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Biomedical subjects

J M Allen

Publications and source records attributed to J M Allen.

At least 19 recordsLinked to original sources
PubMed1992

Functional dissection of the lck proximal promoter.

The lck gene encodes a protein tyrosine kinase that participates in lymphocyte-specific signal transduction pathways. Previous studies have established that lck transcription is regulated by two distinct promoter elements termed proximal (or 3') and distal (or 5'). The proximal promoter is active almost exclusively in thymocytes and becomes inactive later during T-cell maturation. To dissect the mechanisms responsible for lck gene regulation, we generated transgenic animals bearing 5' truncations in the proximal promoter element. Sequences between -584 and +37 with respect to the proximal promoter transcription start site act to direct tissue-specific and temporally correct transcription of either a tagged version of the lck gene itself or a heterologous reporter sequence (lacZ). This region contains binding sites for at least five distinct nuclear proteins, of which one is found only in cells that support proximal lck promoter activity and a second appears only in nonexpressing cells. Interestingly, the transcribed region of the lck gene contains positive control elements that can substantially boost expression from minimal (-130 bp) proximal promoter constructs. These results provide a basis for the biochemical dissection of transcriptional regulators that act at defined points during T-cell development.

Animals↗
PubMed1991

Decreased beta 2-adrenergic receptor mRNA expression in receptor-deficient S49 lymphoma cells.

Many variants of the S49 mouse lymphoma cell have been isolated along the pathway of cyclic AMP generation and response. Two such variants, beta p and beta d, were isolated by Johnson and colleagues and described in 1979 [Mol. Pharmacol. 15:16-27 (1979)]. The beta p and beta d variants express one half and one quarter, respectively, of the wild-type number of beta 2-adrenergic receptors. This observation has now been extended through the use of DNA-excess solution hybridization. Using this exquisitely sensitive technique for quantitation of gene and mRNA, we have been able to demonstrate that the beta 2-adrenergic receptor-deficient variant cells contain the same quantity of the beta 2-adrenergic receptor gene as the wild-type cells. In contrast, the beta 2-adrenergic receptor-deficient variant cells express reduced quantities of beta 2-adrenergic receptor-specific mRNA. The amount of beta 2-adrenergic receptor-specific mRNA correlates very well with the reduction in receptor expression in these cells. Both gene and mRNA in the wild-type and variant cells appear to be the same size, as judged by Southern and Northern analysis. Thus, the diminution of beta 2-adrenergic receptors in the beta p and beta d variants appears to reflect primarily the relative paucity of gene transcripts in the variant cells. These data imply that variations in cellular content of beta 2-adrenergic receptor mRNA, which may occur among closely related cells, is one explanation for differences in receptor number.

Animals↗
PubMed1979

Protection of granulocytes by vitamin E in glutathione synthetase deficiency.

The administration of vitamin E (alpha-tocopherol) was found to improve polymorphonuclear leukocyte function in an infant with congenital deficiency of glutathione synthetase activity. Before therapy with vitamin E the abnormal leukocytes exposed to phagocytic challenge showed oxidant damage. They released 60 per cent more hydrogen peroxide than did normal leukocytes, iodinated only 20 to 25 per cent of the normal number of particles, and were unable to kill bacteria as effectively as normal leukocytes although the rates of phagocytosis were normal. These functional abnormalities disappeared when the patient was placed on 400 IU of alpha-tocopherol daily for three months. Associated with the functional improvement was a normalization of microtubule assembly during phagocytic challenge.

Blood Bactericidal Activity↗
PubMed1979

Oxidative damage to neutrophils in glutathione synthetase deficiency.

Several episodes of neutropenia were observed in a child with glutathione synthetase deficiency (5-oxoprolinuria). Studies of the patient's glutathione-deficient neutrophils were undertaken to examine the responses of the cells to oxidative stress associated with phagocytosis. The patient's neutrophils contained 10--20% of normal glutathione content. Circulating neutrophils in infection-free periods appeared less mature than normal by morphologic criteria, suggesting increased cell turnover. The cells ingested particles, responded to chemotactic stimuli, and oxidized 1-14C glucose normally. However, following ingestion of particles, the cells accumulated excess hydrogen peroxide compared with normal cells, and showed impaired protein iodination and bacterial killing. Electron micrographs revealed damage to microtubules and membranous structures in the patient's neutrophils during phagocytosis. The level of glutathione in the cells appears inadequate to protect against peroxide generated during normal cell function, and the cells are thus damaged and rendered less effective in bacterial killing. The data provide evidence for a protective role of glutathione in normal neutrophil function.

Chemotaxis, Leukocyte↗
PubMed1979

Oxidative metabolic responses of rabbit pulmonary alveolar macrophages.

During phagocytosis of opsonized lipopolysaccharide-coated paraffin oil droplets, rabbit alveolar macrophages reduced nitroblue tetrazolium, which effect was in part inhibitable with the use of superoxide dismutase. Exposure of cytochalasin-B-treated rabbit alveolar macrophages to opsonized zymosan led to the generation of superoxide, as quantitated by ferricytochrome C reduction. It was found that nitroblue tetrazolium in the presence of ferricytochrome C could in turn serve as scavenger of superoxide during stimulation of cytochalasin-B-treated rabbit alveolar macrophages. Following challenge with either opsonized zymosan or the membrane perturbant digitonin, rabbit alveolar macrophages released hydrogen peroxide into the extracellular medium. Employment of the surface membrane stimulant phorbol myristrate acetate led to activation of the hexose monophosphate shunt, which activity could be further enhanced in the presence of superoxide dismutase or attenuated in the presence of catalase. These studies demonstrate that rabbit alveolar macrophages release superoxide and hydrogen peroxide during surface membrane perturbation. In turn, hydrogen peroxide generation can stimulate the hexose monophosphate shunt.

Animals↗
PubMed1978

In vitro functional capabilities of canine polymorphonuclear neutrophils collected simultaneously by continuous-flow centrifugation and continuous-flow filtration leukopheresis.

Polymorphonuclear neutrophils were simultaneously collected from dogs by continuous-flow centrifugation and continuous-flow filtration leukapheresis. In vitro studies were performed on cells obtained by the two methods as well as on control cells. Studies consisted of assessment of phagocytic capacity, degranulation, chemotaxis, hexose monophosphate (HMP) shunt activity, and bacterial killing. The cells obtained from the filter were metabolically more active than those harvested by centrifugation, as evidenced by increase in resting HMP shunt activity and dimunition in total available lysozyme-secreting activity compared to centrifuged cells. Despite their impaired phagocytic capacities, the filtered cells were able to kill Staphylococcus aureus as efficiently as the centrifuged cells. Both cell populations responded to chemotactic gradients equally.

Animals↗
PubMed1978

Improvement of polymorphonuclear leucocyte oxidative and bactericidal functions in chronic granulomatous disease with 4-amino-4'-hydroxylaminodiphenyl sulphone.

In an effort to restore oxidant-dependent capabilities to chronic granulomatous disease (CGD) polymorphonuclear leucocytes (PMN), we studied a dapsone derivative, 4-amino-4'-hydroxylaminodiphenyl sulphone (DDS-NOH), known to generate H2O2. After incubation of CGD PMN with 0.2 and 1.0 mM DDS-NOH for 30 min, the rate of glucose-1-14C oxidation via hexose monophosphate (HMP) shunt increased 2--4-fold and that of iodination of ingested zymosan particles 1.5--2.7-fold. Both effects could be further enhanced by superoxide dismutase (SOD) but inhibited by catalase. In three patients, 0.2 mM DDS-NOH improved in vitro killing of Staph. aureus. DDS-NOH 0.02 mM induced capping of Concanavalin A (Con A) receptor complexes suggesting interference by the drug with microtubule-associated function. Thus, optimal concentrations of DDS-NOH may be employed as an oxidant to improve metabolic and bactericidal functions of PMN from patients with CGD.

Aminobiphenyl Compounds↗
PubMed1978

Role of microtubules in granulocyte adherence.

The adherence of human polymorphonuclear leukocytes (PMN) to nylon fibers is inhibited in a dose-dependent fashion by exposure in vitro of these cells to either colchicine or VM-26, both of which agents prevent microtubule assembly. Mean adherence of human PMN was 48% +/- 2%, following treatment with 10(-5) M colchicine or 10(-4) M VM-26 it was reduced to 31% +/- 2% and 7%, respectively. Peritoneal PMN obtained from mice and mink with Chediak-Higashi syndrome (CHS) thought to have a microtubule-membrane disorder affecting the PMN had a mean adherence of 29% +/- 3% and 40% +/- 8% compared to control values of 46% +/- 5% and 73% +/- 8%, respectively, from the mice and mink. Both ascorbic acid and bethanechol, shown previously to enhance microtubule assembly in humans with CHS, normalized granulocyte adherence in PMN obtained from mice with CHS. Cyclic nucleotide levels were not altered by treatment of human PMN with colchicine, nor did they differ between normal and CHS animals. Thus it appears that the state of microtubule assembly may directly affect the properties of the PMN plasma membrane without requiring alterations of cyclic nucleotides as an intermediary.

Animals↗
PubMed1978

Potentiation of polymorphonuclear leukocyte motile functions by 2,3-dihydroxybenzoic acid.

DHB potentiates PMN phagocytosis of opsonized lipopolysaccharide-coated PO droplets by 53% to 45% and chemotaxis by 36% to 10% at 10-3M and 10-4M concentrations, respectively. The effect is reversible in vitro. In the presence of DHB, PMNs released significantly less superoxide and H2O2 to the extracellular media but were able to oxidize 1-14C-glucose normally during phagocytosis. Although iodination of opsonized particles was decreased in the presence of DHB, the PMNs were able to kill Staphylococcus aureus 502A normally. These results support the hypothesis that directed cell movement and phagocytosis can be enhanced by protecting the PMN membrane against auto-oxidative damage from scavenging superoxide and H2O2 by means of DHB.

Cell Movement↗