Polyamine metabolism in McCoy cells: III. Comparative studies of the metabolic fate of exogenous putrescine in human fibroblasts and McCoy cultures

The fate of exogenously added 14C-putrescine following incubation for 24 hours with McCoy and human skin fibroblast cultures was examined. The nature of the polyamine derivatives found were quite different indicative of a difference in the cellular metabolism of polyamines. Exogenously added putrescine (PUT) was metabolized by both McCoy and human skin fibroblast cultures to form spermidine (SPD), spermine (SPM), gamma-aminobutyric acid (GABA) and some unidentified compounds. Within the experimental period of observation, human cultured fibroblasts metabolized PUT more efficiently than McCoy cells and converted more than 50% of it into SPD, SPM, GABA and unknown compounds. Monoacetyl putrescine (MAP) was formed by human skin fibroblasts. It was mainly identified in the culture medium. No MAP was detectable either intracellularly or extracellularly in McCoy cultures. The percentage of 14C-radioactivity found as PUT in the culture medium was greater in McCoy cells (86.0%) than in human fibroblasts (53.9%). The reverse was true for the percentage distribution of 14C-radioactivity as PUT inside the cells. No low Mr conjugates of SPD or SPM were found in the medium or intracellularly with either culture type. Some low Mr putrescine conjugates were found in the culture media; these were identified by the liberation of PUT upon acid hydrolysis.     

Kinetic studies of enzymatic hydrolysis of insoluble cellulose: analysis of the initial rates

A study was conducted on the kinetics of enzymatic hydrolysis of pure insoluble cellulose using unpurified culture filtrate Trichoderma reesei, with the emphasis on the initial reaction period. The initial hydrolysis rate and extent of enzyme (soluble protein)adsorption, either apparent or initial, were evaluated under various experimental conditions. It has been found that the various mass-transfer steps do not control the overall hydrolysis rate and that the hydrolysis rate is mainly controlled by the surface reaction step promoted by the adsorbed enzyme. It has also been found that the initial hydrolysis rate strongly depends on the initial extent of soluble protein adsorption and the effectiveness of the adsorbed soluble protein to promote the hydrolysis. The initial extent of soluble protein adsorption, in turn, is related to the initial cellulose concentration, enzyme concentration, and specific surface area of cellulose, whereas the effectiveness of the initially adsorbed soluble protein to promote the derived to interrelate these parameters without resorting to the Michaelis-Menten kinetics. The present result appear to imply that the role of enzyme-substrate complex formation should not be ignored in deriving a mechanistic kinetic model for enzymatic hydrolysis of cellulose.     

Growth modulation by epidermal growth factor (EGF) in human colonic carcinoma cells: constitutive expression of the human EGF gene

The functional role of epidermal growth factor (EGF) in epithelium-derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor-responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor-alpha (TGF-alpha) secretion. Growth stimulation and down-modulation of both high and low affinity EGF receptors were observed in the EGF-transfected Moser clones. Results of experiments using anti-EGF and anti-EGF-receptor antibody to block the proliferation of EGF-transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF-alpha were operative in these cells. By comparison, a growth-inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF-transfected KM12SM clones. Both the parental and EGF-transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti-EGF or anti-EGF-receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF-transfected KM12SM clones were non-autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium-derived human colonic carcinoma cells resulted in divergent altered growth characteristics.     

Alkylation of nucleic acids by DNA-targeted 4-anilinoquinolinium aniline mustards: kinetic studies

The rate of constant for hydrolysis of a series of 4-substituted aniline mustards Ar-X-pC6H4-N(CH2CH2Cl)2, where Ar is 4-anilinoquinolinium and X = O, CH2, CONH and CO, have been measured in water and 0.02 M imidazole buffer at 37 degrees C and in 50% aqueous acetone at 66 degrees C. The equilibrium binding constants of the compounds and their hydrolysis products to nucleic acids of differing base composition have been determined at varying ionic strengths, and the results are consistent with the compounds binding as expected in the DNA minor groove. The alkylating reactivity of the mustards towards these nucleic acids has been measured in water at 37 degrees C and in 0.01 M HEPES buffer over a range of temperatures from 25 degrees C to 60 degrees C. Evaluation of the thermodynamic parameters for these kinetic and equilibrium studies suggests that the interaction with nucleic acids is via an internal SN2 mechanism involving an aziridinium ion.     

Characterization of the Bacillus subtilis sporulation gene spoVK.

The sporulation gene spoVK of Bacillus subtilis was cloned by use of the insertional mutation spoVK::Tn917 omega HU8. The spoVK gene was shown to be the site of an incorrectly mapped mutation called spoVJ517. Thus, a separate spoVJ gene as defined by the 517 mutation does not exist and is instead identical with spoVK.     

Up-regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in murine peritoneal exudate macrophages by both GM-CSF and IL-3.

Murine peritoneal exudate macrophages (PEM) display multiple CSF receptors. In this study, the expression of granulocyte-macrophage (GM)-CSF receptors in PEM was studied. PEM displayed over 5000 single type, high affinity GM-CSF receptors/cell with a Kd = 38 to 42 pM and an apparent molecular mass of 86,000 Da. Treatment of PEM with low, but not high, concentrations of recombinant murine (rMu) GM-CSF continuously for 24 h resulted in a marked up-regulation of GM-CSF receptors in PEM. A similar up-regulation of GM-CSF receptors also was detected in PEM cultures treated with rMuIL-3 (1-100 ng/ml) for 24 h or longer, regardless the doses of rMuIL-3 added in this case. Scatchard analysis of equilibrium binding showed that the enhanced binding activities in both cases were due to an increase in total number of GM-CSF receptors rather than changes in receptor affinity. Contrariwise, treatment with recombinant human macrophage-CSF (greater than 100-1000 ng/ml) partially inhibited the expression of GM-CSF receptors in PEM. Removal of rMuGM-CSF from culture medium 24 h after treatment led to a further up-regulation of GM-CSF receptors over a 4 to 24-h period, depending on the doses of initial treatment. On the other hand, removal of rMuIL-3 from culture medium after prolonged treatment did not result in further increase in GM-CSF receptors. The protein synthesis inhibitor cycloheximide abrogated GM-CSF receptor up-regulation induced by both rMuIL-3 and rMuGM-CSF, whereas actinomycin D inhibited only the second (8-24 h) phase of GM-CSF receptor up-regulation induced by exposure to high concentrations rMuGM-CSF (10 ng/ml). These findings suggest that rMuGM-CSF and rMuIL-3 up-regulate GM-CSF receptors in PEM in part through similar or identical metabolic pathways and provide further evidence of a close linkage between IL-3 and GM-CSF receptors.     

Eosin Y staining of proteins in polyacrylamide gels.

A staining method is described in which various proteins in polyacrylamide gels can be stained by using eosin Y. After a brief incubation of a polyacrylamide gel in an acidic solution of 1% eosin Y, various proteins, including human erythrocyte membrane sialoglycoproteins which are not detectable by Coomassie blue R-250 (CB), can be detected with a sensitivity of 10 ng protein. This is far more sensitive than CB staining and is comparable to the sensitivity of silver staining. In a Western blot, the antigenicity of an eosin Y stained protein is retained. In addition, proteins on an immunoblot sheet can be detected by eosin Y staining. The method described is rapid, sensitive, and reproducible with various proteins in polyacrylamide gels and has the added advantage of also staining sialoglycoproteins.     

Characterization of the Ni-Fe-C complex formed by reaction of carbon monoxide with the carbon monoxide dehydrogenase from Clostridium thermoaceticum by Q-band ENDOR.

Q-Band ENDOR studies on carbon monoxide dehydrogenase (CODH) from the acetogenic bacterium Clostridium thermoaceticum provided unambiguous evidence that the reaction of CO with CODH produces a novel metal center that includes at least one nickel, at least three iron sites, and the carbon of one CO. The 57Fe hyperfine couplings determined by ENDOR are similar to the values used in simulation of the M?ssbauer spectra [Lindahl et al. (1989) J. Biol. Chem. 265, 3880-3888]. EPR simulation using these AFe values is equally good for a 4Fe or a 3Fe center. The 13C ENDOR data are consistent with the binding of a carbon atom to either the Ni or the Fe component of the spin-coupled cluster. The 13C hyperfine couplings are similar to those determined earlier for the C0-bound form of the H cluster of the Clostridium pasteurianum hydrogenase, proposed to be the active site of hydrogen activation [Telser et al. (1987) J. Biol. Chem. 262, 6589-5694]. The 61 Ni ENDOR data are the first nickel ENDOR recorded for an enzyme. The EPR simulation using the ENDOR-derived hyperfine values for 61Ni is consistent with a single nickel site in the Ni-Fe-C complex. On the basis of our results and the M?ssbauer data [Lindahl et al. (1989) J. Biol. Chem. 265, 3880-3888], we propose the stoichiometry of the components of the Ni-Fe-C complex to be Ni1Fe3-4S greater than or equal to 4C1, with four acid-labile sulfides.     

香菇和侧耳属间原生质体的电融合研究

从培养在液体培养基中的香菇、美味侧耳和平菇的单核菌丝用酶法分离了原生质体。施加0.5MHz、500PV/cm的正弦波和μs、6000PV/cm方形脉冲的电场诱导下使其电融合。电融合后的融合子和原生质体在固体培养基上植板培养成菌落。在显微镜下检查融合子菌株菌丝的锁状联合选出从融合子长成的菌株。香菇和美味侧耳的融合菌株产生频率为61.53%,香菇和平菇的融合菌株产生频率为32.58%。根据融合菌株与亲本的拮抗作用和他们的过氧化物同工酶和酯酶同工酶的电泳酶谱与其亲本酶谱的不同,证实这些融合菌株是从融合的异核体生长成的。同时讨论了电融合方法和结果。