Calculation of accurate small angle X-ray scattering curves from coarse-grained protein models

Background  

Genome sequencing projects have expanded the gap between the amount of known protein sequences and structures. The limitations of current high resolution structure determination methods make it unlikely that this gap will disappear in the near future. Small angle X-ray scattering (SAXS) is an established low resolution method for routinely determining the structure of proteins in solution. The purpose of this study is to develop a method for the efficient calculation of accurate SAXS curves from coarse-grained protein models. Such a method can for example be used to construct a likelihood function, which is paramount for structure determination based on statistical inference.     

Serum immunoassay of a small cell lung cancer associated ganglioside: development of a sensitive scintillation proximity assay

We here report an enzyme linked immunosorbent assay (ELISA) and a scintillation proximity assay (SPA) for detection of the ganglioside FucGM₁ in sera from small cell lung cancer (SCLC) patients. The SPA was more sensitive and reproducible than the ELISA. In this assay, monoclonal antibodies specific for FucGM₁ were bound to SPA particles and incubated with labelled FucGM₁ and 100 µl test-serum overnight, and counted in a -counter. The sensitivity was 0.2 ng. Seven out of twenty sera from SCLC patients were positive, whereas none of twenty sera from healthy individuals were positive for FucGM₁. The SPA was more sensitive than the previously reported HPTLC as well as a direct ELISA.Abbreviations MAb monoclonal antibody - SPA scintillation proximity assay - HPTLC high performance thin layer chromatography - SCLC small cell lung cancer - FucGM₁ Fuc1-2Gal1-3GalNAc1-4(NeuAc2-3)-Gal1-4Glc1-1Cer - ELISA enzyme linked immunosorbent assay - FCS foetal calf serum - PBS phosphate buffered saline     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.     

Role of guanosine kinase in the utilization of guanosine for nucleotide synthesis in Escherichia coli

Using purine auxotrophic strains of Escherichia coli with additional genetic lesions in the pathways of interconversion and salvage of purine compounds, we demonstrated the in vivo function of guanosine kinase and inosine kinase. Mutants with increased ability to utilize guanosine were isolated by plating cells on medium with guanosine as the sole purine source. These mutants had altered guanosine kinase activity and the mutations were mapped in the gene encoding guanosine kinase, gsk. Some of the mutants had acquired an additional genetic lesion in the purine de novo biosynthetic pathway, namely a purF, a purL or a purM mutation. A revised map location of the gsk gene is presented and the gene order established as proC-acrA-apt-adk-gsk-purE.     

Temporal and spatial development of individual species of plankton algae from European lakes

The temporal and spatial developments of 15 phytoplankton species widely distributed in European and North American lakes, have been studied by informations in the literature and especially by their behaviour during a 5 years development in the Danish lake Grane Langsø. The species were characterized by temporal differences in maximum abundance, the density at the maximum, the number of maxima per year, the length of the vegetative period and the constancy of the annual maximum based on long-term studies. In several cases the phytoplankton species can be characterized by one individual behaviour in one group of lakes, while the same species behaves differently in another group of lakes. The results suggest that several species include two or more types with different tolerances and environmental requirements. Most of the species included are Chrysophytes, mainly species of Uroglena and Dinobryon. A remarkable feature of Grane Langsø's Chrysophytes is their predilection to accumulate in the bottom water during the summer. A similar behaviour was not demonstrated by Chrysophytes in other lakes.     

Chaperone-mediated activation in vivo of a Pseudomonas cepacia lipase

An extracellular Pseudomonas cepacia lipase, LipA, is inactive when expressed in the absence of the product of the limA gene. Evidence has been presented that LimA is a molecular chaperone. The lipA and limA genes have been cloned in separate and independently inducible expression systems in Escherichia coli. These systems were used to test the molecular chaperone hypothesis by investigating whether LimA could activate presynthesized prelipase and whether presynthesized LimA could activate newly synthesized prelipase. The results show that LimA cannot activate presynthesized prelipase and that presynthesized LimA can activate only a limited number of de novo synthesized prelipase molecules. Co-immunoprecipitation of prelipase/lipase with LimA generated a 1:1 complex of prelipase/lipase and LimA. The results suggest that a 1:1 complex of LipA and LimA is required for prelipase processing and secretion of active lipase.     

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.     

Radioiodine treatment of multinodular non-toxic goitre.

OBJECTIVE--To investigate the long term effect of radioactive iodine on thyroid function and size in patients with non-toxic multinodular goitre. DESIGN--Consecutive patients with multinodular non-toxic goitre selected for radioactive iodine treatment and followed for a minimum of 12 months (median 48 months) after an intended dose of 3.7 MBq/g thyroid tissue corrected to a 100% uptake of iodine-131 in 24 hours. PATIENTS--69 patients with a growing multinodular non-toxic goitre causing local compression symptoms or cosmetic inconveniences. The treatment was chosen because of a high operative risk, previous thyroidectomy, or refusal to be operated on. MAIN OUTCOME MEASUREMENTS--Standard thyroid function variables and ultrasonically determined thyroid volume before treatment as well as 1, 2, 3, 6, and 12 months after treatment and then once a year. RESULTS--56 patients were treated with a single dose of 131I, 12 with two doses, and one with four doses. In 45 patients treated with one dose and remaining euthyroid the median thyroid volume was reduced from 73 (interquartile range 50-106) ml to 29 (23-48) ml at 24 months in the 39 patients in whom this was measured during follow up. The median reduction was 40 (22-48) ml (60% reduction, p < 0.0001), half of which occurred within three months. Patients treated with two doses as well as those developing hypothyroidism and hyperthyroidism had a significant reduction in thyroid volume. Eleven patients developed hypothyroidism (cumulative five year risk 22%, 95% confidence interval 4.8% to 38.4%). Side effects were few: three cases of hyperthyroidism and two cases of radiation thyroiditis. Only one patient was dissatisfied with the result; she was referred for operation six months after treatment. CONCLUSIONS--A substantial reduction in thyroid volume accompanied by a low incidence of hypothyroidism and few side effects makes the use of radioactive iodine an attractive alternative to surgery in selected cases of non-toxic multinodular goitre.