Cloning, expression and purification of orthologous membrane proteins: a general protocol for preparation of the histidine sensor kinase ETR1 from different species

Orthologous proteins do not necessarily share the same function in all species and those sharing the same function might employ a modified catalytic mechanism. Thus, comparative analysis of homologous or orthologous proteins from different organisms can provide detailed information on the function and the mechanism of an entire protein family. The sensor kinase ETR1 from Arabidopsis thaliana has been well characterized by genetic, physiological and biochemical studies. However, as further model plants are coming into focus for plant hormone research, a general protocol for isolation and purification of orthologous ETR1 proteins seems instrumental for a detailed molecular analysis of this protein family. In this study, we describe the native purification of recombinant ETR1 from Arabidopsis thaliana by mild solubilization with the zwitter-ionic detergent Fos-Choline-14 and single-step purification by immobilized metal ion affinity chromatography. The same protocol was successfully applied for the purification of the orthologous proteins from the moss Physcomitrella patens subsp. patens and the tomato Lycopersicon esculentum. The successful transfer of the purification protocol to proteins of the same family which share sequence identity of 63-80% only suggests that this protocol presents a general purification strategy which is likely to apply also to the purification of other members of the sensor histidine kinase family.     

The role of thrombin and thrombin receptors in ischemic,hemorrhagic and traumatic brain injury: deleterious or protective?

In the last two decades it has become apparent that thrombin has many extravascular effects that are mediated by a family of protease-activated receptors (PARs). PAR-1, -3 and -4 are activated via cleavage by thrombin. The importance of extravascular thrombin in modulating ischemic, hemorrhagic and traumatic injury in brain has recently become clear. Thus, in vitro, thrombin at low concentration protects neurons and astrocytes from cell death caused by a number of different insults. In vivo, pretreating the brain with a low dose of thrombin (thrombin preconditioning), attenuates the brain injury induced by a large dose of thrombin, an intracerebral hemorrhage or by focal cerebral ischemia. Thrombin may also be an important mediator of ischemic preconditioning. In contrast, high doses of thrombin kill neurons and astrocytes in vitro and cause disruption of the blood-brain barrier, brain edema and seizures in vivo. This review examines the role of thrombin in brain injury and the molecular mechanisms and signaling cascades involved.     

Inhibition of deoxyribonuclease I by actin is to protect cells from premature cell death

Deoxyribonuclease I (Dnase1) is the major extracellular endonuclease. It is secreted by digestive glands into the alimentary tract and into the plasma, lacrimal fluid and urine by hepatocytes, lacrimal glands and renal proximal tubular cells, respectively. In many species the activity of Dnase1 is inhibited by monomeric actin. However, the biological significance of this high affinity interaction is unknown. We generated a Dnase1 mutant with extremely reduced actin binding capacity. EGFP-constructs of wild-type and mutant Dnase1 were transfected into MCF-7 breast cancer cells and apoptosis or necrosis was induced by staurosporine or oxidative stress. During apoptosis faster chromatin fragmentation occurred in cells transfected with mutant Dnase1. When wt (wild-type)- or mutated Dnase1 were added to cells after induction of necrosis, faster chromatin degradation occurred in the presence of mutant Dnase1. Inclusion of actin under these conditions inhibited chromatin degradation by wt- but not by mutated Dnase1. Thus, inhibition of Dnase1 by actin may serve as a self-protection mechanism against premature DNA degradation during cell damage.     

Rate of Belowground Carbon Allocation Differs with Successional Habit of Two Afromontane Trees

Background

Anthropogenic disturbance of old-growth tropical forests increases the abundance of early successional tree species at the cost of late successional ones. Quantifying differences in terms of carbon allocation and the proportion of recently fixed carbon in soil CO₂ efflux is crucial for addressing the carbon footprint of creeping degradation.

Methodology

We compared the carbon allocation pattern of the late successional gymnosperm Podocarpus falcatus (Thunb.) Mirb. and the early successional (gap filling) angiosperm Croton macrostachyus Hochst. es Del. in an Ethiopian Afromontane forest by whole tree ¹³CO₂ pulse labeling. Over a one-year period we monitored the temporal resolution of the label in the foliage, the phloem sap, the arbuscular mycorrhiza, and in soil-derived CO₂. Further, we quantified the overall losses of assimilated ¹³C with soil CO₂ efflux.

Principal Findings

¹³C in leaves of C. macrostachyus declined more rapidly with a larger size of a fast pool (64% vs. 50% of the assimilated carbon), having a shorter mean residence time (14 h vs. 55 h) as in leaves of P. falcatus. Phloem sap velocity was about 4 times higher for C. macrostachyus. Likewise, the label appeared earlier in the arbuscular mycorrhiza of C. macrostachyus and in the soil CO₂ efflux as in case of P. falcatus (24 h vs. 72 h). Within one year soil CO₂ efflux amounted to a loss of 32% of assimilated carbon for the gap filling tree and to 15% for the late successional one.

Conclusions

Our results showed clear differences in carbon allocation patterns between tree species, although we caution that this experiment was unreplicated. A shift in tree species composition of tropical montane forests (e.g., by degradation) accelerates carbon allocation belowground and increases respiratory carbon losses by the autotrophic community. If ongoing disturbance keeps early successional species in dominance, the larger allocation to fast cycling compartments may deplete soil organic carbon in the long run.     

The catalytic cycle of a thiamin diphosphate enzyme examined by cryocrystallography

Enzymes that use the cofactor thiamin diphosphate (ThDP, 1), the biologically active form of vitamin B(1), are involved in numerous metabolic pathways in all organisms. Although a theory of the cofactor's underlying reaction mechanism has been established over the last five decades, the three-dimensional structures of most major reaction intermediates of ThDP enzymes have remained elusive. Here, we report the X-ray structures of key intermediates in the oxidative decarboxylation of pyruvate, a central reaction in carbon metabolism catalyzed by the ThDP- and flavin-dependent enzyme pyruvate oxidase (POX)3 from Lactobacillus plantarum. The structures of 2-lactyl-ThDP (LThDP, 2) and its stable phosphonate analog, of 2-hydroxyethyl-ThDP (HEThDP, 3) enamine and of 2-acetyl-ThDP (AcThDP, 4; all shown bound to the enzyme's active site) provide profound insights into the chemical mechanisms and the stereochemical course of thiamin catalysis. These snapshots also suggest a mechanism for a phosphate-linked acyl transfer coupled to electron transfer in a radical reaction of pyruvate oxidase.     

The phylogeny of Strepsiptera (Hexapoda)

Previous phylogenetic analyses of Strepsiptera have been limited to characters from only males or first instar larvae, and by poor taxonomic sampling. This investigation is the first cladistic analysis to use more than fourfold as many characters as any prior study, and a broader sampling of taxa. The analysis of 189 morphological characters of all stages of representatives of all extant strepsipteran families and characters of adult males of amber fossils results in the following branching pattern: (?Protoxenos+ (?Cretostylops + (?Mengea + (Mengenillidae + (Corioxenidae + (Bohartillidae + (Halictophagidae + (Elenchidae + (?Protelencholax + (Myrmecolacidae + (Callipharixenidae + (Xenidae + Stylopidae)))))))))))). The basal placement of the Baltic amber fossil ?Protoxenos and the Burmese amber fossil ?Cretostylops is well founded. Even though ?Cretostylops is older than ?Protoxenos it is almost certainly not the most basal strepsipteran group but the sister group of a clade comprising the Baltic amber fossil ?Mengea + Strepsiptera s. str. (excl. stemlineage). Monophyly of Mengenillidae, Stylopidia, Stylopiformia s.l., Corioxenidae, Xenidae, and Stylopidae is confirmed. Mengenillidia is paraphyletic (with respect to ?Mengea (Mengeidae)), Elenchidae (with respect to ?Protelencholax) and the genus Stichotrema (with respect to the Baltic amber fossils). Thus Protelencholacidae fam. n. is described, and S. weitschati and S. triangulum are transferred to Palaeomyrmecolax. A ground plan of adult male Strepsiptera is provided and evolutionary interpretations are presented based on the obtained cladograms. © The Willi Hennig Society 2005.