Visuomotor control within a distributed parieto-frontal network

The aim of this functional magnetic resonance imaging study was to investigate differences in visuomotor control with increasing task complexity. Twelve right-handed volunteers were asked to perform their signature under different degrees of visual control: internally generated movement with closed eyes, signing with open eyes, tracking the line of the projected signature forwards, and tracking the line of the projected signature backwards. There was a gradual onset and disappearance of activation within a distributed network. Parietal, lateral and medial frontal brain areas were activated during all conditions, confirming the involvement of a parieto-frontal system. The weight of activation shifted with increasing task complexity. Internally generated movements activated predominantly the inferior parietal lobule and the ventral premotor cortex, as well as the rostral cingulate area, pre-supplementary motor area (pre-SMA) and SMA proper. Opening the eyes reduced SMA and cingulate activation and activated increasingly the occipito-parietal areas with higher task complexity. Visually guided movements produced an activation predominantly in the superior parietal lobule and dorsal premotor cortex. This study bridges human activation studies with the results of neurophysiological studies with monkeys. It confirms a gradual transition of visuomotor control with increasing task complexity within a distributed parieto-frontal network.     

Septotemporal distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in the rat hippocampus

The distribution of glutamate receptors in transverse hippocampal sections has been well investigated. However, in spite of the known septotemporal gradients of hippocampal connectivity no systematic studies exist about the distribution of glutamate receptors along the septotemporal (longitudinal) hippocampal axis. Therefore, in the present study this issue was investigated using receptor autoradiography for the [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites. Hippocampi from 30-day-old rats were sectioned perpendicularly to their longitudinal axis, yielding a total of 25–30 equidistantly spaced autoradiographs for each hippocampus. For each section layer-specific concentrations of binding sites were calculated by the aid of a computerized image analysing system. The dependency of concentrations of binding sites on the septotemporal position was evaluated by regression analysis. Gradients of binding were confined to distinct hippocampal layers. Significant septotemporal gradients of [³H]MK-801 binding were observed in selected layers of CA1 and the dentate gyrus, a septal to temporal decrease of binding in the oriens and radiatum layers of CA1 being most prominent. For [³H]AMPA, significant septotemporal gradients of binding were restricted to layers of CA3, CA4 and the dentate gyrus, with values generally increasing from septal to temporal levels. The observed septotemporal gradients possibly reflect functional segregations along the longitudinal hippocampal axis and could be important for the comparability of ligand binding studies using transverse hippocampal sections or hippocampal slice cultures.     

Mild phenotype in two unrelated patients with a partial deletion of 21q22.2-q22.3 defined by FISH and molecular studies

We describe two unrelated patients with cytogenetically visible deletions of 21q22.2-q22.3 and mild phenotypes. Both patients presented minor dysmorphic features including thin marfanoid build, facial asymmetry, downward-slanting palpebral fissures, depressed nasal bridge, small nose with bulbous tip, and mild mental retardation (MR). FISH and molecular studies indicated common deleted areas but different breakpoints. In patient 1, the breakpoint was fine mapped to a 5.2 kb interval between exon 5 and exon 8 of the ETS2 gene. The subtelomeric FISH probe was absent on one homologue 21 indicating a terminal deletion spanning approximately 7.9 Mb in size. In patient 2, the proximal breakpoint was determined to be 300-700 kb distal to ETS2, and the distal breakpoint 2.5-0.3 Mb from the 21q telomere, indicating an interstitial deletion sized approximately 4.7-7.3 Mb. The 21q- syndrome is rare and typically associated with a severe phenotype, but different outcomes depending on the size and location of the deleted area have been reported. Our data show that monosomy 21q of the area distal to the ETS2 gene, representing the terminal 7.9 Mb of 21q, may result in mild phenotypes comprising facial anomalies, thin marfanoid build, and mild MR, with or without signs of holoprosencephaly.     

Serosal mesothelium retains vasculogenic potential.

Mesothelia comprise the epithelial covering of coelomic organs and line the cavities in which they are housed. Mesothelia contribute to the vasculature of the heart and the intestinal tract by developmental processes of epithelial-mesenchymal transition (EMT), migration, and differentiation into endothelial cells, vascular smooth muscle cells, and pericytes. Here, we establish a novel in vitro system to analyze the differentiative potential of mesothelia. Using explants from serosal mesothelium (the mesothelial covering of the gut), we demonstrate that much of the developmental program observed in embryonic mesothelia is retained in the adult structure. Namely, processes of epithelial spreading, EMT, and differentiation into smooth muscle cells from these cells are observed. Interestingly, we were unable to stimulate endothelial cell differentiation using serum or various signaling factors. Taken together, these data reveal that differentiated serosal cells retain vasculogenic potential and provide a generalizable model for future studies on the developmental and differentiative capacity of the mesothelial cell type.     

Four new adenosine deaminase mutations,altering a zinc-binding histidine,two conserved alanines,and a 5′ splice site

Three new missense mutations (H15D, A83D, and A179D) and a new splicing defect (573 + 1G→A) in the 5′ splice site of intron 5 were among six mutant adenosine deaminase (ADA) alleles found in three unrelated patients with severe combined immunodeficiency disease, the most common phenotype associated with ADA deficiency. When expressed in vitro, the H15D, A83D, and A179D proteins lacked detectable ADA activity. The splicing defect caused skipping of exon 5, resulting in premature termination of translation and a reduced level of mRNA. H15D is the first naturally occurring mutation of a residue that coordinates directly with the enzyme-associated zinc ion. Molecular modeling based on the atomic coordinates of murine ADA suggests that the D15 mutation would create a cavity or gap between the zinc ion and the side chain carboxylate of D15. This could alter the ability of zinc to activate a water molecule postulated to play a role in the catalytic mechanism. A83 and A179 are not directly involved in the active site, but are conserved residues located respectively in a helix 4 and β strand 4 of the α/β barrel. Replacement of these small hydrophobic Ala residues with the charged, more bulky Asp side chain may distort ADA structure and affect enzyme stability or folding.© 1995 wiley-Liss, Inc.     

C4d]FlowPRA screening--a specific assay for selective detection of complement-activating anti-HLA alloantibodies

On waiting lists, transplant candidates are routinely screened for potentially harmful complement-fixing alloantibodies using complement-dependent cytotoxicity (CDC) panel-reactive antibody (PRA) testing. We have recently developed a novel cell-independent assay for assessment of complement-activating panel reactivity ([C4d]FlowPRA), which is based on selective flow-cytometric detection of alloantibody-triggered C4 complement split product deposition to human leukocyte antigen (HLA)-coated FlowPRA beads. Serum specimens selected from 120 transplant candidates were evaluated by [C4d]FlowPRA (HLA class I vs II) in comparison with FlowPRA IgG alloantibody screening (HLA class I vs II), a method detecting both complement- and noncomplement-activating alloantibodies, and with CDC-PRA on separated T (T-CDC) or B cells (B-CDC, evaluation on platelet-absorbed sera). For each assay, >/=10% PRA reactivity was considered positive. Comparing complement-dependent PRA assays with standard FlowPRA, the specificity calculated for [C4d]FlowPRA (HLA class I: 92%; class II: 100%) was found to be superior to that of CDC testing (T-CDC-PRA: 79%; B-CDC-PRA: 86%). Because noncomplement-activating alloreactivities were not detected for both techniques, low sensitivities were calculated ([C4d]FlowPRA HLA class I: 61%; class II: 31%; T-CDC-PRA: 70%; B-CDC-PRA: 55%). Compared with CDC-PRA, [C4d]FlowPRA gave a high specificity (HLA class I compared with T-CDC: 89%, HLA class II compared with B-CDC: 95%) but, at least in part because of false-positive CDC results, a modest sensitivity (66% and 38%, respectively). For both HLA classes, we found a highly significant association between absolute [C4d]FlowPRA and CDC-PRA levels (p < 0.0001). Our results suggest that detection of C4 split product deposition to FlowPRA beads may represent an attractive HLA-specific and time-effective alternative to CDC-PRA screening.     

Double labeling of neurons by retrograde axonal tracing and non-radioactive in situ hybridization in the CNS of adult zebrafish

A number of genes affecting axonal projections are currently being identified in zebrafish mutant screens. Analyzing the expression of these genes in the adult brain in relation to specific neuronal populations could yield insights into new functional contexts, such as the successful axonal regeneration in adult zebrafish. Here, we provide a relatively simple procedure for non-radioactive in situ hybridization in sections of adult zebrafish brains in combination with retrograde axonal tracing using the fluorescent neuronal tracer rhodamine dextran amine (RDA). A lesion is inflicted on the spinal cord of adult zebrafish and a crystal of RDA is then applied to the lesion site resulting in retrograde labeling of neurons in the brain through their spinal axons. Six to eighteen days later fish are perfusion-fixed, and in situ hybridization is carried out on vibratome-cut floating sections using a protocol simplified from that used for whole-mounted zebrafish embryos. This procedure leads to robust double labeling of axotomized neurons with RDA and an in situ hybridization signal for the growth-associated protein 43 (GAP-43). This method can be used to identify gene expression in specific populations of projection neurons and to detect changes in gene expression in axotomized neurons in the CNS of adult zebrafish.     

Opioid dependence prevents the action of pertussis toxin in the guinea-pig myenteric plexus

Summary The longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum has been employed for the study of the effect of pertussis toxin (IAP) on opioid dependence. Guinea-pigs were treated with IAP (120 g/kg, i.p.) either prior to chronic administration of an opioid or after opioid dependence had been established. The isolated preparations were tested in vitro for dependence; that is, the naloxone-precipitated withdrawal contracture. Naloxone almost failed to evoke a sign of dependence in preparations treated with IAP prior to chronic exposure to an opioid. In contrast, IAP failed to affect the withdrawal contracture when applied to an animal after dependence has been established. It is concluded that theN _i-unit, the substrate for IAP, plays a critical function in the development of dependence. The continuous activation of the opioid receptor associated with the development of dependence may induce changes inN _i which in turn prevent the interaction of IAP with its substrate.     

Compressive Strength of Iliac Bone ECM Is Not Reduced in Osteogenesis Imperfecta and Increases With Mineralization

Osteogenesis imperfecta (OI) is an inheritable, genetic, and collagen-related disorder leading to an increase in bone fragility, but the origin of its “brittle behavior” is unclear. Because of its complex hierarchical structure, bone behaves differently at various length scales. This study aims to compare mechanical properties of human OI bone with healthy control bone at the extracellular matrix (ECM) level and to quantify the influence of the degree of mineralization. Degree of mineralization and mechanical properties were analyzed under dry conditions in 12 fixed and embedded transiliac crest biopsies (control n = 6, OI type I n = 3, OI type IV n = 2, and OI type III n = 1). Mean degree of mineralization was measured by microcomputed tomography at the biopsy level and the mineral-to-matrix ratio was assessed by Raman spectroscopy at the ECM level. Both methods revealed that the degree of mineralization is higher for OI bone compared with healthy control. Micropillar compression is a novel technique for quantifying post-yield properties of bone at the ECM level. Micropillars (d = 5 μm, h = 10 μm) were fabricated using focused ion beam milling and quasi-statically compressed to capture key post-yield properties such as ultimate strength. The qualitative inspection of the stress–strain curves showed that both OI and healthy control bone have a ductile response at the ECM level. The quantitative results showed that compressive strength is not reduced in OI bone and is increasing with OI severity. Nanoindentation measurements revealed that OI bone tends to have a higher Young's modulus, hardness, and dissipated energy compared with healthy bone. Micropillar strength and indentation modulus increased linearly and significantly (p < .0001) with mineral-to-matrix ratio. In conclusion, this study indicates that compressive mechanical properties of dry OI bone at the iliac crest are not inferior to healthy control at the ECM level and increase with mineralization. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).