The national medical effectiveness research initiative. The search for what really works in treating common clinical conditions.

In 1989, Congress established the Agency for Health Care Policy and Research within the U.S. Public Health Service and gave it the lead in a national effort to produce reliable evidence of which medical treatments are effective and which are not. The research program addresses the effectiveness of different available strategies for diagnosis, prevention, treatment, and management of common conditions, including diabetes, and emphasizes outcomes that matter to patients. The modest size of this program and the eagerness for its results have led to the development and use of innovative research methods. Illustrations of these methods and early findings are provided.

Spermidine labels proteins during sea urchin embryogenesis.

We have previously described the presence of a protein containing intact, covalently bound spermidine during very early embryogenesis of the sea urchin (Strongylocentrotus purpuratus). Proteins containing other polyamine metabolites also appear as embryogenesis proceeds. These proteins which contain label derived from exogenous radioactive spermidine show a characteristic pattern which changes during the course of embryonic development. We document for the first time that hypusine, the polyamine metabolite which is a characteristic component of the eukaryotic protein translation initiation factor eIF-4D, is present in more than one species of macromolecule. In addition, N1-acetylspermidine has also been identified as a significant intracellular metabolite of spermidine during embryogenesis.

Polyamines and their derivatives as modulators in growth and differentiation.

The polyamines and their derivatives are essential for life in eukaryotic and most prokaryotic cells, but their exact role in preserving cell function is not clear. These polyamines provide endogenous cations and thus participate in regulation of the intracellular pH; in addition, polyamine derivatives modulate cell growth and differentiation. The naturally occurring monoacetyl derivatives can induce increased activity of ornithine decarboxylase, the first enzyme in polyamine synthesis, and thus produce positive feedback to their production. The diacetyl derivatives of putrescine and of the synthetic analogue, 1,6-diaminohexane, induce differentiation and inhibit growth in many types of cells in vitro. In addition, they inhibit the proliferative and secretory response of normal B lymphocytes to B-cell mitogens and reduce production of antibodies in vitro. They also inhibit the proliferation of chronic lymphocytic leukemia cells (a B-lymphocyte leukemia). The parent polyamines are post-translational modifiers of proteins, and hypusine, a derivative of spermidine, is a covalently bound constituent of the eukaryotic protein synthetic initiation factor, eIF-4D. Although these various actions do not at present fall into a coherent pattern, they clearly indicate that polyamines and their derivatives play an important part in modulating cell proliferation and differentiation.

Polyamine metabolism in chronic lymphocytic leukemia.

We have previously shown that polyamines have profound effects on lymphocyte proliferation and function. We now report that the proliferative response of cultured lymphocytes from patients with chronic lymphocytic leukemia is abrogated by the addition of diacetyldiaminohexane (HMBA), the 6-carbon analogue of diacetylputrescine. In addition, this study demonstrates the post-translational modification of proteins by polyamines in CLL-lymphocytes, a decrease in the uptake of exogenous spermidine by CLL-lymphocytes which have been exposed to HMBA, and the significant conversion of spermidine to N1-acetylspermidine in CLL-lymphocytes.

Polyamines in murine splenic lymphocytes.

Diacetyldiamines cause compromised B-lymphocyte function, as evidenced by our previous demonstration of inhibition of mitogen activation and decreased secretion of immunoglobulin in murine spleen cultures. In this study, we report that putrescine and spermidine are differentially metabolized by the cell. Diacetyldiamine, which is also taken up by these cells and metabolized, causes a striking decrease in cell uptake of exogenous putrescine and spermidine. We also report for the first time that several distinct macromolecules containing radioactive polyamines may be resolved, and that hypusine is present in more than one species of macromolecule.

Spermidine is bound to a unique protein in early sea urchin embryos.

Spermidine is rapidly taken up and becomes bound to protein during the very early hours of sea urchin embryogenesis. During the first 6 hr after fertilization of freshly obtained sea urchin eggs (Strongylocentrotus purpuratus), which are incubated in the presence of exogenous [3H]-spermidine, up to 7% of the total cell-associated spermidine appears uniquely as spermidine bound in macromolecular form. This unique protein containing spermidine migrates as a single radioactive band in gel electrophoresis. It has a Mr of approximately equal to 30,000 and is readily distinguishable from the protein initiation factor eIF-4D, which has a Mr of 18,000, the only other identifiable protein known to date to be posttranslationally modified by polyamines.

Differential release of serotonin without comparable histamine under diverse conditions in the rat mast cell.

Pretreatment of mast cells with various psychotropic agents was shown to permit preferential release of serotonin without substantial release of histamine or massive degranulation. Differential release involved both endogenous, granule-stored serotonin, and exogenous radiolabeled serotonin that had been taken up by the cell. This phenomenon occurred in mast cells stimulated to secrete with suboptimal concentrations of the classic mast cell secretagogue compound 48/80, was associated with drugs of several different structures and known mechanisms of action, and could be inhibited by certain prostaglandins. Furthermore, differential release of serotonin occurred in mast cells of retired breeders without the use of drugs or other exogenous agents. Light microscopic studies of mast cells undergoing differential release showed minimal degranulation, indicating that most of the serotonin release did not occur via classic exocytosis. The ability of mast cell to selectively release serotonin, by a mechanism unlike that occurring in allergic anaphylactic secretion, constitutes one of the first instances of differential release from secretory cells, suggests a new mechanism of release of secretory products, and expands the potential role of mast cells in the pathophysiology of the body.

Polyamine metabolism in differentiating Friend erythroleukemia cells.

Changes occur in polyamine metabolism which are associated with differentiation of Friend erythroleukemia cells in culture. The intracellular distribution of the polyamines changes, the absolute level of each of the major polyamine falls, and the uptake and metabolism of exogenous putrescine is altered during the course of differentiation. We have examined the labeling of putrescine metabolites after the administration of radioactive putrescine, either as a single dose added at the onset of induction, or as a pulse label added at various times during the course of differentiation. In these experiments we measured both acid-soluble and acid-precipitable radioactivity. The conversion of putrescine to gamma-aminobutyric acid is enhanced in the cell induced to differentiate, and the acid-precipitable fraction rises as the percentage of total cell-associated radioactivity in the induced cell. We have identified hypusine [N-epsilon-(4-amino-2-hydroxybutyl)lysine] as a major radioactive component of a single radioactive Mr 18,000 protein band in both the noninduced and the induced cells.

Identification of a glycosidase activity with apparent specificity for 2-deoxy-D-glucose in glycosidic linkage.

2-Deoxy-D-glucose (dGlc) is a carbohydrate with significant activity as an inhibitor of glucose metabolism and as a precursor in the synthesis of glycosylated macromolecules; several of the enzymes associated with its metabolism remain uncharacterized. In the present report, the partial purification and some of the properties of a mammalian enzyme that appears to be relatively specific for the hydrolysis of dGlc bound in glycosidic linkage is described. The physiological function of this enzymatic activity is unknown. In addition, dGlc has been shown to be taken up by HTC cells in culture and incorporated into macromolecular bound form, both as dGlc and as 2-deoxygalactose which is formed from dGlc.

Acetylated diamines inhibit endotoxin-induced lymphocyte activation.

Fully acetylated diamines hexamethylenebisacetamide (HMBA) and diacetylputrescine (tetramethylenebisacetamide, TMBA) cause inhibition of murine B lymphocyte activation in the absence of ruminant serum. Mitogenesis induced by LPS, LAP, and 8-BrcGMP was profoundly inhibited in the presence of 3 mM HMBA and TMBA. PHA-stimulated thymidine uptake was not affected. Higher levels of the acetylated polyamines were toxic to lymphocytes. Immunoglobulin secretion by LPS-stimulated spleen cells was inhibited in similar fashion. This inhibition requires the early addition of the polyamines and does not appear to be associated with direct inhibition of DNA synthesis. These data support the hypothesis that polyamines represent immunomodulatory agents and suggest that acetylated derivatives of polyamines may also have activity in regulating B lymphocyte activation.

GABA from putrescine is bound in macromolecular form in keratinocytes.

We report for the first time that the neurotransmitter gamma-aminobutyric acid (GABA) exists in macromolecular form in keratinocytes. GABA derived from putrescine (Pu) has been identified as a component of acid-precipitable material of cultured human keratinocytes. Confluent, stratified cultures of human foreskin keratinocytes exposed to [3H]-Pu for 4 hours took up about 14% of the radioactivity from the medium and 1% of the total cell-associated radioactivity was precipitable by trichloroacetic acid (TCA). Both attached and shed cells were examined by HPLC for Pu and its radioactive metabolites in TCA-insoluble and TCA-soluble fractions. GABA accounted for the major portion (54%) of the radioactivity derived from Pu in the TCA-precipitable material of attached keratinocytes. Pu and spermidine represented lesser amounts, 35% and 9% respectively, of the total TCA-precipitable radioactivity. In addition, a large portion of acid soluble radioactivity derived from Pu (63%) was GABA, whereas Pu and spermidine represented 29% and 6% respectively of the total TCA-soluble radioactivity. The exact origin of GABA in acid-precipitable material, as well as its form of attachment, is currently under investigation.

Endocrine function in small cell undifferentiated carcinoma of the lung.

The endocrine status of 106 patients with undifferentiated small cell carcinoma of the lung was evaluated before treatment was begun. Almost one half of the patients had evidence of abnormal control of the secretion of adrenal cortical steroids, manifested by loss of diurnal rhythmicity or dexamethasone suppressibility. Only two had the clinical syndrome of ectopic ACTH secretion. Evidence of inappropriate secretion of vasopressin was found in 38% of the patients, most of whom also had abnormalities of corticosteroid secretory pattern. About one half of the patients had evidence of abnormal glucose tolerance, and many also had a paradoxical rise of plasma growth hormone concentration after glucose administration. The levels of the other hormones studies were normal. The pattern of hormone abnormality observed in these patients appears to be relatively specific for small cell undifferentiated carcinoma, and is different from that observed in other pulmonary tumors. Patients with abnormal control of plasma cortisol had a worse prognosis than those with normal adrenal function, largely because of decreased response rates to chemotherapy. Other endocrine abnormalities were of no prognostic significance.

Affinity chromatography with specific antibody increases activity and retains antigenicity of ornithine decarboxylase.

An affinity chromatography column with antibody specific to ornithine decarboxylase (ODC) was employed successfully for the retention and subsequent partial elution of ODC with enhanced activity and retention of antigenicity. This may be a useful procedure for further attempts to produce antiserum for identification and characterizations of ODC in vitro and in vivo.

Factors modulating the activity of ornithine decarboxylase in rat HTC cells.

Dibutyryl cyclic AMP (dibu-cAMP), N1-acetylputrescine and both N1-acetylspermidine and N8-acetylspermidine regularly induce ornithine decarboxylase activity in both a time- and a concentration-dependent manner when added to HTC cells in suspension culture. N,N'-Diacetyl-1,6-diaminohexane and N-acetyl-1,3-diaminopropane, aliphatic analogues of acetylated putrescine containing the carboxamide bond but not known to occur naturally in animal tissues, are also effective inducing agents. The addition of 10(-2)M putrescine to the cell culture at the time of maximal induction of ODC in HTC cells by dibu-cAMP causes a marked decrease in the apparent half-life of ODC in less than 10 minutes. Ten-fold concentration of HTC cells at the time of their maximal induction by dibu-cAMP also results in an immediate decrease both in the specific activity of ODC and in its apparent half-life. When the population density of induced HTC cells is increased the relative effect of exposure of cells to increasing levels of putrescine is lessened. Some of the implications of these observations are discussed.