Genetic Analysis of the Chlamydomonas Reinhardtii I-Crei Mobile Intron Homing System in Escherichia Coli

We have developed and used a genetic selection system in Escherichia coli to study functional requirements for homing site recognition and cleavage by a representative eukaryotic mobile intron endonuclease. The homing endonuclease, I-CreI, was originally isolated from the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. I-CreI homing site mutants contained base pair substitutions or single base deletions that altered the rate of homing site cleavage and/or product release. I-CreI endonuclease mutants fell into six phenotypic classes that differed in in vivo activity, toxicity or genetic dominance. Inactivating mutations clustered in the N-terminal 60% of the I-CreI amino acid sequence, and two frameshift mutations were isolated that resulted in premature translation termination though retained partial activity. These mutations indicate that the N-terminal two-thirds of the I-CreI endonuclease is sufficient for homing site recognition and cleavage. Substitution mutations altered in four potential active site residues were examined: D20N, Q47H or R70A substitutions inactivated endonuclease activity, whereas S22A did not. The genetic approach we have taken complements phylogenetic and structural studies of mobile intron endonucleases and has provided new information on the mechanistic basis of I-CreI homing site recognition and cleavage.     

Generation of nonspecific murine cytotoxic T cells in vitro by purified human interleukin 2

Murine spleen cells developed into nonspecific cytotoxic cells within 72 hr of culture in the presence of highly purified sources of human interleukin 2. In whole spleen cell cultures, human interleukin 2 generated effector cells which were Thy 1.2⁺, Lyt 2.2⁺, resistant to γ irradiation (1000 R), and capable of lysing both H-2 compatible and incompatible targets. The effector cells generated in this manner were not restricted to classical natural killer cell-sensitive targets. If thymus-derived cells (T cells) were depleted from the spleen cell population before culture with human interleukin 2, the effector cells generated were enriched in effectors capable of lysing natural killer cell-sensitive targets. Interferon was not produced in interleukin 2-stimulated spleen cell cultures. In addition, heterologous antibody to murine -γ-interferon did not abrogate the generation of cytotoxic cells by human interleukin 2. These and additional data suggest that human interleukin 2 is capable of stimulating γ-irradiation-sensitive Thy 1.2⁺ cell(s) capable of lysing a variety of target cells regardless of inherent sensitivities to classical natural killer cells. Thy 1.2^? cells were also stimulated by human interleukin 2 and lysed only natural killer cell-sensitive targets. Human interleukin 2 caused some Thy 1.2^? cells to become susceptible to lysis by anti-Thy 1.2 serum and complement.     

Aquaporins influence seed dormancy and germination in response to stress

Aquaporins influence water flow in plants, yet little is known of their involvement in the water‐driven process of seed germination. We therefore investigated their role in seeds in the laboratory and under field and global warming conditions. We mapped the expression of tonoplast intrinsic proteins (TIPs) during dormancy cycling and during germination under normal and water stress conditions. We found that the two key tonoplast aquaporins, TIP3;1 and TIP3;2, which have previously been implicated in water or solute transport, respectively, act antagonistically to modulate the response to abscisic acid, with TIP3;1 being a positive and TIP3;2 a negative regulator. A third isoform, TIP4;1, which is normally expressed upon completion of germination, was found to play an earlier role during water stress. Seed TIPs also contribute to the regulation of depth of primary dormancy and differences in the induction of secondary dormancy during dormancy cycling. Protein and gene expression during annual cycling under field conditions and a global warming scenario further illustrate this role. We propose that the different responses of the seed TIP contribute to mechanisms that influence dormancy status and the timing of germination under variable soil conditions.     

Small mouse cholangiocytes proliferate in response to H1 histamine receptor stimulation by activation of the IP3/CaMK I/CREB pathway

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP(3))/Ca(2+)-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca(2+) concentration levels; and 2) increased [Ca(2+)](i) levels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP(3)/Ca(2+)-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24-48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP(3), Ca(2+) and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP(3)/Ca(2+) levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP(3)/Ca(2+)/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.     

Final height in patients with idiopathic short stature and high growth hormone responses to stimulation tests

Children with idiopathic short stature (ISS) may have normal or increased growth hormone (GH) responses to provocation tests and achieve a final height (FH) below -2.0 standard deviation score (SDS) if untreated. FH of subjects with high stimulated GH levels has not been studied in detail. AIM: It was the aim of this study to analyse FH in ISS patients with high GH peak responses to the provocation test. PATIENTS AND METHODS: We studied 16 patients (9 pre-pubertal) with ISS and a GH peak >or=40 mU/l to insulin-induced hypoglycaemia. The patients were recalled at age 19.7 +/- 2.5 years for measurement of FH when blood samples were obtained for serum insulin-like growth factor (IGF)-I, IGF binding protein 3, acid-labile subunit and GH binding protein measurements. GH bioactivity was determined using the Nb2 bioassay. RESULTS: FH was -3.1 +/- 1.0 SDS, being significantly lower than target height (TH). At FH, IGF-I levels were within -1.5 and +1.5 SDS for age and sex in 10 patients and higher than +1.5 SDS in 6 patients. IGF binding protein 3, acid-labile subunit, GH binding protein levels and GH bioactivity values were normal. SUMMARY: These data suggest that patients with ISS and high GH levels during a GH stimulation test may have a more compromised FH. The association of severe ISS with a peak GH >40 mU/l might suggest a degree of insensitivity for the GH-IGF-I axis.     

Production and response to interleukin 2 in vitro and in vivo after bone marrow transplantation in mice

Lymphoid cells from bone marrow radiation chimeras do not produce normal levels of IL 2 but are capable of responding to IL 2 in mitogenic and cytotoxic assays in vitro. The administration of recombinant human IL 2 into host mice that have received allogeneic, H-2-compatible marrow did not enhance mortality.     

The role of anthropogenic influences in a mixed-conifer forest mortality episode

Abstract. Anthropogenic influences on recent tree mortality in mid-montane mixed conifer forests of southern California, USA, and northern Baja California, Mexico, were investigated. The Pinus jeffreyi-Abies concolor phase of the mixed-conifer montane community was sampled at three sites, characterized by: (1) low levels of air pollution and long-term fire suppression (Holcomb Valley in the San Bernardino Mountains, California); (2) chronic, severe air pollution and long-term fire suppression (Barton Flats in the San Bernardino Mountains, California); and (3) little air pollution and no fire suppression (La Corona Arriba in the Sierra San Pedro Martir in Baja California, Mexico). Similar percentages of dead standing trees, around 14 %, were found at both San Bernardino sites, but a comparatively low level, 4 %, was found in the Mexican forest. Anthropogenic effects, in particular fire suppression, may play an important role in enhancing the impact of natural stresses on the dynamics of mixed conifer forests.