Detection and characterization of acidic compartments (vacuoles) in Chlorella vulgaris 11h cells by 31P-in vivo NMR spectroscopy and cytochemical techniques

Acidic inorganic phosphate (Pi) pool (pH around 6) was detected besides the cytoplasmic pool in intact cells of Chlorella vulgaris 11h by ³¹P-in vivo nuclear magnetic resonance (NMR) spectroscopy. It was characterized as acidic compartments (vacuoles) in combination with the cytochemical technique; staining the cells with neutral red and chloroquine which are known as basic reagents specifically accumulated in acidic compartments. Under various conditions, the results obtained with the cytochemical methods were well correlated with those obtained from in vivo NMR spectra; the vacuoles were well developed in the cells at the stationary growth phase where the acidic Pi signal was detected. In contrast, cells at the logarithmic phase in which no acidic Pi signal was detected contained only smaller vesicles that accumulated these basic reagents. No acidic compartment was detected by both cytochemical technique and ³¹P-NMR spectroscopy when the cells were treated with NH₄OH. The vacuolar pH was lowered by the anaerobic treatment of the cells in the presence of glucose, while it was not affected by the external pH during the preincubation ranging from 3 to 10. Possible vacuolar functions in unicellular algae especially with respect to intracellular pH regulation are discussed.Non-standard abbreviations EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MDP methylene diphosphonic acid - NMR nuelear magnetic resonance - PCA perchloric acid - PCV packed cell volume - Pi inorganic phosphate - Pic sytoplasmic inorganic phosphate - Piv vacuolar inorganic phosphate - ppm parts per million - SP sugar phosphates - TCA trichloroacetic acid     

Nuclear-magnetic-resonance imaging of leaves ofMesembryanthemum crystallinum L. plants grown at high salinity

Differences in water binding were measured in the leaf cells ofMesembryanthemum crystallinum L. plants grown under high-salinity conditions by using nuclear-magnetic-resonance (NMR) imaging. The 7-Tesla proton NMR imaging system yielded a spatial resolution of 20·20·100 m³. Images recorded with different spin-echo times (4.4 ms to 18 ms) showed that the water concentrations in the bladder cells (located on the upper and lower leaf surface), in the mesophyll cells and in the water-conducting vessels were nearly identical. All of the water in the bladder cells and in the water-conducting vessels was found to be mobile, whilst part of the water in the mesophyll cells was bound. Patches of mesophyll cells could be identified which bound water more strongly than the surrounding mesophyll cells. Optical investigations of leaf cross-sections revealed two types of mesophyll cells of different sizes and chloroplast contents. It is therefore likely that in the small-sized mesophyll cells water is strongly bound. A long-term asymmetric water exchange between the mesophyll cells and the bladder cells during Crassulacean acid metabolism has been described in the literature. The high density of these mesophyll cells in the lower epidermis is a possible cause of this asymmetry.Abbreviations CAM Crassulacean acid metabolism - NMR nuclear magnetic resonance - T_E spin-echo time     

Analysis of electric and magnetic fields leaking from induction heaters

Results are presented of an investigation on electric and magnetic fields leaking from inductive (magnetic) heaters that are used for thermal processing of high-power electron tubes and lasers in an industrial plant. Measurements of electric and magnetic fields were done using both commercially available and laboratory-developed instrumentation. Isotropic H-field sensors were developed to allow quantitative evaluation of high-intensity magnetic fields. Ten induction heaters with nominal A.C. power ranging from 2.5 kW to 15 kW and operating at frequencies between 300 kHz and 790 kHz were surveyed. Electric field strengths up to 8 kV/m and magnetic field strengths up to 20 A/m were measured.     

Chronic exposure to ELF fields may induce depression

Exposure to extremely-low-frequency (ELF) electric or magnetic fields has been postulated as a potentially contributing factor in depression. Epidemiologic studies have yielded positive correlations between magnetic- and/or electric-field strengths in local environments and the incidence of depression-related suicide. Chronic exposure to ELF electric or magnetic fields can disrupt normal circadian rhythms in rat pineal serotonin-N-acetyltransferase activity as well as in serotonin and melatonin concentrations. Such disruptions in the circadian rhythmicity of pineal melatonin secretion have been associated with certain depressive disorders in human beings. In the rat, ELF fields may interfere with tonic aspects of neuronal input to the pineal gland, giving rise to what may be termed "functional pinealectomy." If long-term exposure to ELF fields causes pineal dysfunction in human beings as it does in the rat, such dysfunction may contribute to the onset of depression or may exacerbate existing depressive disorders.     

Measurement of Free Intracellular Calcium in the Brain by 19F-Nuclear Magnetic Resonance Spectroscopy

We report the first measurement of the free intracellular calcium level in an actively metabolising intact cerebral tissue preparation. To this end, we applied the recently developed 19F-nuclear magnetic resonance calcium chelator, 5,5'-F2-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), in superfused cerebral cortical slices to give values for the intracellular Ca2+ concentration of 350 and 480 nM, at external calcium concentrations of 1.2 and 2.4 mM, respectively. Under both conditions, the intracellular Ca2+ concentration was increased by depolarisation using a high external K+ concentration. Interleaved 31P spectra showed that the presence of the 5FBAPTA had a deleterious effect on the metabolic state of the tissue with an external Ca2+ concentration of 1.2 mM, but normal viability was maintained using 2.4 mM.     

Phosphorus-31 magnetic resonance spectroscopy of forearm flexor muscles in student rowers using an exercise protocol adjusted for differences in cross-sectional muscle area

To assess exercise energy metabolism of forearm flexor muscles in rowers, six male student rowers and six control subjects matched for age and sex were studied using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Firstly, to adjust for the effect of differences in cross-sectional muscle area, the maximal cross-sectional area (CSAmax) of the forearm flexor muscles was estimated in each individual using magnetic resonance imaging. Multistage exercise was then carried out with an initial energy production of 1 J.cm-2 CSAmax for 1 min and an increment of 1 J.cm-2 CSAmax every minute to the point of muscle exhaustion. A series of measurements of 31P-MRS were performed every minute. The CSAmax was significantly greater in the student rowers than in the control subjects [19.8 (SD 2.2) vs 17.1 (SD 1.2) cm2, P less than 0.05]. The absolute maximal exercise intensity (J.min-1) was greater in the rowers than in the control subjects. However, the maximal exercise intensity per unit of muscle cross sectional area (J.min-1.cm-2) was not significantly different between the two groups. During mild to moderate exercise intensities, a decrease in phosphocreatine and an increase in inorganic phosphate before the onset of acidosis were significantly less in the rowers, indicating a requirement of less adenosine 5'-diphosphate to drive adenosine 5'-triphosphate production. The onset of acidosis was also significantly delayed in the rowers. No difference was observed in forearm blood flow between the two groups at the same exercise intensity (J.min-1.cm-2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

Modification of the 1979 "Denver wire code" for different wire or plumbing types.

This article examines "wire configuration coding" as used to estimate relative residential AC magnetic field exposure in epidemiological studies--and the need to alter such coding for time or locations other than those in which the code was developed. Effects of different secondary wire practices are particularly examined.     

High-Resolution Proton Magnetic Resonance Analysis of Human Cerebrospinal Fluid

High-resolution proton magnetic resonance spectroscopy was used to analyze human cerebrospinal fluid obtained from patients with several neurological problems. The major metabolites measured included glucose, lactate, glutamine, citrate, inositol, acetate, creatine, creatinine, beta-hydroxybutyrate, alanine, and pyruvate. A drug vehicle, propylene glycol, was also measured. Alterations in the cerebrospinal fluid of these metabolites provided information concerning metabolism of the brain. Magnetic resonance spectroscopy offered a simple and rapid means of assessing these and other exogenous and endogenous compounds in diseases affecting the nervous system.     

Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain. All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains. These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences. This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals. Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed. It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain. No special role can be attached thus far to individual lipoyl domains. On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain.