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.     

Identical phalangeal defects induced by phenytoin and nifedipine suggest fetal hypoxia and vascular disruption behind phenytoin teratogenicity.

In previous experimental studies in rabbits, we have shown that vasodilating drugs (including nifedipine) cause distal digital defects. These defects were preceded by edema, hemorrhage, and finally necrosis of the developed cartilage in the phalanges. The underlying mechanism is most likely a fetal hypoxic response, secondary to maternal hypotension and decreased uteroplacental blood flow. Since phenytoin is known to cause distal digital defects both in man and rabbits, we decided to compare the defects provoked by oral administration of phenytoin (100 mg/kg) versus nifedipine (8.3 mg/kg) to New Zealand White rabbits on days 6-18 of gestation. In order to investigate phase-specificity, phenytoin (150 mg/kg) was given on days 14-17. The result of single dose administration on day 16 of phenytoin (300 mg/kg) versus nifedipine (33.2 mg/kg) was also studied. In this latter experiment maternal heart rate was measured up to 21 hours after phenytoin administration. Phenytoin induced digital defects identical with those produced by nifedipine and caused marked maternal cardiodepression. The defects consisted of a reduction, absence, or abnormal structure of the distal phalanges. The distal phalanx of the fourth digit on the hindpaw was the first to be affected, with inclusion of other phalanges, both on the hind- and forepaws, with increasing dose. The sensitive period for induction and histological appearance of these defects was identical for phenytoin and nifedipine. These results suggest that vascular disruption due to a fetal hypoxic response lies behind phenytoin teratogenicity, as has been shown for vasodilators. A cardiodepressive action on the maternal and fetal hearts, possibly in combination with decreased uteroplacental blood flow, is discussed as a probable factor behind phenytoin teratogenicity.     

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     

Automated thermometric enzyme immunoassay of human proinsulin produced by Escherichia coli

We have determined and monitored the production and release of human proinsulin by genetically engineered Escherichia coli cells. Several M9 media samples were analyzed sequentially after centrifugation with the aid of a rapid automated flow-through thermometric enzyme-linked immunosorbent assay (TELISA) system. The response time was 7 min after sample injection and a single assay was complete after 13 min. Insulin concentrations in the range of 0.1-50 micrograms/ml could be determined. The TELISA method correlated well with conventional radioimmunoassay determinations. Standard curves were reproducible over a period of several days even when the immobilized antibody column was stored at 25 degrees C in the enzyme thermistor unit. Thus, immediate assay start up was possible.     

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.     

Convective heat transfer measured directly with a heat flux sensor

A heat flux disk has been developed that directly measures the convective heat transfer in W/m2. When the sensor is calibrated on an aluminum cylinder, the calibration constant obtained is greatest in still air. As air movement increases, the calibration constant is reduced with increasing convective heat transfer coefficient, 0.5%.W-1.m2.K. The influence of wind on the calibration value is greatly reduced when the sensor is attached to a surface with lower thermal conductivity. The local convective heat transfer coefficient (hc) of the human body was measured. The leg acts in a manner similar to that of a cylinder, with the highest hc value at the front facing the wind and the lowest approximately 90 degrees from the wind, and in the wake a value is obtained that is close to the average hc value of the leg. When hc is measured at several angles and positions all over the body, the results indicate that the body acts approximately as a cylinder with a hc value related to the wind speed as hc = 8.6.v0.6 W.m-2.K-1, where v is velocity.     

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.     

The synthesis and biochemical evaluation of new A1 selective adenosine receptor agonists containing 6-hydrazinopurine moieties

The synthesis and SAR of a series of novel derivatives of N-aminoadenosine is described, along with their in vitro effects in biochemical assays. The rat brain A₁ adenosine receptor binding of these compounds is very dependent upon the purine 2-substituent. The novel agonist, 2-chloro-N-[4-(phenylthio)-1-piperidinyl]adenosine, exhibits a L_i value for A₁ receptor binding of <1 nM.