A Dual Strategy to Cope with High Light in Chlamydomonas reinhardtii

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.     

The K+ Channel KCa3.1 as a Novel Target for Idiopathic Pulmonary Fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a common, progressive and invariably lethal interstitial lung disease with no effective therapy. We hypothesised that K_Ca3.1 K⁺ channel-dependent cell processes contribute to IPF pathophysiology.

Methods

K_Ca3.1 expression in primary human lung myofibroblasts was examined using RT-PCR, western blot, immunofluorescence and patch-clamp electrophysiology. The role of K_Ca3.1 channels in myofibroblast proliferation, wound healing, collagen secretion and contraction was examined using two specific and distinct K_Ca3.1 blockers (TRAM-34 and ICA-17043 [Senicapoc]).

Results

Both healthy non fibrotic control and IPF-derived human lung myofibroblasts expressed K_Ca3.1 channel mRNA and protein. K_Ca3.1 ion currents were elicited more frequently and were larger in IPF-derived myofibroblasts compared to controls. K_Ca3.1 currents were increased in myofibroblasts by TGFβ1 and basic FGF. K_Ca3.1 was expressed strongly in IPF tissue. K_Ca3.1 pharmacological blockade attenuated human myofibroblast proliferation, wound healing, collagen secretion and contractility in vitro, and this was associated with inhibition of TGFβ1-dependent increases in intracellular free Ca²⁺.

Conclusions

K_Ca3.1 activity promotes pro-fibrotic human lung myofibroblast function. Blocking K_Ca3.1 may offer a novel approach to treating IPF with the potential for rapid translation to the clinic.     

Purification of clinical-grade disulfide stabilized antibody fragment variable—Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli

Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion—toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the V_H or V_L. Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, V_L and V_H PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins—MR1-1 and HA22.     

Early Tertiary fossil plants and paleoclimate of Lanzhou Basin

Fossil plants from the lower part of Xianshuihe Formation in the Lanzhou Basin, Gansu Province were studied. The flora contains 29 species, representing 20 genera and 12 families, which include Lauraceae ( Daphnogene ), Lardizabalaceae ( Akebia ), Berberidaceae ( Berberis ), Ulmaceae ( Planera, Ulmus, Zelkova ), Betulaceae ( Alnus, Carpinus ), Myricaceae( Myrica ), Salicaceae ( Populus, Salix), Myrsinaceae(Ardisia), Rosaceae ( Prunus, Sorbus, Sorbaria, Spiraea ), Leguminosae ( Gleditsia, Sophora), Anacardiaceae (Rhus), Caprifoliaceae(Viburnum). An analysis of the floristic elements and their foliar physiognomy shows that most members of the flora are deciduous broad-leaved trees or shrubs with a few evergreen shrubs. The most noteworthy species is Rhus turcomanica which was present in the Middle Eocene to Late Eocene of Central Asia (Kazakhstan, Turkmenistan). Generally, Rhus turcomanica occurred at the same beds as Palibinia, an extinct fossil plant whose presence indicates a subtropical dry climate. Another species, Sorbaria callicomifolia Kornilova was present from the Early Oligocene to Early Miocene of Central Asia (Kazakhstan and Turkmenistan). According to an analysis of spores and pollen, this flora contains over 20 species. It is predominated by the angiosperm pollen. There appeared Ephedripites and Nitrariadites which were important elements in the dry area. Ephedripites was found from the Upper Cretaceous to Early Tertiary. Nitrariadites occurred in the Late Miocene, whereas Rhus turcomanica and Sorbaria callicomifolia were both reported in the subtropical dry area from the Middle Eocene to Early Oligocene. The latest record of Rhus turcomanica is from the Middle Eocene to Early Oligocene of Central Asia. The presence of this element in the lower part of Xianshuihe Formation may indicate that itsage is the latest stage of the Early Oligocene.     

Structure and chromosomal localization of the human glycogenin-2 gene GYG2

Glycogenin-2 is one of two self-glucosylating proteins involved in the initiation phase of the synthesis of the storage polysaccharide glycogen. Cloning of the human glycogenin-2 gene, GYG2, has revealed the presence of 11 exons and a gene of more than 46 kb in size. The structure of the gene explains much of the observed diversity in glycogenin-2 cDNA sequences as being due to alternate exon usage. In some cases, there is variation in the splice junctions used. Over regions of protein sequence similarity, the GYG2 gene structure is similar to that of the other glycogenin gene, GYG. A genomic GYG2 clone was used to localize the gene to Xp22.3 by fluorescence in-situ hybridization. Localization close to the telomere of the short arm of the X chromosome is consistent with mapping information obtained from glycogenin-2 STS sequences. Glycogenin-2 maps between the microsatellite anchor markers AFM319te9 (DXS7100) and AFM205tf2 (DXS1060), and its 3' end is 34.5 kb from the 3' end of the arylsulphatase gene ARSD. GYG2 is outside the pseudoautosomal region PAR1 but still in a region of X-Y shared genes. As is true for several other genes in this location, an inactive remnant of GYG2, consisting of exons 1-3, may be present on the Y chromosome.     

Glycogen metabolism in tissues from a mouse model of Lafora disease

Laforin, encoded by the EPM2A gene, by sequence is a member of the dual specificity protein phosphatase family. Mutations in the EPM2A gene account for around half of the cases of Lafora disease, an autosomal recessive neurodegenerative disorder, characterized by progressive myoclonus epilepsy. The hallmark of the disease is the presence of Lafora bodies, which contain polyglucosan, a poorly branched form of glycogen, in neurons, muscle and other tissues. Glycogen metabolizing enzymes were analyzed in a transgenic mouse over-expressing a dominant negative form of laforin that accumulates Lafora bodies in several tissues. Skeletal muscle glycogen was increased 2-fold as was the total glycogen synthase protein. However, the -/+glucose-6-P activity of glycogen synthase was decreased from 0.29 to 0.16. Branching enzyme activity was increased by 30%. Glycogen phosphorylase activity was unchanged. In whole brain, no differences in glycogen synthase or branching enzyme activities were found. Although there were significant differences in enzyme activities in muscle, the results do not support the hypothesis that Lafora body formation is caused by a major change in the balance between glycogen elongation and branching activities.     

Pathogen frequency in an age-structured population of<Emphasis Type="Italic"> Plantago lanceolata</Emphasis>

Life-history traits can play important roles in determining the course of ecological species interactions. We explored the consequences of host age on a host-pathogen interaction by quantifying pathogen frequency in an age-structured host population. Our project was motivated by an interest in whether the demographic structure of a host population has consequences for species interactions. In 2 successive years, we planted large cohorts of the perennial herb Plantago lanceolata in its natural environment and observed infection by Fusarium moniliforme, a non-lethal floral fungal pathogen, over 3 years. We documented substantial variation of pathogen frequency across years and between cohorts. Logistic regression revealed that pathogen frequency increased with the number of inflorescences produced and with evidence of prior pathogen presence, whereas it decreased with increasing plant longevity. In addition, interannual variation and an age-year interaction contributed to the observed pathogen frequencies. There was a significant positive effect of age on pathogen frequency overall, but this was not consistent over all ages. Pathogen frequency was higher in 2-year-old plants than in 1-year-olds, suggesting that age-structure can influence the host-pathogen interaction. This pattern did not continue into 3-year-old plants. A possible explanation for this is that selective mortality allows only generally robust plants, and consequently the most resistant plants, to survive to the oldest ages.