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PubMed1986

Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis.

Skin secretions from the South African frog Xenopus laevis have been chromatographed by high performance liquid chromatography (HPLC), fractionated, and analyzed by fast atom bombardment-mass spectrometry (FAB-MS). The HPLC chromatograms showed the secretion to be a complex mixture with over 30 components at similar levels to the four peptides previously isolated from X. laevis skin, i.e. xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa. FAB-MS analysis of the HPLC fractions gave numerous protonated molecular ions ranging from m/z 491 to 2662. Preliminary assignments of these components were made by comparing these experimental molecular weights to those predicted for regions within the xenopsin, caerulein, thyrotropin-releasing hormone, and PGLa precursors. These results suggested that many of these skin secretions were peptides originating from additional processing of the xenopsin, caerulein, and PGLa precursors, primarily involving cleavage at single arginine residues, and a novel cleavage at the NH2-terminal side of single lysines. These assignments were subsequently confirmed by Edman degradation, FAB-MS peptide sequencing, and amino acid analysis. All of these peptides contain one or more lysines and would be expected to have amphiphilic structures. As yet, nothing is known about their activity, although they resemble in composition the mast cell degranulating peptides melittin and the bombolitins. These precursor fragments were also found to have limited sequence homology to bombinin, a hemolytic amphibian peptide isolated from the European Bombina toad.

Amino Acid Sequence↗
PubMed1984

Cytotoxic T-cell precursors revealed in neonatally tolerant mice.

Induction of neonatal tolerance leads to a profound reduction in cytotoxic T-lymphocyte precursor frequencies against the tolerated alloantigen ("tolerogen") as evaluated by limiting-dilution analysis. The curves obtained were linear within the range tested and, thus, did not yield evidence for any dissociation of cytotoxic precursors from regulatory cell populations. However, it could be shown that cytotoxic T-lymphocyte precursor frequencies against the tolerogen increased drastically if the tolerant spleen cells were adsorbed, prior to limiting-dilution culture, on monolayers of syngeneic blasts expressing receptors for the tolerogen but not if they were adsorbed on syngeneic blasts against third-party antigens. This finding implies that cytotoxic precursor cells against the tolerogen are not clonally deleted in tolerant animals but rather are suppressed by a regulatory cell population that is present at high frequency and presumably acts in an anti-idiotypic fashion.

Animals↗
PubMed1989

The TGF-alpha precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction.

The 50 amino acid form of TGF-alpha is cleaved from a conserved integral membrane glycoprotein by a protease that, in many tumor cells, appears to be limiting. To test whether the membrane-bound precursor has biological activity in the absence of processing, we introduced amino acid substitutions at the proteolytic cleavage sites. BHK cells transfected with expression vectors containing these altered sequences do not secrete detectable levels of mature TGF-alpha into the medium, but express high levels of proTGF-alpha at the cell surface. Coincubation of these BHK cells with A431 cells demonstrates that membrane-bound proTGF-alpha may bind to EGF receptors on the surface of contiguous cells, induce receptor autophosphorylation, and thereby produce a rapid rise in A431 intracellular calcium levels. Thus, proTGF-alpha can be biologically active in the absence of processing, a fact that may have implications for the integral membrane precursors of related growth factors.

Amino Acid Sequence↗
PubMed2001

The use of GABA(A) receptors expressed in neural precursor cells for cell-based assays.

GABA(A) receptors are known targets for certain classes of environmental neurotoxins and pharmaceutical compounds. Since few neural cell lines express functional GABA(A) receptors, the capacity to rapidly screen for compounds that affect GABA(A) receptor function is presently limited. Previous work has demonstrated that rat neural precursor cells express functional GABA(A) receptors that can be monitored via Ca(2+) imaging. This study examined GABA(A) receptor subunit expression to determine whether GABA(A) receptor function and its interactions with neurotoxins is preserved after passaging. Neural precursor cells isolated from embryonic day 13 rat brain were expanded in serum-free medium containing basic fibroblast growth factor and passaged three times. Reverse transcription-polymerase chain reaction analysis demonstrated early expression of abundant mRNAs encoding various GABA(A) receptor subunits. Ca(2+) imaging showed that the highly proliferating precursor cells in passaged cultures maintained expression of functional GABA(A) receptors. In addition, we showed that trimethylolpropane phosphate, a neurotoxin generated during partial pyrolysis of a synthetic ester turbine engine lubricant, potently inhibited muscimol (GABA(A) receptor agonist) but not depolarization-induced cytosolic Ca(2+) increase. The findings of this study suggest that neural precursor cells may be well suited for the evaluation of certain environmental neurotoxins with convulsant activity. The potential use of neural precursor cells in high-throughput screens for compounds acting on GABA(A) receptors is discussed.

Animals↗
PubMed2005

Are cementoblasts a subpopulation of osteoblasts or a unique phenotype?

Experimental studies have shown a great potential for periodontal regeneration. The limitations of periodontal regeneration largely depend on the regenerative potential at the root surface. Cellular intrinsic fiber cementum (CIFC), so-called bone-like tissue, may form instead of the desired acellular extrinsic fiber cementum (AEFC), and the interfacial tissue bonding may be weak. The periodontal ligament harbors progenitor cells that can differentiate into periodontal ligament fibroblasts, osteoblasts, and cementoblasts, but their precise location is unknown. It is also not known whether osteoblasts and cementoblasts arise from a common precursor cell line, or whether distinct precursor cell lines exist. Thus, there is limited knowledge about how cell diversity evolves in the space between the developing root and the alveolar bone. This review supports the hypothesis that AEFC is a unique tissue, while CIFC and bone share some similarities. Morphologically, functionally, and biochemically, however, CIFC is distinctly different from any bone type. There are several lines of evidence to propose that cementoblasts that produce both AEFC and CIFC are unique phenotypes that are unrelated to osteoblasts. Cementum attachment protein appears to be cementum-specific, and the expression of two proteoglycans, fibromodulin and lumican, appears to be stronger in CIFC than in bone. A theory is presented that may help explain how cell diversity evolves in the periodontal ligament. It proposes that Hertwig's epithelial root sheath and cells derived from it play an essential role in the development and maintenance of the periodontium. The role of enamel matrix proteins in cementoblast and osteoblast differentiation and their potential use for tissue engineering are discussed.

Cell Differentiation↗
PubMed1997

Lignans in homemade and commercial products containing flaxseed.

Epidemiologic, in vitro, and in vivo studies support the hypothesis that mammalian lignans have cancer protective effects. Flaxseed is the richest source of plant precursors to mammalian lignans. However, there are limited data on the lignans from processed foods containing flaxseed, despite increasing flaxseed use. Thus the objective of this study was to quantify the lignans in flaxseed-containing processed foods and their relationship to the percent flaxseed in the food. Twenty-five foods including raw flaxseed, homemade products containing flaxseed, and commercial breads and breakfast cereals with and without flaxseed were subjected to an in vitro fermentation designed to simulate the colonic environment necessary for the conversion of plant precursors to mammalian lignans. The lignan production from cereals containing flaxseed was significantly greater than that from their component grains (p < or = 0.01), and the lignan production from breads containing > 4% flaxseed was significantly greater than that from breads containing < 4% flaxseed (p < or = 0.05). Lignan production was significantly related to percent flaxseed in homemade products (r = 0.95, p < or = 0.01) and breakfast cereals (r = 0.997, p < or = 0.0001). Although a significant relationship was also observed in commercial breads (r = 0.58, p < or = 0.05), the correlation was lower because of the variability in different flaxseed varieties and the lignan contribution from other grains and oilseeds in these products. In conclusion, the addition of flaxseed to processed foods increased the production of mammalian lignans significantly. The amount of lignan production was linearly dependent on the percent flaxseed but was also influenced by other grains and the variety of flaxseed.

Bread↗
PubMed1999

Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain.

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-kappaB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.

Amino Acid Sequence↗
PubMed1982

The regulation of bone marrow-derived mononuclear phagocyte differentiation and mobilization in syngeneic and allogeneic YAC-lymphoma-bearing mice.

YAC-lymphoma inoculation (ip) into syngeneic A/J mice results in a massive in situ proliferation of the lymphoma cells, with non-significant increases in the peritoneal macrophage population, and also in a remarkable and progressive increase in the bone marrow precursor cells forming both macrophage and fibroblastoid colonies in vitro. In the allogeneic situation (BALB/c mice), YAC-lymphoma cells proliferate to a limited extent before tumour regression. These processes (i.e. proliferation and regression) were accompanied by a progressive increase in the peritoneal macrophage population, which declined to normal after tumour regression. Macrophages accumulating at the regression stage exhibited in vitro cytolytic activity towards YAC-lymphoma cells and cytostatic activity towards unrelated cells. No correlation was observed between the limited asynchronous increase in the bone marrow level of precursor cells for in vitro differentiating macrophage and fibroblastoid colonies, on one hand, and macrophage accumulation at the tumour site in BALB/c mice on the other. Conditioned media from in vitro proliferating YAC-lymphoma cells exhibited high colony-stimulating activity for macrophage colony formation in vitro. Bone marrow cells taken from YAC-lymphoma inoculated A/J mice expressed potent tumorogenic potential at early stages after tumour inoculation. At day 2 after tumour inoculation the bone marrow cells were already tumorogenic and concomitantly expressed augmented levels of macrophage and fibroblastoid colony-forming capacity. The results indicate an inter-relationship between tumour growth and mononuclear phagocyte differentiation and proliferation.

Animals↗
PubMed1966

Gluconeogenesis in the perfused rat liver.

1. A modification of the methods of Miller and of Schimassek for the perfusion of the isolated rat liver, suitable for the study of gluconeogenesis, is described. 2. The main modifications concern the operative technique (reducing the period of anoxia during the operation to 3min.) and the use of aged (non-glycolysing) red cells in the semi-synthetic perfusion medium. 3. The performance of the perfused liver was tested by measuring the rate of gluconeogenesis, of urea synthesis and the stability of adenine nucleotides. Higher rates of gluconeogenesis (1mumole/min./g.) from excess of lactate and of urea synthesis from excess of ammonia (4mumoles/min./g. in the presence of ornithine) were observed than are likely to occur in vivo where rates are limited by the rate of supply of precursor. The concentrations of the three adenine nucleotides in the liver tissue were maintained within 15% over a perfusion period of 135min. 4. Ca(2+), Na(+), K(+), Mg(2+) and phosphate were found to be required at physiological concentrations for optimum gluconeogenesis but bicarbonate and carbon dioxide could be largely replaced by phosphate buffer without affecting the rate of gluconeogenesis. 5. Maximal gluconeogenesis did not decrease maximal urea synthesis in the presence of ornithine and ammonia and vice versa. This indicates that the energy requirements were not limiting the rates of gluconeogenesis or of urea synthesis. 6. Addition of lactate, and especially ammonium salts, increased the uptake of oxygen more than expected on the basis of the ATP requirements of the gluconeogenesis and urea synthesis.

Adenosine Triphosphate↗
PubMed1998

Enhanced TGFbeta1 maturation in high five cells coinfected with recombinant baculovirus encoding the convertase furin/pace: improved technology for the production of recombinant proproteins in insect cells.

One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger inactive precursor proteins. One example is transforming growth factor beta 1 (TGFbeta1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGFbeta1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGFbeta1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGFbeta1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGFbeta1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGFbeta1 by coexpressing prohTGFbeta1 and human furin convertase in High Five cells. Using this system, the production of mature active TGFbeta1 increased in a dose-dependent fashion reaching up to 7. 8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGFbeta1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide.

Animals↗
PubMed1995

Successful therapy with donor buffy coat transfusions in patients with relapsed chronic myeloid leukemia after bone marrow transplantation is associated with high frequencies of host-reactive interleukin 2-secreting T helper cells.

Six patients treated for relapsed chronic myeloid leukaemia after allogeneic bone marrow transplantation with donor buffy coat transfusions were investigated. In the 5 patients who achieved molecular remission high frequencies of host-reactive interleukin 2-secreting T helper cell precursors (Th-p) were detectable by limiting dilution analysis. In four of the patients the presence of Th-p was associated with a clinical syndrome similar to transfusion GVHD suggesting a T cell response to minor histocompatibility antigens (minor H) expressed by both malignant haemopoiesis and host tissues. In the fifth responding patient no GVHD or bone marrow hypoplasia was observed. The nature of the antigens recognised by these donor Th-p remains unknown. No host-reactive Th-p were detectable in the non-responder and host-reactive cytotoxic T cell precursors (CTL-p) were not consistently detectable in the responding patients.

Adult↗
PubMed1998

Tolerance induction by "megadose" hematopoietic transplants: donor-type human CD34 stem cells induce potent specific reduction of host anti-donor cytotoxic T lymphocyte precursors in mixed lymphocyte culture.

BACKGROUND: Recently, the use of megadoses of CD34+ hematopoietic progenitors has been reported to abrogate resistance to engraftment, thus overcoming major histocompatibility barriers in bone marrow transplantation in leukemia patients. METHODS: The ability of human CD34+ cells to possess potent tolerizing activity was studied by limiting dilution analysis of cytotoxic T lymphocyte (CTL) precursors (CTL-p) in human peripheral blood lymphocytes after addition of purified CD34+ cells. RESULTS: The addition of purified human CD34+ cells to primary mixed lymphocyte culture led to a marked reduction of antiallogeneic CTL-p frequency against stimulator cells of the same origin, compared with the response against cells of third-party origin. The CD34+ cells caused a marked inhibition of the CTL activity, when added at an equal number with the responder T cells, and they were still present after the mixed lymphocyte culture, which suggests that no significant killing of CD34+ cells had occurred. The tolerizing activity is abrogated by irradiation and requires cell contact. This pattern of tolerization most closely resembles what has been ascribed to veto cells in other systems. Phenotypic analysis of the purified CD34+ cells showed that they express MHC class I and class II antigens, but do not express costimulatory molecules of the B7 family. CONCLUSIONS: It is possible, that CD34+ cells in the megadose transplants-perhaps by their inability to provide costimulatory molecules-are actively reducing the frequency of CTL-p directed against their antigens, and thereby help to overcome allogeneic rejection, and enhance their own engraftment.

Antigens, CD34↗
PubMed1985

Frequency of herpes simplex virus-specific helper T lymphocyte precursors in the lymph node cells of infected mice.

We have established a limited dilution assay to estimate the frequency of herpes simplex virus type 1 (HSV)-specific, interleukin 2 (IL 2)-producing helper T lymphocyte precursors (HTL-P). The estimated frequency of such cells in suspensions of local lymph node (LN) cells 5 days after in vivo virus infection was 1:2470 to 1:5800. Frequencies of HTL-P in cells from uninfected mice were below levels of detection of our system and were judged to be below 1:100,000. Removal of Lyt-2+ cells from responder LN cells before culture increases HTL-P frequency twofold to threefold, indicating the likely operation of some form of suppression in unseparated cultures. The demonstration of HSV-specific HTL-P required that cells from virus-primed mice be reexposed in vitro to viral antigen. In addition, clones expanded during a 9-day culture period failed to generate IL 2 unless reexposed to specific viral antigen or cross-reactivate HSV-2. Thus, HSV-specific HTL-P were strictly antigen dependent. No evidence was obtained for antigen-independent subpopulations of HTL-P as occurs with viral-specific cytotoxic T lymphocyte precursors. The clonal progeny of HTL-P were of the Thy-1+ Lyt+2- phenotype. Priming in vivo for the subsequent in vitro detection of HTL-P required that mice be exposed to infectious virus. Thus neither UV-inactivated nor heat-inactivated nor extracted viral glycoproteins could prime for HTL-P detection. The relevance of these findings for the future use of subunit vaccines against HSV is briefly discussed.

Animals↗
PubMed1996

Generation of hydroxytrimethyllysine from trimethyllysine limits the carnitine biosynthesis in premature infants.

epsilon-N-Trimethyl-L-lysine (TML) was given orally for 1 day to two groups of premature infants. There was no change in the output or plasma levels of carnitine at a dose of 100 mumol/day; however, the urinary TML increased 17-fold. In the second group, administration of 1 mmol TML increased the plasma levels and urinary output of carnitine; the output of TML increased 62-fold. During a search of the metabolites of carnitine biosynthesis by 1H NMR analysis of urine, only one new resonance (corresponding to the TML) could be identified in both groups. Fast atom bombardment mass spectrometry (FAB-MS) analysis of urine samples indicated an increase in TML in the treated patients; no changes were found in the relative abundance of any other precursors. These data show that a significant limitation of the conversion of hydroxy-TML to carnitine is not likely; rather, the conversion of TML to hydroxy-TML is regulatory in neonatal carnitine biosynthesis.

Carnitine↗
PubMed1999

Intra-A chain disulphide bond forms first during insulin precursor folding.

In this study, we investigated the folding pathway of insulin precursor and compared it with that of insulin-like growth factor I (IGF-I). The intra-A chain disulphide bond was found to form early in insulin precursor folding, whereas the corresponding disulphide bond in IGF-I formed late. Intra-A chain disulphide-bond deleted [A6, A11-Ser] proteins, including proinsulin, insulin, and A chain, were employed for this investigation. Under the same conditions the recombination yield of insulin from S-sulphonates of native A and B chains was 22%, while the yield of [A6, A11-Ser] insulin from S-sulphonates of [A6, A11-Ser] A chain and native B chains was only approx. 7%. This indicated that the intra-A chain disulphide bond may serve to stabilize the A chain folding intermediate so as to facilitate the correct recognition and pairing with the B chain. Time courses of oxidation of reduced insulin A chains, reduced A and B chains, and reduced proinsulins showed that the intra-A chain disulphide bond formed first during insulin precursor folding. The formation of intra-A chain disulphide bond further accelerated the formation of the other two inter-chain disulphide bonds. The time course of helix structure formation of insulin A chains also indicated that the intra-A chain disulphide bond formed first, and could stabilize partially folded A chain helix structure. The rate of intra-A chain disulphide bond formation was almost the same as that for both helix structure formation and insulin molecule formation, indicating that the formation of the intra-A chain disulphide bond was the rate limiting step for the folding of insulin precursor.

Circular Dichroism↗
PubMed1985

Cluster formation in a symmetrical network: a dynamical system for the description of the suppression among non-immune T lymphocytes and its application to the effects of immunization.

A mathematical model has been developed for the description of the suppressive regulation between polyclonally activated normal and immune T cells. The model assumes reversible cell-cell interactions to interpret results from limiting dilution experiments performed to determine the frequencies of precursor cells for antigen-specific T effector lymphocytes and to analyse mechanisms regulating the maturation of precursor into effector T cells. In particular, the model deals with the changes induced in the T lymphocytes population following immunization with antigens. In these limiting dilution experiments, T cells are placed in cultures at varying cell numbers with all other essential culture constituents kept in excess. After polyclonal activation of the T cells in culture they are supplied with growth and maturation factors so that they form daughter clones of functionally active T effector cells. The typical result observed was that effector T cells develop in cultures at low cell input but that this development is totally suppressed at high cell numbers. This result suggested that, at high cell numbers, the effector T cells are exposed to a sufficient number of other T cells of appropriate specificity to permit suppressive interactions. Whereas this is the case for non-immune T cells, T cells after immunization develop into effector cells both at high as well as at low cell concentrations, though with efficiencies less than proportional to their number of precursors. Our mathematical model is made up of a set of first order autonomous ordinary differential equations in many variables permitting the calculations of numbers of free cells and of cells engaged in cellular clusters of varying sizes. Free cells can develop into effector cells whereas cells engaged in clusters cannot. We calculate the consequences of several reasonable hypotheses concerning the effects of immunization. We consider the possibility that immunization modifies the growth behavior of the antigen-specific cells to permit an increased or accelerated clonal expansion in culture. Alternatively, we consider the possibility that immunization changes the interaction strength between cells specific for the immunizing antigen and other cells. Thirdly, we have connected both behaviors by calculating the case of an inverse relationship between growth rates and intensities of interaction between cells. Our model has been inspired by the symmetrical network model and can be interpreted in this framework. It proposes that immune regulation is a consequence of idiotype-anti-idiotype interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Simple mucin-type carbohydrate antigens in pleomorphic adenomas.

Simple mucin-type carbohydrate structures, T, Tn and sialosyl-Tn, are regarded as general markers of carcinomas in several epithelial tissues as a result of incomplete synthesis with precursor accumulation. The structures have a very limited distribution in normal tissues and secretions, including saliva and salivary glands. The expression of simple mucin-type carbohydrate structures and ABH(O) variants was studied in paraffin-embedded and frozen tissue sections from 37 pleomorphic adenomas with associated normal parotid tissue, using immunohistology and a panel of MAbs with well-defined specificity for T, Tn, sialosyl-Tn, and blood group H and A variants hereof. The immature Tn and sialosyl-Tn antigen structures were expressed in the epithelial ductular structures of the tumors, whereas they were almost absent from normal parotid tissue, indicating aberrant glycosylation with accumulation of precursor structures. Furthermore, the tumors showed loss of A antigen. The prognostic significance of these results is discussed. The modified myoepithelial cells in periductular and solid areas expressed T and sialosyl-T antigens, similar to normal myoepithelial cells and basal cells. Thus these modified MEC seem to have retained their normal simple mucin-type glycosylation pattern, suggesting that T antigen may be used as a marker of MEC in salivary gland tumors.

Adenoma, Pleomorphic↗