in the medium because of high osmotic stress. Total production of 5 target compounds (per 1 L medium), eleutheroside B and E, chlorogenic acid, total phenolics, and flavonoids, was decreased with increasing medium salt strength. However, the highest total production of eleutheroside B and E (per 1 L medium), the main bioactive compounds in this plant, were observed at 1/2 and 3/4 MS, respectively. Therefore, 1/2 MS is a suitable medium salt strength for both biomass and bioactive compound productions, and optimization of bioreactor culture conditions will benefit the large-scale production of E. koreanum-derived bioactive compounds for commercialization.  相似文献   

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