Cross-talk between calcium signalling and protein phosphorylation at the thylakoid

The role of protein phosphorylation for adjusting chloroplast functions to changing environmental needs is well established, whereas calcium signalling in the chloroplast is only recently becoming appreciated. The work presented here explores the potential cross-talk between calcium signalling and protein phosphorylation in chloroplasts and provides the first evidence for targets of calcium-dependent protein phosphorylation at the thylakoid membrane. Thylakoid proteins were screened for calcium-dependent phosphorylation by 2D gel electrophoresis combined with phospho-specific labelling and PsaN, CAS, and VAR1, among other proteins, were identified repeatedly by mass spectrometry. Subsequently their calcium-dependent phosphorylation was confirmed in kinase assays using the purified proteins and chloroplast extracts. This is the first report on the protein targets of calcium-dependent phosphorylation of thylakoid proteins and provides ground for further studies in this direction.     

Comment to: “Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro” by Z. Ren et al. Exp. Cell Res. 317 (2011) 2950-2957: Spontaneous transformation of mesenchymal stem cells in culture: Facts or fiction?

There is at present a controversy in the literature whether MSCs are susceptible to spontaneous in vitro transformation or not. Several groups have reported spontaneous transformation of MSCs from various species. However, some of these reports were not true transformations and later proven to be due to cross-contaminating cancer cells. To date there is no solid evidence that MSCs can undergo spontaneous transformation in culture. Only two groups used DNA fingerprinting to authenticate their transformed cells, and both groups later showed cross-contamination of cancer cells in their cultures. In this commentary, we address the paper "Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro" by Z. Ren et al. Exp. Cell Res. 317 (2011) 2950-2957. In this article the authors characterize the transformed mesenchymal cells (TMCs) and claim to have verified their origin. We question the authentication of the TMCs made by the authors and we also believe it is in the interest of the scientific community, that a highly controversial finding, such as spontaneous transformation of MSCs, should be properly verified by stringent methods, preferably DNA fingerprinting, in order to validate if an actual transformation event has occurred.     

Crystallohydrodynamics of protein assemblies: Combining sedimentation, viscometry, and x-ray scattering

Crystallohydrodynamics describes the domain orientation in solution of antibodies and other multidomain protein assemblies where the crystal structures may be known for the domains but not the intact structure. The approach removes the necessity for an ad hoc assumed value for protein hydration. Previous studies have involved only the sedimentation coefficient leading to considerable degeneracy or multiplicity of possible models for the conformation of a given protein assembly, all agreeing with the experimental data. This degeneracy can be considerably reduced by using additional solution parameters. Conformation charts are generated for the three universal (i.e., size-independent) shape parameters P (obtained from the sedimentation coefficient or translational diffusion coefficient), nu (from the intrinsic viscosity), and G (from the radius of gyration), and calculated for a wide range of plausible orientations of the domains (represented as bead-shell ellipsoidal models derived from their crystal structures) and after allowance for any linker or hinge regions. Matches are then sought with the set of functions P, nu, and G calculated from experimental data (allowing for experimental error). The number of solutions can be further reduced by the employment of the D max parameter (maximum particle dimension) from x-ray scattering data. Using this approach we are able to reduce the degeneracy of possible solution models for IgG3 to a possible representative structure in which the Fab domains are directed away from the plane of the Fc domain, a structure in accord with the recognition that IgG3 is the most efficient complement activator among human IgG subclasses.     

Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity

Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin-binding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.     

Perfused hearts from Type 2 diabetic (db/db) mice show metabolic responsiveness to insulin

Diabetic (db/db) mice provide an animal model of Type 2 diabetes characterized by marked in vivo insulin resistance. The effect of insulin on myocardial metabolism has not been fully elucidated in this diabetic model. In the present study we tested the hypothesis that the metabolic response to insulin in db/db hearts will be diminished due to cardiac insulin resistance. Insulin-induced changes in glucose oxidation (GLUox) and fatty acid (FA) oxidation (FAox) were measured in isolated hearts from control and diabetic mice, perfused with both low as well as high concentration of glucose and FA: 10 mM glucose/0.5 mM palmitate and 28 mM glucose/1.1 mM palmitate. Both in the absence and presence of insulin, diabetic hearts showed decreased rates of GLUox and elevated rates of FAox. However, the insulin-induced increment in GLUox, as well as the insulin-induced decrement in FAox, was similar or even more pronounced in diabetic that in control hearts. During elevated FA and glucose supply, however, the effect of insulin was blunted in db/db hearts with respect to both FAox and GLUox. Finally, insulin-stimulated deoxyglucose uptake was markedly reduced in isolated cardiomyocytes from db/db mice, whereas glucose uptake in isolated perfused db/db hearts was clearly responsive to insulin. These results show that, despite reduced insulin-stimulated glucose uptake in isolated cardiomyocytes, isolated perfused db/db hearts are responsive to metabolic actions of insulin. These results should advocate the use of insulin therapy (glucose-insulin-potassium) in diabetic patients undergoing cardiac surgery or during reperfusion after an ischemic insult.     

Evaluation of culture systems for attachment and proliferation of epithelial cells cultured from ovine semen

Different culture systems were evaluated for their ability to support attachment and proliferation of the somatic cells obtained from ovine semen. Ejaculates (n = 14) were collected from eight rams representing three breeds, Dorper, Suffolk and Hampshire. All samples were processed immediately and somatic cells were obtained from 11 of the 14 ejaculates. These cells had classic epithelial morphology and expressed cytokeratin, indicating they were of epithelial origin. Cells from four rams with the greatest growth rates were used for subsequent studies. Cells were cultured in four different media for 5 days and total numbers of attached cells vs. total numbers of seeded cells were counted and compared each day. Four media were evaluated: (1) a supplemented medium composed of DMEM/F12, 10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, 30 μg/ml bovine pituitary extract, 5 μg/ml insulin, 10 ng/ml cholera toxin, and 50 μg/ml gentamycin; (2) sheep fetal fibroblast (SFF)-conditioned medium; (3) swiss 3T3 fibroblast-conditioned medium; and (4) basic medium composed of DMEM/F12, 10% FBS, and 50 μg/ml gentamycin. Cell proliferation was greater in the supplemented medium, SFF-conditioned medium, and 3T3 fibroblast-conditioned medium compared to the basic medium by day 2 of culture (p < 0.05, n = 24), and greater in supplemented medium compared to the SFF-conditioned medium and 3T3 fibroblast-conditioned medium by day 4 of culture (p < 0.05, n = 24). Three different surfaces: (1) Matrigel basement membrane matrix-coated plastic; (2) collagen I-coated plastic; and (3) uncoated plastic were evaluated for their ability to support proliferation and attachment of the cells obtained from semen. Cell proliferation was greater when cells were cultured on the Matrigel-coated compared to the collagen I-coated and uncoated plastic by day 2 of culture (p < 0.05, n = 16). Cell attachment was greater when cells were plated on the Matrigel-coated and collagen I-coated plastic compared to the uncoated plastic (p < 0.05, n = 16). These studies describe an effective system for the culture and proliferation of epithelial cells obtained from ovine semen samples. The system may increase the likelihood of obtaining cells from frozen semen, which could be used for cloning to recover animals of genetic value in which semen is the only material that is available.     

Microbial response to reinjection of produced water in an oil reservoir

The microbial response to produced water reinjection (PWRI) in a North Sea oil field was investigated by a combination of cultivation and culture-independent molecular phylogenetic techniques. Special emphasise was put on the relationship between sulphate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB), and results were used to evaluate the possibility of nitrate treatment as a souring management tool during PWRI. Samples were collected by reversing the flow of the injection water, which provided samples from around the injection area. The backflowed samples were compared to produced water from the same platform and to backflowed samples from a biocide-treated seawater injector, which was the previous injection water treatment of the PWRI well. Results showed that reinjection of produced water promoted growth of thermophilic SRB. Thermophilic fatty acid oxidising NRB and potential nitrate-reducing sulphide-oxidising bacteria were also found. The finding of thermophilic NRB makes nitrate treatment during PWRI possible, although higher nitrate concentration will be necessary to compensate for the increased SRB activity.     

COMPARATIVE EVIDENCE FOR THE EVOLUTION OF SPERM SWIMMING SPEED BY SPERM COMPETITION AND FEMALE SPERM STORAGE DURATION IN PASSERINE BIRDS

Sperm swimming speed is an important determinant of male fertility and sperm competitiveness. Despite its fundamental biological importance, the underlying evolutionary processes affecting this male reproductive trait are poorly understood. Using a comparative approach in a phylogenetic framework, we tested the predictions that sperm swim faster with (1) increased risk of sperm competition, (2) shorter duration of female sperm storage, and (3) increased sperm length. We recorded sperm swimming speed in 42 North American and European free-living passerine bird species, representing 35 genera and 16 families. We found that sperm swimming speed was positively related to the frequency of extrapair paternity (a proxy for the risk of sperm competition) and negatively associated with clutch size (a proxy for the duration of female sperm storage). Sperm swimming speed was unrelated to sperm length, although sperm length also increased with the frequency of extrapair paternity. These results suggest that sperm swimming speed and sperm length are not closely associated traits and evolve independently in response to sperm competition in passerine birds. Our findings emphasize the significance of both sperm competition and female sperm storage duration as evolutionary forces driving sperm swimming speed.     

Microbial analysis of backflowed injection water from a nitrate-treated North Sea oil reservoir

Reservoir souring in offshore oil fields is caused by hydrogen sulphide (H₂S) produced by sulphate-reducing bacteria (SRB), most often as a consequence of sea water injection. Biocide treatment is commonly used to inhibit SRB, but has now been replaced by nitrate treatment on several North Sea oil fields. At the Statfjord field, injection wells from one nitrate-treated reservoir and one biocide-treated reservoir were reversed (backflowed) and sampled for microbial analysis. The two reservoirs have similar properties and share the same pre-nitrate treatment history. A 16S rRNA gene-based community analysis (PCR-DGGE) combined with enrichment culture studies showed that, after 6 months of nitrate injection (0.25 mM NO₃ ⁻), heterotrophic and chemolithotrophic nitrate-reducing bacteria (NRB) formed major populations in the nitrate-treated reservoir. The NRB community was able to utilize the same substrates as the SRB community. Compared to the biocide-treated reservoir, the microbial community in the nitrate-treated reservoir was more phylogenetically diverse and able to grow on a wider range of substrates. Enrichment culture studies showed that SRB were present in both reservoirs, but the nitrate-treated reservoir had the least diverse SRB community. Isolation and characterisation of one of the dominant populations observed during nitrate treatment (strain STF-07) showed that heterotrophic denitrifying bacteria affiliated to Terasakiella probably contributed significantly to the inhibition of SRB. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.