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Autografts using untreated or in vitro manipulated bone marrow and peripheral blood stem cells represent promising approaches to the treatment of malignant diseases. In this work,the collagen gel culture technique was compared with agar and methylcellulose for its capacity to permit the growth of human granulomonocytic (day 14 CFU-GM; collagen vs agar or MTC) or erythroblastic (day 7 CFU-E and day 14 BFU-E; collagen versus methylcellulose) colonies in autologous transplantation products. Our results show that the collagen culture system always gave as many or more colonies than the other techniques. It also allowed harvesting of gels onto glass slides and subsequent May-Grünwald-Giemsa,cytochemical or immunocytochemical staining. We suggest that the collagen assay represents an interesting alternative to the widely used agar or methylcellulose systems for the culture of hematopoietic progenitors because of the equal or higher number of colonies detected,the easy phenotypical identification of colonies in stained gels,and the ability to store high-quality documentation. This technique is particularly attractive for use in the quality control of autologous bone marrow transplantation procedures. View Publication

Ionomycin,a new polyether antibiotic with a high affinity for calcium ions,is obtained in pure form from fermentation broths of Streptomyces conglobatus sp. nov. Trejo by solvent extraction. It is unique amongst known polyether antibiotics in that it has a UV absorption maximum at 300 nm. thereby distinguishing it from other antibiotics of its class. The Ca salt has the molecular formula C41H70O9Ca. Ionomycin is a narrow spectrum antibiotic being active against Gram-positive bacteria. View Publication

By using immunoelectron microscopy,we have localized the binding site on 50S Escherichia coli ribosomal subunits for puromycin,an antibiotic that interacts with the ribosomal peptidyltransferase center. This was achieved by affinity-labeling 50S subunits with N-bromoacetyl puromycin and treating the labeled subunits with an antibody specific for the N6,N6-dimethyladenosine moiety of puromycin. The position of the puromycin binding site was then revealed by localizing the attachment sites of the IgG molecules on the surfaces of the 50S subunits under the electron microscope: it was located at the interface between the subunits,on and around the wider lateral protuberance of the 50S subunit. This localizes directly the peptidyl transferase center on the surface of the large ribosomal subunit. View Publication

A method has been described for the long-term culture of human bone marrow cells in liquid medium. Hematopoiesis,as measured by the production of granulocytic-macrophage progenitor cells (CFUc),continued for at least 20 weeks and was dependent upon the presence of a marrow-derived adherent layer of cells. As in the case of murine marrow liquid cultures,the adherent layer consisted of mononuclear phagocytic cells,endothelial cells,and lipid-laden adipocytes,the latter being essential for long-term hematopoiesis. Optimal growth conditions included McCoy's medium supplemented with fetal bovine serum,horse serum,and hydrocortisone and incubation at 33 degrees C. Horse serum in conjunction with hydrocortisone appeared essential for the growth of adipocytes. View Publication

Serotonin receptor affinity and photelectron spectral data were obtained on a number of substituted N,N-dimethyltryptamines. Evidence is presented that electron-donating substituents in the 5-position lead to enhanced behavioral disruption activity and serotonin receptor affinity as compared to unsubstituted N,N-dimethyltryptamine and analogues substituted in the 4- or 6-position. Some correlation was found between ionization potentials and behavioral activity,which may have implications concerning the mechanism of receptor binding. View Publication

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We measured the free concentration of 1,25-dihydroxyvitamin D (1,25[OH]2D) using centrifugal ultrafiltration,and the level of vitamin D-binding protein (DBP) in 24 normal subjects,17 pregnant subjects,and 25 alcoholic subjects with liver disease. Our objective was to determine whether the increase in total 1,25(OH)2D levels in pregnant women and the reduction in total 1,25(OH)2D levels in subjects with liver disease reflected a true difference in free 1,25(OH)2D levels or whether such differences were due solely to the variations in DBP levels (and thus,the amount of 1,25[OH]2D bound) in these groups. In subjects with liver disease the mean total 1,25(OH)2D concentration (22.6 +/- 12.5 pg/ml) and the mean DBP concentration (188 +/- 105 micrograms/dl) were nearly half the normal values (41.5 +/- 11.5 pg/ml and 404 +/- 124 micrograms/dl,respectively,P less than 0.001),whereas the mean free 1,25(OH)2D level was similar to normal values (209 +/- 91 fg/ml and 174 +/- 46 fg/ml,respectively). In contrast,in pregnant subjects the mean total 1,25(OH)2D level (82 +/- 21 pg/ml) and mean DBP level (576 +/- 128 micrograms/dl) were significantly higher than normal (P less than 0.001). Although the mean percent free 1,25(OH)2D level in pregnant subjects was below normal (0.359 +/- 0.07% vs. 0.424 +/- 0.07%,P less than 0.001),the mean free 1,25(OH)2D level was 69% higher than normal (294 +/- 98 fg/ml vs. 174 +/- 46 fg/ml,P less than 0.001). When data from all three groups were combined,there was a linear correlation between total 1,25(OH)2D and DBP levels but not between DBP and percent free 1,25(OH)2D levels; the increased DBP levels in the pregnant subjects were associated with less of an effect on percent free 1,25(OH)2D than were the reduced DBP levels in the subjects with liver disease. Our data suggest that (a) free 1,25(OH)2D levels appear to be well maintained even in subjects with liver disease and reduced DBP levels,(b) free 1,25(OH)2D levels are increased during pregnancy despite the increase in DBP levels,and (c) free 1,25(OH)2D levels cannot be inferred accurately from measurements of total 1,25(OH)2D and DBP levels alone in subjects with various physiologic and pathophysiologic conditions. View Publication

Tamoxifen is used widely in the treatment of endocrine-responsive breast cancers in humans. Studies were undertaken to examine the biological character (estrogenic-antiestrogenic properties) and estrogen receptor (ER) interaction of the cis- and trans-isomers of tamoxifen and hydroxytamoxifen in MCF-7 human breast cancer cells. For each compound,the following parameters were monitored: affinity for ER and effects on cellular ER levels; stimulation-inhibition of cell growth,plasminogen activator activity,and cellular progesterone receptor levels; and isomer interconversion and metabolism in vitro. The relative binding affinities of the compounds cis-tamoxifen,trans-tamoxifen,cis-hydroxytamoxifen,and trans-hydroxytamoxifen for cytosol ER were 0.3,2.5,1.8,and 310%,respectively,in which the affinity of estradiol is considered 100%. cis-Tamoxifen behaved as a weak estrogen agonist in all assays,while trans-tamoxifen was an effective estrogen antagonist. cis-Tamoxifen behaved like estradiol in stimulating MCF-7 cell growth and increasing plasminogen activator activity and cellular progesterone receptor content,although very much higher concentrations of cis-tamoxifen (10(-6) M) were needed to achieve the levels of stimulation observed with 10(-10) M estradiol. trans-Tamoxifen and trans-hydroxytamoxifen suppressed cell growth,inhibited plasminogen activator activity of control cells,and suppressed estradiol-stimulation of plasminogen activator activity,and they evoked minimal increases in cellular progesterone receptor levels. trans-Hydroxytamoxifen had a 100-fold increased affinity for ER and was approximately 100-times more potent than was trans-tamoxifen in suppressing cell growth and plasminogen activator activity. cis-Hydroxytamoxifen behaved as an estrogen antagonist,suppressing cell growth and plasminogen activator activity,and it elicited submaximal increases in progesterone receptor levels. This apparently paradoxical behavior of cis-hydroxytamoxifen was shown to be due to the fact that the cis- and trans-hydroxytamoxifens readily undergo isomeric interconversion upon exposure to our cell culture conditions,resulting in substantial accumulation of the higher-affinity trans-hydroxytamoxifen in the nuclear ER fraction of cells. In contrast to the facile interconversion of the hydroxytamoxifen isomers,there is no metabolism or interconversion of the parent compounds cis- and trans-tamoxifen in vitro. Hence,by the criteria we have used,the biological characters of trans-tamoxifen and trans-hydroxytamoxifen are similar,the major difference being the approximately 100-fold enhanced potency of the hydroxylated form. In contrast,cis-t View Publication

In HeLa cells which have been exposed to 5 mM sodium butyrate for 21 h,the level of histone acetylation is greatly increased as compared to control cells (Riggs,M.G.,Whittaker,R.G.,Neumann,J.R.,and Ingram,V.R. (1977) Nature 268,462-464). Our experiments indicate that the increase in the relative amounts of multiacetylated forms of histones H4 and H3 following butyrate treatment is the result of an inhibition of histone deacetylase activity. View Publication

Central stimulant actions of 10 methylxanthines in mice correlate with affinities for adenosine receptors labeled with N6-[3H]cyclohexyladenosine. Affinities of methylxanthines for adenosine receptors are consonant with central levels attained at behaviorally effective doses. The much higher concentrations of methylxanthines required to influence benzodiazepine receptor binding do not correlate with behavioral potency. N6-(L-Phenylisopropyl)adenosine (L-PIA),a metabolically stable analog of adenosine with high affinity for adenosine receptors,is an extremely potent behavioral depressant,reducing locomotor activity of mice at doses as little as 0.05 mumol/kg. The D isomer,which has much less affinity for adenosine receptors,is much less active as a central depressant. Theophylline stimulates locomotor activity and reverses depressant effects of L-PIA. Caffeine or 1,7-dimethylxanthine,when administered alone,elicits biphasic effects,with locomotor depression at lower doses and stimulation at higher doses. When administered with L-PIA,even low doses of caffeine produce marked stimulation. 3-Isobutyl-1-methylxanthine given alone elicits only behavioral depression. However,like theophylline and caffeine,isobutylmethylxanthine reverses the L-PIA-evoked depression,converting it into pronounced locomotor stimulation. The data strongly suggest that the behavioral stimulant effects of methylxanthines involve a blockade of central adenosine receptors. View Publication

Bay K8644 increased unidirectional Ca2+ influx and produced tension development in rabbit aorta. Both responses could be evoked in the tissue maximally stimulated with norepinephrine. When the arterial rings were maximally activated by high K+ depolarization,Bay K8644 was without effect. The tension evoked by high K+ and Bay K8644 was more sensitive to the dihydropyridine Ca2+ antagonist PY108-068 than norepinephrine induced tension. These results indicate that Bay K8644 activates only potential operated Ca2+ channels which are opened by high K+ depolarization. View Publication