Speciation of antimony(III) and antimony(V) in cell extracts by anion chromatography/inductively coupled plasma mass spectrometry

An analytical method for the separation and quantification of Sb(III) and Sb(V) using anion chromatography with ICP-MS is presented. The optimum conditions for the separation of the antimony species were established with 15 mmol/L nitric acid at pH 6 as eluent system on a PRP-X100 column. The retention times for antimony(V) and antimony(III) were 85 s and 300 s with detection limits of 0.06 microg/L and 0.29 microg/L, respectively. The proposed method was applied to cell extracts of Leishmania donovani, which were incubated with antimony(III) and antimony(V). Some metabolism seemed to occur within the cells.     

PHOTOINDUCTION OF PHARBITIS FLOWERING: RELATIONSHIP TO RNA SYNTHESIS AND OTHER METABOLIC EVENTS

We have previously shown that a wave of enhanced uridine incorporation into RNA occurs in the more vegetative parts of the plumule at the end of the single dark period that evokes flowering in Pharbitis nil. We demonstrate here that a light break that suppresses flowering suppresses this wave as well. It does not shift the kinetics of the wave of uridine incorporation to a different time. The enhanced incorporation is into all RNA fractions. It had been concluded from excision experiments that the floral stimulus reaches the apex much after photoinduction. There is a metabolic shock caused by such excision of the cotyledons or surgical removal of the plumule that can suppress flowering if it is performed near the end of the inductive dark period. The terminal bud is more affected by this shock than lateral buds. Excision of the cotyledons enhances the rate of incorporation of exogenous uridine into the plumule. We propose that the “floral stimulus” stimulating incorporation into RNA reaches the plumule immediately after the end of the critical dark period.     

High-resolution separation of peptides by sodium dodecyl sulfate-polyacrylamide gel "focusing"

As a follow-up of our previous report (Anal. Chem. 2007, 79, 821-827) on analytical SDS-PAGE focusing, a refinement of the method for separation of peptides in the small to medium M(r) range (0.5-10 kDa) is here reported, based on a shallow gradient of immobilized positive charges (0-10 mM) onto a minimally sieving polyacrylamide gel matrix (4%T, 2.5%C). Unlike conventional SDS-PAGE, which rarely can achieve the separations of polypeptide chains below a critical value of 10 kDa, the present method can be fine-tuned to perform such separations even down to a size of only 500 Da. In the case of larger fragments, the major peptide zones are shown, under microscope observation, to be composed by envelopes of bands as narrow as 20-100 microm, spaced at regular intervals of 100-150 microm. It is hypothesized that such larger peptides could form complexes with rather small SDS micelles and that such peptide-SDS complexes could differ in charge by just a single negative charge.     

Speciation of antimony(III) and antimony(V) in cell extracts by anion chromatography/ inductively coupled plasma mass spectrometry

An analytical method for the separation and quantification of Sb(III) and Sb(V) using anion chromatography with ICP-MS is presented. The optimum conditions for the separation of the antimony species were established with 15 mmol/L nitric acid at pH 6 as eluent system on a PRP-X100 column. The retention times for antimony(V) and antimony(III) were 85 s and 300 s with detection limits of 0.06 μg/L and 0.29 μg/L, respectively. The proposed method was applied to cell extracts of Leishmania donovani, which were incubated with antimony(III) and antimony(V). Some metabolism seemed to occur within the cells.     

Transformation of 1,2,5,6-Tetrahydropyridines with Mycellar Fungi

It has been shown that on biotransformation of a series of 1,2,5,6-tetrahydropyridines with strains of the mycellar fungi Cunninghamella verticillata VKPM F-430, Beauveria bassiana ATCC 7159, and Penicillium simplicissimum KM-16, the culture of Cunninghamella verticillata possesses the greatest transforming activity and selectivity. With the aid of the latter practically quantitative oxidation of 1,2,5,6-tetrahydropyridines occurs into the corresponding trans-diol. The structure and spatial disposition of trans-1-benzyl-3,4-dihydroxypiperidine was demonstrated by data of chromato-mass spectrometric analysis and high resolution NMR spectra and was confirmed by comparison with an authentic sample obtained by an alternate synthesis using the oxidation of 1-benzyl-1,2,5,6-tetrahydropyridine with trifluoroperacetic acid.     

Nonlinear electrophoresis of point-like particles--is it possible?

A new universal method for the generation of nonlinear electrophoretic mobility of a packet of any particles is suggested. The method is based on the investigation of particle packet dynamics under the influence of an external force. The system under consideration is a homogeneous and isotropic medium with traps for these particles. Packet dynamics is described by a linear diffusion equation. The measured packet parameters are the position and the velocity of a packet maximum. It is shown that these parameters are nonlinear in the external field under definite limitations on the trap properties. This statement is proved both theoretically and experimentally for the simple model of diffusive substrate, the so-called comb structure. The prospects of designing new supporting substrates (microfluidic systems) with a nonlinear response are discussed.     

Light-dependent Oxygen Consumption in Bacteriochlorophyll-Serine-treated Melanoma Tumors: On-line Determination Using a Tissue-inserted Oxygen Microsensor

Successful application of anticancer therapy, and especially photodynamic therapy (PDT) mediated by type II (PDTII) processes, depends on the oxygen content within the tumor before, during and after treatment. The high consumption of oxygen during type II PDT imposes constraints on therapy strategies. Although rates of oxygen consumption and repletion during PDTII were suggested by theoretical studies, direct measurements have not been reported. Application of a novel oxygen sensor allowed continuous and direct in situ measurements (up to a depth of 8–9 mm from the tumor surface and for several hours) of temporal variations in the oxygen partial pressure (pO₂) during PDT. Highly pigmented M2R mouse melanoma tumors implanted in CD1 nude mice were treated with bacteriochlorophyll-serine (Bchl-Ser; a new photodynamic reagent) and were subjected to fractionated illumination (700 < λ. < 900 nm) at a fluence rate of 12 mW cm^-2. This illumination led to total oxygen depletion with an average consumption rate of 7.2 uAf(O₂) s^-1. Spontaneous reoxygenation (at an average rate of 2.5 µM(O₂)/s) was observed during the following dark period. These rates are in good agreement with theoretical considerations (Foster et al., Radiat. Res. 126, 296,1991 and Henning et al, Radiat. Res. 142, 221, 1995). The observed patterns of oxygen consumption and recovery during prolonged periods of light/dark cycles were interpreted in terms of vasculature damage and sensitizer clearance. The presented data support the previously suggested advantages of fractionated illumination for type II photodynamic processes.     

Parallel isoelectric focusing II

A miniature electrophoretic device is developed on the basis of a new isoelectric focusing (IEF) method, namely parallel isoelectric focusing. We report here the theory and the results of operation of a new parallel isoelectric device (PID). The main advantages and limitations of the method are discussed for miniaturization purposes. It is shown that the method guarantees the fast and complete separation of any complex protein mixtures under acceptable conditions, such as voltage source, temperature, size of the device, and separation process duration. It is shown that the main problem of PID miniaturization is the buffer design, and the relation between Immobiline buffer capacity and solution buffer capacity. The main experimental limitation of PID resolution is protein sensitivity to pH changes.     

Antivascular treatment of solid melanoma tumors with bacteriochlorophyll-serine-based photodynamic therapy

We describe here a strategy for photodynamic eradication of solid melanoma tumors that is based on photo-induced vascular destruction. The suggested protocol relies on synchronizing illumination with maximal circulating drug concentration in the tumor vasculature attained within the first minute after administrating the sensitizer. This differs from conventional photodynamic therapy (PDT) of tumors where illumination coincides with a maximal concentration differential of sensitizer in favor of the tumor, relative to the normal surrounding tissue. This time window is often achieved after a delay (3-48 h) following sensitizer administration. We used a novel photosensitizer, bacteriochlorophyll-serine (Bchl-Ser), which is water soluble, highly toxic upon illumination in the near-infrared (lambda max 765-780 nm) and clears from the circulation in less than 24 h. Nude CD1 mice bearing malignant M2R melanotic melanoma xenografts (76-212 mm3) received a single complete treatment session. Massive vascular damage was already apparent 1 h after treatment. Changes in vascular permeability were observed in vivo using contrast-enhanced magnetic resonance imaging (MRI), with the contrast reagent Gd-DTPA, by shortening spin-spin relaxation time because of hemorrhage formation and by determination of vascular macromolecular leakage. Twenty-four hours after treatment a complete arrest of vascular perfusion was observed by Gd-DTPA-enhanced MRI. Histopathology performed at the same time confirmed primary vascular damage with occlusive thrombi, hemorrhage and tumor necrosis. The success rate of cure of over 80% with Bchl-Ser indicates the benefits of the short and effective treatment protocol. Combining the sensitizer administration and illumination steps into one treatment session (30 min) suggests a clear advantage for future PDT of solid tumors.