Isolation, characterization and preclinical development of human glial-restricted progenitor cells for treatment of neurological disorders.

Aim: Glial-restricted progenitor cells (GRPs), a neural cell population that gives rise to astrocytes and oligodendrocytes both in vitro and in vivo, hold great promise as a cellular therapeutic for the treatment of demyelinating and neurodegenerative diseases of the CNS. The manufacturing and characterization protocols of human-derived GRPs (hGRPs; trade name Q-Cells) for use in a clinical setting that adhere to rigorous standards for their isolation, propagation, characterization and storage are presented.

Materials & Methods: hGRPs, defined by their immunoreactivity with A2B5 antibodies, were isolated from fetal cadaver forebrain tissue of mice 17-24 weeks gestational age using Miltenyi paramagnetic bead cell separation technology.

Modulation of group III metabotropic glutamate receptors by hydrogen ions.

Protons act as neuromodulators and produce significant effects on synaptic transmission. The molecular basis of neuromodulation by extracellular protons is partially explained by their effects on certain neurotransmitter receptors and ion channels. The metabotropic glutamate receptors (mGluRs) are a family of eight receptor subtypes that are widely expressed throughout the mammalian CNS.

Domains determining ligand specificity for Ca2+ receptors.

The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the level of extracellular Ca2+. The Ca2+ receptor is a member of a family of G protein-coupled receptors that includes metabotropic glutamate receptors (mGluRs), gamma-aminobutyric acidB receptors, and putative pheromone receptors. As a family, these receptors are characterized by limited sequence homology and an unusually large putative extracellular domain (ECD).

Allosteric activation of the Ca2+ receptor expressed in Xenopus laevis oocytes by NPS 467 or NPS 568.

The Ca2+ receptor is a G protein-coupled receptor that enables parathyroid cells and certain other cells in the body to respond to changes in the concentration of extracellular Ca2+. In this study, two novel phenylalkylamine compounds, NPS 467 and NPS 568, were examined for effects on Xenopus laevis oocytes expressing the bovine or human parathyroid Ca2+ receptors. Increases in chloride current (ICl) were elicited in oocytes expressing the bovine Ca2+ receptor when the extracellular Ca2+ concentration was raised above 1.

Colicin E1 binding to membranes: time-resolved studies of spin-labeled mutants.

To investigate the mechanism of interaction of the toxin colicin E1 with membranes, three cysteine substitution mutants and the wild type of the channel-forming fragment were spin labeled at the unique thiol. Time-resolved interaction of these labeled proteins with phospholipid vesicles was investigated with stopped-flow electron paramagnetic resonance spectroscopy. The fragment interacts with neutral bilayers at low pH, indicating that the interaction is hydrophobic rather than electrostatic.

FASTRUN: a special purpose, hardwired computer for molecular simulation.

We describe the design, construction, and performance of a special purpose, hardwired accelerator for molecular mechanical calculations called FASTRUN. The processor was designed at Columbia University in 1984, constructed in the Instrumentation Division of Brookhaven National Laboratory, and delivered to Columbia in final form in 1989. It was rendered functional for molecular mechanics in early 1990.

A single tryptic fragment of colicin E1 can form an ion channel: stoichiometry confirms kinetics.

The molecularity of the ion channel formed by peptide fragments of colicin has taken on particular significance since the length of the active peptide has been shown to be less than 90 amino acids and the lumen size at least 8 A. Cell survival experiments show that killing by colicin obeys single-hit statistics, and ion leakage rates from phospholipid vesicles are first order in colicin concentration. However, interpretation in molecular terms is generally complicated by the requirement of large numbers of colicin molecules per cell or vesicle.

CARTOS II semi-automated nerve tracing: three-dimensional reconstruction from serial section micrographs.

The paper describes the history of tracing activities in the Levinthal lab from 1965 to the present. It also focuses on the evolution of electron micrograph (EM) tracing from early hand tracing reconstruction to the current, nearly fully automated CARTOS II tracing system. The present hardware systems and tracing algorithms are described in detail.

Site-directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation.

Colicin E1 is an E. coli plasmid-encoded water-soluble protein that spontaneously inserts into lipid membranes to form a voltage-gated ion channel. We have employed a novel approach in which site-directed mutagenesis is used to provide highly specific attachment points for nitroxide spin labels.

A Purkinje cell differentiation marker shows a partial DNA sequence homology to the cellular sis/PDGF2 gene.

To search for genes involved in determining the morphology of individual neuronal types, a cDNA library was constructed from postnatal day 13 mouse cerebellum. From this library, 2 clones, L7 and L19, were isolated by a differential hybridization procedure and shown by in situ hybridization to be Purkinje cell-specific within the cerebellum. Both RNAs appear between postnatal days 4 and 8 and continue into adulthood, coinciding with terminal differentiation of the Purkinje cells.

Infancy and early childhood follow-up of neonates with periventricular or intraventricular hemorrhage or isolated ventricular dilation: a case controlled study.

Survivors of periventricular or intraventricular hemorrhage and isolated ventricular dilation showed a higher incidence of major developmental problems in the motor areas than matched control subjects in infancy. This effect is still seen, but less evident in early childhood. Problems were mainly related to grades III and IV periventricular or intraventricular hemorrhage and isolated ventricular dilation.

Predicting antibody hypervariable loop conformations. II: Minimization and molecular dynamics studies of MCPC603 from many randomly generated loop conformations.

We describe a method for predicting the conformations of loops in proteins and its application to four of the complementarity determining regions [CDRs] in the crystallographically determined structure of MCPC603. The method is based on the generation of a large number of randomly generated conformations for the backbone of the loop being studied, followed by either minimization or molecular dynamics followed by minimization starting from these random structures. The details of the algorithm for the generation of the loops are presented in the first paper in this series (Shenkin et al.

A very short peptide makes a voltage-dependent ion channel: the critical length of the channel domain of colicin E1.

Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH-terminal fragments which have channel-forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel-forming fragment obtained by CNBr cleavage of the wild-type molecule consists of the C-terminal 152 amino acids. By the use of oligonucleotide-directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site.

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: translocation of regions outside the channel-forming domain.

C-terminal fragments of colicin E1, ranging in mol wt from 14.5 to 20 kD, form channels with voltage dependence and ion selectivity qualitatively similar to those of whole E1, placing an upper limit on the channel-forming domain. Under certain conditions, however, the gating kinetics and ion selectivity of channels formed by these different E1 peptides can be distinguished.

A graph theory model of the glomerular capillary network and its development.

Graph theory methods were used to analyze the topology of the renal glomerular capillary network using data both from a serial reconstruction of a rat glomerulus and from the literature. The graphs obtained were tested for planarity, and all but one were found to be nonplanar. This result indicated that the development of the glomerular capillary network must include a nonplanar growth process, and new growth models were proposed.

Inhibitory mechanism in zebrafish optic tectum: visual response properties of tectal cells altered by picrotoxin and bicuculline.

In previous work we described 4 types of visual response among tectal cells of the zebrafish. Cells of one class, type I, have no spontaneous activity, but respond phasically at ON and OFF. Their responses to moving edges, to stimuli that grow in size, and to stimuli equal in size and shape to the whole receptive field (RF) suggest that these cells may receive inhibitory input from near neighbor cells of the same type in the tectum, as well as excitatory input from retinal fibers.

Structure-function relationships for a voltage-dependent ion channel: properties of COOH-terminal fragments of colicin E1.

The effects on planar lipid bilayer membranes of carboxyl-terminal fragments derived from the bacteriocin colicin E1 by either proteolysis or CNBr cleavage are indistinguishable from those of the voltage-dependent parent colicin molecule. An upper limit to the length of the COOH-terminal peptide required for channel formation is 152 amino acid residues from the COOH-terminal end, as indicated by the CNBr fragment. In addition, use of carboxypeptidase shows that the COOH-terminal end of the molecule remains on the side of the membrane to which it was added.