Field comparison of disjunct and conventional eddy covariance techniques for trace gas flux measurements.

A field intercomparison experiment of the disjunct eddy covariance (DEC) and the conventional eddy covariance (EC) techniques was conducted over a grass field. The half-hourly water vapor fluxes measured by the DEC were within the estimated uncertainty from the fluxes measured by the EC. On the average there was a slight overestimation (<10%) of the fluxes measured by the DEC during the day and underestimation during the night as compared to the fluxes measured by the EC. As this bias does not appear in the simulated DEC measurements it is likely to be due to instrumental problems. The insensitivity of the quality of the fluxes measured by the DEC method to the deficiencies in the gas analysis shows the robustness of this new approach for measuring the surface-atmosphere exchange of trace gases.

Diffusion of lactate and ammonium in relation to growth of Geotrichum candidum at the surface of solid media.

Geotrichum candidum was cultivated at the surface of solid model media containing peptone to simulate the composition of Camembert cheese. The surface growth of G. candidum induced the diffusion of substrates from the core to the rind and the diffusion of produced metabolites from the rind to the core. In the range of pH measured during G. candidum growth, constant diffusion coefficients were found for lactate and ammonium, 0.4 and 0.8 cm(2) day(-1), respectively, determined in sterile culture medium. Growth kinetics are described using the Verlhust model and both lactate consumption and ammonium production are considered as partially linked to growth. The experimental diffusion gradients of lactate and ammonium recorded during G. candidum growth have been fitted. The diffusion/reaction model was found to match with experimental data until the end of growth, except with regard to ammonium concentration gradients in the presence of lactate in the medium. Indeed, G. candidum preferentially assimilated peptone over lactate as a carbon source, resulting in an almost cessation of ammonium release before the end of growth. On peptone, it was found that the proton transfer did not account for the ammonium concentration gradients. Indeed, amino acids, being positively charged, are involved in the proton transfer at the beginning of growth. This effect can be neglected in the presence of lactate within the medium, and the sum of both lactate consumption and ammonium release gradients corresponded well to the proton transfer gradients, confirming that both components are responsible for the pH increase observed during the ripening of soft Camembert cheese.

A new B-chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B-chain structure.

The solution structure of a new B-chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D-NMR and molecular modeling. This structure is compared with that of the oxidized B-chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res.46, 424-433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B-chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B-chain. 2D-NMR experiments confirmed, among multiple conformations, that the B-chain mutant presents defined secondary structures such as a alpha-helix between residues B9 and B19, and a beta-turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B-chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B-chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B-chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B-chains.

Reconstruction of the biomass history from carbon and nitrogen substrate consumption, ammonia release and proton transfer during solid cultures of Geotrichum candidum and Penicillium camembertii.

Geotrichum candidumand Penicillium camembertii were cultivated on the surface of a gelified medium, simulating the composition of the aqueous phase of a Camembert cheese. The relation of their growth with substrate consumption (carbon or nitrogen), metabolite production (ammonia), or proton transfer (deduced from pH by means of the buffer capacity of the medium) was examined. The coefficients associated with cellular biosynthesis and resulting from cellular maintenance were determined. From these coefficients and the considered substrate utilization or metabolite production kinetics, the growth kinetics were reconstructed until the end of growth. The model allowed analysis of biosynthesis and cellular maintenance contributions to the considered kinetics. At the end of growth, almost all the peptone was used for G. candidum biosynthesis, while most of the lactic acid (62%) was used for cellular maintenance. P. camembertii metabolized fewer amino acids as carbon sources, resulting in use of peptone for maintenance (12%), and lactic acid (80%) for cell biosynthesis. For both microorganisms, ammonia production was growth-associated, since this production resulted from the deamination of carbon- and nitrogen-source amino acids, in close relation with peptone consumption.

Sequences and antimycoplasmic properties of longibrachins LGB II and LGB III, two novel 20-residue peptaibols from Trichoderma longibrachiatum.

Longibrachins are members of the class of natural Aib-containing peptides designated as peptaibols. Six longibrachins, LGA I-IV and LGB II and III, were purified from a Trichoderma longibrachiatum strain by a procedure employing several chromatography steps including reversed-phase HPLC. The amino acid sequence determination was based on a combination of liquid secondary ion mass spectrometry (LSIMS) and two-dimensional 1H and 13C NMR spectroscopy. Longibrachins are 20-residue peptaibols with a C-terminal phenylalaninol and either neutral (LGA; Gln18) or acidic (LGB; Glu18) character. Longibrachins LGB II and III have novel sequences. Both longibrachins LGA and LGB show significant bactericidal activity against mycoplasmas (Acholeplasma, Mycoplasma, and Spiroplasma), with minimal inhibitory concentrations in the range 1.56-12.5 microM (3-25 micrograms/mL), and also perturb the permeability of membrane bilayers. Longibrachin LGA IV is the most potent of the presently known 18-20-residue peptaibols. The antimicrobial and membrane-perturbing properties of longibrachins, which are described here for the first time, were shown to be correlated.

Enantioseparation of aminoglutethimide and thalidomide by high performance liquid chromatography or supercritical fluid chromatography on mono-2 and mono-6-O-pentenyl-beta-cyclodextrin-based chiral stationary phases.

Mono-2 and mono-6-O-pentenyl-beta-cyclodextrin (mono-2-pent-beta-CD and mono-6-pent-beta-CD), covalently linked to mercaptopropylsilica gel (thiol-Si) through thioether or sulfone linkage, reveal differentiated enantioselectivities in the separation of piperidine-2,6-dione-related drugs, namely aminoglutethimide and thalidomide, in supercritical fluid conditions. Supercritical fluid chromatographic resolution on completely defined mono-cyclodextrin derivative-based chiral stationary phases (CSP) is a method of choice for the separation of aminoglutethimide but not effective for thalidomide. For both high performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) conditions, the impact of the position, imposed to be 2 or 6 in our synthetic pathway, of the pentenyl moiety on one of the glucopyranosidics of the CD cage is of crucial importance in the chiral discrimination phenomenon. Additionally, the nature of the heteroatom present in the spacer arm between the CD and the silica gel, in this case thioether or sulfone functionality, is also essential for the chiral recognition mechanism(s) for the solute enantiomer.

A new model for the reconstruction of biomass history from carbon dioxide emission during batch cultivation of Geotrichum candidum.

A non-structured model has been developed to describe the CO2 emission during growth of Geotrichum candidum on a lactate + peptone-based liquid medium. From the nitrogen and carbon mass balances, it was shown that about 50% of the total CO2 released was from the metabolism of the energy supply for biosynthesis, and the remaining from that for maintenance; thus, CO2 production was considered to be partially associated with growth. The model fitted the experimental data as long as a net growth was observed (0-50 h). The coefficients for growth- and non-growth-associated CO2 production were A = 0.646 (dimensionless) and B = 0.017 h(-1), respectively. From the coefficients of the model and the CO2 history data, the biomass kinetics has been reconstructed, and the calculated biomass concentrations agree fairly well with the experimental data. From this, measurement of the CO2 evolved may be used as an indirect and non-intrusive method of monitoring fungal growth during the first 50 h of cultivation.

The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli.

Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non-SOS-dependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol. 174, 7428-7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin-overproducing Escherichia coli strain by reverse-phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two-dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21-residue peptide, cyclo(-Val1-Gly-Ile-Gly-Thr- Pro-Ile-Ser-Phe-Tyr-Gly-Gly-Gly-Ala-Gly-His-Val-Pro-Glu-Tyr-Phe21- ). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21-Val1 bond and led to a single peptide, MccJ25-L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 microgram.mL-1), the thermolysin-linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N-terminal extremity, the present study shows that removal of this extension and head-tail cyclization of the resulting propeptide are the only post-translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.

Expression of glycylated tubulin during the differentiation of spermatozoa in mammals.

Using quantitative immunogold analyses of tubulin isoforms we previously demonstrated a unique differential expression of glutamylated tubulin in the flagellum of mouse and man spermatozoa [Fouquet et al., 1997: Tissue Cell 29:573-583]. We have performed similar analyses for glycylated tubulin using two monoclonal antibodies, TAP 952 and AXO 49, directed to mono- and polyglycylated tubulin respectively. Glycylated tubulin was not found in centrioles and cytoplasmic microtubules (manchette) of germ cells. In mouse and man, axonemal tubulin was first monoglycylated and uniformly distributed in all doublets at all levels of the flagellum in elongating spermatids. In human mature spermatozoa axonemal microtubules were enriched in monoglycylated tubulin from the base to the tip of the flagellum. In mouse sperm flagellum a similar gradient of monoglycylated tubulin was also observed in addition to an opposite gradient of polyglycylated tubulin. In both species, monoglycylated tubulin labeling predominated in doublets 3-8 whereas glutamylated tubulin labeling [Fouquet et al., 1997] predominated in doublets 1-5-6. These differential labelings were suppressed after motility inhibition of mouse spermatozoa by sodium azide treatment and in non-motile human spermatozoa lacking dynein arms. The unique distribution of these tubulin isoforms and the known inhibition of motility induced by their specific antibodies are consistent with a complementary role of tubulin glycylation and glutamylation in the regulation of flagellar beating in mammalian spermatozoa.

Immunoelectron microscopic visualization of intermediate basic proteins HPI1 and HPI2 in human spermatids and spermatozoa.

In humans, intermediate basic proteins HPI1 and HPI2 are considered as common precursors of the P2 protamine family, according to data provided by structural studies of these proteins. The occurrence and fate of proteins HPI1 and HPI2 were investigated in nuclei of human spermatids and spermatozoa by means of immunoelectron microscopy. A specific polyclonal antibody against a synthetic peptide overlapping the N-terminus of HPI1 and HPI2 was prepared and used to detect these proteins on sections of testis and ejaculated sperm. A quantitative analysis of labelling density was performed on micrographs using an interactive image analysis system. The first signs of labelling of intermediate basic proteins appeared in spermatid nuclei at steps 4-5 of spermiogenesis, i.e. during the chromatin condensation process. The nuclear labelling density strongly increased in elongating spermatids (steps 5 and 6) and then sharply decreased from step 6 to step 8 of spermiogenesis. However, weak labelling persisted in the nuclei of mature spermatids and ejaculated spermatozoa. The present results show that the intermediate basic proteins HPI1 and HPI2 are synthesized in large amounts in human spermatids during elongation phase and disappear almost totally in mature spermatids when deposition of protamines is completed in condensed nuclei.

Three-dimensional structure of the ion-channel forming peptide trichorzianin TA VII bound to sodium dodecyl sulfate micelles.

Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the alpha, alpha-dialkylated amino acids, alpha-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignment have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHC alpha H coupling constants, temperature coefficients of amide protons (delta gamma/delta TNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances six phi dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 A for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a beta-turn that leads to an angle about 90 degrees-100 degrees between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.

Expression of tubulin isoforms during the differentiation of mammalian spermatozoa.

Using the GT 335 mAb we have previously demonstrated a differential expression of glutamylated tubulin isoforms during spermatogenesis and in spermatooza of the mouse and human. Moreover, the proximodistal decrease of the immunolabeling and its predominance in doublets 1-5-6, corresponding to the plane of the flagellar wave, suggested that the glutamylated tubulin could be involved in a functional heterogeneity of microtubules in peripheral doublets of the sperm flagellum. In order to characterize further the importance of glutamylated tubulin in the sperm model, we analyzed tubulin isoforms by immunoblotting and quantitative immunogold, using antibodies to the C-terminal domain of both subunits including non-glutamylated and glutamylated epitopes. The unique differential immunolabeling of the glutamylated tubulin was confirmed with three mAbs 406-3, 392-2 and B3, in addition to GT 335. This differential labeling was interpreted as a differential accessibility of tubulin epitopes since it was greatly reduced in human spermatozoa lacking dynein arms and after motility inhibition of normal spermatozoa by azide pretreatment. We suggest that the glutamylated tubulin interacts with other axonemal and/or periaxonemal proteins which could be involved in flagellar beating and its regulation.

Incidence of AZF (azoospermia factor) deletions and familial forms of infertility among patients requiring intracytoplasmic spermatozoa injection (ICSI).

PURPOSE: The aim of our study was to determine the incidence of AZF deletions and familial forms of infertility suggesting autosomal mutations among patients requiring intracytoplasmic sperm injection with ejaculated sperm. METHODS: Cases with obstructive pathologies were excluded; 81 patients were classified according to the numeration of spermatozoa. The distribution was as follows: 10 cases with normal numeration (greater than 20 million/ml) (group 1), 10 cases with between 10 and 20 million/ml (group 2), 6 cases with between 5 and 10 million/ml (group 3), 15 cases with between 1 and 5 million/ml (group 4), 29 cases with less than 1 million/ml (group 5), and 11 azoospermic patients (group 6). The infertility of 11 of the 81 patients might be explained by testicular ectopy. RESULTS: We found two deletions limited to the AZFc region among our 81 infertile patients--one deletion in group 5 and one deletion in group 4 (both groups of oligozoospermic patients)--and no deletion in the groups with normal or subnormal numerations. We found six familial forms of infertility. We did not find any AZF deletion, neither in these 6 patients nor in the 11 with testicular ectopy. The identification of these families of infertile men will allow research of autosomal genes involved in male infertilities. CONCLUSIONS: It is important to test deletions of the AZFc region for oligozoospermic patients, and familial forms of infertility do not seem to concern the same individuals.

Immunoelectron microscopical distribution of histones H2B and H3 and protamines during human spermiogenesis.

The fine structural distribution of histones H2B and H3, and protamines were localized by means of specific antibodies and ultrastructural immunocytochemistry in nuclei of human spermatids and spermatozoa. The antibodies were used to detect the nuclear basic proteins on section of testis and ejaculated spermatozoa by immunoelectron microscopy. A quantitative analysis of labelling density was performed on micrographs using an interactive image analysis system. The labelling density of somatic-type histones H2B and H3 and of their testis-specific variants was constant in the nuclei of young spermatids with round nuclei (stages 1-2), and then increased in intermediate spermatids (stages 3-4). Histone H3 labelling decreased at the end of the elongation phase (stage 5) while histone H2B labelling decreased in mature spermatids (stage 6) only. Spermatozoa were found to be weakly labelled by the anti-histone antibodies. The first signs of labelling of protamines and basic intermediate proteins appeared in spermatid nuclei at stage 4, increased further in stage 6 spermatids and persisted in all sperm nuclei. The present work shows that histone-to-protamine replacement occurs at the beginning of the spermatid maturation phase in human. However, histones are partially retained in mature spermatids and sperm nuclei.

Glutamylated tubulin as a marker of microtubule heterogeneity in the human sperm flagellum.

Four site-directed monoclonal antibodies (mAbs) to tubulin: DM1A and DM1B general anti-alpha and anti-beta tubulin, 6-11B-1 anti-acetylated alpha tubulin and GT335 anti-glutamylated alpha and beta tubulin were used to study the distribution of tubulin isoforms in the human sperm flagellum. Since indirect immunofluorescence (IIF) did not give reliable results, a quantitative study of the immunogold labelling of the flagellum was performed at five levels: the mid-piece, three successive regions of the principal piece and the terminal piece. A uniform labelling was observed with DM1A, DM1B and 6-11B-1 mAbs. In contrast, the labelling of glutamylated tubulin detected with GT335 mAb decreased from the middle piece to the terminal piece both for peripheral doublets and the central pair. The changes in labelling of peripheral doublets were related to the pattern of outer dense fibre (ODF) changes. Thus doublets 1-5-6, associated with the largest number of ODF, were the most heavily labelled. This predominant labelling corresponded to the plane of flagellar beating suggesting a functional heterogeneity of peripheral doublets.

Behaviour of the yeast Kluyveromyces marxianus var. marxianus during its autolysis.

The lactic yeast Kluyveromyces marxianus var.marxianus (formerly K. fragilis) autolyzates at faster rate than Saccharomyces cerevisiae. During K. marxianus autolysis, quite similar release kinetics were observed for intracellular space markers (potassium ions, nucleotides), cell-wall components (polysaccharides, N-acetyl-D-Glucosamine) and non specific products (amino nitrogen). By Scanning Electronic Microscopy examination, no cell burst was observed, but a variation in cell shape (from ellipsoîdal to cylindrical), as well as a 43% decrease in the internal volume were observed. The mechanism proposed for S. cerevisiae autolysis appeared also likely for K. marxianus.

Comparative immunogold analysis of tubulin isoforms in the mouse sperm flagellum: unique distribution of glutamylated tubulin.

The distribution of different tubulin isoforms in the mouse sperm flagellum was studied using four site-directed antibodies to tubulin: DM1A and DM1B general anti alpha and beta-tubulin, 6-11B-1 anti-acetylated alpha-tubulin, and GT335 anti-glutamylated alpha and beta-tubulin. Quantitative immunogold analyses were performed on five regions of the flagellum: the middle piece, three successive regions of the principal piece, and the terminal piece. A uniform labeling was observed with DM1A and DM1B along the entire flagellum both for peripheral doublets and the central pair. Similar results were obtained with 6-11B-1 directed to acetylated alpha-tubulin, an N-terminal-modified tubulin isoform. In contrast, the labeling for glutamylated alpha and beta-tubulin, C-terminal modified isoforms, was not uniform. The highest intensity was found in the middle piece and the terminal piece. The labeling which decreased significantly both for peripheral doublets and central pair along the principal piece was considered as a loss of glutamylated tubulin accessibility. From the middle piece to the end of the principal piece, this labeling was predominant in doublets 1-5-6, corresponding to the plane of the flagellar wave. However, the labeling for doublets 2-3-4-7-8-9 was heterogeneous, showing an increasing asymmetry. These results suggest that in the mammalian sperm cell model, the glutamylated tubulin might be involved in a functional heterogeneity among peripheral doublets of the flagellum.

Fluorescence in situ hybridization analysis of minute marker chromosomes in leukemia with monosomy 7.

Monosomy 7 was detected in bone marrow cells from three patients, one with myeloid leukemia, and two others with myelodysplastic syndrome following previous chemotherapy. Fluorescence in situ hybridization (FISH), carried out with an alphoid DNA probe specific for chromosome 7 centromere, showed that a small marker chromosome present in the tumor cells' karyotype of the three patients, was derived from the missing chromosome 7. In two cases, the marker was a ring chromosome, whereas in the third case it was a tiny dot-like chromosome, unnoticed at first examination on R-banded metaphases. In the three cases, the marker was lost in a proportion of tumor cells. FISH experiments suggested that the marker centromere had undergone structural alterations, with a fluorescence pattern distinct from a normal one. On the whole, these data suggest that: firstly, leukemia-associated monosomy 7 results, in a proportion of cases, from a structural event rather than from simple loss of a whole chromosome 7; secondly, interpretation of interphase FISH must be cautious in monosomy 7 evaluation; and thirdly structural alteration of the chromosome 7 derivative alphoid DNA could explain its propensity to segregate unequally and to be lost at mitosis.

Differential distribution of glutamylated tubulin in the flagellum of mouse spermatozoa.

Using a monoclonal antibody (GT 335) we previously demonstrated that glutamylation is a predominant posttranslational modification of alpha and beta tubulin isoforms in the axoneme of mouse spermatozoa (Fouquet et al., Cell Motil. Cytoskel. 27, 49, 1994). However, we noted that the staining intensity and/or distribution of glutamylated tubulin were not identical using either indirect immunofluorescence (IIF) or immunoelectron microscopy. To test this discrepancy various permeabilization procedures were performed for IIF: methanol or acetone alone or in combination, including freezing pretreatment and with or without paraformaldehyde fixation. Each procedure gave a particular labeling of sperm axoneme. The diversity of axoneme labeling in mouse spermatids and spermatozoa appeared dependent both on the absence or presence of periaxonemal sheaths and permeabilization procedures. For comparison with IIF and to avoid problematic premeabilization treatments a quantitative postembedding immunogold approach was preferred. In these conditions the labeling predominated in the middle piece of the sperm flagellum and decreased progressively in the principal piece. However, the labeling of the terminal piece was similar to that of the middle piece. These results suggested a differential glutamylated tubulin distribution along the axoneme of the mouse sperm flagellum.