Determination of trace levels of fatty acid metal salts by high-performance liquid chromatography with fluorescence prelabeling

A simple method is described for picomole determinations of fatty acid metal salts. Fatty acid salts are directly labeled with 4-bromomethyl-7-methoxycoumarin in the presence of excess ethylenediaminetetraacetic acid tripotassium salt without any solvent extractions. The fluorescence derivatives of fatty acids are separated by reverse-phase high-performance liquid chromatography followed by fluorometric detection. The response of each fatty acid (C8-C18) calcium salt is linear from 1 to 50 micrograms/ml of samples. The detection limit is about 7 pmol. Good recoveries are obtained for the calcium salts of myrystic acid and soap (C8-C18, C18:1,2). The new method is successfully applied to the study on biodegradation of fatty acids in river water.     

Kinetic characteristics of lipids of mammalian tissues in autooxidation reactions

We generalize the results of multiyear studies of the level of antioxidative activity of lipids isolated from tissues of laboratory rodents of different species and lines. A classification of lipids according to their ability to inhibit thermal autooxidation of methyl oleate is proposed. The involvement of lipids in low-temperature autooxidation reactions at the radical initiation and chain propagation stages was proved using the model developed. In addition to antioxidative activity, the initial content of peroxides in lipids, determined by their degree of unsaturation, and the antiperoxide activity of lipids are proposed for quantitative estimation of the kinetic characteristics of lipids of mammalian tissues. The dependence of effects on the rate of radical initiation in the system is shown, which is determined by the influence of the physicochemical properties of lipids on the coordination of relationships and balance of biochemical functions in biological objects differing in the intensity of oxidation processes.     

Real-time measurement of the rate of sarcoplasmic reticulum calcium pumping in skinned muscle fibers

A method and a device had been developed to directly measure the accumulation of calcium in the sarcoplasmic reticulum and its release from the sarcoplasmic reticulum, depending on the free Ca²⁺ concentration in the solution. The sarcoplasmic reticulum occupies to 30% of the volume of the swim bladder muscles of the oyster toadfish Opsanus tau. To isolate and skin muscle fibers and to remove the accumulated calcium from the sarcoplasmic reticulum, a set of solutions containing EGTA as a pCa buffer was used. To measure the calcium exchange between a fiber ～10 nl in volume and the solution in a 5-μl cuvette, instead of EGTA, 50–100 μM FURA2 or bisFURA2 was used both as pCa buffer and as a fluorescent indicator of the calcium concentration in the cuvette. An increase in fluorescence intensity meant an increase in the free FURA concentration in the solution surrounding the fiber since the calcium entering the sarcoplasmic reticulum was taken from this solution. The slope of the fluorescence curve corresponded to a rate of calcium accumulation in the sarcoplasmic reticulum of 1.6 μmol per second per liter of the solution in the cuvette or 2.6 mmol per second per liter of the sarcoplasmic reticulum. A solution without oxalate and ruthenium red may exhibit oscillations of the free FURA concentration, which can be explained by calcium-activated calcium release from the sarcoplasmic reticulum.     

pH-sensitive pHLIP® coated niosomes

Nanomedicine is becoming very popular over conventional methods due to the ability to tune physico-chemical properties of nano vectors, which are used for encapsulation of therapeutic and diagnostic agents. However, the success of nanomedicine primarily relies on how specifically and efficiently nanocarriers can target pathological sites to minimize undesirable side effects and enhance therapeutic efficacy. Here, we introduce a novel class of targeted nano drug delivery system, which can be used as an effective nano-theranostic for cancer. We formulated pH-sensitive niosomes (80–90?nm in diameter) using nonionic surfactants Span20 (43–45?mol%), cholesterol (50?mol%) and 5?mol% of pH (Low) insertion peptide (pHLIP) conjugated with DSPE lipids (DSPE-pHLIP) or hydrophobic fluorescent dye, pyrene, (Pyr-pHLIP). In coating of niosomes, pHLIP was used as an acidity sensitive targeting moiety. We have demonstrated that pHLIP coated niosomes sense the extracellular acidity of cancerous cells. Intravenous injection of fluorescently labeled (R18) pHLIP-coated niosomes into mice bearing tumors showed significant accumulation in tumors with minimal targeting of kidney, liver and muscles. Tumor-targeting niosomes coated with pHLIP exhibited 2–3 times higher tumor uptake compared to the non-targeted niosomes coated with PEG polymer. Long circulation time and uniform bio-distribution throughout the entire tumor make pHLIP-coated niosomes to be an attractive novel delivery system.     

Variation of Pleurotus ostreatus (Jacq. Ex Fr.) P. Kumm. (1871) performance subjected to differentdoses and timings of nano-urea

Supplementation of the growing substrate by nitrogenous additives has been known to improve the production of oyster mushroom (Pleurotus ostreatus (Jacq. ex Fr.) P. Kumm. (1871)). However, the application of nano-additives has not been reported in such cultivation yet. The study investigated the effect of nano-urea added in two different doses (3 g and 5 g per kg substrate), once (at spawning or after first flush) or twice (at spawning and after first flush) to the growing substrate consisting of wheat straw and spent oyster substrate (1:1, w/w). Results showed that the application of nano-urea once has induced the highest number of mushroom flushes (four flushes) despite the dose applied. Contrarily to early findings, where high doses of nitrogen have caused inhibition of mushroom growth and production, nano-urea application has had better effects when applied twice. With 5 g/kg, it induced the shortest period between the first and the third flush (15 days). With 3 g/kg, it resulted in the highest biological and economic yields at the third flush (332.7 g/bag and 283.1 g/bag respectively), in total (973.4 g/bag and 854.0 g/bag respectively), the highest biological efficiency (109.6%), and pileus diameter/stipe length ratio (2.8). Experimental findings of the current study may be potentially applied at commercial scale.     

A novel Golgi mannosidase inhibitor: Molecular design,synthesis, enzyme inhibition,and inhibition of spheroid formation

Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell–cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.     

Nicotine inhibition of carotenoid cyclization in Cucurbita ficifolia cotyledons

Nicotine inhibits carotenoid cyclization in greening cucurbit cotyledons resulting in the accumulation of acyclic and monocyclic carotenes. Chlorophyll synthesis is also inhibited by the alkaloid.     

Lipid and pigment changes during shoot initiation in cultured explants of Pinus radiata

Cotyledon explants of radiata pine contain principally lipid and protein reserve materials, which decline during shoot initiation. This process was followed phytochemically and ultrastructurally. Fatty acid and sterol analyses indicated that there were both quantitative and qualitative changes in the different classes of lipid. The most pronounced changes were an increase in the linolenic acid content of the polar lipids and the appearance of stigmasterol during shoot initiation. There was also a continued increase in chlorophyll and carotenoid levels, which paralleled chloroplast development. It appears that the changes observed were similar to those that occur in cotyledons during normal seedling development.     

Precision and accuracy of indole-3-acetic acid analyses performed with the 2-methylindolo-α-pyrone fluorescence assay and with a high performance liquid chromatography technique with spectrofluorimetric detection, exemplified on pine tissue (Pinus sylvestris L.)

The precision and accuracy of two versions of the 2-methylindolo-α-pyrone (2-MIP) assay and of a high performance liquid chromatography (HPLC) technique was assessed when applied to analysis of indole-3-acetic acid (IAA) in pine ( Pinus sylvestris ) tissue. In one version of the 2-MIP assay total fluorescence at wavelengths > 450 nm was recorded, whereas in the other version fluorescence spectra were recorded and analyzed. Such analysis revealed the occurrence in some samples of fluorescent contaminants that impaired both precision and accuracy. The precision and accuracy of the 2-MIP assay could be improved by additional sample purification, and the over-all accuracy of the assay was finally verified, at a high level of sample purity, by successive approximation. The precision of the assay was then 14%. Successive approximation also verified that the HPLC technique was accurate at a less advanced stage of sample purity than the 2-MIP assay, and had a precision of 14%.