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PubMed1985

Transport and proteolytic processing of rabbit cardiac cathepsin D.

Rabbit cardiac cathepsin D is synthesized as a 53,000-mol wt precursor that undergoes limited proteolysis at an unknown intracellular site to a 48,000-mol wt active form. To examine the site of proteolytic processing, isolated perfused rabbit hearts were fractionated by differential centrifugation 150 or 300 min after pulse labeling with [35S]methionine. Newly synthesized precursor and processed cathepsin D were quantitatively isolated from each fraction by extraction, immunoadsorption, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After 30-min pulse perfusions, all of the 35S-labeled cathepsin D was present as precursor, with the greatest amounts found in low-density subcellular fractions. Proteolytic processing of cathepsin D precursor occurred after chase perfusions that were coincident with the subcellular redistribution of newly synthesized enzyme from sites of synthesis to heavier subcellular structures. Pulse-chase perfusions with chloroquine (10 microM) inhibited precursor proteolytic processing and the time-dependent subcellular redistribution of newly synthesized cathepsin D. The data are consistent with a model for cardiac lysosomal enzyme maturation in which limited proteolytic processing occurs coincident with or soon after the transport of precursors to an acidic intracellular compartment. The results thus suggest that cathepsin D proteolytic processing occurs within cardiac lysosomes.

Animals↗
PubMed2002

Hedgehog-Gli signalling and the growth of the brain.

The development of the vertebrate brain involves the creation of many cell types in precise locations and at precise times, followed by the formation of functional connections. To generate its cells in the correct numbers, the brain has to produce many precursors during a limited period. How this is achieved remains unclear, although several cytokines have been implicated in the proliferation of neural precursors. Understanding this process will provide profound insights, not only into the formation of the mammalian brain during ontogeny, but also into brain evolution. Here we review the role of the Sonic hedgehog-Gli pathway in brain development. Specifically, we discuss the role of this pathway in the cerebellar and cerebral cortices, and address the implications of these findings for morphological plasticity. We also highlight future directions of research that could help to clarify the mechanisms and consequences of Sonic hedgehog signalling in the brain.

Animals↗
PubMed1995

Estrogen suppresses stromal cell-dependent lymphopoiesis in culture.

Numbers of pre-B cells change dramatically and reciprocally in response to estrogen levels in mice, suggesting that normal lymphopoiesis may be under hormonal control. However, little is known of the mechanisms involved in this process. We found that estrogen receptor mRNA was detectable by RT-PCR in lymphocyte supporting stromal cells as well as B lymphocyte precursors. Unlike glucocorticoids, estrogen did not induce apoptosis in isolated B lineage lymphocytes or interfere with their responsiveness to IL-7 in semisolid agar. Estrogen did inhibit clonal expansion of B cell precursors in a limiting dilution-type assay when the lymphocytes were cultured on a stromal cell clone. In other experiments, B cell precursors at particular stages of differentiation were isolated by cell sorting and cocultured with stromal cells for 4 days. This revealed that some subsets were more sensitive to an estrogen-containing environment than others. Although numbers of recovered cells were greatly reduced, the remaining lymphocytes had undergone relatively normal differentiation. The surviving population was enriched in cells that had acquired cytoplasmic mu chains, BP-1 Ag, and clonability with IL-7. Hormone-mediated inhibition occurred in serum and phenol-red free medium, and in cultures replete with IL-7. Direct contact between stromal cells and lymphocytes was not required. Furthermore, suppression resulted when stromal cells alone were treated with the hormone. These findings indicate that estrogen may regulate B lymphopoiesis via its influence on the microenvironment and that estrogen-induced stromal cell genes merit further study.

Animals↗
PubMed1979

Partial characterization of a tropoelastin precursor isolated from chick aorta.

Evidence is presented that indicates tropoelastin is derived from a soluble elastin with a molecular weight of 95000. Tropoelastin and its proposed precursor were isolated from the aortas of copper-deficient chicks. Although it is doubtful that the proposed precursor is an initial product of elastin translation, i.e., a proelastin, it is proposed to be at least a truncated form of proelastin that is converted to tropoelastin. The key to its isolation was the presence of alpha 1-antitrypsin at each step in the purification procedure. The first 11 amino acid residues at the NH2 terminal of the proposed tropoelastin precursor (GGVPGVAVPGGV) are the same as those for tropoelastin. Its amino acid composition is similar to that of tropoelastin, except for higher amounts of acidic amino acid residues. Further, the proposed precursor contains a limited number of aldehydic functions, presumably in the form of peptidyl allysine. This was taken as an indication that the proposed precursor serves as a substract for lysyl oxidase. Under the conditions used for the isolation, the precursor appeared to be in higher concentrations than tropoelastin in aorta extracts from copper-deficient chicks.

Amino Acid Sequence↗
PubMed1986

Cytotoxic T-cell precursors against non-immunogenic rat tumors: limiting dilution analysis.

After in vitro stimulation of lymphocytes with syngeneic tumor cells in the presence of interleukin 2 (IL-2), a cytotoxic T-cell response was observed against these spontaneously arising BDX tumors, which are non-immunogenic in the syngeneic host. The response was due to cytotoxic T-cells (CTL), which were specific for syngeneic tumor cells, i.e. neither syngeneic lymphoblasts, allogeneic lymphoblasts, allogeneic tumor cells, nor NK targets were lysed. The frequencies of anti-tumor lymph-node (LN) and spleen CTL precursors (CTLp) against a panel of syngeneic tumors of different histology varied between 1/6,000 and 1/16,000. Further analysis of the reactivity pattern of anti-tumor CTL revealed some degree of cross-reactivity within the syngeneic system. By a limiting dilution (LD) split culture approach in combination with cold target (CT) inhibition, it could be shown that tumors carry a panel of tumor-associated antigens (TAAs). Some tumors express individually specific as well as cross-reactive TAAs, while others carry cross-reactive TAAs only.

Animals↗
PubMed1983

Frequency of cytotoxic T lymphocyte precursors to herpes simplex virus type 1 as determined by limiting dilution analysis.

The conditions for establishing a limiting dilution assay to measure cytotoxic T lymphocyte precursors (CTL-P) against herpes simplex virus type 1 (HSV-1) were determined. Analysis by Poisson statistics demonstrated that the estimated frequency of HSV-1-reactive cells in the spleens of normal mice was less than 1/250,000. In contrast, mice immunized previously with infectious HSV-1 demonstrated a CTL-P frequency between 1/3,500 and 1/15,670. The generation of a maximum cytotoxic T lymphocyte response required that mice be primed in vivo with infectious virus. Immunization with inactivated virus either failed to elicit detectable CTL-P frequencies or gave frequencies markedly less than those induced with infectious virus. To obtain positive cultures, the responder cell population had to be exposed to stimulator splenocytes expressing viral antigens. Normal splenocytes without virus or normal splenocytes with T cell growth factor did not result in significant cytotoxicity. Split culture analysis comparing cytotoxicity against syngeneic and allogeneic virus-infected targets provided evidence for specificity, H-2 restriction, and the T cell nature of the CTL-P. It was determined that precursors were eliminated by treatment with anti-Thy 1, Lyt 2.1, or Lyt 1.1, indicating the CTL-P were Lyt 1(+)2(+) cells. Cytotoxicity was reduced after treatment of the responders with anti-Lyt 2 plus complement, which gave further evidence of the T cell nature of the cytotoxic T lymphocytes. These experiments demonstrated the feasibility of using the limiting dilution approach as a highly sensitive and quantitative means to measure the cell-mediated immune response to HSV-1 antigens.

Animals↗
PubMedDate not supplied

Induction of limited growth and differentiation of early thymic precursor cells by thymic epithelial cell lines.

The early thymic precursor population of adult mice (low CD4 precursor) has the potential to produce T cells, B cells and dendritic cells if transferred into the appropriate inductive environment of an irradiated recipient. To assess its developmental potential in vitro, this population was isolated and cultured, alone and with various stromal cell lines. Cultured alone, these precursor cells all died rapidly. Co-culture with 3T3 fibroblasts gave good survival but no growth. Co-culture with thymic cortical epithelial cell lines induced significant proliferation after an initial 50% cell loss. However, the supernatant of these cortical epithelial cell lines caused only limited proliferation after very extensive cell death. Examination of the surface phenotype of the cultures on the cortical epithelial layer showed some changes which were compatible with very early steps of thymocyte development, but none of the features of more developed T cells were seen. A proportion of the proliferating cells developed some of the surface markers and morphology of dendritic cells. Immature myeloid cells also grew in these cultures; these appeared to derive from a small number of myeloid progenitors, possibly contaminants within the preparation, and their outgrowth required only soluble factors released by the cortical epithelial cells.

Animals↗
PubMed2026

Genome-scale overexpression screening identifies product tolerance and efflux transport as key determinants of high-level L-tryptophan production in Escherichia coli.

L-tryptophan is a high-value aromatic amino acid widely used in the food, feed, and pharmaceutical industries. However, large-scale microbial production is constrained by insufficient precursor supply and limited strain tolerance to high product concentrations. In this study, modular metabolic engineering was first employed to enhance the availability of key precursors, including shikimate, serine, and glutamine, yielding strain TRPJ-13 with a 34.6% increase in L-tryptophan titer. To enhance strain tolerance, an indigo-based high-throughput reporter system was constructed and coupled with genome-scale overexpression library screening, leading to the identification of soxS as a tolerance-conferring target. Mechanistic analysis demonstrated that soxS upregulated lpxC to enhance lipopolysaccharide biosynthesis, thereby reinforcing membrane integrity and improving L-tryptophan tolerance. Combinatorial engineering of soxS and lpxC generated strain TRPJ-23, which increased L-tryptophan tolerance by 74.8% and L-tryptophan titer by 10.3%. Furthermore, YicL was identified as a novel transmembrane protein involved in L-tryptophan transport that effectively promoted L-tryptophan efflux, further increasing the titer by 9.0%. After fermentation optimization, strain TRPJ-28 produced 74.3 g/L L-tryptophan in a 5-L bioreactor, with a yield of 0.26 g/g and a productivity of 1.24 g/L/h. In a 1000-L pilot-scale bioreactor, TRPJ-28 reached a titer, yield, and productivity of 70.4 g/L, 0.25 g/g, and 1.17 g/L/h, respectively. This study provides new engineering insights for developing industrially promising L-tryptophan-producing strains.

Genome-scale overexpression screening↗
PubMed1989

Differential in vitro activation of CD4+CD8- and CD8+CD4- herpes simplex virus-specific human cytotoxic T cells.

In order to clarify the differential activation of CD4+ and CD8+ HSV-specific CTL, we compared the characteristics of CTL generated by different methods of in vitro HSV stimulation by treatment of effectors with anti-CD4 and anti-CD8 mAb and C after the elimination of nonspecific cytotoxic effector cells. Cell-free HSV mainly activated CD4+ CTL precursors, whereas HSV-infected fibroblasts were more effective in activating CD8+ CTL precursors than CD4+ CTL precursors. In addition, limiting dilution analyses with enriched T cells from two HSV-seropositive donors revealed that the frequency of HSV-specific CD4+ CTL precursors responsive to stimulation with free HSV was approximately 1/4,000 to 6,000 CD4+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/19,000 to 22,000 CD4+ T cells. Conversely, the frequency of CD8+ CTL precursors in peripheral blood responsive to stimulation with free HSV was approximately 1/28,000 to 30,000 CD8+ T cells, whereas that of precursors responsive to stimulation with HSV-infected fibroblasts was approximately 1/10,000 to 11,000 CD8+ T cells. The present data suggest that generalized viral infection due to cell-free viruses is fought mainly by CD4+ CTL, which have previously been reported to possess both cytotoxicity and helper function, and that localized viral infection on HLA class II-negative fibroblasts is prevented from spreading to adjacent cells mainly by CD8+ CTL. Such differential activation of CD4+ and CD8+ CTL seems probable when considering the protective mechanisms against viral infection.

Antibodies, Monoclonal↗
PubMed1998

Morphological analysis of the early stages of oligodendrocyte development in the vertebrate central nervous system.

Oligodendrocytes are responsible for myelin formation in the vertebrate central nervous system. While in vitro analysis have provided critical information on the cellular properties of oligodendrocyte precursors, they provide limited information on the morphological development of these cells in the intact CNS. Recent studies have begun to provide insights into when and where oligodendrocyte precursors arise in the neural tube. In the chick CNS, the monoclonal antibody O4 selectively labels oligodendrocyte lineage cells both in vitro and in vivo and here we discuss the characteristics of O4+ oligodendrocyte precursors during development of the chick CNS. The earliest oligodendrocytes initially develop in restricted locations in the CNS. In the spinal cord, for example, oligodendrocyte precursors arise in the ventral ventricular zone dorsal to the floor plate. These early oligodendrocyte precursors are integral components of the lining of the central canal and have an embryonic neuroepithelial cell morphology, suggesting that commitment to the oligodendrocyte lineage occurs in the ventricular zone. With maturation these early oligodendrocyte precursors lose their ventricular connection, adopt a uni- or-bipolar morphology, and migrate throughout the CNS. When these cells reach presumptive white matter, they stop migrating, become multiprocessed, and differentiate into immature oligodendrocytes. The maturation of these newly formed oligodendrocytes results in the upregulation of expression of a variety of myelin specific genes such as MBP and PLP and the subsequent elaboration of the myelin organelle. In the developing optic nerve, the onset of myelination occurs several days after oligodendrocyte precursors populate the nerve, suggesting that additional signals are required to induce myelin formation.

Animals↗
PubMed1999

Regulation of bone morphogenetic protein activity by pro domains and proprotein convertases.

Bone morphogenetic proteins (BMPs) are derived from inactive precursor proteins by endoproteolytic cleavage. Here we show that processing of Nodal and Myc-tagged BMP4 is significantly enhanced by SPC1/Furin or SPC4/PACE4, providing direct evidence that regulation of BMP signaling is likely to be controlled by subtilisin-like proprotein convertase (SPC) activities. Nodal processing is dramatically enhanced if two residues adjacent to the precursor cleavage site are substituted with amino acids found at the equivalent positions of Activin, demonstrating that structural constraints at the precursor cleavage site limit the processing efficiency. However, in transfection assays, mature Nodal is undetectable either in culture supernatants or in cell lysates, despite efficient cleavage of the precursor protein, suggesting that mature Nodal is highly unstable. Domain swap experiments support this conclusion since mature BMP4 or Dorsalin are also destabilized when expressed in conjunction with the Nodal pro domain. By contrast, mature Nodal is stabilized by the Dorsalin pro domain, which mediates the formation of stable complexes. Collectively, these data show that the half-life of mature BMPs is greatly influenced by the identity of their pro regions.

Amino Acid Sequence↗
PubMed1985

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone.

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.

Amino Acid Sequence↗
PubMed2003

Dopaminergic neuronal differentiation from rat embryonic neural precursors by Nurr1 overexpression.

In vitro expanded CNS precursors could provide a renewable source of dopamine (DA) neurons for cell therapy in Parkinson's disease. Functional DA neurons have been derived previously from early midbrain precursors. Here we demonstrate the ability of Nurr1, a nuclear orphan receptor essential for midbrain DA neuron development in vivo, to induce dopaminergic differentiation in naïve CNS precursors in vitro. Independent of gestational age or brain region of origin, Nurr1-induced precursors expressed dopaminergic markers and exhibited depolarization-evoked DA release in vitro. However, these cells were less mature and secreted lower levels of DA than those derived from mesencephalic precursors. Transplantation of Nurr1-induced DA neuron precursors resulted in limited survival and in vivo differentiation. No behavioral improvement in apomorphine-induced rotation scores was observed. These results demonstrate that Nurr1 induces dopaminergic features in naïve CNS precursors in vitro. However, additional factors will be required to achieve in vivo function and to unravel the full potential of neural precursors for cell therapy in Parkinson's disease.

Animals↗
PubMed2002

The NEDD8 pathway is essential for SCF(beta -TrCP)-mediated ubiquitination and processing of the NF-kappa B precursor p105.

The p50 subunit of NF-kappaB is generated by limited processing of the precursor p105. IkappaB kinase-mediated phosphorylation of the C-terminal domain of p105 recruits the SCF(beta-TrCP) ubiquitin ligase, resulting in rapid ubiquitination and subsequent processing/degradation of p105. NEDD8 is known to activate SCF ligases following modification of their cullin component. Here we show that NEDDylation is required for conjugation and processing of p105 by SCF(beta-TrCP) following phosphorylation of the molecule. In a crude extract, a dominant negative E2 enzyme, UBC12, inhibits both conjugation and processing of p105, and inhibition is alleviated by an excess of WT- UBC12. In a reconstituted cell-free system, ubiquitination of p105 was stimulated only in the presence of all three components of the NEDD8 pathway, E1, E2, and NEDD8. A Cul-1 mutant that cannot be NEDDylated could not stimulate ubiquitination and processing of p105. Similar findings were observed also in cells. It should be noted that NEDDylation is required only for the stimulated but not for basal processing of p105. Although the mechanisms that underlie processing of p105 are largely obscure, it is clear that NEDDylation and the coordinated activity of SCF(beta-TrCP) on both p105 and IkappaBalpha serve as an important regulatory mechanism controlling NF-kappaB activity.

Animals↗
PubMed2003

Local cooperation dominates over competition between CD4+ T cells of different antigen/MHC specificity.

Interactions between CD4(+) T cells in vivo are controlled by a balance between cooperation and competition. In this study the interaction between two populations of CD4(+) T cells of different MHC/peptide specificity was probed at different precursor frequencies, delivering one or both Ags to APC using particle-mediated DNA delivery. Expansion of clonal populations of Ag (OVA and pigeon cytochrome c-specific) CD4(+) T cells was limited at higher precursor frequencies, presumably reflecting intraclonal competition. In contrast, a strong enhancement of the number of cells expressing IFN-gamma, IL-4, and IL-2 was observed in populations of cells at low precursor frequency in the presence of a high frequency of activated cells of a different Ag specificity. The helper effect was most potent when both Ags were delivered to the same dendritic cell (i.e., linked). This reflects the requirement of epicrine or paracrine help for optimal activation of T cell clones at low frequency. A measure of help was also delivered in an endocrine manner (unlinked), especially for Th1 responses, suggesting that there is also limited diffusion of cytokines between dendritic cell clusters. The dominant effects of cooperation over competition between CD4(+) T cells responding to different Ags may have important implications in terms of the efficacy of multivalent vaccines.

Adoptive Transfer↗
PubMed2006

Limited influence of olanzapine on adult forebrain neural precursors in vitro.

We evaluated the activity of the atypical antipsychotic drug olanzapine on differentiation and gene expression in adult neural precursor cells in vitro. Neural precursors obtained from forebrain subventricular zone (SVZ)-derived neurospheres express a subset (13/24) of receptors known to bind olanzapine at high to intermediate affinities; in contrast, all 24 are expressed in the SVZ. In the presence of 10 nM, 100 nM or 1 microM olanzapine, there is no significant change in the frequency of oligodendrocytes, neurons, GABAergic neurons and astrocytes generated from neurosphere precursors. In parallel, there is no apparent change in cell proliferation in response to olanzapine, based upon bromodeoxyuridine incorporation. There are no major changes in cytological differentiation in response to the drug; however, at one concentration (10 nM) there is a small but statistically significant increase in the size of glial fibrillary acidic protein-labeled astrocytes derived from neurosphere precursors. In addition, olanzapine apparently modulates expression of one serotonin receptor -- 5HT2A -- in differentiating neurosphere cultures; however, it does not modify expression of several other receptors or schizophrenia vulnerability genes. Thus, olanzapine has a limited influence on differentiation and gene expression in adult neural precursor cells in vitro.

Animals↗
PubMed1986

Effects of antineoplastic agents on growth, morphology and metabolism of Torulopsis glabrata.

The effect of treatment by a number of antineoplastic agents on the growth, ultrastructure and macromolecular synthesis of T. glabrata was studied. Many differences were noted in the response of this yeast to these agents. Thiotepa and methotrexate inhibited the growth of T. glabrata, while it was resistant to endoxan-asta and vincristine sulphate. A variation of morphological response of T. glabrata was also observed. Methotrexate enhanced filamentation while thiotepa influenced the surface structures of the cells, resulting in loss of cytoplasmic materials and cell collapse. The other two drugs had little or no effect on the morphology of the yeast tested. The incorporation of precursors for macromolecular synthesis of T. glabrata in the presence of thiotepa and methotrexate was restricted. Thiotepa affected the uptake of precursors of RNA, DNA and protein limiting them to between 62 and 66% of the control values. In contrast, methotrexate limited the uptake of macromolecular precursors to a lesser extent. The possible mechanism of action of antineoplastic agents against yeast and the clinical implications of these findings are discussed.

Amino Acids↗
PubMedDate not supplied

Early communication development in children with Rett syndrome.

This retrospective pilot study investigated the course of pre-linguistic and linguistic communication development in children with Rett syndrome (RS) from birth to 24 months of age. Parents of 17 children with RS completed a questionnaire based on the Clinical Linguistic Auditory Milestone Scale. No child exceeded single word use and most children had begun to regress. Pre-regression delay was in evidence along with limited intentional gestural communication as a precursor to single word use. Limited use of communicative gestures is suggested as a possible early predictor of RS pending further investigation.

Child, Preschool↗