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PubMed1991

Proton transfer is rate-limiting for translocation of precursor proteins by the Escherichia coli translocase.

The protonmotive force stimulates translocation in vivo, in crude in vitro reactions, and in a purified, reconstituted reaction. Translocation activity is a function of the pH at the inner face of the membrane. Both the transmembrane pH gradient and the transmembrane electrical potential stimulate translocation. A late-stage translocation intermediate of the proOmpA preprotein completes its translocation in the absence of ATP when a protonmotive force is imposed. This completion of translocation is retarded by a factor of greater than 3 in deuterium oxide relative to water, demonstrating that translocation involves proton-transfer reactions in rate-limiting steps.

Adenosine Triphosphatases↗
PubMed1988

Antibody-forming capacity of B cell-deficient chickens reconstituted with limiting numbers of B cell precursors.

To examine the antibody-forming capacity of neonatally cyclophosphamide-treated chickens reconstituted with limiting numbers of B cell precursors, we analyzed their antibody responses to six unrelated antigens. Our results demonstrate that about 10 x 10(6) bursal cells are needed in this adoptive cell transfer model to restore normal immune competence to B cell-deficient birds. From our earlier data we know that with low repopulating cell numbers (less than 10 x 10(6) cells) developing bursal follicles are of clonal origin. Ten million cells repopulate about 40%, i.e. about 4 x 10(3) of the bursal lymphoid follicles. Assuming the clonal origin of the follicles these results imply that the B cell system of birds receiving this dose is derived from less than 5 x 10(3) precursor cells. At the lowest reconstituting dose (1.25 x 10(6) cells) most birds do not respond to the antigens studied. However, their B cell system is derived from only about 500 precursor cells. Because the antibody repertoire of a normal chicken was estimated to be at least 10(6) our results suggest that each precursor gives rise to a large number (greater than 200) of immunoglobulin V-region gene variants during its clonal proliferation in the bursa. Our results are thus consistent with the proposed "hyperconversion" mechanism of generation of antibody diversity in the chicken and provide quantitative data useful for estimating what such somatic modification rates might be.

Animals↗
PubMed1986

Estimation of the B lymphocyte precursor frequencies to herpes simplex type 1 glycoproteins by a limiting dilution assay.

The precursor frequency of B lymphocytes from Balb/c mice producing HSV-1 glycoprotein B (gB), glycoprotein C (gC), and glycoprotein D (gD) antibody was determined by limiting dilution analysis under conditions to detect antibody from the clonal progeny of a single B cell precursor. In spleens of naive mice the average gC frequency was 1/48,917 +/- 5,550, while gD was 1/73,330 +/- 15,898, and gB frequency was in excess of 1/100,000. Immunization with live HSV-1 (KOS) increased the B cell frequencies of all three glycoproteins to approximately 1:3,000; however, the serum gB antibody ELISA titer was fivefold higher than gC or gD.

Animals↗
PubMed1990

Heterogeneity of mouse helper T cells. Evidence from bulk cultures and limiting dilution cloning for precursors of Th1 and Th2 cells.

Many long term mouse Th clones express either the type 1 or type 2 Th cell (Th1 or Th2) cytokine secretion phenotype. In this report we present two lines of evidence for the existence of additional Th differentiation states. Lectin-stimulated spleen cells secreted moderate levels of IL-2 compared with long term Th1 clones, whereas the levels of other cytokines were more than 100-fold lower than those produced by either Th1 or Th2 clones. This suggests that many spleen cells produce substantial amounts of IL-2 but little or no IL-4, IL-5, IFN-gamma, IL-3, and granulocyte/macrophage-CSF. In contrast to long term Th clones, many short term alloreactive clones displayed cytokine secretion phenotypes intermediate between the Th1 and Th2 patterns. The proportion of recognizable Th1 and Th2 clones at early times in culture was greatly increased by immunization of the mice from which the responder and stimulator cells were derived; Brucella abortus immunization resulted in the isolation of exclusively Th1 clones, whereas infection with Nippostrongylus brasiliensis resulted in a strong trend toward the isolation of Th2 clones. The immunization of mice from which responder cells were derived strongly affected the type of Th clone obtained, whereas the source of stimulator cells had much less effect, suggesting that the commitment of Th cells to the Th1 or Th2 phenotypes occurred mainly in vivo. A model for the possible relationships of the various Th cells is presented.

Animals↗
PubMed1997

Active unfolding of precursor proteins during mitochondrial protein import.

Precursor proteins made in the cytoplasm must be in an unfolded conformation during import into mitochondria. Some precursor proteins have tightly folded domains but are imported faster than they unfold spontaneously, implying that mitochondria can unfold proteins. We measured the import rates of artificial precursors containing presequences of varying length fused to either mouse dihydrofolate reductase or bacterial barnase, and found that unfolding of a precursor at the mitochondrial surface is dramatically accelerated when its presequence is long enough to span both membranes and to interact with mhsp70 in the mitochondrial matrix. If the presequence is too short, import is slow but can be strongly accelerated by urea-induced unfolding, suggesting that import of these 'short' precursors is limited by spontaneous unfolding at the mitochondrial surface. With precursors that have sufficiently long presequences, unfolding by the inner membrane import machinery can be orders of magnitude faster than spontaneous unfolding, suggesting that mhsp70 can act as an ATP-driven force-generating motor during protein import.

Bacterial Proteins↗
PubMed1998

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.

Journal Article↗
PubMed1996

Frequency analysis of recipient-reactive helper and cytotoxic T lymphocyte precursors using a combined single limiting dilution assay.

In the present study, in order to simplify the procedure of helper T lymphocyte precursor (HTLp) and cytotoxic T lymphocyte precursor (CTLp) assays, reduce the sample size required and compare the sensitivity of HTLp and CTLp assays in the detection of alloreactive responses, the same set of limiting dilution mixed lymphocyte cultures was used and evaluated for both HTLp and CTLp frequencies. Ninety-four normal volunteer or patient/donor pairs were tested. High frequencies of HTLp (1:385 to 1:28,571) and CTLp (1:554 to 1:25,000) were detected for HLA unmatched unrelated individuals. The range of HTLp and CTLp frequencies of HLA identical siblings was 1:67,000 to 1:1,000,000 and 1:3700 to 1:1,000,000, respectively. In HLA matched unrelated pairs both HTLp and CTLp frequencies ranged from 1:1000 to 1:500,000, overlapping the values obtained from the former two groups. High CTLp and HTLp frequencies were found in 73% and 84% of HLA matched unrelated patient/donor pairs, respectively, suggesting that these in vitro functional assays may provide additional and essential information for the selection of unrelated bone marrow donors. The results of this study support the hypothesis that the combined single limiting dilution system is an efficient and informative method to quantitatively analyse HTLp and CTLp frequencies against major and minor histoincompatibilities with the advantage of being less time-consuming, less laborious and requiring fewer lymphocytes. The results also suggest that the HTLp assay is at least as efficient as the CTLp assay for the detection of alloreactivity other than of HLA identical siblings and may be useful for predicting graft-versus-host disease after bone marrow transplantation using HLA matched unrelated donors. The clinical value of this modified limiting dilution assay to predict graft-versus-host disease in bone marrow transplantation using HLA identical sibling donors and matched unrelated donors is being assessed.

Blood Donors↗
PubMed1999

Human CD28-CD8+ T cells contain greatly expanded functional virus-specific memory CTL clones.

At birth, almost all human peripheral blood CD8+ T cells express the costimulatory molecule CD28. With increasing age, the proportion of CD8+ T cells that lack CD28 increases. Because the Ag specificity of CD28-CD8+ T cells has not previously been defined, we studied the contribution of CD28-CD8+ T cells to the memory CD8+ CTL response against two human persistent viruses, human CMV (HCMV) and HIV. From PBMC of healthy virus carriers we generated multiple independent CTL clones specific for defined viral peptides and sequenced their TCR beta-chains. We designed clonotypic oligonucleotides complementary to each beta-chain hypervariable sequence and quantified the size of individual immunodominant CTL clones in PBMC. Some individual CTL clones were very large, comprising up to 3.1% of all CD8+ T cells in PBMC, and were generally maintained at a stable level for months. Individual virus-specific CTL clones were consistently more abundant in purified CD28- cells than in the CD8+ population as a whole. Because CD28-CD8+ cells as a population have been reported to proliferate poorly in response to mitogen, we studied the function of these virus-specific CD28- CTL clones by quantifying the frequency of peptide-specific CTL precursors using limiting dilution analysis. CD28-CD8+ T cells contained high frequencies of functional memory CTL precursors specific for peptides of HCMV or HIV, generally higher than in the CD8+ T cell population as a whole. We conclude that in asymptomatic HCMV and HIV infection, human CD28-CD8+ T cells contain high frequencies of functional virus-specific memory CTL clones.

Amino Acid Sequence↗
PubMed1996

Cytokine regulation of apoptosis in hematopoietic precursor cells.

Cytokines are known to influence hematopoietic precursor cell survival. Apoptosis (a programmed cell death pathway) has been identified as the key factor limiting hematopoietic precursor cell survival and cytokines have been demonstrated to induce or prevent apoptosis in a variety of hematopoietic precursor cell populations. Whether a specific cytokine inhibits or suppresses apoptosis depends on cytokine-mediated modulation of target cell cytokine receptors, cell death regulator genes such as bcl-2 family members, Fas receptor, and other pathways. Finally, the intracellular pathways of cytokine receptor-mediated control of apoptosis have begun to be unraveled, implicating specific intracellular receptor domains and protein kinases in the regulation of apoptosis and hematopoietic precursor cell survival.

Animals↗
PubMed1994

The relative radioresistance of interleukin-2 production by human peripheral blood lymphocytes: consequences for the development of a new limiting dilution assay for the enumeration of helper T lymphocyte precursor frequencies.

We describe a limiting dilution assay for the enumeration of alloreactive helper T lymphocyte precursor frequencies in human peripheral blood. The proliferation rate of the murine indicator cell line, cytotoxic T lymphoblastic line 2 (CTLL-2) induced by interleukin-2 (IL-2) culture supernatants was determined by staining with the fluorescent DNA dye propidium-iodide. Lymphocytes from healthy individuals as well as from patients with end stage kidney disease and no previous allosensitization exhibited a relative radioresistance of their IL-2 production up to gamma irradiation doses of 40-60 Gy. This differs from previous findings in the literature, showing a total inhibition of the IL-2 production in unsensitized individuals using a gamma irradiation dose of 20 Gy. The consequences of this relative radioresistance are that for a reliable stimulator cell inactivation in assays for the enumeration of helper T lymphocyte precursors gamma irradiation doses of at least 50 (-60) Gy are needed. Increasing the gamma irradiation dose for the inactivation of the stimulator cells can result in a decrease of the antigen presenting capacity of these cells.

Animals↗
PubMed1973

Biosynthesis of serum albumin in rat liver. Evidence for the existence of 'proalbumin'.

1. A protein(s) of rat liver (precipitated from soluble extracts of the microsomal fraction by anti-albumin) yields albumin after limited hydrolysis by trypsin. 2. Evidence that the product of limited tryptic hydrolysis is albumin, is based upon ion-exchange chromatography, electrofocusing and peptide ;mapping'. 3. The albumin ;precursor' is recognized by anti-albumin and is apparently not distinguished from albumin by anti-albumin. 4. A small peptide is liberated from the presumptive albumin precursor during limited tryptic hydrolysis. This peptide is labelled by arginine, but not by leucine, lysine or methionine. 5. These results support our previous suggestion based on kinetic evidence that the albumin-like protein(s), in the anti-albumin precipitate from rat liver, is an albumin precursor.

Animals↗
PubMed1995

Restoration of urine concentrating ability and accumulation of medullary osmolytes after chronic diuresis.

Restoration of urine osmolality (Uosm) and medullary osmolyte contents after chronic diuresis was studied in rats infused for 6 days with furosemide and subsequently given the vasopressin analogue, 1-desamino-8-D-arginine vasopressin (DDAVP). Papillary tip intra- and extracellular electrolyte concentrations were measured by electron microprobe analysis, tissue contents of methylamines (glycerophosphorylcholine, betaine), polyols (myo-inositol, sorbitol), and several amino acids in different kidney zones by high-performance liquid chromatography. Administering DDAVP continuously after diuresis increased Uosm from (means +/- SE) 348 +/- 8 to 1,265 +/- 127 after 1 day and 2,485 +/- 186 mosmol/kgH2O after 3 days. The sum of all osmolytes at the papillary tip rose from 309.2 +/- 28.9 to 690.9 +/- 105.8 and 1,282.8 +/- 21.0 mmol/kg protein after days 1 and 3, respectively. Although interstitial tonicity (sum of Na, Cl, and K concentrations) was increased by 116 and 223% after 1 and 3 days DDAVP, intracellular tonicity was similar in chronic diuresis and following 1 or 3 days DDAVP. Coadministration of DDAVP with betaine, myo-inositol, and choline ("osmolyte treatment") did not accelerate the restoration of Uosm but caused significantly higher contents of osmolytes (except myo-inositol) in inner medulla and/or papilla after 3 days. In a minority of animals, restoration of Uosm and reaccumulation of medullary osmolytes were impeded in both DDAVP- and DDAVP/osmolyte-treated rats. These data indicate that, after chronic diuresis, accumulation of organic osmolytes and restoration of Uosm proceed in parallel. Capacity for transport and/or synthesis of organic osmolytes, rather than their availability, appear to limit reaccumulation on the first day of recovery. By the third day, delivery of some osmolytes or their precursors may limit the restoration of medullary osmolyte content. The failure of some rats to attain sufficient concentrating ability within this time period may be related to deficient reaccumulation of medullary osmolytes.

Amino Acids↗
PubMed1993

T cell-dependent differentiation of human B cells into IgM, IgG, IgA, or IgE plasma cells: high rate of antibody production by IgE plasma cells, but limited clonal expansion of IgE precursors.

The development of human functional Ig precursors into plasma cells expressing IgM, IgG, IgA, or IgE was compared. Purified human B cells were stimulated at limiting dilution with irradiated EL4 helper cells, IL-2, and IL-4. B cells proliferated exponentially until Day 8 of culture. Nondividing plasma cells of all isotypes were detectable in ELISPOT assays between Days 8 and 10 and secreted 1.8 +/- 0.7 ng antibody per cell within 24 hr. This indicates that plasma cells of all isotypes, including IgE, bear a comparable potential to secrete antibody. It further shows that Ig switching does not delay the development into IgE plasma cells, despite that switching from IgM to IgE in vitro required 6 days of IL-4 action. The proliferation and Ig production by B cells readily declined after Days 8 and 10, respectively, and could not be prolonged by restimulating B cells with fresh helper cells and lymphokines in secondary cultures. This indicates that B cells have developed into nondividing, high rate Ig-secreting plasma cells within 9 days, and that they do not differentiate any further under the applied conditions. In contrast to IgM, IgG, and IgA committed B cells, IgE switched cells did not undergo clonal expansion, since the numbers of functional IgE precursors corresponded to the maximal numbers of IgE-secreting plasma cells, whereas the numbers of IgM-, IgG-, or IgA-secreting cells exceeded the number of functional precursors 15-fold. The results demonstrate that human B cells of all isotypes, including IgE, have the potential to secrete antibody at a comparably high rate, and that the IL-4-induced switch process does not delay the differentiation into plasma cells.

Animals↗
PubMed2007

Ester-based precursors to increase the bioavailability of quercetin.

Plant polyphenols exhibit a variety of potentially useful biochemical properties in vitro, but their evaluation and clinical exploitation in vivo is hampered by their limited bioavailability. Precursors exhibiting resistance to phase II metabolism during absorption are therefore desirable. We report here the synthesis as well as stability and solubility studies of several ester derivatives of quercetin (3,3',4',5,7-pentahydroxy flavone), most of which comprise an aminoacyl group. To model transepithelial absorption, we tested transport across supported tight monolayers of MDCK-1, MDCK-2, and Caco-2 cells. Quercetin itself was extensively conjugated by all three types of cells. A few of our precursors did not cross the monolayers, but others did, undergoing partial deacylation. No phase II conjugation was observed during transport of these compounds across MDCK or some Caco-2 clones. With other Caco-2 lines complete deacylation occurred, followed by metabolism of quercetin. Since elimination of residual acyl groups is expected to take place in vivo, ester derivatives of polyphenols may constitute a useful method to increase systemic aglycone concentrations.

Adsorption↗
PubMed1987

Proteolytic enzymes in the post-translational processing of polypeptide hormone precursors.

Selective and limited proteolysis is a key step in the post-translational modification of peptide hormone precursors. This process appears to involve a proteolytic machinery including highly specific endoproteases. Some of the enzyme systems possibly involved in the processing of pro-neuropeptides will be described and their mechanism of action discussed. Special emphasis will be on the following: i) the physico-chemical characteristics of proteolytic enzymes which are believed to be involved in the processing of some of these polypeptide hormone precursors; ii) the bio-specificity of these enzymes toward the substrates; iii) the importance of both secondary and tertiary structures of the cleavage domain in recognition by the selective proteases. These properties will be discussed in connection with the possible importance of the maturation enzymes in the in vivo regulation of hormone biosynthesis.

Animals↗
PubMed1997

Intraembryonic hematopoietic stem cells.

Intraembryonic hematopoietic stem cells (HSC) were first detected in avian chimeras associating an embryo with a yolk sac (YS). Cell markers were used to construct chimeras. The results showed that YS blood precursors undergo primitive erythropoiesis and become extinct, whereas intraembryonic precursors colonize rudiments of blood-forming organs and settle in the bone marrow as self-renewable HSC. The model is valid in the mouse as shown by in vitro cultures of cells obtained from embryo structures or YS separated prior to circulation. This approach, as well as restoration of irradiated adults, demonstrates that YS precursors have a limited potential compared with embryo precursors. The emergence of hematopoietic precursors in both YS and embryos is closely linked to the emergence of the endothelial network and is restricted to the mesoderm layer associated with endoderm.

Animals↗
PubMed1995

Molecular analysis of TCR junctional variability in individual infiltrated islets of non-obese diabetic mice: evidence for the constitution of largely autonomous T cell foci within the same pancreas.

Insulitis develops in non-obese diabetic (NOD) mice as a multicentric and asynchronous process. In an effort to understand how this T cell mediated process expands within each islet and propagates between the islets of the same pancreas, we have analyzed the junctional diversity of TCR V beta 6 and V beta 8.2 transcripts cloned from infiltrated islets. The material examined was obtained from individual islets of 8 and 12 week old NOD mice or from pooled islets of 4 week old individual mice. Compared with spleen transcripts, where every V beta 6 or V beta 8.2 clone displays a different junction, islet transcripts are considerably less diverse. Each islet harbors from one to a maximum of six independent CDR3 sequences out of 10 or more analyzed colonies. On the other hand, there is an overall diversity of sequences when comparing the islets of the same pancreas or individual mice at 4 weeks. Altogether, these results support the idea that TCR repertoires are already divergent at the very early onset of insulitis and that each islet-centered infiltrate develops rather autonomously from the oligoclonal expansion of a limited set of precursors. Recirculation between islets is limited and does not seem to be the main mode of propagation of insulitis. Finally, a close analysis of J beta usage and N additions in beta chain transcripts from infiltrating cells reveals definite biases suggestive of an ongoing selective process imposed upon intra-pancreatic T lymphocytes.

Amino Acid Sequence↗