An improved method for the generation and transfection of protoplasts from Cucumis sativus Cotyledons

Summary A protocol was developed for the preparation of Cucumis sativus var Straight 8 protoplasts that incorporates a two-step Ficoll^® gradient and results in a high percentage of viable, debris-free protoplasts suitable for the transient expression of foreign genes. Polyethylene glycol and electroporation were compared for their effect on protoplast transfection with commonly used reporter genes. Using a polyethylene glycol method, cucumber protoplasts transfected with a plasmid containing the -glucuronidase gene showed high expression levels, while protoplasts transfected with a plasmid containing the chloramphenicol acetyl transferase gene showed levels of activity that were barely distinguishable from mock-transfected controls. Tomato ringspot virus genomic RNA was also transfected into the protoplasts, and the assembly of viral particles was confirmed.     

Glutamine synthetase (GS) expression is reduced in senile dementia of the Alzheimer type

Glutamine synthetase (GS), a metabolic marker of the mature astrocyte, was investigated in the temporal neocortex of postmortem brain samples of 8 cases, either not demented or affected by senile dementia of the Alzheimer type. A negative correlation between the GS protein level and the density of both classical A4 deposits and senile plaques was evidenced. Such a correlation for GS underlies a dysfunction of the astroglial metabolism and particularly of the glutamate and ammonia neutralization. Since GS is sensitive to oxidative lesioning, the changes in GS level that were observed, occurring at the posttranslational stage, might reflect oxidative damage and have severe consequences on the pathological cascade of events.     

Subregional mapping of the human gonadotropin-releasing hormone receptor (GnRH-R) gene to 4q between the markers D4S392 and D4S409

We have isolated nine yeast artificial chromosomes (YACs) containing the gene that encodes the human gonadotropin-releasing hormone receptor (GnRH-R) gene by screening the YAC library of the Centre d'Etude du Polymorphisme Humain (Hôpital Saint-Louis, Paris, France) by the use of the polymerase chain reaction. We defined the location of the GnRH-R gene relative to 4q microsatellite markers D4S392 and D4S409. The genetic positions of these markers on chromosome 4 are 76 and 77 cM, respectively. This location was further established by chromosomal in situ hybridization.     

Excess of deletions of maternal origin in the DiGeorge/Velo-cardio-facial syndromes. A study of 22 new patients and review of the literature

We have determined the parental origin of the deleted chromosome 22 in 29 cases of DiGeorge syndrome (DGS) using a CA-repeat mapping within the commonly deleted region, and in one other case by using a chromosome 22 short arm heteromorphism. The CA-repeat was informative in 21 out of 29 families studied and the deleted chromosome was of maternal origin in 16 cases (72%). When these data are pooled with recent results from the literature, 24 de novo DGS, velo-cardio-facial syndrome (VCFS) and isolated conotruncal cardiac disease deletions are found to be of maternal origin and 8 of paternal origin, yielding a ² of 8 with a probability level lower than 0.01. These data, and review of the literature on familial DGS/VCFS and isolated conotruncal cardiopathies suggest that there is a strong tendency for the 22q11.2 deletions to be of maternal origin.     

Isolation and characterization of a new hydrogen-utilizing bacterium from the rumen

Abstract A new H2 CO2-utilizing acetogenic bacterium was isolated from the rumen of a mature deer. This is the first report of a spore-forming Gram-negative bacterial species from the rumen. The organism was a strictly anaerobic, motile rod and was able to grow autotrophically on hydrogen and carbon dioxide. Acetate was the major product detected. Glucose, fructose and lactate were also fermented heterotrophically. The optimum pH for growth was 7.0–7.5, and the optimum temperature was 37–42 °C. Yeast extract was required for growth and rumen fluid was highly stimulatory. The DNA base ratio was 52.9 ± 0.5 mol% G + C. On the basis of these characteristics and fermentation products, the isolate was considered to be different from acetogenic bacteria described previously.     

Adsorption equilibrium and dynamics of lactase/CM-Sephadex system

Summary Partitioning behaviour and adsorption isotherms of lactase/CM-Sephadex system at equilibrium were investigated together with the adsorption kinetics in this study. Maximum adsorption was obtained at the pH values between 5.5–6.0. Adsorption isotherm was a close fit to the Langmuir model.Nomenclature a specific mass transfer area - D_m molecular diffusion coefficient (m²/sec) - e₁, e₂ charge of the protein and the gel - k apparent mass transfer coefficient (s^-1) - ka global mass transfer coefficient - f partition coefficient - K_p dissociation constant for adsorbent-adsorbate complex, (mg/mL solvent) - p equilibrium concentration of free enzyme, (mg free enzyme/mL solution) - q equilibrium concentration of adsorbed enzyme, (mg ads./mL gel) - q_m maximum adsorption capacity, (mg ads./ml gel) - Re particle Reynolds number - Sh Sherwood number - V_g/V gel volume (mL)/bulk solvent volume (mL) - Z dimensionless extent of adsorption - K_p/P_o , model parameter - (/) +1 , model parameter - V_g q_m / V P_o , model parameter     

Entrapment of l-tryptophan producing Escherichia coli in different matrices: activity of immobilized cells

Cells of Escherichia coli induced for l-tryptophan synthase [l-serine hydro-lyase (adding indole-glycerol-phosphate), EC 4.2.1.20] have been assayed in DMF and DMSO aqueous solvents as reaction medium. Up to 20% DMF/water, cells retained 90% of their tryptophan synthase activity. Concentrations of 20 mM indole, which did not inhibit this reactivity, could be reached with 5% DMF/water. Four matrices were compared for cell immobilization: polyacrylamide, foam particles of bovine seum albumin, alginate and κ-carrageenan. The best activity was retained with the latter matrix, and the preparations thus obtained allowed high productivity of l-tryptophan. Various systems of production of l-tryptophan with κ-carrageenan and DMF/water were studied.     

Clonal analysis of the T lymphocytes involved in parent versus Fn1 graft-versus-host reaction

A systemic graft-versus-host reaction (GVHR) leading to 50% mortality by day 20 was elicited by the injection of CBA (10⁵) or B10 (10⁶) parental T lymphocytes into irradiated (750 rad) and bone marrow protected (CBA x B10)F₁ recipients. Between days 12 and 28 the spleens of the sick mice were analyzed by limiting dilution, performed with irradiated F₁ cells and a source of interleukin-2 (IL-2), to determine the frequency of cells with an antihost proliferative or cytolytic activity and to derive T lymphocyte clones. The frequency of cells with antihost proliferative or cytolytic activity was approximately 10^–3 in either combination. In the CBA vs F₁ GVHR, all eight clones isolated with anti-F₁ activity were Lyt-2^–, noncytolytic, mixed lymphocyte reaction (MLR) responders and IL-2 producers, three of which mapped to the A ^b locus, while in the B10 anti-F1 combination, eight of the nine anti-F₁ clones isolated were Lyt-2⁺, poor MLR responders and non-IL-2 producers, but cytolytic and mapping to K _k . These findings suggest a much higher frequency of T cells recognizing the A-locus antigens in the CBA than in the B10 strain.     

Project Cassini: A Saturn Orbiter/Titan Probe mission proposal

Titan is the only moon in the solar system with a substantial atmosphere. The organic chemistry of its N₂–CH₄ atmosphere may resemble that of the earth's primitive atmosphere before life arose. The investigation of the synthesis of prebiotic molecules in Titan's atmosphere and the atmospheric and surface environments of this planet-sized moon will be the focal point of the Cassini Project proposed to the European Space Agency for an international Saturn Orbiter/Titan Probe mission.     

Comparative use of fructose and glucose in human liver and fibroblastic cell cultures

Summary The effect of fructose as a substitute for glucose in cell culture media was investigated in human skin fibroblast and liver cell cultures. Cells were grown for between 2 and 10 days in identical flasks in four different media, containing 5.5, mmol·1⁻¹ and 27.5 mmol·I⁻¹ glucose and fructose, respectively. In the presence of fructose, cell growth was stimulated, but less in liver cells than fibroblasts. At Day 6, increases were observed in [³H]thymidine incorporation, protein levels, and amino acid consumption, and a reduction was noted in ATP levels. In media containing 5.5, mmol·1⁻¹ glucose or fructose, consumption of fructose was four times lower than that of glucose at Day 3 and did not rise until Day 6. In fructose media, the lactate production was very low (four to five times less than that of glucose) and the pH values were always higher. Some findings were different for the fibroblasts and liver cells, owing to the specific characteristics of these two cell types in culture; this applied especially to the effects of glucose and fructose concentrations of 27.5 mmol·1⁻¹. Several possible explanation for the stimulation of cell growth in fructose medium were discussed. This work was supported by grants for the Institut National de la Santé et de la Recherche Médicale (ATP 82-79-114) and the Unité d'Enseignement et de Recherche, Le Kremlin-Bicêtre, Université Paris-Sud (C. R. 848).