Effects of temperature and CO2 enrichment on kinetic properties of phospho-enol-pyruvate carboxylase in two ecotypes of Echinochloa crus-galli (L.) Beauv., a C4 weed grass species

Summary Two populations of Echinochloa crus-galli (Québec, Mississippi) were grown at the Duke University Phytotron under 2 thermoperiods (28°/22°C, 21°/15°C day/night) and 2 CO₂ regimes (350 and 675 l l^-1). Thermostability, energy of activation (E _a ),K _m (PEP), K _m (Mg⁺⁺), and specific activity of phospho-enol-pyruvate carboxylase (PEP_c) were analyzed in partially purified enzyme preparations of plants grown for 5 weeks. Thermostability of PEP_c from extracts (in vitro) and leaves (in situ) was significantly higher in Mississippi plants. In vitro denaturation was not appreciably modified by thermal acclimation but CO₂ enrichment elicited higher thermostability of PEP_c. In situ thermostability was significantly higher than that of in vitro assays and was higher in Mississippi plants acclimated at 28°/22°C and in plants of the two ecotypes grown at 675 l l^-1 CO₂. E _a (Q ₁₀ 30°/20°C) for PEP_c was significantly lower in Québec plants as compared to Mississippi and no acclimatory shifts were observed. Significantly higher K _m's (PEP) in 20°C assays were obtained for Mississippi as compared to Québec plants but values were similar at 30°C and 40°C assays. K _m (Mg⁺⁺) decreased at higher assay temperatures and were significantly lower for PEP_c of the Québec ecotype. No significant changes in K _m (Mg⁺⁺) values were associated with modifications in temperature on CO₂ regimes. PEP_c activity measured at 30°C was significantly higher for Québec plants when measured on a leaf fresh weight, leaf area or protein basis but not on a chlorophyll basis. Significantly higher PEP_c activity for both genotypes was observed for plants acclimated at 21°/15°C or grown at 675 l l^-1 CO₂. Net photosynthesis (Ps) and net assimilation rates (NAR) were higher in Québec plants and were enhanced by CO₂ enrichment. NAR was higher in plants acclimated at low temperature, while an opposite trend was observed for Ps. PEP_c activities were always in excess of the amounts required to support observed rates of CO₂ assimilation.     

Evaluation of H2O activity in the free or phosphorylated catalytic site of Ca2+-ATPase

The sarcoplasmic reticulum Ca2+-ATPase catalyses a reversible calcium transport coupled to phosphate transfer between ATP and water. It has been proposed [Biochemistry (1980) 19, 4252-4261] that the reactivity of the acyl-phosphate bond is dependent on the water activity within the catalytic site. We have tested this hypothesis and found that the polarity in the free catalytic site is lower than that of water, a further and large decrease is observed when the enzyme is phosphorylated by Pi. Phosphorylation by ATP indicates that this polarity change is specifically associated with the formation of the ADP-insensitive phosphoenzyme.     

Glutamate synthase in rice roots. Studies on the electron donor specificity

Rice root glutamate synthase activity was assayed with various reducing systems. Ferredoxin-dependent glutamate synthase (EC 1.4.7.1) and pyridine nucleotide-dependent glutamate synthase (NADH, EC 1.4.1.14; or NADPH, EC 1.4.1.13) exhibited a strict specificity for the electron donor. The ferredoxin-dependent glutamate synthase from rice roots could accept electrons from photoreduced ferredoxin in an illuminated reconstituted spinach chloroplast system. Thioredoxin, a potent electron carrier, was not able to provide either ferredoxin-dependent or pyridine nucleotide-dependent glutamate synthase with electrons as no glutamate formation was detected in the presence of reduced thioredoxin f or m.     

Interleukin 2-dependent natural killer (NK) cell lines from patients with primary T cell immunodeficiencies

Bulk cultured cell lines with natural killer (NK) activity were derived by in vitro culture with interleukin 2-containing conditioned medium (IL 2-CM) of peripheral blood mononuclear leukocytes (PBL) from patients with primary T cell deficiencies. Lines were developed from three patients with severe combined immunodeficiency (SCID) and one patient with Nezelof's syndrome and contained several populations of cells with distinct phenotypes. All lines contained a cell population expressing the Leu-5 (50K) (sheep red blood cell receptor), 3A1 (40K), and OKT10 antigens, but lacking the pan T cell antigens Leu-1 (67K) and Leu-4 (19K) as well as the markers of T cell subsets Leu-2a (32K) and Leu-3a (56K). These cells failed to express the Leu-7 antigen and only weakly expressed OKM1. In addition, one line contained a population of Leu-5+, 3A1+, OKT10+, Leu-2a+, Leu 1-, and Leu 4- cells. Three of the lines also contained populations with classic T cell (Leu-1 and-Leu 4+) phenotypes. The lines were enriched in NK activity compared with the PBL from which they were derived. Their growth was strictly dependent on IL 2-CM. Highly purified IL 2, lacking any other detectable protein contaminants or lymphokine activities, was capable of supporting the growth of the Leu-5+, 3A1+ "null" cell populations from these lines without alteration in their functional activity or phenotype. Thus, studies of in vitro expanded cell lines from patients with severe disorders of T cell function and thymic involution indicate that this "null" cell population does not require thymic maturation to develop its effector function. This "null" cell population can be maintained in vitro in the presence of IL 2. This finding is analogous to the data obtained from study of NK cells in athymic (nude) mice.     

The utilization ofL-amino acids as carbon source by yeasts of the generaHansenula andTrichosporon

The assimilation of singleL-amino acids as carbon source in the absence of added nitrogen has been studied for all the described species in the generaHansenula andTrichosporon. The range in the number of amino acids used by individual species varied in the two genera, inHansenula from 0 to 6, inTrichosporon from 0 to 16. These characters were considered in relation to the phylogenetic scheme and ecological origin ofHansenula.     

Effects of Electrical Stimulation on Molecular Forms of Butyrylcholinesterase in Denervated Fast and Slow Latissimus Dorsi Muscles of Newly Hatched Chicken

The effects of denervation and direct electrical stimulation upon the activity and the molecular form distribution of butyrylcholinesterase (BuChE) were studied in fast-twitch posterior latissimus dorsi (PLD) and in slow-tonic anterior latissimus dorsi (ALD) muscles of newly hatched chicken. In PLD muscle, denervation performed at day 2 substantially reduced the rate of rapid decrease of BuChE specific activity which takes place during normal development, whereas in the case of ALD muscle little change was observed. Moreover, the asymmetric forms which were dramatically reduced in denervated PLD muscle were virtually absent in denervated ALD muscle at day 14. Denervated PLD and ALD muscles were stimulated from day 4 to day 14 of age. Two patterns of stimulation were applied, either 5-Hz frequency (slow rhythm) or 40-Hz frequency (fast rhythm). Both patterns of stimulation provided the same number of impulses per day (about 61,000). In PLD muscle, electrical stimulation almost totally prevented the postdenervation loss in asymmetric forms and led to a decrease in BuChE specific activity. In ALD muscle, electrical stimulation partially prevented the asymmetric form loss which occurs after denervation. This study emphasizes the role of evoked muscle activity in the regulation of BuChE asymmetric forms in the fast PLD muscle and the differential response of denervated slow and fast muscles to electrical stimulation.     

Extensive changes in cytokeratin expression patterns in pathologically affected human gingiva

The stratified squamous epithelium of the oral gingiva and the hard palate is characterized by a tissue architecture and a cytoskeletal composition similar to, although not identical with, that of the epidermis and fundamentally different from that of the adjacent non-masticatory oral mucosa. Using immunocytochemistry with antibodies specific for individual cytokeratins, in situ hybridization and Northern blots of RNA with riboprobes specific for individual cytokeratin mRNAs, and gel electrophoresis of cytoskeletal proteins of microdissected biopsy tissue samples, we show changes in the pattern of expression of cytokeratins and their corresponding mRNAs in pathologically altered oral gingiva. Besides a frequently, although not consistently, observed increase in the number of cells producing cytokeratins 4 and 13 (which are normally found as abundant components in the sulcular epithelium and the alveolar mucosa but not in the oral gingiva) and a reduction in the number of cells producing cytokeratins 1, 10 and 11, the most extensive change was noted for cytokeratin 19, a frequent cytokeratin in diverse one-layered and complex epithelia. While in normal oral gingiva cytokeratin 19 is restricted to certain, sparsely scattered cells of --or near--the basal cell layer, probably neuroendocrine (Merkel) cells, in altered tissue of inflamed samples it can appear in larger regions of the basal cell layer(s) and, in apparently more advanced stages, also in a variable number of suprabasal cells. Specifically, our in situ hybridization experiments show that this altered suprabasal cytokeratin 19 expression is more extended at the mRNA than at the protein level, indicating that cytokeratin 19 mRNA synthesis may be a relatively early event during the alteration. These changes in cytokeratin expression under an external pathological influence are discussed in relation to other factors known to contribute to the expression of certain cytokeratins and with respect to changes occurring during dysplasia and malignant transformation of oral epithelia.