E2F-1 binding affinity for pRb is not the only determinant of the E2F-1 activity

E2F-1 is the major cellular target of pRB and is regulated by pRB during cell proliferation. Interaction between pRB and E2F-1 is dependent on the phosphorylation status of pRB. Despite the fact that E2F-1 and pRB have antagonistic activities when they are overexpressed, the role of the E2F-1-pRB interaction in cell growth largely remains unknown. Ideally, it would be better to study the properties of a pRB mutant that fails to bind to E2F, but retains all other activities. To date, no pRB mutation has been characterized in sufficient detail to show that it specifically eliminates E2F binding but leaves other interactions intact. An alternative approach to this issue is to ask whether mutations that change E2F proteins binding affinity to pRB are sufficient to change cell growth in aspect of cell cycle and tumor formation. Therefore, we used the E2F-1 mutants including E2F-1/S332-7A, E2F-1/S375A, E2F-1/S403A, E2F-1/Y411A and E2F-1/L132Q that have different binding affinities for pRB to better understand the roles of the E2F-1 phosphorylation and E2F-1-pRB interaction in the cell cycle, as well as in transformation and gene expression. Data presented in this study suggests that in vivo phosphorylation at amino acids 332-337, 375 and 403 is important for the E2F-1 and pRB interaction in vivo. However, although E2F-1 mutants 332-7, 375 and 403 showed similar binding affinity to pRB, they showed different characteristics in transformation efficiency, G₀ accumulation, and target gene experiments.     

Mixed lymphocyte reactivity in spleen cells from F-9 tumor-bearing mice. II. Functional cross-reactivity between F-9 cells and testicular cells

Previous serological analysis has revealed cross-reactive antigens on F-9 cells and mouse germ cells. Therefore, we investigated whether suppressor activity in spleen cells from F-9 tumor-bearing mice can be restimulated in vitro by adding F-9 cells or testicular cells to mixed lymphocyte cultures. We found equal potentiation of suppressor T-cell activity with F-9 cells and with syngeneic testicular cells; as shown by sensitivity to anti-Lyt 2.2 plus complement treatment. Suppressor activity of spleen cells from other tumor systems tested was not enhanced. The data suggest that the same or cross-reactive antigens on F-9 teratocarcinoma cells and testicular cells may have similar regulatory functions.     

Overexpression of E2F-1 inhibits progression of gastric cancer in vitro

E2F-1 plays a critical role in cell cycle regulation and other biological processes in cells. E2F-1 mediates apoptosis and suppresses tumorigenesis in many tissue types, but there are few data available on E2F-1 expression and its relationship to tumor kinetics in gastric cancer. To gain better insight into the involvement of E2F-1 in the biological characteristics of gastric tumors, we investigated the effect of E2F-1 overexpression on the progression of gastric carcinoma cells. A gastric cancer cell line stably overexpressing E2F-1 (MGC-803/E2F-1) was established. The influence of E2F-1 overexpression on in vitro cell growth was assessed by measuring cell survival, colony formation, and cell cycle progression. The results clearly show that overexpression of E2F-1 significantly inhibits cell growth and proliferation, blocking entry into the S-phase of the cell cycle. MGC-803/E2F-1 cells also had a higher apoptotic rate than control cells. In addition, E2F-1 reduced the motility and invasion of gastric cancer cells.     

Effect of volatile substances of Streptomyces platensis F-1 on control of plant fungal diseases

Headspace volatile substances (VS) produced by Streptomyces platensis F-1 were preliminarily identified using GC–MS. The effects of VS released by S. platensis F-1 on the control of leaf blight/seedling blight of rice caused by Rhizoctonia solani, leaf blight of oilseed rape caused by Sclerotinia sclerotiorum and fruit rot of strawberry caused by Botrytis cinerea, as well as on the growth of these three pathogenic fungi, were investigated. Results showed that sixteen volatile compounds were tentatively identified in 1-week-old cultures of S. platensis F-1 grown on autoclaved wheat seeds. They could be chemically grouped into alcohols, esters, acids, alkanes, ketones and alkenes. The most abundant composition in volatiles of S. platensis F-1 is geosmin, an earthy-muddy–smelling compound. Two antifungal compounds, phenylethyl alcohol and (+)-epi-bicyclesesquiphellandrene, were detected in the volatile profile of S. platensis F-1. Consistent fumigation of healthy tissues of rice, oilseed rape and strawberry to VS of S. platensis effectively reduced the incidence and/or the severity of leaf blight/seedling blight of rice (R. solani), leaf blight of oilseed rape (S. sclerotiorum) and fruit rot of strawberry (B. cinerea). A significant (P < 0.05) suppression of the mycelial growth of R. solani, S. sclerotiorum and B. cinerea by the VS of S. platensis was observed. The potential of using VS of S. platensis F-1 as a biofumigant to control plant fungal diseases is discussed.     

H(2) and CO(2) Evolution by Anaerobically Adapted Chlamydomonas reinhardtii F-60

Using manometric and enzymic techniques, H₂ and CO₂ evolution in darkness and light has been studied in the green alga Chlamydomonas reinhardtii F-60. F-60 is a mutant strain characterized by an incomplete photosynthetic carbon reduction cycle but an intact electron transport chain.     

Investigation of an F-18 oxytocin receptor selective ligand via PET imaging

The compound 1-(1-(2-(2-(2-fluoroethoxy)-4-(piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydroquinolin-2(1H)-one (1) was synthesized and positively evaluated in vitro for high potency and selectivity with human oxytocin receptors. The positron emitting analogue, [F-18]1, was synthesized and investigated in vivo via PET imaging using rat and cynomolgus monkey models. PET imaging studies in female Sprague–Dawley rats suggested [F-18]1 reached the brain and accumulated in various regions of the brain, but washed out too rapidly for adequate quantification and localization. In vivo PET imaging studies in a male cynomolgus monkey suggested [F-18]1 had limited brain penetration while specific uptake of radioactivity significantly accumulated within the vasculature of the cerebral ventricles in areas representative of the choroid plexus.     

Spontaneous variability of glucose oxidase-producing fungus <Emphasis Type="Italic">Penicillium adametzii</Emphasis> LF F-2044

The glucose oxidase-producing fungus Penicillium adametzii LF F-2044 was studied for natural variability. Four variants of the fungus differed in morphological characteristics and glucose oxidase synthesis. The synthesis of extracellular glucose oxidase and the productivity of morphological variants P. adametzii LF F-2044.1 and P. adametzii LF F-2044.2 were 127–146 and 95–159% higher, respectively, than the control. Highly active morphological variants of the fungus were chosen for further selection experiments.     

Mechanism of activation and spectral shift of the F-695 emission band in chloroplasts as induced by 1,10-phenanthroline

1. Changes in the fluorescence emission spectrum of chloroplast, at 77 °K, induced by chaotropic reagents and 1,10-phenanthroline, were analyzed.2. Fourth-derivative analysis of the emission spectra identified the exact location of a new band (referred to as “F-700”) at 700 nm and showed that the conversion of F-695 into F-700 does not occur by a gradual red-shift of the F-695 band, but by the appearance of a new band at 700 nm at the expense of an intensity decrease in the F-695 emission.3. F-700 shows two distinct fluorescence characteristics, namely the wavelength of its emission maximum and its intensity, but still retains the principal properties of F-695 such as steep temperature dependence at low temperatures, transient phenomena at 77 °K, and an excitation spectrum of the Photosystem II type. Thus F-700 is concluded to be a modified state of F-695.4. In addition to the compounds of the urea-guanidine class, inorganic anions such as SCN^?, I^? and ClO₄^? were active in the transformation. The specificity and theorder of effectiveness of these reagents indicated that their action is that of chaotropic reagents. Transformation was inhibited by the presence of compounds such as sugars, salts, alcohols and dimethylsulfoxide which seem to affect the activity of water.5. 5-Methyl-1,10-phenanthroline partly substituted for the action of 1,10-phenanthroline, while the other six different derivatives of 1,10-phenanthroline and a few other bifunctional ligands were inactive. The structure-activity relations and the effective concentrations in the transformation differed greatly from those of the inhibition of the electron transport chain, suggesting that the action of 1,10-phenanthroline in the transformation is a yet unrecognized action of this reagent on Photosystem II.6. Transformation was generally observed in chloroplast preparations from 11 different higher plants and two species of algae tested. In Lolium sp. the transformation was partly attained by 1,10-phenanthroline alone.7. From these results, the state of F-695 in chloroplast membranes and the mechanism of transformation into F-700 are discussed.     

Isolation and characterization of extracellular glucose oxidase from Penicillium adametzii LF F-2044.1

Hydroxides of magnesium and zinc, aluminum oxide, zinc phosphate, and co-precipitated Ca₃(PO₄)₂ and Mg(OH)₂ were efficient in binding extracellular glucose oxidase (GO) of P. adametzii LF F-2044.1 in a culture liquid filtrate (CLF). Basic Al₂O₃ was the most appropriate adsorbent for GO isolation from the CLF of the fungus. A GO isolation method was developed, which allowed for obtaining an enzyme with a high degree of purification. Spectral properties of the enzyme, its catalytic activity, and stability were characterized. The GO of P. adametzii LF F-2044.1 exhibited high pH stability, retaining activity within the range 4.5–9.0. The rate that GO-catalyzed D-glucose oxidation increased as the temperature increased (up to approximately 60°C). The catalytic activity and thermal stability of GO depended on its concentration in the medium. Under optimum conditions, the fractions GO-1 and GO-2 were characterized by K _M values of 1.56 × 10^?2 and 2.19 × 10^?2 M, respectively; the corresponding values of k _cat equaled 235.1 and 318.2 s^?1.     

Purification and characterization of two constitutive cytochromes P-450 (F-1 and F-2) from adult female rats: identification of P-450F-1 as the phenobarbital-inducible cytochrome P-450 in male rat liver

Two hepatic microsomal cytochromes P-450, P-450F-1 and P-450F-2 were purified to electrophoretic homogeneity from untreated adult female rats by high-performance liquid chromatography (HPLC) with anion-exchange, cation-exchange, and hydroxyapatite columns. Cytochromes P-450F-1 and P-450F-2 were not adsorbed with the anion-exchange column, but were retained on a cation-exchange column and were separated poorly. These forms separated on hydroxyapatite HPLC. The molecular weights of cytochromes P-450F-1 and P-450F-2 were 50,000 and 49,000, respectively. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of cytochromes P-450F-1 and P-450F-2 were at 450 and 448 nm, respectively. Both forms catalyzed the N-demethylation of benzphetamine and had low catalytic activity for 7-ethoxycoumarin. Cytochrome P-450F-1 had low 2 alpha-hydroxylation activity toward testosterone. Cytochrome P-450F-2 had low 15 alpha-hydroxylation activity. On the basis of these results and those of NH2-terminal sequence analysis, cytochrome P-450F-2 seemed to be the typical female-specific cytochrome P-450. The NH2-terminal sequence of cytochrome P-450F-1 was identical to that of cytochrome P-450PB-2 purified from hepatic microsomes of male rats treated with phenobarbital. Cytochromes P-450F-1 and P-450PB-2 had identical chromatographic properties, minimum molecular weight, spectral properties, and peptide maps. Furthermore, the antibody to phenobarbital-inducible cytochrome P-450PB-2 gave a single immunoprecipitin band with cytochrome P-450F-1 by Ouchterlony double-diffusion analysis.     

Effect of mutation on synthesis of alkaloid byPenicillium roquefortii VKM F-141 andP. fellutanum VKM F-1073

Mutant strains ofPenicillium roquefortii VKM F-141 andP. fellutanum VKM F-1073 were obtained by mutagenesis induced by ultraviolet irradiation,N-methyl-N-nitronitrosoguanidine, and bromouracil. By the rates of alkaloid production, the mutant strains can be divided into three groups: (1) unable to synthesize alkaloids; (2) with a high rate of biosynthesis; (3) with changed alkaloid composition. Compounds not characteristic of wild-type cultures were found in alkaloid fractions of some mutant strains.     

Effects of urea and o-phenanthroline on F-695 emission in chloroplasts

To clarify the nature of the chlorophyll species which fluorescesat about 695 nm in vivo (F-695), effects of the addition ofurea and related compounds and of urea plus o-phenanthrolineon the emission spectra of spinach chloroplast fragments, at77°K, were examined. F-695 emission was partially decreased by the presence of alow concentration (0.1 M) of urea, thiourea, guanidine hydrochloride,methylguanidine hydrochloride, acetamide, N-methylurea, or dimethylurea. The concurrent addition of o-phenanthroline with the reagents(0.1–1 M) caused a marked increase in F-695 emission anda decrease in F-685 emission. Methyl-substituted ureas and acetamide,however, were less effective. The effect was largely dependenton the pH of the sample. These two effects, the decrease and increase in F-695 emission,were reversible and were inhibited by the presence of ethanol. The phenomena are probably due to specific interactions betweenadded reagents and the component(s) in chloroplasts which havean intimate connection with a postulated energy trap; an invivo species of chlorophyll responsible for the fluorescenceemission near 695 nm. (Received August 17, 1971; )