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PubMed1995

cry IA(b) transcript formation in tobacco is inefficient.

Chimaeric PCaMV35Scry genes direct in tobacco mesophyll protoplasts mRNA levels of less than one transcript per cell. We provide evidence that this low cytoplasmic cry IA(b) mRNA level is not due to a rapid turnover but rather results from a marginal import flow of cry messenger into the cytoplasm. Run-on assays indicate that the frequency of transcription initiation is not limiting. However, the cry precursor mRNA carries at least three regions that are recognized as introns. The absence of high cytoplasmic levels of spliced cry mRNAs suggests that these mRNAs are unstable and/or not efficiently made. Point mutations in the 5' splice site of the most distal intron allows high accumulation levels of the full-length mRNA. This implies that the inefficient formation of full-size mRNA is a major cause of the low expression level of chimaeric cry IA(b) genes in tobacco.

Bacillus thuringiensis Toxins↗
PubMed1979

Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum.

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.

Animals↗
PubMed1975

Control of composition and activity of the photosynthetic apparatus of Rhodopseudomonas capsulata grown in ammonium-limited continuous culture.

Rhodopseudomonas capsulata was grown either phototropically in the light or chemotrophically in the dark at oxygen tensions of 5 mm and 3 mm Hg in ammonium-limited continuous culture. During growth limitation bacteriochlorophyll content of cells and membranes varied dependent on growth rate drastically in chemotrophic cultures. Concomittantly, the ratio of membrane protein to total protein varied in the range of 30-41%. This dependence of membrane differentiation on growth rate was less evident in phototrophically grown cells. The incorporation of the bulk of bacteriochlorophyll was shown to be quantitatively correlated to the incorporation of 1-3 low molecular weight proteins with molecular weights in the range of 14 to less than 10 k daltons. Supported by similar findings of other authors it is proposed, that these proteins are to be attributed to the species of antenna bacteriochlorophyll and represent components of the photosynthetic apparatus. With decreasing growth rates the size of the photosynthetic unit with respect to the population of bacteriochlorophyll- and protein molecules was reduced subsequent to a reduction in the rate of incorporation of antenna-bacteriochlorophyll and the low molecular weight proteins, the reaction-center bacteriochlorophyll content of the membranes remaining constant. A parallel decrease in potential phosphorylating capacity was observed. It is concluded, that under these conditions, primary photochemical reactions in the reaction center were not the rate-limiting step in photophosphorylation. The interaction of growth limitation by an anabolic precursor (NH+4) and control of membrane differentiation by light intensity or oxygen tension is discussed.

Adenosine Triphosphate↗
PubMed1990

Stimulation of rheumatoid synovial and blood T cells and lines by synovial fluid and interleukin-2: characterization of clones and recognition of a co-stimulatory effect.

Rheumatoid arthritis (RA) is characterized by the presence of interleukin-2 (Il-2) receptor-positive T cells in the peripheral blood and synovial compartments. Utilizing the limiting dilution technique, the precursor frequencies of Il-2 responsive T cells were determined in peripheral blood and synovial sites from RA patients and in the blood of normal donors. The frequencies of Il-2 responsive T cells were significantly higher in RA patients (range from 1/180 to 1/7432) compared to normal donors (range from 1/400 to 1/8163). T-cell clones raised by the addition of Il-2 alone were predominantly of the CD4-positive phenotype. Peripheral blood T cells, synovial T-cell clones and lines derived from RA patients were co-stimulated with Il-2 and synovial fluid or supernatants from cultured synovial lining cells. This co-stimulation induced a strikingly enhanced proliferative T-cell response while synovial fluid alone was without effect. This stimulatory activity was found in the high molecular weight range (approximately 150 kDa) and could not be attributed to the action of immunoglobulins or known cytokines such as Il-2 or interleukin-1 (Il-1), suggesting the activity of a material that modulates the Il-2-dependent growth of T cells. The co-stimulatory capacity of synovial fluid with Il-2 may be relevant to the activated state, especially of synovial T cells.

Adult↗
PubMed2002

Method optimization for the determination of carbonyl compounds in disinfected water by DNPH derivatization and LC-ESI-MS-MS.

A method has been developed for quantitative determination of carbonyl disinfection by-products (DBP) from aqueous samples by derivatization with 2,4-dinitrophenylhydrazine combined with high-performance liquid chromatography (HPLC) and electrospray ionization (ESI) tandem mass spectrometry (MS-MS). The effect of excess of derivatization reagent and derivatization time, the effect of buffer and dry-gas temperature in the ESI process, and the effect of focus potential and collision energy in MS measurement are shown. Major fragment ions for compound identification on the basis of collision-induced dissociation (CID) mass spectra (MS) are given, as are common fragments for screening analyses by MS experiments such as the use of precursor ion scans. Detection limits in the microg x L(-1) range could be achieved by selected ion monitoring measurements without sample preconcentration. Solid-phase extraction improved the sensitivity by a factor of 25 to 250. The applicability of the method is illustrated by DBP analyses of samples from outdoor swimming pools after chlorination. Several carbonyl compounds, e.g. aldehydes, ketones, hydroxybenzaldehyde, and dicarbonyl compounds were identified.

Journal Article↗
PubMed1984

An in vitro amino acid incorporation method for assessing the status of in vivo protein synthesis.

A quantitative in vitro amino acid incorporation assay is described which can be used to assess the status of in vivo protein synthesis. The preparation and incubation conditions employed result in constant precursor specific activity and limit amino acid incorporation to completion of nascent peptide chains. Results obtained with this method correlate well with measurements of polyribosome profiles using sucrose gradient centrifugation. The assay is easily applied to a large number of samples, and requires only a fraction of the time and tissue necessary for conventional measures of polysome aggregation. The method has been found suitable for studies of protein synthesis in mouse brain and liver, and in gerbil brain, but not in mouse kidney. Products of in vitro protein synthesis can be separated by standard electrophoretic techniques, allowing a characterization of proteins whose mRNAs are actively translated in vivo.

Amino Acids↗
PubMed1991

Immobilized anti-CD3-induced T cell growth: comparison of the frequency of responding cells within various T cell subsets.

Human T cells can be divided into subsets based on the expression of CD29, CD45RA, CD45RO, LFA-3, or CD11a. It has been suggested that the subset of CD4+ T cells that expresses high densities of CD29, CD11a, CD45RO, and LFA-3 contains "memory" T cells, whereas the subset of cells that expresses CD45RA contains "naive" T cells. In order to obtain a more complete picture of the functional capacities of human naive and memory CD4+ and CD8+ T cell subsets, highly purified T cells were activated with a uniform stimulus and responses were examined in bulk cultures and under limiting dilution conditions. T cell activation was achieved with an immobilized mAb to the CD3 molecular complex, 64.1. In bulk cultures, immobilized 64.1 stimulated a vigorous response. Moreover, the number of cells entering the cell cycle, the magnitude of the [3H]thymidine incorporation, and the growth of the cells over 6 days in culture by naive and memory CD4+ T cells was comparable. To delineate the frequency of responsive cells in each subset more precisely, cells were cultured with immobilized 64.1 at limiting dilution and the precursor frequency of responding cells was assessed by examining wells microscopically for visible growth. Immobilized 64.1 was able to induce some T cells from each subset to grow in the complete absence of AC, when exogenous IL2 was present. The number of responding CD4+ and CD8+ cells was comparable. The percentage of naive cells responding in each population was approximately three times greater than the frequency of memory cells. IL4 could also support the growth of immobilized 64.1-activated CD4+ T cells, but the frequency of responding cells was much lower than that supported by IL2. The vast majority of the IL-4 responsive CD4+ cells resided within the naive cell subset. The data indicate that the response of CD4+ and CD8+ naive and memory T cell subsets to immobilized anti-CD3 depends on the density of responding cells. Naive T cells have an enhanced capacity to grow when cultured in the absence of other T cells or accessory cells. This ability may facilitate their expansion during primary immune responses.

Antibodies, Monoclonal↗
PubMed1989

The biosynthetic pathway of pyrimidine (deoxy)nucleotides: a sensor of oxygen tension necessary for maintaining cell proliferation?

Culture experiments on Ehrlich ascites tumor cells revealed that a low oxygen tension (about 20% in normoxic atmosphere) induced an increase in the length of the growth cycle. The relative growth of aerobic control cells after transfer to the second in vitro passage was 145% within 24 h, and reduced to 50% at 1% O2 and about 30% at 0.1% O2. The increase in protein and DNA content of these hypoxic cultures was equally impaired. Also, the cell cycle traverse as analyzed by flow cytometry was affected predominantly at the G1/early S stage. Uptake of labeled thymidine into acid-insoluble material of hypoxic cells was below that of controls whereas incorporation of uridine exceeded that of normoxic controls. Supplementation of cells cultured under 0.1 and 1% O2 with 0.1 mM uridine or 0.1 mM deoxycytidine + 0.01 mM deoxyadenosine and deoxyguanosine improved all growth parameters; deoxynucleosides were more effective than uridine in cells under 0.1% O2 whereas in cells cultured under 1% O2 similar effects of both were observed. This points to an insufficient supply of nucleic acid precursors even under moderate limitations of oxygen tension and not only under strict hypoxia. Whereas a 12-h cultivation time at 0.1% O2 hardly impaired cell growth after reoxygenation, a cultivation time of 24 h considerably reduced the cellular capability to recover. This was alleviated by addition of (deoxy)nucleosides from the beginning of hypoxic culture. The results are interpreted as supporting the concept that the biosynthetic pathway of pyrimidine (deoxy)nucleotides--because of two oxygen-dependent enzymes, dihydroorotate dehydrogenase and ribonucleotide reductase--is a potential transducer of environmental limitations in oxygen tension to the proliferative capacity of cells.

Animals↗
PubMed1996

Analysis of alloreactive helper T lymphocyte precursor frequencies. Influence of interleukin-2 produced by the stimulating cells.

In the determination of alloreactive helper T lymphocyte precursor frequencies (HTLpf) by limiting dilution analysis (LDA), peripheral blood mononuclear cells (PBMC) are used as stimulating cells. Interleukin-2 (IL-2) production by stimulating T cells constitute a source of error in these assays. We found that 100-150 Gy of gamma irradiation was required to abrogate IL-2 production by stimulating PBMC. This dose of irradiation, however, greatly reduced their allostimulatory capability. Here we describe how non-irradiated PBMC, immunomagnetically depleted of T cells and thus of IL-2 producing cells, can be used as stimulators in assays to determine alloresponsive HTLpf.

CD4-Positive T-Lymphocytes↗
PubMed1988

In vivo immunopotentiating activity of thymopentin in aging humans: modulation of IL-2 receptor expression.

The effect of thymopentin treatment was investigated in immunocompromised elderly subjects. Thymopentin was able to increase IL-2 production and IL-2 receptor expression, as assessed on PHA-activated blasts by percentage of Tac-positive cells and response to exogenous IL-2. After treatment, an increased precursor frequency, estimated by limiting dilution analysis, of PHA-responding lymphocytes was observed in two out of six subjects tested. In vitro experiments with thymopentin show that the drug was able to enhance blastogenesis by PHA of elderly lymphocytes but not of adult cells. These results indicate that (a) the increased IL-2 synthesis/IL-2 receptor expression may be the crucial mechanism of the immunopotentiating activity of the drug in elderly subjects and (b) an increased intrinsic T-cell responsiveness seems to be responsible for this immunopotentiating activity, although an increase in the size of the responsive T-cell pool could not be excluded.

Adjuvants, Immunologic↗
PubMed1984

Abnormal T-lymphocyte proliferation and cytotoxic responses induced by a neonatal injection of hydrocortisone: normalization by interleukin-2 addition.

Spleen cells from mice neonatally treated with hydrocortisone hemisuccinate (HHC) had an abnormal pattern of cell-mediated immunity when tested two weeks later. Mitogen-induced proliferation was reduced. The proliferative responses to allo-antigens were normal but the cytotoxic response thereby induced was impaired. The mechanisms of these anomalies are complex: reduction of cytotoxic precursors as measured by limiting dilution assays; enhancement of suppressor cells which are able to reduce the cytotoxic response of normal spleen cells; reduction of interleukin-2 (Il-2) production. The addition of exogenous Il-2 restores normal proliferative and cytotoxic responses.

Animals↗
PubMed1993

Induction of CD8+ cytotoxic T cells by immunization with killed influenza virus and effect of cholera toxin B subunit.

The MHC class I cytotoxic T-lymphocyte (CTL) response in mice given formalin-inactivated influenza whole-virus vaccine (WVV) with or without cholera toxin B subunit (CTB) was studied. Intraperitoneal injection of Balb/c (H-2d) mice with high doses of A/Taiwan/1/86 (H1N1) WVV stimulated influenza A virus-specific CTL response in a dose-dependent manner. A dose of 4.4 or 44 micrograms induced CTL response equal to or greater than live influenza virus infection. Coadministration of vaccine with 5 or 25 micrograms of CTB resulted in a higher level of CTL than with vaccine alone. CTL lysed A/Taiwan and A/Shanghai (H3N2) virus-infected class I-expressing P815 (H-2d) but not virus-infected EL-4 (H-2b) target cells nor B/Yamagata virus-infected target cells. Virus-infected MHC class II- and class I-expressing A20 (H-2d) targets were also lysed. Depletion of Lyt-2+ (CD8+) T cells with monoclonal antibody completely abrogated lysis of P815 target cells and resulted only in a slight reduction of lysis of A20 target cells. Depletion of L3T4+ (CD4+) T cells or NK cells had minimal effect on lysis of either P815 or A20 target cells. Using limiting dilution analysis, the precursor CTL (pCTL) frequency paralleled CTL activity. Significant CTL activity was detected 7 months after immunization. These results demonstrate that adequate doses of influenza WVV with or without CTB can induce long-lasting influenza A cross-reactive MHC class I-restricted CD8+ CTL response in mice. Thus, coadministration of influenza WVV with CTB may lead to an effective vaccine that stimulates both CTL and antibody responses.

Animals↗
PubMed1989

Mechanisms by which a phorbol ester and a diacylglycerol analog inhibit hen granulosa cell steroidogenesis.

We have previously reported that treatment of hen granulosa cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), or the diacylglycerol analog, 1-oleoyl-2-acetylglycerol (OAG), attenuates the steroidogenic response to luteinizing hormone (LH) at sites both prior and distal to the formation of cyclic 3',5'-adenosine monophosphate (cAMP). The present study was designed to determine the site(s) of inhibition within the steroidogenic pathway by evaluating the effects of OAG and PMA on key enzyme systems involved in hen granulosa cell steroidogenesis: adenylyl cyclase, phosphodiesterase, the cholesterol-side-chain-cleavage (CSCC) complex and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). The adenylyl cyclase activator, forskolin (0.1 mM), stimulated a 3.3-fold increase in granulosa cell cAMP formation, and this increase was inhibited by the presence of OAG (2.5, 25 and 63 microM) in a dose-dependent manner. By contrast, a 1.8-fold increase in cAMP accumulation induced by the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM), was not altered by OAG at any dose (2.5, 25 and 63 microM). Inclusion of 25-hydroxycholesterol (2500 ng/tube) in the incubation medium in the presence of 1.0 microM cyanoketone resulted in a 10-fold increase in pregnenolone production. Increasing concentrations of OAG (2.5, 25 and 63 microM) caused a dose-dependent suppression of the conversion of 25-hydroxycholesterol to pregnenolone. On the other hand, granulosa cells incubated with 200 ng/tube pregnenolone increased progesterone production 100-fold, but this increase was not inhibited by either PMA (3.2, 32, 8.1 and 162 nM) or OAG (2.5, 25 and 63 microM). The results indicate that activation of protein kinase C can suppress the function of at least two key enzymes involved in hen granulosa cell steroidogenesis. Inhibition of adenylyl cyclase greatly reduces the steroidogenic response of granulosa cells to endocrine factors that act via increasing levels of cAMP (i.e. LH). Furthermore, a reduction in CSCC activity limits the availability of precursor required for progesterone production. These data provide additional evidence of a role for protein kinase C in modulating ovarian function in the domestic hen.

Animals↗
PubMed1995

Oligodendrocyte precursors originate from both the dorsal and the ventral parts of the spinal cord.

Oligodendrocytes have been recently claimed to originate from bilateral columns of precursors whose extension is limited to the ventral region of the neuroepithelium. We designed an experiment in which the developmental capabilities of the different regions of the ventricular epithelium could be tested. We exchanged isotopically and isochronically defined sectors of the E2 spinal cord between quail and chick embryos and followed the production and migration of oligodendrocytes by using a quail-specific cDNA probe encoding the oligodendrocyte marker Schwann cell myelin protein. We showed that oligodendrocytes are generated in vivo from both ventral and dorsal halves of the neural tube. Moreover, extensive ventrodorsal, as well as dorsoventral, migrations of oligodendrogenic cells take place during spinal cord differentiation.

Animals↗
PubMed1992

Rheumatoid arthritis synovial fluid enhances T cell effector functions.

Rheumatoid arthritis is a chronic autoimmune joint disease of unknown etiology. T cells are believed to be important in the pathogenesis of rheumatoid arthritis since they infiltrate the joints and express several activation markers, such as MHC class II and IL-2R. In this study we have elucidated the effect on freshly isolated T cells of rheumatoid arthritis synovial fluid (RA-SF), which contains in vivo produced cytokines and enzymes. The mouse mixed lymphocyte culture (MLC) has been used as a model and specific cytotoxicity was evaluated against 51Cr-labelled sensitive target cells. Studies have shown that RA-SF contains a B cell differentiation activity that can cross-react between the human and murine species. Here we have shown that the addition of RA-SF strongly potentiates cytotoxic activity as well as lymphokine production by allogeneic activated effector T cells. The enhanced cytotoxicity induced by RA-SF was found to be due to a combined effect of increased cytotoxic T lymphocyte (CTL) precursor frequency, measured by limiting dilution analysis, and a more efficient killing on a per cell basis. Kinetic studies show that RA-SF must be added within 48 h after initiation of the MLC, otherwise the effect is lost. The target cell specificity of RA-SF was studied, using enriched CD4+ or CD8+ responder cells in the MLC. It was found that RA-SF could act directly on the CD8+ cells and potentiate their development to cytotoxic effector cells: this activity was not found when CD4+ responder cells were used instead. RA-SF could, on the other hand, greatly enhance IL-2 production by CD4+ responder cells. We suggest that B and T cell activity in RA-SF is important in the propagation of chronic inflammation in the joints of patients with rheumatoid arthritis.

Animals↗
PubMed1989

Activation of masked neural determinants in amphibian eggs and embryos and their release from the inducing tissue.

The neural-archencephalic (forehead) inducing activity of the microsomes and the high speed supernatant from Xenopus laevis ovaries and eggs was tested on gastrula ectoderm of Triturus alpestris. Both fractions have a very small inducing activity which increases considerably after autolysis or treatment with dissociating agents. A comparison with the inducing activity of the high speed supernatant from gastrulae suggests that neural inducing factors are synthesized in the ovary, stored in a masked form and activated, in part, during gastrulation. When the supernatant proteins from eggs and gastrulae were subjected to size-exclusion HPLC the neural inducing activity was eluted in different size classes, suggesting a limited proteolysis of a precursor during early embryogenesis. Further experiments have shown that treatment of the blastoporal lip from early Triturus gastrulae with actinomycin D or cycloheximide which inhibit the synthesis of RNA and protein respectively, diminishes the neural-archencephalic (forehead) inducing activity of the blastoporal lip. Treatment with actinomycin D or cycloheximide followed by ethanol, which, besides other structures, impairs the plasma membrane, does, however, not reduce the inducing activity. This may suggest that components which are needed for the transport of the neuralizing factor are diminished, when the synthesis of mRNA and protein is inhibited. Such components are probably needed for the release of the neuralizing factor from the blastoporal lip, the inducer tissue.

Animals↗
PubMed1995

Role of inflammation in respiratory tract infections.

In respiratory tract infections, leukocytes are recruited to the lungs, where they help remove invading organisms through phagocytic clearance. In some instances, however, the normal inflammatory response may be as destructive as it is defensive. The neutrophils secrete a proteolytic enzyme, neutrophil elastase, which has emerged as a major culprit in the pathogenesis of inflammatory airway disease. This enzyme has been found to strip the bronchial epithelium, reduce ciliary beating, and stimulate excess mucus secretion, leading to mucus retention, bacterial proliferation, and recurrent infections. Neutrophil elastase also stimulates epithelial cell interleukin-8 secretion and produces other chemoattractant cleavage products that lead to further neutrophil recruitment. It also impairs host defenses by damaging the major opsonophagocytic receptor on the neutrophil and by weakening the efficacy of immunoglobulins. For this reason, an important goal in the treatment of respiratory tract infections should be to prevent or shorten the duration of neutrophil elastase release. A number of different approaches have been proposed or attempted in an effort to modulate the proteinase burden. These include direct inhibition of elastase and interference with the recruitment, adherence, and degranulation of neutrophils. An even more fundamental approach would start in the bone marrow, where attempts might be made to modulate or modify neutrophil precursors so as to limit the destructive potential of the mature neutrophils.

Bone Marrow Cells↗
PubMed1998

Optimisation of the conditions for generating human DC initiated antigen specific T lymphocyte lines in vitro.

Naive T lymphocytes specific for a given primary antigen occur in low frequencies and require the relevant antigen to be presented by specialist antigen presenting cells (APC), i.e., dendritic cells (DC). For these reasons, the in vitro induction of primary T lymphocyte responses remains a significant technical challenge. We have attempted to improve current strategies for generating in vitro responses by optimising (i) isolation and concomitant activation of DC from peripheral blood, (ii) uptake, processing and presentation of antigen by DC and (iii) antigen driven T lymphocyte proliferation. We established that RPMI 1640 media supplemented with 10% autologous serum resulted in the best yield of CMRF-44+, CD14-, CD19- DC after enrichment over a Nycodenz gradient. Optimal presentation of whole protein and peptide antigen was achieved by addition after the purification of the APC, i.e., at the start of the T lymphocyte proliferation assay. RPMI 1640 supplemented with 10% autologous serum or plasma supported the best antigen driven specific T lymphocyte responses. Using these optimised conditions, we compared the efficacy of PBMC and purified blood DC for priming T lymphocyte responses to the chronic myeloid leukaemia (CML) specific bcr-abl (b3a2) peptide. Peptide specific T lymphocyte responses were generated with both purified DC and whole PBMC, suggesting that T lymphocyte precursor frequency was the limiting factor in these experiments. These results will aid in the generation of human T lymphocyte lines to primary antigens, for in vitro and therapeutic applications.

Animals↗