R. H. Wasserman

Rapamycin is active against B-precursor leukemia in vitro and in vivo, an effect that is modulated by IL-7-mediated signaling.

A balance between survival and apoptotic signals regulates B cell development. These signals are tightly regulated by a host of molecules, including IL-7. Abnormal signaling events may lead to neoplastic transformation of progenitor B cells. Signal transduction inhibitors potentially may modulate these abnormal signals. Inhibitors of the mammalian target of rapamycin (mTOR) such as rapamycin have been used as immunosuppressive agents. We hypothesized that rapamycin might demonstrate activity against B-precursor acute lymphoblastic leukemia. We have found that rapamycin inhibited growth of B-precursor acute lymphoblastic leukemia lines in vitro, with evidence of apoptotic cell death. This growth inhibition was reversible by IL-7. One candidate as a signaling intermediate cross-regulated by rapamycin and IL-7 was p70 S6 kinase. Rapamycin also demonstrated in vivo activity in Eμ-ret transgenic mice, which develop pre-B leukemia/lymphoma: Eμ-ret transgenic mice with advanced disease treated daily with rapamycin as a single agent showed a >2-fold increase in length of survival as compared with symptomatic littermates who received vehicle alone. These results suggest that mammalian target of rapamycin inhibitors may be effective agents against leukemia and that one of the growth signals inhibited by this class of drugs in precursor B leukemic cells may be IL-7-mediated.

Intestinal calcium transport and calcium extrusion processes at the basolateral membrane

The intestinal absorption of calcium has been proposed to occur by the transcellular transfer of Ca2+ through the enterocyte proper and between the cells of the intestinal epithelium, i.e., the paracellular path. Attention in this report is given to the transcellular models of Ca2+ absorption and, more specifically, the Ca2+ extrusion events occurring at the basolateral membrane. These extrusion processes include the operation of an ATP-dependent Ca2+ pump and a Na+/Ca2+ exchanger, as well as exocytosis as the terminal event in a proposed vesicular transport mechanism. Evidence for the presence of an ATP-dependent Ca2+ pump at the basolateral membrane is documented and illustrated with biochemical and immunological data from studies on the avian intestinal basolateral membrane. As shown immunohistochemically, the Ca2+ pump was primarily localized on the enterocyte basolateral membrane. The ATP-dependency and vitamin D enhancement of Ca2+ uptake by isolated basolateral membrane vesicles are shown. Western blot analysis of intestinal mucosa, by using a monoclonal antibody produced against the erythrocyte Ca2+ pump, indicated that the number of pump units is increased by 1,25-dihydroxycholecalciferol. The possible involvement of calbindin-D28K as a direct stimulator of the Ca2+ pump is discussed, and the quantitative relationship between Ca2+ transport rates and Ca2+ pumping activity has been estimated. Information related to the basolateral membrane Na+/Ca2+ exchanger and the vesicular transport model of Ca2+ absorption is also briefly reviewed.

Vitamin D-induced calcium binding factor in rat intestinal mucosa.

Summary Oral administration of 500 IU of vitamin D2 or vitamin D3 to rachitic rats resulted in the elaboration of a Ca-binding factor which could be detected in the supernatant fraction of duodenal mucosal homogenates at 72 hours after administration of the vitamin. This factor was found to be associated with the initial or protein containing portion of the supernatants after percolation through Sephadex G-25; this indicates that the factor is a macromolecule, presumably a protein. Further separation of the supernatants by Sephadex G-100 gel filtration showed that the increased binding activity seen after vitamin D administration was mainly associated with only one of the protein peaks present in the Sephadex G-100 fractions. This particular peak was much less prominent in the fractionated supernatant of the mucosal homogenates from rachitic rats. The results suggest that the vitamin D-dependent binding activity is due to the presence of a specific calcium binding protein in the supernatant fraction of duodenal mucosal homogenates from vitamin D-treated rachitic rats. The authors gratefully acknowledge the technical assistance of Mr. Philip Shapiro and Mrs. Gabriele E. Collins.

Active Transport of Calcium by Rat Duodenum in vivo

In studies of the living rat, it was observed that ionic calcium is transferred against a concentration gradient and an electropotential gradient by the duodenal membrane; this would constitute evidence for the active transport of calcium in intestinal absorption and corroborates earlier observations made in vitro. The significance of this process in regard to total calcium absorption is unknown at present.

Vitamin D3: induction of calcium-binding protein in embryonic chick intestine in vitro.

Induction of the synthesis of calcium-binding protein in chick embryonic intestine maintained in vitro was accomplished by simply adding vitamin D3 to the culture medium. Accompanying the induction of this protein, there was enhanced radiocalcium uptake by the intestine. These observations represent the first demonstration of an in vitro physiological effect of vitamin D3 on the calcium absorptive mechanism of the intestine.

1,25-Dihydroxyvitamin D3-glycoside: Identification of a calcinogenic principle of solanum malacoxylon

Abstract The water soluble calcinogenic factor present in the plant Solanum malacoxylon is partially purified by selective extraction and chromatography on silicic acid and then hydrolyzed with a mixed preparation of glycosidases from the sea worm, Charonia lampus . Hydrolysis produces a chloroform soluble factor with biologic characteristics of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), the hormonal form of vitamin D. Purification of this factor is accomplished by chromatography on Sephadex LH-20, silicic acid, and Celite columns, yielding 3 μg of active material. During the isolation, bioactivity (as assessed by the ability of fractions to compete with labeled 1,25-(OH) 2 D 3 for binding to a specific intestinal receptor protein) migrates exactly with authentic tritiated 1,25-(OH) 2 D 3 . The purified factor has an ultraviolet absorption spectrum identical to that of 1,25-(OH) 2 D 3 and analysis via direct probe mass spectrometry yields a parent molecular ion of m/e 416 and a fragmentation pattern indistinguishable from synthetic 1,25-(OH) 2 D 3 hormone. We therefore conclude that the vitamin D-like principle in Solanum malacoxylon is a sterol-glycoside which contains the 1,25-(OH) 2 D 3 molecule as its active sterol component.

Calcium absorption and calcium-binding protein synthesis: solanum malacoxylon reverses strontium inhibition.

The ingestion of diets containing high concentrations of stable strontium inhibits calcium absorption and intestinal calcium-binding protein synthesis and, as shown by others, does so by inhibiting the conversion of 25-hydroxycholecalciferol to 1,25-dihydroxycholecalciferol, the active form of vitamin D. The addition of the South American plant Solanum malacoxylon to strontium-containing diets counteracts the inhibitory action of dietary strontium, thereby indicating that the plant contains a factor which can mimic the action of 1,25-dihydroxycholecalciferol and representing the first such factor identified in a botanical source.

Embryonic chick intestine in organ culture: stimulation of calcium transport by exogenous vitamin D-induced calcium-binding protein.

Abstract Vitamin D 3 or a potent metabolite, 1,25-dihydroxycholecalciferol, induces calcium-binding protein (CaBP) synthesis and stimulates transmucosal calcium transport in embryonic chick duodena maintained in novel organ culture apparatus. When added to the sterol-free culture medium, highly purified chick intestinal CaBP, similarly and specifically, stimulates calcium transport in the cultured duodena. These results clearly demonstrate the involvement of CaBP in intestinal calcium transport.

Control of calcium absorption and intestinal calcium-binding protein synthesis☆

A current hypothesis suggests that the degree of Ca absorption is hormonally controlled via the feed-back regulation of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) production from 25-hydroxycholecalciferol (25-OHD3) by kidney 1-hydroxylase. To test this hypothesis, dihydrotachysterol3 (DHT3), a steroid not requiring 1-hydroxylation for biological activity, was given to chicks as the only source of vitamin D-activity. As expected, DHT3-treated chicks did not adapt to a calcium-deficient diet. However, both the efficiency of Ca absorption and net synthesis of CaBP were stimulated in DHT3-treated chicks by a low phosphorus intake, providing evidence for an alternate pathway of control.

Some aspects of vitamin D action; calcium absorption and the vitamin D-dependent calcium-binding protein

Publisher Summary This chapter examines calcium absorption and the vitamin D-dependent calcium-binding protein (CaBP). The investigations on the physiology of calcium absorption on CaBP indicated that there is nearly a 1:1 correspondence between the concentration of intestinal CaBP and the capacity of the intestine to absorb calcium. These correlations strongly indicate that CaBP has a significant role in the calcium translocation process. Calcium-binding protein of calf kidney was found exclusively in the cortex. Quantitative measurements on a single animal indicated that the concentration in the kidney cortex was about one-sixth that of the duodenum when expressed on the basis of total protein. Studies have shown that diphenylhydantoin and phenobarbital, the drugs used for anticonvulsant therapy of epileptics, are associated with the production of bone lesions in such patients if inadequate levels of vitamin D are provided. A unique system for the study of the intestinal calcium absorptive mechanism has been developed utilizing the embryonic chick duodenum maintained in organ culture.