T Cell Specific Adapter Protein (TSAd) Interacts with Tec Kinase ITK to Promote CXCL12 Induced Migration of Human and Murine T Cells

Background

The chemokine CXCL12/SDF-1α interacts with its G-protein coupled receptor CXCR4 to induce migration of lymphoid and endothelial cells. T cell specific adapter protein (TSAd) has been found to promote migration of Jurkat T cells through interaction with the G protein β subunit. However, the molecular mechanisms for how TSAd influences cellular migration have not been characterized in detail.

Principal Findings

We show that TSAd is required for tyrosine phosphorylation of the Lck substrate IL2-inducible T cell kinase (Itk). Presence of Itk Y511 was necessary to boost TSAd''s effect on CXCL12 induced migration of Jurkat T cells. In addition, TSAd''s ability to promote CXCL12-induced actin polymerization and migration of Jurkat T lymphocytes was dependent on the Itk-interaction site in the proline-rich region of TSAd. Furthermore, TSAd-deficient murine thymocytes failed to respond to CXCL12 with increased Itk phosphorylation, and displayed reduced actin polymerization and cell migration responses.

Conclusion

We propose that TSAd, through its interaction with both Itk and Lck, primes Itk for Lck mediated phosphorylation and thereby regulates CXCL12 induced T cell migration and actin cytoskeleton rearrangements.     

Electronic spectrum and magnetic properties of a dinuclear nickel(II) complex with two nickel(II) ions of C2-twisted octahedral geometry

A dinickel(II) complex [Ni₂(sym-hmp)₂](BPh₄)₂·3.5DMF·0.5(2-PrOH) (1) was synthesized with a dinucleating ligand, 2,6-bis[(2-hydroxyethyl)methylaminomethyl]-4-methyl-phenol [H(sym-hmp)]. The complex 1 (C₉₀H_118.50B₂N_7.50Ni₂O₁₀) crystallized in the triclinic space group with dimensions a = 14.7446(4) Å, b = 15.4244(4) Å, c = 18.7385(6) Å, α = 86.9495(9)°, β = 76.7263(10)°, γ = 86.5370(8)°, and V = 4136.8(2) Å³ and with Z = 2; this is isomorphous to a previous cobalt(II) complex [Co₂(sym-hmp)₂](BPh₄)₂. Single-crystal X-ray analysis revealed a bis(μ-phenoxo)dinickel(II) core structure containing two distorted octahedral nickel(II) ions of C₂ symmetry. The order of the coordination bond lengths is Ni-O(phenoxo) < Ni-O(hydroxy) < Ni-N. The electronic spectrum of 1 was typical for the octahedral nickel(II) complexes, but the axial elongation and the C₂-twist of the equatorial plane were found after a detailed analysis. The bond angles obtained by the electronic spectrum agreed with the crystallographically obtained bond angles within 2.3°. The order of the AOM parameters was e_{σ,O(phenoxo)} > e_{σ,O(hydroxy)} > e_σ,N, which was consistent with the order of the coordination bond lengths. Magnetic susceptibility data for 1 were fitted well with the parameters 2J = −69.7 cm⁻¹, D = 0.00 cm⁻¹, g = 2.17, and TIP = 265 × 10⁻⁶ cm³ mol⁻¹. The result indicates significant antiferromagnetic exchange interaction and negligible zero-field splitting, while the isostructural cobalt(II) complex showed an anisotropic behavior.     

Transformation and DNA repair: linkage by DNA recombination

The stability of microbial genomes is constantly challenged by horizontal gene transfer, recombination and DNA damage. Mechanisms for rapid genome variation, adaptation and maintenance are a necessity to ensure microbial fitness and survival in changing environments. Indeed, genome sequences reveal that most, if not all, bacterial species have numerous gene functions for DNA repair and recombination. These important topics were addressed at the Second Genome Maintenance Meeting (GMM2).     

Identification of metabolites from 2D 1H-13C HSQC NMR using peak correlation plots

Background

Identification of individual components in complex mixtures is an important and sometimes daunting task in several research areas like metabolomics and natural product studies. NMR spectroscopy is an excellent technique for analysis of mixtures of organic compounds and gives a detailed chemical fingerprint of most individual components above the detection limit. For the identification of individual metabolites in metabolomics, correlation or covariance between peaks in ¹H NMR spectra has previously been successfully employed. Similar correlation of 2D ¹H-¹³C Heteronuclear Single Quantum Correlation spectra was recently applied to investigate the structure of heparine. In this paper, we demonstrate how a similar approach can be used to identify metabolites in human biofluids (post-prostatic palpation urine).

Results

From 50 ¹H-¹³C Heteronuclear Single Quantum Correlation spectra, 23 correlation plots resembling pure metabolites were constructed. The identities of these metabolites were confirmed by comparing the correlation plots with reported NMR data, mostly from the Human Metabolome Database.

Conclusions

Correlation plots prepared by statistically correlating ¹H-¹³C Heteronuclear Single Quantum Correlation spectra from human biofluids provide unambiguous identification of metabolites. The correlation plots highlight cross-peaks belonging to each individual compound, not limited by long-range magnetization transfer as conventional NMR experiments.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0413-z) contains supplementary material, which is available to authorized users.     

Hypothermia Modulates Cytokine Responses After Neonatal Rat Hypoxic-Ischemic Injury and Reduces Brain Damage

While hypothermia (HT) is the standard-of-care for neonates with hypoxic ischemic injury (HII), the mechanisms underlying its neuroprotective effect are poorly understood. We examined ischemic core/penumbra and cytokine/chemokine evolution in a 10-day-old rat pup model of HII. Pups were treated for 24 hr after HII with HT (32℃; n = 18) or normothermia (NT, 35℃; n = 15). Outcomes included magnetic resonance imaging (MRI), neurobehavioral testing, and brain cytokine/chemokine profiling (0, 24, 48, and 72 hr post-HII). Lesion volumes (24 hr) were reduced in HT pups (total 74%, p < .05; penumbra 68%, p < .05; core 85%, p = .19). Lesion volumes rebounded at 72 hr (48 hr post-HT) with no significant differences between NT and HT pups. HT reduced interleukin-1β (IL-1β) at all time points (p < .05); monocyte chemoattractant protein-1 (MCP-1) trended toward being decreased in HT pups (p = .09). The stem cell signaling molecule, stromal cell-derived factor-1 (SDF-1) was not altered by HT. Our data demonstrate that HT reduces total and penumbral lesion volumes (at 24 and 48 hr), potentially by decreasing IL-1β without affecting SDF-1. Disassociation between the increasing trend in HII volumes from 48 to 72 hr post-HII when IL-1β levels remained low suggests that after rewarming, mechanisms unrelated to IL-1β expression are likely to contribute to this delayed increase in injury. Additional studies should be considered to determine what these mechanisms might be and also to explore whether extending the duration or degree of HT might ameliorate this delayed increase in injury.     

Technical Complications during Veno-Venous Extracorporeal Membrane Oxygenation and Their Relevance Predicting a System-Exchange – Retrospective Analysis of 265 Cases

Objectives

Technical complications are a known hazard in veno-venous extracorporeal membrane oxygenation (vvECMO). Identifying these complications and predictive factors indicating a developing system-exchange was the goal of the study.

Methods

Retrospective study on prospectively collected data of technical complications including 265 adult patients (Regensburg ECMO Registry, 2009-2013) with acute respiratory failure treated with vvECMO. Alterations in blood flow resistance, gas transfer capability, hemolysis, coagulation and hemostasis parameters were evaluated in conjunction with a system-exchange in all patients with at least one exchange (n = 83).

Results

Values presented as median (interquartile range). Patient age was 50(36–60) years, the SOFA score 11(8–14.3) and the Murray lung injury Score 3.33(3.3–3.7). Cumulative ECMO support time 3411 days, 9(6–15) days per patient. Mechanical failure of the blood pump (n = 5), MO (n = 2) or cannula (n = 1) accounted for 10% of the exchanges. Acute clot formation within the pump head (visible clots, increase in plasma free hemoglobin (frHb), serum lactate dehydrogenase (LDH), n = 13) and MO (increase in pressure drop across the MO, n = 16) required an urgent system-exchange, of which nearly 50% could be foreseen by measuring the parameters mentioned below. Reasons for an elective system-exchange were worsening of gas transfer capability (n = 10) and device-related coagulation disorders (n = 32), either local fibrinolysis in the MO due to clot formation (increased D-dimers [DD]), decreased platelet count; n = 24), or device-induced hyperfibrinolysis (increased DD, decreased fibrinogen [FG], decreased platelet count, diffuse bleeding tendency; n = 8), which could be reversed after system-exchange. Four MOs were exchanged due to suspicion of infection.

Conclusions

The majority of ECMO system-exchanges could be predicted by regular inspection of the complete ECMO circuit, evaluation of gas exchange, pressure drop across the MO and laboratory parameters (DD, FG, platelets, LDH, frHb). These parameters should be monitored in the daily routine to reduce the risk of unexpected ECMO failure.     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Airborne <Emphasis Type="Italic">Legionella</Emphasis> bacteria from pulp waste treatment plant: aerosol particles characterized as aggregates and their potential hazard

In response to reported outbreaks of Legionnaires’ disease near a biological waste treatment plant, Legionella-containing aerosols were characterized at this location. Culturable bacteria-containing particles were assessed with slit-to-agar samplers and liquid impingement. Results showed that the air at this location contained a higher level of airborne Legionella particles compared to that of a control clean location 90 km away. On average, about 1 of 50 culturable particles contained Legionella (2%). The median particle size was estimated to 3.5 μm permitting an estimation of a maximum about 147 Legionella cells per aggregate. This implies that in <11 h, a human under such conditions (worse-case), may inhale an estimated lethal dose of individual Legionella cells (1,000 cells, 100% alveolar retained deposition level). Additionally, 44 taxonomically different airborne bacterial genera were measured at the treatment plant using denaturing gradient-gel electrophoresis (DGGE) and 16S rDNA analysis of air samples. Of these, 64% of the genera were not detected at the clean location. Our findings suggest that the treatment facility may emit respirable particles, as complex aggregates, containing a mixture of several Legionella cells and other bacterial cells. These findings can potentially have an impact on assessing environmental risk exposure and health hazard prediction modeling.     

CP-60,993, a new dianemycin-like ionophore produced byStreptomyces hygroscopicus ATCC 39305: fermentation,isolation and characterization

Summary CP-60,993, 19-epi-dianemycin, is a novel polycyclic ether antibiotic produced byStreptomyces hygroscopicus ATCC 39305. Fermentation recovery, purification and crystallization were achieved using standard procedures. CP-60,993 was characterized as a monocarboxylic acid. Elemental analysis suggested a molecular formula of C₄₇H₇₈O₁₄ for the free acid and C₄₇H₇₇O₁₄ Na for the sodium salt. Crystalline form CP-60,993 sodium salt shows the following properties: m.p. 193205°C, E _{1 cm} ^1% =157 at 232 nm, [] _D ^25°C +11.0 (c 1, methanol). The structure, determined by MS, PMR and CMR, differs from dianemycin only in the stereochemistry at position 19. This was confirmed by X-ray crystallography carried out on the rubidium salt of CP-60,993. It exhibited activity in vitro against Gram-positive and anaerobic bacteria, efficacy againstEimeria coccidia in vivo in poultry, and stimulation in vitro of rumen propionic acid production.