Multimeric system of 99mTc-labeled gold nanoparticles conjugated to c[RGDfK(C)] for molecular imaging of tumor α(v)β(3) expression

Integrin α(V)β(3) plays a critical role in tumor angiogenesis and metastasis. Suitably radiolabeled cyclic RGD peptides can be used for noninvasive imaging of α(V)β(3) expression. The aim of this research was to prepare a multimeric system of technetium-99m-labeled gold nanoparticles conjugated to c[RGDfK(C)] and to evaluate its biological behavior as a potential radiopharmaceutical for molecular imaging of tumor angiogenesis. Hydrazinonicotinamide-GGC (HYNIC-GGC) and c[RGDfK(C)] peptides were synthesized and conjugated to gold nanoparticles (AuNP, 20 nm) by means of spontaneous reaction of the thiol groups of cysteine. The nanoconjugate was characterized by TEM, FT-IR, UV-vis, XPS, and Raman spectroscopy. To obtain (99m)Tc-HYNIC-GGC-AuNP-c[RGDfK(C)] ((99m)Tc-AuNP-RGD), the (99m)Tc-HYNIC-GGC radiopeptide was first prepared and added to 1.5 mL of AuNP solution (1 nM) followed by c[RGDfK(C)] (10 μL, 50 μM) at 18 °C with stirring for 15 min. Radiochemical purity (RP) was determined by size-exclusion HPLC and ITLC-SG analyses. In vitro binding studies were carried out in α(V)β(3) receptor-positive C6 glioma cancer cells. Biodistribution studies were accomplished in athymic mice with C6-induced tumors with blocked and nonblocked receptors, and images were obtained using a micro-SPECT/CT. TEM and spectroscopy techniques demonstrated that AuNPs were functionalized with peptides. RP was 96 ± 2% without postlabeling purification. (99m)Tc-AuNP-RGD showed specific recognition for α(V)β(3) integrins expressed in C6 cells, and 3 h after i.p. administration in mice, the tumor uptake was 8.18 ± 0.57% ID/g. Micro-SPECT/CT images showed evident tumor uptake. (99m)Tc-AuNP-RGD demonstrates properties suitable for use as a target-specific agent for molecular imaging of tumor α(V)β(3) expression.     

Genotoxic and cytotoxic effects induced by aluminum in the lymphocytes of the common carp (Cyprinus carpio)

Few studies have been made in regard to the effect of aluminum on the molecular and cellular structure and function of aquatic organisms; therefore, in the present report we determined the genotoxic and cytotoxic effects induced by the metal on the lymphocytes of carp (Cyprinus carpio). Three groups of fish were exposed to 0.05, 120, and 239 mg/L of aluminum (Al), respectively, by using Al₂ (SO₄)₃·7H₂O, and another group was included as control. The cells obtained were studied with the comet assay, flow cytometry, and the TUNEL method. With the first method we found a concentration and time dependent, significant increase in the amount of DNA damage induced by Al, and a higher damage when we evaluated the level of oxidized DNA. By applying flow cytometry we established that the metal induced a DNA content increase and ploidy modifications as well as apoptosis and disturbances of the cell cycle progression. With the last method we determined a significant increase in the amount of apoptotic cells, mainly in the 72–96 h period. Our results established that Al caused deleterious DNA and cellular effects in the tested organism, and they suggested the pertinence of evaluating toxicity induced by the metal in organisms living in contaminated water bodies.     

Induction of laccases in Trametes versicolor by aqueous wood extracts

The induction of laccase isoforms in Trametes versicolor HEMIM-9 by aqueous extracts (AE) from softwood and hardwood was studied. Samples of sawdust of Pinus sp., Cedrela sp., and Quercus sp. were boiled in water to obtain AE. Different volumes of each AE were added to fungal cultures to determine the amount of AE needed for the induction experiments. Laccase activity was assayed every 24 h for 15 days. The addition of each AE (50 to 150 μl) to the fungal cultures increased laccase production compared to the control (0.42 ± 0.01 U ml^?1). The highest laccase activities detected were 1.92 ± 0.15 U ml^?1 (pine), 1.87 ± 0.26 U ml^?1 (cedar), and 1.56 ± 0.34 U ml^?1 (oak); laccase productivities were also significantly increased. Larger volumes of any AE inhibited mycelial growth. Electrophoretic analysis revealed two laccase bands (lcc1 and lcc2) for all the treatments. However, when lcc2 was analyzed by isoelectric focusing, inducer-dependent isoform patterns composed of three (pine AE), four (oak AE), and six laccase bands (cedar AE) were observed. Thus, AE from softwood and hardwood had induction effects in T. versicolor HEMIM-9, as indicated by the increase in laccase activity and different isoform patterns. All of the enzymatic extracts were able to decolorize the dye Orange II. Dye decolorization was mainly influenced by pH. The optimum pH for decolorization was pH 5 (85 %), followed by pH 7 (50 %) and pH 3 (15 %). No significant differences in the dye decolorizing capacity were detected between the control and the differentially induced laccase extracts (oak, pine and cedar). This could be due to the catalytic activities of isoforms with pI 5.4 and 5.8, which were detected under all induction conditions.     

A simple protocol for extraction and detection of pyrazines produced by fungi in solid state fermentation

Summary A protocol for the extraction and detection of pyrazines produced by fungi in Solid State Fermentation that gave good recovery of these compounds was devised together with specific techniques for detection by Gas Chromatography (GC). The protocol involved solid-liquid and liquid-liquid extractions, concentration of the extracts and GC analysis. Extraction yields were measured from a model system in which synthetic pyrazines were added. Extracts were analysed with a gas chromatograph equipped with a thermo-ionic detector to allow specific pyrazine detection.     

Laccase–luminol chemiluminescence system: an investigation of substrate inhibition

Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase–luminol system, determine its kinetic parameters, and analyze the effects of protein and OHˉ concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase–luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 μM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase–luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification.     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-¹⁴C]linoleic acid or [5-¹⁴C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [¹⁴C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [¹⁴C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-¹⁴C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species.     

Rhizoctonia solani, an elicitor of 6-pentyl-α-pyrone production by Trichoderma harzianum in a two liquid phases, extractive fermentation system

6-pentyl-alpha-pyrone (6PP) production by Trichoderma harzianum, in an extractive fermentation system, was elicitated by Rhizoctonia solani. The extent of 6PP elicitation was related to the state of Rhizoctonia and to the Trichoderma inoculum type. The use of non-viable Rhizoctonia solani mycelium in mycelium-inoculated Trichoderma harzianum culture, yielded the maximal 6PP production (474 mg l(-1)) compared to control cultures (147 mg l(-1)) and decreased the process time from 192 to 96 h.     

Suppression Effects on Pineapple Soil-Borne Pathogens by <i>Crotalaria juncea</i>, Dolomitic Lime and Plastic Mulch Cover on MD-2 Hybrid Cultivar

The development and implementation of sustainable and environmentally friendly agricultural practices are indispensable as alternatives to pesticide use and to keep populations of soil-borne plant pathogens at levels that do not affect crop productivity. The present research evaluates the incidence of soil-borne phytopathogens on the pineapple variety MD-2, which was subjected to different treatments: Incorporation of Crotalaria juncea into the soil (organic amendment), application of dolomitic lime to soil (inorganic amendment), and the use of plastic mulch covering the soil. During the crop cycle (15 months), the following variables were evaluated: plant height (cm), fruit weight (kg·plant⁻¹ ), crop yield (ton·ha⁻¹ ), the bud root disease incidence caused by Phytophthora nicotianae, number of soil phytoparasitic nematodes and colony-forming-units (CFUs) of soil fungi and oomycetes. The results indicate that Crotalaria juncea treatment reduced the pathogen population (nematode and oomycetes) at levels that did not affect crop development, so that yield increased (18–20%). The incorporation of C. juncea into the soil as an organic amendment favors the populations of fungi disease suppressors (Trichoderma-Aspergillus). The phytoparasitic nematodes (Meloidogyne sp., Pratylenchus sp., and Mesocriconema sp.) and oomycetes (Phytophthora spp., and Pythium spp.) showed a reduction of their population levels by effects of organic amendment (C. juncea). The plastic mulch was also effective, probably due to the maintenance of optimal condition to crop growth and weed control. However, the dolomitic lime application had the poorest effect under the conditions of the study area on the variables analyzed. The described observations are characteristics of a system-based approach for the potential management of soil-borne pathogens of pineapple MD-2 in Veracruz, México.