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.     

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%.     

Structural Characterization of Tau in Fuzzy Tau:Tubulin Complexes

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Transients of delayed fluorescence induction signal and photosynthetic antennas: A possible relationship. Mathematical modeling approach

A mathematical model was developed for resolved temporal transients of experimentally recorded delayed fluorescence (DF) induction signal. During an intermittent light regime, antennas of the photosynthetic apparatus were treated as targets, repeatedly hit by potentially absorbable photons within a series of consecutive light flashes. Formulas were derived for the number of antennas, cumulatively hit by a specific number of photons, as a function of the flash serial number (time). Model parameters included number of absorbable photons in one flash, antenna sizes, and their number. A series of induction curves were analyzed, obtained from a Zea mays leaf segment and differing in the previous dark period (t _d). Each curve, consisting of the two most prominent DF transients (C and D), was fitted with several model types, differing in the number of absorbed photons. For both transients, the best fitting result was achieved when DF induction was linked to the second absorbed photon. As expected, model parameters related to antenna sizes showed weaker dependence on t _d than those referring to antenna number. With restrictions applied to this model, the two DF induction transients may be related to two classes of photosynthetic antennas. Their different sizes may have a predominant influence on the efficiency of photon absorption and possibly time-dependent appearance of DF transients. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 325–335. The text was submitted by the authors in English.     

Application of a photosystem II model for analysis of fluorescence induction curves in the 100 ns to 10 s time domain after excitation with a saturating light pulse

A mathematical model of photosystem II (PSII) events was used to analyze chlorophyll fluorescence transients in the time domain from 100 ns to 10 s after excitation with a saturating 10-ns flash, applied as a part of specialized illumination protocol, using preparations of a thermophilic strain of the unicellular green alga, Chlorella pyrenoidosa Chick (using both intact and diuron-treated cells). Analysis of simulation results has proven that particular attention should be given to flash-induced recombination processes, including nonradiative recombination in PSII, while subsequent charge transfer along the electron transport chain of thylakoid membrane can be adequately described by a single reaction of quinone reoxidation. The PSII model was extended by taking inhibition by diuron of the electron transport in the acceptor side of PSII into account, which allowed simulation of fluorescence induction curves observed in the presence of this inhibitor. The model parameters were determined (stromal pH, rate constants of nonradiative recombination, and the initial reduction state of the quinone pool) which provided adequate simulation of experimentally observed ratios of the maximal and initial fluorescence levels (F _m/F ₀).