The CD85J/leukocyte inhibitory receptor-1 distinguishes between conformed and beta 2-microglobulin-free HLA-G molecules

For a proper development of the placenta, maternal NK cells should not attack the fetal extravillous cytotrophoblast cells. This inhibition of maternal NK cells is partially mediated via the nonclassical MHC class I molecule HLA-G. Recently, we demonstrated that HLA-G forms disulfide-linked high molecular complexes on the surface of transfected cells. In the present study, we demonstrate that HLA-G must associate with beta(2)m for its interaction with CD85J/leukocyte Ig-like receptor-1 (LIR-1). Although HLA-G free H chain complexes are expressed on the surface, they are not recognized and possibly interfere with CD85J/LIR-1 and HLA-G interaction. The formation of these complexes on the cell surface might represent a novel mechanism developed specifically by the HLA-G protein aimed to control the efficiency of the CD85J/LIR-1-mediated inhibition. We also show that endogenous HLA-G complexes are expressed on the cell surface. These findings provide novel insights into the delicate interaction between extravillous cytotrophoblast cells and NK cells in the decidua.     

Analysis of the minimal amyloid-forming fragment of the islet amyloid polypeptide. An experimental support for the key role of the phenylalanine residue in amyloid formation.

The development of type II diabetes was shown to be associated with the formation of amyloid fibrils consisted of the islet amyloid polypeptide (IAPP or amylin). Recently, a short functional hexapeptide fragment of IAPP (NH(2)-NFGAIL-COOH) was found to form fibrils that are very similar to those formed by the full-length polypeptide. To better understand the specific role of the residues that compose the fragment, we performed a systematic alanine scan of the IAPP "basic amyloidogenic units." Turbidity assay experiments demonstrated that the wild-type peptide and the Asn(1) --> Ala and Gly(3) --> Ala peptides had the highest rate of aggregate formation, whereas the Phe(2) --> Ala peptide did not form any detectable aggregates. Dynamic light-scattering experiments demonstrated that all peptides except the Phe(2) --> Ala form large multimeric structures. Electron microscopy and Congo red staining confirmed that the structures formed by the various peptides are indeed amyloid fibrils. Taken together, the results of our study provide clear experimental evidence for the key role of phenylalanine residue in amyloid formation by IAPP. In contrast, glycine, a residue that was suggested to facilitate amyloid formation in other systems, has only a minor role, if any, in this case. Our results are discussed in the context of the remarkable occurrence of aromatic residues in short functional fragments and potent inhibitors of amyloid-related polypeptides. We hypothesize that pi-pi interactions may play a significant role in the molecular recognition and self-assembly processes that lead to amyloid formation.     

Identification of new mutations in Israeli patients with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (ALD) is a peroxisomal disorder characterized by impaired peroxisomal betaoxidation of very-long-chain fatty acids (VLCFAs). This is probably due to reduced activation of the VLCFAs and results in demyelination of the nervous system and adrenocortical insufficiency. The ALD gene is localized on Xq28, has 10 exons and encodes a protein of 745 amino acids with significant homology to the membrane peroxisomal protein PMP70. Characterizing the disease causing mutations is of importance in prenatal diagnosis because 12-20% of women who are obligatory carriers show false-negative results when tested for VLCFA in plasma. We have analyzed DNA from blood samples of 7 Jewish (5 Sephardi and 2 Ashkenazi) and 3 Arab Israeli families suffering from ALD. Five missense-type mutations were identified: R104H, Y174C, L229P, R401Q, and G512C. A single mutation, R464X, was nonsense, and two, Y171 frameshift and E471 frameshift, were frameshift. Interestingly, a single mutation was identified in three families of Moroccan Jewish descent, probably due to a founder effect.     

High-throughput assay for temporal kinetic analysis of lytic coliphage activity

Plaque analysis allows the determination of phage titer and multiplicity of infection. Yet, this overnight assay provides only endpoint results, ignoring kinetic aspects. We introduce an alternative high-throughput and rapid method for kinetic analysis of lytic coliphage activity. Escherichia coli was infected with serial dilutions of MS2 coliphage, and bacterial growth was monitored using a multi-well plate reader providing within hours the equivalent data as obtained overnight. Additional information is yielded, including phage replication rate, progeny size per cycle, and viral propagation during bacterial growth. This method offers further insights into physicochemical mechanisms of lytic coliphage infection and temporal control. It also provides a virus–host interaction acumen.     

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro–computed tomography (µCT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and µCT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, µCT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7^th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7^th and 10^th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.     

Remating by female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae): Temporal patterns and modulation by male condition

We determined the temporal pattern of female remating in the Mediterranean fruit fly, Ceratitis capitata, and how mating with sterile males affects remating. In addition, we examined the hypotheses that sterile male nutrition and age affect the subsequent receptivity of their mates. Temporally, female receptivity varied significantly throughout the experimental period. Relatively high levels of remating (14%) on the days following the first copulation were followed by a decline, with a significantly low point (4.1%) 2 weeks after mating. Subsequently, receptivity is gradually restored (18%) 3 and 4 weeks after the initial copulation. When females were first mated to sterile males, significantly higher remating percentages were recorded. The ability of sterile males to inhibit receptivity of both wild and laboratory reared females on the day of first mating was significantly improved when they were fed a nutrient rich diet. Male age at first mating also affected female receptivity: sterile males of intermediate age (11 days old) inhibited female remating significantly more than younger or older flies. Although further studies are needed to determine the relative roles of natural and sexual selection in modulating patterns of female sexual receptivity, the Sterile Insect Technique may be improved by releasing well nourished, older sterile males.     

Microcomputed tomography-based structural analysis of various bone tissue regeneration models

Microcomputed tomography (microCT) analysis is a powerful tool for the evaluation of bone tissue because it provides access to the 3D microarchitecture of the bone. It is invaluable for regenerative medicine as it provides the researcher with the opportunity to explore the skeletal system both in vivo and ex vivo. The quantitative assessment of macrostructural characteristics and microstructural features may improve our ability to estimate the quality of newly formed bone. We have developed a unique procedure for analyzing data from microCT scans to evaluate bone structure and repair. This protocol describes the procedures for microCT analysis of three main types of mouse bone regeneration models (ectopic administration of bone-forming mesenchymal stem cells, and administration of cells after both long bone defects and cranial segmental bone defects) that can be easily adapted for a variety of other models. Precise protocols are crucial because the system is extremely user sensitive and results can be easily biased if standardized methods are not applied. The suggested protocol takes 1.5-3.5 h per sample, depending on bone tissue sample size, the type of equipment used, variables of the scanning protocol and the operator's experience.