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PubMed1993

Limiting dilution analysis of the allo-MHC anti-paternal cytotoxic T cell response. I: Normal primigravid and multiparous pregnancies.

Anti-paternal cytotoxic T lymphocyte precursor frequencies (CTLpF) were determined by limiting dilution analysis (LDA) in the peripheral blood of eight primigravid and seven multiparous women during the three trimesters of pregnancy. In five of these women the responses to cord blood lymphocytes (CBL) and paternal lymphocytes were also determined at the time of delivery and at 6 weeks post delivery. As controls, CTLpF against unrelated third party donors were determined. A wide range of CTLpF against all three groups of targets was found in both the primigravid and multiparous women, reflecting the wide range of frequencies found in random populations. These frequencies remained fairly constant during and 6 weeks after the pregnancy. Splitwell analysis demonstrated that the responses generated in our culture system were specific to the stimulator. The LDA data conform to single-hit kinetics, indicating that only cytotoxic T cells were limiting in the assay. Proliferative responses of maternal lymphocytes to paternal, cord blood and third party MHC antigens also remained unchanged as determined by time-course mixed lymphocyte reactions (MLR). Our data suggest that there is no significant allo-stimulation or suppression of the maternal immune system during normal pregnancy. The mother remains immunocompetent and is capable of both cytotoxic and proliferative responses to paternally-derived fetal MHC antigens. Our findings confirm that in normal pregnancy the trophoblast, which is devoid of classical MHC antigens, forms an effective immune barrier which prevents interaction of the maternal and fetal immune systems.

Cells, Cultured↗
PubMed1977

Rapidly metabolized glycoproteins in a neuroblastoma cell line.

The metabolism of neuroblastoma cell glycoproteins was examined using L-E13H]fucose. Incubation of monolayer cultures with [3H]fucose resulted in a rapid uptake of the radioactive precursor and its incorporation into acid-insoluble macromolecules. Less than 3% of the [3H]fucose that was isolated from neuroblastoma cells by trichloroacetic acid precipitation was associated with glycolipids. The metabolism of fucosylated macromolecules was studied in cells which were labelled to a steady state, and then reincubated under conditions which limited reutilization of the radioactive precursor (40 mM unlabelled fucose). During reincubation of the cells, we observed a rapid metabolism (27% by 2 h) of the prelabelled macromolecules which stabilized within a cell generation time to give an overall rate of turnover of 9%. This rapid loss of radioactivity from the cells was not due to exocytosis since less than 4% of the [3H]-fucose was lost into the media as macromolecules during a 5 h reincubation period. The presence of 40 mM fucose in the media did not affect cell growth until after 24 h of incubation or cellular protein synthesis until after 15 h of incubation. When the metabolism of neuroblastoma cell glycoproteins was measured in the presence of 1.8 - 10(-4) M cycloheximide, there appeared to be a less rapid decrease in cell-associated specific activity, and an increased reutilization of [3H]fucose. Although the major proportion of the radioactivity remained as e13H]fucose, extensive incubation of neuroblastoma cells with this radioactive precursor led to increased amounts of tritium associated with other cellular components; However, a rapid rate of glycoprotein metabolism could also be demonstrated with cells incubated with [14C]fucose. This eliminated the possibility that the above results were restricted to the tritiated precursor and merely a reflection of hydrogen-tritium exchange.

Cell Line↗
PubMed2002

ELISA analysis of beta-secretase cleavage of the Swedish amyloid precursor protein in the secretory and endocytic pathways.

Limiting beta amyloid (Abeta) production could become an important therapeutic target in Alzheimer's disease (AD). Abeta is derived by the sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. A double missense mutation in APP found in a Swedish pedigree (APPsw) elevates Abeta40 and Abeta42 production. Abeta production and, therefore, beta-secretase cleavage of APPsw reportedly occur in the endoplasmic reticulum (ER), Golgi and endocytic compartments. However, the relative contribution of beta-secretase cleavage occurring in each compartment has not been determined. Experiments described here use a novel ELISA to measure the beta-cleaved product, APPswbeta. Using this ELISA, we provide new information regarding the relative amount of beta-secretase cleavage of APPsw that occurs in secretory and endocytic pathways. Using a dilysine retrieval motif to retain APPsw in the ER, we discovered that less than 15% of the beta-secretase cleavage was still detected. Experiments utilizing endocytosis-impaired mutants of APPsw revealed that little or no beta-secretase cleavage of APPsw appears to take place through endocytosis. Surprisingly, deletion of the entire cytoplasmic tail increased both APPswbeta and Abeta secretion, suggesting that protein interactions with this region normally impede beta-secretase cleavage. These results suggest that APPsw is cleaved by beta-secretase late in the secretory pathway.

Alzheimer Disease↗
PubMed1999

Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain.

We have explored the plastic ability of neuronal precursors to acquire different identities by manipulating their surrounding environment. Specifically, we sought to identify potential signals involved in the specification of forebrain dopaminergic neurons. Here we describe culture conditions under which tyrosine hydroxylase (TH) expression is induced in neuronal precursors, which were derived directly from the embryonic striatum and adult subependyma (SE) of the lateral ventricle or generated from multipotent forebrain stem cells. TH was successfully induced in all of these cell types by 24 hr exposure to basic fibroblast growth factor (FGF2) and glial cell conditioned media (CM). The greatest magnitude of the inductive action was on embryonic striatal precursors. Although FGF2 alone induced limited TH expression in striatal cells (1.1 +/- 0.2% of neurons), these actions were potentiated 17.5-fold (19.6 +/- 1.5% of neurons) when FGF2 was coadministered with B49 glial cell line CM. Of these TH-immunoreactive cells, approximately 15% incorporated bromodeoxyuridine (BrdU), indicating that they were newly generated, and 95% coexpressed the neurotransmitter GABA. To investigate whether precursors of the adult forebrain subependyma were competent to respond to the instructive actions of FGF2+CM, they were first labeled in vivo with a pulse of BrdU. Although none of the cells expressed TH in control, 0.2% of total cells showed TH immunoreactivity in FGF2+CM-treated cultures. Under these same conditions only, in vitro-generated precursors from epidermal growth factor-responsive stem cells exhibited TH expression in 10% of their total neuronal progeny. Regulation of neurotransmitter phenotype in forebrain neuronal precursors, by the synergistic action of FGF2 and glial-derived diffusible factors, may represent a first step in understanding how these cells are generated in the embryonic and adult brain and opens the prospect for their manipulation in vitro and in vivo for therapeutic use.

Animals↗
PubMed2004

Developmental consequences of abnormal folate transport during murine heart morphogenesis.

BACKGROUND: Folic acid is essential for the synthesis of nucleotides and methyl transfer reactions. Folic acid-binding protein one (Folbp1) is the primary mediator of folic acid transport into murine cells. Folbp1 knockout mouse embryos die in utero with multiple malformations, including severe congenital heart defects (CHDs). Although maternal folate supplementation is believed to prevent human conotruncal heart defects, its precise role during cardiac morphogenesis remains unclear. In this study, we examined the role of folic acid on the phenotypic expression of heart defects in Folbp1 mice, mindful of the importance of neural crest cells to the formation of the conotruncus. METHODS: To determine if the Folbp1 gene participates in the commitment and differentiation of the cardiomyocytes, relative levels of dead and proliferating precursor cells in the heart were examined by flow cytometry, Western blot, and immunohistostaining. RESULTS: Our studies revealed that impaired folic acid transport results in extensive apoptosis-mediated cell death, which concentrated in the interventricular septum and truncus arteriosus, thus being anatomically restricted to the two regions of congenital heart defects. Together with a reduced proliferative capacity of the cardiomyocytes, the limited size of the available precursor cell pool may contribute to the observed cardiac defects. Notably, there is a substantial reduction in Pax-3 expression in the region of the presumptive migrating cardiac neural crest, suggesting that this cell population may be the most severely affected by the massive cell death. CONCLUSIONS: Our findings demonstrate for the first time a prominent role of the Folbp1 gene in mediating susceptibility to heart defects.

Animals↗
PubMed1993

Analysis of human blood cell development in the SCID-hu mouse, an in vivo replica of the human fetal hematopoietic system.

Human fetal blood tissues implanted in the SCID immuno-deficient mouse are tolerated and develop considerably. The bone marrow maintains its autonomous hematopoietic function for several months. Conversely, thymic lymphopoiesis can continue over the long term only in the presence of an appropriate source of stem cells. This in vivo replica of the human blood system can be used to test the hematogenic abilities of candidate populations of human hematopoietic stem cells, as suggested by the hematogenic reconstitution of human thymus and marrow in SCID mice obtained from purified CD34+ precursors. In the same model, hematogenic activity of CD34+ cells was limited to the sub-population of these precursors coexpressing the antigen Thy-1. These results, combined with those of in vitro analyses, suggest that the population of CD34+ Thy-1+ components of fetal liver and of the bone marrow is extremely rich in human hematopoietic stem cells.

Animals↗
PubMed2001

Endosomal sorting of amyloid precursor protein-P-selectin chimeras influences secretase processing.

Amyloid beta protein, the major component of the senile plaques in Alzheimer's disease, is generated by secretory and endocytic processing of amyloid precursor protein. Internalized amyloid precursor protein either recycles to the plasma membrane, where alpha-secretase resides, or moves to acidic compartment(s) for beta-secretase exposure. While the trans-Golgi network contains beta-secretase activity, recent examination of the subcellular distribution of this proteinase, called BACE, has led to the suggestion that beta-secretase activity might also reside at the plasma membrane and in endosomes. To examine the role of endocytic compartments in beta-secretase processing of amyloid precursor protein, the wild-type and endosomal sorting mutant P-selectin cytoplasmic domains were used to control movement of amyloid precursor protein through endosomes. Amyloid precursor protein/P-selectin, which is sorted from early to late endosomes, undergoes significantly less alpha-secretase cleavage, and more beta-secretase cleavage, than amyloid precursor protein/P-selectin768A, a mutant that recycles more efficiently to the cell surface. Our results demonstrate that endosomal sorting influences relative exposure of the amyloid precursor protein/P-selectin chimeras to alpha- and beta-secretase activities, and suggest that, because delivery to late endocytic compartments favors beta-secretase processing of amyloid precursor protein, there is likely limited beta-secretase activity in early endosomes or at the cell surface. We propose that the trans-Golgi network may be involved in both secretory and endocytic generation of amyloid beta protein.

Alzheimer Disease↗
PubMed1987

Maturation-associated changes in the peripheral cytoplasm of human neutrophils: a review.

The process of hemopoietic stem cell differentiation and proliferation leads to a large number of precursor cells committed to the neutrophil lineage. These precursor cells undergo a further limited degree of proliferation but, most importantly, undergo a dramatic process of maturation which alters the phenotype of the cell from a myeloblast, which is incapable of normal circulation and function into a segmented neutrophil capable of chemokinesis, chemotaxis, particle ingestion, microbicidal action, and other functions required to subserve the inflammatory process. This review describes the changes in the cell surface and cell cytoplasm that occur during precursor cell maturation and, to the extent possible, correlates molecular and macromolecular changes during maturation with the development of functional capacity.

Antigens, Surface↗
PubMed1979

Repair and survival after UV in quiescent and proliferating Microtus agrestis cells: different rates of incision and different dependence on DNA precursor supply.

Cultured cells of Microtus agrestis, the common field vole, perform unscheduled DNA synthesis after UV irradiation. They respond to incubation with a DNA synthesis inhibitor (1-beta-D-arabinofuranosylcytosine) following UV in ways typical of cells capable of excision repair, with reduced survival and an accumulation of breaks in pre-existing DNA. Microtus cells irradiated with UV in a quiescent pre-S-phase state are more sensitive to UV than are proliferating cells, in terms of survival. Adding DNA precursors (deoxyribonucleosides), and--in case of proliferating cells--growing in complete rather than dialysed serum, enhance UV survival. Quiescent cells show a higher rate of endonucleolytic incision of DNA after UV than do proliferating cells. The balance between incision (producing single-strand DNA breaks) and repair DNA synthesis (leading to rejoining of breaks) is shifted by the addition of deoxyribonucleosides, which suggests that DNA precursor supply is a rate-limiting factor in repair. The lower survival of quiescent cells (in the absence of added deoxyribonucleosides) may be due to insufficient precursor supply to meet the demands of the high incision rate.

Animals↗
PubMed1984

Recent research on the behavioral effects of tryptophan and carbohydrate.

Animal and human studies indicate that diet can alter plasma and brain concentrations of neurotransmitter precursors, with possible implications for the synthesis and release of brain neurotransmitters. The best known example is serotonin, whose synthesis is limited by the availability of its precursor, tryptophan, in the brain. Consuming tryptophan or a carbohydrate-rich, protein-poor meal increases brain levels of tryptophan and serotonin. Although a carbohydrate meal itself lacks tryptophan, the meal causes insulin to be secreted. Insulin, in turn, decreases plasma levels of large neutral amino acids that would ordinarily compete with tryptophan for transport across the blood-brain barrier. Resulting brain changes in serotonin provide a plausible mechanism whereby diet could affect behaviour. Research on human subjects suggests that ingesting tryptophan or carbohydrate can reduce subjective alertness and possibly influence some aspects of objective performance. Effects on sleep latency and on pain perception have also been detected. Behavioral effects may come about via the action of tryptophan on brain serotoninergic pathways, although other mechanisms may operate and must still be ruled out before the mechanism is certain.

Adult↗
PubMed1988

Limiting-dilution analysis of human CTL differentiation. Requirement for a lymphokine-mediated differentiation signal.

The induction of human influenza virus-specific memory cytotoxic T lymphocytes (CTL) from CTL precursors (CTLp) was investigated using limiting-dilution cultures and cell lines. Differentiation of maximal numbers of CTLp in limiting-dilution cultures required at least three signals: antigen stimulation, interleukin-2 (IL-2), and a differentiation factor distinct from IL-2. Antigen-specific CTLp proliferated in response to antigen stimulation and recombinant DNA-derived IL-2, but often failed to acquire cytolytic activity unless conditioned medium (CM) from mitogen-stimulated peripheral blood mononuclear cell (PBMC) cultures was added to the cultures. Temporal analysis of the requirement for CM indicated that it was providing a late signal for CTLp differentiation. This analysis was confirmed by developing CTLp cell lines, which were found to proliferate in response to IL-2 and antigen but not to exhibit influenza virus-specific cytotoxicity until CM was added.

Cell Differentiation↗
PubMed1976

Transfer ribonucleic acid biosynthesis. Substrate specificity of ribonuclease P.

Bacteriophage T4 synthesizes proline and serine tRNA species which are derived from a common precursor RNA. The processing of this precursor RNA involves the replacement of a U-A-A terminus in serine tRNA by C-C-A prior to precursor cleavage. In the present work we have examined in detail the cleavage of T4 proline-serine precursor RNA by the previously identified ribonuclease P. Ribonuclease P accurately cleaves precursor RNA terminating in either C-C-A or U-A-A to generate the 5' termini characteristic of both mature tRNA species. These cleavages do not depend solely on the nucleotide sequence of the precursor RNA since isolated oligonucleotides spanning the cleavage sites are not substrates for the enzyme. Two types of experiments show that RNase P kinetically favors precursor RNA ending C-C-A over that ending U-A-A. Isolated preparations of precursor RNA containing the C-C-A sequence were cleaved more rapidly by RNase P than precursor RNA ending U-A-A. In addition, the serine tRNA generated by limited cleavage of a mixed population of precursor RNA ending C-C-A or U-A-A was enriched 3-fold in the C-A-A sequence relative to the starting material. Bacteriophage T4 proline-serine precursor RNA, in contrast to other tRNA precursors, accumulates in measurable amounts in wild type cells. This accumulation would appear to be a consequence of the requirement for the generation of the C-C-A sequence prior to RNase P cleavage. The enzymic specificity of RNase P in vitro therefore reflects the in vivo pathway for serine tRNA biosynthesis, where the C-C-A sequence is synthesized while the serine tRNA sequence is still a part of the large precursor RNA.

Base Sequence↗
PubMed2006

The genetics of the amyloidoses: interactions with immunity and inflammation.

Historically, the amyloidoses have been associated with inflammation and the immune response. From Virchow's original description in human pathologic inflammatory states through their identification in horses used to produce antitoxin to their frequent occurrence in the course of multiple myeloma and a somewhat abortive designation as 'gammaloid', the disorders were felt to have an inflammatory origin. These presumptive associations antedated the availability of a reliable method for tissue extraction that would allow chemical analysis of the major deposited molecules. With the identification of the multiple precursors and the realization that most were not intrinsic elements of immune/inflammatory pathways, the investigative emphasis shifted to the analysis of the biophysical events involved in aggregation and fibril formation. As more in vivo models and better tools for examination of tissues have become available, it appears as if inflammation may participate as both a response to, and an amplifier of, the effects of the fibrillar aggregates. Hence, while only a limited number of amyloid protein precursors are involved in immunity and inflammation per se, host defense, in its broadest sense, is likely to be involved in the clinically relevant amyloidoses. Further it now appears that harnessing the immune response in an appropriate fashion may be able to play a role in treatment.

Amyloid↗
PubMed1986

Compartmentation of guanine nucleotide precursors for DNA synthesis.

We have studied the kinetics of guanine incorporation into DNA in mouse T-lymphoma (S-49) mutant cells [PNPase (purine-nucleoside phosphorylase)- and HGPRTase (hypoxanthine: guanine phosphoribosyltransferase)-deficient] that are incapable of converting dGuo (deoxyguanosine) to Gua (guanine) ribonucleotides. Of the two possible pathways for an exogenous guanine source to reach DNA, firstly: dGuo----dGMP----dGDP----dGTP and secondly: Gua----GMP----GDP----dGDP----dGTP only the second pathway was found to be functional in providing guanine for DNA replication, although deoxyguanosine readily produced toxic cellular dGTP levels via the first pathway. The functional guanine-nucleotide-precursor pools for DNA are rather small; further, the depletion of the small GMP pool, but not that of GDP, GTP and dGTP, correlated well with the inhibition of DNA synthesis by mycophenolic acid, an IMP dehydrogenase inhibitor. These results support the hypothesis that guanine-nucleotide incorporation into DNA is highly compartmentalized and that a small functional guanine-nucleotide pool, e.g., the GMP pool, may serve a crucial role in limiting the availability of DNA precursor substrate.

Animals↗
PubMed1981

Quantitative studies on T cell diversity. II. Determination of the frequencies and Lyt phenotypes of two types of precursor cells for alloreactive cytotoxic T cells in polyclonally and specifically activated splenic T cells.

Two different limiting dilution systems have been applied to compare precursor frequencies of alloreactive cytotoxic T cells (CTL-P) in the polyclonally and specifically activated lymphocyte populations and in selected Lyt T cell subsets. Both systems make use of T cell growth factor for T cell expansion but differ with respect to the activation step in that lymphocytes are either activated directly with allogenetic stimulator cells or are sensitized polyclonally with concanavalin A (Con A) in bulk culture before their expansion under limiting dilution conditions. In polyclonally activated C57BL/6 lymphocyte populations, two types of CTL-P specific for H-2d alloantigens could be identified: a frequent set with a frequency of 1/100-1/300, and a rare set with a frequency of 1/2,000-1/8,000. In contrast, only a single CTL-P set was found in specifically activated populations with a frequency similar to that of the frequent CTL-P found on Con A blasts. In Con A blasts, the frequent at higher cell concentrations by suppressor T cells, whereas rare CTL-P were insensitive to this suppressive mechanism. Whereas in specifically activated T cells, the predominant CTL-P phenotype was Lyt-123, the predominant Lyt phenotypes for the frequent and the rare CTL-P found in Con A blasts were Lyt-123 and Lyt-123, respectively, which suggests that they represent primary and secondary CTL-P, respectively. The results are discussed with respect to previous reports on the involvement of Lyt T cell subsets in the generation of cytotoxic responses and their regulation by T suppressor cells.

Animals↗
PubMed1996

Systemin--a polypeptide defense signal in plants.

Insect and pathogen attacks activate plant defense genes within minutes in nearby cells, and within hours in leaves far distant from the sites of the predator attacks. A search for signal molecules involved in both the localized and distal signalling has resulted in the identification of an 18-amino-acid polypeptide, called systemin, that activates defense genes in leaves of tomato plants when supplied at levels as low as fmols/plant. Several lines of evidence support a role for systemin as a wound hormone. As with animal polypeptide hormones, systemin is derived from a larger precursor protein, called prosystemin, by limited proteolysis. Systemin has been shown by autoradiography to be phloemmobile and, by antisense technology, to be an essential component of the wound-inducible, systemic signal transduction system leading to the transcriptional activation of the defensive genes. A search for the receptor of systemin has led to the identification in plant plasma membranes of a systemin-binding protein. However, this protein has properties not of a receptor, but of a furin-like proteinase that cleaves systemin into smaller polypeptides. Systemin and its precursor prosystemin provide prototypes for the emerging possibilities that polypeptide hormones may have broad roles in signalling environmental stress responses, and in regulating plant growth and development as well.

Abscisic Acid↗
PubMed1998

Axonal outgrowth of cultured neurons is not limited by growth cone competition.

We have examined the question of scarcity-driven competition for outgrowth among growth cones of a single neuron. We measured spontaneous neurite elongation rates from 85 hours of videotape of the arbors of 31 chick sensory neurons in culture. These rate measurements were analyzed in ten minute periods that allowed cell bodies to be classified as to the number of their growth cones and the elongation to be analyzed as a series of discrete events. Comparing periods in which neurons maintained simple bipolar morphology we find no temporal competition between the two growth cones. That is, periods of above-average growth by one growth cone are not compensated by below-average growth during the same period by its sibling growth cone. Analyzing all outgrowth from a neuron based on its number of growth cones shows that net elongation rate from a single cell body is a linear function of the number of growth cones from 1 to 11. These observations suggest that growth cones behave independently and are not limited by availability of structural precursors. A surplus pool of structural precursors available for normal growth is also indicated by the high capacity for growth from single neurites when experimentally stimulated by mechanical tension. In addition, towing one or more neurites at above average rates does not cause any decline in simultaneous growth cone-mediated outgrowth from a single neuron compared to the 2-3 hour period prior to experimentally induced elongation. This high capacity for growth combined with the often observed, intermittant growth behavior of individual growth cones suggests that neurite outgrowth is intrinsically limited primarily by poor growth cone 'performance,' not scarcity-driven competition. We postulate that growth cones are poor 'tractors,' exerting too little tension to exploit the available capacity for axonal elongation.

Animals↗
PubMed2006

Origin and evolution of the vertebrate vomeronasal system viewed through system-specific genes.

Tetrapods have two distinct nasal chemosensory systems, the main olfactory system and the vomeronasal system (VNS). Defined by certain morphological components, the main olfactory system is present in all groups of vertebrates, while the VNS is found only in tetrapods. Previous attempts to identify a VNS precursor in teleost fish were limited by functional and morphological characters that could not clearly distinguish between homologous and analogous systems. In the past decade, several genes that specifically function in the VNS have been discovered. Here we first describe recent evolutionary studies of mammalian VNS-specific genes. We then review evidence showing the presence and tissue-specific expression of the VNS-specific genes in teleosts, as well as co-expression patterns of these genes in specific regions of the teleost olfactory epithelium. We propose that a VNS precursor exists in teleosts and that its evolutionary origin predated the separation between teleosts and tetrapods.

Animals↗