Measuring antibody affinity and performing immunoassay at the single molecule level

Fluorescence correlation spectroscopy (FCS) enables direct observation of the translational diffusion of single fluorescent molecules in solution. When fluorescent hapten binds to antibody, analysis of FCS data yields the fractional amounts of free and bound hapten, allowing determination of the equilibrium binding constant. Equilibrium dissociation constants of anti-digoxin antibodies and corresponding fluorescein-labeled digoxigenin obtained by FCS and fluorescence polarization measurements are identical. It is also possible to follow a competitive displacement of the tracer from the antibody by unlabeled hapten using FCS in an immunoassay format. The fluorescence polarization immunoassay for vancomycin detection was used to test the FCS approach. Fitting of the FCS data for the molar fractions of free and bound fluorescein-labeled vancomycin yielded a calibration curve which could serve for determination of the vancomycin concentration in biological samples.     

SHP-1- and phosphotyrosine-independent inhibitory signaling by a killer cell Ig-like receptor cytoplasmic domain in human NK cells

Killer cell Ig-like receptors (KIR) are MHC class I-binding immunoreceptors that can suppress activation of human NK cells through recruitment of the Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) to two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains. KIR2DL4 (2DL4; CD158d) is a structurally distinct member of the KIR family, which is expressed on most, if not all, human NK cells. 2DL4 contains only one ITIM in its cytoplasmic domain and an arginine in its transmembrane region, suggesting both inhibitory and activating functions. While 2DL4 can activate IFN-gamma production, dependent upon the transmembrane arginine, the function of the single ITIM of 2DL4 remains unknown. In this study, tandem ITIMs of KIR3DL1 (3DL1) and the single ITIM of 2DL4 were directly compared in functional and biochemical assays. Using a retroviral transduction method, we show in human NK cell lines that 1) the single ITIM of 2DL4 efficiently inhibits natural cytotoxicity responses; 2) the phosphorylated single ITIM recruits SHP-2 protein tyrosine phosphatase, but not SHP-1 in NK cells; 3) expression of dominant-negative SHP-1 does not block the ability of 2DL4 to inhibit natural cytotoxicity; 4) surprisingly, mutation of the tyrosine within the single ITIM does not completely abolish inhibitory function; and 5) this correlates with weak SHP-2 binding to the mutant ITIM of 2DL4 in NK cells and a corresponding nonphosphorylated ITIM peptide in vitro. These results reveal new aspects of the KIR-inhibitory pathway in human NK cells, which are SHP-1 and phosphotyrosine independent.     

Comparative subcellular immunolocation of polypeptides associated with xylan and callose synthases in French bean (Phaseolus vulgaris) during secondary wall formation

The Golgi apparatus of plant cells is thought to be the main site of synthesis of cell wall matrix polysaccharides and the terminal glycosylation of glycoproteins. Much of this evidence still depends on earlier biochemical studies employing subcellular fractionation. However acquiring pure Golgi membranes is still difficult and the question of spatial organisation of glycosyl transferases can be addressed by immunolocation of the enzymes. An antibody to a xylan synthase-associated polypeptide from French bean, the enzyme which synthesises the core polysaccharide for secondary wall xylan, has been raised and shown to inhibit its activity. Xylan is deposited in secondary thickenings and the xylan synthase was only detected in appreciable amounts in developing xylem cells. The location within the Golgi stack was observed throughout the dictyosomes. Some enzyme subunits were also detected in post-Golgi vesicles. A second antibody to a non-catalytic M(r) 65000 subunit of beta 1,3- glucan (callose) synthase was used for a comparative study. Although the bulk of this enzyme has been detected in previous studies at plasmamembrane-wall interfaces in sieve plates and stressed tissue, a Golgi-location can be observed in root tip meristematic cells during cell plate formation. The enzyme was present throughout the stacks. Callose was also immunolocated in a similar manner to xylan in secondary walls and thickenings and in pits in developing xylem. In these cells, the callose synthase was detected at the surface of the growing thickenings and the plasmamembrane within the pits.     

AGAP1, an endosome-associated,phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Detection and quantification of Plectosphaerella cucumerina,a potential biological control agent of potato cyst nematodes,by using conventional PCR,real-time PCR,selective media,and baiting

Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.     

A new HPLC-ELSD method to quantify indican in Polygonum tinctorium L. and to evaluate beta-glucosidase hydrolysis of indican for indigo production

A method to quantify the indigo precursor indican (indoxyl-beta-D-glucoside) in Polygonum tinctorium L. has been developed. Plant material was extracted in deionized water, and indican was identified and quantified using high performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD). Results confirmed that with this method it is possible to measure indican content in a short time, obtaining reliable and reproducible data. Using this method, leaf indican content was quantified every 15 days during the growing season (from May to October) in P. tinctorium crops grown in a field experiment in Central Italy. Results showed that indican increased along the growing season until flowering and was positively affected by photosynthetic active radiation (PAR). Indican is naturally hydrolyzed by native beta-glucosidase to indoxyl and glucose, the indoxyl yielding indigo. The activity of two enzymes, sweet almond beta-glucosidase and Novarom G preparation, were compared with P. tinctorium native beta-glucosidase to evaluate indigo production. Results showed that the ability to promote indigo formation increased as follows: almond beta-glucosidase 相似文献   

A pyoverdin from Pseudomonas sp. CFML 95-275

From Pseudomonas sp. CFML 95-275 a pyoverdin was isolated with a cyclopeptidic substructure. It could be shown that this pyoverdin is identical with one obtained from Pseudomonas fluorescens BTP 7 for which a lactone structure had been deduced from the interpretation of a FAB spectrum. The elucidation of the correct structure of the pyoverdin is described.     

Disinsertion of the levator aponeurosis from the tarsus in growing children

To confirm when the levator aponeurosis is disinserted and how the disinsertion is compensated for in growing children, the earliest and latest photographs of the same children were the subjects of a retrospective comparative study regarding upward displacement of the superior palpebral crease and the eyeball in the palpebral fissure. Ninety-four children (48 boys and 46 girls) were selected from 615 patients with cleft lip and palate who were followed for more than several years at our outpatient clinic and whose 58,000 photographs were digitized. The earliest and latest photographs of the patients were taken in primary gaze position; the former, taken at less than 3 years of age, and the latter, taken at more than 6 years of age, were selected for this study. The intervals between the two photographs ranged from 3 to 14 years (mean, 9.61 years; SD, 3.11). The superior palpebral crease moved upward parallel with the growth of the children (p < 0.0001) as well as with the length of the growth period (p = 0.0141). The lower eyelid did not move downward (p < 0.0001). The eyeball also displaced upward parallel with growth (p < 0.0001) and with the length of the growth period (p = 0.0302). The more the superior palpebral crease was displaced upward, the more the eyeball was displaced upward (p = 0.0005). The levator aponeurosis may be likely to disinsert from the tarsus in growing children, thus requiring compensatory, excessive contraction of the levator muscle, which may cause upward displacement of the superior palpebral crease. Subsequently, excessive contraction of the superior rectus muscle in conjunction of the levator muscle may rotate the eyeball upward, which may incline the head. When the head is not inclined in the primary gaze position, compensatory contraction of the inferior rectus muscle to maintain the horizontal visual axis may displace the eyeball upward in the orbit by means of the inferior suspensory ligament of Lockwood.