bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibodies bound to lipid haptens in model membranes diffuse as rapidly as the lipids themselves.
Authors: Smith L M , Parce J W , Smith B A , McConnell H M
Published: May 2006

A pattern photobleaching method has been used to measure the rates of lateral diffusion of fluorescent-labeled specific anti-nitroxide IgG bound with both combining sites to nitroxide-containing phospholipids in liposomal membranes composed of dimyristoyl phosphatidylcholine at 28 degrees C ("fluid"), dipalmitoyl phosphatidylcholine at 32 degrees C ("Solid"), and dipalmitoyl phosphatidylcholine containing 15 or 25 mol% cholesterol ("solid" or "fluid," respectively, at 32 degrees C). The diffusion coefficients of the bound immunoglobulin were found to be the same as those of fluorescent-labeled phospholipids in each case even though these diffusion coefficients range from 10(-11) to 10(-8) cm2/sec. Hapten-containing liposomal membranes of the type studied here have previously been shown to elicit a number of antibody-dependent immune responses. Therefore, this work indicates that membrane-bound but otherwise freely diffusing antibodies are sufficient for these reponses.
pH-dependent migration of copper(II) to the vacant zinc-binding site of zinc-free bovine erythrocyte superoxide dismutase
Authors: Valentine Joan S. , Pantoliano Michael W. , McDonnell Peter J. , Burger Allan R. , Lippard Stephen J.
Published: May 2006

Bovine erythrocyte superoxide dismutase (Cu
2
Zn
2
SODase; superoxide:superoxide oxidoreductase, EC 1.15.1.1) consists of two identical subunits each containing Cu
2+
and Zn
2+
in close proximity. We describe here electron spin resonance (ESR) and visible absorption spectroscopic studies of the zinc-free derivative of this protein, Cu
2
E
2
SODase (E = empty) over the pH range 6-10. The ESR spectrum of the zinc-free protein at 77 K is markedly pH dependent. At pH < 8.0 the ESR spectrum is axial in appearance. At pH > 8.0, the lineshape becomes increasingly distorted with increasing pH until, at pH = 9.5, the spectrum is very broad and resembles that of the four-copper derivative Cu
2
Cu
2
SODase and of model imidazolate-bridged binuclear Cu(II) complexes. ESR spectra at 30°C are also consistent with formation of Cu(II)-Im-Cu(II). A plot of changes in the signal amplitude of g⊥ for Cu
2
E
2
SODase as a function of pH gives an apparent p
K
a
of 8.2 for the transition. The long-wavelength absorption with λ
max
= 700 nm characteristic of Cu
2
E
2
SODase shifts with increasing pH to 800 nm and the resulting visible spectrum is identical to that of Cu
2
Cu
2
SODase. All of the above-mentioned spectroscopic changes induced by additions of NaOH are reversed when the pH is decreased with HNO
3
, although the approach to equilibrium is slow in the latter case. The results of these experiments are consistent with a reversible, pH-dependent migration of Cu
2+
from the native copper site of one subunit of the zinc-free protein to the empty zinc site of another subunit. By contrast, native protein, Cu
2
Zn
2
SODase, and the four-copper protein, Cu
2
Cu
2
SODase, show no variation in visible or ESR spectral properties in this pH range. Some previous results concerning the activity of Cu
2
E
2
SODase and its thermal stability are reexamined in light of these new findings.
Differences in mutagenicity and cytotoxicity of (+)- and (-)-benzo[a]pyrene 4,5-oxide: a synergistic interaction of enantiomers.
Authors: Chang R L , Wood A W , Levin W , Mah H D , Thakker D R , Jerina D M , Conney A H
Published: May 2006

In order to study the biological effects of (+)- and (-)-benzo[a]pyrene 4,5-oxide, a synthesis of these molecules has been developed based on the resolution of (+/-)-cis-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene. The (-) enantiomer of benzo[a]pyrene 4,5-oxide was 1.5- to 5.5-fold more mutagenic than the (+) enantiomer in strains TA 98, TA 100, TA 1537, and TA 1538 of Salmonella typhimurium and in Chinese hamster V79 cells. In studies with V79 Cells, the (-) enantiomer of benzo[a]pyrene 4,5-oxide was also more cytotoxic than the (+) enantiomer. When mixtures of the enantiomers were studied in V79 cells, synergistic cytotoxic and mutagenic responses were observed. The greatest cytotoxic and mutagenic effects occurred with a 3:1 mixture of the (-) and (+) enantiomers of benzo[a]pyrene 4,5-oxide, respectively.
Identification of a Rous sarcoma virus transformation-related protein in normal avian and mammalian cells.
Authors: Rohrschneider L R , Eisenman R N , Leitch C R
Published: May 2006

Avian sarcoma viruses (ASV) contain a gene (src) whose protein product mediates sarcomagenic transformation. This product is a 60,000-Mr phosphoprotein designated pp60src. We have found that normal uninfected frog, chicken, rat, and human cells contain a 60,000-Mr phosphoprotein related to the product of the ASV src gene and have designated that protein pp60. A phosphoprotein of similar size was not detectable in Drosophila cells. The pp60 proteins were detected by immunoprecipitation with rabbit antitumor serum containing broad spectrum antibodies to pp60src. Peptide maps of [35S]methionine-labeled pp60 and pp60src indicated major similarities as well as some differences in amino acid composition. Peptide maps of the 32P-labeled proteins demonstrated that the phosphopeptides of all endogenous pp60 molecules tested were identical. However, some differences were noted between the phosphopeptide patterns of pp60 and viral pp60src. The kinase activity associated with pp60src was measured in the immunocomplex and resulted in the transfer of radioactive phosphorus from [gamma-32P]ATP to the immunoglobulin heavy chain as well as to an 80,000-Mr phosphoprotein. The pp60 of chicken, rat, and human origin also contained an associated kinase activity. These results are consistent with the notion that the pp60 molecules are the protein products of endogenous sarc sequences found in vertebrate cells.
Endogenous digitalis-like activity in mammalian brain.
Authors: Fishman M C
Published: May 2006

A fraction of brain has been prepared that contains a substance mimicking actions of the digitalis glycosides. It both blocks the binding of [3H]ouabain to Na+,K+-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and inhibits the uptake of 86Rb+ into human erythrocytes. Partial isolation was achieved by Sephadex G-10 fractionation and subsequent desalting. This activity is potentially capable of regulating ionic fluxes and concentration gradients across the cell membrane.
Methylation of integrated adenovirus type 12 DNA sequences in transformed cells is inversely correlated with viral gene expression.
Authors: Sutter D , Doerfler W
Published: May 2006

The adenovirus type 12 (Ad12) DNA sequences integrated into the DNA of four lines of Ad12-transformed hamster cells are extensively methylated. Methylation in mammalian cell DNA is believed to occur predominantly at 5'-C-G-3' sequences. The majority, although not all, of the 5'-C-C-G-G-3' sequences present in integrated Ad12 DNA are methylated. Ad12 DNA isolated from purified virions, on the other hand, is not methylated to any significant extent. The segments of the integrated viral DNA comprising early genes, which are expressed as mRNA in two lines of Ad2-transformed hamster cells, are undermethylated in comparison to late viral segments, which are not expressed and are extensively methylated. In contrast, in two lines of Ad12-induced rat brain tumor cells, some of the late viral genes have been shown to be expressed as mRNA. The segment of the integrated Ad12 DNA that comprises these late genes, the EcoRI B fragment, is undermethylated in comparison to the extensive methylation of the same fragment in Ad12-transformed hamster cells. Thus, there appears to exist a striking inverse correlation between the levels of methylation of specific DNA segments and the extent to which these segments are expressed as mRNA. The functional significance of this correlation remains to be determined. It may provide a clue to understanding the regulation of gene expression in transformed cells and perhaps in eukaryotic cells in general.
Conductimetric determination of thermodynamic pairing constants for symmetrical electrolytes
Authors: Fuoss Raymond M.
Published: May 2006

Earlier theories of electrolytic conductance are reviewed; all of these, with the exception of the Arrhenius-Ostwald theory, are based on physical models. Their theory failed to describe the conductance of strong electrolytes because it did not include the effects (then unsuspected) of long-range forces on mobility. Thermodynamic derivations are independent of model; applied to the postulated equilibrium A
+
+ B
-
⇄ A
+
B
-
between free ions and nonconducting paired ions, the thermodynamic pairing constant

K
a

equals

a
p

/(
a
±)
2
, and Δ
G
, the difference in free energy between paired ions (activity =

a
p

) and free ions (activity =
a
±
), equals (-
RT
ln

K
a

). Converting to the molarity scale,

K
a

= (1000 ρ/
M
)[1 - γ)/
cy
2
(y
±
)
2
]. Here ρ is the density of the solvent of molecular weight
M
,
c
is stoichiometric concentration of electrolyte (mol/liter), γ is the fraction of solute present as unpaired ions, and
y
±
is their activity coefficient. The corresponding conductance function Λ = Λ(
c
;Λ
0
,
R
,△
G
)involves three parameters: Λ
0
, the limiting equivalent conductance;
R
, the sum of the radii of the cospheres of the ions; and Δ
G
. Conductance data for cesium bromide and for lithium chloride in water/dioxane mixtures and for the alkali halides in water are analyzed to determine these parameters. Correlations between the values found for
R
and Δ
G
and properties characteristic of salt and solvent are then discussed.
Expression of Epstein-Barr virus genes in different cell types after microinjection of viral DNA.
Authors: Graessmann A , Wolf H , Bornkamm G W
Published: May 2006

Gene expression of Epstein-Barr virus (EBV) was studied after microinjection of viral DNA into different types of cells. Raji TK- cells, known to express viral gene functions after superinfection with the EBV-P3HR-1 virus strain, were attached to plastic dishes by using anti-lymphocyte IgG, phytohemagglutinin, or concanavalin A as a ligand. It was difficult to inject DNA into the small and fragile Raji cells. After formation of polykaryons by cell fusion, microinjection became more efficient. At 24 hr after injection of P3HR-1 virus DNA, 90-100% of the injected cells expressed the early antigen complex as observed by immunofluorescence staining; 70-80% of the cells simultaneously incorported [3H]thymidine, indicating that thymidine kinase is expressed after injection of viral DNA. Additionally, synthesis of the virus capsid antigen was demonstrated in 20-30% of the recipient Raji cells. Human diploid fibroblasts, African green monkey kidney cells, and rat fibroblasts, which do not represent natural target cells for EBV, could also be induced to synthesis of early antigen complex by injection of P3HR-1 virus DNA.
Akvr-1, a dominant murine leukemia virus restriction gene, is polymorphic in leukemia-prone wild mice.
Authors: Gardner M B , Rasheed S , Pal B K , Estes J D , O'Brien S J
Published: May 2006

We describe a restriction gene (Akvr-1, for AKR virus restriction) that is polymorphic for two alleles, Akvr-1R (restrictive) and Akvr-1r (susceptible), in a feral population of mice (Mus) musculus domesticus) at a squab farm near Lake Casitas (LC) in southern California. Akvr-1k is a dominant allele that exhibits 100% penetrance in prevention of viremia of AKR endogenous retrovirus and of virus-mediated lymphoma in LC (Akvr-1RR) X AKR F1 hybrids. The restriction phenotype segregates as a single Mendelian locus in backcrosses to AKR mice. Akvr-1R likewise is effective in restriction of NB-tropic Moloney murine leukemia virus-induced viremia and NB-tropic Friend virus-induced splenomegaly but fails to restrict expression or pathogenesis of LC-derived amphotropic retrovirus. Pleiotropic restriction of AKR, Friend, and Moloney ecotropic viruses, but not of amphotropic virus, suggests that the viral targets of Akvr-1 in the three ecotropic viruses are similar to each other and distinct from the target in the LC-amphotropic virus. The relationship of Akvr-1 to previously reported murine restriction loci Fv-1, Fv-2, and Fv-4 is discussed.
Amino acid side chain conformation in angiotensin II and analogs: Correlated results of circular dichroism and
1
H nuclear magnetic resonance
Authors: Piriou F. , Lintner K. , Fermandjian S. , Fromageot P. , Khosla M. C. , Smeby R. R. , Bumpus F. M.
Published: May 2006

[1-Sarcosine,8-isoleucine]angiotensin II (Sar-Arg-Val-Tyr-Ile-His-Pro-Ile) has been shown to be a potent antagonist of the pressor action of angiotensin II. With a view to increase half-life
in vivo
of this peptide, the amino acid residue at position 4 (tyrosine) or position 5 (isoleucine) was replaced with the corresponding
N
-methylated residue. This change drastically reduced the antagonistic properties of this analog. The present work was therefore undertaken to investigate the effect of
N
-methylation on overall conformation of these peptides and to determine the conformational requirements for maximum agonistic or antagonistic properties. Conformation studies were carried out by circular dichroism and proton nuclear magnetic resonance spectroscopy in aqueous solution as a function of pH. The results indicated that: (
i
) angiotensin II and [1-sarcosine,8-isoleucine]angiotensin II gave practically identical spectroscopic data; and (
ii
)
N
-methylation in either position 4 or position 5 resulted in remarkable changes in the peptide backbone and a severe limitation in rotational freedom of side chains in tyrosine, isoleucine, and histidine residues. However, rotational restriction of the tyrosine side chain was found to be less pronounced in [1-sarcosine,4-
N
-methyltyrosine,8-isoleucine]angiotensin II than in [1-sarcosine,5-
N
-methylisoleucine,8-isoleucine]angiotensin II. Thus, these results suggest that: (
i
) the backbone and side chain structure of a potent angiotensin II antagonist should resemble that of the hormone, angiotensin II, so that it can mimic the hormone in recognizing and binding with the receptor on the cell membrane; and (
ii
) greater impact of
N
-methylation in position 5 on the overall conformation of these peptides points to the controlling influence of position 5 (isoleucine) in aligning the residues in the central segment (tyrosine-isoleucine-histidine) of angiotensin II and its potent agonist and antagonist analogs in a nearly extended structure. Any change in this arrangement may lead to reduced biological activity.
Tyrosine hydroxylase: a substrate of cyclic AMP-dependent protein kinase.
Authors: Vulliet P R , Langan T A , Weiner N
Published: May 2006

Data demonstrating the direct phosphorylation of tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2] purified from rat pheochromocytoma by ATP, Mg2+ and cyclic AMP-dependent protein kinase catalytic subunit are presented. The incorporation of phosphate is highly correlated with the activation of the hydroxylase when either the time of preincubation or the amount of protein kinase subunit is varied. The rate of phosphorylation of tyrosine hydroylase compares favorably with that of H1 histone, a known substrate of protein kinase. Lineweaver-Burk analysis of crude or purified rat pheochromocytoma tyrosine hydroxylase activity, as a function of pterin cofactor concentration, in the absence of ATP, Mg2+, and protein kinase catalytic subunit, yields a curvilinear relationship which can be resolved into two lines, suggesting two enzyme forms with different affinities for pterin cofactor. A fraction of the hydroxylase present in the tumor exists in the activated state, presumably due to the presence of ATP and endogenous protein kinase activity. When the solubl enzyme is activated by cyclic AMP, ATP, Mg2+, and protein kinase, virtually all of the enzyme is converted to the low Km state. We conclude that tyrosine hydroxylase is a substrate of cyclic AMP-dependent protein kinase in vitro and, presumably, in vivo.
NH2-terminal amino acid sequence of human fibroblast interferon.
Authors: Stein S , Kenny C , Friesen H J , Shively J , Del Valle U , Pestka S
Published: May 2006

The purification of human fibroblast interferon by chromatography on Blue Sepharose and high-performance liquid chromatography is described. The amino acid composition and a partial sequence of the homogeneous protein are reported. The NH2 terminus was determined to be NH2-Met1-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-Phe-Gln-X-Gln-Lys.
Immunological identification of the human erythrocyte glucose transporter.
Authors: Sogin D C , Hinkle P C
Published: May 2006

A rabbit antibody against the human erythrocyte glucose transporter was purified by affinity chromatography and used to determine the distribution of transporter on polyacrylamide gels after electrophoresis in sodium dodecyl sulfate. Fresh erythrocyte ghosts showed transporter only at the broad 55,000 Mr band, as did the isolated transporter. HeLa cell plasma membranes showed a similar band of crossreacting material at Mr 55,000. The amount of crossreacting material in human erythrocyte ghosts and in plasma membranes from human HeLa cells and mouse L-1210 cells was determined in an enzyme-linked immunosorbent assay which gave results consistent with the extent of glucose-reversible binding of cytochalasin B.
Studies on the active center of D- and L-lactate dehydrogenases using oxamate-diaminohexyl-Sepharose affinity chromatography.
Authors: Tuengler P , Stein T N , Long G L
Published: May 2006

Vertebrate and invertebrate L-lactate dehydrogenases (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) are effectively bound to oxamate-diaminohexyl-Sepharose, whereas several D-lactate dehydrogenases (D-lactate:NAD+ oxidoreductase, EC 1.1.1.28) do not bind to the same Sepharose. One explanation for our findings is that the enzymes' substrate is oriented in a reversed manner in the active center of the D- and L-lactate dehydrogenases.
Inheritance of chloroplast DNA in
Chlamydomonas reinhardtii
Authors: Grant David M. , Gillham Nicholas W. , Boynton John E.
Published: May 2006

Two symmetrically located deletions of approximately 100 base pairs each have been identified in chloroplast DNA of
Chlamydomonas reinhardtii
. Although present in a mutant strain that requires acetate for growth, both deletions have been shown to be distinct from the nonphotosynthetic phenotype of this strain. These physical markers in the chloroplast genome and maternally inherited genetic markers showed strict cotransmission in reciprocal crosses. Thus, our results are consistent with the location of the well-characterized maternally inherited genetic markers in chloroplast DNA of
C. reinhardtii
.
A defect in the structure of type I procollagen in a patient who had osteogenesis imperfecta: excess mannose in the COOH-terminal propeptide.
Authors: Peltonen L , Palotie A , Prockop D J
Published: May 2006

Fibroblasts from normal human subjects and from a patient who had osteogenesis imperfecta were incubated with [3H]mannose, and types I and III procollagens were isolated from the culture medium. The type I procollagen from the patient's fibroblasts contained 2-3 time more [3H]mannose than the type I procollagen from the normal fibroblasts. In contrast, there was no difference in the [3H]mannose content of the type III procollagen simultaneously synthesized and secreted by the same cells. Isolation of a collagenase-resistant peptide fragment from the type I procollagen showed that the excess mannose was located in the COOH-terminal propeptide of the protein. Radioimmunoassays of the medium and the cell layer showed that the type I procollagen synthesized by the patient's fibroblasts was secreted into the medium more slowly than the type I procollagen synthesized by normal fibroblasts. These results appear to provide evidence for an alteration in the structure of procollagen in osteogenesis imperfecta.
Immunoreactive dynorphin in pituitary and brain.
Authors: Goldstein A , Ghazarossian V E
Published: May 2006

Distribution of the potent opioid peptide dynorphin has been determined in pituitary gland (pig, beef, rat), in the various regions of rat brain, and in rat spinal cord, by using a highly specific antiserum. By gel permeation chromatography in 4 M guanidine, the porcine pituitary immunoreactivity is found in a major peak of apparent molecular weight about 1700 and a minor peak of about 3400. Similar peaks are found in rat pituitary extracts, whereas rat brain contains, in addition, two peaks of larger apparent molecular weight. In the pituitary, immunoreactive dynorphin is found predominantly in pars nervosa. In the central nervous system, it is distributed widely, with highest concentrations in hypothalamus, medulla-pons, midbrain, and spinal cord. Although dynorphin contains leucine-enkephalin, the regional distribution of dynorphin is different from that of enkephalin or of any other known opioid peptide.
Proteolytic inactivation of luciferases from three species of luminous marine bacteria, Beneckea harveyi, Photobacterium fischeri, and Photobacterium phosphoreum: evidence of a conserved structural feature.
Authors: Holzman T F , Baldwin T O
Published: May 2006

Upon limited proteolysis of luciferases from the luminous marine bacteria Photobacterium fischeri, Photobacterium phosphoreum, and Beneckea harveyi, the rate of loss of luciferase activity is the same as the rate of loss of the heavier subunit of all three enzymes. It thus appears that the larger subunit of the luciferase from P. phosphoreum should be designated alpha based on its apparent homology with the alpha subunits of the luciferases from B. harveyi and P. fischeri. The luciferase from B. harveyi is more sensitive to chymotrypsin than to trypsin; the luciferases of the Photobacterium species are more sensitive to trypsin than to chymotrypsin. Proteolytic inactivation of all three luciferases results from hydrolysis of a few peptide bonds in the alpha subunit; the proteolytic fragments from the three luciferases in 0.50 M phosphate are approximately the same size, indicating that the three enzymes have a protease-labile region at about the same position in the primary structure of their alpha subunits. Phosphate stabilizes all three luciferases against inactivation by proteases. Formation and degradation of intermediate species derived from the alpha subunits are readily observable in all three luciferases. Phosphate alters both the rate of product formation and the sites of peptide bond scission. The beta subunits of the luciferases from the two Photobacterium species, unlike the enzyme of B. harveyi, appear to be degraded in buffers containing low concentrations of phosphate; in high-phosphate buffers, the beta subunits of all three luciferases appear to resist proteases. Analysis of native and chymotrypsin-inactivated P. fischeri and P. phosphoreum luciferases in the analytical ultracentrifuge indicates that, as with B. harveyi luciferase, the products of limited proteolysis do not dissociate under nondenaturing conditions. The fact that the luciferases from evolutionarily diverse species of luminous bacteria have protease-sensitive bonds in the same region of the alpha subunit that are stabilized by anions strongly suggests that the protease-labile region of the alpha subunit is either an integral component of or in close proximity to the active center.
In vitro comparison of initiation properties of bacteriophage lambda wild-type PR and x3 mutant promoters.
Authors: Hawley D K , McClure W R
Published: May 2006

The in vitro initiation properties of the PR promoter of bacteriophage lambda and of a PR mutant, x3, were compared. Using the abortive initiation reaction, we measured the lags in the approach to a final steady-state rate when dinucleotide synthesis was initiated with RNA polymerase. These lags corresponded to the average times required for the formation of transcriptionally active open complexes. By measuring the lags at different RNA polymerase concentrations, we could separate open complex formation into two steps, based on a simple model in which the initial bimolecular association of free promoter and polymerase in a closed complex is followed by an isomerization to the open complex. The contribution of each step to the overall rate of open complex formation was quantitated for both promoters. We found that the x3 mutation, which is located in the -35 region of PR, resulted in a decrease in the association constant for the initial binding to the closed complex to 5% of its wild-type value and a decrease in the rate of the isomerization to 20%. The lifetimes and abortive initiation characteristics of the mutant and wild-type promoters were similar. We concluded that the main effect of the x3 mutation was to increase the average time of open complex formation and that the functional properties of the open complexes did not differ significantly between the two promoters.
A highly polymorphic locus in human DNA.
Authors: Wyman A R , White R
Published: May 2006

A locus in the human genome, not associated with any specific gene, has been found to be a site of restriction fragment length polymorphism. The polymorphism was found by hybridizing a 16-kilobase-pair segment of single-copy human DNA, selected from the human genome library cloned in phage lambda CH4A, to a Southern transfer of total human DNA digested with EcoRI. DNAs from a number of individuals from within Mormon pedigrees as well as random individuals have been examined. The locus is highly variable, with at least eight alleles present, homozygotes accounting for less than 25% of the individuals examined. The polymorphism appears to be the result of DNA rearrangements rather than base-pair substitutions or modifications. Examination of the DNA from seven members of a family revealed fragment lengths that are consistent with their inheritance as Mendelian alleles through three generations.
Isolation and characterization of the amyloid-related apoprotein (SAA) from human high density lipoprotein.
Authors: Eriksen N , Benditt E P
Published: May 2006

Two apoproteins immunologically related to the 9000-dalton abnormal tissue constituent known as amyloid protein AA were isolated from the lipoprotein density interval 1.125-1.21 g/cm3 (HDL3) of a pool of human serums by delipidation, gel filtration, and ion-exchange chromatography. Lesser amounts of the same apoproteins were isolated from the density interval 1.063-1.125 g/cm3 (HDL2). These apoproteins, designated apoSAA1 and apoSAA2, have molecular weights near 11,500, almost identical amino acid compositions, and slightly different isoelectric points. Their amino acid sequences are identical as far as determined (30 residues), except that apoSAA2 lacks the NH2-terminal arginine found in apoSAA1. The sequence is homologous with that of amyloid protein AA, which thus has residing in the plasma high density lipoproteins a potential precursor whose biological significance and function remain to be determined.
Specific in vitro transcription of conalbumin gene is drastically decreased by single-point mutation in T-A-T-A box homology sequence.
Authors: Wasylyk B , Derbyshire R , Guy A , Molko D , Roget A , Téoule R , Chambon P
Published: May 2006

A single-point mutation, consisting of a T-to-G transversion, was made in the third nucleotide of the conalbumin gene T-A-T-A-A-A-A homology sequence (the T-A-T-A or Goldberg-Hogness box). In an in vitro system, specific transcription of the mutant DNA was drastically decreased compared to the normal gene. This down-mutation is consistent with the idea that the T-A-T-A box is an important element for specific initiation of transcription.
Enhancement of N-methyl-N'-nitro-N-nitrosoguanidine transformation of Syrian hamster cells by a phorbol diester is independent of sister chromatid exchanges and chromosome aberrations.
Authors: Popescu N C , Amsbaugh S C , DiPaolo J A
Published: May 2006

12-O-Tetradecanoylphorbol 13-acetate (TPA), a known tumor promoter, enhances the morphologic transformation of Syrian hamster embryo cells induced by low transforming concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0.025-0.1 microgram/ml) without potentiation of cell lethality or of changes in sister chromatid exchanges (SCEs) or chromosomal aberration frequencies. When MNNG was added to logarithmically growing cultures and TPA was added 2, 24, or 48 hr later, no changes in SCE frequency relative to MNNG alone occurred. Similar results were obtained with TPA in cells that had been exposed to MNNG in stationary growth phase. Whereas no transformation occurred with TPA alone and pretreatment with TPA did not affect MNNG transformation, its addition 2 hr after MNNG reduced transformation frequency but addition 24-72 hr after MNNG increased the transformation frequency up to 6-fold. TPA had a minimal effect on increasing the transformation frequency (2-fold) induced by MNNG at 0.25 micrograms/ml, a high concentration. Of three polycyclic hydrocarbons, perylene, benzo[g,h,i]perylene, and benz[a]anthracene, known as weak or noncarcinogens, only benz[a]anthracene induced a very low transformation frequency; however, after TPA, transformation occurred with all three. Because the number of cells whose transformation was initiated by low doses of carcinogen is much larger than the number of cells giving rise to transformed colonies in the absence of TPA, the frequency of the initial event is greater than can be expected from point mutations. Furthermore, the promotional aspect of transformation is not accompanied by a parallel increase in SCE.
Cell cycle and the differential expression of HLA-A,B and HLA-DR antigens on human B lymphoid cells.
Authors: Sarkar S , Glassy M C , Ferrone S , Jones O W
Published: May 2006

Monoclonal antibodies specific to HLA antigens and the fluorescence-activated cell sorter were used to analyze the changes in the density of human histocompatibility antigens HLA-A,B and HLA-DR on the surface of synchronously growing WI-L2 cells (a human B cell line) progressing through the cell cycle. The WI-L2 cells were synchronized by density-dependent arrest in G1, and samples from G0, G1, late S and late G2 phases were used to determine the frequency distribution of cell volume, DNA content, and the relative amounts of cell surface HLA antigens; the observed density changes were calculated from these values. The HLA-A,B density remained nearly constant throughout the cell cycle, whereas the HLA-DR density increased sharply at the G2-M stage. These results suggest a cell cycle-dependent differential control of the expression of these two sets of histocompatibility antigens on B cells.
Reversible growth arrest in simian virus 40-transformed human fibroblasts.
Authors: Hoffman R M , Jacobsen S J
Published: May 2006

A reversible growth arrest of simian virus 40-transformed human fibroblasts has been produced by replacement of methionine in the growth medium by its immediate metabolic precursor, homocysteine, Although these arrested cells exhibit a greatly reduced cloning efficiency when plated in methionine-supplemented medium, they resume rapid proliferation without a lag when subconfluent cells are refed with methionine-supplemented medium. This growth arrest is accompanied by a reduction in the percentage of mitotic cells in the cell population. Furthermore, data obtained using fluorescence-activated cell sorting techniques indicate that the cells are arrested i the S and G2 phases of the cell cycle. This is in contrast to a G1-phase accumulation of cells, which occurs only in methionine-supplemented medium at very high densities and which is similar to the G1 block seen in cultures of normal fibroblasts at high density. The apparent relationship between specific events in the DNA-synthetic and premitotic phase of the cell cycle and methionine dependence in these transformed cultures is discussed.
Barbiturate receptor sites are coupled to benzodiazepine receptors.
Authors: Leeb-Lundberg F , Snowman A , Olsen R W
Published: May 2006

Barbiturates enhance the binding of [3H]diazepam to benzodiazepine receptor sites in rat brain. This effect occurs at pharmacologically relevant concentrations of barbiturates, and the relative activity of a series of compounds correlates highly with anesthetic activity of the barbiturates and with their ability to enhance postsynaptic inhibitory responses to the neurotransmitter gamma-aminobutyric acid. Barbiturate enhancement of benzodiazepine binding is stereospecific, with the more active anesthetic isomers of N1-methylbarbiturates being also more active than their stereoisomers in enhancing benzodiazepine binding. The active barbiturates produce a reversible enhancement in the affinity of specific benzodiazepine binding with no effect on the number of binding sites. The barbiturate enhancement, but not the baseline benzodiazepine binding, is competitively inhibited by the convulsant picrotoxinin (at 1-10 microM), a drug that has been shown to label barbiturate-sensitive brain membrane sites related to the gamma-aminobutyric acid receptor-ionophore complex. The barbiturate effect is also dependent upon the presence of certain anions, and only those anions, that penetrate the chloride channels regulated by gamma-aminobutyric acid receptors. These results suggest that picrotoxin-sensitive barbiturate binding sites are coupled to benzodiazepine receptors in the gamma-aminobutyric acid receptor-ionophore complex, and that these binding sites have the properties of pharmacologically relevant receptors that mediate at least part of the action of various nervous system depressant and excitatory drugs.
Amplification of genes for chorion proteins during oogenesis in
Drosophila melanogaster
Authors: Spradling Allan C. , Mahowald Anthony P.
Published: May 2006

The endochorion and exochorion of
Drosophila
eggs are synthesized by the ovarian follicle cells during a brief period of about 5 hr. In this terminal phase of egg chamber development, the structural genes for several abundant chorion proteins are expressed at high levels according to a temporally regulated program. The female-sterile mutation
ocelliless
maps at the site of the genes for two of these proteins, the 36,000- and 38,000-dalton chorion proteins (c36 and c38), which are closely linked. The mutation results in a
cis
-acting reduction in the amounts of c36 and c38 that accumulate in late-stage egg chambers. We have investigated the mechanism that underlies this decreased production by using cDNA clones complementary to these gene sequences. Unexpectedly, it was found that, in normal females, the genes for c36, c38, and at least one other chorion protein are specifically amplified more than 10-fold in the DNA of late-stage egg chambers. The extra replication involves at least some adjacent chromosomal sequences and begins prior to the onset of mRNA and protein synthesis. The additional DNA remains stable after gene expression has ceased. The behavior of these genes is thus reminiscent of the properties of the DNA puffs that have been described in several groups of Diptera. The extent of amplification of c36 and c38, but not of the 18,000-dalton chorion protein c18 (which is unlinked), was decreased in the egg chambers of flies homozygous for
ocelliless
, suggesting that altered gene dosage may be responsible for the decreased synthesis of chorion proteins in the mutant.
Free radicals and inflammation: superoxide-dependent activation of a neutrophil chemotactic factor in plasma.
Authors: Petrone W F , English D K , Wong K , McCord J M
Published: May 2006

The intravenous administration of superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) to animals with induced inflammation suppresses the inflammatory response and inhibits leukocyte infiltration into the challenged site, suggesting that neutrophil-generated superoxide reacts with an extracellular precursor to generate a substance chemotactic for neutrophils. Plasma exposed to superoxide in vitro becomes potently chemotactic. The appearance of chemotactic activity is inhibited by superoxide dismutase but not by catalase. The chemotactic factor does not stimulate superoxide production or degranulation in neurtrophils. Intradermal injection of superoxide-treated plasma or of a superoxide-generating system causes heavy infiltration of neutrophils to the injection site but does not cause overt signs of inflammation. The chemotactic factor consists of a chloroform-extractable component bound to serum albumin. The superoxide-dependent chemotactic factor appears to play a major role in communication in neutrophil-mediated inflammatory events. Prevention of production of this factor appears to be the major anti-inflammatory action of superoxide dismutase.
Clumping of acetylcholinesterase activity in the developing striatum of the human fetus and young infant
Authors: Graybiel Ann M. , Ragsdale Clifton W.
Published: May 2006

The distribution of acetylcholinesterase activity (acetylcholine acetylhydrolase, EC 3.1.1.7) in the developing human striatum has been studied histochemically in autopsy material from fetal brains of estimated gestational ages 16-29 weeks (180-1000 g) and from the brains of infants 2 days to 4 months old. The findings provide evidence that striatal acetylcholinesterase activity in the human fetus and neonate is concentrated in a network of densely stained zones that appear in cross section as variably shaped 0.5- to 1.0-mm-wide dark patches distributed in a lighter background matrix. An orderly arrangement of the patches seemed well established in the putamen by the 16th-18th week of gestation (crown-rump length 14-15 cm) but in the caudate nucleus the pattern was still not fully elaborated at these ages. The lateroventral part of the caput was mainly dark and its rostromedial margin, though rich early on in pseudocholinesterase activity, was still without strong acetylcholinesterase activity as late as 20-21 weeks (crown-rump length 16-20 cm). The ganglionic eminence at these ages was sharply divided into a dorsal part with little cholinesterase activity and a ventral part with a high content of pseudocholinesterase. Little information was gained about striatal development during late stages of gestation, but in three 5- to 7-month fetal specimens not only dark patches but also patches with dark perimeters and pale centers were present. Clumping of cholinesterase activity appeared at birth and up to the third month of postnatal life. The patches in both caudate nucleus and putamen were dark and of fairly uniform tint in the striatum of the young infant and the matrix staining was darker than in the fetuses. Around the fourth postnatal month hints of the mature pattern were present, with zones of low cholinesterase activity appearing against a dark background in the caudate nucleus and (in one case) a nearly homogeneous staining pattern appearing in the putamen.
Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits.
Authors: Tzartos S J , Lindstrom J M
Published: May 2006

Seventeen cell lines producing monoclonal antibodies against Torpedo californica (torpedo) acetylcholine receptor (AcChoR) and its subunits were established. By using these antibodies as probes, we identified: (i) a similar antigenic determinant on alpha and beta torpedo subunits, (ii) a similar antigenic determinant on gamma and delta subunits, (iii) antigenic determinants unique for alpha or beta torpedo AcChoR subunits, (iv) a small region on the alpha subunit that dominates the immunogenicity of native torpedo AcChoR in rats (a monoclonal antibody directed at this region could bind to rat AcChoR in vivo and cause passive experimental autoimmune myasthenia gravis), and (v) antigenic determinants on torpedo subunits recognized in AcChoR from other species. The unexpected similarities between alpha and beta and between gamma and delta subunits raise the possibility that the complex four-subunit structure of AcChoR was derived from a simpler precursor and suggests that these antigenic similarities might reflect some structural and functional homologies.
Presence of cobalamin analogues in animal tissues
Authors: Kondo Haruki , Kolhouse Fred , Allen Robert H.
Published: May 2006

Cobalamin (Cbl, vitamin B-12) has been extracted and isolated from a number of animal tissues by using (
i
) reverse-affinity chromatography on R protein-Sepharose followed by adsorption to and elution from charcoal-coated agarose and (
ii
) paper chromatography. Radioisotope dilution assays showed that only 75-97% of the Cbl chromatographed in the position of crystalline Cbl. The remaining 3-25% was present in a number of slower and faster moving fractions. This suggested that Cbl analogues are present in animal tissues because appropriate controls ruled out the possibility that this material was artifactually derived from Cbl during the extraction and purification procedures. With a large-scale isolation from rabbit kidney, the material in five such fractions contained cobalt and had absorption spectra that were similar to but different from the spectrum of Cbl, indicating that they were Cbl analogues. Compared to Cbl, these Cbl analogues had decreased but definite affinities for Cbl-binding proteins with the following order of strength of binding: R protein > transcobalamin II > intrinsic factor. Compared to Cbl, they also had decreased but definite growth-promoting activity for two microorganisms,
Euglena gracilis
and
Lactobacillus leichmannii
, which require Cbl for growth. These Cbl analogues differed from each other and from 18 synthetic Cbl analogues, including the most common Cbl analogues synthesized by microorganisms, in at least one of the above features. These studies indicate that animal tissues contain a number of Cbl analogues whose origins, structures, and biologic activities remain to be determined.
Crystallization of part of the mitochondrial electron transfer chain: cytochrome c oxidase--cytochrome c complex.
Authors: Ozawa T , Suzuki H , Tanaka M
Published: May 2006

Cytochrome c oxidase (ferrocytochrome c oxygen oxidoreductase, EC 1.9.3.1) was purified from beef heart mitochondria by affinity chromatography. Phospholipids were removed by washing the oxidase with detergent on the affinity column; 1 mole of cardiolipin remained per mole of heme a. The oxidase was mixed with excess cytochrome c in 1.5% (wt/vol) cholate to form a complex. Slow removal of the detergent from the mixture by dialysis resulted in crystallization of cytochrome oxidase in the form of a 1:1 complex with cytochrome c. The chemical composition and spectrophotometric properties of the crystal are described. Increasing the solubility of a hydrophobic membrane protein by combination with hydropholic ligand is demonstrated as a maneuver for crystallizing the membrane protein.
There is a correlation between the DNA affinity and mutagenicity of several 3-amino-1-methyl-5H-pyrido[4,3-b]indoles.
Authors: Pezzuto J M , Lau P P , Luh Y , Moore P D , Wogan G N , Hecht S M
Published: May 2006

3-Amino-1-methyl-5H-pyrido[4,3-b]indole, previously reported to be a component of tryptophan pyrolysates, is an intensely mutagenic compound requiring microsomal activation for expression of mutagenicity. We have found that this species and several synthetic analogs interact noncovalently with calf thymus DNA, as judged by both fluorescence quenching and differential dialysis. Remarkably, this noncovalent interaction correlated with the mutagenic potential of the compounds in a bacterial mutagenesis assay. Therefore, it is suggested that the mechanism of mutagenesis involves metabolic activation followed by physicochemical interaction with DNA; for these compounds, the latter step may be limiting for the expression of mutagenicity.
Isolation of two closely related vitellogenin genes, including their flanking regions, from a Xenopus laevis gene library.
Authors: Wahli W , Dawid I B
Published: May 2006

A gene library of Xenopus laevis was constructed from embryonic DNA partially digested with restriction endonucleases Hae III and Alu I and joined to the phage lambda Charon 4 cloning vector with EcoRI linkers. Nucleotide sequences from three of the four related vitellogenin genes have been isolated. Two of the genes (called A1 and A2) were isolated in their entirety together with long stretches of flanking sequences. These two closely related vitellogenin genes have lengths of about 21 and 16 kilobases, but both produce a vitellogenin mRNA of 6.3 kilobases.
Synthesis and maturation of lambda receptor in Escherichia coli K-12: in vivo and in vitro expression of gene lamB under lac promoter control.
Authors: Marchal C , Perrin D , Hedgpeth J , Hofnung M
Published: May 2006

The lambda receptor is an outer membrane protein from Escherichia coli K-12 lamB, its structural gene, is part of the maltose regulon. We have cloned this gene in a phage so that it is under the control of the lac promoter. The phage was devised in such a way that it can infect lamB mutants and that chromosomal lamB mutations can be transferred to it. In vivo, the lambda receptor is expressed under lac promoter control and is exported normally to the outer membrane, independently of the expression of the other genes of the maltose regulon. In vitro, DNA of the phage allows efficient synthesis of the lamB product. The protein--or pre-lambda-receptor--made in vitro contains an NH2-terminal sequence of about 25 amino acids not found in the lambda receptor. We have detected no inactivation of phage lambda by the pre-lambda-receptor. Conversion of the pre-lambda-receptor to a form that has the apparent molecular weight of the mature lambda receptor was achieved. A lamB mutation that blocks export in vivo also blocks conversion in vitro.
A general method, employing arsenazo III in liposomes, for study of calcium ionophores: results with A23187 and prostaglandins.
Authors: Weissmann G , Anderson P , Serhan C , Samuelsson E , Goodman E
Published: May 2006

Multilamellar (MLV) and large unilamellar (LUV) lipid vesicles (liposomes) trap the metallochromic dye arsenazo III [2,7-bis(arsonophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid ] in their aqueous compartments. When ionophore A23187 was preincorporated into either MLV or LUV above 0.001 mol%, addition of Ca to the outside of liposomes produced spectral shifts characteristic of the Ca . AIII2 complex. The method permitted detection of two molecules of A23187 per liposome. Liposomes with A23187 were permselective: divalent cations were translocated in the order Mn greater than Ca greater than Sr greater than Mg congruent to Ba. Because prostaglandins (PGs) may act as Ca ionophores, we have incorporated into MLVs and LUVs stable prostaglandins (PGE2, PGI2, PGB1), endoperoxide analogs, and a water-soluble, polymeric derivative of PGB1:PGBx. None acted as ionophore. In contrast, when added to the outside of preformed MLV or LUV, PGBx, at concentrations above 1 micro M, provoked permselective uptake of Ca equivalent to that induced by 10 nM A23187. These studies demonstrate not only that liposomes containing arsenazo III may be employed in a sensitive asssay for agents that translocate divalent cations, but that a water-soluble derivative of a naturally occurring fatty acid, PGBx, is a potent ionophore.