共找到 20 条结果
The recently developed stereospecific sodium salt glycosylation procedure has been successfully applied to the synthesis of the beta-D-2'-deoxyribofuranosides of benzimidazole, 5,6-dihalogeno benzimidazoles, and some 2-substituted analogues in high yield. The 5,6-dibromo analogue was obtained by bromination of the parent nucleoside. These have all been characterized by spectroscopic methods, including 1H NMR, which permitted analyses of their solution conformations and comparison with those of the corresponding ribofuranosides. Some biological aspects, including preliminary results on cytotoxicity and antiviral activity, are briefly considered.
Phytochemical and microscopial analysis of leaves and stems of various species of Encelia showed a strict correlation between the presence of resin ducts and the accumulation of benzopyrans and benzofurans. Fluorescence microscopy of Encelia farinosa proved unambigously that these compounds are stored exclusively in the resin ducts and the surrounding cells.
There are non-random features in the occurrences of nucleotides in the DNA's of certain organisms which are detectable by statistical analyses of the entire sequence. Earlier, using the bacteriophage Phi-X 174 DNA sequence, we had reported that the self-information values for one type of dinucleotide association showed a marked periodicity when their autocorrelation coefficients were graphed. A similar, but computationally simpler, analysis has been developed which gives a comparable indication of periodicity. The difference, in average autocorrelation coefficients obtained with this analysis, between the peak values and all others has been used as an index to compare the extent of periodic non-randomness for a series of natural DNA sequences and for various artificial sequences. Calculations show that triplet periodicity, the relationship between dinucleotides separated by a single nucleotide, is characteristic only of the natural sequences of certain filamentous phages and is not found prominently in any other DNA analyzed (including sequences of similar length from plasmids, yeast, bacteria and higher animals). By shuffling nucleotides in a given sequence or by substituting selected nucleotides to alter various positions in both periodic and aperiodic sequences, we have found that an excess or deficiency of a given nucleotide at one of the three positions in a triplet reading frame can simulate the periodic characteristic. Thus, it appears that this global statistical analysis detects the tendency for single-strand phages to utilize a specific nucleotide, rather than one randomly selected, to constitute codons.
The influence of Mg2+ ions on the secondary and tertiary structure of the RNA from bacteriophage MS2 was investigated by small-angle X-ray scattering and light scattering and by sedimentation experiments. The analysis of the outer part of the X-ray scattering curve obtained at low temperature in the absence of Mg2+ yielded a cross-section radius of gyration of 0.88 nm and a mass per unit length of 1720 g mol-1 nm-1. Very similar values for these parameters, which refer to the secondary structure of the RNA molecule, were also derived from the X-ray scattering curves obtained in the presence of different amounts of Mg2+ (0.07 to 1 ions per nucleotide). On the contrary, the inner part of the X-ray scattering curves turned out to be highly dependent on the Mg2+ concentration: the cross-section radius of gyration and the mass per unit length, which were determined from the scattering curves at small angles as parameters related to the tertiary structure of the RNA, amounted to 3.11 nm and 4000 g mol-1 nm-1, respectively, in the absence of Mg2+ and increased significantly upon raising the concentration of Mg2+. The increase of these structural parameters was found to be accompanied by a decrease of the overall radius of gyration (as revealed indirectly by X-ray scattering and directly by light scattering measurements) and by an increase of the sedimentation coefficient. The results from the investigations of the RNA at low temperature clearly establish the existence of double-stranded structures down to very low Mg2+ concentrations as well as the occurrence of Mg2+ induced changes of the tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)
This study was aimed at investigating whether cells of CFU-C derived colonies could form secondary colonies. Bone marrow cultures of volumes of agar medium between 25 microliter and 75 microliter contained in glass capillaries were stimulated with mouse lung-conditioned medium (MLCM) containing granulocyte/macrophage colony-stimulating factor (GM-CSF). Agar gels with colonies of up to greater than or equal to 20 were blown out into identical culture medium, completely dispersed on a whirl-mix to single cell suspensions, and used for establishing secondary agar cultures. In these secondary cultures considerable numbers of secondary granulocytic, mixed granulocytic/macrophage and macrophage colonies as well as numerous clusters arose. In contrast, when single colonies were recultured, only few secondary cell aggregates were formed. When primary cultures containing up to greater than or equal to 20 cell aggregates were used for serial reculture at intermittent intervals of 3 and 4 days, a 2-7-fold increase of colony-forming cells was found in tertiary cultures as was monitored by 7 day colony counts. And by use of different kinds of CSF-containing media, an over 4-fold increase of secondary over primary colonies was obtained with bovine lung-conditioned medium (BLCM) in primary and L-cell-conditioned medium (LCCM) in secondary 7 day cultures. Primary capillary cultures were found to be devoid of CFU-S. Also, setting up bone marrow cultures in petri dishes and stimulating with MLCM, growth of primary as well as secondary colonies was obtained. The results indicate some self-renewal potential of CFU-C in vitro.
We have analysed spermatogenetic cells by flow cytometry to quantify effects of ionizing radiation. The radiation-induced reductions of testicular DNA-synthesizing cells, primary spermatocytes, haploid round and elongated spermatids as well as the increases of numerical chromosome aberrations (abnormal diploid spermatids and aneuploidies) in NMRI inbred mice are described. Testicular weights were determined as a parameter of germ cell decrease, and histologic cross sections of the testes were analysed. Since even an exposure of 0.05 Gy (= 5 rad) may be detected by a reduction of DNA-synthesizing cells (Acta Radiol. Oncol. Radiat. Phys. Biol. 21, 349-351 (1982) [1]), the use of the in vivo system "spermatogenesis" as a biological dosimeter to monitor low dose effects and to determine RBE values of different radiation qualities is suggested.
A high yielding production of the trichothecene mycotoxin 3-acetyldeoxynivalenol (3-AcDON) in cultures of Fusarium culmorum is described. By supplying [14C]acetate, 14C-labelled 3-AcDON suitable for further metabolic studies has been obtained. The pattern of labelling has been ascertained by using 13C-labelled acetate precursors, and is in line with established biosynthetic data. A second trichothecene produced in significant amounts by F. culmorum has been identified as 3 alpha, 15-diacetoxy-7 alpha, 8 alpha-dihydroxy-12, 13-epoxytrichothec-9-ene (7 alpha, 8 alpha-dihydroxycalonectrin).
The properties of the sarcoplasmic reticulum membranes isolated from slow-twitch type I soleus and fast-twitch type II psoas muscles of control and thyroxine treated rabbits were comparatively studied. Membrane yield, maximal calcium storing capacity, ATP-supported calcium uptake, calcium-dependent ATPase activity and calcium-dependent phosphoprotein formation were found to be 3-10 fold higher in psoas than in soleus preparations. Membrane yield, calcium-dependent ATPase activity, ATP-supported calcium transport and calcium-dependent phosphoprotein are at least twice enhanced in the membranes from soleus muscles of animals treated for 14-21 days with thyroxine. The corresponding capacities of the membranes from psoas muscles are not further augmented by the same thyroxine treatment. The maximal calcium storing capacity of the psoas membranes is their sole specific property which is significantly increased. The changes in the properties of the soleus muscles' sarcoplasmic reticulum membranes are engendered by an increase from 5 to 30-50% in the number of type II fibres. Since the calcium transporting properties of the sarcoplasmic reticulum membranes from type II fibres qualitatively differ from those of type I fibres, thyroxine does not only affect quantitative but also qualitative parameters of the muscles' sarcoplasmic reticulum membrane system.
The kinetics of thermal denaturation of ceruloplasmin in water and in water with different percentage of D2O in the temperature range 25-85 degrees C, following the optical density change of the 610 nm charge transfer band of the protein has been investigated. A temperature Tt approximately equal to 65 degrees C above which an irreversible denaturation process of the protein active site takes place has been found. The kinetics of the denaturation process of the protein are fitted by two first order reactions, which have been assigned to a different thermal denaturation behaviour of the two Cu2+ type I sites existing in the protein. Addition of D2O to the protein solution affects the two kinetics in a different way, i.e. the rate of one of them is increased whilst that of the other is decreased. The different effect of D2O on the kinetics of disruption of the two copper sites is discussed in terms of different location and degree of hydrophobicity of the two Cu2+ type I sites.
In a simple phosphate buffer or a sodium chloride solution resting cells of Pseudomonas spec. DSM 2874 produced up to 15 g/l of different rhamnolipids. The rhamnolipid composition of the organic crude extract depended on the temperature during the cultivation and on the C-source. The optimal sodium chloride concentration for rhamnolipid formation was about 100 mM/l and the optimal phosphate buffer concentration about 65 mM/l. The optimal pH-value for the production of rhamnolipids from n-alkanes or glycerol was in the range pH 6.0-7.2. While rhamnolipid formation with glycerol as the sole C-source showed a wide optimum ranging from 27 degrees up to 37 degrees C, production of rhamnolipids from n-alkanes had a sharp optimum at 37 degrees C. The addition of multivalent cations, different N-sources and EDTA caused an inhibition of rhamnolipid formation, while the n-alkane concentration had no influence. Specific rhamnolipid formation decreased with increasing cell concentration. Various C-sources were suitable for the formation of rhamnolipids by resting cells of Pseudomonas spec. DSM 2874. Yields, which were comparable to those obtained on n-alkanes or glycerol, were found for stearic acid, fatty alcohols and vegetable oils. A study of the time course of glycolipid production of resting cells was carried out in a 20 1-bioreactor with an intensor system and with n-tetradecane as the sole C-source.
The phospholipase C-activity in crude extracts of bovine blood platelets is strongly inhibited by the calmodulin-inhibitors fluphenazine and calmidazolium in the mM range, and activated by ATP and ADP, but not by AMP. The activating effect is also shown by the nonhydrolysable ATP- and ADP-analogs alpha,beta- and beta,gamma-methyleneadenosine 5'-triphosphate and alpha,beta-methyleneadenosine 5'-diphosphate, thus indicating that it is an allosteric effect. The stimulation of the phospholipase C-activity by ATP is also detectable in some partially purified fractions of the crude platelet extract, but it is abolished on further purification of the enzyme.
Incubation of synaptosomal plasma membranes (SPM) with liposomes of phosphatidylserine (PS), phosphatidylinositol (PIN) or phosphatidylglycerol (PGL), led to an increase of acetylcholinesterase (AchE) activity at concentrations of 0.1-1 mumol phospholipids per mg SPM protein. The use of higher concentrations (1-7 mumol/mg protein), however, led to a progressive inhibition of the activity with respect to the maximal percentage of enzyme stimulation. To explain the enzyme stimulation by the acidic phospholipids, AchE was solubilized with the detergent Lubrol-PX and showed no change in the enzyme activity at any PS, PIN or PGL concentration used, indicating that these compounds do not act on the protein molecule directly. Arrhenius plots of AchE activities in untreated SPM (control), exhibited a break point at 23 degrees C, which was decreased to 16-17 degrees C in PS-treated SPM. Moreover, the Arrhenius activation energy (Ea) value in PS-treated SPM was increased related to the Ea below the break point in the control. These results indicate that acidic phospholipids do not act on AchE directly, but indirectly, affecting the membrane fluidity probably. Such modifications of interactions between lipid and AchE may control physiological processes in the central nervous system.
The effects of osmolarity, calcium concentration and cell-size liposomes in the subphase on the surface tension of phospholipid monolayers were investigated. The monolayers were spread from chloroform solutions of phosphatidic acid at air/water solution interface. The liposomes (of average diameter 3 micron) were formed from phosphatidic acid/egg lecithin (1:2) mixtures in water or 0.1 M water solutions of sucrose. For this system there were critical concentrations of calcium ions to produce a large reduction of the monolayer surface tension. The threshold calcium concentrations depended upon the sucrose concentration in the subphase. Without sucrose the threshold calcium concentration was 8 mM, while for isoosmotic sucrose solutions (0.1/0.1 M in/out of liposome) it was 14 mM. It sharply increased to 28 mM CaCl2 at sucrose concentration difference across the liposome membrane 0.02 M and decreased to 26 mM, 19 mM, and 18 mM with further increase of that difference to 0.04 M, 0.06 M, and 0.08 M, respectively. The rate of monolayer surface tension decrease was measured as a function of time at 30 mM CaCl2 and different sucrose concentrations in the subphase solution. The initial rates at first decreased with increasing the osmotic pressure and after that they increased. The minimum occurred at sucrose concentration gradient across the liposome membrane 0.02 M, i.e., at the point of maximum threshold calcium concentration required for large decrease of the monolayer surface tension. These facts may be explained by recent theories of dynamics of adhesion, instability and fusion of membranes modeled as thin films.
The sulfhydryl enzyme malate synthase from baker's yeast was X-irradiated with 6 kGy in air-saturated aqueous solution (enzyme concentration: congruent to 10 mg/ml; volume: 120 microliters), in the absence or presence of the specific scavengers formate, superoxide dismutase, and catalase. After X-irradiation, a small aliquot of the irradiated solutions was tested for enzymic activity while the main portion was investigated by means of small-angle X-ray scattering. Additionally, an unirradiated sample without additives was investigated as a reference. Experiments yielded the following results: X-irradiation in the absence of the mentioned scavengers caused considerable aggregation, fragmentation, and inactivation of the enzyme. The dose Dt37 for total (= repairable + non-repairable) inactivation resulted as 4.4 kGy. The mean radius of gyration was found to be about 13 nm. The mean degree of aggregation was obtained as 5.7, without correction for fragmentation. An estimation based on the thickness factor revealed that about 19% of material might be strongly fragmented. When this amount of fragments was accordingly taken into account, a value of 7.1 was obtained as an upper limit for the mean degree of aggregation. The observed retention of the thickness factor and the finding of two different cross-section factors are in full accord with the two-dimensional aggregation model established previously (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99-121 (1980)). The presence of catalytic amounts of superoxide dismutase and/or catalase, in the absence of formate, during X-irradiation reduced both aggregation and inactivation significantly. The presence of formate (10 or 100 mM) during X-irradiation led to a strong decrease of aggregation and inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)
Oxidation of methionine, 4-(methylthio)-2-oxobutyric acid (KMB), or 1-aminocyclopropane carbonic acid (ACC) are indicator reactions for activated oxygen species such as singlet oxygen (1O2), OH.-radical like oxidants, superoxide anion (O2.-), hydrogen peroxide (H2O2) or activated hemo-iron complexes like peroxidase- or catalase-"compound I". Methionine is oxidized by OH. as well as by 1O2 forming ethylene, but not by tetrachloro-decaoxygen complex (TCDO) in the absence or presence of catalytic hemoproteins such as peroxidase, hemoglobin or myoglobin. Both KMB and ACC are oxidized by TCDO under the catalysis of the above hemo-proteins where neither catalase nor superoxide dismutase are inhibitors. TCDO hemo-protein complex is an oxidant with similar properties as peroxidase-compound I and can clearly be differentiated from O2.-, H2O2, OH. and 1O2.
Hydrogen atoms from the radiolysis of water at pH 1.6 add to the 5,6-double bond of pyrimidines. The preferential site of attack is the C(5) position (values in brackets) in the case of 6-methyluracil (87%), 1,3-dimethyluracil (71%), uracil (69%) and poly(U) (60%). This reaction yields a radical of reducing properties which can be monitored by its reaction with tetranitromethane in a pulse radiolysis experiment. In thymine (37%), thymidine (32%) and 1,3-dimethylthymine (25%) H-addition no longer preferentially occurs at C(5), but addition is now mainly at C(6). Hydrogen abstraction from the methyl groups or the sugar moiety is negligible (less than or equal to 5.5%). A comparison is made with literature values for the equivalent reactions of OH radicals.
An enzymatic assay system for nitrate employing the membrane-bound nitrate reductase (EC 1.7.99.4) of E. coli is described. Contrary to previous enzymatic assay systems, the present method is a kinetic one, i.e. the substrate, nitrate, is assayed by measuring the reaction rate of the nitrate reductase-catalyzed reaction. Based on the observation that the nitrate reductase-catalyzed reaction obeys pseudo-first order kinetics, a test system is described allowing the assay of nitrate at a concentration as low as 1 ppm. The relatively high Michaelis-Menten constant for nitrate (0.3 mM) of the E. coli nitrate reductase favours nitrate assay by the kinetic method.
[14C]penicillin binding experiments and membrane analysis were carried out with cell envelope preparations from Escherichia coli and Proteus mirabilis. After incubation with [14C]penicillin G labeled free lipoprotein could be identified. The analysis of the isolated lipoprotein by SDS polyacrylamide gel electrophoresis indicates that there is only one protein with an apparent molecular weight of 7000. The amino acid composition of isolated labeled free lipoprotein from E. coli was identical to the lipoprotein already found in E. coli. It is a point of interest that the amino acid composition of the isolated labeled free lipoprotein from P. mirabilis D 52 differs from that found in other mutants of this strain. The free form of lipoprotein from P. mirabilis D 52 is composed of 61 amino acids and has glycine, phenylalanine and proline as specific components.
The effect of L-norleucine, an isomer of leucine, on protein metabolism in vivo was studied in suckling rats. Rats were injected subcutaneously with various doses of L-norleucine (0.5 and 5.0 mumol/g body wt.) every 12 h from 3 to 15 days post partum. Protein concentration, amino acid concentrations, and incorporation of [3H]tyrosine into protein were analyzed in liver, muscles of thigh and small intestine. Amino acid concentrations and insulin levels in serum were also measured. At 5 days of age, norleucine induced an increase in protein concentration of skeletal muscle with an increased incorporation of [3H]tyrosine into protein indicating an accelerated protein synthesis. Changes in protein metabolism were paralleled by alterations in the amino acid pattern of this tissue. When protein concentration and protein synthesis were increased in skeletal muscle, protein concentration of small intestine was decreased, accompanied by elevated levels of amino acids in tissue. Protein synthesis of small intestine was not altered by the norleucine treatment. The results suggest a close interrelationship between skeletal muscle and small intestine with respect to protein turnover. The effects of norleucine were less pronounced at 10 and 15 days of age, which indicates a metabolic adaptation to the treatment. Alterations in amino acid concentrations of tissue due to changes in protein metabolism were not uniform but tissue-specific. Current concepts for explaining the effects of branched-chain amino acids (BCAA) on protein turnover in skeletal muscle are based on the assumption that the BCAA or leucine alone might become rate-limiting for protein synthesis in muscle under catabolic conditions. The amino acid analogue norleucine, however, cannot replace any of the BCAA in protein. Additionally, norleucine affected protein metabolism in highly anabolic organisms. Therefore, the present thoughts on this issue appear to be incomplete.
The fatty acyl specificity of phospholipase A1 and A2 in homogenates of mouse bone marrow-derived macrophages was determined using phosphatidylcholine and phosphatidylethanolamine of different acyl chain composition. Phosphatidylcholine with arachidonoyl at position 2 was cleaved preferentially by an alkaline phospholipase A2 (pH-optimum 9.0) leading to selective liberation of arachidonic acid. In contrast, phosphatidylcholines with oleoyl or linoleoyl at position 2 were degraded mainly by an acid phospholipase A1 (pH-optimum 4-5) resulting in a conservation of these fatty acids esterified in lysophosphatides. Substrate kinetics of the alkaline phospholipase A2 revealed a 30 fold higher affinity (Km = 3.8 X 10(-7) M) for 1-acyl-2-arachidonoyl-glycerophosphocholine compared to 1-acyl-2-oleoyl-glycerophosphocholine. The kinetic data were not influenced by endogenous lipids indicating that exogenous substrates do not equilibrate with cellular lipids. These results are suitable to explain a selective liberation of arachidonic acid from a mixture of phospholipids.