PTPBR7 Binding Proteins in Myelinating Neurons of the Mouse Brain

Mouse protein tyrosine phosphatase PTPBR7 is a receptor-like, transmembrane protein that is localized on the surface of neuronal cells. Its protein phosphatase activity is reduced upon multimerization, and PTPBR7-deficient mice display motor coordination defects. Extracellular molecules that may influence PTPBR7 activity, however, remain to be determined. We here show that the PTPBR7 extracellular domain binds to highly myelinated regions in mouse brain, in particular the white matter tracks in cerebellum. PTPBR7 deficiency does not alter this binding pattern, as witnessed by RAP in situ staining of Ptprr-/- mouse brain sections. Additional in situ and in vitro experiments also suggest that sugar moieties of heparan sulphate and chondroitin sulphate glycosaminoglycans are not critical for PTPBR7 binding. Candidate binding proteins were affinity-purified exploiting the PTPBR7 extracellular domain and identified by mass spectrometric means. Results support the suggested link between PTPRR isoforms and cerebellar calcium ion homeostasis, and suggest an additional role in the process of cell-cell adhesion

Targeting CD44v6 for fluorescence-guided surgery in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is an often highly invasive tumor, infiltrating functionally important tissue areas. Achieving complete tumor resection and preserving functionally relevant tissue structures depends on precise identification of tumor-free resection margins during surgery. Fluorescence-guided surgery (FGS), by intraoperative detection of tumor cells using a fluorescent tracer, may guide surgical excision and identify tumor-positive resection margins. Using a literature survey on potential surface molecules followed by immunohistochemical validation, we identified CD44 variant 6 (CD44v6) as a constitutively expressed antigen in the invasion zone of HNSCC lesions. The monoclonal anti-CD44v6 antibody BIWA was labeled with both a near-infrared fluorescent dye (IRDye800CW) and a radioactive label (Indium-111) and dual-modality imaging was applied in a locally invasive tumor mouse model. BIWA accurately detected human HNSCC xenografts in mice with a tumor uptake of 54 ± 11% ID/g and invasion regions with an accuracy of 94%. When dissected under clinical-like conditions, tumor remnants approximately 0.7 mm in diameter consisting of a few thousand cells were identified by fluorescence imaging, resulting in reliable dissection of invasive microregions. These data indicate that CD44v6 is a suitable target for reliable near-infrared detection and FGS of invasive HNSCC lesions in vivo.</p

PTPBR7 Binding Proteins in Myelinating Neurons of the Mouse Brain

Abstract Mouse protein tyrosine phosphatase PTPBR7 is a receptor-like, transmembrane protein that is localized on the surface of neuronal cells. Its protein phosphatase activity is reduced upon multimerization, and PTPBR7-deficient mice display motor coordination defects. Extracellular molecules that may influence PTPBR7 activity, however, remain to be determined. We here show that the PTPBR7 extracellular domain binds to highly myelinated regions in mouse brain, in particular the white matter tracks in cerebellum. PTPBR7 deficiency does not alter this binding pattern, as witnessed by RAP in situ staining of Ptprr -/-mouse brain sections. Additional in situ and in vitro experiments also suggest that sugar moieties of heparan sulphate and chondroitin sulphate glycosaminoglycans are not critical for PTPBR7 binding. Candidate binding proteins were affinity-purified exploiting the PTPBR7 extracellular domain and identified by mass spectrometric means. Results support the suggested link between PTPRR isoforms and cerebellar calcium ion homeostasis, and suggest an additional role in the process of cell-cell adhesion

Identification of a Rat Model for Usher Syndrome Type 1B by N-Ethyl-N-nitrosourea Mutagenesis-Driven Forward Genetics

The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat

Plasmodium berghei Deltap52&p36 Parasites Develop Independent of a Parasitophorous Vacuole Membrane in Huh-7 Liver Cells

Contains fulltext : 109076.pdf (publisher's version ) (Open Access)The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Deltap52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Deltap52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Deltap52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Deltap52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Deltap52&p36 parasites were able to fully mature and produced infectious merozoites. These Deltap52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Deltap52&p36 liver stages were infectious for C57BL/6 mice

Intranuclear development of <i>Δp52&p36 P. berghei</i> parasites.

<p>A) Pie diagrams of intranuclear and cytosolic wildtype and mutant replicating parasites at 24, 36 and 48 hours post invasion in Huh-7 cells. The diameter of the circles represents the relative number of replicating parasites observed per coverslip, where the wildtype circle at 24 hour represent 100% and all other circles are deduced (wildtype  = 1300–1500 and Δp52&p36 = 20–40 replicating parasites per coverslip at 24 hours post infection. Absolute numbers are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050772#pone.0050772.s003" target="_blank">Table S1</a> B) UIS-4 and HSP70 expression on an intranuclear <i>P. berghei</i> parasite 44 hours post infection (Bar  = 10 µm). C) MSP-1 expression on intranuclear (<i>Δp52&p36)</i> and cytocolic (wildtype) <i>P. berghei</i> parasites 52 hours post infection (Bar  = 10 µm).</p

<i>Δp52&p36</i> merozoites are capable of inducing a blood-stage infection.

<p>Huh-7 cells were infected with <i>Δp52&p36</i> and WT parasites and cultured for 65 hours. After 65 hours, culture supernatant containing merozoites was collected and injected i.v in C57BL/6 mice. Regular Giemsa staining was performed in all groups, 2–14 days post i.v injection in mice, to control for asexual parasites.</p

Cell Membrane Integrity in Myotonic Dystrophy Type 1: Implications for Therapy

<div><p>Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a <i>DMPK</i> gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.</p></div