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.     

Anterior hip joint force increases with hip extension, decreased gluteal force, or decreased iliopsoas force

Abnormal or excessive force on the anterior hip joint may cause anterior hip pain, subtle hip instability and a tear of the acetabular labrum. We propose that both the pattern of muscle force and hip joint position can affect the magnitude of anterior joint force and thus possibly lead to excessive force and injury. The purpose of this study was to determine the effect of hip joint position and of weakness of the gluteal and iliopsoas muscles on anterior hip joint force. We used a musculoskeletal model to estimate hip joint forces during simulated prone hip extension and supine hip flexion under four different muscle force conditions and across a range of hip extension and flexion positions. Weakness of specified muscles was simulated by decreasing the modeled maximum force value for the gluteal muscles during hip extension and the iliopsoas muscle during hip flexion. We found that decreased force contribution from the gluteal muscles during hip extension and the iliopsoas muscle during hip flexion resulted in an increase in the anterior hip joint force. The anterior hip joint force was greater when the hip was in extension than when the hip was in flexion. Further studies are warranted to determine if increased utilization of the gluteal muscles during hip extension and of the iliopsoas muscle during hip flexion, and avoidance of hip extension beyond neutral would be beneficial for people with anterior hip pain, subtle hip instability, or an anterior acetabular labral tear.     

Autosomal recessive ichthyosis with hypotrichosis caused by a mutation in ST14, encoding type II transmembrane serine protease matriptase

In this article, we describe a novel autosomal recessive ichthyosis with hypotrichosis syndrome, characterized by congenital ichthyosis associated with abnormal hair. Using homozygosity mapping, we mapped the disease locus to 11q24.3-q25. We screened the ST14 gene, which encodes matriptase, since transplantation of skin from matriptase(-/-)-knockout mice onto adult athymic nude mice has been shown elsewhere to result in an ichthyosislike phenotype associated with almost complete absence of erupted pelage hairs. Mutation analysis revealed a missense mutation, G827R, in the highly conserved peptidase S1-S6 domain. Marked skin hyperkeratosis due to impaired degradation of the stratum corneum corneodesmosomes was observed in the affected individuals, which suggests that matriptase plays a significant role in epidermal desquamation.     

Multiplexed immunoassays for proteins using magnetic luminescent nanoparticles for internal calibration

Suspension arrays present a promising tool for multiplexed assays in large-scale screening applications. A simple and robust platform for quantitative multiprotein immunoanalysis has been developed with the use of magnetic Co:Nd:Fe(2)O(3)/luminescent Eu:Gd(2)O(3) core/shell nanoparticles (MLNPs) as a carrier. The magnetic properties of the MLNPs allow their manipulation by an external magnetic field in the separation and washing steps in the immunoassay. Their optical properties enable the internal calibration of the detection system. The multiplexed sandwich immunoassay involves dual binding events on the surface of the MLNPs functionalized with the capture antibodies. Secondary antibodies labeled with conventional organic dyes (Alexa Fluor) are used as reporters. The amount of the bound secondary antibody is directly proportional to the concentration of the analyte in the sample. In our approach, the fluorescence intensity of the reporter dye is related to the luminescence signal of the MLNPs. In this way, the intrinsic luminescence of the MLNPs serves as an internal standard in the quantitative immunoassay. The concept is demonstrated for a simultaneous immunoassay for three model proteins (human, rabbit, and mouse IgGs). The method uses a standard bench plate reader. It can be applied to disease diagnostics and to the detection of biological threats.     

Identification of key tissue type for antler regeneration through pedicle periosteum deletion

Epimorphic regeneration is the “holy grail” of regenerative medicine. Research aimed at investigating the various models of epimorphic regeneration is essential if a fundamental understanding of the factors underpinning this process are to be established. Deer antlers are the only mammalian appendages that are subject to an annual cycle of epimorphic regeneration. In our previous studies, we have reported that histogenesis of antler regeneration relies on cells resident within the pedicle periosteum (PP). The present study elaborates this finding by means of functional studies involving the deletion of PP. Four yearling and four 2-year-old stags were selected for total PP deletion or partial PP deletion experiments. Of the animals in the total PP deletion group, one showed no signs of antler regeneration throughout the antler growth season. Two showed substantial and one showed marginal delays in antler regeneration (at 34, 20 and 7 days, respectively) compared with the corresponding sham-operated sides. Histological investigation revealed that the delayed antlers were derived from regenerated PP. Unexpectedly, the regenerative capacity of the antler from the total periosteum-deleted pedicles depended on antler length at surgery. Of the four deer that had partial PP deletion, two regenerated antlers exclusively from the left-over PP on the pedicle shafts in the absence of participation from the pedicle bone proper. The combined results from the PP deletion experiments convincingly demonstrate that the cells of the PP are responsible for antler regeneration. The authors thank the New Zealand Foundation of Research, Science and Technology and Deer Industry New Zealand for funding their research.     

Comparison of naturally acquired antibody responses against the C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 under low transmission and unstable malaria conditions in Sri Lanka

We report here, for the first time, a comparison of naturally acquired antibody responses to the 42 and 19 kDa C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 assayed by ELISA using p42 and p19 baculovirus-derived recombinant proteins, respectively. Test populations comprised patients with microscopy confirmed acute P. vivax infections from two regions endemic for vivax malaria where low transmission and unstable malaria conditions prevail, and a non-endemic urban area, in Sri Lanka. The antibody prevalence to the two proteins, both at the individual and population levels, tend to respond more to p42 than to p19 in all test areas, where >14% of individuals preferentially recognized p42, compared with <2% for p19. In patients with no previous exposure to malaria, 21% preferentially recognized p42, whereas none exclusively recognized p19. A significantly lower prevalence of anti-p19 IgM, but not anti-p42 IgM, was observed among residents from endemic areas compared with their non-endemic counterparts. Individuals from both endemic areas produced significantly less anti-p19 IgM compared with anti-p42 IgM. IgG1 was the predominant IgG isotype for both antigens in all individuals. With increasing exposure to malaria in both endemic areas, anti-p19 antibody responses were dominated by the functionally important IgG1 and IgG3 isotypes, with a concurrent reduction in IgM that was lacking in the non-endemic residents. This antibody switch was also reflected for PvAMA-1 as we previously reported with the identical battery of sera. In contrast, the antibody switch for p42 was restricted to endemic residents with more extensive exposure. These results suggest that an IgM-dominated antibody response against the p42 polymorphic region in endemic residents may interfere with the development of an IgG-dominated "protective" isotype shift to p19, that may complicate vaccine development.