bluuebunny/crossref_metadata_2025 huggingface dataset:
Membrane ultrastructural changes during calcium phosphate-induced fusion of human erythrocyte ghosts.
Authors: Zakai N , Kulka R G , Loyter A
Published: May 2006

Nascent calcium phosphate promotes the agglutination and fusion of human erythrocyte ghosts. Membrane phospholipids of erythrocyte ghosts treated with Ca2+ and phosphate ions become exposed to attack by phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) (Bacillus cereus). Freeze-fracture pictures of fused erythrocyte ghosts show the presence of regions deficient in intramemebrane particles in the protoplasmic face which we believe to be regions of fusion. Discontinuous regions of the protoplasmic and exoplasmic faces are observed, which are apparently intermediate stages in the process of fusion. TH-in-section electron micrographs reveal deposits of calcium phosphate in areas of contact and fusion of ghosts. Ca2+ in the presence of N-[tris(hydroxymethyl)methyl]glycine (Tricine) buffer causes the formation of blebs in the membrane but does not cause changes in the intramembrane particle pattern or induce fusion. It is suggested that nascent calcium phosphate acts by forming protein-free regions of phospholipid bilayer which can fuse readily.
Isolation and preliminary characterization of 10-nm filaments from baby hamster kidney (BHK-21) cells.
Authors: Starger J M , Goldman R D
Published: May 2006

A procedure for isolating 10-nm filaments as juxtanuclear caps from normal and colchicine-treated BHK-21 cells is described. These aggregates of 10-nm filaments retain their birefringence and their structural integrity. The major proteins comprising the filament preparations can be resolved as two bands on sodium dodecyl sulfate gels, corresponding to approximate molecular weights of 54,000 and 55,000.
Gene activation in somatic nuclei after injection into amphibian oocytes.
Authors: De Robertis E M , Gurdon J B
Published: May 2006

Genes that are unexpressed in somatic cells have been activated by injecting cultured cell nuclei of the frog Xenopus laevis into oocytes of the newt Pleurodeles waltlii. The genes that were activated are normally expressed in oocytes but not in cultured cells. Conversely, genes that are normally expressed in cultured cells but not in oocytes became inactive when cultured cell nuclei were injected into oocytes. These changes in gene activity were detected by two-dimensional gel electrophoresis of proteins synthesized by oocytes injected with nuclei. Controls, which included the use of alpha-amanitin, showed that these changes in protein synthesis are dependent on gene transcription. We conclude that genes that become inactive during cell differentiation can be reactivated, in the absence of cell division, by normal components of oocyte cytoplasm.
Isolation of virus-free Herpesvirus saimiri antigen-positive plasma membrane vesicles.
Authors: Pearson G R , Scott R E
Published: May 2006

Experiments were undertaken to determine whether virus-free, antigen-positive plasma membrane vesicles could be isolated from owl monkey kidney cells infected with Herpesvirus saimiri. The results demonstrate that vesicles can be produced by using a vesiculation fluid containing 25 mM formaldehyde/2 mM dithiothreitol. Electron microscopy revealed that these vesicles were free of detectable virus particles. Vesicles prepared from the infected cells contained virus-induced membrane antigens as shown by membrane immunofluorescence and by inhibition of antibody-dependent lymphocyte cytotoxicity. Nonhuman primates immunized with vesicles produced antibodies to these membrane antigens, late cytoplasmic antigens, and neutralizing antibodies. Infectious virus was not demonstrated in these vesicles by cocultivation with owl monkey kidney cells or by the inoculation of cottontop marmosets. Furthermore, no DNA could be demonstrated in vesicles prepared from Herpesvirus-infected owl monkey kidney cells. The implication of these findings in relation to the question of a virus-free membrane vaccine against Herpesvirus infections is discussed.
Adsorption inhibition as a mechanism of freezing resistance in polar fishes.
Authors: Raymond J A , DeVries A L
Published: May 2006

Polar fishes are known to have serum proteins and glycoproteins that protect them from freezing, by a noncolligative process. Measurements of antifreeze concentrations in ice and scanning electron micrographs of freeze-dried antifreeze solutions indicate that the antifreezes are incorporated in ice during freezing. The antifreezes also have a pronounced effect on the crystal habit of ice grown in their presence. Each of four antifreezes investigated caused ice to grow in long needles whose axes were parallel to the ice c axis. Together these results indicate the antifreezes adsorb to ice surfaces and inhibit their growth. A model in which adsorbed antifreezes raise the curvature of growth steps on the ice surface is proposed to account for the observed depression of the temperature at which freezing occurs and agrees well with experimental observations. The model is similar to one previously proposed for other cases of crystal growth inhibition.
Sequence specificity of mutagen-nucleic acid complexes in solution: Intercalation and mutagen-base pair overlap geometries for proflavine binding to dC-dC-dG-dG and dG-dG-dC-dC self-complementary duplexes
Authors: Patel Dinshaw J. , Canuel Lita L.
Published: May 2006

The complex formed between the mutagen proflavine and the dC-dC-dG-dG and dG-dG-dC-dC self-complementary tetranucleotide duplexes has been monitored by proton high resolution nuclear magnetic resonance spectroscopy in 0.1 M phosphate solution at high nucleotide/drug ratios. The large upfield shifts (0.5 to 0.85 ppm) observed at all the proflavine ring nonexchangeable protons on complex formation are consistent with intercalation of the mutagen between base pairs of the tetranucleotide duplex. We have proposed an approximate overlap geometry between the proflavine ring and nearest neighbor base pairs at the intercalation site from a comparison between experimental shifts and those calculated for various stacking orientations. We have compared the binding of actinomycin D, propidium diiodide, and proflavine to self-complementary tetranucleotide sequences dC-dC-dG-dG and dG-dG-dC-dC by UV absorbance changes in the drug bands between 400 and 500 nm. Actinomycin D exhibits a pronounced specificity for sequences with dG-dC sites (dG-dG-dC-dC), while propidium diiodide and proflavine exhibit a specificity for sequences with dC-dG sites (dC-dC-dG-dG). Actinomycin D binds more strongly than propidium diiodide and proflavine to dC-dG-dC-dG (contains dC-dG and dG-dC binding sites), indicative of the additional stabilization from hydrogen bonding and hydrophobic interactions between the pentapeptide lactone rings of actinomycin D and the base pair edges and sugar-phosphate backbone of the tetranucleotide duplex.
Mutagenicity and cytotoxicity of benz[
a
]anthracene diol epoxides and tetrahydro-epoxides: Exceptional activity of the bay region 1,2-epoxides
Authors: Wood Alexander W. , Chang Richard L. , Levin Wayne , Lehr Roland E. , Schaefer-Ridder Maria , Karle Jean M. , Jerina Donald M. , Conney Allan H.
Published: May 2006

Three diastereomeric pairs of diol epoxides, two tetrahydro-epoxides, and the K-region oxide of the polycyclic aromatic hydrocarbon benz[
a
]anthracene were evaluated for mutagenic activity in strain TA 100 of
Salmonella typhimurium
and in line V79-6 of Chinese hamster lung cells. The two diastereomeric 1,2-epoxides of the
trans
-3,4-dihydrodiol of benz[
a
]anthracene are 15 to 35 times more mutagenic to the bacteria and 65 to 125 times more mutagenic to the mammalian cells than are the diastereomeric pairs of benz[
a
]anthracene-8,9-diol-10,11-epoxides or benz[
a
]anthracene-10,11-diol-8,9-epoxides. 1,2-Epoxy-1,2,3,4-tetrahydrobenz[
a
]anthracene is the most mutagenic and cytotoxic of the nine derivatives and is 5 and 25 times more mutagenic than 3,4-epoxy-1,2,3,4-tetrahydrobenz[
a
]anthracene in bacterial and mammalian cells, respectively. In either test system, benz[
a
]anthracene 5,6-oxide (K-region oxide) has less than 10% of the activity of any of the 1,2-epoxides derived from benz[
a
]anthracene. The relative stabilities of the derivatives in aqueous solution do not account for the differences in mutagenic activity because the more mutagenic derivatives tend to be less stable. The benz[
a
]anthracene diol epoxides, like the benzo[
a
]pyrene diol epoxides, are refractory to the action of epoxide hydrase. The exceptional mutagenic activity of the 1,2-epoxide derivatives of benz[
a
]anthracene is consistent with and supportive of the hypothesis that bay region epoxides on saturated, angular benzo-rings of unsubstituted polycyclic aromatic hydrocarbons are ultimate carcinogens.
Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo.
Authors: Tanaka K , Hayakawa H , Sekiguchi M , Okada Y
Published: May 2006

The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by UV light. A clear dose response was observed between the level of UV-induce unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4V1, a mutant defective in the endonuclease V gene, showed no ability to restore the UV-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by UV light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after UV irradiation.
Mutants of Escherichia coli lacking in highly penicillin-sensitive D-alanine carboxypeptidase activity.
Authors: Matsuhashi M , Takagaki Y , Maruyama I N , Tamaki S , Nishimura Y , Suzuki H , Ogino U , Hirota Y
Published: May 2006

Mutants of Escherichia coli lacking in the highly penicillin-sensitive enzyme activities of D-carboxy-peptidase, transpeptidase, and endopeptidase, and with the concomitant absence of penicillin-binding protein 4 of B.G. Spratt and A.B. Pardee [(1975) Nature 254, 516-517] were isolated. The defect of these mutants is ascribed to the lack of an enzyme, D-alanine carboxypeptidase Ib. Genetic mapping studies show the mutation (dacB) to be located at 68 min on the E. coli chromosome map. The dacB mutation results in the simultaneous loss of D-alanine carboxypeptidase and penicillin-binding protein 4. The mutants grew normally under a wide range of growth conditions. We conclude that the enzyme is not a necessary component for normal peptidoglycan biosynthesis in E. coli.
Fatty liver induced in Zucker "fatty" (ff) rats by a semisynthetic diet rich in sucrose.
Authors: Novikoff P M
Published: May 2006

A new fatty liver model is described in which heredity plays a major role. A marked fatty liver develops when the homozygous mutant Zucker rat (ff or "fatty") is fed a semisynthetic diet enriched in sucrose. Lean littermates do not develop fatty liver on this experimental diet. Even old ff rats do not develop fatty livers on chow; the experimental diet is required. The fatty liver is described, including light microscopic and preliminary electron microscopic observations.
Coordination of ribosome content and polysome formation during estradiol stimulation of vitellogenin synthesis in immature male chick livers.
Authors: Bast R E , Garfield S A , Gehrke L , Ilan J
Published: May 2006

To elucidate the mechanisms by which protein synthesis is affected by estradiol, we characterized cockerel liver polysomal profiles during hormone induction and withdrawal. We describe a method for isolating intact polysomes which results in preparations that are stable even after storage in solution at 10 degrees for 16 hr. In addition, our procedure eliminates the necessity for starving animals prior to experiments. Recovery of radioactive polysomes indicated that yield is about 90% and that our polysomal preparations appear to represent polysome distribution in vivo. Using this approach we show that estradiol injection stimulates ribosome content 6-fold and that formation of polysomes is coincident with the induction of vitellogenin synthesis. We also demonstrate that the size and number of polysomes increase and decrease in a coordinated fashion with the rate of vitellogenin synthesis. The kinetics of ribosome synthesis and the fact that at least 80% of the newly synthesized ribosomes are directly recruited into polysomes indicate that ribosomes might be limiting the rate of protein synthesis during the stimulatory phase of the hormone cycle.
Nitric oxide activates guanylate cyclase and increases guanosine 3′:5′-cyclic monophosphate levels in various tissue preparations
Authors: Arnold William P. , Mittal Chandra K. , Katsuki Shoji , Murad Ferid
Published: May 2006

Nitric oxide gas (NO) increased guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] activity in soluble and particulate preparations from various tissues. The effect was dose-dependent and was observed with all tissue preparations examined. The extent of activation was variable among different tissue preparations and was greatest (19- to 33-fold) with supernatant fractions of homogenates from liver, lung, tracheal smooth muscle, heart, kidney, cerebral cortex, and cerebellum. Smaller effects (5- to 14-fold) were observed with supernatant fractions from skeletal muscle, spleen, intestinal muscle, adrenal, and epididymal fat. Activation was also observed with partially purified preparations of guanylate cyclase. Activation of rat liver supernatant preparations was augmented slightly with reducing agents, decreased with some oxidizing agents, and greater in a nitrogen than in an oxygen atmosphere. After activation with NO, guanylate cyclase activity decreased with a half-life of 3-4 at 4° but re-exposure to NO resulted in reactivation of preparations. Sodium azide, sodium nitrite, hydroxylamine, and sodium nitroprusside also increased guanylate cyclase activity as reported previously. NO alone and in combination with these agents produced approximately the same degree of maximal activation, suggesting that all of these agents act through a similar mechanism. NO also increased the accumulation of cyclic GMP but not cyclic AMP in incubations of minces from various rat tissues. We propose that various nitro compounds and those capable of forming NO in incubations activate guanylate cyclase through a similar but undefined mechanism. These effects may explain the high activities of guanylate cyclase in certain tissues (e.g., lung and intestinal mucosa) that are exposed to environmental nitro compounds.
Isolation of an iron-molybdenum cofactor from nitrogenase
Authors: Shah Vinod K. , Brill Winston J.
Published: May 2006

A method for the isolation of an iron-molybdenum cofactor (FeMoCo) from component I of nitrogenase is described. This method is used to isolate FeMoCo from aerobic, anaerobic, facultative, and photosynthetic nitrogen-fixing organisms. The Fe/Mo ratio in the FeMoCo from
Azotobacter vinelandii
and
Clostridium pasteurianum
is 8:1. The FeMoCo contains six atoms of acid-labile sulfide per eight Fe atoms. Crystalline component I from
A. vinelandii
contains 2 Mo, 33 Fe, and 27 acid-labile sulfide atoms per molecular weight of 250,000. The specific activity of FeMoCo is 425 nmol of C
2
H
4
formed/min per nmol of Mo. There is better than 98% reconstitution between FeMoCo and inactive component I in
A. vinelandii
mutant strain UW45. The FeMoCo yield from component I is about 90%. FeMoCo from nitrogenase component I of
C. pasteurianum, Klebsiella pneumoniae, Bacillus polymyxa
, and
Rhodospirillum rubrum
activates inactive component I in an extract from mutant strain UW45 and follows saturation kinetics. The FeMoCo in various nitrogen-fixing organisms seems to be very similar. Wild-type
A. vinelandii
derepressed for nitrogenase synthesis in tungsten-containing medium and
K. pneumoniae
mutant strain UN109 are also activated
in vitro
by FeMoCo.

The FeMoCo is very sensitive to oxygen, but is stable even at room temperature as long as it is kept anaerobic and in
N
-methylformamide, the solvent used for its isolation. FeMoCo is unstable in an aqueous environment, even though it is kept strictly anaerobic. Knowledge of the structure of this cofactor should be useful for understanding the role of molybdenum at the active site of nitrogenase, role of ligands close to molybdenum in electron and proton transfer, and the catalytic mechanism of nitrogen fixation. The FeMoCo might be used as a model for synthesizing catalysts for chemical nitrogen fixation.
Conformational alteration of protein synthesis elongation factor EF-Tu by EF-Ts and by kirromycin.
Authors: Blumenthal T , Douglass J , Smith D
Published: May 2006

Alterations of the structure of EF-Tu have been investigated by using the rate of EF-Tu cleavage by trypsin as a conformational probe. The presence of EF-Ts bound to EF-Tu results in a 10-fold increase in the cleavage rate. The antibiotic kirromycin, which inhibits protein synthesis by virtue of its interaction with EF-Tu, mimics this effect of EF-Ts. Both kirromycin and EF-Ts also facilitate the exchange of free GDP with GDP bound to EF-Tu. The results suggest that EF-Ts and kirromycin induce a similar conformational change in EF-Tu, thereby "opening" the guanine nucleotide binding site. The trypsin-cleaved EF-Tu still can bind GDP and EF-Ts and can function in Qbeta replicase, but it no longer spontaneously renatures following denaturation in urea.
Identification of synaptic acetylcholine receptor sites in retina with peroxidase-labeled α-bungarotoxin
Authors: Vogel Zvi , Maloney Greg J. , Ling Alice , Daniels Mathew P.
Published: May 2006

An α-bungarotoxin-horseradish peroxidase conjugate, which binds specificity to nicotinic acetylcholine receptors, was synthesized. This conjugate was bound by 5-7% of the synapses in the inner plexiform layer of the chicken retina. Bipolar ribbon synapses as well as amacrine synapses bound the conjugate.
Opiate-dependent modulation of adenylate cyclase.
Authors: Sharma S K , Klee W A , Nirenberg M
Published: May 2006

Reactions mediated by the opiate receptors that inhibit adenylate cyclase (EC 4.6.1.1) are closely coupled to subsequent reactions that gradually increase adenylate cyclase activity of neuroblastoma X glioma NG108-15 hybrid cells. Opiate-treated cells have higher basal-, prostaglandin E1-, and 2-chloroadenosine-stimulated activities than do control cells. However, NaF or guanosine 5'-(beta, gamma-imido)triphosphate abolishes most of the differences in adenylate cyclase activity observed with homogenates from control and opiate-treated cells. Cycloheximide blocked some, but not all, of the opiate-dependent increase in adenylate cyclase activity. These results suggest that the opiate-dependent increase in adenylate cyclase is due to conversion of adenylate cyclase to a form with altered activity. Protein synthesis also is required for part of the opiate effect. We propose that activity of adenylate cyclase determines the rate of conversion of the enzyme from one form to the other and that opiates, by inhibiting adenylate cyclase, alter the relative abundance of low- and high-activity forms of the enzyme.
Abnormal lithium and sodium transport in erythrocytes of a manic patient and some members of his family
Authors: Pandey G. N. , Ostrow D. G. , Haas M. , Dorus E. , Casper R. C. , Davis J. M. , Tosteson D. C.
Published: May 2006

This paper compares the transport of Li
+
and Na
+
in erythrocytes from a patient with mania and from members of his family to that in erythrocytes from normal humans. In normal human erythrocytes, Li
+
is transported by at least three operationally distinct pathways: one inhibited by ouabain (ouabain-sensitive), one by phloretin (phloretin-sensitive), and one not inhibited by either compound (insensitive). Li
+
can be driven up its electrochemical potential gradient by an oppositely directed electrochemical potential gradient for Na
+
—i.e., Li
+
/Na
+
counterflow can occur—through the phloretin-sensitive pathway but not through the other two pathways. Because ouabain-sensitive Li
+
transport is negligible under physiological conditions, Li
+
distribution between erythrocytes and plasma
in vivo
depends mainly on the balance between Li
+
/Na
+
counterflow and the insensitive pathway(s) of Li
+
transport. The steady-state ratio of Li
+
concentration in the erythrocytes to that in the plasma of the patient was between 2 and 3 times higher than the comparable ratio in normal persons. The phloretin-sensitive Li
+
/Na
+
counterflow system was almost absent in the erythrocytes of the patient. Furthermore, unlike those from normal individuals, the patient's erythrocytes showed no external Li
+
-stimulated, phloretin-sensitive, ouabain-insensitive Na
+
efflux. The magnitudes of the ouabain-sensitive and insensitive pathways for Li
+
transport in the patient's erythrocytes were within normal limits. The decreased Li
+
/Na
+
counterflow in the patient's erythrocytes was probably not due to the presence of an inhibitor in the plasma of the patient but rather to an intrinsic defect in the erythrocytes. Because the father and several siblings of the patient showed a similar abnormality in erythrocyte Li
+
/Na
+
transport, it is probable that this defect is inherited.
Purification and properties of human erythrocyte pyrimidine 5'-nucleotidase.
Authors: Torrance J D , Whittaker D , Beutler E
Published: May 2006

A 250,000-fold purification of pyrimidine 5'-nucleotidase from human erythrocytes has been achieved using a combination of DEAE-cellulose chromatography, ammonium sulfate fractionation, gel filtration, and isoelectric focusing. Polyacrylamide disc and starch gel electrophoresis of the purified material show two strong protein bands. On starch gel these bands exhibited pyrimidine 5'-nucleotidase activity. Two faint protein bands devoid of enzyme activity were also found in the case of polyacrylamide electrophoresis. The enzyme has a pH optimum at 7.5 and is most stable between pH 6 and 7.5. The enzyme has pI of 5.0 and a molecular weight of 28,000 by gel filtration. The Km of the purified enzyme was 10 muM, compared to 40 muM when measured in hemolysate. The higher Km in the hemolysate is due to the presence of an inhibitor. Inorganic phosphate was shown to be a competitive inhibitor of pyrimidine 5'-nucleotidase and inorganic phosphate in the hemolysate may be responsible for increasing the Km of the enzyme for the substrate cytidine monophosphate.
High aerobic glycolysis of rat hepatoma cells in culture: Role of mitochondrial hexokinase
Authors: Bustamante Ernesto , Pedersen Peter L.
Published: May 2006

A tumorigenic anchorage-dependent cell line (H-91) was established in culture from an azo-dye-induced rat ascites hepatoma. When grown in a glucose-containing medium the cells exhibit high rates of lactic acid production characteristic of rapidly growing tumor cells. However, when glucose is replaced with galactose the cells grow equally well but exhibit only moderately elevated rates of lactic acid production. The molecular basis for this observation cannot be attributed to differences in permeability because initial rates of glucose and galactose entry into hepatoma cells are identical. Rather, the activity of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is found to be high in hepatoma cells, about 20-fold higher than that of control and regenerating rat liver. Moreover, tumor hexokinase activity is not inhibited by low concentrations (<0.6 mM) of the reaction product glucose 6-phosphate. Additionally, 50% of the hexokinase activity of hepatoma cells is found associated with the mitochondrial fraction. This fraction is 3-fold enriched in hexokinase activity relative to the homogenate and 4-fold enriched relative to the nuclear and postmitochondrial fractions. Tumor mitochondrial hexokinase appears to be coupled directly to oxidative phosphorylation, because addition of glucose to respiring hepatoma mitochondria (after a burst of ATP synthesis) results in stimulation of respiration. In contrast, glucose has no effect on the respiration of mitochondria from control and regenerating liver. These results suggest that the high glycolytic capacity of H-91 hepatoma cells is due, at least in part, to an elevated form of hexokinase concentrated in the mitochondrial fraction of the cell.
Fundamental aspects of electron transfer: experimental verification of vibronically coupled electron tunneling.
Authors: Potasek M J , Hopfield J J
Published: May 2006

A detailed investigation of the applicability of the vibronically coupled electron tunneling theory in biomolecules can be made by a quantitative study of a weak charge-transfer optical absorption band that has been predicted by this theory. The measurement of the position, width, and molar extinction coefficient of this band is examined in the bound model system cytochrome c-Fe(CN)6 at room temperature and demonstrates that the theory is quantitatively applicable in this system. The size of the parameters measured is typical of those relevant for biological electron transfers. The comparisons lend credibility to the generality of vibronically coupled electron theory in biomolecules and its short transfer distances.
Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts.
Authors: Kaufman S J , Parks C M
Published: May 2006

A nonfusing variant, fu-1, of the L8 line of rat myoblasts was isolated and characterized with respect to its growth in vitro and developmental properties. Comparative analyses of density-dependent inhibition of growth, serum requirements, cell adhesiveness, colony formation in soft agar, and hexose transport in L8 and fu-1 cells support the conclusion that the fu-1 cells are transformed. In addition, fu-1, but not L8, cells promote the development of tumors in athymic nude mice. fu-1 cells also do not make increased levels of creatinekinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) or myosin and they express an endogenous type-C virus. Both L8 and fu-1 cells express myokinase (ATP:AMP phosphotransferase, EC 2.7.4.3) activities in single cells. In contrast to fu-1 cells, the parent L8 line has increased creatine kinase and myosin after fusion and spontaneously contracts; expression of an endogenous virus could not be detected in these cells. These results suggest that loss of the ability to differentiate normally is associated with the loss of the normal control of cell division of myoblasts grown in vitro and in vivo.
Synthesis of fibrinolytic activator and inhibitor by endothelial cells
Authors: Loskutoff David J. , Edgington Thomas S.
Published: May 2006

Vascular endothelial cells derived from rabbit vena cava and maintained in continuous culture exhibited properties characteristic of the intact endothelium. These cells were used as a model for characterizing the fibrinolytic components specified by the endothelium. Endothelial cells in culture digested radiolabeled fibrinogen. Digestion resulted from the synthesis and secretion of a plasminogen activator. Fibrinolysis was not detected when cells were grown in medium lacking plasminogen, indicating the absence of plasminogen-independent fibrinolytic enzymes. Phorbol-myristate-acetate increased extracellular plasminogen activator activity dramatically. This increase was prevented when actinomycin D or cycloheximide was included in the growth medium, indicating that new gene expression was required for it. Intracellular plasminogen activator could not be detected unless the cell extracts were exposed briefly to mildly acidic conditions. Mixing experiments between acid-treated and untreated extracts suggested that the cells contained a potent, acid-labile inhibitor of fibrinolysis. As little as 10 μg of protein from whole cell extracts inhibited both cell and urokinase-mediated fibrinolysis by more than 70%. Cell fractionation studies localized the inhibitor to the cytosol whereas plasminogen activator activity was restricted to the membrane-rich fraction. This membrane fraction did not require acidification for activity, suggesting that the inhibitor had been removed and that acidification did not activate a plasminogen proactivator. These observations demonstrate that regulation of endothelial fibrinolytic activity is far more complex than had been anticipated and raise several uncertainties in regard to detecting the presence of plasminogen activators in cells and tissues.
Mutator action by
Escherichia coli
strains carrying
dnaE
mutations
Authors: Sevastopoulos C. G. , Glaser D. A.
Published: May 2006

Several newly isolated temperature-sensitive
dnaE
mutants of
Escherichia coli
exhibit powerful mutagenic action at permissive temperatures. Mutation rates for the two most active mutants were assayed at four different temperatures and compared to wild-type behavior. Temperature-resistant revertants of the original temperature-sensitive
dnaE
mutants exhibited lower, nearly normal, mutation rates, but no antimutator strains were found.
Amyloid protein SAA is associated with high density lipoprotein from human serum.
Authors: Benditt E P , Eriksen N
Published: May 2006

Human serums contain a protein antigenically related to protein AA, the principal protein of a major class of amyloid substance. The serum antigen, SAA, occurs mainly in a high molecular weight form, 1 to 2 X 10(5). This work shows that the bulk of the SAA sediments at density 1.12 g/cm3 and floats at density 1.21 g/cm3, as does the high density lipoprotein HDL3. SAA is associated with the apolipoproteins ApoA-I and ApoA-II. The total cholesterol:total protein ratio of the fraction with density 1.12-1.21 g/cm3 is 0.2:1, consistent with that of SAA constituent of the HDL3 fraction into low molecular weight species of the order of 13,000. The quantity of SAA may vary from 0.1% or more of the total protein of HDL3.
Identification of a host protein necessary for bacteriophage morphogenesis (the groE gene product).
Authors: Georgopoulos C P , Hohn B
Published: May 2006

Mutations in the groE gene of Escherichia coli, which block the correct assembly of the phage lambda head, have been previously described. Many groE mutations exert pleiotropic effects, such as inability to propagate phages T4 and T5 and inability to form colonies at 43 degrees. With the help of the EcoRI and HindIII restrictionenzymes and the appropriate phage vectors, we have constructed two lambda transducing phages, called W3 and H18, that carry the groE+ bacterial gene. Upon lysogenization by phage H18 the groE bacterial mutants recover their gro+ phenotype for both phage growth and the ability to form colonies at 43 degrees. We have identified the groE+ bacterial gene product as a protein of 65,000 molecular weight. Mutants of the W3 transducing phage that were selected on the basis of their ability to propagate on some groE mutant hosts induce the synthesis of a groE protein with altered electrophoretic mobility.
Similarity of nucleotide sequences around the origin of DNA replication in mouse polyoma virus and simian virus 40.
Authors: Soeda E , Kimura G , Miura K
Published: May 2006

The nucleotide sequence around the origin of replication in DNA of mouse polyoma virus was determined by 32P labeling of the 3' terminus of the Hap II-5/Alu I-1 DNA fragment, with the use of DNA polymerase. The result coincided with our previous report on the 32P labeling, with the use of polynucleotide kinase, of the 5' terminus of the Hap II-5/Hha I-1 DNA fragment, which corresponds to the large part of the present fragment, Hap II-5/Alu I-1. A symmetrical (A+T)-rich region containing a five-A stretch (or a five-T stretch) was flanked by two small regions with a 2-fold rotational axis of symmetry. On comparison of the sequence near the replication origin of polyoma DNA with that in the corresponding region of simian virus 40 DNA, which was included in the EcoRI-G fragment sequenced by Weissman's group (Subramanian K.N., Dahr, R. & Weissman, S. M. (1977) J. Biol. Chem. 252, 355--367), a considerable similarity was detected. Several possible common sequences for important biological activities such as the starting of DNA replication and RNA synthesis were suggested.
Isotopic (
18
O) shift in
31
P nuclear magnetic resonance applied to a study of enzyme-catalyzed phosphate—phosphate exchange and phosphate (oxygen)—water exchange reactions
Authors: Cohn Mildred , Hu Angela
Published: May 2006

An isotopic shift of the
31
P nuclear magnetic resonance due to
18
O bonded to phosphorus of 0.0206 ppm has been observed in inorganic orthophosphate and adenine nucleotides. Thus, the separation between the resonances of
31
P
18
O
4
and
31
P
16
O
4
at 145.7 MHz is 12 Hz and, in a randomized sample containing ∼50%
18
O, all five
16
O-
18
O species are resolved and separated from each other by 3 Hz. Not only does this yield the
18
O/
16
O ratio of the phosphate but, more important, the
18
O-labeled phosphate in effect can serve as a double label in following phosphate reactions, for oxygen in all cases and for phosphorus, provided the oxygen does not exchange with solvent water. Thus, it becomes possible to follow labeled phosphorus or labeled oxygen continuously as reactions proceed. Rate studies involving (
i
) phosphorus and (
ii
) oxygen are illustrated by continuous monitoring of the exchange reactions between (
i
) the β phosphate of ADP and inorganic phosphate catalyzed by polynucleotide phosphorylase and (
ii
) inorganic orthophosphate and water catalyzed by yeast inorganic pyrophosphatase. In the ADP—P
i
exchange, the P
i
(
18
O
4
) yielded an α P(
16
O
3
18
O) and a β P(
18
O
4
), proving that bond cleavage occurs between the α P and the α-β bridge oxygen. Among the many additional potential uses of this labeling technique and its spectroscopic observation are: (
i
) different labeling of each phosphate group of ATP, (
ii
) to follow rate of transfer of
18
O from a nonphosphate compound such as a carboxylic acid to a phosphate compound, and (
iii
) to follow the rate of scrambling (for example, of the β-γ bridge oxygen of ATP to nonbridge β P positions) and simultaneously the rate of exchange of the γ P nonbridge oxygens with solvent water in various ATPase reactions.
Carcinogens enhance survival of UV-irradiated simian virus 40 in treated monkey kidney cells: induction of a recovery pathway?
Authors: Sarasin A R , Hanawalt P C
Published: May 2006

Treatment of monkey kidney cells with low doses of carcinogen enhances the survival of UV-irradiated simian virus 40 (SV40). This is true for compounds with UV-like effects (metabolites of aflatoxin B1, N-acetoxyacetylaminofluorene) and compounds with x-ray-like effects (methyl methanesulfonate, ethyl methanesulfonate). This phenomenon resembles the UV-reactivation of viruses in eukaryotic cells. The carcinogen-induced enhancement of the survival of UV-irradiated SV40 is correlated with the inhibition of host-cell DNA synthesis, suggesting that the inhibition is an inducing agent. An enhancement of UV-irradiated SV40 survival is also obtained in cells treated with hydroxyurea or cycloheximide for long enough that there is still inhibition of host DNA synthesis during the early stage of SV40 infection. We hypothesize that treatment of host cells with carcinogens induces a new recovery pathway that facilitates the replication of damaged DNA, bypassing the lesions and resulting in the enhanced survival of UV-irradiated SV40. This inducible process might represent the expression of "SOS repair" functions in eukaryotic cells analogous to the previously demonstrated induction of SOS repair in bacteria after UV or carcinogen treatment.
Role of contact inhibition in the regulation of receptor-mediated uptake of low density lipoprotein in cultured vascular endothelial cells.
Authors: Vlodavsky I , Fielding P E , Fielding C J , Gospodarowicz D
Published: May 2006

Bovine vascular endothelial cells during logarithmic growth bind, internalize, and degrade low density lipoprotein (LDL) via a receptor-mediated pathway. However, contact-inhibited (confluent) monolayers bind but do not internalize LDL. This is in contrast to aortic smooth muscle cells or endothelial cells that have lost the property of contact inhibition. These cells internalize and degrade LDL at both high and low cell densities. The LDL receptors of smooth muscle and sparse endothelial cells down-regulate in response to LDL. In contrast, normal endothelial cells at confluency show little response. When contact inhibition in endothelial monolayers was locally released by wounding, and LDL was present, only cells released from contact inhibition accumulated LDL cholesterol. In smooth muscle cells under the same conditions, the entire culture interiorized lipid. It thus appears that in endothelial cells, unlike smooth muscle cells, contact inhibition is the major factor regulating cellular uptake of LDL cholesteryl ester. Reversal of contact inhibition by wounding provides a mechanism by which the endothelium could be the primary initiator of the atherosclerotic plaque.
Structural flexibility of isozyme variants: genetic variants in Drosophila disguised by cofactor and subunit binding.
Authors: Johnson G B
Published: May 2006

Wild populations of Drosophila mojavensis exhibit considerable conformational variation in the NAD+-free form of alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1). The variation appears genetic, as it does not occur within an inbred strain. The NAD+-bound form of alcohol dehydrogenase, present in the same individuals, does not exhibit the variation, suggesting that the binding of NAD+ acts to stabilize conformation. Such cofactor binding to enzymes may thus conceal considerable variation. A similar effect is suggested for binding of esterase subunits.
Increased turnover of arterial collagen in hypertensive rats.
Authors: Nissen R , Cardinale G J , Udenfriend S
Published: May 2006

The turnover of total collagen in several tissues of 12-week-old normotensive and hypertensive rats was estimated by using tritium-labeled proline as a precursor. The effect of reutilization of the label was minimized by treatment with large doses of unlabeled proline subsequent to administering the radioactive imino acid. The collagen from skin, tail tendon, aorta, and mesenteric artery in normotensive animals had a half-life of about 60--70 days. In hypertensive animals the half-lives of skin and tail tendon collagen were unchanged but the half-lives of collagen in the aorta and mesenteric artery were reduced to 17 days.
Experimental evidence for secondary protein-chromophore interactions at the Schiff base linkage in bacteriorhodopsin: Molecular mechanism for proton pumping
Authors: Lewis A. , Marcus M. A. , Ehrenberg B. , Crespi H.
Published: May 2006

Resonance Raman spectroscopy of the retinylidene chromophore in various isotopically labeled membrane environments together with spectra of isotopically labeled model compounds demonstrates that a secondary protein interaction is present at the protonated Schiff base linkage in bacteriorhodopsin. The data indicate that although the interaction is present in all protonated bacteriorhodopsin species it is absent in unprotonated intermediates. Furthermore, kinetic resonance Raman spectroscopy has been used to monitor the dynamics of Schiff base deprotonation as a function of pH. All our results are consistent with lysine as the interacting group. A structure for the interaction is proposed in which the interacting protein group in an unprotonated configuration is complexed through the Schiff base proton to the Schiff base nitrogen. These data suggest a molecular mechanism for proton pumping and ion gate molecular regulation. In this mechanism, light causes electron redistribution in the retinylidene chromophore, which results in the deprotonation of an amino acid side chain with pK >10.2 ± 0.3 (e.g., arginine). This induces subsequent retinal and protein conformational transitions which eventually lower the pK of the Schiff base complex from >12 before light absorption to 10.2 ± 0.3 in microseconds after photon absorption. Finally, in this low pK state the complex can reprotonate the proton-deficient high pK group generated by light, and the complex is then reprotonated from the opposite side of the membrane.
DNA sequence of the araBAD promoter in Escherichia coli B/r.
Authors: Greenfield L , Boone T , Wilcox G
Published: May 2006

The L-arabinose operon in Escherichia coli is a model system for the study of the control of gene expression. Maximal expression of the araBAD operon requires two positive control components: the araC protein-L-arabinose complex and the cyclic AMP receptor protein-cyclic AMP complex. Both araC protein and cyclic AMP receptor protein are required for the initiation of transcription of araBAD mRNA. We have used the plasmid pBR322 as a vector for cloning DNA fragments that contain the araBAD promoter. The cloned ara fragments were identified by both physical and genetic tests. A restriction map was constructed and the DNA sequence of the promoter was determined. The promoter contains a site that is similar to the RNA polymerase recognition sites in the galactose and lactose operons. It also contains a region similar to the known cyclic AMP receptor protein binding sites in the galactose and lactose operons.
An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function
Authors: Reynafarje Baltazar , Lehninger Albert L.
Published: May 2006

This paper describes the properties and a possible biological role of a transport process across the inner membrane of rat liver mitochondria resulting in the exchange of ATP
4-
(out) for ADP
3-
(in) + 0.5 phosphate
2-
(in). This transmembrane exchange reaction, designated as the ATP-ADP-phosphate exchange, is specific for the ligands shown, electroneutral, insensitive to
N
-ethylmaleimide or mersalyl, inhibited by atractyloside, and appears to occur only in the direction as written. It is thus distinct from the well-known phosphate-hydroxide and phosphate-dicarboxylate exchange systems, which are inhibited by mersalyl, and from the ATP-ADP exchanger, which does not transport phosphate. During ATP hydrolysis by mitochondria, half of the phosphate formed from ATP passes from the matrix to the medium by the mersalyl-insensitive ATP-ADP-phosphate exchange and the other half by the well-known mersalyl-sensitive phosphate-hydroxide exchange. These and other considerations have led to a hypothesis for the pathway and stoichiometry of ATP-dependent reverse electron transport, characterized by a requirement of 1.33 molecules of ATP per pair of electrons reversed and by the utilization of a different membrane transport pathway for phosphate and adenine nucleotides than is taken in forward electron flow and oxidative phosphorylation. The possible occurrence of independent pathways for ATP-forming forward electron flow and ATP-consuming reverse electron flow is consonant with the fact that the opposing degradative and synthetic pathways in the central routes of cell metabolism generally have different pathways that are independently regulated.
Serine-borate complex as a transition-state inhibitor of gamma-glutamyl transpeptidase.
Authors: Tate S S , Meister A
Published: May 2006

gamma-Glutamyl transpeptidase, a membrane-bound enzyme, functions in the gamma-glutamyl cycle to catalyze utilization of glutathione. It has been postulated that the amino-acid-stimulated utilization of glutathione by gamma-glutamyl transpeptidase reflects an aspect of amino acid translocation. As one approach to the effective in vivo inhibition of this enzyme, the inhibition of the enzyme by L-serine in the presence of borate buffers [Revel, J.P. & Ball, E.G. (1959) J. Biol. Chem. 234, 577-582] was reinvestigated. Inhibition by L-serine, D-serine, and alpha-methyl-DL-serine in the presence of borate is competitive with respect to gamma-glutamyl substrate and such inhibition is parallel to the activity of transpeptidase toward L-gamma-glutamyl, D-gamma-glutamyl, and L-gamma-(alpha-methyl)glutamyl derivatives. L-Serine and borate effectively protect against inactivation of the enzyme by the gamma-glutamyl analogs, 6-diazo-5-oxonorleucine and azaserine, which bind to the gamma-glutamyl site of the enzyme. These studies, kinetic investigations, equilibrium dialysis experiments, and other data support the view that inhibition is produced by formation of serine-borate complex which binds at the gamma-glutamyl binding site of the light subunit of gamma-glutamyl transpeptidase. The data indicate that serine-borate complex is a transition state inhibitor of gamma-glutamyl transpeptidase.
Detection of
31
P nuclear magnetic resonance signals in brain by
in vivo
and freeze-trapped assays
Authors: Chance Britton , Nakase Yuzo , Bond Meredith , Leigh John S. , McDonald George
Published: May 2006

The
31
P NMR spectrum of energy-related metabolites under strictly aerobic conditions in rapidly respiring tissues under physiological conditions has been approached by the study of the
31
P NMR signals
in vivo
and in freeze-trapped organs. Freezing the head of the anesthetized animal by liquid N
2
, excision of the brain tissue (white and gray matter) at -196°, and transfer to the NMR tube occurs without alteration of the metabolite concentrations. The sample is warmed to the region -15° to -10°, at which temperatures there is sufficient mobility for recording
31
P NMR at concentrations characteristic of brain tissues (∼5 mM) with an adequate signal to noise ratio in 10 min but insufficient mobility for significant enzymatic activity. A ∼0.4-sec acquisition time is adequate for nuclear relaxation and a 10-min scan gives an adequate signal to noise ratio. Metabolism of creatine phosphate, P
i
, and sugar phosphates occurs by 1 hr at -10° and 2 hr at -12°. Extrapolation of the approximately zero order kinetics of disappearance of creatine phosphate and appearance of P
i
suggests that <10% of these two metabolites has been altered in the time of the first measurement.

A comparison of the freeze-trapped state and the
in vivo
metabolite pattern is afforded in preliminary experiments on the head of the living mouse (brain and skeletal tissue) in aerobic and anaerobic states. Longer relaxation times and mild hypoxia due to the restricted diameter of the NMR tube gives significantly lower creatine phosphate/ATP values for this condition. Both direct
in vivo
and freeze-trapped assays of energy-related metabolites afford excellent approaches to the detection of anoxia and to the evaluation of metabolic control in hypoxic conditions.
Reaction to phytotoxins in a potato population derived from mesophyll protoplasts
Authors: Matern Ulrich , Strobel Gary , Shepard James
Published: May 2006

Alternaria solani
, the causal agent of early blight disease in potato, produces two host-specific, lipidlike toxins in culture. Both compounds are required in the leaf bioassay for the elicitation of typical early blight symptoms, but the compounds are individually inactive. The procedures for the preparation of both compounds are outlined. These compounds can be used effectively to select for toxin-insensitive and sensitive clones of a Russet Burbank potato cultivar that have been regenerated from single mesophyll protoplasts. Furthermore, both sensitivity and insensitivity to the toxins in these clones is well correlated with susceptibility and resistance to
A. solani
. Potato clones that have been produced by somatic cell regeneration techniques maintain their reaction type to these fungal toxins for at least two generations of vegetative propagation. The genetic basis for this variation among these potato clones remains to be explained.