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Biomedical subjects

D A Chambers

Publications and source records attributed to D A Chambers.

At least 55 records · Page 3Linked to original sources
PubMed1983

A kinetic study of rat salivary gland alkaline phosphatase and its inhibition by cadmium.

Alkaline phosphatase, confined to myoepithelial cells and blood capillaries in rat submandibular salivary gland (SSG), may participate in the regulation of salivary flow. To determine whether the alkaline phosphatase of SSG has unique properties, comparative kinetic and inhibition studies on enzymes from SSG, intestine and kidney were performed. The Km values (at optimal pH for each tissue) of 0.34, 0.55 and 0.49 mM with p-nitrophenylphosphate for the enzymes from SSG, kidney and small intestine respectively were similar. However, in the presence of cadmium the Ki values of 0.08 and 0.12 microM for the enzymes from SSG and kidney respectively were different from the value of 1.86 microM for the enzyme from small intestine. Differences in Ki values suggest differences in biochemical properties between the enzyme from small intestine and that isolated from SSG or kidney.

Acetates↗
PubMed1982

Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling.

Cyclic AMP inhibits platelet aggregation induced by physiological agents. 8 Azido [32P]cyclic AMP (N3 cyclic AMP) has been utilized as a photoaffinity probe to define the cyclic AMP-binding proteins present in unperturbed human platelets and their subcellular fractions. Specificity of cyclic AMP binding was determined by contrasting binding in the presence and absence of excess unlabelled cyclic AMP, cyclic GMP and 5'-AMP. Binding was unaffected by 5'-AMP and obliterated by cyclic AMP. Four major species of binding proteins, 49 000, 42 000, 39 000, 37 000, were obtained in all platelet fractions (crude homeogenate, cytosol, membranes and granules). Two-dimensional gel electrophoresis of platelet cytosol resolved the major molecular weight species into 15 specific cyclic AMP binding proteins of four molecular weight classes differing by charge density. These studies suggest that platelets contain an array of specific cyclic AMP-binding proteins which may function in hemostatic regulation.

Affinity Labels↗
PubMed1982

A role for humoral factors in psoriasis.

Because a large body of evidence suggests that the presence of circulating humoral factors could play a role in the induction/maintenance/resolution of psoriasis and because few investigators have explored their potential, we elected to conduct a survey analysis of the effects of the noncellular component of blood, serum, from normal and psoriatic subjects on assays of the inflammatory/immune system and on cell proliferation. Sera from psoriatic subjects are less chemotactic for monocytes than normal; this does not appear to be secondary to excess inhibitors for the chemoattractants, ZAS and bacterial factor. Preincubation of monocytes in psoriasis sera and normal sera results in an equal degree of inhibition of subsequent chemotaxis by monocytes. Relative to media alone, both normal and psoriasis sera equally inhibit DNA synthesis of HeLa cells. The addition of psoriasis sera to mitogenically stimulated lymphocytes will selectively suppress the blastogenic response in comparison to normal sera. These differences prompted a 2-d gel electrophoresis analysis of sera of psoriatic subjects. Specific differences are noted in the group of proteins migrating in the area of alpha 1 antitrypsin. These findings suggest that the noncellular component of the humoral system must also be factored into considerations of the etiopathology of psoriasis.

Blood Protein Electrophoresis↗
PubMed1981

Involved and uninvolved skin from psoriatic subjects: are they equally diseased? Assessment by skin transplanted to congenitally athymic (nude) mice.

A highly significant, but unanswered, question in the pathogenesis of psoriasis relates to how normal appearing and diseased skin can coexist, undergo spontaneous flares and remissions, and yet appear to be genetically acquired. A plausible explanation for these disparate observations is that there is a basic defect in epidermal proliferation of skin of subjects with psoriasis and that disease expression is governed by other host factors. To address this question, we compared epidermal proliferation of skin involved and uninvolved with psoriasis with normal skin before and after transplantation to congenitally athymic (nude) mice, a biologic milieu free of humoral factors unique to the donor host. Results demonstrated that (a) before transplant, synthesis of DNA by the epidermal cells from skin uninvolved and involved with psoriasis is significantly higher than normal, 1.6 and 3.6 times, respectively; (b) 6 wk after transplantation, synthesis of DNA by epidermal cells is unchanged for normal skin, increased for uninvolved skin, and decreased for involved skin. These increases and decreases are of such a magnitude that at 6 wk the number of epidermal cells synthesizing DNA per 1,000 basal cells is identical, and is 2.2 times that of normal skin. When removed from the milieu of the afflicted host, skin involved and uninvolved with psoriasis appear equally "diseased." These data support the notion that there is aberrant epidermal proliferation in skin of patients with psoriasis and that host factors appear to play a role both in the expression and nonexpression of this disease.

Adult↗
PubMed1980

Epidermal proliferation of nude mouse skin, pig skin, and pig skin grafts. Failure of nude mouse skin to respond to the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate.

Human skin transplanted to nude mice offers a possible experimental system for the study of normal epidermal proliferation and differentiation, and for their pathological counterparts. Crucial to the development of such a system is the demonstration that such grafts retain the responsive features of donor skin. To document that donor proliferative characteristics are maintained in the grafts, a comparative analysis of agents that induce proliferation was made on skin of mice homozygous and heterozygous for nude, on pig skin, and on pig skin transplanted onto nude mice. A wave of epidermal proliferation could be induced in pig skin and pig skin grafted onto nude mice, but not in nude mouse skin after the topical application of 10 ng 12-O-tetradecanoyl phorbol 13-acetate (TPA). A 10-fold greater concentration of TPA or 5% croton oil induced proliferation in all species of epidermis studied. Mice, heterozygous for nude, showed a normal response to 10 ng TPA, suggesting that the ability to respond to TPA may be related, in part, to a recessive genetic trait. Nude mouse skin transplanted to a heterozygous littermate capable of responding to 10 ng TPA does not respond. These observations argue that: the graft retains its donor proliferative characteristics when transplanted to the nude, and the inability of the nude mouse to respond to lower doses of TPA may be related to absorption, the nude gene(s), or an inherent threshold to response. The lack of response to the promoter TPA provides a plausible explanation for the decreased incidence of tumors arising in nude mice during two-stage carcinogenesis experiments.

Animals↗
PubMed1980

Identification and characterization of the adenosine 3',5'-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum.

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3',5'-[32P]monophosphate (8-N3-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5'-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.

Affinity Labels↗
PubMed1978

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate on concanavalin A-stimulated sterol and fatty acid synthesis in mouse spleen lymphocytes.

Substantial increases in both 3beta-OH sterol and fatty acid synthesis were observed after concanavalin A addition to mouse spleen lymphocytes cultured in serum-free media. The rate of sterol synthesis increased linearly up to 60 h. The rate of fatty acid synthesis increased up to 20 h, reaching a plateau in synthetic activity which was the maintained. CO2 production from acetate was slightly stimulated by concanavalin A. In contrast to sterol and fatty acid synthesis, the rate of CO2 production in both mitogen-stimulated and resting cultures declined with time. Dibutyryl cyclic AMP had a strong inhibitory effect on concanavalin A-stimulated sterol and fatty acid synthesis from acetate, but only a slight effect on CO2 production. Delayed addition of dibutyryl cyclic AMP resulted in reduced inhibition. The data suggest a sequence of initiation for fatty acid and sterol synthesis prior to DNA synthesis and a possible regulatory role of cyclic AMP in this initiation. The results support the hypothesis that lymphocyte activation is sequential within the spleen cell population and is accompanied by fatty acid and sterol synthesis.

Bucladesine↗
PubMed1978

Cyclic nucleotide-binding proteins detected by photoaffinity labeling in nucleus and cytoplasm of bovine liver.

A photoaffinity labeling method was used to characterize and compare cyclic nucleotide-binding proteins of bovine liver cytosol with binding proteins of the nucleus. After photoaffinity labeling of cytosol with 8-azido cyclic [(32)P]AMP, autoradiographs of sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed two major labeled proteins of 47,000 and 52,000-55,000 daltons. DEAE-cellulose column-derived fractions suggested that the larger protein was the regulatory subunit of peak II cyclic AMP-dependent protein kinase and the smaller protein was the regulatory subunit of peak I kinase. The smaller protein was largely present as the free regulatory subunit. The two binding proteins differed in their ability to bind cyclic GMP. Binding to both proteins was abolished by excess unlabeled cyclic AMP but not by 5'-AMP. Photoaffinity labeling of a 0.14 M salt extract of nuclei and a nonhistone chromosomal protein preparation revealed two major binding proteins with the same molecular weight and competition profiles as those of the cytosol. Detergent-washed nuclei gave similar results. Several minor binding proteins were observed in both cytosol and nucleus. One protein (36,000 daltons) was unique to the nucleus and had low affinity for 8-azido cyclic AMP. Photoaffinity labeling with cyclic [(3)H]GMP revealed a cytosol protein, absent from the nucleus, of 31,000 daltons and the ligand was competed for by both cyclic GMP and 5'-GMP. These studies suggest that the major specific cyclic AMP-binding proteins of bovine liver are the type I and type II regulatory subunits of cyclic AMP-dependent protein kinase and are present in both nucleus and cytoplasm.

Affinity Labels↗
PubMed1976

Regulation of cell cycles.

Of the major achievements in cell biology during the last 25 years, none is more important than the understanding of regulation of cell cycles. In 1953 two fundamental observations concerning DNA were made. Watson and Crick suggested that the three-dimensional structure of DNA exists as a double helix with specific base pairings, and Howard and Pelc observed that DNA is replicated during a specific phase in the mitotic cycle. Thus developed the theory of cell cycles. Next, investigators explored which events occur during each phase of the cycle and what controls the readout of the genes of proliferation or differentiation. In 1961, Jacob and Monod proposed that for prokaryotic cells the operon is the mechanism which controls the readout of the genes; and by the end of the 1960s, several investigators had defined the role of cyclic AMP and its mechanism of action at the gene level. The control mechanisms of eukaryotic cells are less well defined. Basically there are two types of regulatory molecules: those that arrive at the cell surface and send messages inside the cell; and those that enter the cell, bind to receptors, and then enter the nucleus to interact with the genes. During the past five to ten years, the cell surface and its receptors have received considerable attention as the recognition and control areas for cell proliferation and differentiation, and currently the role of the cyclic nucleotides and prostaglandins is being investigated. Various model systems are now available for detailed studies of these control mechanisms.

Animals↗
PubMed1969

The stimulatory effect of cyclic adenosine 3'5'-monophosphate on DNA-directed synthesis of beta-galactosidase in a cell-free system.

A cell-free system allowing for synthesis of beta-galactosidase enzymatic activity has been developed. This system requires DNA containing the beta-galactosidase gene, a cell-free extract of Escherichia coli bacteria, and the low-molecular-weight components necessary for transcription of the DNA and translation of the resulting messenger RNA. Such a system is useful for studying enzyme synthesis, as well as its regulation. The gene for beta-galactosidase is part of the lac operon whose expression is under the control of the lac repressor. In whole cells the lac repressor inhibits almost all of the gene expression for beta-galactosidase. In the cell-free system, we had previously been able to repress about half the gene expression. Adding cyclic adenosine 3'5'-monophosphate to the cell-free system improved the yield of beta-galactosidase enzymatic activity by 8 to 30 times and the efficiency of repression from 50 to 95 per cent.

Adenine Nucleotides↗