A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins

A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.     

Magnesium supplementation and deoxycorticosterone acetate--salt hypertension: effect on arterial mechanical properties and on activity of endothelin-1

The aim of this study was to show whether the decrease in blood pressure induced by Mg supplementation in deoxycorticosterone acetate - salt (DOCA-salt) hypertensive rats is associated with mechanical modifications of blood vessels and (or) changes in tissular production and (or) vasoconstrictor activity to endothelin-1. DOCA-salt treatment increased blood pressure, media thickness, cross-sectional area, and lumen diameter of carotid arteries. Distensibility and incremental elastic modulus versus stress were not altered in carotid arteries, suggesting that the DOCA-salt vessel wall adapts structurally to preserve its blood pressure buffering capacity. Magnesium supplementation attenuated DOCA-salt hypertension. In comparison with normotensive rats, systolic, mean, and pulse pressures were higher whereas diastolic pressure was not different in Mg-supplemented DOCA-salt rats. Magnesium supplementation did not significantly modify the elastic parameters of carotid arteries. In resistance mesenteric arteries, DOCA-salt hypertension induces an inward hypertrophic remodeling. Magnesium supplementation attenuates wall hypertrophy and increases lumen diameter to the normotensive diameter, suggesting a decrease in peripheral resistance. Magnesium supplementation normalizes the altered vasoconstrictor activity of endothelin-1 in mesenteric arteries and attenuates endothelin-1 overproduction in kidney, left ventricle, and aorta of DOCA-salt rats. These findings suggest that Mg supplementation prevents blood pressure elevation by attenuating peripheral resistance and by decreasing hypertrophic effect of endothelin-1 via inhibition of endothelin-1 production.     

Evidence consistent with human L1 retrotransposition in maternal meiosis I

We have used a unique polymorphic 3' transduction to show that a human L1, or LINE-1 (long interspersed nucleotide element-1), retrotransposition event most likely occurred in the maternal primary oocyte during meiosis I. We characterized a truncated L1 retrotransposon with a 3' transduction that was inserted, in a Dutch male patient, into the X-linked gene CYBB, thereby causing chronic granulomatous disease. We used the unique flanking sequence to localize the precursor L1 locus, LRE3, to chromosome 2q24.1. In a cell culture assay, the retrotransposition frequency of LRE3 is greater than that for any other element that has been tested to date. The patient's mother had two LRE3 alleles that differed slightly in the 3'-flanking genomic DNA. The patient had a single LRE3 allele that was identical to one of the maternal alleles; however, the patient's insertion matched the maternal LRE3 allele that he did not inherit. Other data indicate that there is only a small chance that the father (unavailable for analysis) carries the precursor LRE3 allele. In addition, paternal origin of the insertion would have required that an LRE3 mRNA transcribed before meiosis II be carried separately from its precursor LRE3 allele in the fertilizing sperm. Since the mother carries a potential precursor allele and the insertion was on the patient's maternal X chromosome, it is highly likely that the insertion originated during maternal meiosis I.     

Low proliferative and high migratory activity in the area of Brachyury expressing mesoderm progenitor cells in the gastrulating rabbit embryo

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that centre on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridisation revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which we call the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation.     

Geometric algorithms for the analysis of 2D-electrophoresis gels.

In proteomics, two-dimensional gel electrophoresis (2-DE) is a separation technique for proteins. The resulting protein spots can be identified either by using picking robots and subsequent mass spectrometry or by visual cross inspection of a new gel image with an already analyzed master gel. Difficulties especially arise from inherent noise and irregular geometric distortions in 2-DE images. Aiming at the automated analysis of large series of 2-DE images, or at the even more difficult interlaboratory gel comparisons, the bottleneck is to solve the two most basic algorithmic problems with high quality: Identifying protein spots and computing a matching between two images. For the development of the analysis software CAROl at Freie Universit?t Berlin, we have reconsidered these two problems and obtained new solutions which rely on methods from computational geometry. Their novelties are: 1. Spot detection is also possible for complex regions formed by several "merged" (usually saturated) spots; 2. User-defined landmarks are not necessary for the matching. Furthermore, images for comparison are allowed to represent different parts of the entire protein pattern, which only partially "overlap." The implementation is done in a client server architecture to allow queries via the internet. We also discuss and point at related theoretical questions in computational geometry.     

Transbilayer movement of monohexosylsphingolipids in endoplasmic reticulum and Golgi membranes

The transbilayer movement of glycosphingolipids has been characterized in Golgi, ER, plasma, and model membranes using spin-labeled and fluorescent analogues of the monohexosylsphingolipids glucosylceramide and galactosylceramide and of the dihexosylsphingolipid lactosylceramide. In large unilamellar lipid vesicles, monohexosylsphingolipids underwent a slow transbilayer diffusion (half-time between 2 and 5 h at 20 degrees C). Similarly, the inward redistribution of these sphingolipids in the plasma membrane of the hepatocyte-like cell line HepG2 and of erythrocytes was slow. However, in rat liver ER and Golgi membranes, we found a rapid transbilayer movement of spin-labeled monohexosylsphingolipids (half-time of approximately 3 min at 20 degrees C), which suggests the existence of a monohexosylsphingolipid flippase. The transbilayer movement of glucosylceramide in the Golgi and the ER displayed a saturable behavior, was inhibited by proteolysis, did not require Mg-ATP, and occurs in both directions. Treatment with DIDS inhibited the flip-flop of glucosylceramide but not that of phosphatidylcholine. These data suggest that the transbilayer movement of monoglucosylceramide in the ER and in the Golgi involves a protein that could be distinct from that previously evidenced for glycerophospholipids in the ER. In vivo, transbilayer diffusion should promote a symmetric distribution of monohexosylsphingolipids which are synthesized in the cytosolic leaflet. This should allow glucosylceramide rapid access to the lumenal leaflet where it is converted to lactosylceramide. No significant transbilayer movement of lactosylceramide occurred in both artificial and natural membranes over 1 h. Thus, lactosylceramide, in turn, is unable to diffuse to the cytosolic leaflet and remains at the lumenal leaflet where it undergoes the subsequent glycosylations.     

'Small' talk: Opa proteins as mediators of Neisseria-host-cell communication

Opa proteins are variable outer membrane proteins of Neisseria gonorrhoeae and Neisseria meningitidis that mediate tight interaction of these pathogens with human cells. They have emerged as a paradigm of a bacterial toolbox allowing recognition of different host receptors and orchestrating the cell type tropism displayed by pathogenic Neisseriae. Recent work has highlighted the molecular basis of Opa-protein-host-receptor interaction and has shed new light on the functional consequences of this interaction with regard to bacterial attachment, invasion, and responses elicited in particular host cells.     

Comparison of the radiotoxicity of two alpha-particle-emitting immunoconjugates,terbium-149 and bismuth-213, directed against a tumor-specific,exon 9 deleted (d9) E-cadherin adhesion protein

We investigated the effects of the alpha-particle emitters (149)Tb and (213)Bi coupled to a tumor-specific antibody targeting the mutated delta 9 E-cadherin (d9 E-Cad) on single cells and cell pellets. The d9 mutation of the adhesion molecule E-cadherin is found in 10% of diffuse-type gastric cancers and is not expressed in normal tissue. Human breast cancer cells (MDA-MB-435S) transfected with d9 E-Cad or the wild-type E-cadherin gene were used to study the effects of anti-d9 E-Cad MAb coupled to (149)Tb and (213)Bi ((149)Tb-d9 MAb and (213)Bi-d9 MAb). The density of binding sites determined on transfected MDA tumor cells by Scatchard analysis and flow cytometry varied from 4 x 10(4) to 6 x 10(4) antigens per cell. Internalization of radioimmunoconjugates by cells expressing d9 E-Cad was less than 10% of bound antibody within 240 min. The effect of the radioimmunoconjugates on cell suspensions and cell pellets was quantified by [(3)H]thymidine incorporation, and the dose to the cell nuclei was determined using microdosimetric calculations. (149)Tb and (213)Bi immunoconjugates affected cells in suspension similarly. Significant differences in the proliferation capacity of d9 E-cadherin- and wild-type E-cadherin-expressing cells were observed at activity concentrations around 185 kBq/ml, corresponding to antibody concentrations between 200 ng/ml and 1000 ng/ml. Proliferation after incubation with (213)Bi-d9 MAb was 50% greater in pelleted wild-type E-Cad-expressing cells compared to wild-type E-Cad cells in suspension. In contrast, the proliferation of pelleted d9 E-Cad cells was similar to that of d9 E-Cad cells in suspension. For (149)Tb-d9 MAb, no significant difference was found between pelleted cells and cells in suspension for low activity concentrations. However, at high activity concentrations, (149)Tb-d9 MAb had only a small effect on pelleted cells. These in vitro studies demonstrate different effects of (149)Tb and (213)Bi conjugated to a tumor-specific antibody toward single cells and tumor cell pellets. Microdosimetric simulation of single cell survival after alpha-particle irradiation modeled the experimental results with reasonable accuracy.     

Evidence for the existence of psychrophilic methanogenic communities in anoxic sediments of deep lakes

In order to obtain evidence for the existence of psychrophilic methanogenic communities in sediments of deep lakes that are low-temperature environments (4 to 5 degrees C), slurries were first incubated at temperatures between 4 and 60 degrees C for several weeks, at which time they were amended, or not, with an additional substrate, such as cellulose, butyrate, propionate, acetate, or hydrogen, and further incubated at 6 degrees C. Initial methane production rates were highest in slurries preincubated at temperatures between 4 and 15 degrees C, with maximal rates in slurries kept at 6 degrees C. Hydrogen-amended cultures were the only exceptions, with the highest methane production rates at 6 degrees C after preincubation at 30 degrees C.