Modalités du transport et du métabolisme de l'AIA 14C ou AIA 3H en relation avec la morphogenèse des bourgeons axillaires chez le Scrophularia arguta

Modes of transport and metabolism of ¹⁴ C-IAA and ³ H-IAA in relation to morphogenesis of axillary buds in Scrophularia arguta. The main objectives of this study were to investigate the morphogenetic role of IAA on the growth and development of axillary buds. After foliar applications of radioactive IAA for 6 h on intact plants of Scrophularia arguta Sol. the characteristics of auxin transport were studied by liquid scintillation counting, thin layer chromatography and microautoradiography. The main part of the radioactivity moved at a mean rate of 7 mm/h. Over long periods of transport, the tracers accumulated at the base of the axis and in the roots. The nodes were a little richer in ³H or ¹⁴C than the internodes. This fact seemed to be correlated with the vascular organization of this part of the stem. A very weak proportion of tracers was found in axillary buds. The radioactivity was to about 50% associated with the IAA molecule; the rest corresponded essentially to indolyl-4-acetyl-l-aspartic acid and indolyl-3-aldehyde. Tracers were mainly concentrated in the phloem along the whole axis and, to a lesser extent in some of the young differentiating metaxylem vessels, and in the medullary rays. No radioactivity was found in the cambial zone and in the mature xylem, nor in the parenchymas. These results support the view of an indirect role of IAA on the axillary bud growth and morphogenesis.     

Decarboxylation and Transport of Auxin in Segments of Sunflower and Cabbage Roots. II. A Chromatographic Study Using IAA-1-14C and IAA-5-3H

The polar movement of IAA has been examined in 5-mm root segments of Brassica oleracea and Helianthus annum. The movement was studied partly with IAA-1-¹⁴C and partly with IAA-5-³H. In both plants a slight acropetal flux of ¹⁴C and IAA-³H was found through the segments. The recovered radioactivity in the agar receiver blocks and in the receiver end of the segments increased as a function of time. A large portion of the applied IAA was converted on the cut surfaces and in the tissues of the segments. Chromatographic analysis indicated different destruction products when estimated by scintillation counting and by spraying with in-dole reagent (DMCA). Chromatograms run in isopropanol: ammonia: water, 8:1:1, yielded three different substances, one spot near the starting line and one near the front, neither of which has been identified. Finally there was a spot with R_f 0.4–0.6, probably representing IAA.     

Evaluation of <Emphasis Type="Italic">Isochrysis galbana</Emphasis> (clone T-ISO) cell numbers by digital image analysis of color intensity

In microalgal cultivation, measuring cell numbers as a means to monitor growth rates is a long-standing problem. Many automated counting systems and schemes have been developed; among these are image analysis systems. However, such imaging systems have presented difficulties in dealing with the complexities of computer recognition of individual microscopic cells. It is known that the coloration of microalgae suspension is species specific and that color intensity increases are typically associated with increasing numbers. Using this qualitative insight, the present work describes the design, construction, and comparative performance of an inexpensive digital imaging system optimized for counting microalgal cells. The system circumvents the need to count individual cells and extracts cell numbers directly from the macroscopic color intensity of a microalgal suspension. The results suggest, using Isochrysis galbana (T-ISO) as an illustrative example, that this scheme is potentially useful for inexpensive and automated biomonitoring of microalgal cell numbers. Percentage difference comparisons with a standard Coulter Counter indicated that the three algorithms tested provided better than 10% accuracy over density thresholds of 1.52 × 10⁶ to 8.10 × 10⁶ cells mL⁻¹ with precision of 4% attainable at high density concentrations.     

Silver staining test of nucleolar suppression in the Lilium hybrid ‘Black Beauty’

Nucleolar organizing regions in mitotic chromosomes from Lilium longiflorum, L. henryi and L. speciosum, have been silver banded. Nucleoli in interphase cells were also silver stained. Silver banding and nucleolar counting in Lilium cv ‘Black Beauty’, a hybrid between L. speciosum and L. henryi, indicate that nucleolar suppression is not occurring.     

Microwave Assisted Alcoholysis of Isocyanates Derived from N<Superscript>α</Superscript>-[(9-fluorenylmethyl)oxy]carbonyl Amino Acids: Synthesis of N-Fmoc-N<Superscript>1</Superscript>-Z-/Boc-/Alloc-/Bsmoc-gem-diamines

A general and efficient method has been developed for the alcoholysis of isocyanates derived from the N^α-[(9-fluorenylmethyl)oxy]carbonyl amino acids with various alcohols including hindered ones assisted by MW irradiation. Thus, the synthesis of N, N¹-diurethane protected gem-diamines wherein Fmoc protection on one of the amino groups and Z-/Boc-/Alloc or Bsmoc group on the other amino function has been accomplished. All the new orthogonally diurethane protected gem-diamines have been obtained as crystalline solid powders in 80 to 94% yield. The bisprotected gem-diamines have been fully characterized by IR, ¹H NMR, ¹³C NMR as well as by mass spectrometry.     

Liquid scintillation counting of aqueous solutions of potassium salts in a Triton X-100/toluene scintillant

The use of a toluene/Triton X-100 scintillant for counting ¹⁴C in aqueous solutions of potassium salts including potassium hydroxide has been investigated. Suitable conditions for the counting of CO₂ entrapped in potassium hydroxide are described. Quench correction by automatic external standard channels ratio procedures has been found to be of value with optimised conditions of counting.     

The two‐phase partitioning system – a powerful technique to purify integral membrane proteins of Corynebacterium glutamicum for quantitative shotgun analysis

We established a single consecutive strategy which assigned the most comprehensive number of integral membrane proteins from Gram‐positive bacteria to date. For this purpose, we adapted a biphasic partitioning system for the biotechnologically intensively used Corynebacterium glutamicum and proved for the first time that such a system is well suited for quantitative comparison. 297 integral membrane proteins were identified by our integrated approach, which depletes stringently cytosolic proteins. In combination with our previously developed SIMPLE strategy, our data comprise 61% (374 integral membrane proteins) of the entire membrane proteome, which aims towards an almost comprehensive coverage. Wild type and a production strain of C. glutamicum were compared by ¹⁵N metabolic labelling and quantitation was obtained by spectral counting and peak areas. Both quantification strategies display a consistent trend in up or downregulation of proteins. Nevertheless, spectral counting often provides results indicating a much stronger regulation compared to ProRata values. Either spectral counting seems to exaggerate protein regulation or ProRata tends to attenuate the information about the regulation level. We highlight some of the biologically relevant candidates, which prove that our approach helps to give a deeper quantitative insight towards the understanding of transport and other membrane associated processes, important for strain development of C. glutamicum.     

Modified two-dimensional electrophoresis for identification and autoradiography of ribosomal proteins reacted withp-chloromercuribenzoate

Ribosomal proteins labelled with [¹⁴C]PCMB, a reversibly reacting sulfhydryl reagent, lose most of their radioactivity when separated by the conventional two-dimensional polyacrylamide gel electrophoresis. Therefore, the two-dimensional method was modified to insure stability of the PCMB-protein complex during the procedure. Proteins ofEscherichia coli ribosomes were separated by the modified method, and those which reacted with [¹⁴C]PCMB were identified by counting of stained spots and by autoradiography of dried gels.     

Mechanism of nitrate uptake into Chara corallina cells: lack of evidence for obligatory coupling to proton pump and a new NO3−/NO3− exchange model

Abstract. Nitrate uptake into Chara corallina cells at different external pH (pH_o) after different NO₃⁻ pretreatment conditions has been investigated. Following NO₃⁻ pretreatment (0.2 mol m⁻³ NO₃⁻) there was little effect of pH_o on subsequent net NO₃⁻ uptake into Chara cells. After N deprivation (2 mmol m⁻³ NO₃⁻) there was a pronounced effect of pH_o on nitrate uptake, the maximum rate occurring at pH_o 4.7. There was no consistent relationship between OH⁻ efflux and NO₃⁻ uptake in short term experiments (< 1 h). NO₃⁻ efflux was also sensitive to pH_o, the maximum rate occurring at ∼ pH_o 5.0. An inhibitor of the proton pump, DES, immediately stimulated NO₃⁻ uptake into cells pretreated with NO₃⁻ and prevented the time-dependent decrease in NO₃⁻, uptake into Chara cells that had been previously treated with low N (2 mmol m⁻³ NO₃⁻). NO₃⁻ efflux was found to be very sensitive to DES with K_i= 0.7 mmol m⁻³. At the optimum pH_o for NO₃⁻ uptake the effect of DES on membrane potential (ψ_m) were slight, and only apparent after 30 min. The results are interpreted in context of current models relating NO₃⁻ uptake and H⁺ pump activity. A new model for NO₃⁻ uptake is proposed which involves NO₃⁻/NO₃⁻ exchange at steady state.     

NhaK,a novel monovalent cation/H<Superscript>+</Superscript> antiporter of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Four Na⁺/H⁺ antiporters, Mrp, TetA(L), NhaC, and MleN have so far been described in Bacillus subtilis 168. We identified an additional Na⁺/H⁺ antiporter, YvgP, from B. subtilis that exhibits homology to the cation: proton antiporter-1 (CPA-1) family. The yvgP-dependent complementation observed in a Na⁺(Ca²⁺)/H⁺ antiporter-defective Escherichia coli mutant (KNabc) suggested that YvgP effluxed Na⁺ and Li⁺. In addition, effects of yvgP expression on a K⁺ uptake-defective mutant of E. coli indicated that YvgP also supported K⁺ efflux. In a fluorescence-based assay of everted membrane vesicles prepared from E. coli KNabc transformants, YvgP-dependent Na⁺ (K⁺, Li⁺, Rb⁺)/H⁺ antiport activity was demonstrated. Na⁺ (K⁺, Li⁺)/H⁺ activity was higher at pH 8.5 than at pH 7.5. Mg²⁺, Ca²⁺ and Mn²⁺ did not serve as substrates but they inhibited YvgP antiport activities. Studies of yvgP expression in B. subtilis, using a reporter gene fusion, showed a significant constitutive level of expression that was highest in stationary phase, increasing as stationary phase progressed. In addition, the expression level was significantly increased in the presence of added K⁺ and Na⁺.     

Seasonal variation in δ13C and δ18O of cellulose from growth rings of Pinus radiata

Seasonal variation in δ¹³C and δ¹⁸O of cellulose (δ¹³C_c and δ¹⁸O_c) was measured within two annual rings of Pinus radiata growing at three sites in New Zealand. In general, both δ¹³C_c and δ¹⁸O_c increased to a peak over summer. The three sites differed markedly in annual water balance, and these differences were reflected in δ¹³C_c and δ¹⁸O_c. Average δ¹³C_c and δ¹⁸O_c from each site were positively related, so that the driest site had the most enriched cellulose. δ¹³C_c and δ¹⁸O_c were also related within each site, although both the slope and the closeness of fit of the relationship varied between sites. Supporting the theory, the site with the lowest average relative humidity also had the greatest change in δ¹⁸O_c‰ change in δ¹³C_c. Specific climatic events, such as drought or high rainfall, were recorded as a peak or a trough in enrichment, respectively. These results suggest that seasonal and between‐site variation in δ¹³C_c and δ¹⁸O_c are driven by the interaction between variation in climatic conditions and soil water availability, and plant response to this variation.     

Autonomous and non-autonomous traits mediate social cooperation in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

In the trishanku (triA ⁻ ) mutant of the social amoeba Dictyostelium discoideum, aggregates are smaller than usual and the spore mass is located mid-way up the stalk, not at the apex. We have monitored aggregate territory size, spore allocation and fruiting body morphology in chimaeric groups of (quasi-wild-type) Ax2 and triA ⁻ cells. Developmental canalisation breaks down in chimaeras and leads to an increase in phenotypic variation. A minority of triA ⁻ cells causes largely Ax2 aggregation streams to break up; the effect is not due to the counting factor. Most chimaeric fruiting bodies resemble those of Ax2 or triA ⁻ . Others are double-deckers with a single stalk and two spore masses, one each at the terminus and midway along the stalk. The relative number of spores belonging to the two genotypes depends both on the mixing ratio and on the fruiting body morphology. In double-deckers formed from 1:1 chimaeras, the upper spore mass has more Ax2 spores, and the lower spore mass more triA ⁻ spores, than expected. Thus, the traits under study depend partly on the cells’ own genotype and partly on the phenotypes, and so genotypes, of other cells: they are both autonomous and non-autonomous. These findings strengthen the parallels between multicellular development and behaviour in social groups. Besides that, they reinforce the point that a trait can be associated with a genotype only in a specified context.     

Investigation of some impurity profiles by (n,α) reactions

To investigate some impurity profiles by (n,α) reactions for destructive and nondestructive analysis, two experimental facilities on a horizontal channel of the well-moderated research reactor have been developed. In accordance with an α-particle registration geometry two different measurement techniques based on using a surfacebarrier Si(Au) detector and a counting ionization chamber have been used. The determination limit of boron is ofn x 10¹⁶ cm^-3 or (1 /dv 5) x 10^-5 % with a depth resolution of ±0.03 Μm in semiconductors. A solid-state nuclear track detector (SSNTD) has also been used to determine the boron surface and deep distributions in semiconductors, plants, and other materials.     

The behavior of <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> as a parent of somatic hybrid introgressed lines is associated with UV resistance of its chromosomes

Protoplasts of Bupleurum scorzonerifolium irradiated with 380 μW/cm² UV for 5 min were fused by the PEG-mediated method with untreated protoplasts of Arabidopsis thaliana. The fusion products were cultured in the P5 liquid medium for single hybrid cell clone formation. As a total, 81 independent putative hybrid clones (cell lines) were obtained, and seventeen of them were identified as somatic hybrids by chromosome counting, GISH, RAPD, and SSR analyses. More than 80 B. scorzonerifolium-like green introgressed plants and leaves were regenerated from 49 somatic hybrid cell lines, which contained chromatin and DNA characteristic of A. thaliana. To assess the UV tolerance of both parents with chromatin exclusion and introgression, their protoplasts were UV-irradiated (380 μW/cm² for 0 and 5 min), and the protoplasts of A. thaliana were more sensitive to UV than those of B. scorzonerifolium as judged by Single Cell Gel Electrophoresis analysis. The possible relationship between UV resistance of B. scorzonerifolium and A. thaliana chromosome elimination and the formation of somatic introgressed hybrid plants is discussed.     

A new method for analysis of heart rate variability: counting statistics of 1/f fluctuations

1/f fluctuations in heart rate are usually measured by spectral analysis using Fast Fourier Transform. Here, we introduce a new method based on counting the number of QRS complexes in time intervals t of various length. For a 1/f ^b spectrum the variance of counts in t follows a power-law as gDt ^1+b. This method is applied to analyze long term fluctuations in heart rate variability in 10 healthy men. The results show periods of 1/f fluctuations with interpolated periods of white noise during night hours.     

Diurnal variation of Δ13CO2, ΔC18O16O and evaporative site enrichment of δH218O in Piper aduncum under field conditions in Trinidad

Concurrent measurements of gas exchange, instantaneous isotope discrimination (Δ) against ¹³CO₂ and C¹⁸O¹⁶O, and extent of ¹⁸O enrichment in H₂O at the evaporative sites, were followed in a tropical forest pioneer, Piper aduncum, on two different days in Trinidad during February 1995. Δ¹³CO₂ differed from that predicted from measurements of internal:external CO₂ concentration (C_i/C_a) and showed a wide range of values which decreased throughout the course of the day. Derivation of C_c (the CO₂ concentration at the carboxylation site) was not possible using carbon isotope discrimination under field conditions in situ and was derived assuming a constant value of internal transfer conductance (g_w). Under low rates of assimilation the derived C_c/C_a, like C_i/C_a, remained relatively stable over the course of both days and ΔC¹⁸O¹⁶O followed evaporative demand. Lower values of ΔC¹⁸O¹⁶O on day 2 occurred in response to the indirect effect of increased leaf-to-air vapour pressure deficits (VPD) and reduced stomatal conductance. For the first time, direct determination of the δH₂¹⁸O of transpired water vapour (δ_t) allowed derivation of evaporative site enrichment without the prerequisite of isotopic steady state (ISS) defined in the Craig and Gordon model. Generally, δ_t was less enriched than the source water (δ_s) in the morning and more enriched in the afternoon, which would be predicted from an increase and decrease in ambient VPD, respectively. On both days, leaves of P. aduncum approached ISS (indicated where δ_t≈δ_s) between 1300 and 1500 h. Evaporative site enrichment was maintained into the late afternoon, despite a decrease in ambient VPD. The data presented provide a greater insight into the natural variation in isotopic discrimination under field conditions, which may help to refine models of terrestrial biome discrimination.     

NMR,HS‐SPME‐GC/MS,and HPLC/MSn Analyses of Phytoconstituents and Aroma Profile of Rosmarinus eriocalyx

In this work, a comprehensive study on the chemical constituents of the aerial parts of Rosmarinus eriocalyx (Lamiaceae), an aromatic shrub traditionally consumed as a food and herbal remedy in Algeria, is presented. The aroma profile was analysed by headspace solid phase microextraction (HS‐SPME) coupled with gas chromatography‐mass spectrometry (GC/MS), whereas the crude extract constituents were analyzed by ¹H‐NMR and by high performance liquid chromatography coupled with mass spectrometry (HPLC/MSⁿ). Thirty‐nine volatile compounds, most of them being monoterpenes, have been identified, with camphor, camphene, and α‐pinene as the most abundant constituents. ¹H‐NMR analysis revealed the presence of phenolic compounds and betulinic acid while HPLC/MSⁿ allowed the identification of glycosilated and aglyconic flavonoids as well as phenylpropanoid derivatives. Some of these constituents, namely as betulinic acid, rosmanol, and cirsimaritin were reported for the first time in R. eriocalyx.     

In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor

Summary 80dargECBH DNA has been used to direct cell-free synthesis of argininosuccinase, the argH gene product in Escherichia coli K12. In vitro enzyme synthesis is sensitive to repression by partially purified preparations from an argR ⁺ strain but not by corresponding preparations from an argR ^- strain. Using DNA-cellulose chromatography, approximately seventyfold purification of repressor has been obtained. The partially purified preparation represses argininosuccinase synthesis but has no effect on -galactosidase synthesis.