Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.     

Occurrence of ceramide-glycanase in the earthworm, Lumbricus terrestris

We have detected the presence of ceramide-glycanase in the earthworm, Lumbricus terrestris. We have also devised a simple method for the preparation of this enzyme from the earthworm. This enzyme cleaved the linkage between the ceramide and the glycan chain in LacCer, GbOse3Cer, GbOse4Cer, GbOse5Cer, GM3, GM2, GM1 and GD1a. By using tritium-labeled GM2 as substrate, the optimum pH of this enzyme was found to be between pH 4 and 4.5. In the earthworm, the ceramide-glycanase was mainly found in the muscle. The intestine was found to contain a very low level of this enzyme. Because of their easy availability, earthworms should become a convenient source for the preparation of ceramide-glycanase.     

Reduction of tumor growth following treatment with a glutamine antimetabolite

Assessment of arterial-venous differences across transplanted methylcholanthrene-induced sarcomas in rats revealed significant decreases in plasma concentrations of glutamine, serine and glucose. Treatment with the glutamine antimetabolite, acivicin, significantly reduced tumor weights by 65% at the conclusion of the experiment 34 days after tumor induction. These results suggest that glutamine is an essential metabolic substrate for tumor growth and that blockade of glutamine utilization can inhibit the growth of these transplantable sarcomas.     

Sodium-dependent lysine flux across bullfrog alveolar epithelium

Amino acid transport across the alveolar epithelial barrier was studied by measuring radiolabeled lysine fluxes across bullfrog lungs in an Ussing chamber. In the absence of a transmural electrical gradient, L-[14C]lysine was instilled into the upstream reservoir and the rate of appearance of the radiolabel in the downstream reservoir was determined. Two lungs from the same animal were used simultaneously to determine tracer fluxes both into and out of the alveolar bath. Results showed that the radiolabel flux measured in the alveolar to the pleural direction was greater than that measured in the opposite direction in the presence of sodium in the bathing fluids. The net flux of L-[14C]lysine was saturable with [Na+], with an apparent transport coefficient (Kt) of 28 mM for Na+. Hill analysis of [14C]lysine flux vs. [Na+] indicated a coupling ratio of 1:1 between sodium and radiolabeled L-lysine. Total L-lysine flux as a function of [L-lysine] was also saturable, with Kt of 7.3 mM for L-lysine. Ouabain significantly decreased absorptive (alveolar-to-pleural) radiolabel flux, while slightly increasing the flux observed in the opposite direction. L-leucine completely inhibited absorptive net flux of L-[14C]lysine. alpha-Methylaminoisobutyric acid (MeAIB), on the other hand, only slightly reduced net flux of L-[14C]lysine from the control value. The presence of a net absorptive, Na+-dependent amino acid flux across the alveolar epithelial barrier indicates that the tissue is capable of removing amino acids and sodium from the alveolar fluid by a coupled cotransport mechanism, which may be important for both protein metabolism and fluid balance by alveolar epithelium.     

固定化酶的微环境效应—载体离子基团性质的影响

作者合成了阴离子型和阳离子型葡聚糖,以此为载体,用CNBr活化其剩余羟基,固定化了葡萄糖淀粉酶和葡萄糖异构酶。就离子型载体对固定化酶的蛋白载量、最适pH和热稳定性等的影响做了考察。发现固定化酶的蛋白载量不仅与载体的电性质有关,也与酶分子自身的电性质有关。当载体电性质与酶蛋白电性质相反时,固定化酶的蛋白载量增加,热稳定性提高、载体电性质与酶蛋白电性质相同时,固定化酶的蛋白载量不变或下降,其热稳定性不变。作者还发现当离子型载体孔度和体系缓冲液浓度一定时,酶分子能否进入多孔性载体内部,对其最适pH是否变化影响极大。若酶分子仅被连接在载体的外表层,其最适pH不发生变化,反之亦然。作者还观察到当多糖类载体引入氨基或羧基后,大大增强了其抵抗微生物侵蚀的能力。     

Characterization and chromosomal mapping of the gene encoding the cellular DNA binding protein ILF.

Recently we isolated a cellular DNA binding protein, designated interleukin enhancer binding factor (ILF), that binds to purine-rich regulatory motifs in both the HIV-1 LTR and the IL2 promoter. Further analysis of the ILF gene reveals the existence of two mRNA species, both of which encode proteins containing the recently described fork head DNA binding domain. Gel retardation analysis demonstrates that the portion of the ILF protein with homology to the fork head domain is sufficient to mediate DNA binding to a number of related purine-rich sequences. ILF mRNA is expressed constitutively in both lymphoid and nonlymphoid tissues. Chromosomal mapping localizes the ILF gene to human chromosome 17q25, which is a site of chromosomal translocations in some cases of human acute myelogous leukemias. These studies further characterize the structure of the cellular DNA binding protein ILF and may prove valuable in the molecular analysis of possible translocations affecting this gene.     

Metabolism of [4-14C]estrone in hamster and rat hepatic and renal microsomes: species-, sex- and age-specific differences.

The metabolism of [4-14C]estrone (E1) was examined in liver and kidney microsomes of adult castrated male and ovariectomized female hamsters and rats and in neonatal and immature hamster renal microsomes. In castrated male hamster liver microsomes, E1 was metabolized extensively to six major metabolites; 15 beta-hydroxyestrone, 7 alpha-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone, 2-hydroxyestrone, and delta(9,11)-dehydroestrone, and a nonpolar fraction. Six minor metabolites of E1 were also detected. In contrast, kidney microsomes derived from castrated male hamsters metabolized E1 to mainly 17 beta-estradiol, 2- and 4-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone and one monohydroxyestradiol metabolite. However, 16 alpha-hydroxyestrone was not detected. A variable, but low amount of estriol was also found. Interestingly, the quantity of 2-hydroxyestrone found in kidney microsomes of the hamster represented 26% of the total amount of metabolites formed, whereas in liver microsomes, only 9% of the overall metabolism resulted in the formation of 2-hydroxyestrone. The ability of kidney microsomes of female ovariectomized hamsters and two different rat strains to metabolize E1 was 5.9- and 9.4-fold lower, respectively, compared to renal microsomes of male castrated hamsters. The onset of oxidative metabolism in newborn hamster kidneys during development was also assessed. The results indicate that the oxidative metabolism of [14C]E1 in renal microsomes of newborn hamsters was 20-fold less than in kidney microsomes of adult hamsters. While catechol E1 metabolites were essentially negligible in hamster kidneys of these ages, it was evident that the conversion of E1 to estradiol via 17 beta-hydroxysteroid dehydrogenase resembles levels seen in the adult animals. Between the age of one and two months, the male hamster kidney exhibited the capacity to metabolize E1 at levels seen in fully mature adult hamsters.     

Probing the opioid receptor complex with (+)-trans-superfit. I. Evidence that [D-Pen2,D-Pen5]enkephalin interacts with high affinity at the delta cx binding site.

A variety of data support the existence of an opioid receptor complex composed of distinct but interacting mu cx and delta cx binding sites, where "cx" indicates "in the complex." The ability of subantinociceptive doses of [Leu5]enkephalin and [Met5]enkephalin to potentiate and attenuate morphine-induced antinociception, respectively, is thought to be mediated via their binding to the delta cx binding site. [D-Pen2,D-Pen5]Enkephalin also modulates morphine-induced antinociception, but has very low affinity for the delta cx binding site in vitro. In the present study, membranes were depleted of their delta ncx binding sites by pretreatment with the site-directed acylating agent, (3S,4S)-(+)-trans-N-[1-[2-(4-isothiocyanato)phenyl)-ethyl]-3-methy l-4- piperidyl]-N-phenylpropaneamide hydrochloride, which permits selective labeling of the delta cx binding site with [3H][D-Ala2,D-Leu5]enkephalin. The major findings of this study are that with this preparation of rat brain membranes: a) there are striking differences between the delta cx and mu binding sites; and b) both [D-Pen2,D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin exhibit high affinity for the delta cx binding site.     

Urea-induced transformation of native estrogen receptor and evidence for separate DNA-and ATP-binding sites

We have investigated the involvement of hydrophobic receptor domains during transformation of the native estrogen receptor to a form(s) with high affinity for immobilized DNA and ATP. In the presence of 6 M urea the intact estrogen-receptor complex was completely (greater than 90%, n = 12) transformed into a DNA-binding configuration but only partially (35-45%, n = 8) transformed into an ATP-binding state. Similar experiments performed with unliganded receptor preparations further distinguished the receptor's DNA and ATP binding properties. While the urea-induced increase in receptor affinity for DNA-agarose was estrogen-dependent, the urea-induced increase in affinity for ATP-agarose was steroid-independent. This is the first direct evidence that hydrophobic receptor domains may be involved in the steroid-dependent exposure of the DNA binding site. This event is partially reversible and suggests that electrostatic interactions alone may not be sufficient to accurately describe receptor recognition of specific DNA acceptor sites.