Development of a noncompetitive phage anti-immunocomplex assay for brominated diphenyl ether 47

We present a new application of the noncompetitive phage anti-immunocomplex assay (PHAIA) by converting an existing competitive assay to a versatile noncompetitive sandwich-type format using immunocomplex binding phage-borne peptides to detect the brominated flame retardant, brominated diphenyl ether 47 (BDE 47). Three phage-displayed 9-mer disulfide-constrained peptides that recognize the BDE 47-polyclonal antibody immunocomplex were isolated. The resulting PHAIAs showed variable sensitivities, and the most sensitive peptide had a dose-response curve with an SC₅₀ (concentration of analyte producing 50% saturation of the signal) of 0.7 ng/ml BDE 47 and a linear range of 0.3-2 ng/ml, which was nearly identical to the best heterologous competitive format (IC₅₀ of 1.8 ng/ml, linear range of 0.4-8.5/ml). However, the PHAIA was 1400-fold better than homologous competitive assay. The validation of the PHAIA with extracts of house furniture foam as well as human and calf sera spiked with BDE 47 showed overall recovery of 80-113%. The PHAIA was adapted to a dipstick format (limit of detection of 3.0 ng/ml), and a blind test with six random extracts of local house furniture foams showed that the results of the PHAIA and dipstick assay were consistent, giving the same positive and negative detection.     

An open-flower mutant of Melilotus alba: Potential for floral-dip transformation of a papilionoid legume with a short life cycle?

This paper describes an open-flower mutant, designated opf, that we discovered in a genetic screen of fast neutron bombardment mutants in an attempt at floral-dip transformation of Melilotus alba (Fabaceae; white sweetclover), an alternative papilionoid legume host for Sinorhizobium meliloti. The opf mutant developed flowers with reflexed sepals and petals, thereby exposing the stamens and carpel, whereas wild-type sweetclover inflorescences developed closed flowers where the young stamens and carpel remain covered during the early stages of flower development. Based on crosses with the wild type, the mutant segregated as a single, Mendelian recessive. Crosses were successful only when the opf mutant served as the female parent, suggesting that the mutant was male sterile. However, no obvious differences from wild-type stamen development were observed in the opf mutant. The anther defect was due to indehiscence. However, as the plants approached the end of their life cycle, the frequency of selfing increased. We also investigated whether the opf mutant could be transformed via Agrobacterium tumefaciens floral-dip infiltration because open flowers like those of Arabidopsis appear to be more readily transformable. However, similar to wild-type M. alba, the opf mutant is refractory to floral-dip transformation by Agrobacterium tumefaciens.     

Open Capture–Recapture Models with Heterogeneity: II. Jolly–Seber Model

Summary Estimation of abundance is important in both open and closed population capture–recapture analysis, but unmodeled heterogeneity of capture probability leads to negative bias in abundance estimates. This article defines and develops a suite of open population capture–recapture models using finite mixtures to model heterogeneity of capture and survival probabilities. Model comparisons and parameter estimation use likelihood‐based methods. A real example is analyzed, and simulations are used to check the main features of the heterogeneous models, especially the quality of estimation of abundance, survival, recruitment, and turnover. The two major advances in this article are the provision of realistic abundance estimates that take account of heterogenetiy of capture, and an appraisal of the amount of overestimation of survival arising from conditioning on the first capture when heterogeneity of survival is present.     

Identification of recurring protein structure microenvironments and discovery of novel functional sites around CYS residues

Background  

The emergence of structural genomics presents significant challenges in the annotation of biologically uncharacterized proteins. Unfortunately, our ability to analyze these proteins is restricted by the limited catalog of known molecular functions and their associated 3D motifs.     

Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, <Emphasis Type="Italic">Butyrivibrio fibrisolvens</Emphasis>

Background  

Health-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA.     

Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.     

Inflorescence and floral development of Dahlstedtia species (Leguminosae: Papilionoideae: Millettieae)

Inflorescence and floral development of two tropical legume trees, Dahlstedtia pinnata and Dahlstedtia pentaphylla, occurring in the Atlantic Forest of south-eastern and southern Brazil, were investigated and compared with other papilionoids. Few studies have been made of floral development in tribe Millettieae, and this paper is intended to fill that gap in our knowledge. Dahlstedtia species have an unusual inflorescence type among legumes, the pseudoraceme, which comprises axillary units of three or more flowers, each with a subtending bract. Each flower exhibits a pair of opposite bracteoles. The order of flower initiation is acropetal; inception of the floral organs is as follows: sepals (5), petals (5), carpel (1) plus outer stamens (5) and finally inner stamens (5). Organ initiation in sepal, petal and inner stamen whorls is unidirectional; the carpel cleft is adaxial. The vexillum originates from a tubular-shaped primordium in mid-development and is larger than other petals at maturity, covering the keels. The filament tube develops later after initiation of inner-stamen primordia. Floral development in Dahlstedtia is almost always similar to other papilionoids, especially species of Phaseoleae and Sophoreae. But one important difference is the precocious ovule initiation (open carpel with ovules) in Dahlstedtia, the third citation of this phenomenon for papilionoids. No suppression, organ loss or anomalies occur in the order of primordia initiation or structure. Infra-generic differences in the first stages of ontogeny are rare; however, different species of Dahlstedtia are distinguished by the differing distribution pattern of secretory cavities in the flower.     

Plasmid profile and construction of a small shuttle vector in <Emphasis Type="Italic">Laribacter hongkongensis</Emphasis>

Among 21 human strains of Laribacter hongkongensis, small plasmids were observed in four strains, and large ones in six strains. The smallest, 3264-bp plasmid, pHLHK19, has only one ORF that encodes a putative replication initiator protein and a predicted origin of replication (ori) with a DnaA box, three 18-bp direct repeats and five pairs of inverted repeats. An Escherichia coli-L. hongkongensis shuttle vector was constructed by ligating the HindIII-digested pHLHK19, containing the replication initiator protein and ori of pHLHK19, to HindIII-digested pBK-CMV. This shuttle vector can propagate in E. coli and L. hongkongensis with good transformation efficiencies.     

The insulin-specific T cells of nonobese diabetic mice recognize a weak MHC-binding segment in more than one form

Several naturally occurring anti-insulin CD4 T cells were isolated from islet infiltrates of NOD mice. In accordance with the results of others, these T cells recognized the segment of the beta-chain from residues 9-23. Peptides encompassing the B:(9-23) sequence bound weakly to I-Ag7 in two main contiguous registers in which two residues at the carboxyl end, P20Gly and P21Glu, influenced binding and T cell reactivity. Naturally occurring insulin-reactive T cells exhibited differing reactivities with the carboxyl-terminal amino acids, although various single residue changes in either the flanks or the core segments affected T cell responses. The insulin peptides represent another example of a weak MHC-binding ligand that is highly immunogenic, giving rise to distinct populations of autoimmune T cells.