A simple and rapid protein array based method for the simultaneous detection of biowarfare agents

A protein chip has been developed that allows the simultaneous detection of a multitude of different biowarfare agents. The chip was developed for the ArrayTube platform providing a cheap and easy to handle technology solution that combines a microtube-integrated protein chip with the classical procedure of a sandwich-enzyme-linked immunosorbent assay and signal amplification by streptavidin-poly-horseradish peroxidase. Specific immunoassays for Staphylococcus enterotoxin B, ricin, Venezuelan equine encephalitis virus, St. Louis encephalitis virus, West Nile virus, Yellow fever virus, Orthopox virus species, Francisella tularensis, Yersinia pestis, Brucella melitensis, Burkholderia mallei and Escherichia coli EHEC O157:H7 were developed and optimized. All assays could be completed within 1 to 1 1/2 h and detection levels were demonstrated to be as low as in well established ELISAs. Most interesting, as a result of careful antibody screening and testing, it is currently possible to analyse at least five of the "dirty dozen" agents on one single protein chip in parallel.     

High resolution ion chamber array delivery quality assurance for robotic radiosurgery: Commissioning and validation

PurposeHigh precision radiosurgery demands comprehensive delivery-quality-assurance techniques. The use of a liquid-filled ion-chamber-array for robotic-radiosurgery delivery-quality-assurance was investigated and validated using several test scenarios and routine patient plans.Methods and materialPreliminary evaluation consisted of beam profile validation and analysis of source–detector-distance and beam-incidence-angle response dependence. The delivery-quality-assurance analysis is performed in four steps: (1) Array-to-plan registration, (2) Evaluation with standard Gamma-Index criteria (local-dose-difference ⩽ 2%, distance-to-agreement ⩽ 2 mm, pass-rate ⩾ 90%), (3) Dose profile alignment and dose distribution shift until maximum pass-rate is found, and (4) Final evaluation with 1 mm distance-to-agreement criterion. Test scenarios consisted of intended phantom misalignments, dose miscalibrations, and undelivered Monitor Units. Preliminary method validation was performed on 55 clinical plans in five institutions.ResultsThe 1000SRS profile measurements showed sufficient agreement compared with a microDiamond detector for all collimator sizes. The relative response changes can be up to 2.2% per 10 cm source–detector-distance change, but remains within 1% for the clinically relevant source–detector-distance range. Planned and measured dose under different beam-incidence-angles showed deviations below 1% for angles between 0° and 80°. Small-intended errors were detected by 1 mm distance-to-agreement criterion while 2 mm criteria failed to reveal some of these deviations. All analyzed delivery-quality-assurance clinical patient plans were within our tight tolerance criteria.ConclusionWe demonstrated that a high-resolution liquid-filled ion-chamber-array can be suitable for robotic radiosurgery delivery-quality-assurance and that small errors can be detected with tight distance-to-agreement criterion. Further improvement may come from beam specific correction for incidence angle and source–detector-distance response.     

The estimation of micro-algal protein content and its meaning to the evaluation of algal biomass I. Comparison of methods for extracting protein

The spectrophotometric evaluation of micro-algal protein needs a prior extraction from cells in order to liberate protein for measurement. The conditions of extraction (temperature, duration, normality of sodium hydroxide, pretreatment) which yield optimal protein content are tested with three algal cultures (Scenedesmus, Synechococcus, Asterionella). A standard method of extraction is presented. Comparison of this method with nine published methods reveals markedly lower protein yields for easy extractable (43–100%) and hard extractable (5–75%) algal species, relative to this method, depending on ease of cell wall breakage. The application of this standard method to field investigations is demonstrated and compared to other biochemical parameters. The advantages of this method over other protein extraction methods, with respect to field material, are discussed.     

Protein sorting and expression of a unique soybean cotyledon protein,GmSBP, destined for the protein storage vacuole

The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and -conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, -conglycinin (/ subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitative differences between GmSBP and glycinin may be attributed to the unconserved order and spacing of cis-acting regulatory elements present within the promoter regions. Ultimately, GmSBP is transported to the mature protein storage vacuole. The biological function of GmSBP within the protein storage vacuole remains uncertain, but its localization is a remnant of its evolutionary link to a globulin-like or vicilin-like ancestor that gave rise to the 7S family of storage proteins.     

Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding.

Proteins destined for the secretory pathway must first fold and assemble in the lumen of endoplasmic reticulum (ER). The pathway maintains a quality control mechanism to assure that aberrantly processed proteins are not delivered to their sites of function. As part of this mechanism, misfolded proteins are returned to the cytosol via the ER protein translocation pore where they are ubiquitinated and degraded by the 26S proteasome. Previously, little was known regarding the recognition and targeting of proteins before degradation. By tracking the fate of several mutant proteins subject to quality control, we demonstrate the existence of two distinct sorting mechanisms. In the ER, substrates are either sorted for retention in the ER or are transported to the Golgi apparatus via COPII-coated vesicles. Proteins transported to the Golgi are retrieved to the ER via the retrograde transport system. Ultimately, both retained and retrieved proteins converge at a common machinery at the ER for degradation. Furthermore, we report the identification of a gene playing a novel role specific to the retrieval pathway. The gene, BST1, is required for the transport of misfolded proteins to the Golgi, although dispensable for the transport of many normal cargo proteins.     

Protein accumulation,RNA and soluble amino nitrogen content in developing endosperm of two varieties of Triticum aestivum with high and low protein seed

Summary The kinetics of protein accumulation, the variation in RNA, the soluble amino nitrogen content of developing endosperm of two varieties of Triticum aestivum, with high and low protein content in the mature seed, suggest a possible relation between maintenance of the RNA content and the ability to synthesize protein. A sudden halt in protein accumulation is observed as the RNA starts to decrease. The hypothesis is also advanced that maintenance of the RNA content might, in turn, be dependent on the presence, in the endosperm of developing wheat seed, of a certain level of soluble amino nitrogen which could then play the role of limiting factor for protein synthesis.Publication No. 491 from the Divisione Applicazione delle Radiazioni del C.N.E.N., SCN Casaccia, S.M. di Galeria, Rome, Italy.     

Der1p, a protein required for degradation of malfolded soluble proteins of the endoplasmic reticulum: topology and Der1-like proteins

The endoplasmic reticulum (ER) contains a highly effective protein quality control system eliminating malfolded proteins by a mechanism called ER-associated protein degradation (ERAD). Here, we unravel the topology of Der1p, a previously identified component of the ERAD system. Der1p contains four transmembrane domains, its N- and C-terminus protrude into the cytoplasm and contribute to its function. Additionally, we describe a yeast homologue of Der1p, Dfm1p, which does not seem to be involved in ERAD. In contrast, a Caenorhabditis elegans orthologue of Der1p, R151.6, is capable of complementing der1-defective phenotypes in yeast.     

Mapping quantitative trait loci for milling quality, protein content and color characteristics of rice using a recombinant inbred line population derived from an elite rice hybrid

Milling properties, protein content, and flour color are important factors in rice. A marker-based genetic analysis of these traits was carried out in this study using recombinant inbred lines (RILs) derived from an elite hybrid cross ’Shanyou 63’, the most-widely grown rice hybrid in production in China. Correlation analysis shows that the traits were inter-correlated, though the coefficients were generally small. Quantitative trait locus (QTL) analysis with both interval mapping (IM) and composite interval mapping (CIM) revealed that the milling properties were controlled by the same few loci that are responsible for grain shape. The QTL located in the interval of RM42-C734b was the major locus for brown rice yield, and the QTL located in the interval of C1087-RZ403 was the major locus for head rice yield. These two QTLs are the loci for grain width and length, respectively. The Wx gene plays a major role in determining protein content and flour color, and is modified by several QTLs with minor effect. The implications of the results in rice breeding were discussed. Received: 15 September 2000 / Accepted: 31 March 2001     

Effect of irradiation on the protein profile,protein content,and quality of agar from <Emphasis Type="Italic">Gracilaria asiatica</Emphasis> Zhang et Xia (Rhodophyta)

There are about 15 species of Gracilaria reported in Vietnam. Of these, Gracilaria asiatica Zhang et Xia is being cultivated on a large scale in Northern Vietnam, which has a subtropical climate. During the rainy season, from May to October, the growth of G. asiatica is drastically reduced or even ceases due to very low salinity and high temperature. Therefore, it is important to improve the tolerance of G. asiatica to a wide range of salinity and temperatures. This paper presents the results of research on strain improvement of G. asiatica using irradiation and selection media. Three irradiation doses of 20, 60, and 100 krad were tested against the control (with no irradiation). Afterward, the seaweed biomass was cultivated on a selected medium, ESS-1, containing NaCl in concentrations of 23‰ (C1) and 0‰ (C2). The results showed that a higher survival rate of G. asiatica was observed with the 20- and 60-krad doses. The protein content and composition of selected seaweeds were analyzed and compared with the control. SDS-PAGE showed no remarkable difference in the protein composition between the control and irradiated samples. However, the 67-kDa protein band of seaweed treated with 20 and 60 krad, then grown on ESS-1 medium with 23% NaCl, had a higher density than other samples. This protein was reported to play an important role in G. asiatica, by enhancing its tolerance to variable salinity and temperature. Although the organic and inorganic content of all samples remained almost the same, the content and quality of agar extracted from irradiated seaweeds were higher than those of the controls. Due to the high amount of 3.6 anhydro-α-L-galactose combined with low amounts of sulfate found in irradiated seaweeds, the freezing and melting points of extracted agar were lower. Eventually, this resulted in higher condensation and better quality of agar, such as in its gel-forming ability. The quality of fluid agar extracted from selected seaweeds improved as shown in the remarkable decrease in Ca²⁺, Mg²⁺, and total Fe ion content, thus lowering its melting point compared with the control. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Development,validation and characterization of an analytical method for the quantification of hydrolysable urinary metabolites and plasma protein adducts of 2,4- and 2,6-toluene diisocyanate, 1,5-naphthalene diisocyanate and 4,4'-methylenediphenyl diisocyanate

Occupational exposure to diisocyanates within the plastic industry causes irritation and disorders in the airway. The aim of this study was to develop, validate and characterize a method for the determination of 2,4-toluenediamine (2,4-TDA), 2,6-toluenediamine (2,6-TDA), 1,5-diaminonaphthalene (1,5-NDA) and 4,4′-methylenedianiline (4,4′-MDA) in hydrolysed urine and plasma, and to study the correlation between the plasma and urinary levels of these potential biomarkers of 2,4-toluene diisocyanate (2,4-TDI), 2,6-toluene diisocyanate (2,6-TDI), 1,5-naphthalene diisocyanate (1,5-NDI) and 4,4′-methylenediphenyl diisocyanate (4,4′-MDI), respectively. Samples were hydrolysed with 0.3 M NaOH at 100°C for 24 h. The diamines were extracted, derivatized with pentafluoropropionic acid anhydride, and quantified by selected ion monitoring on gas chromatography-mass spectrometry. The repeatability and reproducibility of the method were 7-18% and 7-19%, respectively. Dialysis experiments showed that the metabolites of 2,4-TDI, 2,6-TDI, 1,5-NDI and 4,4′-MDI in plasma were exclusively protein adducts. No free diamines were found in urine, indicating that all diisocyanate-related metabolites were in a conjugated form. For each diisocyanate-related biomarker, there were strongly significant correlations (p&lt;0.001) between individual levels of metabolites in plasma and urine, with Spearman's rank correlation coefficient (r_s) values of 0.74-0.90. The methods presented here will be valuable for the development of biological monitoring methods for diisocyanates.     

Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling

The secretion of Wnt signaling proteins is dependent upon the transmembrane sorting receptor, Wntless (Wls), which recycles between the trans-Golgi network and the cell surface. Loss of Wls results in impairment of Wnt secretion and defects in development and homeostasis in Drosophila, Caenorhabditis elegans, and the mouse. The sorting signals for the internalization and trafficking of Wls have not been defined. Here, we demonstrate that Wls internalization requires clathrin and dynamin I, components of the clathrin-mediated endocytosis pathway. Moreover, we have identified a conserved YXXφ endocytosis motif in the third intracellular loop of the multipass membrane protein Wls. Mutation of the tyrosine-based motif YEGL to AEGL (Y425A) resulted in the accumulation of human mutant Wls on the cell surface of transfected HeLa cells. The cell surface accumulation of Wls^AEGL was rescued by the insertion of a classical YXXφ motif in the cytoplasmic tail. Significantly, a Drosophila Wls^AEGL mutant displayed a wing notch phenotype, with reduced Wnt secretion and signaling. These findings demonstrate that YXXφ endocytosis motifs can occur in the intracellular loops of multipass membrane proteins and, moreover, provide direct evidence that the trafficking of Wls is required for efficient secretion of Wnt signaling proteins.     

Improvement of quality control methods for augmentative releases of the fruit fly parasitoids,Diachasmimorpha longicaudata and Psyttalia fletcheri (Hymenoptera: Braconidae)

Criteria were established to improve quality control methods for augmentative releases of the opiine parasitoids, Diachasmimorpha longicaudata (Ashmead) and Psyttalia fletcheri (Silvestri). These included correlating puparial weight with adult emergence and sex ratio at the Honolulu rearing facility, and determining the effect of air shipments and field releases on parasitoid emergence and subsequent mortality. There was a positive relationship between the weight of 7–10‐day‐old host puparia and percentage of emergence for both D. longicaudata and P. fletcheri. Standardization of shipping methods included placement of ice blocks in the top levels of the ice chests, prompt shipment and pick‐up of samples, and cooling of puparia before shipment to minimize metabolic heat generated in the samples, and to delay emergence of samples. Before standardization, emergence losses of up to 58% were observed for D. longicaudata and 18% for P. fletcheri. When shipping methods were standardized, emergence was no longer reduced. In addition, low emergence rates were associated with reduced flight propensity of D. longicaudata (R_s = ‐ 0.45 at Kilauea and ‐ 0.54 at Kealia). At the two release sites, 88–95% of adult D. longicaudata that did not escape the release containers were males.     

Investigations on the effects of rape oil quality,choline and methionine concentration in diets for laying hens on the trimethylamine content of the eggs,on trimethylamine metabolism and on laying performance

Abstract

An experiment was conducted to study the effects of graded levels of choline addition (0, 500, 1000 and 4000 mg/kg diet) in laying hen diets prepared either with degummed or refined rape oil on the performance, sensory properties and trimethylamine (TMA) contents of the eggs. Furthermore, the diets containing no supplemented choline or 4000 mg choline/kg diet were tested with adequate or inadequate methionine supply (4.2 vs. 2.8 g methionine/kg diet). TMA metabolism and N-balance were measured for the latter diet types, but only with the diets containing refined rape oil. Therefore, a total of 12 and 4 diets were tested in the feeding (n = 60) and balance study (n = 9). Laying performance (23 – 75 weeks of age) was not significantly influenced by increasing choline additions with the exception of feed-to-egg mass ratio which decreased significantly linearly (p _linear = 0.003). However, a significant interaction between choline addition and laying month was detected which was caused by a depression of performance of the unsupplemented control group occurring from the sixth laying month. The most obvious effect of an inadequate methionine supply was a temporary drop in performance between the third and sixth laying months. The mean TMA-concentration in pooled egg yolks [μg/g] increased with dietary choline concentration [mg/kg] in an exponentially related fashion (y = 1.14 + 4E^?10 ? x^2.71, r² = 0.962) and suggested only a minor influence of total dietary choline on TMA content up to approximately 2000 mg choline/kg. Individual TMA-concentrations varied greatly from 0.4 – 1.5 μg/g, from 2.2 – 34 μg/g and from 18.4 – 75 μg/g for eggs with a normal, aberrant and heavily aberrant odour, respectively. It is concluded that a total choline concentration of at least approximately 1500 mg/kg is necessary to maintain a maximal laying performance. An inadequate methionine supply cannot be compensated by an increased addition of choline. Neither degummed nor refined rape oil influenced the TMA content of eggs.