Enhanced Ca2+ storage in sphingosine-1-phosphate lyase-deficient fibroblasts

Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration and adhesion in many cell types. S1P is generated by sphingosine kinases (SphKs), and dephosphorylated by phosphatases or cleaved by S1P lyase. Extracellular S1P activates specific G protein-coupled receptors while intracellular S1P can mobilize Ca²⁺ from thapsigargin-sensitive stores. Here, we have studied Ca²⁺ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase. In these cells, S1P and sphingosine concentrations were elevated about 6-fold and 2-fold, respectively, as measured by liquid chromatography/tandem mass spectrometry. Measurements with fura-2-loaded cells in suspension revealed that resting [Ca²⁺]_i was elevated and agonist-induced [Ca²⁺]_i increases were augmented in S1P lyase-deficient MEFs both in the presence and absence of extracellular Ca²⁺. Importantly, [Ca²⁺]_i increases and Ca²⁺ mobilization induced by the SERCA inhibitor, thapsigargin, were augmented, indicating enhanced Ca²⁺ storage in S1P lyase-deficient MEFs. Measurements with single cells expressing the calmodulin-based Ca²⁺ sensor, cameleon, revealed that at least two cell types could be distinguished in both MEF cell populations, one with a rapid and transient [Ca²⁺]_i increase and the other with a slower and prolonged [Ca²⁺]_i elevation upon stimulation with thapsigargin. The area under the time course of thapsigargin-induced [Ca²⁺]_i increases, reflecting overall Ca²⁺ release, was significantly increased by more than 50% in both rapidly and slowly responding S1P lyase-deficient cells. It is concluded that elevated concentrations of S1P and/or sphingosine lead to enhanced Ca²⁺ storage and elevated basal [Ca²⁺]_i. S1P metabolism thus plays a role not only in acute Ca²⁺ mobilization but also in long-term regulation of Ca²⁺ homeostasis.     

Phylogenetic reconstruction of vertebrate Hox cluster duplications

In vertebrates and the cephalochordate, amphioxus, the closest vertebrate relative, Hox genes are linked in a single cluster. Accompanying the emergence of higher vertebrates, the Hox gene cluster duplicated in either a single step or multiple steps, resulting in the four-cluster state present in teleosts and tetrapods. Mammalian Hox clusters (designated A, B, C, and D) extend over 100 kb and are located on four different chromosomes. Reconstructing the history of the duplications and its relation to vertebrate evolution has been problematic due to the lack of alignable sequence information. In this study, the problem was approached by conducting a statistical analysis of sequences from the fibrillar-type collagens (I, II, III, and IV), genes closely linked to each Hox cluster which likely share the same duplication history as the Hox genes. We find statistical support for the hypothesis that the cluster duplication occurred as multiple distinct events and that the four-cluster situation arose by a three- step sequential process.      

Analysis of surface wave direction by the lateral line system of Xenopus: Source localization before and after inactivation of different parts of the lateral line

The turning responses of clawed toads (Xenopus laevis) to surface waves were examined in animals with an intact lateral line or with different combinations of lateral lines reversibly inactivated by CoCl₂. The responses were characterized with respect to response frequency, turning accuracy, turning side, response time, and swim distance. After the inactivation most animals still responded to surface waves but the responses were different from those of animals with an intact lateral line. They also differed according to the combination of inactivated lines. In all experiments the responses for stimuli in some sectors of the surface did not differ from controls. The location of these sectors co-varied with the position of the intact lines, i.e., normal responses were found for frontal stimulus directions when head lines were intact and for caudolateral stimulus directions when trunk lines were intact. Their size was larger when lines on both sides of the body were intact and smaller when only lines on one side were intact. When the number of functional lines was reduced to one or two on one side of the body the turning angles shown within the sector of normal responses were maintained for stimulus directions outside these sectors. These results can be interpreted as indicating that head and trunk lines represent different position values. When only a single line was functional the toads still turned towards the stimulus source more often than by chance.It is hypothesized that Xenopus uses two mechanisms to determine the direction of surface waves. One uses the position values of head and trunk lines; this mechanism is comparable to the place value postulated for individual head neuromasts of surface feeding fish. The other uses the information encoded in the activity pattern that is elicited in one line when the surface wave travels over the line. This second mechanism yields information about stimulus side but not about stimulus angle.     

Binding of the Receptor Tyrosine Kinase TrkB to the Neural Cell Adhesion Molecule (NCAM) Regulates Phosphorylation of NCAM and NCAM-dependent Neurite Outgrowth

Recognition molecules and neurotrophins play important roles during development and maintenance of nervous system functions. In this study, we provide evidence that the neural cell adhesion molecule (NCAM) and the neurotrophin receptor TrkB directly interact via sequences in their intracellular domains. Stimulation of TrkB by brain-derived neurotrophic factor leads to tyrosine phosphorylation of NCAM at position 734. Mutation of this tyrosine to phenylalanine completely abolishes tyrosine phosphorylation of NCAM by TrkB. Moreover, the knockdown of TrkB in hippocampal neurons leads to a reduction of NCAM-induced neurite outgrowth. Transfection of NCAM-deficient hippocampal neurons with mutated NCAM carrying an exchange of tyrosine by phenylalanine at position 734 leads to promotion of NCAM-induced neurite outgrowth in comparison with that observed after transfection with wild-type NCAM, whereas a reduction of neurite outgrowth was observed after transfection with mutated NCAM, which carries an exchange of tyrosine by glutamate that mimics the phosphorylated tyrosine. Our observations indicate a functional relationship between TrkB and NCAM.     

Improving the accounting of field emissions in the carbon footprint of agricultural products: a comparison of default IPCC methods with readily available medium-effort modeling approaches

Purpose

The estimations of greenhouse gas (GHG) field emissions from fertilization and soil carbon changes are challenges associated with calculating the carbon footprint (CFP) of agricultural products. At the regional level, the IPCC Guidelines for National Greenhouse Gas Inventories (2006a) Tier 1 approach, based on default emission factors, insufficiently accounts for emission variability resulting from pedo-climatic conditions or management practices. However, Tier 2 and 3 approaches are usually considered too complex to be practicable. In this paper, we discuss different readily available medium-effort methods to improve the accuracy of GHG emission estimates.

Methods

We present four case studies—two wheat crops in Germany and two peach orchards in Italy—to test the performance of Tier 1, 2, and 3 methodologies and compare the estimated results with available field measurements. The methodologies selected at Tier 2 and Tier 3 level are characterized by simple implementation and data collection, for which only a medium level of effort for stakeholders is required. The Tier 2 method consists of calculating direct and indirect N₂O, emissions from fertilization with a multivariate empirical model which accounts for pedo-climatic and crop management conditions. The Tier 3 method entails simulation of soil carbon stock change using the Rothamsted carbon model.

Results and discussion

Relevant differences were found among the tested methodologies: in all case studies, the Tier 1 approach exceeded the Tier 2 estimations for fertilizer-induced emissions (up to +50 %) and the measurements. Using this higher Tier approach reduced the estimated CFP calculation of annual crops by 4 and 21 % and that of the perennial crop by 7 %. Removals related to positive soil carbon change calculated using the Tier 1 approach also exceeded the Tier 3 calculations for the studied annual crops (up to +90 %) but considerably underrated the Tier 3 estimations and measurements for perennial crops (?75 %). In this case, the impact of the selected Tier method on the final CFP results was even more relevant: an increase of 194 and 88 % for the studied annual crops and a decrease of 67 % for the perennial crop case study.

Conclusions

The use of higher Tiers for the estimation of land-based emissions is strongly recommended to improve the accuracy of the CFP results. The suggested medium-effort methods tested in this study represent a good compromise between complexity reduction and accuracy improvement and can be considered reliable for the assessment of GHG mitigation potentials.

     

Renal graft dysfunction during infection with cytomegalovirus: association with IgM lymphocytotoxins and HLA-DR3 and DR7.

Of 121 consecutive adult recipients of cadaver renal transplants who were treated with low dose steroids and azathioprine, 23 developed active cytomegalovirus infections. These 23 patients were divided into three groups on the basis of their symptoms related to the infection: five patients had no renal, respiratory, or haematological abnormalities; seven had renal dysfunction; and nine had renal dysfunction plus respiratory or haematological abnormalities. Two patients were regarded as a separate group because their infections occurred two to four weeks after graft nephrectomy. All but three of the patients produced IgM or IgG lymphocytotoxins during their infections. In the patients with mild infections and in control patients without infections, however, these lymphocytotoxins were predominantly IgG antibodies that were not precipitated by 3.5% macrogol (polyethylene glycol). In contrast, 12 of the 16 patients with renal dysfunction during their infections had broadly reactive IgM lymphocytotoxins. These IgM lymphocytotoxins lysed T as well as B lymphocytes at 22 degrees C and were precipitated by 3.5% macrogol, suggesting that they were circulating as immune complexes. Rheumatoid factors were found in sera from nine patients with cytomegalovirus infections, seven of whom developed leukopenia or pneumonia, or both, in addition to renal dysfunction. Some of these immune responses associated with cytomegalovirus infection in transplant recipients may be genetically controlled since 10 of 11 patients positive for HLA-DR3 or DR7 produced IgM lymphocytotoxins.