Quantitation of aspartate and glutamate in HPLC analysis of phenylthiocarbamyl amino acids

In the quantitation of amino acids by precolumn derivatization with phenylisothiocyanate, the yields of N'-phenylthiocarbamyl (PTC)-aspartate and PTC-glutamate from protein hydrolysates are often suboptimal, particularly in analyses following rapid hydrolysis at 160 degrees C. In this paper we show that these losses are due to the presence of materials extracted from the glass container during hydrolysis. In the presence of these extracts, the repeated drying and neutralization steps which precede phenylthiocarbamylation result in samples not fully solubilized by the presently used derivatizing mixtures. Thus the coupling yields for the acidic residues are highly variable. A coupling buffer with the composition 35% H2O, 30% acetonitrile, 25% pyridine, and 10% triethylamine (v/v/v/v) is an efficient solvent for all amino acids in hydrolysates and permits consistent, quantitative derivatization of all amino acids, including aspartate and glutamate.     

Semiautomatic procedure for the investigation of synchronized activity of EEG and heart rate--examination of preterm births]

Recordings of the electroencephalogram (EEG) and of the heart rate variability (HRV) of preterm neonates can give important information on the actual state of the nervous system. Both signals, EEG and HRV, are affected by parameters such as gestational age, stage of maturation and behavioral state. This work describes a method for automatic detection of slow wave EEG-bursts and a tool to average changes in the EEG and the corresponding heart rate. The detection is based on the hjorth activity (HA), calculated from the EEG. HA spikes (HAS) are identified by the determination of the beginning and end of existing spikes. HAS maxima and the time between two consecutive HAS are the basis for the triggering of the bursts. EEG power and time synchronized HR changes are averaged with a time window length of 20 s. Resultant, HR increase and duration are determined. These parameters, obtained by the automatic detection, proved to be comparable to the results of an expert.     

Pilot Preclinical and Clinical Evaluation of (4S)-4-(3-[18F]Fluoropropyl)-L-Glutamate (18F-FSPG) for PET/CT Imaging of Intracranial Malignancies

Purpose

(S)-4-(3-[¹⁸F]Fluoropropyl)-L-glutamic acid (18F-FSPG) is a novel radiopharmaceutical for Positron Emission Tomography (PET) imaging. It is a glutamate analogue that can be used to measure x_C^- transporter activity. This study was performed to assess the feasibility of 18F-FSPG for imaging orthotopic brain tumors in small animals and the translation of this approach in human subjects with intracranial malignancies.

Experimental Design

For the small animal study, GS9L glioblastoma cells were implanted into brains of Fischer rats and studied with 18F-FSPG, the 18F-labeled glucose derivative 18F-FDG and with the 18F-labeled amino acid derivative 18F-FET. For the human study, five subjects with either primary or metastatic brain cancer were recruited (mean age 50.4 years). After injection of 300 MBq of 18F-FSPG, 3 whole-body PET/Computed Tomography (CT) scans were obtained and safety parameters were measured. The three subjects with brain metastases also had an 18F-FDG PET/CT scan. Quantitative and qualitative comparison of the scans was performed to assess kinetics, biodistribution, and relative efficacy of the tracers.

Results

In the small animals, the orthotopic brain tumors were visualized well with 18F-FSPG. The high tumor uptake of 18F-FSPG in the GS9L model and the absence of background signal led to good tumor visualization with high contrast (tumor/brain ratio: 32.7). 18F-FDG and 18F-FET showed T/B ratios of 1.7 and 2.8, respectively. In the human pilot study, 18F-FSPG was well tolerated and there was similar distribution in all patients. All malignant lesions were positive with 18F-FSPG except for one low-grade primary brain tumor. In the 18F-FSPG-PET-positive tumors a similar T/B ratio was observed as in the animal model.

Conclusions

18F-FSPG is a novel PET radiopharmaceutical that demonstrates good uptake in both small animal and human studies of intracranial malignancies. Future studies on larger numbers of subjects and a wider array of brain tumors are planned.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01186601","term_id":"NCT01186601"}}NCT01186601     

Wheat cultivar reactions to deleterious rhizosphere bacteria under gnotobiotic conditions

With the aim of elucidating mechanisms behind bacteria-induced deleterious effects and differential cultivar responses to bacterial inoculations, wheat seedlings were subjected to various tests under gnotobiotic conditions. Inoculation with two deleterious Pseudomonas isolates, Å 112 (fluorescent) and Å 313 (nonfluorescent), induced leaf symptoms and shoot and root growth inhibition, while inoculation with growthneutral bacteria (Serratia liquefaciens andEscherichia coli) had no such effects. Deleterious effects were induced at low inoculum densities (<10³ cells per plant), but required addition of nutrient broth in small amounts for consistency. Effects similar to those obtained with living inoculum could be induced by treating plants with sterile culture filtrates from isolate Å 313 or volatile bacterial metabolites from isolate Å 112. Wheat cultivars previously found to differ in their reaction to inoculation under non-sterile conditions, responded differentially to Å 112 and Å 313 also in the gnotobiotic assay. The results agree with the hypothesis that neither cultivar reaction nor the bacterial effects as such are mediated by interactions with an indigenous rhizosphere microflora.     

TERMS,STUDIES AND EXPERIMENTS ON THE PROBLEMS OF BIRD DISPERSION*

A terminology of the dispersion of birds, and of the ways in which it comes about, is proposed and explained. An examination of recoveries of ringed female Pied Flycatchers Ficedula hypoleuca show that many first breed a considerable distance from their birthplace, in contrast to the situation in the Blue Tit Parus caeruleus and Nuthatch Sitta europaea. The degree of such dispersion in these species, and presumably therefore the gene flow, is inversely correlated with the number of geographical races they contain. Results of experiments designed to investigate the types of dispersion mechanism occurring in F. hypoleuca indicate that both “dispersal” and “spacing” occur in birds of all ages, and that they are usually not oriented, but that the spacing of older birds which have not been constantly faithful to place usually leads to a previously known place.     

Kurze Mitteilungen

Ohne Zusammenfassung     

The cytological localization of rat pancreas ribonuclease by means of fluorescent antibodies

Summary The occurrence of ribonuclease in murine and bovine pancreas has been studied with fluorescent antibodies. Results are identical in the two species. Staining occurred in the ergastoplasmic (basal) zone and in the nucleoli of the acinar cells. In the islets of Langerhans, none or only very faint nucleolar staining occurred. Extraction experiments indicate that ribonuclease might be present in at least two forms in the pancreas: one readily dissolvable and one which is more strongly bound to the cytoplasm.     

Fluorescence lifetime imaging.

We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.     

Structure of the glycoprotein Ib.IX complex from platelet membranes

The glycoprotein Ib.IX complex is a major component of the platelet membrane. It mediates the adhesion of platelets to exposed subendothelium and provides an attachment site for the membrane skeleton on the plasma membrane. The present study was designed to characterize the structure of the glycoprotein Ib.IX complex. Electron microscopy of purified glycoprotein Ib.IX complex in detergent showed that each complex existed as a flexible rod with a globular domain on either end. The overall length of the complex was approximately 59.5 nm. The smaller globular domain had a diameter of approximately 8.9 nm; the larger, a diameter of approximately 15.9 nm. In the absence of detergent, the glycoprotein Ib.IX complexes tended to self-associate through the larger globular domain, suggesting that this domain contained the hydrophobic region that inserts into the membrane. Proteases known to cleave glycoprotein Ib alpha close to its membrane-insertion site released the larger globular domain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that this domain was composed of glycoprotein Ib beta, glycoprotein IX, and a Mr = 25,000 fragment of glycoprotein Ib alpha. Proteolysis at the external end of glycoprotein Ib alpha reduced the size of the smaller globular domain. This study shows that the glycoprotein Ib.IX complex has an elongated shape, with a globular domain on the end that inserts into the membrane and a smaller globular domain on the end of glycoprotein Ib alpha that is oriented external to the plasma membrane.