Selection of fatty acid auxotrophs from the oleaginous yeast Cryptococcus curvatus and production of cocoa butter equivalents in batch culture

Summary A search was carried out for mutants, defective in the conversion of stearic acid to oleic acid in effort to improve the quality of lipids produced by Cryptococcus curvatus ATCC, 20509. Mutants were selected as unsaturated fatty acid (Ufa) auxotrophs. After treatment of parent organism with Ethyl methanesulfonate (EMS), 11 oleate-requiring auxotrophs were isolated. Only 3 of them were real unsaturated fatty acid (Ufa) mutants, while the other 8 were designated as fatty acid synthetase (Fas) mutants. The amount of saturated fatty acid (SFA) was about 65.2 % in the lipids extracted from an Ufa mutant named UfaM3 and it was significantly higher than that of the wild-type (WT) (46.6 %) and similar to that of cocoa butter (60.4 %).     

Effect of taurine,l-glutamine and l-histidine addition in an amino acid glucose solution on the cellular bioavailability of zinc

Radioactive zinc was used to study the effect of a binary parenteral nutrient solution, composed of amino acids and glucose, on zinc uptake by fibroblasts. The influence of addition of taurine, l-glutamine and of the increase in l-histidine content of the admixture was assessed. The pure mixture was highly toxic for cells and so it was diluted 1/5 in tyrode buffer with 2% albumin. As compared with cells incubated in the buffer containing albumin, zinc absorption was significantly higher (P < 0.05) in the presence of the amino acids of the mixture. Amino acids thus increased bioavailability by displacing zinc bound to albumin. When the histidine concentration in the nutrient medium (4.2 mm) was doubled, inhibition was noted after 30 min of incubation and zinc uptake thereafter remained comparable to that in histidine-free medium. The addition of glutamine (4.2 mm), usually not present in binary mixtures, resulted in significant differences as compared with glutamine-free control medium. Taurine (0.8 mm), led to a constant increase in zinc uptake by fibroblasts as compared with that obtained with taurine-free mixture. However, ultrafiltration showed that taurine was not able to displace zinc from albumin.     

Enantioselective inhibitory effect of nicardipine on the hepatic clearance of propranolol in man

The influence of a single oral dose of 30 mg nicardipine on the pharmacokinetics of (R)- and (S)-propranolol, given orally as rac-propranolol 80 mg, was studied in 12 healthy volunteers. The plasma concentrations were higher for the (S)-enantiomer than for the (R)-enantiomer. The Cl_o and the Cl′_intr of (S)-propranolol were significantly lower than the Cl_o and Cl′_intr of (R)-propranolol. The unbound fraction of (R)-propranolol was significantly higher than that of (S)-propranolol. Coadministration of nicardipine significantly increased the AUC and C_max and significantly decreased the Cl_o and Cl′_intr for unbound drug of (R)- and (S)-propranolol. These changes were more important for (R)- than for (S)-propranolol. The protein binding was not altered by nicardipine. The enantioselective effect of nicardipine on the metabolic clearance of propranolol appears to be due to an interaction at the level of the metabolizing enzymes. The effect on blood pressure of rac-propranolol was little affected when nicardipine was coadministered with rac-propranolol, and its bradycardic effect was reduced. © 1994 Wiley-Liss, Inc.     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

Bayesian signal extraction from noisy FT NMR spectra

Summary The statistical interpretation of the histogram representation of NMR spectra is described, leading to an estimation of the probability density function of the noise. The white-noise and Gaussian hypotheses are discussed, and a new estimator of the noise standard deviation is derived from the histogram strategy. The Bayesian approach to NMR signal detection is presented. This approach homogeneously combines prior knowledge, obtained from the histogram strategy, together with the posterior information resulting from the test of presence of a set of reference shapes in the neighbourhood of each data point. This scheme leads to a new strategy in the local detection of NMR signals in 2D and 3D spectra, which is illustrated by a complete peak-picking algorithm.     

Reversal of selenium and zinc deficiencies in chronic hemodialysis patients by intravenous sodium selenite and zinc gluconate supplementation

In six chronic dialyzed uremic patients, an intravenous sodium selenite (Se 50 μg during 5 wk and then 100 μg) and zinc gluconate (Zn 5 mg) supplementation was performed during 20 wk at each dialysis session three times weekly. Before supplementation, plasma Se and Zn, plasma and erythrocytes (RBC) antioxidant metalloenzymes glutathione peroxidase (GPX), and superoxide dismutase (SOD) were significantly decreased, whereas lipid peroxidation (as thiobarbituric acid reactants TBARs) was increased. To obtain a significative change in plasma selenium, we had to use an Se dose of 100 μg/dialysis session. Then, treatment-increased plasma Se (from 0.58 ±0.09 to 0.89±0.16 μmol/L) led to a repletion of RBC-GPX (from 29.6±6 to 43±5.8 U/g Hb) and increased plasma GPX levels (from 62±13 to 151±43 U/L). Plasma Zn and RBC-SOD did not vary significantly. The change of TBARs was not observed between wk 1 and 4. They decreased significantly between wk 4 (4.80±0.21μmol/L) and wk 20 (4.16±0.26 μmol/L). We noted a low correlation between TBARs and plasma GPX. A strong correlation was observed between Se and plasma GPX. The reversal of Se deficiencies should reduce oxidative damage observed in these patients.     

Arrest of somatic embryo development in grapevine: histological characterization and the effect of ABA,BAP and zeatin in stimulating plantlet development

In an effort to understand the causes of arrest of somatic embryo development, generally observed in grapevine (Vitis sp.), histological studies were undertaken, using two cultivars (CH76 and 41B) which differ in their ability to develop into plants. Embryos with a high conversion rate (70%; CH76) formed a well-structured and functional shoot apex between two thread-like cotyledons. In contrast, embryos with a low conversion rate (10%; 41B) formed a normal root apex but lacked a well-structured shoot apex and developed a wide range of aberrant forms in the intercotyledonary area: uncontrolled cellular proliferation, formation of adventitious buds, over-growth of cotyledonary or leaf meristems. ABA increased the conversion rate of 41B embryos from 10% to 20%, but failed to improve embryo morphology. Zeatin and BAP promoted growth of 41B somatic embryos, but generated a high level of abnormalities and failed to improve conversion rate. Applied in combination with ABA, these PGRs increased the frequency of cotyledonary embryos, but decreased the conversion rate.     

Assay of the antiangiogenic compound TNP-470, and one of its metabolites, AGM-1883, by reversed-phase high-performance liquid chromatography in plasma

This paper describes a reversed-phase, high-performance liquid chromatographic (HPLC) method for the isolation, detection, and quantification of TNP-470 (I) and one of its active metabolites, AGM-1883 (II), from plasma. These compounds are initially extracted from plasma with an organic solvent and then separated from one another on a C₁₈ column. Those fractions eluting from the C₁₈ column and containing either I or II are then derivatized through their epoxide moieties with sodium 8-quinolinethiolate (SQT). This derivatization produces fluorescent species that are isolated and quantified by a second reversed-phase HPLC analysis. The assay yields a lower limit of reliable quantification of 2.5 ng/ml and is linear to a concentration at least as high as 160 ng/ml. The inter-assay percent coefficient of variation is less than 18%.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

The influence of the type of contraction on the masseter muscle EMG power spectrum

Different behaviours of the EMG power spectrum across increasing force levels have been reported for the masseter muscle. A factor that could explain these different behaviours may be the type of contraction used, as was recently shown for certain upper limb muscles⁵. The purpose of this study was to compare, between two types of isometric contractions, the behaviour of EMG power spectrum statistics (median frequency (MF) and mean power frequency (MPF)) obtained across increasing force levels. Ten women exerted, while biting in the intercuspal position, three 5 s ramp contractions that increased linearly from 0 to 100% of the maximal voluntary contraction (MVC). They also completed three step contractions (constant EMG amplitude) at each of the following levels: 20, 40, 60 and 80% MVC. EMG signals from the masseter muscle were recorded with miniature surface electrodes. The RMS, as well as the MPF and MF of the power spectrum were calculated at 20, 40, 60 and 80% MVC for each type of contraction. As expected, the RMS values showed similar increases with increasing levels of effort for both types of contractions. Different behaviours for both MPF (contraction^*force interaction, ANOVA, P<0.05) and MF (contraction^*force interaction, ANOVA, P>0.05) across increasing levels of effort were found between the two types of contraction. The use of step contractions gave rise to a decrease of both MPF and MF with increasing force, while the use of ramp contractions gave rise to an increase in both statistics up to at least 40% MVC followed by a decrease at higher force levels. These findings suggest that the type of contraction used does influence the behaviour of the spectral statistics across increasing force levels and that this could explain the differences obtained in previous studies for the masseter muscle.