Conformation of full-length Bruton tyrosine kinase (Btk) from synchrotron X-ray solution scattering

Brutons's tyrosine kinase (Btk) is a non-receptor protein tyrosine kinase (nrPTK) essential for the development of B lymphocytes in humans and mice. Like Src and Abl PTKs, Btk contains a conserved cassette formed by SH3, SH2 and protein kinase domains, but differs from them by the presence of an N-terminal PH domain and the Tec homology region. The domain structure of Btk was analysed using X-ray synchrotron radiation scattering in solution. Low resolution shapes of the full-length protein and several deletion mutants determined ab initio from the scattering data indicated a linear arrangement of domains. This arrangement was further confirmed by rigid body modelling using known high resolution structures of individual domains. The final model of Btk displays an extended conformation with no, or little, inter-domain interactions. In agreement with these results, deletion of non-catalytic domains failed to enhance the activity of Btk. Taken together, our results indicate that, contrary to Src and Abl, Btk might not require an assembled conformation for the regulation of its activity.     

Influence of nitrogen and sulphur form on manganese acquisition by barley (<Emphasis Type="Italic">shape Hordeum vulgare</Emphasis>)

The influence of various nitrogen (N) and sulphur (S) forms on the uptake of manganese (Mn) in young spring barley (Hordeum vulgare L cv Golf) plants was examined in both a hydroponic system and in a soil-based system. The soil was a typical Danish Mn-deficient soil viz. a sandy loam soil developed on old marine sediments. Plants growing in solution culture with NO₃^– as the only N source had a higher Mn uptake than plants receiving mixtures of NO₃^– and NH₄⁺. These findings were opposite to the results obtained in the soil-based experiments, where plants fertilized with NO₃^– as the only N source accumulated much less Mn than plants fertilized with NH₄⁺. Combining the results of these experiments confirmed that NH₄⁺ acted as a powerful antagonist to Mn²⁺ during uptake but that this antagonistic effect was more than compensated for by the influence of NH₄⁺ in reducing plant-unavailable Mn(IV) to plant-available Mn(II) in the soil. Furthermore the soil experiments showed that fertilizers containing sulphur in the form of reduced S (thiosulphate) had a strong mobilizing effect on Mn, and enabled the plants to accumulate large amounts of Mn in the biomass compared with oxidized S (sulphate). Thus, fertilization with thiosulphate may be very effective in alleviating Mn-deficiency in soils developed on old marine sediments where Mn availability is limiting plant growth.     

Nitrate utilization in barley: relations to nitrate supply and light/dark cycles

Uptake and utilization of nitrate were investigated in Hordeum vulgare L. cvs Mette and Golf in the vegetative stage, 2 and 4 weeks after sowing. The plants were subjected to a light/dark cycle of 16/8 h (18/12°C). Results obtained with the two genotypes were essentially similar. In the light, xylem nitrate transport and shoot nitrate reduction approximately equalled the amount of nitrate absorbed by the root. A drastic decline in translocation to the shoot in darkness was entirely attributable to decreased transpiration since no major changes in xylem nitrate concentration were observed. Darkening caused only a slight decrease in nitrate uptake, while root nitrate reduction was enhanced. Nitrate starvation for 2 days did not significantlly affect dry matter increment, but resulted in a drastic drop in previously accumulated nitrate, indicating that the stored nitrate is accessible and can sustain unrestricted growth. Uptake increased upon re-addition of nitrate and after 8 h it was about twice that of non-starved plants. During recovery, restoration of root nitrate pools and root nitrate reduction took precedence over shoot nitrate accumulation and reduction. Net nitrate uptake and removal of nitrate from the root to the transpiration stream seem to be decisive for the rate of root nitrate reduction.     

Physiological significance of ECL-cell histamine.

In the oxyntic mucosa of the mammalian stomach, histamine is stored in ECL cells and in mucosal mast cells. In the rat, at least 80 percent of oxyntic mucosal histamine resides in the ECL cells. Histamine is a key factor in the regulation of gastric acid secretion. Following depletion of ECL-cell histamine by treatment with alpha-fluoromethylhistidine (alpha-FMH), basal acid secretion was reduced, and gastrin-stimulated acid secretion was abolished. Vagally-induced acid secretion (by insulin injection or pylorus ligation) was unaffected by alpha-FMH treatment but inhibited by an H2 antagonist. These results suggest that gastrin stimulates acid secretion via release of ECL-cell histamine, whereas vagally-induced acid secretion--although histamine-dependent--does not rely on ECL-cell histamine. Gastrin is known to have a trophic effect on the oxyntic mucosa. By combining long-term hypergastrinemia with continuous infusion of alpha-FMH, we were able to show that gastrin-evoked trophic effects in the stomach do not depend on ECL-cell histamine.     

A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation

Epstein–Barr virus (EBV)-related malignancies such as post-transplant lymphoproliferative disease (PTLD) are severe complications after allogeneic stem cell transplantation and solid-organ transplantation. In immunosuppressed transplant recipients, the activity of EBV-specific CTLs are often decreased or absent which leads to an increased risk of developing PTLD. If primary treatment modalities of PTLD fail, the most efficient way of treating the malignancy is adopting EBV-specific CTLs from the donor or, more recently, third-party donors. However, both are time consuming and expensive and often it is too late to administer cells to the patient. We have for the first time, using a rapid isolation protocol of EBV-specific T cells, treated and cured a patient suffering from PTLD with multiple-associated tissue lesions, using her haplo-identical mother as a donor. This treatment approach paves way for a new possibility to within-days treat patients with life-threatening EBV-associated malignancies.     

Differences in the release ofl-glutamate andd-aspartate from primary neuronal chick cultures

Primary neuronal cultures were made from eight-day-old embryonic chick telencephalon. Ten-day-old cultures were used to study the release ofd-[³H]aspartate andl-[³H]glutamate. Thed-[³H]aspartate release was stimulated by increasing potassium concentrations, but it was not calcium dependent. In contrast, the potassium dependentl-[³H]glutamate release was calcium dependent, and furthermorel-[³H]glutamate release was optimal at potassium concentrations<30 mM. The inhibitors of glutamate uptake, dihydrokainate and 1-aminocyclobutane-trans-1,3-dicarboxylic acid (CACB), also referred to as cis-1-aminocyclobutane-1,3-dicarboxylate, were used in the release experiments. Dihydrokainate had no effect on aspartate release, whereas CACB increased both the basal efflux ofd-[³H]aspartate and the potassium evoked release. CACB had no effect on the potassium stimulatedl-glutamate release. We believe thatl-glutamate is released mainly by a vesicular mechanism from the presumably glutamatergic neurons present in our culture.d-aspartate release observed by us, could be mediated by a transporter protein. The cellular origin of this release remains to be assessed.     

Household salt as a retrospective dosemeter using optically stimulated luminescence

The aim of this work was to investigate the potential of a selection of household salts (NaCl) as a retrospective dosemeter for ionising radiation using optically stimulated luminescence (OSL). The OSL-response of five brands of salt to an absorbed dose in the range from 1 mGy to 9 Gy was investigated using a Risø TL/OSL-15 reader and a ⁶⁰Co beam, allowing low dose-rate irradiations. The salt was optically stimulated with blue light (λ = 470 ± 30 nm) at a constant stimulation power (CW-OSL) of 20 mW cm^?2. A linear dose response relationship was found in the dose range from 1 mGy to about 100 mGy and above that level, the relationship becomes moderately supra-linear, at least up to 9 Gy. Depending on the sensitivity and background signal, the minimum detectable absorbed dose (MDD) for the household salt when kept at sealed conditions varied from 0.2 to 1.0 mGy, for the household salts investigated. In addition to its widespread abundance and availability, the low MDD suggests that household salt should seriously be considered as an emergency dosemeter. However, the OSL-properties of NaCl under normal household usage need to be more properly investigated as well as the variation in sensitivity by the quality of the radiation. A further optimisation of the read-out sequence for various brands of commercially available salt may further improve the sensitivity, in terms of luminescence yield, and the signal reproducibility.     

Identification of phosphorylation sites within the SH3 domains of Tec family tyrosine kinases

Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation mechanism, which for Bruton's tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3 domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains, but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however, one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other through transphosphorylation.     

The use of hair as an indicator of occupational 14C contamination

This paper presents a study in which the specific activity of ¹⁴C in hair has been investigated as an easily determined bio-indicator of the integrated ¹⁴C exposure (over several months). The study includes 28 Swedish workers handling ¹⁴C-labelled compounds, or working in a ¹⁴C-enriched environment. Hair samples from personnel at a Swedish nuclear power plant showed very low levels of ¹⁴C contamination, if any. In contrast, personnel at the investigated research departments showed ¹⁴C levels in hair of up to 60% above the natural specific activity of ¹⁴C. Much higher levels, up to 80 times the natural specific activity of ¹⁴C, were found in hair from individuals working at a pharmaceutical research laboratory. This contamination was, however, not solely an internal contamination. There were indications that most of the ¹⁴C in the hair originated from airborne ¹⁴C-compounds, which were adsorbed onto the hair. The difficulties in removing this external ¹⁴C contamination prior to analysis are discussed, as are the possibilities of using accelerator mass spectrometry to analyse various types of samples for retrospective dose assessment.