Essential oil composition and total phenolic content in Cupressus arizonica G. in response to microbial inoculation under water stress conditions.

Arizona Cypress is one of the drought-resistant, aromatic, and aesthetically pleasing trees having several pharmacological uses. Certain microorganisms contribute to the secondary metabolism and synthesis of bioactive compounds in aromatic and medicinal plants. This study aimed to determine the photosynthetic pigments, total phenolic content, antioxidant capacity, and essential oil composition of Arizona cypress under two irrigation regimes and microbial inoculations. We established a factorial experiment with three mycorrhizae inoculations (Rhizophagus irregularis, Funneliformis mosseae, and a mixture of R. irregularis and F. mosseae), a rhizobacterium inoculation (Pseudomonas fluorescens), and two irrigation regimes (well-watered and water stress). Under the water stress regime, seedlings inoculated with F. mosseae (0.46%) and non-inoculated control plants (0.29%) had the highest and lowest essential oil contents, respectively. GC-MS analysis revealed that limonen, a-pinene, terpinen-4-ol, and umbellulone were the most abundant compounds in the seedlings and treatments under study. The water stress regime had a significant and dominant effect on essential oil and antioxidant capacity, whereas seedling growth and photosynthetic pigments tended to decrease under stress conditions. However, co-inoculation of seedlings with mycorrhizae and the bacterium resulted in an increase in phenolic compounds and carotenoids. Under conditions of water stress and mycorrhizal symbiosis, the results of the current study may help increase the level of valuable compounds in Arizona cypress for further pharmaceutical applications.

Antimicrobial activity of biosynthesized silver nanoparticles, amoxicillin, and glass-ionomer cement againstStreptococcus mutansandStaphylococcus aureus.

Background. The development of dental caries is associated with various microorganisms and secondary caries formation is the main cause of restorations failure. The advice for restorative dental materials that have antimicrobial properties has stimulated the introduction of materials containing different antibacterial agents.Objectives. The present study has been designed to synthesize silver nanoparticles (AgNPs) and incorporate AgNPs and amoxicillin into glass ionomer cement (GIC) to synergize its effect on oral microbes. The effect of the added antimicrobial agents on compressive strength (CS) of GIC was also evaluated.Material and methods. Biosynthesis of AgNPs was done usingCupressus macrocarpaextract and AgNPs were characterized. A total of 120 disc-shaped specimens were prepared and classified into 4 main groups where Group A includes conventional GIC, Groups B and C include GIC with AgNPs or amoxicillin, respectively, while Group D included GIC with both AgNPs and amoxicillin. Each group was tested for the antimicrobial activity against bothStreptococcus mutans(S. mutans) andStaphylococcus aureus(S. aureus). The distribution of biofilm was examined via a scanning electron microscope. The CS of the tested material was measured using a Material Test System.Results. The UV-visible spectrum showed a peak of 429 nm. Transmission electron microscopy, x-ray diffraction pattern and Fourier transform infrared analysis confirmed the formation of AgNPs with spherical to oblong polydispersed particles of diameter in the range of 13.5-25.8 nm. The maximum inhibitory zone was recorded for group D against both tested bacteria with a mean of 29 mm at first 24 h period to 15 mm at three weeks and showed antimicrobial rate 92.2% and 92.56%, against both strains, respectively. Additionally, group D disintegrated the structure ofS. aureusbiofilm and even kill bacteria in the biofilms. The addition of AgNPs and amoxicillin caused an insignificant effect on CS of GIC.Conclusion.TheAgNPs showed a synergistic effect in combination with amoxicillin and GIC dental restorative material against studied microorganisms. The agents can be safely added with minimal effect on the mechanical properties of the original cement.

Ethanol-based organosolv treatment with trace hydrochloric acid improves the enzymatic digestibility of Japanese cypress (Chamaecyparis obtusa) by exposing nanofibers on the surface.

The effects of adding trace acids in ethanol based organosolv treatment were investigated to increase the enzymatic digestibility of Japanese cypress. A high glucose yield (60%) in the enzymatic hydrolysis was obtained by treating the sample at 170 °C for 45 min in 50% ethanol liquor containing 0.4% hydrochloric acid. Moreover, the enzymatic digestibility of the treated sample was improved to ∼70% by changing the enzyme from acremonium cellulase to Accellerase1500. Field emission scanning electron microscopy revealed the presence of lignin droplets and partial cellulose nanofibers on the surface of the treated sample. Simultaneous saccharification and fermentation of the treated samples using thermotolerant yeast (Kluyveromyces marxianus NBRC1777) was tested. A high ethanol concentration (22.1 g/L) was achieved using the EtOH50/W50/HCl0.4-treated sample compared with samples from other treatments.

A rapid biosynthesis route for the preparation of gold nanoparticles by aqueous extract of cypress leaves at room temperature.

In the present study, green synthesis of gold nanoparticles was reported using the aqueous extract of cypress leaves. The reduction of gold salt with the extract of cypress leaves resulted in the formation of gold nanoparticles. Effects of extract concentration and extract pH were investigated on the size of the nanoparticles. It was found that the average particle size of synthesized gold nanoparticles depends strongly on extract concentration and extract pH. FT-IR spectroscopy showed that bioorganic capping molecules were bound to the surface of particles. X-ray techniques confirmed the formation of gold nanoparticles and their crystalline structure. The inductively coupled plasma atomic emission spectroscopy analysis displayed that the reaction progress is higher than 90% at room temperature. Gold nanoparticles were mostly spherical in shape along with some irregular shapes. Cypress is an evergreen plant and its leaves are easily available in all four seasons. Also, the rate of the reaction was high and it was completed in only 10min. For these reasons, this method is cost-effective and environmentally friendly. Thus, it can be used in the synthesis of gold nanoparticles instead of chemical methods and other biosynthesis approaches.

Insights into a hydration regulating system in Cupressus pollen grains.

BACKGROUND AND AIMS: Hydration, rupture and exine opening due to the sudden and large expansion of intine are typical of taxoid-type pollen grains. A hemispheric outgrowth external to the exine was observed on Cupressus and Juniperus pollen grains before the intine swelling and exine release. However, the actual existence of this permanent or temporary structure and its precise role in pollen hydration is still being debated. The aim of this paper is to collect information on the actual presence of this peculiar outgrowth on the surface of the Cupressus pollen grain, its structure, composition and function. METHODS: Pollen grains of several Cupressus species were observed using various techniques and methodologies, under light and fluorescence microscopy, phase-contrast microscopy, confocal microscopy, scanning electron microscopy, and an environmental scanning electron microscope. Observations were also performed on other species with taxoid-type pollen grains. KEY RESULTS: A temporary structure located just above the pore was observed on Cupressus pollen grains, as well as on other taxoid-type pollens. It is hemispheric, layered, and consists of polysaccharides and proteins. The latter are confined to its inner part. Its presence seems to regulate the entrance of water into the grains at the beginning of pollen hydration. CONCLUSIONS: The presence of a temporary structure over the pore of taxoid-type pollen grains was confirmed and its structure was resolved using several stains and observation techniques. This structure plays a role in the first phases of pollen hydration.

A modified protocol for RNA isolation from high polysaccharide containing Cupressus arizonica pollen. Applications for RT-PCR and phage display library construction.

RNA isolation is the first step in the study of gene expression and recombinant protein production. However, the isolation of high quantity and high-quality RNA from tissues containing large amounts of polysaccharides has proven to be a difficult process. Cupressus arizonica pollen, in addition to containing high polysaccharide levels, is a challenging starting material for RNA isolation due to the roughness of the pollen grain's walls. Here, we describe an improved technique for RNA isolation from C. arizonica pollen grains. The protocol includes a special disruption and homogenization process as well as a two-step modified RNA isolation technique which consists of an acid phenol extraction followed by a final cleanup using a commercial kit. Resulting RNA proved to be free of contaminants as determined by UV spectrophotometry. The quality of the RNA was analyzed on a bioanalyzer and showed visible 25S and 18S bands. This RNA was successfully used in downstream applications such as RT-PCR and phage display library construction.

Comparative study of the pollen protein contents in two major varieties of Cupressus arizonica planted in Tehran.

During past few years, the Cupressus arizonica has been abundantly planted in Tehran, causing a significant increase of allergic diseases from the middle of winter to the beginning of spring. The aim of this study was the comparison of pollen protein content in two major varieties of C. arizonica planted in Tehran, including C. arizonica var. arizonica and C. arizonica var. glabra, in order to determine pollen's specificity of each variety and also to find out whether environmental conditions can influence pollen protein contents and its allergenic components. Pollen grains were directly collected from mature male cones of trees planted in different areas of the city. Pollen's proteins were extracted, and were analyzed by SDS PAGE. Total protein content of pollen extracts was measured by Bradford assay. Our investigations revealed noticeable differences in protein content of each variety. Bradford protein assay showed a higher total protein content in C. arizonica var. arizonica pollen extracts. A new major protein, with an approximate molecular weight of about 35 kDa was detected in both varieties. Immunoblotting using the serum of a cypress allergic subject showed that the protein with 35 kDa was also the major allergen of both varieties in pollen extracts. These results showed that there are some intraspecie specificities in Arizona cypress pollens. The major allergen of Cupresuss arizonica pollen, Cup a 1 (45 kDa), has been reported as the most representative protein in pollen extracts of Mediterranean countries, but in our autochthon extracts of both varieties, a protein band at 35 kDa was more representative. These observations seem to indicate that C. arizonica pollen protein content may be influenced by environmental conditions. Moreover, Immunoblot results provided a reliable indication on the allergenic activity of this new major protein band at 35 kDa. The confirmation of these aspects would facilitate the preparation of an effective extract, improving the diagnosis of the allergy to the Cupressus arizonica pollen.

Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli.

The cDNA encoding an isoform of the cypress major pollen allergen, Cup a1.02, has been cloned and expressed in Escherichia coli as a N-terminal 6x His-tagged protein. To increase recovery, Cup a1.02 was expressed at high levels exploiting the T5 strong promoter and led to accumulate as inclusion bodies. The insoluble purified aggregates were solubilized in 6 M guanidine hydrochloride, immobilized using nickel-chelating affinity chromatography, and successfully refolded by controlled removal of the chaotropic reagent. Enhanced protein refolding was observed by reducing the protein concentration at 0.6-0.8 mg/ml. SDS-PAGE and gel filtration chromatography indicated an apparent molecular mass of approximately 40 kDa and the occurrence of the protein as monomers. The reconstituted fusion protein displayed the same immunological properties of the native Cup a1.02 protein as proven by IgE immunoreactivity. Immunoblotting, ELISA, and histamine release test showed that the tag did not preclude the protein functionality hence validating its correct three-dimensional folding. The protein fold was also assessed by CD spectroscopy and deconvolution of the spectrum allowed to estimate the secondary structure as a prevalence of beta structures (higher than 60%) and a small contribution from alpha helices (less than 12%). The reported procedure has proven to be useful for the production of multi-milligrams of recombinant Cup a1.02 allergen suitable for structural biology studies and for the molecular and functional characterization of the IgE binding sites.

Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated Cupressus lusitanica cell cultures.

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] rapidly accumulates in elicited Cupressus lusitanica Mill. cultured cells by 4- to 5-fold over the control, and then it is metabolized. Correspondingly, phospholipase C (PLC) activity toward phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is stimulated to high levels by the elicitor and then decreases whereas Ins(1,4,5)P(3) phosphatase activity declines at the beginning of elicitation and increases later. These observations indicate that elicitor-induced biosynthesis and dephosphorylation of Ins(1,4,5)P(3) occur simultaneously and that the Ins(1,4,5)P(3) level may be regulated by both PtdIns(4,5)P(2)-PLC and Ins(1,4,5)P(3) phosphatases. Studies on the properties of PLC and Ins(1,4,5)P(3) phosphatases indicate that PLC activity toward PtdIns(4,5)P(2) was optimal at a lower Ca(2+) concentration than activity toward phosphatidylinositol whereas Ins(1,4,5)P(3) phosphatase activity is inhibited by high Ca(2+) concentration. This suggests that Ins(1,4,5)P(3) biosynthesis and degradation may be regulated by free cytosolic Ca(2+). In addition, a relationship between Ins(1,4,5)P(3) signaling and accumulation of a phytoalexin (beta-thujaplicin) is suggested because inhibition or promotion of Ins(1,4,5)P(3) accumulation by neomycin or LiCl affects elicitor-induced production of beta-thujaplicin. Moreover, ruthenium red inhibits elicitor-induced accumulation of beta-thujaplicin while thapsigargin alone induces beta-thujaplicin accumulation. These results suggest that Ca(2+) released from intracellular calcium stores may mediate elicitor-induced accumulation of beta-thujaplicin via an Ins(1,4,5)P(3) signaling pathway, since it is widely accepted that Ins(1,4,5)P(3) can mobilize Ca(2+) from intracellular stores. This work demonstrates an elicitor-triggered Ins(1,4,5)P(3) turnover, defines its enzymatic basis and regulation, and suggests a role for Ins(1,4,5)P(3) in elicitor-induced phytoalexin accumulation via a Ca(2+) signaling pathway.

Toxic effects of six plant oils alone and in combination with controlled atmosphere on Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Six plant essential oils alone as repellent and fumigant, and in combination with the controlled atmosphere against Liposcelis bostrychophila Badonnel were assessed in the laboratory. These essential oils were extracted from the leaves of six source plants: Citrus tangerina Tanaka, Citrus aurantium L., Citrus bergamia Risso et Poiteau, Pinus sylvestris L., Cupressus funebris End]., and Eucalyptus citriodora Hook. The repellency test indicated that L. bostrychophila adults were repelled by filter paper strips treated with six essential oils. Of these essential oils, the C. funebris oil was most effective followed by that of F. sylvestris, C. tangerina, C. bergamia, and E. citriodora. The average repellency of the C. aurantium oil against L. bostrychophila adults was significantly lower than other five test oils by day 14. These essential oils had a high level of toxicity in the fumigation assay against L. bostrychophila adults at both 10 and 20 ppm. When combined with two controlled atmosphere treatments (12% CO2 + 9% O2, and 10% CO2 + 5% O2, balanced N2), the toxicity of plant oils was enhanced significantly.