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Biomedical subjects

M Hill

Publications and source records attributed to M Hill.

At least 307 records · Page 17Linked to original sources
PubMed1990

Autocrine factor-independent growth of mammalian fibroblasts established in fully synthetic medium: no v-onc requirement in establishment.

In a previous study Chinese hamster fibroblasts carrying a partially deleted v-src were established in a synthetic medium lacking macromolecular supplements and shown to possess a particular serum-free phenotype hereafter designated sf. In cloning efficiency assays, sf, unlike wild-type, fibroblasts required a threshold cell density to grow from single cells, suggesting autocrine stimulation. In the present study a conditioned medium harvested from sf cells was added to the same sf cells, and the resulting cloning density was found to markedly diminish rather than increase. Sf cells were found to be unable to grow at cloning density because of trypsin damage: sf cells seeded into trypsin inhibitor-containing medium cloned with no requirement for threshold cells and were therefore independent of autocrine secretion from neighboring cells. Their cloning efficiency reached 7.7%; this value could not be improved by subcloning the sf culture, and it diminished when selenium was not added to the assay medium. To determine whether v-src is involved in the sf phenotype, five clones of the parental Chinese hamster fibroblast line not infected with Rous sarcoma virus were explanted into serum-free cultures with no macromolecular additives as in the case of v-src-containing cells. Each clone gave rise to an sf cell line growing indefinitely in synthetic medium like the v-src-containing sf cells, showing that the v-src gene is not required either for the establishment or maintenance of the sf phenotype.

Animals↗
PubMed1990

Transformation-defective mutants with 5' deletions of the src gene are frequently generated during replication of Rous sarcoma virus in established quail fibroblasts.

Replication of Rous sarcoma virus (RSV) in avian fibroblasts leads to the generation of replication-competent variants that are defective for cell transformation (td virus). These td variants contain deletions affecting various portions of the v-src gene. We compared the rate of td virus production in Q3B cells, a quail cell line established by mutagen treatment, and in normal quail fibroblasts. Twenty-five days after infection with an RSV stock containing only transforming virions, Q3B cells harbor similar amounts of v-src-containing and v-src-deleted proviruses. However, these cells synthesize very low levels of p60v-src and generate large excess of td variants, as determined by biological assays. Unlike Q3B cells, normal quail fibroblasts infected with the same virus stock produce td variants only after multiple passages of undiluted virus on fresh cells. Restriction analysis showed that the td virus produced by Q3B cells is composed of two types of genomes: one lacking the entire v-src gene and the other carrying partial deletions of this gene predominantly located in the amino-terminal portion of the coding region of v-src. To study the mechanisms of these partial deletions, we molecularly cloned and sequenced the v-src genes of several td proviruses. We show that these mutants carry single or multiple v-src deletions of limited size, presumably generated by multiple mechanisms. Two deletions of 170 and 112 bp located in the 5' portion of v-src are frequently generated during RSV replication in Q3B cells and may represent preferential sites for v-src deletion in these cells.

Animals↗
PubMed1990

Clock mutations alter developmental timing in Drosophila.

The developmental time of period mutants in Drosophila melanogaster was monitored under different environmental conditions. We observed that the pers mutants, which have short 19 h circadian cycles, develop faster from eggs to adult than the wild-type: perL mutants, which have long 28 h circadian rhythms, complete development more slowly than the wild-type. These results suggest that endogenous timers may be involved in regulating the development time of D. melanogaster.

Analysis of Variance↗
PubMed1990

Nucleotide sequence of mouse L19 ribosomal protein cDNA isolated in screening with tre oncogene probes.

A cDNA library was prepared from cytoplasmic poly(A)RNA from mouse NIH-3T3 cells carrying a transfected human tre oncogene. Screening with tre gene probes identified a tre cDNA clone 11-4 and a co-purifying weakly hybridizing cDNA clone 11-5. The 11-5-specific RNA was expressed in both nontransfected and tre-transfected NIH-3T3 cells, showing it is of mouse rather than tre gene origin. Its nucleotide sequence was 717 bp long and contained, starting from the first nucleotide, an open reading frame of 588 bp followed by a 3' noncoding region and 26 A residues at the 3' terminus. Comparison with the GenBank data base revealed 93.7% homology with cDNA encoding the rat L19 ribosomal protein. Furthermore, the 196-amino-acid polypeptide deduced from 11-5 was of the same length and contained only one amino acid difference compared with the rat L19 protein. Comparison with the weakly hybridizing tre gene probe showed stretches of homology that were, however, too short to be taken into consideration. We conclude that the 11-5 sequence encodes the mouse L19 ribosomal protein.

Amino Acid Sequence↗
PubMed1990

Ascitic fluid from human ovarian cancer patients contains growth factors necessary for intraperitoneal growth of human ovarian adenocarcinoma cells.

Human ovarian cancer, the leading cause of death from gynecologic malignancy, tends to remain localized to the peritoneal cavity until late in the disease. In established disease, ascitic fluid accumulates in the peritoneal cavity. We have previously demonstrated that this ascitic fluid is a potent source of in vitro mitogenic activity including at least one unique growth factor. We now report that the human ovarian adenocarcinoma line, HEY, can be induced to grow intraperitoneally in immunodeficient nude mice in the presence (23/28 mice), but not absence (0/21 mice) of ascitic fluid from ovarian cancer patients. Ascitic fluid from patients with benign disease did not have similar effects on intraperitoneal growth of HEY cells (1/15 mice). Once tumors were established by injections of exogenous ascitic fluid, they could progress in the absence of additional injections of ascitic fluid. The mice eventually developed ascitic fluid which contained potent growth factor activity, suggesting that the tumors eventually produced autologous growth factors. This nude mouse model provides a system to study the action of ovarian cancer growth factors on tumor growth in vivo and to evaluate preclinically, therapeutic approaches designed to counteract the activity of these growth factors.

Animals↗
PubMed1990

Halothane increases cytosolic Ca2+ and inhibits Na+/H+ exchange in L6 muscle cells.

The effects of the general anesthetic halothane on the concentration of cytosolic free calcium ([Ca2+]i) and cytosolic pH (pHi), were investigated in L6 rat skeletal muscle cells. Basal [Ca2+]i was 169 +/- 8 nM, measured with the fluorescent Ca2(+)-indicator 1-[2-amino-5-(6-carboxyindol-2-yl)phenoxy]-2-(2'-amino-5- methylphenoxy)ethane-N,N,N',N'-tetra-acetate. Halothane (5.7 mM) increased [Ca2+]i to 225 +/- 15 nM in the presence of extracellular Ca2+, and from 137 +/- 6 nM to 179 +/- 9 nM in Ca2+ absence. This increase was dose-dependent. The anesthetic released about 50% of the releasable Ca2+ from intracellular stores. The resting pHi of L6 cells was 7.24 +/- 0.04, measured with the fluorescent pH indicator bis-carboxyethylcarboxyfluorescein. Halothane did not affect resting pHi, but inhibited cytoplasmic alkalinization by hypertonicity or cytoplasmic acidification: (1) The hypertonicity-induced alkalinization via activation of Na+/H+ exchange (to 7.50 +/- 0.08, initial rate 0.10 +/- 0.02 pH U/min) was inhibited with 5.7 mM halothane by 67%. (2) Acid-loaded cells (pHi 6.43 +/- 0.01 in cells) recovered towards neutrality via activation of Na+/H+ exchange (rate 0.47 pH U/min), and halothane inhibited the rate of pHi recovery by 50%. The halothane-mediated inhibition of alkalinizations after hypertonic exposure or acid-loading was also observed in bis-(o-amino-phenoxy)ethane-N,N,N',N'-tetra-acetate-loaded cells in Ca2(+)-free medium. Therefore, halothane increases [Ca2+]i and in parallel inhibits Na+/H+ exchange, compromising the ability of muscle cells to recover from imposed acidification.

Animals↗
PubMed1989

Activation of electropermeabilized neutrophils by adenosine 5'-[gamma-thio]triphosphate (ATP[S]). Role of phosphatases in stimulus-response coupling.

Electrically permeabilized human neutrophils were used to study the mechanism of activation of the NADPH oxidase by chemotactic factors. The respiratory burst elicited by formyl-methionyl-leucyl-phenylalanine (fMLP) was strictly dependent on the addition of ATP. The response was also supported by adenosine 5'-[gamma-thio]triphosphate (ATP[S]), but not by the non-hydrolysable analogue (p[NH]ppA). In the presence of ATP, displacement of fMLP from its receptor by antagonist peptides resulted in the abrupt termination of the O2-consumption burst. In contrast, the response persisted after displacement of fMLP when ATP[S] was present. This finding is consistent with the formation of biologically active thiophosphoproteins which are resistant to cleavage by cellular phosphatases. Accordingly, lower concentrations of ATP[S], as compared with ATP, were required to support the fMLP response. The data indicate that protein phosphatases control the extent and duration of the response in cells stimulated with chemoattractants. Unlike ATP, sub-millimolar concentrations of ATP[S] elicited a spontaneous respiratory burst in the absence of fMLP or other stimuli. This effect was inhibited by p[NH]ppA and was not observed in intact (non-permeabilized) cells, indicating interaction of ATP[S] with an intracellular adenine-nucleotide-binding site, possibly a protein kinase. These results suggest that protein kinases are active in neutrophils in the absence of exogenous stimuli, but that accumulation of the essential phosphoprotein(s) is normally prevented by the ongoing vigorous phosphatase activity. It is conceivable that control of the respiratory burst is exerted by inhibition of phosphatase activity, instead of or in addition to the more commonly postulated activation of protein kinases.

Adenosine Triphosphate↗
PubMed1989

Physiologic activation of protein kinase C limits IL-2 secretion.

Interaction of Ag, antibodies against the T cell receptor complex, or mitogenic lectins with T lymphocytes induces hydrolysis of membrane phospholipids leading to the production of diacylglycerol (DAG). DAG then activates the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Increases in DAG concentrations are transient as is the increase in PKC activity. Phorbol esters, which induce potent, prolonged activation of PKC, augment many T lymphocyte responses, including cell proliferation and secretion of the T cell growth factor IL-2. Therefore, it has been suggested that activation of PKC is a positive regulatory signal in T lymphocytes. We have determined the consequences of transient stimulation of PKC, and of depletion of PKC, on early cell activation signals and on production of IL-2 by the murine lymphoma line LBRM 331A5. When this cell line is depleted of PKC overnight incubation in high concentrations of phorbol esters, lectin-induced IL-2 secretion is augmented. Similarly, mitogen-induced changes in [Ca2+]i and phosphoinositide metabolism were augmented in these cells. In contrast, a short preactivation of PKC abrogated these early transmembrane signaling events. This suggested that normal physiologic activation of PKC may limit cell activation and decrease IL-2 production. We compared the effects of phorbol esters and mezerein, which produce prolonged activation of PKC, with those of diacylglycerol analogs, which induce transient activation of PKC. At concentrations that give similar levels of PKC activation, phorbol esters and mezerein, but not DAG analogs, increased IL-2 secretion. This suggests that prolonged, nonphysiologic activation of PKC is required to augment IL-2 secretion. Therefore, physiologic activation of PKC may not augment T cell activation but instead may function to decrease cell activation and limit IL-2 secretion.

Animals↗
PubMed1989

Recruitment of alloreactive cytotoxic T lymphocytes by an antigenic peptide.

To investigate the requirements for induction of cytotoxic T lymphocytes (CTL) by peptides we chose the 16-residue nucleoprotein peptide (NPP; 365-380) from the influenza virus A/NT/60/68 as model substrate that is recognized in conjunction with major histocompatibility complex H-2d. Here we present that CTL can be raised from naive animals by repeated in vitro stimulation with high concentrations of peptide. The frequency of this response can be boosted by immunization of the animals with NPP-conjugated to ovalbumin as a carrier. However, in contrast to NPP-specific CTL lines raised from virus-primed animals none of the peptide-induced CTL lines were able to lyse virus-infected targets. Although they did not show an apparent difference in fine specificity of the peptide recognized, their affinity to the target cells was 100-fold lower than that of CTL from virus-primed animals as estimated from the peptide concentration needed to achieve significant lysis. In addition, the activity of peptide-induced CTL was very sensitive to blocking by anti-CD8 antibodies as compared to virus-specific CTL. Furthermore, all peptide-induced CTL showed a high second reactivity for allogeneic H-2k targets. Therefore, it is argued that high epitope density achieved by high peptide concentrations can in vitro recruit lymphocytes of another specificity. For the tested peptide the reactive T lymphocytes showed high alloreactivity.

Amino Acid Sequence↗
PubMed1989

Role of protein kinase C in interleukin 1, anti-T3, and mitogenic lectin-induced interleukin 2 secretion.

Activation of T-lymphocytes by antigen, mitogenic lectins, or antibodies against the T-cell receptor complex, particularly in the presence of IL1, induces the secretion of the T-cell growth factor IL2. IL2 then has a major role in regulating the duration and magnitude of the immune response. Interaction of antigen, antibodies against the T-cell receptor complex, or mitogenic lectins with T-lymphocytes also induces hydrolysis of membrane phospholipids, leading to the production of diacylglycerol, an activator of the Ca2+- and phospholipid-dependent phosphotransferase, protein kinase C (PKC). Phorbol esters, potent activators of PKC, augment secretion of the T-cell growth factor, interleukin 2 (IL2). Activation of PKC may therefore serve as an important early event in the production and secretion of IL2. We have determined whether IL2 secretion can be induced in the murine cell T-lymphocyte line LBRM 331A5, where PKC is inhibited by staurosporine or sphingosine or in cells where PKC is depleted by prolonged incubation with high concentrations of phorbol esters. In cells in which PKC was either inhibited or depleted, antibodies against the T3 portion of the T-cell receptor complex and the mitogenic lectin phytohemagglutinin (PHA) still triggered IL2 secretion. In addition, the monokine IL1 augmented this IL2 secretion irrespective of whether PKC was inhibited or depleted. These data indicate that activation of PKC is not an obligatory step for IL2 secretion in LBRM 331A5 murine T-lymphocytes.

Alkaloids↗
PubMed1989

Ability of mammalian fibroblasts to grow in synthetic medium containing neither serum nor exogenously added macromolecules.

Rous sarcoma virus transformed Chinese hamster fibroblasts, clone CHR1-3, were established at high temperature, then subcloned. Six subclones with round and flat morphology harboring undeleted and partially deleted RSV proviruses, respectively, were seeded into serum-free synthetic medium with no macromolecular additives, and maintained for 2 mo. One flat subclone no. 14, fully designated sfCHR1-3.14 for its serum-free phenotype, was further propagated in the same medium. The cells grew exponentially in loosely attached monolayers and could be serially passaged on bare polystyrene with an average population doubling time of 46 h. Cell attachment could be improved by using collagen-coated polystyrene or by adding a methionine supplement to the culture medium. Furthermore, the sfCHR1-3.14 cells could be subcloned and further grown in nonselective medium. The reversion rate of the sf phenotype was estimated to be 1 to 2%/cell generation. Evidence for an autocrinal stimulation was obtained by cloning efficiency assays showing a requirement for a threshold cell density. Slight growth stimulation could also be detected in assays using conditioned medium from sfCHR1-3.14 cells and serum-restricted wild-type (wt)NIH3T3, but not wtCHR1-3.14, cells as indicator cells. Finally, wtNIH3T3 cells used in these assays were assayed for serum-free growth and found to be able to develop their own sf phenotype; in this respect they resemble the previously established sfCHR1-3.14 cells.

Animals↗
PubMed1989

Immunocytochemical demonstration of myosin heavy chain expression in human muscle.

Three new monoclonal antibodies are shown by immunocytochemical techniques to recognise the adult fast, slow and neonatal myosin heavy chain (MHC) isoforms in adult and fetal human muscle. In fetal muscle of 17-20 weeks of gestation, slow MHC was present only in primary myotubes. Secondary myotubes contained neonatal MHC with different levels of fast and some embryonic MHC. We confirmed the presence of tertiary myotubes in the fetal muscle (Draeger et al. (1987) J. Neurol. Sci., 81: 19-43) and show that these contained fast, neonatal and possibly some embryonic MHC. Fast MHC was therefore present in secondary and tertiary myotubes at least as early as 17 days of gestation.

Adult↗
PubMed1989

Myosin heavy chain expression in human muscle cocultured with mouse spinal cord.

Monoclonal antibodies which specifically recognise the neonatal or adult fast (type 2A and 2B) or slow myosin heavy chain (MHC) were used to investigate the myosin composition of human muscle which had regenerated in culture in the presence or absence of embryonic mouse spinal cord tissue. Adult fast MHC was found in an average of 75% of the cultured fibres, irrespective of the time in culture, the source of the muscle, the presence of neurones or modifications to the growth medium. It was not found alone but was always in association with the neonatal and/or slow myosin isoforms. The expression of fast MHC in human muscle therefore differs from that in mouse muscle in this culture system (Ecob-Prince et al. 1986), but is still strong evidence for the development of at least one aspect of an adult phenotype in culture.

Adolescent↗
PubMed1989

Are theophylline "levels" a reliable indicator of compliance?

Clinicians frequently rely on serum theophylline concentrations (STCs) as an indicator of compliance for asthma medications. Most patients with good compliance do not have excessive fluctuations during routine STC monitoring. However, our experience is that in certain patients, persistently low or erratic STCs may be a sign of abnormal theophylline disposition. With careful analysis of theophylline absorption (STC every 2 hours for 24 hours after oral theophylline doses) and elimination (serial STC for 12 hours after an intravenous dose of aminophylline), we identified several patients with previously unrecognized anomalies of theophylline pharmacokinetics. These include (1) a 16-year-old girl with consistent temporal fluctuation in STCs during administration of a sustained-release formulation every 8 hours because of delayed absorption and enhanced elimination of theophylline at night, (2) a 13-year-old girl with markedly delayed absorption of a once-daily preparation administered in the evening, (3) a 5-year-old boy with erratic absorption of a liquid theophylline preparation with significantly increased STCs during the night, and (4) a 49-year-old man with 60% bioavailability of aminophylline tablets. Based on these observations, we suggest that clinicians carefully consider the possibility of abnormalities in theophylline disposition before assuming unexpected deviations in STCs are solely the result of noncompliance.

Administration, Oral↗
PubMed1989

Regression of the malignant aspects of intraepithelial neoplasias following an LH-RH agonist treatment and detection of human papillomavirus by molecular hybridization.

Patients presenting genital intraepithelial neoplasia and/or flat condyloma were treated with DTrp6-LH-RH (triptorelin) to induce a transitory suppression of estrogens. This treatment led in some cases to a complete clinical and histological regression accompanied by a disappearance of human papillomavirus sequences as detected by molecular hybridization.

Antineoplastic Agents↗
PubMed1989

Measurement of plasma prednisolone level to evaluate a prednisone treatment failure in an adolescent with Crohn's disease.

A 14-year-old boy experienced multiple hospitalizations because of symptoms due to Crohn's disease involving the stomach, duodenum, and ileum. He maintained that oral corticosteroids were not effective for control of his symptoms. However, i.v. corticosteroids always relieved his symptoms. To resolve the question of noncompliance versus altered corticosteroid absorption or metabolism, our patient underwent an oral prednisone absorption study. Prednisolone, the active metabolite of prednisone, was measured in his plasma using a high-pressure liquid chromatography technique. The results led to the discovery of an elaborate deception by the patient and his subsequent need for psychotherapy. This report documents the importance of measuring plasma prednisolone concentrations to diagnose noncompliance, especially in adolescents who are overly concerned about their body image.

Adolescent↗
PubMed1989

Stimulation of macrophage phagocytic but not bactericidal activity by colony-stimulating factor 1.

The ability of mouse peritoneal cells to phagocytose and lyse Listeria monocytogenes was measured after the cells were incubated with purified murine macrophage-specific colony-stimulating factor (CSF-1). Activation of combined phagocytic and bacteriolytic ability required 24 h, with an optimal dose of 1,000 U of CSF-1 per ml. No activation was achieved with a shorter period of incubation, known to be sufficient for GM-CSF to stimulate phagocytosis by granulocytes, and there was no advantage in longer exposure. After 24 h in 1,000 U of CSF-1, macrophages showed visibly increased spreading on the plastic petri dish. Activated cells examined microscopically showed an increase in the number of phagocytic cells and in the numbers of bacteria per phagocytic cell. This increased phagocytic ability was evident also in the increase in the amount of radioactivity associated with the cells following a 30-min incubation with radiolabeled bacteria. When these cells were carefully washed, the percentage of this initial uptake released during the next 2 h was not increased by pretreatment of the cells with CSF-1, showing that the effect of this growth factor was on phagocytosis of the bacteria not on the killing mechanisms per se.

Animals↗