R-loop hybridization of collagen DNA: Separation in Cs2SO4 gradients

High molecular weight poly(A)⁺ RNA (26–35 S) from chicken embryo trunks which is enriched for collagen mRNA was iodinated and hybridized to DNA under conditions of R-loop formation. The R-loops were separated in Cs₂SO₄ gradients from the bulk of DNA yielding double stranded DNA enriched for collagen genes.     

An analysis of the bovine genome by Cs2SO4-Ag density gradient centrifugation

Calf DNA preparations having molecular weights of 5 to 7 × 10⁶ have been fractionated by preparative Cs₂SO₄—Ag⁺ density gradient centrifugation into a number of components. These may be divided into three groups: (1) the main DNA component (1.697 g/cm³; all densities quoted are those determined in CsCl density gradients), the 1.704 and 1.709 g/cm³ components form about 50, 25 and 10% of the genome, respectively; they are characterized by having symmetrical CsCl bands and melting curves, both of which have standard deviations close to those of bacterial DNAs of comparable molecular weight, and by their G + C contents being equal to 39, 48 and 54%, respectively; after heat-denaturation and reannealing, their buoyant densities in CsCl are greater than native DNA by 12, 10 and 3 mg/cm³, respectively. (2) The 1.705, 1.710, 1.714 and 1.723 g/cm³ components represent 4, 1.5, 7 and 1.5% of the DNA, respectively, and exhibit the properties of “satellite” DNAs; their CsCl bands and melting curves have standard deviations lower than those of bacterial DNAs; after heat-denaturation and reannealing, their buoyant densities are identical to native DNA, except for the 1.705 g/cm³ component, which remains heavier by 5 mg/cm³; in alkaline CsCl, only the 1.714 g/cm³ component shows a strand separation. (3) A number of minor components, forming 1% of the DNA, have been recognized, but they have not been investigated in detail; two of them (1.719 and 1.699 g/cm³) might correspond to ribosomal cistrons and mitochondrial DNA, respectively.     

Large-scale purification of viroid RNA using Cs2SO4 gradient centrifugation and high-performance liquid chromatography

A procedure for the purification of viroid RNA from tomato plants is described which yields up to a milligram of viroid RNA of gel electrophoretic homogeneity within 2 days. This technique is at least three times as fast as previous methods and is generally applicable to other RNA species. Plant material was homogenized and phenol extracted. In a Cs₂SO₄ density gradient, viroid RNA together with low-molecular-weight RNA, was separated from large single-stranded RNA, DNA, polysaccharides, polyphenols, and other compounds. The separation is based on the differences in the buoyant density and on the selective precipitation of large single-stranded RNA in Cs₂SO₄. Further purification of viroid RNA was achieved by HPLC over a weak anion exchanger linked to silica gel of optimized pore size. The elution was carried out by a salt gradient with complete exclusion of divalent metal ions. The procedures were applied to whole plants, leaves, stems, roots, cells, and protoplasts. The yields of nucleic acids at the different steps of purification are given for leaves, stems, and roots.     

Buoyant densities of natural and synthetic DNA's in CsCl and Cs2SO4 considered as sequence-dependent properties

The buoyant densities of natural and synthetic DNA's can be accurately interrelated if second-neighbor influences are taken into account. We derive the following expressions, based partly on the buoyant densities of six synthetic DNA's, for the buoyant densities ρ (g/cm³) of DNA's having random sequences. In CsCl, and in Cs₂SO₄, . In these equations, H_G is the mole fraction of G : C base pairs in the DNA and the buoyant densities are calculated relative to densities for E. coli DNA of 1.703 and 1.426 (g/cm³) in CsCl and Cs₂SO₄, respectively.     

Fractionation of L-cell chromatin into DNA, RNA, and protein fractions on Cs2SO4 equilibruim density gradients

A new procedure is described for fractionation of chromatin into DNA, RNA, and total chromatin protein. By isopycnic gradient centrifugation of chromatin preparations in Cs₂SO₄ solutions containing dimethylsulfoxide and sodium sarcosyl it is possible to obtain highly purified fractions of these components. The method gives a very high yield of these chromatin fractions unlike some other methods, where irreversible binding to columns occurs. Also with this method it is possible to obtain highly concentrated fractions, which after a simple dialysis step, can be conveniently analyzed by polyacrylamide gel electrophoresis.Nuclei from L-929 cells were isolated by a method involving citric acid or by a method using a nonionic detergent. The yields of DNA obtained by both methods was compared. Chromatin was isolated from purified nuclei (prepared in either of the above ways) in two different ways also. In one method, chromatin was extracted from nuclei with 1 m NaCl. A second method involving fractionation of lysed nuclei in sucrose and metrizamide solutions was also used. The yields of DNA obtained by both methods was compared. There appears to be little nuclear membrane contamination of any of these chromatin preparations.A preliminary analysis of L-929 cell chromatin total RNA and protein fractions on polyacrylamide and agarose gels has been made. Both fractions appear to be quite complex with a wide spectrum of subcomponents of differing S values.     

NMR reveals a novel glutaredoxin-glutaredoxin interaction interface

Glutaredoxins (Grx) represent a large family of glutathione (GSH)-dependent oxidoreductases that catalyse the reduction of disulfides or glutathione mixed disulfide. Grx domains from pathogenic bacteria and plant Grxs have been recently reported to target specific peroxiredoxins (Prxs). The specificity that triggers the interaction between Grx and Prx is poorly understood and is only based on the structure of Haemophilus influenzae Prx-Grx hybrid (hyPrx5). We report here an NMR study of the Populus tremula Grx C4 that targets a P.tremula D-type II Prx. We show that Grx C4 specifically self-associates in a monomer-dimer equilibrium with an apparent K(d) of ca 2.6 mM. Grx C4 homodimer was docked under experimental restraints. The results reveal a novel Grx-Grx interface that is unrelated to the hyPrx5 Grx-Grx dimer interface. Chemical-shift perturbations and 15N spin-relaxation measurements show that the auto-association surface comprises both the active site and the GSH binding site. Reduced GSH is demonstrated to bind reduced Grx with a K(d) of ca 8.6 mM. The potential biological significance of the new Grx-Grx interaction interface is discussed.     

酒精-沼气双发酵耦联工艺中SO42-的控制

SO2-4是酒精-沼气双发酵耦联工艺稳定运行的重要抑制物.在耦联工艺中,以中温厌氧出水代替自来水配料进行酒精发酵.通过无预糖化工序的同步糖化发酵技术研究,将酒精发酵的初始pH提高到6.0,H2SO4的消耗降低了50%,使SO2-4的质量浓度维持在3g/L的沼气发酵安全范围内.在进行高浓度酒精发酵时,糖化酶的添加量为每克木薯添加140U,发酵54h,最终酒精体积分数达14.7%.糖化酶添加量与常规酒精发酵用量相比,每克木薯增加了糖化酶20 U.     

Cloning of human PRP4 reveals interaction with Clk1.

Prp4 is a protein kinase of Schizosaccharomyces pombe identified through its role in pre-mRNA splicing, and belongs to a kinase family including mammalian serine/arginine-rich protein-specific kinases and Clks, whose substrates are serine/arginine-rich proteins. We cloned human PRP4 (hPRP4) full-length cDNA and the antiserum raised against a partial peptide of hPRP4 recognized 170-kDa polypeptide in HeLa S3 cell extracts. Northern blot analysis revealed that hPRP4 mRNA was ubiquitously expressed in multiple tissues. The extended NH(2)-terminal region of hPRP4 contains an arginine/serine-rich domain and putative nuclear localization signals. hPRP4 phosphorylated and interacted with SF2/ASF, one of the essential splicing factors. Indirect immunofluorescence analysis revealed that endogenous hPRP4 was distributed in a nuclear speckled pattern and colocalized with SF2/ASF in HeLa S3 cells. Furthermore, hPRP4 interacted directly with Clk1 on its COOH terminus, and the arginine/serine-rich domain of hPRP4 was phosphorylated by Clk1 in vitro. Overexpression of Clk1 caused redistribution of hPRP4, from the speckled to the diffuse pattern in nucleoplasm, whereas inactive mutant of Clk1 caused no change of hPRP4 localization. These findings suggest that the NH(2)-terminal region of hPRP4 may play regulatory roles under an unidentified signal transduction pathway through Clk1.     

Conformational changes in bovine-liver glutamate dehydrogenase: a spin-label study.

A spin-labelled analogue of p-chloromercuribenzoate reacts specifically with glutamate dehydrogenase. The most marked change in the properties of the spin-labelled enzyme is a fivefold decrease in the rate of reduction of the coenzyme by L-glutamate and no change in the rate of oxidation by 2-oxoglutarate. The electron spin resonance spectrum is a sensitive probe for the conformational state of the enzyme. Spin-labelled glutamate dehydrogenase in the presence of saturating concentrations of NADPH and 2-oxoglutarate or L-glutamate shows a complete conformational change while in the presence of NADP+ and 2-oxoglutarate only half of the protomers have changed conformation. The conformational change upon addition of NADPH to the spin-labelled glutamate dehydrogenase in the presence of 2-oxoglutarate happens in a concerted way between 20 and 80% saturation with NADPH. One of the conformations is favoured by the activator ADP while the other is favoured by the inhibitor GTP.     

Molecular interaction between 4-aminoantipyrine and catalase reveals a potentially toxic mechanism of the drug

4-Aminoantipyrine (AAP) is scarcely administered as an analgesic drug because of the potential side effects. The residue of AAP in the environment possesses a potential threat to human health. In this article, the binding mode of AAP with the important antioxidant enzyme catalase (CAT) was investigated using spectroscopic and molecular docking methods. AAP can interact with CAT to form an AAP-CAT complex. The binding constant, number of binding sites and thermodynamic parameters were measured, which indicated that AAP could spontaneously bind with CAT through electrostatic forces with one binding site. Molecular docking results revealed that AAP bound into the CAT central cavity. UV-visible absorption, synchronous fluorescence and circular dichroism (CD) results provide data concerning conformational and some microenvironmental changes of CAT. Furthermore, the binding of AAP can inhibit CAT activity in erythrocytes. The present study provides direct evidence at a molecular level to show that exposure to AAP could induce changes in the enzyme CAT structure and function. The estimated methods in this work can be applied to characterize interactions of enzyme systems and other pollutants and drugs.     

Genetic interaction between Bmp2 and Bmp4 reveals shared functions during multiple aspects of mouse organogenesis

Vertebrate Bmp2 and Bmp4 diverged from a common ancestral gene and encode closely related proteins. Mice homozygous for null mutations in either gene show early embryonic lethality, thereby precluding analysis of shared functions. In the current studies, we present phenotypic analysis of compound mutant mice heterozygous for a null allele of Bmp2 in combination with null or hypomorphic alleles of Bmp4. Whereas mice lacking a single copy of Bmp2 or Bmp4 are viable and have subtle developmental defects, compound mutants show embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, skeleton, eye and heart. Within the heart, BMP2 and BMP4 function coordinately to direct normal lengthening of the outflow tract, proper positioning of the outflow vessels, and septation of the atria, ventricle and atrioventricular canal. Our results identify numerous BMP4-dependent developmental processes that are also very sensitive to BMP2 dosage, thus revealing novel functions of Bmp2.     

Subcellular distribution of 35S-sulfur in spinach leaves after application of 35SO 4 2- , 35SO 3 2- , and 35SO2

³⁵SO₂, ³⁵SO ₃ ^2- , and ³⁵SO ₄ ^2- , respectively, were applied to leaves of Spinacia oleracea L. for 60 min in the light. Thereafter, the specific activity was determined in the organelles separated by means of sucrose density gradient centrifugation. In mitochondria and peroxisomes, the specific activity was equally distributed in their protein moieties. After application of ³⁵SO₂ or ³⁵SO ₃ ^2- , the chloroplast lamellae are characterized by elevated specific activity, which is not found after application of ³⁵SO ₄ ^2- . Chloroplast stroma shows a low specific incorporation rate after application of either compound, which may be due to the low turnover rate of Fraction I protein.     

Detoxification of SO2 in conifers differing in SO2-tolerance

Contents of organic sulfur, sulfate and the inorganic cations K⁺, Ca²⁺, Mg²⁺, Mn²⁺ and Na⁺ were compared in needles of three conifer species differing in tolerance to chronic SO₂ immissions. Sulfate and organic sulfur compounds were also measured in bark and wood. Field material was collected from Norway Spruce (Picea abies (L.) Karst.), Colorado Spruce (Picea pungens Engelm.) and Scots Pine (Pinus sylvestris L.) at sites where the SO₂ concentration in air was high, and at another site where it was low. In general, sulfate contents were higher but cation contents lower at the sites where SO₂ concentrations were high than where they were low. Up to 114mmol · (kg DW)^–1 sulfate was measured in fouryear-old needles of Norway Spruce from the Erzgebirge (annual mean of SO₂ in air 32 nl · 1^–1). Sulfate accumulation in this SO₂-sensitive conifer increased with SO₂ concentration in ambient air and with needle age, indicating that the main part of the sulfate resulted from the oxidative detoxification of SO₂. Loss of inorganic cations from ageing needles was reduced, or cation levels even increased, with increasing needle age, while sulfate accumulated. Apparently, cations served as counter-ions for sulfate, which is sequestered in the vacuoles. Individual trees differed in regard to the nature of cations which accumulated with sulfate. Calcium, potassium and magnesium were the dominating cations. Sodium levels were very low. Needles of the SO₂-tolerant conifers Colorado Spruce and Scots Pine growing next to Norway Spruce in the Erzgebirge did not accumulate, or accumulated less, sulfate with increasing needle age as compared to needles of Norway Spruce. However, somewhat more sulfate was found in the bark of the SO₂-tolerant species than in the bark of Norway Spruce. Scots Pine contained distinctly more sulfate in the wood than the other conifers. Since accumulation of organic sulfur compounds could not be observed with increasing needle age, or in bark and wood, reduction does not appear to play a major role in the detoxification of SO₂ by the investigated species. Physiological mechanisms permitting Colorado Spruce and Scots Pine to avoid the sulfate accumulation in the needles and the accompanying sequestration of cations that are observed in neighbouring Norway Spruce are discussed on the basis of the obtained data.Abbreviations S_org organic sulfur compounds Died June 10, 1991, aged 29, in a traffic accident. He initiated this work.This work was supported by the Sonderforschungsbereich 251 of the University of Würzburg and by the Projektgruppe Bayern zur Erforschung der Wirkung von Umweltschadstoffen (PBWU). The authors with to thank Prof. Dr. W Kaiser and Prof. Dr. W. Urbach (both Julius-von-Sachs-Institut, University of Würzburg, Germany) for HPLC-analysis and ICP-analysis.     

Characterization of a 12-kilodalton rhodanese encoded by glpE of Escherichia coli and its interaction with thioredoxin

Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from Escherichia coli. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The glpE gene, a member of the sn-glycerol 3-phosphate (glp) regulon of E. coli, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. The K(m)s for SSO(3)(2-) and CN(-) were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited a k(cat) of 230 s(-1). Thioredoxin 1 from E. coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent K(m) of 34 microM when thiosulfate was near its K(m), suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited ( approximately 17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (http://www.ncbi.nlm.nih.gov/COG/) for which sulfurtransferase activity has been confirmed.     

Structure of an Xrcc4-DNA ligase IV yeast ortholog complex reveals a novel BRCT interaction mode

DNA ligase IV catalyses the final ligation step in the non-homologous end-joining (NHEJ) DNA repair pathway and requires interaction of the ligase with the Xrcc4 'genome-guardian', an essential NHEJ factor. Here we report the 3.9 A crystal structure of the Saccharomyces cerevisiae Xrcc4 ortholog ligase interacting factor 1 (Lif1p) complexed with the C-terminal BRCT domains of DNA ligase IV (Lig4p). The structure reveals a novel mode of protein recognition by a tandem BRCT repeat, and in addition provides a molecular basis for a human LIG4 syndrome clinical condition.     

Control of 35SO 4 2- and 35SO 3 2- incorporation into spinach chloroplasts during photosynthetic CO2 fixation

In addition to membrane translocation, measured in the dark, it was found that pre-illumination of the chloroplasts resulted in an enhancement of sulfate uptake by 25% and of sulfite uptake by 55% as soon as the concentration of the ion in the incubation medium exceeded 2 mmol l^-1. This amount which is additionally taken up after pre-illumination is less readily exchanged for other ions. Kinetics of the uptake in relation to pre-illumination time and to light intensity closely parallel those of titration of SH-groups by 5,5-dithiobis (2-nitrobenzoic acid). As a consequence, 10^-6 mol l^-1 DCMU completely inhibits the light triggered increase of uptake of both ions. Uncoupling with 10^-6 mol l^-1 CCCP increases the light induced ³⁵SO ₃ ^2- binding, but decreases that of ³⁵SO ₄ ^2- , demonstrating the need of ATP formation to initiate sulfate reduction. Rates of uptake, measured at different intensities of pre-illumination under nitrogen or in the presence of bicarbonate, suggest that the presence of a carbon skeleton increases the binding rate for both ions. With respect to ³⁵SO ₄ ^2- , the data further indicate a rate limiting step (ATP sulfurylase or adenosine 5-phosphosulfate sulfotransferase) which is activated by light, thus representing a control step to harmonize the rate of CO₂ fixation and of sulfate incorporation. On the contrary, ³⁵SO ₃ ^2- is directly bound in relation to the amount of SH-groups, which in turn are created by the photosynthetic electron transport, resulting in Car-S-SO ₃ ^- . Since the formation of SH-groups is maximal already at low light intensities, no effective control step for SO ₃ ^2- incorporation is indicated.     

Preparation of TiO2 anatase nanocrystals by TiCl4 hydrolysis with additive H2SO4

A new methodology was developed to synthesize uniform titania anatase nanocrystals by the hydrolysis of titanium chloride in sulfuric acid aqueous solutions at 0-90°C. The samples were characterized by Raman spectroscopy, UV-visible spectroscopy, transmission electron microscopy (TEM), electron diffraction (ED), and an Energy dispersive X-ray spectroscopy (EDS). The effects of the reaction temperature, mole ratio of SO(4)(2-) to Ti(4+), and the calcinations temperature on the particle size and crystal phase were investigated. Depending on the acidity, the hydrolysis temperature, and the calcination temperature, rhombic anatase nanocrystals sizes in the range of 10 nm to 50 nm were obtained. In the additive of sulfuric acid, Raman spectra and electron diffraction confirmed that the nanoparticles are composed of anatase TiO(2). No other titania phases, such as rutile or brookite, were detected.