Regeneration in the spinal cord

Important advances have been made in our understanding of conditions that influence the intrinsic capacity of mature CNS neurons to initiate and maintain a regrowth response. The combination of exogenous neurotrophic support with strategies to alter the terrain at the injury site itself suggests that there are important interactions between them that lead to increased axonal regeneration. The ability of chronically injured neurons to initiate a regeneration response is unexpected. Our view of the role that inhibitors play in restricting axonal growth has also expanded. The findings indicate that the windows of opportunity for enhancing growth after spinal cord injury may be more numerous than previously thought.     

The glutathione-deficient, cadmium-sensitive mutant, cad2–1 , of Arabidopsis thaliana is deficient in γ-glutamylcysteine synthetase

This paper reports that the glutathione (GSH)-deficient mutant, cad2–1 , of Arabidopsis is deficient in the first enzyme in the pathway of GSH biosynthesis, γ-glutamylcysteine synthetase (GCS). The mutant accumulates a substrate of GCS, cysteine, and is deficient in the product, γ-glutamylcysteine. In vitro enzyme assays showed that the cad2–1 mutant has 40% of wild-type levels of GCS activity but is unchanged in the activity of the second enzyme in the pathway, GSH synthetase. The CAD2 locus maps to chromosome 4 and is tightly linked to a gene, GSHA , identified by a previously isolated cDNA. A genomic clone of GSHA complements both the phenotypic and biochemical deficiencies of the cad2–1 mutant. The nucleotide sequence of the gene has been determined and, in the mutant, this gene contains a 6 bp deletion within an exon. These data demonstrate that the CAD2 gene encodes GCS. The cad2–1 mutation is close to the conserved cysteine which is believed to bind the substrate glutamate and the specific inhibitor L-buthionine-[S,R] sulfoximine (BSO). Both root growth and GCS activity of the cad2–1 mutant was less sensitive than the wild-type to inhibition by BSO, indicating that the mutation may alter the affinity of the inhibitor binding site.     

Regulation of Adherence and Virulence by the Entamoeba histolytica Lectin Cytoplasmic Domain, Which Contains a β2 Integrin Motif

Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the β2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin’s role in virulence.     

Isolation and characterization of cytoskeletons from cotton fiber cytoplasts

Summary Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to technical advances in the isolation and purification of anucleate cytoskeletons from animal cells. As a step towards characterizing protein components of the plant cytoskeleton, we have isolated cytoskeletons from cytoplasts (anucleate protoplasts) prepared from cotton fiber cells grown in ovule culture. Cytoplasts isolated into a hypertonic, Ca²⁺-free medium at pH 6.8 retained internal structures after extraction with the detergent, Triton X-100. These structures were shown to include microtubule and microfilament arrays by immunofluorescence and electron microscopy. Actin and tubulin were the only abundant proteins in these preparations, suggesting that microfilaments and microtubules were the major cytoskeleta elements in the isolated cytoskeletons. The absence of additional, relatively abundant proteins suggests that (a) other cytoskeletal arrays potentially present in fiber cells (e.g., intermediate filaments) were either lost during detergent extraction or were minor components of the fiber cell cytoskeleton; and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubulin were not required to maintain microtubules and microfilaments in organized structures.     

Cryopreservation and long-term storage of pear germplasm

Summary Germplasm collections of vegetatively propagated crops are usually maintained as plants in fields or potted in greenhouses or screened enclosures. Safety duplication of these collections, as duplicate plants or separate collections, is costly and requires large amounts of space. Cryopreservation techniques which were recently developed for long-term storage of pear germalasm may offer an efficient alternative to conventional germplasm collection maintenance. Pear (Pyrus L.) germplasm may now be stored as seeds (species), dormant buds or pollen from field-grown trees, or shoot tips fromin vitro-grown plants (cultivars). Pear germplasm may now be cryopreserved and stored for long periods (> 100 yr) utilizing slow-freezing or vitrification ofin vitro-grown shoot-tips. Dormant bud freezing, pollen, and seed cryopreservation of other lines are being developed to complete the base collection forPyrus. This cryopreserved collection provides base (long-term) storage for the field-grown pear germplasm collection at the National Clonal Germplasm Repository, Corvallis, Oregon. Based on a presentation at the 1997 Congress on In Vitro Biology held in Washington, D.C., June 14–18, 1997.     

Lumenal and Transmembrane Domains Play a Role in Sorting Type I Membrane Proteins on Endocytic Pathways

Previous studies have shown that when the cytosolic domains of the type I membrane proteins TGN38 and lysosomal glycoprotein 120 (lgp120) are added to a variety of reporter molecules, the resultant chimeric molecules are localized to the trans-Golgi network (TGN) and to lysosomes, respectively. In the present study we expressed chimeric constructs of rat TGN38 and rat lgp120 in HeLa cells. We found that targeting information in the cytosolic domain of TGN38 could be overridden by the presence of the lumenal and transmembrane domains of lgp120. In contrast, the presence of the transmembrane and cytosolic domains of TGN38 was sufficient to deliver the lumenal domain of lgp120 to the trans-Golgi network. On the basis of steady-state localization of the various chimeras and antibody uptake experiments, we propose that there is a hierarchy of targeting information in each molecule contributing to sorting within the endocytic pathway. The lumenal and cytosolic domains of lgp120 contribute to sorting and delivery to lysosomes, whereas the transmembrane and cytosolic domains of TGN38 contribute to sorting and delivery to the trans-Golgi network.     

The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G0₀ arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.