Structure and expression of a gene from Arabidopsis thaliana encoding a protein related to SNF1 protein kinase

The AKin10 gene from Arabidopsis thaliana encoding a putative Ser/Thr protein kinase (PK) has been isolated and characterized. The AKin10-encoding gene is located on a genomic 5.4-kb BamHI fragment and contains ten introns, one being located in the 5' untranslated region. The deduced amino acid sequence of AKin10 is 65% identical over the catalytic domain to the yeast PK (SNF1). SNF1 is essential for the derepression of many glucose-repressible genes, including Suc2 which encodes invertase. Southern blot hybridization experiments suggested the presence of one copy of the gene per haploid genome of A. thaliana. Northern hybridization experiments indicated that this gene is expressed in roots, shoots and leaves. AKin10 may play an important role in a signal transduction cascade regulating gene expression and carbohydrate metabolism in higher plants.     

The modification of a radiation lesion in animals with an aqueous extract of Hypericum perforatum L. 2.]

The preparation Hypericum perforatum L. has been shown to decrease the intensity level of enzymatic and non-enzymatic processes of lipid peroxidation of rat liver microsomes in vitro and in vivo. In the mucus of the this intestine in vivo the increase of the number of non-enzymatic SH-groups is observed. Possible mechanisms of radioprotective action of the preparation is under discussion.     

An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.     

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.     

Determination of DNA ploidy in archival tissue from non-Hodgkin's lymphoma using flow and image cytometry.

Paraffin-embedded tissue from a series of 40 cases of diffuse, large cell lymphoma was analyzed by both flow and image cytometry to compare the ability of these techniques to detect DNA aneuploid populations. Image cytometry (ICM) was performed both on nuclear suspensions and tissue sections. Twenty cases (50%) were non-diploid by at least one method of analysis. Twenty-five percent of the cases were aneuploid by flow cytometry (FCM) alone. The majority of these cases were near-diploid tumors which could not be resolved by ICM. Peri-tetraploid peaks were identified by ICM of tissue sections alone in 15% of the cases. There was an apparent loss of these peri-tetraploid cells during the preparation of the nuclear suspensions. The remaining cases showed a good correlation between all three methods in the determination of DNA ploidy. Flow and image cytometry are complimentary techniques when applied to archival tissue, however aneuploid populations may be missed if ICM is not performed on tissue sections.     

In situ hybridization as a rapid means to assess meiotic pairing and detection of alien DNA transfers in interphase cells of wide crosses involving wheat and rye

Summary The objectives of this study were to determine if biotin-labelled total genomic DNA of rye (Secale cereale L.) could be used to (i) preferentially label rye meiotic chromosomes in triticale and (ii) detect translocation stocks at interphase and/or early prophase by in situ hybridization. Welsh triticale, a wheat-rye segmental amphiploid, and Kavkaz wheat, a wheat-rye translocation were used. The results indicated that labelled chromosomes of rye and unlabelled chromosomes of wheat could be observed throughout all meiotic stages in the triticale. For Kavkaz wheat, the presence of the translocated 1RS chromosome arm of rye was detected at the interphase or very early prophase stage. Rapid assessment of feasibility of gene transfers and detection of alien DNA in somatic cells at the interphase stage by in situ hybridization allows for rapid decision-making and saves time and expense in plant breeding programs.Plant Research Centre Contribution No. 1276     

Survival of inoculated Laccaria bicolor in competition with native ectomycorrhizal fungi and effects on the growth of outplanted Douglasfir seedlings

The survival, development and mycorrhizal efficiency of a selected strain of Laccaria bicolor along with naturally occurring ectomycorrhizal fungi in a young plantation of Douglas fir was examined. Symbionts were identified and their respective colonization abilities were determined. Eight species of symbiotic fungi, which may have originated in adjacent coniferous forests, were observed on the root systems. Mycorrhizal diversity differed between inoculated (5 taxa) and control (8 taxa) seedlings. Ectomycorrhizal fungi which occurred naturally in the nursery on control seedlings (Thelephora terrestris and Suillus sp.) did not survive after outplanting. Both inoculated and naturally occurring Laccaria species, as well as Cenococcum geophilum, survived on the old roots and colonized the newly formed roots, limiting the colonization by other naturally occurring fungi. Other fungi, such as Paxillus involutus, Scleroderma citrinum and Hebeloma sp. preferentially colonized the old roots near the seedling's collar. Russulaceae were found mainly in the middle section of the root system. Mycorrhizal colonization by Laccaria species on inoculated seedlings (54%) was significantly greater than on controls (13%) which were consequently dominated by the native fungi. Significant differences (up to 239%) were found in the growth of inoculated seedlings, especially in root and shoot weight, which developed mainly during the second year after outplanting. Seedling growth varied with the species of mycorrhizae and with the degree of root colonization. Competitiveness and effectiveness of the introduced strain on improving growth performances of seedlings are discussed.     

Carrier-Mediated Uptake of Abscisic Acid by Suspension-Cultured Amaranthus tricolor Cells

Abscisic acid (ABA) uptake by Amaranthus tricolor cell suspensions was found to include both a nonsaturable component and a saturable part with K_m of 3.74 ± 0.43 micromolar and an apparent V_max of 1.5 ± 0.12 nanomoles per gram per minute. These kinetic parameters as well as the uptake by intact cells at 0°C or by frozen and thawed cells, are consistent with operation of a saturable carrier. This carrier-mediated ABA uptake was partially energized by ΔpH: it increased as the external pH was lowered to pH 4.0; it decreased after the lowering of the ΔpH by the proton ionophore carbonylcyanide-m-chlorophenylhydrazone or after the altering of metabolically maintained pH gradient by metabolic inhibitors (KCN, oligomycin). The carrier is specific for ABA among the plant growth regulators tested, is unaffected by (RS)-trans-ABA and was inhibited by (S)-ABA, (R)-ABA, and also by the ABA analog LAB 173711.     

The candidate gene for the X-linked Kallmann syndrome encodes a protein related to adhesion molecules

Kallmann syndrome associates hypogonadotropic hypogonadism and anosmia and is probably due to a defect in the embryonic migration of olfactory and GnRH-synthesizing neurons. The Kallmann gene had been localized to Xp22.3. In this study 67 kb of genomic DNA, corresponding to a deletion interval containing at least part of the Kallmann gene, were sequenced. Two candidate exons, identified by multiparameter computer programs, were found in a cDNA encoding a protein of 679 amino acids. This candidate gene (ADMLX) is interrupted in its 3' coding region in the Kallmann patient, in which the proximal end of the KAL deletion interval was previously defined. A 5' end deletion was detected in another Kallmann patient. The predicted protein sequence shows homologies with the fibronectin type III repeat. ADMLX thus encodes a putative adhesion molecule, consistent with the defect of embryonic neuronal migration.