Biomedical subjects
Publications and source records attributed to L Sachs.
Obstruction of the common bile duct secondary to hydronephrosis is uncommon. We observed common bile duct dilatation secondary to marked right hydronephrosis. Following nephrectomy the common bile duct returned to normal size, confirming the suspicion that the dilatation was due to extrinsic compression.
Synaptic membrane (SM) and postsynaptic density (PSD) fractions isolated from the cerebral cortex (CTX) and cerebellum (CL) of the canine brain were found to contain one class of specific nitrendipine binding sites. The specific binding constants were: CTX-SM, Kd = 110 pM (Bmax = 126 fmol/mg protein); CTX-PSD, Kd = 207 pM (Bmax = 196 fmol/mg); CL-SM, Kd = 100 pM (Bmax = 65 fmol/mg); CL-PSD, Kd = 189 pM (Bmax = 80 fmol/mg). Treatment of the CTX-SM and CTX-PSD fractions with 0.5% deoxycholate and 1.0% N-lauroyl sarcosinate removed 88-91% and 42-51% of the nitrendipine binding, respectively, indicating that the major nitrendipine binding present in the SM fractions are of non-synaptic origin. Moreover, the percentages of total protein and specific nitrendipine binding removed from PSDs by these detergents were similar, indicating no preferential dissociation of the latter, and suggesting that the receptor protein is firmly bound and is probably an intrinsic component of the PSD fraction. Both Ca2+ and calmodulin were found to be important for the binding of nitrendipine to the CTX-SM and CTX-PSD fractions since: R24571, a calmodulin antagonist, was found to inhibit nitrendipine binding to the CTX-SM and CTX-PSD fractions with IC50 values of 1.1 microM and 0.9 microM, respectively; removal of Ca2+ from the CTX-SM and CTX-PSD fractions with 0.2 mM EGTA resulted in losses of specific nitrendipine binding of 80 and 90%, respectively; Ca2+ alone restored nitrendipine binding to EGTA-pretreated CTX-SM fractions and not to CTX-PSD fractions, with the latter needing both Ca2+ and calmodulin to restore nitrendipine binding; EGTA treatment removed 14-16% and 89-91% of nitrendipine bound to the CTX-SM and CTX-PSD fractions, respectively, suggesting that calmodulin (but not Ca2+) is needed to maintain the nitrendipine-nitrendipine receptor-calmodulin complex; Ca2+-reconstituted EGTA-pretreated CTX-SM fractions and the Ca2+ plus calmodulin-reconstituted EGTA-pretreated CTX-SM and CTX-PSD fractions were found to have similar binding constants to those for the corresponding native, untreated fractions; and the Ca2+/calmodulin dependency on nitrendipine binding was similar to the well-known Ca2+/calmodulin dependency on phosphorylation in EGTA-pretreated PSD fractions. It needed much less Ca2+ to saturate Ca2+/calmodulin-dependent phosphorylation of the pretreated CTX-PSD fractions than the nitrendipine binding. Yet, less calmodulin was needed to saturate nitrendipine binding than the phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)
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Apamin, a 18-amino acid neurotoxin isolated from bee venom, is a specific blocker of one class of the Ca2+-dependent K+ channels. The monoiodo derivative of the toxin with high specific radioactivity (1600 Ci/mmol) has been used to study its binding to synaptic membrane (SM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brains. The Bmax (30.2 fmol/mg protein) for CTX-PSD is about twice that for CTX-SM (17.3 fmol/mg protein), suggesting a concentration of the apamin receptor protein in CTX-PSD over CTX-SM fractions. The lower value of Bmax for CL-PSD (12.3 fmol/mg protein), and the higher Kd value (51 pM) than for CTX-SM (33 pM), CTX-PSD (24 pM), and CL-SM (39 pM), may reflect the disruptive effect of Triton X-100 on these thin structures. The values of Bmax and Kd for CTX-SM are similar to those (22.0 fmol/mg protein and 33 pM) for rat CTX-SM. Both Ca2+ and Na+ inhibit apamin binding to CTX-PSD with K0.5 values of 14 and 31 mM, respectively, while the optimum concentration of KCl for activation is 5 mM. All these values are similar to those found for rat synaptosomes. Covalent labeling of the apamin binding protein, using the non-cleavable cross-linker, disuccinimidyl suberate, reveals an apamin binding polypeptide of 27 kdaltons under reducing and denaturing conditions in both the CTX-SM and CTX-PSD preparations, similar to that (28 kdaltons) reported for rat CTX-SM fractions. Prior phosphorylation of isolated CTX-PSD had no effect on apamin binding, nor did apamin binding influence subsequent phosphorylation of CTX-PSD. Calmodulin, an intrinsic PSD protein, may not play a role in apamin binding to PSD, since addition of calmodulin, or removal of the calmodulin by EGTA treatment, resulted in no change in the binding capacity of the PSD. The apamin binding protein seems to be bound quite firmly in the CTX-PSD fraction since treatments with 0.5% deoxycholate, 1% N-lauroyl sarcosinate, 4 M guanidine-HCl, pH 7.0, 0.5 M KCl and 1.0 M KCl, could only remove the apamin-receptor complexes from CTX-PSD by 40, 55, 52, 12 and 15%, respectively. These results contrast with the findings that the two detergents mentioned solubilize 80-93% of the receptor from synaptosomal or synaptic membrane fractions, indicating that a good deal of the receptor in these fractions is membrane-bound and not connected to the PSD.(ABSTRACT TRUNCATED AT 400 WORDS)
Regulation of the in vivo production of myeloid cell growth-inducing (MGI-1) and differentiation-inducing (MGI-2) proteins has been studied in mice injected with the inflammation-inducing compounds sodium caseinate, thioglycollate and bacterial lipopolysaccharide. The results indicate that these inflammation-inducing compounds can induce in vivo production of MGI-1 and MGI-2; that different inducing agents can cause a different body-distribution of MGI-1 and MGI-2; that there is an independent regulation of in vivo production and distribution of MGI-1 and MGI-2; and that there is a granulocyte growth-inducing protein (MGI-IG = G-CSF) that is not identical to the differentiation-inducing protein (MGI-2). Resident peritoneal macrophages produce MGI-1 and MGI-2 in vitro, but inflammatory macrophages show a reduced ability to spontaneously produce these proteins after in vivo injection of caseinate or thioglycollate. The results thus also indicate that macrophage activation can affect the ability of macrophages to produce the myeloid cell regulatory proteins MGI-1 and MGI-2.
The use of hydrocortisone and timed delivery was compared to expectant management of pregnancies complicated by preterm prematurely ruptured membranes in a prospective randomized trial of 73 patients. The incidence of maternal and neonatal complications was compared. There was no significant difference in the incidence of respiratory distress syndrome. Steroid-treated mothers had an increased incidence of postpartum febrile morbidity. We conclude that treatment of women with preterm prematurely ruptured membranes with hydrocortisone and timed delivery offers no advantage over expectant management.
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Regulation of in-vivo differentiation of myeloid leukemic cells by response to antigen was analysed with different clones of mouse myeloid leukemic cells and human myeloid leukemic cells (HL-60). Differentiation was studied in diffusion chambers implanted into the peritoneal cavity of mice and the antigens used were bovine serum albumin and chicken ovalbumin. It is shown that the presence of either of these antigens in the diffusion chambers can induce differentiation in MGI+D+ mouse and human myeloid leukemic cells, and that pre-immunization with antigen enhanced this in-vivo differentiation. This enhancement showed immunological specificity and was transferred from immunized to non-immunized mice by spleen cells enriched for T lymphocytes. In contrast to these results with MGI+D+ clones of myeloid leukemic cells, clones of WEHI-3B myeloid leukemic cells were induced to differentiate in vivo to the same extent either in the presence or absence of antigen. The results indicate: that in-vivo differentiation of MGI+D+ clones of myeloid leukemic cells can be induced by response to antigen and that in-vivo differentiation of different clones of myeloid leukemic cells can be regulated in different ways.
Bone marrow cells from 9 patients with acute myeloid leukemia and 1 patient with a blast crisis of chronic myeloid leukemia were cultured to determine their ability to be induced to differentiate by different chemotherapeutic compounds. Five of these 10 patients showed differentiation to granulocytic and/or monocytic cells by culture with medium containing the myeloid cell differentiation-inducing protein MGI-2. Actinomycin D induced differentiation in cells from 2 of the patients who did not show differentiation with MGI-2 containing medium. In these 7 patients there was an increase in the ratio of differentiated myeloid cells to blasts. None of these 10 patients showed induction of differentiation by cytosine arabinoside, adriamycin, or daunomycin, but treatment with these compounds showed in some patients an increase in the ratio of differentiated myeloid cells to blasts. The results indicate that this ratio can be increased by differentiation and also in some patients by toxicity to blast cells. With dexamethasone or vinblastine there was no induction of differentiation and no increase in this ratio in any of the 10 patients tested. After in vivo chemotherapy with low dose cytosine arabinoside, cells from one patient showed a similar response in culture to actinomycin D as cells before chemotherapy, whereas in another patient the cells had acquired the ability to respond to actinomycin D. In contrast, after high-dose in vivo chemotherapy with cytosine arabinoside and daunomycin, cells from a third patient seemed to have lost the ability to differentiate in vitro by MGI-2 containing medium or actinomycin D. The results indicate that pre-screening for differentiation-inducing compounds and compounds that show toxicity to blast cells should be useful to select the appropriate compounds to be used for therapy, and that it is advisable to screen the cells both before and after initiation of therapy.
The development of systems for the cloning and clonal differentiation of normal hematopoietic cells in culture made it possible to study the controls that regulate normal growth and differentiation and the changes in these controls in leukemia. Identification of the changes in normal controls that occur in leukemia reveals that the stopping of growth by inducing differentiation of malignant cells can by-pass genetic changes that produce the malignant phenotype. The induction of differentiation in leukemic cells can therefore be a useful approach to therapy.
Myeloid hematopoietic precursor cells are induced to differentiate by the macrophage and granulocyte differentiation-inducing protein MGI-2 (DF). This differentiation-inducing protein bound to double-stranded but not to single-stranded mammalian DNA. The bound MGI-2 was not eluted by high salt, but was eluted by sodium dodecyl sulfate (SDS). MGI-2 also bound to double-stranded E. coli DNA, but with this DNA the bound MGI-2 was eluted by high salt. This indicated a difference in the binding affinities of MGI-2 to mammalian and E. coli DNA. MGI-2 bound to DNA was examined by electron microscopy. The results indicate that MGI-2 formed a multimeric complex with double-stranded DNA and that the size of the complex was correlated with the strength of protein binding to the DNA. The multimeric complex bound to DNA was disrupted by deoxyribonuclease. The data indicated that binding of this differentiation-inducing protein to DNA involves the formation of a multimeric complex in which the monomers are held together by DNA. It is suggested that the formation of such multimeric complexes of MGI-2 and DNA may allow activation of the multiple pathways of gene expression that is required for differentiation.
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It is shown that a 5-day schedule of two injections per day of the myeloid differentiation-inducing protein MGI-2 inhibited the in vivo development of leukemia in SL and SJL/J mice with different syngeneic MGI+D+ clones of myeloid leukemic cells. With this schedule of treatment high levels of MGI-2 were maintained in the serum for long periods. In contrast to these results with MGI-2, the same schedule of injections of the myeloid growth-inducing protein MGI-I did not affect the in vivo development of leukemia in mice with MGI+D+ myeloid leukemic cells, but stimulated normal myelopoiesis in the bone marrow. Different forms of MGI-I including MGI-IM and MGI-IG had different serum half-lives, and the form of MGI-I with the shortest serum half-life showed the smallest in vivo effect on normal myelopoiesis. MGI-2 injections did not inhibit the in vivo development of differentiation-defective WEHI-3B myelomonocytic leukemic cells or YAC lymphoma cells. The results indicate that the in vivo inhibitory effect of MGI-2 on the development of myeloid leukemia correlated with its differentiation-inducing potential on the leukemic cells. It is concluded that this approach of inhibiting leukemia development by inducing differentiation should also be applied to human leukemic patients, whose cells have been shown to be inducible for differentiation in culture by human MGI-2 or by other differentiation-inducing compounds.
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Cells from a clone of mouse myeloid leukemic cells that can be induced to differentiate in vitro to mature cells by the normal macrophage- and granulocyte-inducing protein, MGI-2, were injected into mouse embryos at 10 days gestation. The leukemic cells, derived from an SJL/J mouse, were microinjected into the placentae of C3HeB fetuses and the viable progeny were tested for chimerism by analysis of glucose phosphate isomerase isozymes. Fifty-five percent out of 201 viable progeny died prior to weaning due to tumors derived from the injected cells. Of the remaining 91 apparently healthy adult animals, 2 had chimeric populations. One mouse had a chimeric population of granulocytes and the other mouse a chimeric population of macrophages. The animal with a chimeric population of macrophages was chimeric at 1 and 2 months of age and had lost any detectable donor derived contribution by the age of 3 months. The chimeric granulocyte population in the other animal was still stable at 3 months. The myeloid leukemic cells injected into midgestation embryos thus participated in the development of two different cell types in apparently healthy adult animals. Our results indicate that malignant cells of more restricted developmental potential than teratocarcinoma cells may participate in normal development. It remains to be determined whether this participation in normal development was due to normal differentiation of the malignant cells, or the production of nonmalignant segregants derived from the malignant cells.
Bone marrow cells from 2 patients over 60 years of age with acute myeloblastic (AML) or monoblastic (AMoL) leukemia were cultured in the presence of a low dose of cytosine arabinoside. In the cells from the AML patient this treatment induced differentiation to metamyelocytes and a decrease in the number of blasts, so that there was an 11-fold increase in the ratio of differentiated myeloid cells to blasts. In the patient with AMoL there was differentiation to monocytes and macrophages and only a 3-fold increase in the ratio of differentiated myeloid cells to blasts. In the latter patient actinomycin D was a more potent inducer of differentiation than cytosine arabinoside, daunomycin was similar to cytosine arabinoside and adriamycin showed the lowest response. Four courses of low dose treatment with cytosine arabinoside produced remission in the patient with AML and in another patient with AMoL whose cells were not tested in culture. No remission was induced by this low dose treatment in the patient with AMoL whose cells showed only a small decrease in blast cells in culture with cytosine arabinoside. It is suggested that prescreening for effective compounds in patients with myeloid leukemias and the use of low dose therapy can be of help in obtaining remission without serious side effects. This could be especially useful in patients where there may be severe toxic effects after high dose chemotherapy.
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