A re-evaluation of evidence attributing the difference in cleavage rates of restriction endonuclease at different sites in the substrate to differences in Km values.

The only result supporting the hypothesis that the differences in restriction endonuclease cleavage rates at various target sites are caused by differences in Km values was reported by Forsblom, Rigler, Ehrenberg, Petterson & Philipson [(1976) Nucleic Acids Res. 3, 3255-3269]. The present work shows that the kinetic analysis in that paper is based on incorrect derivation and in fact provides no support for the hypothesis mentioned.     

Dynamic bending rigidity of DNA

Rapidly relaxing components in the decay of the transient electric dichroism of DNA restriction fragments were reported by Diekmann et al. [(1982) Biophys. Chem. 15, 263-270] and P?rschke et al. [(1987) Biopolymers 26, 1971-1974]. These are analyzed using a new normal mode theory for weakly bending rods and assigned to bending. The longest bending relaxation times for fragments with 95-250 base pairs coincide with the theoretical curve calculated for a dynamic bending rigidity corresponding to a dynamic persistence length Pd = 2100 A. Analysis of the relative amplitudes of fast and slow components following weak orienting pulses is also consistent with a rather large dynamic persistence length. The enhancement of the relative amplitude of the fast component in large electric fields is attributed to steady-state bending of initially perpendicular DNAs by the field. Several reasons are proposed why the dynamic bending rigidity is 4 times larger than the apparent static bending rigidity inferred from equilibrium persistence length measurements on the same fragments.     

Endonuclease (R) subunits of type-I and type-III restriction-modification enzymes contain a helicase-like domain.

A statistically significant amino acid sequence similarity is demonstrated between the endonuclease (R) subunit of EcoK restriction-modification (R-M) enzyme, and RNA and DNA helicases of the so-called 'DEAD' family. It is further shown that all three known sequences of R subunits of type-I and type-III R-M enzymes contain the conserved amino acid sequence motifs typical of the previously described helicase superfamily II [(1989) Nucleic Acids Res. 17, 4713-4730]. A hypothesis is proposed that these enzymes may exert helicase activity possibly required for local unwinding of DNA in the cleavage sites.     

Two separable functional domains in the sigma-subunit of RNA polymerase in Bacillus subtilis?

The sigma-subunit of RNA polymerase is responsible for promoter recognition in prokaryotes [(1969) Nature 221, 43-46]. Alterations in the sigma-subunit are thought to be involved in controlling 'global' changes in gene expression, such as those involved in differentiation in the spore-forming bacterium Bacillus subtilis [(1981) Cell 25, 582-584]. Stragier et al. [(1985) FEBS Lett. 195, 3-11] have proposed that sigma-factors are composed of two domains: a C-terminal domain involved in promoter recognition and an N-terminal domain involved in interactions with RNA polymerase. We have sequenced another developmental gene from B. subtilis, spoIIIC, and the strong homology of its predicted product suggests that it too may be a sigma-factor. However, the spoIIIC product is small and lacks completely the conserved N-terminal domain of the sigma-subunits. I propose that the product of the spoIIIC gene may carry out the DNA-recognition functions of a sigma-factor but that it probably requires an auxiliary factor to interact with core RNA polymerase.     

Erythrocyte membrane acyl:CoA synthetase activity

The presence of long-chain acyl:CoA synthetases in mammalian microsomes and mitochondria has been established previously [(1971) Biochim. Biophys. Acta 231, 32-47]. The presence of a plasma membrane-associated enzyme was investigated in human erythrocyte ghost plasma membranes, where an enzyme exhibiting high activity, and with a preferred substrate of 18 carbon chain length, was discovered. The results are consistent with the presence of a single enzyme. The effect of the degree of unsaturation of the fatty acid substrates was not as pronounced as that arising from the length of the carbon chain. The pattern of substrate preference of the enzyme was omega 3 polyenoics greater than omega 6 polyenoics greater than omega 9 monoenoics greater than saturated fatty acids. This may relate to the similar substrate preference pattern exhibited by the fatty acyl desaturase enzymes. However, the role played by long-chain acyl:CoA synthetase in erythrocyte metabolism is uncertain, but may relate to the transportation of polyenoic fatty acids in the circulation.     

Synergistic enhancement of type I and III collagen production in cultured fibroblasts by transforming growth factor-β and ascorbate

Transforming growth factor-β (TGF-β) is a prototype of a family of polypeptides that regulates cellular growth and phenotypic differentiation [(1986) Science 233, 532-534; (1987) Cell 49, 437-438]. TGF-β injection induces angiogenesis and fibrosis locally [(1986) Proc. Natl. Acad. Sci. USA 83, 4167-4171; (1987) Science 237, 1333-1336] and stimulates the synthesis of extracellular matrix proteins, fibronectin, collagens, and proteoglycans in vitro in many cell types [(1986) J. Biol. Chem. 261, 4337-4345; (1987) Biochem J. 247, 597-604]. Ascorbate is also known to induce collagen synthesis and to promote wound healing [(1988) J. Invest. Dermatol. 90, 420-424; (1986) Coll. Rel. Res. 6, 455-466]. We report that in cultured human skin fibroblasts, ascorbate and TGF-β synergistically enhance the biosynthesis of type I and III collagens and their steady-state mRNAs. TGF-β alone has no enhancing effect on type III collagen synthesis. The cooperation between ascorbate and TGF-β may be of significance in wound healing and in disorders of fibrosis.     

Analysis of a circular code model

A circular code has been identified in the protein (coding) genes of both eukaryotes and prokaryotes by using a statistical method called trinucleotide frequency (TF) method [Arquès & Michel (1996). J. theor. Biol. 182, 45-58]. Recently, a probabilistic model based on the nucleotide frequencies with a hypothesis of absence of correlation between successive bases on a DNA strand, has been proposed by Koch & Lehmann [(1997). J. theor. Biol. 189, 171-174] for constructing some particular circular codes. Their interesting method which we call here nucleotide frequency (NF) method, reveals several limits for constructing the circular code observed with protein genes.     

The proteasome/multicatalytic-multifunctional proteinase. In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes.

Proteinase yscE, the proteasome/multicatalytic-multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555-562]. A well-defined set of proteins degraded via ubiquitin-mediated proteolysis are the substrates of the N-end rule pathway [(1986) Science 234, 179-186; (1989) Science 243, 1576-1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N-end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin-mediated proteolysis.     

匙吻鲟仔稚鱼消化酶发育的研究

对出膜后0—53d匙吻鲟的酸性蛋白酶、碱性蛋白酶、α-淀粉酶、脂肪酶以及磷酸酶的活性变化进行了测定。匙吻鲟出膜后饲养于室内水泥培育池中,从第3天开始投喂枝角类,之后于第40天将试验鱼转移至池塘。试验材料为受精卵及出膜后第3、第6、第12、第20、第30、第40、第44、第47、第53天仔稚鱼样品。研究发现主要消化酶在出膜时或卵黄期即可检测出活力。碱性蛋白酶和酸性蛋白酶分别在出膜后3d(3DAH)和刚出膜时(0DAH)检测出活力。碱性蛋白酶活力在44DAH达到最大值[(1.96±0.09)U/fish],47DAH出现下降,但在53DAH开始上升,比活力在53DAH达到最大值[(8.84±0.59)U/mg protein]。酸性蛋白酶在44DAH达到最大值[(0.52±0.05)U/fish],比活力在6DAH出现第一个峰值[(2.08±0.09)U/mg protein],并在30DAH出现最小值[(0.83±0.06)U/mg protein]。试验期间碱性蛋白酶活力高于酸性蛋白酶。在12DAH—40DAH期间α-淀粉酶活力相对稳定,并在47DAH达到最大值[(0.42±0.03)U/fish],比活力在12DAH出现一个峰值[(1.18±0.12)U/mg protein],并于47DAH出现最大值[(1.94±0.16)U/mg protein]。发育早期脂肪酶活力较高,活力和比活力分别在30DAH[(0.20±0.02)U/fish]和6DAH[(2.28±0.22)U/mg protein]出现最大值。碱性磷酸酶活力变化趋势与比活力变化趋势相似,但是最大值分别出现在44DAH[(0.08±0.00)U/fish]和30DAH[(1.96±0.15)U/mg protein]。酸性磷酸酶活力在3DAH出现一个峰值[(0.01±0.00)U/fish],之后显著升高,并在44DAH达到最大值[(0.05±0.00)U/fish],其比活分别在30DAH[(1.19±0.10)U/mg protein]和44DAH[(1.10±0.08)U/mg protein]出现两个峰值。结果表明,蛋白酶、α-淀粉酶和磷酸酶随个体发育活力增加,碱性蛋白酶在个体发育早期对蛋白质的消化具有重要作用。养殖环境发生改变时,酸性蛋白酶、α-淀粉酶、碱性磷酸酶和酸性磷酸酶活力在生长减慢时增加,生长加快时降低,而脂肪酶活力则维持稳定。     

Gamma-COP, a coat subunit of non-clathrin-coated vesicles with homology to Sec21p.

Constitutive secretory transport in eukaryotes is likely to be mediated by non-clathrin-coated vesicles, which have been isolated and characterized [(1989) Cell 58, 329-336; (1991) Nature 349, 215-220]. They contain a set of coat proteins (COPs) which are also likely to exist in a preformed cytosolic complex named coatomer [(1991) Nature 349, 248-250]. From peptide sequence and cDNA structure comparisons evidence is presented that one of the subunits of coatomer, gamma-COP, is a true constituent of non-clathrin-coated vesicles, and that gamma-COP is related to sec 21, a secretory mutant of the yeast Saccharomyces cervisiae.     

Segregation into separate rouleaux of erythrocytes from different species. Evidence against the agglomerin hypothesis of rouleaux formation.

Erythrocytes from one species were labelled with fluorescein isothiocyanate and mixed with unlabelled erythrocytes from another species. Albumin polymers were added to generate rouleaux. The species of origin of erythrocytes in rouleaux was determined by fluorescence microscopy. Erythrocytes from different species segregated into independent rouleaux. However, fluorescent and non-fluorescent erythrocytes from one individual were mixed randomly in rouleaux. These results confirm, using a novel experimental approach, previous observations of Sewchand & Canham [(1976) Can. J. Physiol. Pharmacol. 54, 437-442]. Since rouleaugenic agents are not species-specific, under the 'agglomerin' hypothesis of rouleau formation they would be expected to form bridges between cells from different species. It follows that either the agglomerin hypothesis is incorrect, or additional species-specific surface components are involved in the aggregation of agglomerin-cross-bridged cells.     

Electrogenic proton exchange between cytochrome a3 active center and M-aqueous phase. Evidence for cytochrome a3-associated input proton well

The rate of cyanide binding with the oxidized cytochrome-c oxidase in proteoliposomes is controlled by ionization of a protein group with pK approximately 6.7, the ligand reacting with the protonated enzyme only [(1983) Bioorg. Chem. (USSR) 9, 216-227]. As shown here, the kinetics of cyanide binding depends on the pH inside the proteoliposomes. The reaction rate is affected by the electrical potential difference across the proteoliposome membranes as if the a3-linked ionizable group exchanged H+ with the proteoliposome interior electrogenically. The data corroborate a hypothesis on the existence of a proton well communicating cytochrome oxidase O2-reducing center with the M-aqueous phase.     

Drug protein conjugates--III. Inhibition of the irreversible binding of ethinylestradiol to rat liver microsomal protein by mixed-function oxidase inhibitors, ascorbic acid and thiols

The metabolism of [6,7-3H]ethinylestradiol [( 3H]EE2) by rat liver microsomes was studied in vitro. After incubation of [3H]EE2 with rat liver microsomes for 20 min, 90% of the substrate was metabolised and 18% of the 3H-labelled material irreversibly bound to microsomal protein. Ascorbic acid (1 mM) decreased irreversible binding of 3H and produced an accumulation of 2-hydroxyethinylestradiol (2OH-EE2), while mixed-function oxidase inhibitors (0.5 mM) decreased binding of 3H to protein by inhibiting EE2 2-hydroxylation. Addition of thiols gave water-soluble metabolites which were characterised as 1(4)-thioether derivatives of 2OH-EE2 by co-chromatography with synthetic products. The results are consistent with the hypothesis that the chemically reactive metabolite of EE2 formed in vitro is either a quinone or o-semiquinone derived from 2OH-EE2 [1].     

Dextran sulfate and heparin sulfate inhibit platelet-activating factor-induced pulmonary edema.

We tested the hypothesis that dextran sulfate and heparin sulfate inhibit platelet-activating factor- (PAF) induced pulmonary edema in the isolated perfused guinea pig lung via a charge-dependent mechanism. Dextran sulfate prevented the changes in pulmonary capillary pressure (Ppc, 7.8 +/- 0.9 vs. 14.0 +/- 0.7 cmH2O), lung weight gain (dW, +0.48 +/- 0.29 vs. +8.41 +/- 2.07 g), and pulmonary edema formation or wet-to-dry weight ratio [(W-D)/D, 6.5 +/- 0.3 vs. 13.2 +/- 2.6] occurring 60 min after PAF infusion (10(-11) M) into an isolated lung. The unsulfated form of dextran had no protective effect [Ppc, dW, and (W-D)/D, 11.9 +/- 1.4 cmH2O, +5.33 +/- 2.18 g, and 11.2 +/- 3.2, respectively]. The unrelated anionic compound, heparin sulfate, also inhibited the PAF response [Ppc, dW, and (W-D)/D, 7.0 +/- 0.5 cmH2O, +0.61 +/- 0.32 g, and 6.1 +/- 0.2, respectively], whereas the partially desulfated form of heparin was not effective in inhibiting PAF-induced edema [Ppc, dW, and (W-D)/D, 15.1 +/- 0.7 cmH2O, +6.07 +/- 1.58 g, and 10.0 +/- 1.2, respectively]. When the metachromatic dye crystal violet was used as an indicator of charge interactions, the sulfated compounds interacted with PAF in vitro. The data indicate that PAF-induced pulmonary edema is inhibited by sulfated polysaccharides, possibly via a charge interaction between negatively charged compounds and PAF.     

The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a DD-carboxypeptidase in vitro

A chemiluminescence method for determining acetylcholinesterase activity is described. It is an adaptation of the chemiluminescence assay of acetylcholine described by Israël & Lesbats [(1981) Neurochem. Int. 3, 81-90; (1981) J. Neurochem. 37, 1475-1483]. The acetylcholinesterase activity is measured by monitoring the increase in light emission produced by the accumulation of choline or by determining the amount of choline generated after a short interval. The assay is rapid and sensitive, and uses the natural substrate of the enzyme. Kinetic data obtained with this procedure for acetylcholinesterase from Torpedo and Electrophorus electric organs were comparable with those obtained by using the method of Ellman, Courtney, Andres & Featherstone [(1961) Biochem. Pharmacol. 7, 88-95]. In addition, it was shown that sodium deoxycholate totally inactivated Torpedo acetylcholinesterase but not the Electrophorus enzyme. Competitive inhibitors of acetylcholinesterase protected the enzyme from inactivation.     

Improvement on the competitive binding assay for the measurement of cyclic AMP by using ammonium sulphate precipitation.

The protein-binding assay developed by Brown, Albano, Ekins, Sgherzi & Tampion [(1971) Biochem. J. 121, 561-562] and Brown, Ekins & Albano [(1972) Adv. Cyclic Nucleotide Res. 2, 25-40] was modified by using precipitation with (NH4)2SO4 of the protein-cyclic AMP complex instead of adsorption of the free nucleotide on charcoal. The half-life of the protein-cyclic AMP complex obtained in the presence of charcoal was lower than that of the (NH4)2SO4-precipitated complex. In consequence, owing to the great stability of the precipitated protein-cyclic AMP complex, this method allows more accurate and reproducible determinations.     

Facile, alternative route to lubeluzole, its enantiomer, and the racemate

Lubeluzole [(S)-9] has been synthesized by a convergent synthesis, alkylation of N-methyl-N-piperidin-4-yl-1,3-benzothiazol-2-amine (4) with (+)-(R)-1-chloro-3-(3,4-difluorophenoxy)propan-2-ol [(+)-(R)-8] being the key step. Alcohol (+)-(R)-8 was obtained from commercially available (R)-epichlorohydrin [(R)-6], while the thiazole derivative 4 was easily obtained starting from N-protected piperidin-4-one (1) in a three-step procedure. The same method was used in order to obtain both the (R)-stereoisomer of lubeluzole [(R)-9] and its racemate [(RS)-9]. Overall yields ranged from 20% to 35%. The enantiomeric excess values for (S)-9 and (R)-9 were 97% and 94% respectively, as analyzed by chiral HPLC.     

Functional characterization of Asp-317 mutant of human renin expressed in COS cells

Renin is an unique aspartyl (acid) protease with optimal activity at neutral pH. It has been suggested that Ala-317 of human renin contributes to neutral optimum pH of the enzyme [(1984) FEBS Lett. 174, 102–111]. The hypothesis was verified by the characterization of mutant renin in which Ala-317 was replaced with Asp by a site-directed mutagenesis. Wild-type and mutant renins, which were expressed in COS cells, exhibited different pH-activity profiles and optimum pH of the mutant enzyme was lower than that of the wild-type enzyme. This result suggests that Ala-317 of human renin plays an important role in the determination of optimum pH of the enzyme.     

Gamma-subunit of mouse retinal cyclic-GMP phosphodiesterase: cDNA and corresponding amino acid sequence

The cDNA nucleotide and corresponding amino acid sequences of the gamma-subunit of cyclic-GMP phosphodiesterase (cGMP-PDE gamma) from mouse retina have been determined. The cDNA translated region was found to be 91.5% homologous to the cDNA coding region for the enzyme from bovine retina [(1986) FEBS Lett. 204, 288-292]. On Northern blots of normal mouse retinal RNAs this cDNA hybridized the cGMP-PDE gamma mRNA which is 900 bp long. The mouse gamma-subunit contains 87 amino acid residues which share 97.7% homology with the bovine polypeptide [(1986) FEBS Lett. 204, 288-292]. Only two amino acids have been changed, Ala 8 to Gly and Met 17 to Ile.     

Acoustic microscope study of the elastic properties of fluorapatite and hydroxyapatite, tooth enamel and bone.

Measurements of Rayleigh velocity and attenuation were taken in single mineral crystals of hydroxyapatite and fluorapatite at angular intervals relative to their c axes, using an acoustic microscope. These results are compared with the values that were calculated using the elastic constants of apatite from Yoon and Newnham [(1969) Am. Miner. 54, 1193-1197.] and Katz and Ukraincik [(1971) J. Biomechanics 4, 221-227.]. The slowness curves of various wave modes are plotted and discussed in relation to cross-coupling effects that were found to cause instability in measurements of attenuation for the c axes direction. Velocity measurements were taken in specimens of tooth enamel and bone. Here comparisons are made with the values that were calculated by modelling the 'z' scan response of the microscope, using published data for the elastic and acoustic properties. Comparisons are also made with the measurements on single crystals, since apatite is a major component of enamel and bone.