bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of Escherichia coli mutants defective in enzymes of membrane lipid synthesis.
Authors: Raetz C R
Published: May 2006

A new method has been developed which permits the rapid screening of E. coli colonies for mutants with defective enzymes of phospholipid metabolism. In this procedure, a disc of filter paper is pressed down on an agar plate containing several hundred colonies of mutagen-treated cells, after which the paper is lifted off. In the process the colonies are transferred to the paper, giving rise to a replica print of the master plate. The few cells from each colony left on the master keep growing in the original pattern. The pattern of colonies is also retained on the filter paper, even after the cells are rendered permeable with lysozyme and EDTA. Colonies treated in this manner remain absorbed to the paper, where they can convert sn-(U-14-C)glycero-3-P to phosphatidyl(U-14-C)glycerophosphate, dependent on added CDP-diglyceride. Unrelated reactions of sn-(U-14-C)glycero-3-P that may obscure the synthesis of phosphatidyl-glycerophosphate are inhibited by the addition of reagents poisoning energy generation. The radioactive phospholipid that forms around each colony on the paper is precipitated in situ with trichloroacetic acid, and unreacted sn-(U-14-C)glycero-3-P is washed away. After autoradiography, the colonies on the filter paper are stained with Coomassie blue. When the autoradiogram is superimposed on the strained paper, mutants are identified as blue colonies lacking a black halo. With this method, 20,000 colonies were screened in several days. Four mutants were identified with low levels of CDP-diglyceride:snglycero-3-P phosphatidyl transferase (EC 2.7.8.5, GLYCEROL-PHOSPHATE PHOSPHATIDYLTRANSFERASE, PHOSPHATIDYLGLYCEROPHOSPHATE SYNTHETASE) IN EXTRACTS. With a similar assay, 10,000 additional colonies were screened for mutants with altered CDP-diglyceride:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase), and four strains were found in which the enzyme is thermolabile. The screening technique described here is termed replica printing and should be applicable not only to studies of phospholipid metabolism but also to nucleic acid and protein synthesis.
A p60 polypeptide in the feline leukemia virus pseudotype of Moloney sarcoma virus with murine leukemia virus p30 antigenic determinants.
Authors: Oskarsson M K , Robey W G , Harris C L , Fischinger P J , Haapala D K , Vande Woude G F
Published: May 2006

A 60,000-dalton polypeptide (p60) has been identified in the feline leukemia virus (FeLV) pseudotype of Moloney sarcoma virus [MSV(FeLV)]. This polypeptide is present in the purified virus complex in concentrations greater than either the murine p30 or the feline p27. Purified p60 crossreacts immunologically with murine p30 group antiserum and contains several interspecies determinants, whereas the group specific determinant of FeLV p27 is not detected. Comparison of peptide fingerprints of p60 and murine p30 show many peptides in common. Limited digestion of p60 with either trypsin or chymotrypsin produced p30-35 and p20 peptides which retain the MuLV p30 group and interspecies antigenic activities. The p30 produced by both enzymes comigrates in polyacrylamide gels with the murine p30 of MSV(FeLV), thus suggesting that p60 may be an uncleaved precursor to p30.
Potential for Nitrogen Fixation in Maize Genotypes in Brazil
Authors: Von Bülow Joachim F. W. , Döbereiner Johanna
Published: May 2006

N
2
fixation in field-grown maize (
Zea mays
L.) plants was estimated by a nondestructive acetylene reduction method which permitted the plants to continue growing and produce seeds. Samples from six areas revealed mean nitrogenase activities of 74-2167 nmol of C
2
H
4
/(g of dry roots × hr) for 10 plants. Among 276 S
1
lines planted in two field experiments, 17 lines were selected for further nitrogenase activity assays after prescreening. Variability within lines was high but significant differences among lines were obtained in one experiment. The best lines showed mean nitrogenase activities of 2026, 2315, and 7124 nmol of C
2
H
4
/(g of dry roots × hr), whereas the original cultivar reduced only 313 nmol. The highest value approaches the nitrogenase activity of soybean. If the theoretical 3:1 (C
2
H
4
/N
2
reduced) conversion factor is used, a potential daily N
2
fixation of 2 kg of N
2
/hectare can be calculated. Periodic sampling within a brachytic maize cultivar revealed that maximum nitrogenase activity occurred at about the 75% silking stage. Soil effects also were pronounced. N
2
-fixing
Spirillum sp.
could be isolated from all active root pieces when they were surface sterilized. These organisms appear to be primarily responsible for root nitrogenase activity in maize.
Interaction of hemoglobin with three ligans: organic phosphates and the Bohr effect.
Authors: Benesch R E , Rubin H
Published: May 2006

The assumption that the Bohy coefficient (less than log p 50)/(less than pH) is equal to the haldane coefficient (less than h minus plus) of hemoglobin is shown to be incorrect in the presence of allosteric effectors such as 2,3-diphosphoglycerate. The theoretical relation between the two coefficients in the presence of 2.3-diphosphoglycerate as derived. Experimental data on the variation of both coefficients with diphosphoglycerate concentration are presented and shown to be in agreement with prediction.
Iron-sulfur clusters II: Kinetics of ligand exchange studied on a water-soluble Fe
4
S
4
(SR)
4

n
-

cluster
Authors: Job Robert C. , Bruice Thomas C.
Published: May 2006

The water-soluble tetranuclear iron-sulfur cluster ion Fe
4
S
4
(SCH
2
CH
2
CO
2
-
)
4
6-
(II) has been prepared. The stability of II in water is sufficient to allow the spectrotitrimetric determination of the pK
a
of its Fe
4
S
4
core as 7.4. In our hands the one-electron reductions of compounds I [Fe
4
S
4
(SR)
4
2-
, R = alkyl or aryl,] are thermodynamically irreversible with associated E
1/2
values greater than those for one-electron reduction of ferredoxins. In contrast, the one-electron reduction of II is thermodynamically reversible and the associated potential (-0.58 V versus hydrogen electrode) approaches closely that of the ferredoxins. The kinetics for ligand exchange of II as a function of pH and thiol concentration are in accord with four reversible mercaptan/lyate species exchange reactions followed by product formation via specific acid and base catalysis. Preliminary experiments indicate the nucleophilic order towards II to be Cl
-
[unk] Br
-
< HO
-
< CN
-
.
Characterization of purified DNA-relaxing enzyme from human tissue culture cells.
Authors: Keller W
Published: May 2006

Superhelical simian virus 40 DNA migrates more rapidly during electrophoresis in agarose gels than covalently closed DNA free of superhelical turns (relaxed DNA). The difference in electrophoretic mobility between superhelical and relaxed DNA was used to monitor the activity of a protein from human tissue culture cells which converts superhelical DNA into relaxed DNA. Purified DNA-relaxing protein removes both negative and positive superhelical turns and acts in a catalytic manner. The relaxation of DNA proceeds in a stepwise fashion and DNA intermediates with decreasing numbers of superhelical turns are seen during the course of the reaction.
Crosslinking and labeling of membrane proteins by transglutaminase-catalyzed reactions.
Authors: Dutton A , Singer S J
Published: May 2006

Transglutaminase enzymes catalyze for the formation of epsilon(gamma-glutamyl)lysyl crosslinks, or the substitution of a variety of primary amines for the amide function of protein-bound glutaminyl residues. These enzymes should therefore be useful in crosslinking the proteins of membranes and in attaching a variety of chemical probes and labels to these proteins. This usefulness is demonstrated in experiments with the enzyme liver transglutaminase and the membranes with the enzyme liver transglutaminase and the membranes of mouse erythrocytes and of rabbit skeletal muscle sarcoplasmic reticulum.
Amino-acid sequence of activation cleavage site in plasminogen: homology with "pro" part of prothrombin.
Authors: Sottrup-Jensen L , Zajdel M , Claeys H , Petersen T E , Magnusson S
Published: May 2006

A 38-residue fragment is isolated from carboxymethylated plasminogen. Residues 29-38 have the same sequence as the amino-terminal end of the light chain of plasmin. The sequence 1-28 is therefore the sequence of the carboxyl-terminal end of the heavy chain and contains the specific sequence at which urokinase (EC 3.4.99.26) and other plasminogen-activating serine proteases split. Two of the five carboxymethyl-cysteine residues in the isolated fragment are situated close to the cleavage site and the fragment is not itself a substrate for plasminogen-activators. Residues 1-11 show extensive sequence homology with residues 137-147 and 242-252 in prothrombin, which are located in corresponding regions of the two internally homologous 83-residue structures in the non-thrombin part of the molecule, indicating that such structures may be a common feature of the non-protease part of the larger serine protease zymogens.
Origin of hemopoietic stem cells in embryonic bursa of Fabricius and bone marrow studied through interspecific chimeras.
Authors: Le Douarin N M , Houssaint E , Jotereau F V , Belo M
Published: May 2006

The histogenesis of the bursa of Fabricius and of bone marrow was studied by a biological cell marking technique based on differences in the nuclear structure of two species of birds, Japanese quail (Coturnix coturnix japonica) and chick (Gallus gallus). In quail cells the nucleus contains a large amount of heterochromatin associated with the nucleolus. That makes it possible to distinguish them from chick cells after Feulgen-Rossenbeck staining and by electron microscopy. By grafting bursal rudiments and limb buds of quail into chick and inversely it was possible to demonstrate that the whole hemopoietic population of the bursa of Fabricius and of bone marrow is derived from bloodborne extrinsic stem cells. Neither endoderm nor mesoderm of the bursal rudiments is capable of differentiating into lymphoid cells. Combinations of quail bursal endoderm with chick homologous mesenchyme showed that the reticular cells of the follicles are the only endodermal derivatives of the bursa. The mesenchymal bursal component gives rise to the interfollicular connective cells. The contribution to bone marrow histogenesis of cells of vascular and blood origin, on one hand, and of the elements of the cartilaginous model, on the other hand, was analyzed. It appeared that osteoblasts, osteocytes, and stromal cells of marrow are derived from the perichondrium. In contrast, the endothelium of the vascular buds and the hemopoietic cells which invade the diaphysal cartilage during the endochondral ossification process do not belong to the mesenchymal bone primordium but have a fully extrinsic origin.
Inducible error-prone repair in Escherichia coli.
Authors: Sedgwick S G
Published: May 2006

A hypothesis that ultraviolet-induced mutagenesis arises from the induction of an error-prone mode of postreplication repair that requires the exrA+ recA+ genotype has been tested with alkaline sucrose gradient centrifugation coupled with assays of fixation determined by loss of photoreversibility. The inhibitor of protein synthesis, chloramphenicol, added before irradiation, prevented a small amount of postreplication repair and completely eliminated mutation fixation in E. coli WP2s uvrA. However, chloramphenicol did not affect strand joining: (a) in uvrA bacteria allowed 20 min of growth between irradiation and antibiotic treatment; (b) in nonmutable uvrA exrA bacteria; and (c) in uvrA tif bacteria grown at 42 degrees for 70 min before irradiation. These observations indicate that an inducible product is involved in a fraction of postreplication repair and is responsible for induced mutagenesis.
A rapid and specific method for isolation of thiol-containing peptides from large proteins by thiol-disulfide exchange on a solid support.
Authors: Egorov T A , Svenson A , Rydén L , Carlsson J
Published: May 2006

Activated thiol-Sepharose [agarose-(glutathione-2-pyridyl disulfide) conjugate] has been used to immobilize proteins with a single or a few thiol groups via disulfide bridges. The immobilized proteins were subsequently proteolytically degraded. After washing, the thiol-containing peptides were eluted with a reducing agent. A single preparative paper electrophoresis, occasionally after a modification such as oxidation, was sufficient to obtain pure peptides in good yields. The method was applied to the major parvalbumin from hake muscle (a protein with 108 amino acid residues and one cysteine residue), to mercaptalbumin from bovine serum (565 residues and one cysteine), and to human serum ferroxidase [EC 1.16.3.1; iron (II):oxygen oxidoreductase] (ceruloplasmin) (1065 residues and three cysteines). The use of the technique, e.g., as a simple means of obtaining homologous peptides in related proteins, is discussed.
Absence of polyadenylate segment in globin messenger RNA accelerates its degradation in Xenopus oocytes.
Authors: Marbaix G , Huez G , Burny A , Cleuter Y , Hubert E , Leclercq M , Chantrenne H , Soreq H , Nudel U , Littauer U Z
Published: May 2006

Rabbit globin poly(A)-free and native mRNA preparations were microinjected into Xenopus oocytes. The amount of globin message remaining after incubation of injected oocytes was determined by molecular hybridization with a radioactive complementary DNA probe, synthesized by copying globin mRNA with purified RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus. The results show that 56 hr after injection about 85% of the poly(A)-free mRNA molecules are degraded, while native poly(A)-containing mRNA chains are almost completely preserved during the same period of time.
Mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrin), vinyl chloride, and cyclophosphamide.
Authors: McCann J , Simmon V , Streitwieser D , Ames B N
Published: May 2006

We have previously described a very sensitive and efficient bacterial test designed to detect chemical carcinogens as mutagens. Chloroacetaldehyde is mutagenic in this system and is of interest because it is a possible metabolite in mammals of the large volume industrial chemicals 1,2-dichloroethane (ethylene dichloride) (3.5 billion kg/yr, U.S.) and vinyl chloride (2.5 billion kg/yr, U.S.), and of the antineoplastic agent cyclophosphamide. Chloroacetaldehyde reverts a new Salmonella bacterial tester strain (TA100). Chloroacetaldehyde is shown to be hundreds of times more effective in reversion of TA100 than is chloroethanol (ethylene chlorohydrin), a known metabolic precursor of chloroacetaldehyde and a possible metabolite of dichloroethane and vinyl chloride, or than vinyl chloride, which is itself mutagenic for TA100. Chloroethanol is shown to be activated by rat (or human) liver homogenates to a more highly mutagenic form with reversion properties similar to chloroacetaldehyde. Reversion properties of cyclophosphamide after in vitro metabolic activation suggest that chloroacetaldehyde is not the active mutagenic form of this antineoplastic drug.
Dynamics of a protein matrix revealed by fluorescence quenching.
Authors: Eftink M R , Ghiron C A
Published: May 2006

The fluorescence of the supposedly buried tryptophan in ribonuclease T1 has been found to be collisionally quenched by acrylamide with a rate constant of 3 X 10(8) M--1 sec--1. Only a slight decrease in the quenching rate is observed upon a 5-fold increase in the viscosity of the solution. For this to be the case, the diffusion of the quencher must be limited by the protein matrix. To explain the process of diffusion through this complex material, the formation of "holes" in the lattice of a protein due to nanosecond fluctuations must be invoked. Thus, the dynamic character of a protein molecule is revealed. The quenching rate constant has an activation energy of 9 kcal/mol which can be used to characterize the nature of the cohesive forces in the microenvironment about the indole ring. The mechanical properties of a portion of a protein matrix can, therefore, be described as one would for a fluid.
Fluorescence polarization and viscosities of membrane lipids of 3T3 cells.
Authors: Fuchs P , Parola A , Robbins P W , Blout E R
Published: May 2006

Studies of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in membranes of normal, transformed, and revertant 3T3 cells allowed estimation of microviscosities (eta) of the lipid bilayers of these cells. Fluorescence polarization values observed with normal cells were significantly lower than those observed with cells transformed either by polyoma virus or by simian virus 40. The values of fluorescence polarization obtained with revertant Py6R1 cells were lower than those obtained with the normal cells. The calculated microviscosities (eta) show a 50% increase upon transformation. Possible correlations between the bilayer viscosity and the mobility of the receptors in the membrane are discussed.
Histidine regulation in Salmonella typhimurium: an activator attenuator model of gene regulation.
Authors: Artz S W , Broach J R
Published: May 2006

An activator-attenuator model of positive control, a s opposed to the classic repressor-operator model of negative control, is proposed for the major operon-specific mechanism governing expression of the histidine gene cluster of Salmonella typhimurium. Evidence for this mechanism is derived from experiments performed with a coupled in vitro transcription-translation system, as well as with a minimal in vitro transcription system [Kasai, T. (1974) Nature 249, 523--527]. The product (G enzyme, or N-1-[5'-phosphoribosyl]adenosine triphosphate:pyrophosphate phosphoribosyltransferase; EC 2.4.2.17) of the first structural gene (hisG) of the histidine operon is not involved in the positive control mechanism. However, a possible role for G enzyme as an accessory negative control element interacting at the attenuator can be accommodated in our model. The operon-specific mechanism works in conjunction with an independent mechanism involving guanosine 5'-diphosphate 3'-diphosphate (ppGpp) which appears to be a positive effector involved in regulating amino-acid-producing systems, in general [Stephens, J.C., Artz, S.W. & Ames, B.N. (1975) Proc. Nat. Acad. Sci. USA, in press].
Degradation of penicillin G to phenylacetylglycine by D-alanine carboxypeptidase from Bacillus stearothermophilus.
Authors: Hammarström S , Strominger J L
Published: May 2006

D-Alanine carboxypeptidase from Bacillus stearothermophilus is a membrane-bound enzyme which is inhibited by covalent interaction with penicillin G. The penicilloyl enzyme spontaneously reactivates and simultaneously releases a penicillin G degradation product; 0.2 mumol of the latter was isolated after incubation of 4.2 mumol of [8-14C]penicillin G with 10 g of membrane protein. It was identified as phenylacetylglycine by chromatographic techniques, infrared spectroscopy, and mass spectrometry. A mechanism for the degradation is proposed in which the remaining part of penicillin G would be released as 5,5-dimethyl-delta2-thiazoline-4-carboxylic acid. The implications of this finding are discussed.
Stimulation of epinephrine-sensitive fat cell adenylate cyclase by cytosol: effect of cholera toxin.
Authors: Ganguly U , Greenough W B
Published: May 2006

Cytosol prepared from rat epididymal fat cells by centrifugation at 100,000 X g for 1 hr was found to enhance the basal and epinephrine-sensitive adenylate cyclase [EC 4.6.1.1; ATP pyrophosphate-lyase (cyclizing)] of fat cell ghosts. Cholera toxin also stimulated adenylate cyclase and increased the response to epinephrine in fat cells. A possible relationship between the adenylate cyclase modifying activities of cytosol and the effects of cholera toxin was sought. Cytosol from freshly prepared fat cells added to ghosts prepared from cells that had been exposed to toxin for varying periods showed a progressive loss of responsiveness to cytosol epinephrine-enhancing activity. The effect appeared within 15 min after toxin exposure, a full 30 min before any direct effect of toxin on adenylate cyclase was seen. Since exposure to toxin decreased membrane response to cytosol epinephrine-enhancing activity, the possibility that epinephrine-enhancing activity in cytosol might be altered by toxin was explored. Cytosol from cells exposed to toxin for varying periods lost epinephrine-enhancing activity to an appreciable degree within 15 min. Examination of these early events after exposure to toxin should clarify the way in which this bacterial substance affects mammalian cells. The cytosol epinephrine-enhancing activity was destroyed by boiling for 3 min and was partially inactivated by trypsin. It was nondialyzable and stable at -70 degrees.
Parallel changes in puffing activity and patterns of protein synthesis in salivary glands of Drosophila.
Authors: Lewis M , Helmsing P J , Ashburner M
Published: May 2006

The changes in protein synthesis of salivary glands of Drosophila resulting from a brief exposure at 37 degrees have been analyzed on sodium dodecyl sulfate--acrylamide gels. In D. melanogaster and D. hydei this treatment induces nine and six new puffs, respectively, in the polytene chromosomes. After 20 min treatment seven new proteins are synthesized by the glands of D. melanogaster and six by those of D. hydei as detected by [35S]methionine labeling. Other agents, e.g., recovery from anaerobiosis, induce the same puffs and the same proteins. The extent of protein induction and the degree of puff induction are related to the severity of the temperature treatment. The new proteins are detected after 10 min treatmene at 37 degrees and their synthesis is inhibited by actinomycin D. Actinomycin D added 5 min after the start of temperature treatment has little effect on subsequent protein synthesis. The induced proteins are not tissue specific. Electrophoretic differences of two proteins exist between D. melanogaster and D. simulans, encouraging attempts to map the proteins' gene loci and to test directly whether or not the puffs code for them.
A mechanism for initiation of genetic recombination.
Authors: Wagner R E , Radman M
Published: May 2006

A mechanism for the initiation of genetic recombination is proposed. Its key features are the pairing, nicking, and cross-annealing of palindromic loops, i.e., structures formed by DNA with sequences of inverted complementary repeats. This mechanism may provide a simple, yet specific means of producing crossed strand connections between homologous DNA duplexes to form structures which can be intermediates in the process of genetic recombination.
An endotoxin-induced serum factor that causes necrosis of tumors.
Authors: Carswell E A , Old L J , Kassel R L , Green S , Fiore N , Williamson B
Published: May 2006

In studying "hemorrhagic necrosis" of tumors produced by endotoxin, it was found that the serum of bacillus Calmette--Guerin (BCG)-infected mice treated with endotoxin contains a substance (tumor necrosis factor; TNF) which mimics the tumor necrotic action of endotoxin itself. TNF-positive serum is as effective as endotoxin itself in causing necrosis of the sarcoma Meth A and other transplanted tumors. A variety of tests indicate that TNF is not residual endotoxin, but a factor released from host cells, probably macrophages, by endotoxin. Corynebacteria and Zymosan, which like BCG induce hyperplasia of the reticulo-endothelial system, can substitute for BCG in priming mice for release of TNF by endotoxin. TNF is toxic in vitro for two neoplastic cell lines; it is not toxic for mouse embryo cultures. We propose that TNF mediates endotoxin-induced tumor necrosis, and that it may be responsible for the suppression of transformed cells by activated macrophages.
Bacteriophage T4 whiskers: a rudimentary environment-sensing device.
Authors: Conley M P , Wood W B
Published: May 2006

The 400 A filaments or "whiskers," which extend outward from the collar region of the phage, control retraction and extension of the tail fibers in response to certain environmental conditions. The tail fibers of normal phage retract in the absence of a required adsorption cofactor, at low pH, at low ionic strength, at low temperature, and at high concentrations of polyethylene glycol. The tail fibers of mutant whiskerless (wac) phage still retract under the first two conditions, but not the last three. Antibodies to whiskers neutralize T4, probably by fixing tail fibers in the retracted configuration. Phage with retracted tail fibers adsorb poorly to host bacterial cells, and their adsorption rate increases as the fibers become extended. These results suggest that one function of the whiskers is to retract the tail fibers and thereby prevent adsorption to host cells under certain conditions that might be unfavorable for production of phage progeny following infection.
Polarity of actin filaments in Characean algae.
Authors: Kersey Y M , Hepler P K , Palevitz B A , Wessells N K
Published: May 2006

Heavy meromyosin from rabbit muscle combines with oriented Nitella and Chara actin in vitro to form arrowhead structures directed opposite to the cytoplasmic flow in the living plant cell. All filaments and all bundles of filaments in the apically directed stream are similarly oriented; polarity with respect to the axis of the thallus is reversed in the downward stream. The actin filaments are attached to the chloroplasts at the ectoplasm-endoplasm interface, where the motive force for streaming is known to be generated.
DNA single-strand breaks during repair of UV damage in human fibroblasts and abnormalities of repair in xeroderma pigmentosum.
Authors: Fornace A J , Kohn K W , Kann H E
Published: May 2006

The method of DNA alkaline elution was applied to a study of the formation and resealing of DNA single-strand breaks after irradiation of human fibroblasts with ultraviolet light (UV). The general features of the results were consistent with current concepts of DNA excision repair, in that breaks appeared rapidly after UV, and resealed slowly in normal fibroblasts, whereas breaks did not appear in those cells of patients with xeroderma pigmentosum (XP) that are known to have defects in DNA repair synthesis. The appearance of breaks required a short post-UV incubation, consistent with the expected action of an endonuclease. Cells of the variant form of XP characterized by normal DNA repair synthesis exhibited normal production of breaks after UV, but were slower than normal cells in resealing these breaks. This difference was enhanced by caffeine. A model is proposed to relate this finding with a previously described defect in post-replication repair in these XP variant cells. DNA crosslinking appears to cause an underestimate in the measurement of DNA breakage after UV.
Molecular cloning of DNA from F sex factor of Escherichia coli K-12.
Authors: Skurray R A , Nagaishi H , Clark A J
Published: May 2006

We describe the molecular cloning of various DNA segments generated by partial EcoRI endonuclease digestion of the sex factor F. These segments have been analyzed by agarose gel electrophoresis of EcoRI digests and were arranged in a series of overlapping fragments using the EcoRI fragment map of F established by H. Ohtsubo and E. Ohtsubo. The clones isolated demonstrate one or more of the following F-specified functions: inhibition of female-specific phage (T7) multiplication, formation of F pili, surface exclusion, or immunity to lethal zygosis. These properties are discussed in terms of the EcoRI fragments of F that specify them.
Nature of O2 and CO binding to metalloporphyrins and heme proteins.
Authors: Collman J P , Brauman J I , Halbert T R , Suslick K S
Published: May 2006

The O2 vibration of dioxygen adducts of Fe and Co model complexes of alpha,alpha,alpha,alpha-tetrapivalamidophenylporphyrin ("picket fence" porphyrin, TpivPP) with 1-methylimidazole and 1-tritylimidazole as axial bases are reported, obtained with difference techniques between 16O2, 18O2, 169-18O, and NO with a Fourier transform infrared spectrometer. Assignments of upsilono2 are (O2)Fe(TpivPP) 1-methylimidazole, 1159 cm-1 in Nujol; (O2)Fe(TpivPP) 1-tritylimidazole, 1163 in benzene; (O2)Co(TpivPP) 1-methylimidazole, 1150 in Nujol; (O2)Co(TpivPP) 1-tritylimidazole, 1153 in benzene. Comparisons with other known Fe, Co, Cr, and Ti dioxygen complexes are made, and it is concluded that the bent dioxygen ligand is best viewed as bound superoxide, O2-. The CO affinities of various hemoproteins and model systems are discussed. A correlation between the CO stretching frequency and its binding constant is described. The drastically lowered affinity of hemoproteins for CO compared with unencumbered models is attributed to steric hindrance in the distal binding site, which allows discrimination between the already bent FeIII-O2- and the normally linear FeII-CO systems. If the affinity of hemoproteins in living systems for CO relative to O2 were not decreased, then massive poisoning would result from endogenous CO.
Ethidium bromide-(dC-dG-dC-dG)2 complex in solution: intercalation and sequence specificity of drug binding at the tetranucleotide duplex level.
Authors: Patel D J , Canuel L L
Published: May 2006

The binding of ethidium bromide (EtdBr) to the dC-dG-dC-dG self-complementary duplex has been monitored at the resolvable drug and nucleic acid protons and backbone phosphates at high nucleotide/drug (N/D) ratios by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. We observe averaged resonances (25 degrees-95 degrees) for the nucleic acid and drug nonexchangeable protons in the presence of excess tetranucleotide (N/D = 24), indicative of rapid exchange relative to the chemical shifts in the free and complexed states. Complex formation results in upfield shifts for the base protons at the terminal and internal base pairs and an increase in the transition midpoint for the duplex-to-strand conversion. We observe upfield chemical shift changes of 1.2 ppm at the Watson-Crick guanine N-1 proton(s) on complex formation (N/D = 24), with slow exchange between (dC-dG-dC-dG)2 and EtdBr-(dC-dG-dC-dG)2 relative to this chemical shift difference at-5 degrees. The EtdBr phenanthridine ring protons shift upfield by about 0.9 ppm (H-2, H-4, H-7, H-9) and greater than 0.5 ppm (H-1, H-10) on complex formation, with the chemical shifts versus temperature plots (25 degrees-95 degrees) monitoring the dissociation of the EtdBr-(dC-dG-dC-dG)2 structure. These upfield shifts at the exchangeable and nonexchangeable base protons and phenanthridine ring (but not side chain) protons demonstrate intercalation of the phenanthridine ring of EtdBr into the dC-dG-dC-dG duplex in solution. The intercalation model may be supported by the observation of downfield shifts (up to 1ppm) at the internucleotide phosphate(s) of the tetranucleotide duplex on addition of EtdBr at low temperatures. We observe stronger binding of EtdBr to the self-complementary dC-dG-dC-dG (2 dC-dG intercalation sites) and dC-dC-dG-dG (1 dC-dG site) duplexes compared to the dG-dG-dC-dC (no dC-dG sites) as monitored by UV absorbance changes at 480 nm. These studies extend to the tetranucleotide duplex level earlier observations that EtdBr exhibits a selectivity for formation of complexes to dinucleoside monophosphates with a pyrimidine (3'-5') purine sequence in the crystal and in solution. The experimental proton NMR upfield shifts at the phenanthridine protons on formation of the EtdBr-(dC-dG-dC-dG)2 complex compare favorably with calculated values (atomic diamagnetic anisotropy and ring current contributions) based on the overlap geometry for EtdBr intercalated into the pyrimidine (3'-5') purine dinucleoside monophosphate duplex in the crystal.
Methods for identification of recombinants of phage lambda.
Authors: Sanzey B , Mercereau O , Ternynck T , Kourilsky P
Published: May 2006

Two methods are described which allow the screening of a large number of phage plaques for a specific DNA sequence carried by the phage or a specific antigen produced within the phage plaque. These methods were set up with lambda and lambdalac phages. Phage plaques were transferred onto nitrocellulose filters by desiccation in 0.1 M NaOH, and the lac sequence was detected by hybridization to radioactive lac mRNA. Beta-Galactosidase was detected by reaction with anti-beta-galactosidase immune serum included in the soft agar of the titration plates; the precipitate thus formed was revealed by means of enzyme-coupled antibodies and in situ coloration. These methods are potentially useful for the identification of lambda transducers, including those which are generated by in vitro recombination with eukaryotic DNA.
ATP-PPi exchange activity of progesterone receptor.
Authors: Moudgil V K , Toft D O
Published: May 2006

Progesterone receptor preparations from avian oviduct catalyze a pyrophosphate (PPi)-exchange reaction between ATP and 32P-labeled PPi. The reaction requires ATP exclusively and is Mn++-dependent. This enzyme activity is detectable in receptor preparations that have been purified extensively by chromatography on ATP-Sepharose and DEAE-Sephadex columns. Polyacrylamide gel electrophoresis of purified preparations reveals a comigration of [3H]progesterone-receptor complex and the enzyme activity. The PPi-exchange reaction is inhibited by both o-phenanthroline and rifamycin AF/013, which also block the nuclear binding of progesterone receptor. These findings indicate that progesterone receptor may be an enzyme or a subunit of an enzyme that is active in nucleotide metabolism.
Evidence for an intermediate with a single-strand break in the reaction catalyzed by the DNA untwisting enzyme.
Authors: Champoux J J
Published: May 2006

The DNA untwisting enzyme relaxes covalently closed circylar DNAs by the sequential breaking (nicking) and closure of one strand of the duplex. The use of highly purified enzyme from rat liver nuclei at very high protein concentrations has permitted the detection of the nicked intermediate in the reaction. The nicking of closed circular simian virus 40 DNA was measured by alkaline sucrose gradient sedimentation or by equilibrium centrifugation in CsCl gradients containing propidium diiodide. The following observations support the hypothesis that the nicked DNA represents an intermediate in the untwisting reaction. The extent of nicking does not increase with time. Nicking is observed in the range of salt concentrations where the enzyme is active (0.01-0.25 M KCl), but is not observed at 0.50 Mkdl, where enzyme activity is undetectable. The nicked DNA that is generated during the reaction carried out in low salt rapidly disappears if the KCl concentration is raised to 0.50 M. At constant enzyme concentration, the number of nicks in the reaction mixture is independent of DNA concentration in the range from 3 to 14 mug/ml. The addition of an excess of unlabeled DNA to a reaction initially containing labeled nicked DNA partially chases the label from the nicked intermediate into covalently closed circular DNA.
Amplification in Escherichia coli of enzymes involved in genetic recombination: construction of hybrid ColE1 plasmids carrying the structural gene for exonuclease I.
Authors: Vapnek D , Alton N K , Bassett C L , Kushner S R
Published: May 2006

Endo-R-HindIII restriction endonuclease fragments obtained from F30 and pMB9 plasmid DNAs were ligated in vitro and used to transform a recB21 recC22 sbcB15 strain of E. coli K-12. The inability of this strain to stably maintain pMB9 alone permitted the isolation of transformants that carried hybrid plasmids containing the sbcB+ allele. These transformants became sensitive to ultraviolet light and recombination defieient and showed a 25-fold increase in the level of exonuclease I activity. The stability of the sbcB hybrid plasmids and their effects on exonuclease I activity have also been determined in wild-type and recA1 genetic backgrounds. The presence of the plasmids results in a 7-fold increase in the level of exonuclease I in a wild-type strain and 15-fold increase in a recA1 strain. The increased activity in the recA1 mutant appears to be a result of increased plasmid stability in this genetic background.
Regulation of ornithine decarboxylase activity by guanine nucleotides: in vivo test in potassium-depleted Escherichia coli.
Authors: Sakai T T , Cohen S S
Published: May 2006

The observation that guanosine 5'-triphosphate (GTP) is an activator and guanosine 5'-diphosphate-3'-diphosphate (ppGpp) is an inhibitor of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) of Escherichia coli [E. Hölttä et al. (1974) Biochem. Biophys. Res. Commun. 59, 1104-1111] has been confirmed. The hypothesis that synthesis of both polyamine and RNA in E. coli is regulated in vivo by these nucleotides was tested in E. coli B-207. On transfer of this K+-requiring, amino-acid-deficient strigent strain from K+-medium to Na+-medium, the organism stops protein synthesis, maintains a high rate of RNA synthesis, and increases putrescine synthesis from ornithine manyfold. Under these conditions, the cells do not markedly change their contents of GTP and ppGpp. The proposed mechanism of regulation of RNA and putrescine synthesis by guanine nucleotides does not appear to explain the metabolic phenomena observed in this organism during K+ deficiency. Nevertheless, amino acid depletion in K+-medium does result in a marked increase in ppGpp.
Erythrocyte membranes undergo cooperative, pH-sensitive state transitions in the physiological temperature range: evidence from Raman spectroscopy.
Authors: Verma S P , Wallach D F
Published: May 2006

We have examined the Raman scattering from erythrocyte ghosts at 2700 to 3000 cm-1 (CH-stretching region). Plots of the intensity (I) of the 2930 cm-1 band relative to the intensity of the thermally stable 2850 cm-1 band, i.e., the [I2930/I2850] ratio, as a function of temperature reveal a sharp discontinuity, which at pH 7.4 has a lower limit of 38 degrees and is irreversible above 42 degrees. [I2930/I2850] is stable between pH 7.0 and pH 7.4, but increases or decreases sharply below pH 7.0 or above pH 7.5, respectively. Reduction of pH to 6.5 lowers the transition temperature by about 16 degrees, and a shift to pH 6.0 drops the transition range to 0 to 7 degrees. The above effects of temperature and pH on Raman scattering closely correspond to those detected by studies on the interaction of membrane protein fluorophores and lipid-soluble fluorescence quenchers [Bieri, V. and Wallach, D.F.H. (1975) Biochim. Biophys. Acta 406, 415-423]. Taken together, these results suggest that the transitions represent concerted process, involving hydrophobic amino acid residues and lipid chains at apolar protein-lipid boundaries.
Negative homotropic cooperativity and affinity heterogeneity: preparation of yeast glyceraldehyde-3-phosphate dehydrogenase with maximal affinity homogeneity.
Authors: Gennis L S
Published: May 2006

A three-step procedure including affinity chromatography on NAD+-azobenzamidopropyl-Sepharose has been designed for the purification of yeast glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] with maximized specific activity and maximized homogeneity with respect to affinity for the coenzyme, NAD+.Binding isotherms allow the analysis of cooperativity patterns that disclose both the average ligand affinity in the system and the distribution of ligands among the sites, only for systems with complete affinity homogeneity. The presence of affinity heterogeneity, resulting from multiple oligomeric species differing only in their affinity for coenzyme, gives rise to isotherms which falsely manifest apparent negative cooperativity. A method for distinguishing negative homotropic cooperativity from affinity heterogeneity is suggested.
Presence of diadenosine 5',5''' -P1, P4-tetraphosphate (Ap4A) in mamalian cells in levels varying widely with proliferative activity of the tissue: a possible positive "pleiotypic activator".
Authors: Rapaport E , Zamecnik P C
Published: May 2006

An accurate assay of diadenosine 5',5'''- P1,P4-tetraphosphate [A(5') pppp(5')A], which was shown to be formed in vitro in the backreaction of the amino acid activation step, has been developed in various cell lines in culture and in normal mouse liver or hepatoma in vivo. Use of radioactive labeling of acid-soluble nucleotides to high specific activity followed by chromatographic separation techniques yielded levels of Ap4A varying from 5 to 0.05 muM (from 30 pmol/mg of protein to 0.15 pmol), depending on the doubling time of the cell line or the proliferative state of the cells. The levels of Ap4A incells is inversely related to their doubling time, varying from 0.1 X 10(-4) of the cellular ATP levels in slowly growing cells to 20 X 10(-4) of the ATP levels of cells with rapid doubling times. The steady-state levels of ATP of different cell lines, although showing some fluctuations, are not related to the doubling time of the cells. Arrest of cellular proliferation by serum deprivation or amino acid starvation, which does not alter the cellular ATP levels more than 2-fold, does nevertheless cause a decrease of 30 to 50-fold in the Ap4A levels. Inhibition of protein synthesis by pactamycin or puromycin, or inhibition of DNA synthesis by hydroxyurea, leads to a more dramatic decrease of 50 to 100-fold in intracellular Ap4A levels. The metabolic lability of Ap4A is also demonstrated by its rapid depletion after decreases in the ATP/ADP ratio. The possibility of Ap4A being a metabolic "signal nucleotide" that is formed at the onset of protein synthesis and is active in positive growth regulation (positive pleiotypic activation) is discussed.
Embryonal carcinoma antigen and the T/t locus of the mouse.
Authors: Kemler R , Babinet C , Condamine H , Gachelin G , Guenet J L , Jacob F
Published: May 2006

The presence of the F9 antigen and of four other antigens related to the T/t locus of the mouse was investigated by immunofluorescence on preimplantation embryos. In morulae heterozygous for any of these t haplotypes, both the appropriate t antigen and the F9 antigen are expressed. The F9 antigen segregates among the progeny of crosses producing embryos homozygous for some (tw32 and tw5) but not for other haplotypes. It is concluded that (i) whatever the time of action of a t haplotype, its corresponding antigen is expressed during cleavage and (ii) the F9 antigen is specified by a gene(s) in the region of the T/t locus.
Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic, and dual-function neurons.
Authors: Furshpan E J , MacLeish P R , O'Lague P H , Potter D D
Published: May 2006

Electrophysiological studies were made on microcultures (300-500 mum in diameter) in which solitary sympathetic principal neurons from newborn rats grew on previously dissociated rat heart cells.Some neurons inhibited,some excited, and others first inhibited and then excited the cardiac myocytes. Application of drugs provided evidence for secretion of acetylcholine by the first group, catecholamines by the second, and both acetylcholine and catecholamines by the third. Solitary neurons which inhibited themyocytes usually excited themselves at nicotinic synapses (autapses).
Viscosity of dilute gases and vapors
Authors: Hildebrand J. H.
Published: May 2006

The well-known formula for calculating the viscosity of hard sphere gases, η

o

= 2(
mkT
)
1/2
/3π
3/2
σ
2
, where σ
2
is molecular cross section, is altered to η

o

=
K
(
MT
)
1/2
/

V
t

2/3
, where

V
t

is the molal volume of a liquid that has expanded sufficiently to permit mean free paths long enough to have significant fractions of the random thermal momenta of molecules in free flight and
M
is molal mass.

This formulation places upon a single straight line points for all nonpolar molecules, mono- and polyatomic molecules alike, over long ranges of temperature.
Dopamine receptors in human and calf brains, using [3H]apomorphine and an antipsychotic drug.
Authors: Seeman P , Chau-Wong M , Tedesco J , Wong K
Published: May 2006

In order to develop a better dopamine receptor radioligand, [3H[apomorphine was prepared and tested for dopamine-like binding properties in both calf and human brain tissues. Specific binding of [3H]apomorphine was defined as that binding which occurred in the presence of 1 muM (-)-butaclamol (an inactive neuroleptic) minus that occurring in the presence of 1 muM (+)-butaclamol (active neuroleptic). The specific binding was saturable, the number of sites being double that of specific [3H]dopamine binding, and occurred primarily in dopamine-rich regions of postmortem human brains. The binding had a dissociation constant of 0.9 nM for human caudate (2 nM for calf caudate) and was blocked by dopamine and norepinephrine, but not by isoproterenol or (-)-propranolol, distinguishing it from a beta-adrenergic receptor. Since there was little desorption of [3H]apomorphine, the ligand permits extensive washing during routine assays for dopamine receptors, and facilitates biochemical purification of the receptor.
Conversion of 3T3 fibroblasts into adipose cells: triggering of differentiation by prostaglandin F2alpha and 1-methyl-3-isobutyl xanthine.
Authors: Russell T R , Ho R
Published: May 2006

Green and Kehinde [(1974) cell 1, 113-116] have isolated clones of Swiss 3T3 fibriblasts that are able to convert to adipose cells. In this paper we report on two chemicals (prostaglandin F2alpha, 0.1 mug ml, and 1-methyl-3-isobutyl xanthine, 0.5 mM) that are able to rapidly and irreversibly program the fibroblasts to differentiate into adipose cells. Confluent cultures treated with prostaglandin F2alpha and insulin for 3-5 days, followed by insulin alone for 7-48 hr, yield numerous adipocyte colonies compared to control dishes and dishes treated with insulin alone. Cells treated with prostaglandin F2alpha or 1-methyl-3-isobutyl xanthine alone, rinsed, and then exposed to insulin gave similar results, indicating that the continuous presence of the triggering agent is not required to elicit the conversion of the fibroblasts to adipocytes. Agents that raise intracellular levels of 3':5'-cyclic AMP. 1.0 mM; 8-bromo-cyclic AMP,0.5 mM; and prostaglandin E1, 0.1 mug/ml) do not trigger the conversion process, suggesting that cyclic AMP may not be the mediator of differentiation in these cells. 8-Bromo-cyclic AMP, however, does induce thase; 3':5'-nucleotidohydrolase; EC 3.1.4.17) in these cells; the induction appears to be mediated by increases in intracellular cyclic AMP levels. These results indicate that this cell line might offer a system for studying the regulation of a type of cellular differentiation.