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PubMed1991

HLA-B8,DR3 T cell impairment is completely restored by in vitro treatment with interleukin-2.

The activity of recombinant interleukin-2 (rIL-2) on the in vitro lymphocyte proliferative response to phytohemagglutinin mitogen was investigated in healthy HLA-B8,DR3 positive and negative subjects. The response to mitogen, significantly decreased in HLA-B8,DR3 positive subjects, was completely restored by adding rIL-2. Moreover, in HLA-B8,DR3 positive subjects the in vitro treatment with rIL-2 significantly increased the reduced frequency of mitogen responsive T lymphocyte precursors, as assessed by limiting dilution analysis. These data suggest that a decrease in the size of the pool of T cell precursors able to produce IL-2 is responsible for the impairment of T cell function observed in HLA-B8,DR3 positive subjects. Since in autoimmune diseases it is possible to show the same impairment(s) of T cell functions which can be observed in HLA-B8,DR3 positive subjects, these results could be of practical value for the understanding of pathogenetic mechanism(s) of autoimmune diseases and, in case, for therapeutical purposes.

Adult↗
PubMed1998

Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats.

In vitro expansion of central nervous system (CNS) precursors might overcome the limited availability of dopaminergic neurons in transplantation for Parkinson's disease, but generating dopaminergic neurons from in vitro dividing precursors has proven difficult. Here a three-dimensional cell differentiation system was used to convert precursor cells derived from E12 rat ventral mesencephalon into dopaminergic neurons. We demonstrate that CNS precursor cell populations expanded in vitro can efficiently differentiate into dopaminergic neurons, survive intrastriatal transplantation and induce functional recovery in hemiparkinsonian rats. The numerical expansion of primary CNS precursor cells is a new approach that could improve both the ethical and the technical outlook for the use of human fetal tissue in clinical transplantation.

Animals↗
PubMed2000

Analysis of the biologic properties of p230 Bcr-Abl reveals unique and overlapping properties with the oncogenic p185 and p210 Bcr-Abl tyrosine kinases.

The reciprocal translocation between chromosomes 9 and 22 that fuses coding sequences of the Bcr and Abl genes is responsible for a remarkably diverse group of hematologic malignancies. A newly described 230-kd form of Bcr-Abl has been associated with an indolent myeloproliferative syndrome referred to as chronic neutrophilic leukemia. We have cloned the corresponding gene and examined the biologic and biochemical properties of p230 Bcr-Abl after retroviral-mediated gene transfer into hematopoietic cell lines and primary bone marrow cells. p230 Bcr-Abl-expressing 32D myeloid cells were fully growth factor-independent and activated similar signal transduction pathways as the well-characterized p210 and p185 forms of Bcr-Abl. In contrast, primary mouse bone marrow cells expressing p230 required exogenous hematopoietic growth factors for optimal growth, whereas p185- and p210-expressing cells were independent of growth factors. The 3 Bcr-Abl proteins exerted different effects on differentiation of bone marrow cells. p185 induced outgrowth of lymphoid precursors capable of tumor formation in immunodeficient mice. In contrast, p210- and p230-expressing bone marrow cells caused limited outgrowth of lymphoid precursors that failed to form tumors in immunodeficient mice. Removal of cytokines and autologous stroma from Bcr-Abl-expressing bone marrow cultures produced the expansion of distinct lineages by the various Bcr-Abl proteins. p185 drove expansion of cytokine-independent lymphoid progenitors, while p210 and p230 generated cytokine-independent monocyte/myeloid cells. These findings suggest that the different Bcr-Abl fusion proteins drive the expansion of different hematopoietic populations, which may explain the association of the various Bcr-Abl oncoproteins with different spectra of human leukemias. (Blood. 2000;95:2913-2921)

Animals↗
PubMed1980

Limiting dilution analysis of alloantigen-reactive T lymphocytes. IV. High frequency of cytolytic T lymphocyte precursor cells in MLC blasts separated by velocity sedimentation.

A sensitive limiting dilution microculture system was used to obtain minimal estimates of the frequency of cytolytic T lymphocyte precursor cells (CTL-P) directed against DBA/2 alloantigens, after priming of spleen cells in unidirectional mixed leukocyte cultures (MLC, C57BL/6 anti-DBA/2). The mean CTL-P frequency in day 4 to 5 MLC populations was found to be approximately 50- to 100-fold greater than the frequency in normal spleen, and up to 25% of the cells present in such MLC could be identified operationally as CTL-P. Even higher frequencies (up to 50%) of CTL-P were obtained in a population of large-sized cells separated from day 4 MLC by velocity sedimentation. Furthermore, since a strikingly quantitative correlation was observed between CTL activity and CTL-P frequency in such separated MLC populations, it is likely that mature CTL in MLC are not end cells, but can further proliferate and thus behave operationally as CTL-P.

Animals↗
PubMed1992

Secretion of human blood coagulation factor XIIIa by the yeast Yarrowia lipolytica.

The industrial yeast, Yarrowia lipolytica, secretes high yields of an alkaline extracellular protease (AEP), which is synthesized as a preproprotein encoded by the XPR2 gene. We investigated the possibility of using this system for the secretion of human coagulation factor XIII subunit a (FXIIIa). This protein is naturally secreted in the plasma by an unknown, signal peptide-independent mechanism and has so far been found to be nonsecretable in yeast. We have designed six hybrid genes encoding fusion proteins between increasing portions of the AEP preprodomain and the precursor or mature forms of FXIIIa. All constructs directed translocation of the FXIIIa precursor into the endoplasmic reticulum. Transport of the translocated and core-glycosylated hybrid precursor to the Golgi apparatus appeared to be strongly rate limiting, and most of the precursors appeared to be partially proteolysed. One of these constructs directed the extracellular secretion of a low amount of hyperglycosylated FXIIIa. These results indicate that fusion to the yeast AEP signal peptide and dipeptide stretch allows FXIIIa to be translocated, albeit inefficiently, through the endoplasmic reticulum and to follow a classical secretory transit.

Amino Acid Sequence↗
PubMed2005

Regulation of CXCR3 and CXCR4 expression during terminal differentiation of memory B cells into plasma cells.

C-X-C motif chemokine receptor 3 (CXCR3) and CXCR4 expressed on immunoglobulin G (IgG)-plasma-cell precursors formed in memory immune responses are crucial modulators of the homing of these cells. Here, we studied the regulation of the expression of these chemokine receptors during the differentiation of human memory B cells into plasma cells. We show that CXCR3 is absent on CD27- naive B cells but is expressed on a fraction of memory B cells, preferentially on those coexpressing IgG1. On differentiation into plasma-cell precursors, CXCR3+ memory B cells maintain the expression of this chemokine receptor. CXCR3- memory B cells up-regulate CXCR3 and migrate toward concentration gradients of its ligands only when costimulated with interferon gamma (IFN-gamma), but not interleukin 4 (IL-4), IL-1beta, IL-6, IFN-alpha, IFN-beta, or tumor necrosis factor alpha (TNF-alpha). In contrast, the differentiation of CXCR4- B cells into plasma cells is generally accompanied by the induction of CXCR4 expression. These results show that lack of CXCR4 expression on plasma-cell precursors is not a limiting factor for plasma-cell homing and that the expression of CXCR3 on memory B cells and plasma-cell precursors is induced by IFN-gamma, provided in human T helper type 1 (Th1)-biased immune responses. Once induced in memory B cells, CXCR3 expression remains part of the individual cellular memory.

B-Lymphocytes↗
PubMed1985

Processing of precursor ribosomal RNA and the presence of a modified ribosome assembly scheme in Escherichia coli relaxed strain.

An electrophoretic system capable of separating 25 S, 23 S, 17.5 S and 16 S ribosomal RNA (rRNA) species was used to study the synthesis and fate of rRNA during amino acid starvation and resupplementation of E. coli relaxed strain KL99. This E. coli relAl strain responded to an amino acid starvation by increasing the rate of synthesis of 25 S and 17.5 S precursor rRNA. When the limiting amino acid was resupplemented, a previously observed 40-fold increase in the cellular guanosine 5'-diphosphate, 3'-diphosphate content [Mol. Gen. Genet. (1983) 192, 5-9] appeared to cause a reduction in new rRNA synthesis. Following amino acid resupplementation, the precursor 25 S and 17.5 S rRNA accumulated during the amino acid starvation were conserved and processed to 23 S and 16 S rRNA species, respectively. This suggests that a modified ribosome assembly scheme involving stable precursor rRNA exists in relAl bacteria during periods of amino acid limitation and resupplementation.

Amino Acids↗
PubMed1986

Distribution of terminal transferases of acylglycerol synthesis in cell fractions from lactating mammary gland.

Cytoplasmic lipid droplets and microlipid droplets, intracellular precursors of milk lipid globules, had little ability to incorporate radioactivity from glycerol 3-phosphate or palmitoyl-CoA into triacylglycerols. The limited incorporation of these precursors by micro- and cytoplasmic lipid droplets from rat and cow mammary gland was into phospholipids primarily. Acyltransferases catalyzing incorporation of glycerol 3-phosphate into acylglycerols were concentrated in a relatively high buoyant density class of rough microsomes. Palmitoyl-CoA-sn-1,2-diacylglycerol acyltransferase activity was distributed heterogeneously among fractions obtained by equilibrium density gradient fractionation of mammary homogenates. Observations suggest that terminal steps of acylglycerol synthesis are localized primarily in rough endoplasmic reticulum of milk secreting mammary epithelial cells. There appears to be a heterogeneous distribution of acyltransferases along the reticular network.

Acyltransferases↗
PubMed1985

New concepts in management of neutropenia.

Neutropenia is a life-threatening sequel of hematological disorders and a dominant factor limiting the dosage of cytotoxic chemotherapy. The role of the neutrophil is of such importance in defence against microbial invasion that measures that modify the behaviour of residual hemopoietic tissue to promote a modest increase in neutrophils, can confer considerable benefit by reducing the frequency and severity of infection. Such a change can be mediated in bone marrow depression by diversion of more progeny of immature precursors into the neutrophil series, or by enhancement of the stimulatory drive operating on neutrophil production. The former effect can be achieved by hypertransfusion of red cells to reduce the demand on the limited precursor population for cells of the erythroid series. The latter effect can be achieved by administration of lithium carbonate. Neutropenia caused by autoimmune injury to the neutrophil series can also be successfully modified by measures which suppress the underlying immune dyscrasia or the function of the reticulo-endothelial system. Corticosteroid administration and splenectomy can be helpful in certain specific types of neutropenia. Administration of cyclophosphamide and azathioprine has both mutagenic and marrow suppressive potential, but can induce remissions in severe chronic isolated neutropenia and in systemic lupus erythematosis.

Agranulocytosis↗
PubMed1980

Size separation and polyclonal activation to immunoglobulin secretion of early precursors of B lymphocytes.

In adult mammals the bone marrow is a major site of B lymphocyte production, which occurs through the differentiation of pluripotent stem cells. In vivo studies have permitted the description of intermediate stages of differentiation. Following the recent development of culture conditions and the discovery of many polyclonal activators, it is now possible to carry out in vitro a finer analysis of B lymphocyte precursors differentiation. Because in vivo studies have shown that precursors vary in size, adult murine bone marrow cells were separated according to size. Fractions were cultured with polyclonal mitogens and tested for their capacity to develop into Ig-secreting cells. With this assay system, up to six populations were detected in adult bone marrow that can differentiate along the B cell lineage. The characterization of those populations was made according to size, mitogen(s) responsiveness, and kinetics of maturation. Precursor frequencies estimated by limiting dilution analysis further suggest that in cultures of some fractions of the gradient, new lymphocytes are produced through differentiation of immature precursors, although in cultures of some other fractions B lymphocytes may arise by self-renewal of mature cells.

Animals↗
PubMed1997

Oligodendrocyte precursor cells count time but not cell divisions before differentiation.

During vertebrate development, many types of precursor cell divide a limited number of times before they stop and terminally differentiate. It is unclear what limits cell proliferation and causes the cells to stop dividing when they do. The stopping mechanisms are important as they influence both the number of differentiated cells generated and the timing of differentiation. We have been studying the 'stopping' problem in the oligodendrocyte cell lineage [1] [2], which is responsible for myelination in the vertebrate central nervous system. Previous studies demonstrated that the proliferation of oligodendrocyte precursor cells isolated from the developing rat optic nerve is limited by an intrinsic 'clock' mechanism [3], which consists of two components: a counting mechanism that counts time or cell divisions, and an effector mechanism that arrests the cell cycle and initiates cell differentiation when the appropriate time is reached [4] [5]. In the present study, we address the question of whether the counting mechanism operates by counting cell divisions. We show that precursor cells cultured at 33 degrees C divide more slowly but stop dividing and differentiate sooner, after fewer cell divisions, than when they are cultured at 37 degrees C, indicating that the counting mechanism does not count cell divisions but measures time in some other way. In addition, we show that the levels of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) rise faster at 33 degrees C than at 37 degrees C, consistent with previous evidence [6] that the accumulation of p27 may be part of the counting mechanism.

Animals↗
PubMed1998

Improved L-lysine yield with Corynebacterium glutamicum: use of dapA resulting in increased flux combined with growth limitation.

The amino acid L-lysine is produced on a large scale using mutants of Corynebacterium glutamicum. However, as yet recombinant DNA techniques have not succeed in improving strains selected for decades by classic mutagenesis for high productivity. We here report that seven biosynthetic enzymes were assayed and oversynthesis of the dihydrodipicolinate synthase resulted in an increase of lysine accumulation from 220 mM to 270 mM. The synthase, encoded by dapA, is located at the branch point of metabolite distribution to either lysine or threonine and competes with homoserine dehydrogenase for the common substrate aspartate semialdehyde. When graded dapA expression was used, as well as quantification of enzyme activities, intracellular metabolite concentrations and flux rates, a global response of the carbon metabolism to the synthase activity became apparent: the increased flux towards lysine was accompanied by a decreased flux towards threonine. This resulted in a decreased growth rate, but increased intracellular levels of pyruvate-derived valine and alanine. Therefore, modulating the flux at the branch point results in an intrinsically introduced growth limitation with increased intracellular precursor supply for lysine synthesis. This does not only achieve an increase in lysine yield but this example of an intracellularly introduced growth limitation is proposed as a new general means of increasing flux for industrial metabolite over-production.

Corynebacterium↗
PubMed1982

Effects of GM-CSF deprivation on precursors of granulocytes and macrophages.

Culture of C57BL bone marrow cells in the absence of GM-CSF led to a loss of recoverable granulocyte-macrophage colony-forming cells of 2% per hour. The rate of loss of progenitor cells in cultures of CBA fetal liver cells was 5-6% per hour. Surviving colony-forming cells exhibited a normal responsiveness to GM-CSF but generated smaller colonies than normal when subsequently stimulated by GM-CSF. Transfer of washed individual day-3 granulocyte-macrophage colony cells to cultures lacking GM-CSF indicated that most cells were unable to survive or proliferate in the absence of GM-CSF. Death of transferred cells was rapid and invariable when the cells were from macrophage-forming colonies. However some cells from 40-70% of granulocyte-forming colonies were able to undergo one or two divisions in the absence of GM-CSF. This phenomenon was seen most often with cells from colonies where matching colony cells exhibited a higher-than-average proliferative capacity in parallel stimulated cultures. The results indicate the difficulty that will be encountered in obtaining valid metabolic data from unstimulated populations of granulocyte-macrophage precursor cells. The ability of some granulocyte precursor cells to exhibit limited proliferation following GM-CSF deprivation suggests that significant amounts of GM-CSF may be bound to or be internalized in some precursor cells and result in cell division in the absence of GM-CSF from culture medium.

Animals↗
PubMed1987

The pathogenesis and biochemistry of amyloidosis.

The transformation of serum proteins into Congo red-sensitive fibrillar material is requisite for the onset and progression of amyloid disease. All the mechanisms which lead to the disease itself have not been elucidated, but our knowledge has increased significantly. It is apparent that in all types of amyloid fibrils, three common features are displayed by the major protein constituents. These are that the fibril protein has a serum precursor, a high degree of anti-parallel beta-sheet conformation and a distinctive ultrastructure on electron microscopy. In the AL and AA forms of amyloidosis, the putative precursors appear to undergo limited degradation to form the protein component of amyloid fibrils. It has been suggested that there may be certain primary structural characteristics inherent in precursor molecules which make them amyloidogenic, thus predisposing them to amyloid fibril formation. This would include certain subtypes of immunoglobulin light chains, possibly kappa I and lambda VI, in the AL type of amyloidosis and one of the polymorphic SAA species, SAA2, which has been identified as the predominating isotype found in AA amyloid fibrils. In AH amyloidosis, the mechanism of amyloid fibril formation appears to be simply a concentration phenomenon where elevated concentrations of B2-M are not handled normally and amyloid deposition is the result. Amyloidogenesis in the hereditary form of systemic amyloidosis may involve other factors in addition to the presence of a variant precursor prealbumin as indicated by the delayed onset of the disease. It is evident that the elucidation of the mechanism(s) which governs the onset and progression of the amyloidoses will allow future regulation and treatment of these all too often complex disorders.

Amyloid↗
PubMed2000

Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells.

During animal development, cells become progressively more restricted in the cell types to which they can give rise. In the central nervous system (CNS), for example, multipotential stem cells produce various kinds of specified precursors that divide a limited number of times before they terminally differentiate into either neurons or glial cells. We show here that certain extracellular signals can induce oligodendrocyte precursor cells to revert to multipotential neural stem cells, which can self-renew and give rise to neurons and astrocytes, as well as to oligodendrocytes. Thus, these precursor cells have greater developmental potential than previously thought.

Animals↗
PubMed1982

The effect of x-rays on the precursors of antibody forming cells (B cells) as measured with the in vitro limiting dilution assay.

The effect of X-irradiation upon murine antibody-forming cell precursors (B cells) was established in cultures of spleen cells stimulated with the B cell mitogen lipopolysaccharide (LPS). At day 5 and 7 the numbers of IgM- and IgG2-secreting cells were determined in cultures of irradiated and nonirradiated spleen cells. From these numbers a Do of 0.6-1.2 Gy for the IgM, and of 0.9-2.1 Gy for the IgG2 response was calculated. Similar Do values were obtained under limiting dilution culture conditions. In the limiting dilution assay the effect of irradiation upon the size of the IgM-producing clones could also be determined. It was found that irradiation reduced the number of LPS-reactive B cells without affecting the size of the clones produced by the surviving cells.

Animals↗
PubMed1999

Mutation of K234 and K236 in the voltage-dependent anion channel 1 impairs its insertion into the mitochondrial outer membrane.

Previous in vitro studies indicated that mutation of both K234 and K236 to arginine, glutamine, or glutamic acid impaired the ability of the voltage-dependent anion channel (VDAC1) to insert into the outer membrane of the mitochondria (Smith et al. 1995). These same mutants were expressed in a strain of Saccharomyces cerevisiae with a disruption in the VDAC1 gene. The mutant VDAC1 forms were found in the mitochondria suggesting that they were correctly sorted to the outer membrane. However, only very small amounts of the mutants were inserted into the mitochondrial membranes. Mitochondria isolated from the strains expressing the mutants were capable of catalyzing the translocation of both wild-type VDAC1 and pre-alcohol dehydrogenase III indicating that the translocation apparatus was functional. These results confirm the previously drawn conclusion that K234 and K236 are part of a membrane insertion motif. The failure of the mutant VDAC1 forms to insert did not cause VDAC1 precursors to accumulate in the soluble cell cytoplasm or in the microsomal fraction. The apparent lack of a "precursor pool" suggested that a post-transcriptional control mechanism might limit the amounts of VDAC1 precursors in the cell. Such a control mechanism is consistent with the observation that the amount of VDAC1 was very similar after epichromosomal (gene in a 2u plasmid controlled by a Gal1 promoter) and chromosomal expression (endogenous gene controlled by the endogenous promoter).

Amino Acid Substitution↗
PubMed1997

The influence of alanine infusion on glucose production in 'malnourished' African children with falciparum malaria.

By US standards, about half of African children are malnourished, although most appear clinically normal. It is possible that precursor supply for gluconeogenesis is limited to a greater extent in these seemingly malnourished African children than in healthy children, consequently limiting glucose production. Since in malaria peripheral glucose utilization is increased, precursor supply could play an even more critical role in maintaining glucose production in African children suffering from falciparum malaria. We studied the effect of alanine infusion (1.5 mg/kg/min) on glucose production (measured by infusion of [6,6-2H2]glucose) and plasma glucose concentration in 10 consecutive children with acute, uncomplicated falciparum malaria. By US standards, six children were below the 10th percentile of weight for height and seven were below the 10th percentile of height for age. Plasma concentrations of alanine increased during alanine infusion from 153 +/- 21 to 468 +/- 39 mumol/l, whereas plasma lactate concentrations did not change (1.4 +/- 0.2 vs. 1.3 +/- 0.2 mmol/l). Plasma glucose concentration and glucose production did not change during alanine infusion: 4.6 +/- 0.3 vs. 4.5 +/- 0.3 mmol/l and 5.8 +/- 0.4 vs. 5.7 +/- 0.3 mg/kg/min, respectively. Gluconeogenic precursor supply is sufficient for maintainance of glucose production in African children with uncomplicated malaria who are malnourished by US standards.

Acute Disease↗