bluuebunny/crossref_metadata_2025 huggingface dataset:
Resolution of products of the duplicated hemoglobin alpha-chain loci by isoelectric focusing.
Authors: Whitney J B , Copland G T , Skow L C , Russell E S
Published: May 2006

Using a high-resolution isoelectric focusing system, we have been able to separate the products of the individual "nonallelic+ mouse hemoglobin alpha-chain genes. The hemoglobins of mice of inbred strains that make two structurally different, but electrophoretically identical, alpha chains resolve into two bands on isoelectric focusing. The hemoglobin from other strains that make only one type of alpha chain forms a single band. Two new "alleles" of haplotypes of Hba (the alpha-chain complex locus) are described that code for hemoglobins with solubilities similar to that of the hemoglobin of strain C57BL/6J, but that give demonstrably different isoelectric focusing patterns. Because this method allows Hba typing of strains with the Hbbd or "diffuse" allele at the hemoglobin beta-chain locus, we have also been able to classify a number of previously untyped stocks, including some recently derived from feral populations. Among these mice we have found new representatives of most of the extant Hba genotypes. The demonstration that it is possible to detect genetic differences involving only neutral amino acid substitutions between proteins suggest that careful application of appropriate isoelectric focusing systems to the analysis of other proteins will greatly increase the sensitivity of our ability to detect genetic variation.
Maximum geographic range of a mutant allele considered as a subtype of a Brownian branching random field
Authors: Sawyer Stanley , Fleischman Joseph
Published: May 2006

A rare allelic type is modeled as a field of individuals diffusing independently in
d
-dimensional space (
d
= 1,2,...), in which individuals are replaced by random numbers of offspring at a constant rate. In an infinite-allele model with selectively identical alleles, the offspring distribution would have mean 1 —
u
, in which
u
is the mutation rate; otherwise mean (
1
—
u
)
w
, in which
w
is the relative fitness of the allele. Let
p
(
x
) be the probability that some descendent of an individual initially at zero diffuses unilaterally as far as
x
(
d
= 1), or else the probability that some descendent of an individual at
x
diffuses within
a
> 0 of the origin (
d
≥ 2). A nonlinear differential equation is found for
p
(
x
) that is solvable for
d
= 1 and related to Emden's equation for
d
≥ 2. For
p
(
x
) ≥ 10
-4
and
u
≤ 10
-5
, genetic drift is more important than mutation in the behavior of
p
(
x
) (
d
= 1). If
u
= 0 and
w
= 1,
p
(
x
) ∼
C
/
x
2
as
x
→ ∞ for
d
≤ 3. As a mathematical application, it is shown that if the initial distribution is uniform Poisson, a bounded open set
K
is visited by individuals in the field at arbitrarily large times if
d
≥ 2 but not if
d
= 1.
Comparison of structure and function of human erythrocyte and human muscle actin.
Authors: Nakashima K , Beutler E
Published: May 2006

Human erythrocyte actin and human skeletal muscle actin were purified by acetone powder extraction and gel filtration. Pure human erythrocyte actin resembles muscle actin in its polymerization and depolymerization by phalloidin, cytochalasin B, and DNase I, in its peptide mapping pattern, and in the amino acid composition of corresponding peptides. Isoelectric focusing gel analysis showed that human erythrocyte actin exists in the beta/gamma form, but muscle actin is in the alpha form. Abnormal deformability of resealed erythrocyte membranes was observed after incorporation of the actin-specific agents, phalloidin and DNase I, suggesting that erythrocyte actin might function as a membrane structural element to maintain erythrocyte membrane deformability.
Quantitative studies of ferritinlike iron in erythrocytes of thalassemia, sickle-cell anemia, and hemoglobin Hammersmith with Mössbauer spectroscopy.
Authors: Bauminger E R , Cohen S G , Ofer S , Rachmilewitz E A
Published: May 2006

By using the technique of recoil-free absorption (Mössbauer effect) in iron, we found large amounts of iron, yielding a well-defined spectrum different from that of oxy- or deoxyhemoglobin, in whole erythrocytes of 13 patients with beta-thalassemia major and intermedia, 3 with hemoglobin H disease, 2 with sickle-cell anemia, and 1 with unstable hemoglobin Hammersmith. The Mössbauer spectra at various temperatures of this additional component of iron were found to be identical to spectra obtained from isolated ferritin or hemosiderin. This observation, together with additional arguments, strongly suggests that the compound responsible for the additional subspectrum is an iron storage protein, ferritin or hemosiderin. The amounts of ferritinlike iron were comparable to those of hemoglobin iron and were particularly large in reticulocytes. No ferritinlike iron was detected in patients with severe autoimmune hemolytic anemia and pernicious anemia. The large quantities of ferritinlike iron in hemoglobinopathies are probably due to intracellular hemoglobin denaturation and the consequent release of excess iron.
Identification of inosine and hypoxanthine as endogenous ligands for the brain benzodiazepine-binding sites.
Authors: Asano T , Spector S
Published: May 2006

Two endogenous ligands for the brain benzodiazepine-binding sites were isolated from bovine brain through gel filtration, paper electrophoresis, and paper chromatography. These ligands were identified as inosine and hypoxanthine, and both had a higher affinity for the brain benzodiazepine-binding sites than for benzodiazepine sites in some peripheral tissues. They did not bind to any other receptors tested, such as the opiate, muscarinic cholinergic, gamma-aminobutyric acid, and beta-adrenergic receptors. Both inosine and hypoxanthine competitively inhibited the binding of [3H]diazepam to the brain binding site.
High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules.
Authors: Struhl K , Stinchcomb D T , Scherer S , Davis R W
Published: May 2006

A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, three modes of yeast transformation are defined. (i) Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5) transform yeast cells at low frequency (1--10 colonies per microgram) and integrate into the genome by homologous recombination; this recombination is reversible. (ii) Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6) transform yeast cells at much higher frequency (5000--20,000 colonies per microgram). Such molecules replicate autonomously with an average copy number of 5--10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. (iii) Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7) transform yeast with an efficiency of 500--5000 colonies per microgram; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The uses of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events are presented.
Asymmetric lateral distribution of unshielded phosphate groups in nucleosomal DNA and its role in DNA bending.
Authors: Mirzabekov A D , Rich A
Published: May 2006

We suggest that an asymmetric charge neutralization of DNA phosphate groups produces part of the driving force for nucleosome folding. In nucleosome core particle DNA, many of the phosphate groups are neutralized by histones, and a lateral alignment of these histones along the core DNA has been demonstrated [Mirzabekov A. D., Shick, V. V., Belyavsky, A. V. & Bavykin, S. G. (1978) Proc. Natl. Acad. Sci. USA 75, 4184--4189]. Histones appear to shield DNA phosphatases asymmetrically at one side of the surface of the DNA double helix along all its length inside the core. The external side of the DNA helix remains unneutralized. The electrostatic repulsion between negatively charged unneutralized phosphates may fold the nucleosomal DNA towards the side occupied by histones.
Specialized transducing phage lambda carrying the genes for coupling factor of oxidative phosphorylation of Escherichia coli: increased synthesis of coupling factor on induction of prophage lambda asn.
Authors: Kanazawa H , Miki T , Tamura F , Yura T , Futai M
Published: May 2006

Studies were made of the synthesis of the coupling factor complex (F1--F0) of oxidative phosphorylation after prophage induction of a set of Escherichia coli strains lysogenic for defective transducing phage lambda asn, lambda uncA, or lambda bglC. The transducing phages had been isolated from a strain of E. coli carrying prophage lambda cI857 S7 within the bglB gene located near the unc gene cluster [Miki, T., Hiraga, S., Nagata, T. & Yura, T. (1978) Proc. Natl. Acad. Sci. USA 75, 5099--5103]. When lysogenic cells carrying lambda asn and lambda cI857 S7 were induced at high temperature, synthesis of the F1-ATPase portion of the complex increased to severalfold that of the noninduced cells. In contrast, no increase was observed upon thermoinduction of cells carrying lambda uncA or lambda bglC. The number of membrane sites that could bind purified F1-ATPase also increased significantly upon induction by lambda asn but not by lambda uncA or lambda bglC. In addition, F1-depleted membranes prepared from lambda asn-induced bacteria required more dicyclohexylcarbodiimide to seal the proton pathway than did those from noninduced bacteria. These results strongly suggest that lambda asn carries a set of bacterial genes coding for all the F1 polypeptides (the alpha, beta, gamma, delta, and probably the epsilon subunits) and at least some of the genes involved in formation of F0 polypeptides. Although lambda uncA carries the structural gene (uncA) for the alpha subunit of F1-ATPase, it apparently does not carry the whole set of F1--F0 genes.
Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane.
Authors: Yankner B A , Shooter E M
Published: May 2006

Cells of a continuous line of rat pheochromocytoma (PC12) were incubated with 125I-labeled beta nerve growth factor (beta NGF), and at given time intervals the cell nuclei were isolated by a procedure that used the detergent Triton X-100. NGF was detectable in the nucleus after 20 min and continued to accumulate in a linear fashion for several hours after the total binding to the cell had reached steady state. After 17 hr at 37 degrees C, about 60% of the NGF bound to the cell was in the nucleus, NGF was not translocated to the nucleus at 4 degrees C. When nuclei were purified from PC12 cells and incubated with 125I-labeled beta NGF, specific binding sites were found. Binding was saturable and consistent with two sites: a high-affinity site with a Kd of 0.08 nM (+/- nM) and a lower-affinity site with a Kd of 9.0 nM (+/- 2.0 nM). The receptors in the nucleus were shown to be localized to the nuclear membrane. Membrane-free chromatin did not bind NGF specifically. The translocation of NGF to the nucleus was accompanied by a commensurate decrease in the cell-surface binding capacity. In the nucleus, however, the receptor capacities of both sites were increased when PC12 cells were grown in the presence of NGF.
Reversion to methionine independence in simian virus 40-transformed human and malignant rat fibroblasts is associated with altered ploidy and altered properties of transformation
Authors: Hoffman Robert M. , Jacobsen Stephen J. , Erbe Richard W.
Published: May 2006

Many transformed and malignant cells, unlike normal cells, do not grow when methionine in the growth medium is replaced by its immediate precursor homocysteine [Chello, P. & Bertino, J. (1973)
Cancer Res
. 33, 1898-1904 and Hoffman, R. & Erbe, R. (1976)
Proc. Natl. Acad. Sci. USA
73, 1523-1527]. Rare cells from those populations revert to methionine independence [Hoffman, R., Jacobsen, S. & Erbe, R. (1978)
Biochem. Biophys. Res. Commun
. 82, 228-234]. We report here that methionine-independent revertants of both human fibroblasts transformed by simian virus 40 and malignant rat fibroblasts concomitantly revert for some of the properties associated with the transformed state. Of the 13 methionine-independent revertants described here, 5 showed increased anchorage dependence as reflected by reduced cloning efficiences in methylcellulose; 8 showed an increased serum requirement for optimal growth; 8 showed decreased cell density in medium containing high serum; and 3 altered their cell morphology significantly. Eight of the 13 have increased chromosome numbers. All lines tested contained immunologically identifiable tumor antigen of simian virus 40. Thus by selecting for methionine independence it is possible to select for heterogeneous transformation revertants, indicating further a relationship between altered methionine metabolism and oncogenic transformation. Therefore a positive metabolic method to select for transformation revertants has been developed, and its use has resulted in selection of human transformation revertants.
Allelic pairing and gene regulation: A model for the zeste—white interaction in
Drosophila melanogaster
Authors: Jack Joseph W. , Judd Burke H.
Published: May 2006

Data are presented that indicate that the
z
locus of
Drosophila melanogaster
represses
w
locus activity, but the repression is effective only on paired or physically adjacent
w
loci. Various mutant alleles of
z
and
w
were combined in a series of different doses to determine the effect of dosage and physical position in the nucleus on gene expression. In
z/z
individuals, paired white alleles fail to be expressed, while unpaired alleles are expressed normally. The results are discussed in terms of a model postulating that the
z
gene product represses the transcription of
w
+
by complexing with an RNA produced by part of the white locus itself. In order to be effective in repression, there must be two
w
+
genes producing the RNA in a limited volume in the nucleus. Such a model necessarily imposes a specific architecture on the chromatin of interphase nuclei.
13
C NMR studies of gluconeogenesis in rat liver cells: Utilization of labeled glycerol by cells from euthyroid and hyperthyroid rats
Authors: Cohen S. M. , Ogawa S. , Shulman R. G.
Published: May 2006

The gluconeogenic pathway from [2-
13
C]glycerol and [1,3-
13
C]glycerol has been followed in suspensions of isolated rat hepatocytes at 25°C by
13
C NMR at 90.5 MHz. The flow of label through the major pathway from glycerol to L-glycerol 3-phosphate and into glucose was followed in cells from control and triiodothyronine-treated rats. Treatment increased the rates of glucose formation and glycerol consumption 2-fold and decreased the αGP level to 40%. We calculate that ≈60% of the flux is through the mitochondrial glycerol phosphate dehydrogenase in cells from triiodothyronine-treated rats, compared with ≈15% in cells from the controls. Equal distribution of label between the trioses of glucose was obtained and, because the C
3
-C
4
spin-spin coupling gives the distribution of labeled carbons in the
same
molecule, it was possible to measure the amount of triose from unlabeled fructose incorporated into the glucose labeled at carbons 1, 3, 4, and 6. About 10% of the hexoses had flowed through the pentose cycle and back into the hexose pathway in cells from fasted rats. From the distribution of label at glucose carbons not labeled via the major pathway and from the carbon spin-spin splitting patterns observed, we conclude that transketolase is reversible whereas transaldolase is essentially irreversible in the nonoxidative pentose branch.
Biochemical studies of taste sensation: binding to taste tissue of 3H-labeled monellin, a sweet-tasting protein.
Authors: Cagan R H , Morris R W
Published: May 2006

Binding of 3H-labeled methylated monellin to taste receptor tissue was demonstrated in vitro. Preparation of bovine and human circumvallate (taste) papillae bound more of the ligand than did lingual and nonlingual epithelial preparations devoid of taste buds. Binding to the taste preparations saturated at high ligand concentrations. Furthermore, sugars and other sweet-tasting molecules appeared to compete to some extent with this sweet-tasting protein for its binding sites. These binding measurements of the intensity sweet-tasting protein monellin to taste receptor preparations help to establish the binding interactions as an initial step taste sensation.
Direct measurement of changes in sodium pump current in canine cardiac Purkinje fibers
Authors: Gadsby David C. , Cranefield Paul F.
Published: May 2006

Purkinje fibers from dog hearts may have either a “high“ resting potential of about -90 mV or a “low“ resting potential of about -40 mV when immersed in low-Cl
-
solution containing 4 mM K
+
. Brief exposure of Purkinje fibers at the low level of resting potential to K
+
-free fluid causes further depolarization, and return to K
+
-containing solution elicits a transient hyperpolarization which reaches a peak within a few seconds and then declines within a few minutes. Repeating these changes in K
+
concentration after clamping the membrane potential at its steady resting level in K
+
-containing fluid allows the changes in net membrane current presumably underlying the depolarization and transient hyperpolarization to be measured. Net inward current is recorded when the fiber is exposed to K
+
-free solution, and a transient net outward current arises when it is returned to K
+
-containing solution. The transient net outward current reflects a temporary increase in the rate of electrogenic Na
+
extrusion caused by the rise in intracellular Na
+
concentration that occurs while the sodium pump is slowed in K
+
-free fluid. Sodium extrusion remains enhanced, presumably until the internal Na
+
concentration has been brought back to its resting level. The transient outward current is completely abolished by the cardiac steroid acetylstrophanthidin, and its amplitude is increased as the prior exposure to K
+
-free fluid is prolonged. The decay of the transient outward current and the decline in intracellular Na
+
concentration both appear to follow first-order kinetics.
Induction of rat sarcomas in rats treated with antithymocyte sera after transplantation of human cancer cells.
Authors: Huebner R J , Fish D C , Djurickovic D , Trimmer R W , Bare A L , Bare R M , Smith G T
Published: May 2006

Human cancer cells that had had high (greater than 160) tissue culture passages, when transplanted into antithymocyte-treated F344 newborn rats, caused induction of rat sarcomas in the rats within 2 or 3 subcultures, whereas human cancer cells with low (5-33) passages in vitro did not cause overt induction of rat sarcomas until after 5-10 subtransplantations. Because oncornavirus activity was not detected in either rat or human tumors, it is suggested that transforming sequences located on the human tumor cells may have been transferred to supporting rat reticulum cells in close contact with the human cancer cells.
Role of Thy-1 antigen in the in vitro differentiation of a rat mammary cell line.
Authors: Dulbecco R , Bologna M , Unger M
Published: May 2006

A line of rat mammary cells, LA7, undergoes differentiation into domes or ridges or projections under partial medium control. Immunofluorescent studies show that at the time of dome formation the LA7 cultures express the Thy-1 antigen at the borders between cells. Exposure of the cultures to certain monoclonal anti-Thy-1.1 sera prevents formation of new domes and causes disappearance of preexisting ones; antisera to whole cells produce no visible effect. Cultures exposed to anti-Thy-1 sera at effective concentrations of antibody against domes show morphological changes and ridge formation. It seems that the specific interactions of these antisera with the Thy-1 antigen redirects the differentiation program of the cells.
Effect of tumor promoters, protease inhibitors, and repair processes on x-ray-induced sister chromatid exchanges in mouse cells.
Authors: Nagasawa H , Little J B
Published: May 2006

The induction of sister chromatid exchanges (SCE) in the second postirradiation mitosis was studied in mouse 10T1/2 cells irradiated with 400 rads (4 grays) and maintained in stationary growth for several hours after x-ray exposure (similar to liquid holding recovery experiments in bacterial cells). X-irradiation with no recovery period induced few SCE. With short recovery intervals, however, the SCE frequency rose in parallel with the increase in survival, reaching a maximum increase of 2-fold after 4 hr; SCE declined with longer recovery intervals. The influence of postirradiation incubation with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and with the protease inhibitors antipain and leupeptin was studied on spontaneous, x-ray-induced (no recovery), and recovery-induced (4 hr) SCE. TPA (0.1 microgram/ml and 1.0 microgram/ml) increased the frequency of both spontaneous and direct x-ray-induced SCE, but not of recovery-induced SCE. Incubation with the protease inhibitors suppressed both TPA- and recovery-induced SCE, but had no effect on direct x-ray-induced SCE. These results are discussed in relation to the hypothesis that promotional events in carcinogenesis may involve the expression of mutational damage in cells by mitotic segregation.
Selective toxicity of purine deoxynucleosides for human lymphocyte growth and function
Authors: Gelfand Erwin W. , Lee Jacob J. , Dosch Hans-Michael
Published: May 2006

A role for the enzymes adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyl-transferase, EC 2.4.2.1) in the functional maturation of lymphoid cells has been revealed by the association of inherited deficiencies of these enzymes and profound immune deficiency. Previous studies have suggested that the selective toxicity for lymphocytes may be mediated by the accumulation of toxic deoxynucleoside metabolites, likely through the action of specific kinases enriched in lymphoid cells. In order to study possible mechanisms whereby lymphocyte function may be impaired in these disorders, we have studied the effect of nucleosides and their deoxy analogues on both T and B lymphocyte growth and function. In the presence of deoxyguanosine, there was marked inhibition of T lymphoblast growth, phytohem-agglutinin-induced cell proliferation, and T suppressor cell activity. T helper cell activity and the differentiation of B cells to an antibody-secreting stage were unaffected. Deoxyadenosine was much less inhibitory, but in the presence of an inhibitor of adenosine deaminase, its effects on lymphocyte growth and function were markedly potentiated. The addition of deoxycytidine prevented deoxyadenosine toxicity in all assays, whereas it only interfered with deoxyguanosine effects on T lymphoblast growth. These studies provide some initial understanding for the selective loss of specific lymphocyte functions in individuals with inborn errors of purine metabolism.
Translocation of glutathione from lymphoid cells that have markedly different gamma-glutamyl transpeptidase activities.
Authors: Griffith O W , Novogrodsky A , Meister A
Published: May 2006

Translocation of intracellular glutathione to the medium was studied in lymphoid cells (grown in tissue culture) that have very high, very low, or intermediate levels of membrane-bound gamma-glutamyl transpeptidase, in the absence and presence of various inhibitors of this enzyme. The data show that glutathione is translocated to the medium by all of the cell lines studied, but that glutathione does not accumulate in the medium unless the cellular transpeptidase activity is either very low or substantially inhibited. Translocation of glutathione does not seem to be directly related to the activity of gamma-glutamyl transpeptidase. The present and previous [Griffith, O.W. & Meister, A. (1979) Proc. Natl. Acad. Sci. USA 76, 268--272] findings suggest that translocation of intracellular glutathione is a general property of many mammalian cells. Glutathione exported from cells that have membrane-bound transpeptidase may be recovered by the cell in the form of transpeptidation or degradation products. Translocation of glutathione may also reflect operation of a rather general mechanism that protects and maintains the integrity of cell membranes.
Fine specificity of antibodies to poly(Glu60Ala30Tyr10) produced by hybrid cell lines.
Authors: Pierres M , Ju S T , Waltenbaugh C , Dorf M E , Benacerraf B , Germain R N
Published: May 2006

The polyethylene glycol-mediated cell fusion technique has been used to analyze the diversity of the antibody response to the terpolymer poly(Glu60Ala30Tyr10)(GAT). Nine stable clones (all producing IgM K anti-GAT antibodies) were isolated from a fusion between P3-X63-Ag8 myeloma cells and spleen cells from a DBA/2 mouse sensitized to GAT 5 days earlier. Seven other clones (producing IgG K anti-GAT antibodies) were derived from another fusion between NSI myeloma cells and spleen cells of (C57BL/6 X DBA/2)F1 hybrid mice hyperimmunized with GAT. These 16 anti-GAT antibodies were grouped according to their pattern of reactivity with GAT and the two related polymers of poly(Glu60Ala40) (GA) and poly(GLU50Tyr50) (GT). Two monoclonal anti-GAT antibodies (IgM F9-102.2 and IgG F17-148.3) demonstrated crossreactivity with GA but failed to crossreact with GT determinants. In contrast, the remaining 14 hybridoma antibodies demonstrated preferential reactivity with GAT but also exhibited crossreactive binding to GT and in some cases GA. There was a correlation between the fine specificity pattern and the presence of a common anti-GAT idiotype on these antibodies. Thus, the hybridoma anti-GAT antibodies which reacted with GT shared crossreactive idiotypic determinants (CGAT) present in mouse anti-GAT immune sera. In contrast, the monoclonal F9-102.2 and F17-148.3 antibodies that failed to bind to GT lacked the major CGAT idiotypic determinants.
Integrated spatial electron populations in molecules: The electron projection function
Authors: Streitwieser Andrew , Collins John B. , McKelvey John M. , Grier David , Sender John , Toczko A. Glenn
Published: May 2006

A “projection function,”
P(x,z)
, is defined as the partial integral of the molecular electron density, ρ(
x,y,z
), over the region -∞ <
y
< +∞. The projection provides a three-dimensional representation of molecular electron distributions. Chemically useful information can be discerned from graphical displays in either perspective plot or contour format. Numerical integration of the function gives the integrated spatial electron population for any region of interest. The use of the projection function and difference functions is exemplified by application to acetaldehyde.
Measured work of deformation and repulsion of lecithin bilayers.
Authors: Parsegian V A , Fuller N , Rand R P
Published: May 2006

We used three complementary techniques to vary the chemical potential of water in lipid/water mixtures; we measured the work of removing water from the multilayer lattice formed in water by the zwitterionic phospholipid egg lecithin. By x-ray diffraction, we observed the structural consequences of water removal. There are no discrete classes of "bound water" in this system; the work of removal is a continuous function of water content and lattice repeat spacing. From 30 to 3 A separation between bilayers there exists an exponential "hydration force" repulsion with a 2.6 A decay length. This interaction translates into a very large force to prevent contact between vesicles and planar membranes. It may be an important feature in controlling vesicle-to-cell fusion. As water is removed, bilayers not only move closer, but thicken as the lipid polar groups on the same bilayer move closer together. It is possible to divide the applied work into that of direct bilayer repulsion and that of bilayer deformation. We thus obtained a first determination of the lateral pressure required to create large increases in bilayer thickness and concomitant decreases in bilayer area. The lateral pressure reaches 25 dynes/cm for a 25% decrease in bilayer area. Systematic measurements of the mechanical properties of bilayers suffering such large deformation will allow critical tests of theories on bilayer stability and phase transition.
Simian virus 40 recombinants are produced at high frequency during infection with genetically mixed oligomeric DNA.
Authors: Wake C T , Wilson J H
Published: May 2006

Classical approaches to analysis of mitotic recombination by use of simian virus 40 (SV40) are limited in usefulness because of low frequencies of recombination. To bypass the apparent rate-limiting step in normal SV40 recombination, oligomeric SV40 was constructed in vitro by ligation of mixtures of pairs of linear DNAs carrying genetically distinct temperature-sensitive mutations. Cultured monkey cells infected with the unfractionated ligation products yielded frequencies of nonparental recombinant progeny that were increased up to 500-fold relative to cells infected with a mixture of the untreated circular molecules. Pairwise crosses were performed with tsB4, tsB8, and tsBC11 DNAs, using unfractionated oligomers constructed from linear molecules cleaved by EcoRI or BamHI. In each cross the fraction of progeny with nonparental genotypes was roughly proportional to the physical distances between the mutant sites. These results suggest a random, rather than site-specific, conversion of oligomers to monomers. Somewhat surprisingly, nonligated mixtures of linear tsB4 and tsB8 DNAs, created by EcoRI digestion, produced a 40-t to 100-fold increase in the frequency of nonparental progeny. These results indicate that intermolecular associations must occur with fairly high efficiency between these linear molecules.
beta 0 thalassemia, a nonsense mutation in man.
Authors: Chang J C , Kan Y W
Published: May 2006

We determined the complete nucleotide sequence of the 5' noncoding region and the first 74 amino acids of the nonfunctional beta-globin mRNA in a patient with homozygous beta 0 thalassemia. We identified the molecular defect as a single nucleotide substitution in the coding region of the mRNA. At the position corresponding to amino acid 17, replacement of an adenine by a uracil changes the triplet AAG, which codes for lysine in the normal beta chain, to an amber termination codon, UAG. This type of beta 0 thalassemia represents an example of a nonsense mutation in man.
Immunochemical characterization of surface antigens of TerC, a teratocarcinoma-derived cell line.
Authors: Larraga V , Edidin M
Published: May 2006

Rabbit and mouse antisera prepared against teratocarcinoma cells precipitate both glycoproteins and glycolipids from detergent extracts of radiolabeled cells. Extracts of immunoprecipitates with chloroform/methanol, 2:1 (vol/vol) have been resolved on thin-layer gels into multiple peaks. There are more species seen in extracts of teratocarcinoma cells than in extracts of the crossreacting cultured cell line, cl 1d. The teratocarcinoma antigens may be extracted out of chloroform/methanol into buffered saline. Incubation in these secondary extracts converts unreactive cells (lymphocytes to cells reactive with antisera against teratocarcinoma. Furthermore, the coated cells absorb at least 80% of the activity of antisera against teratocarcinoma targets.
Cell surface antigens of human melanoma identified by monoclonal antibody.
Authors: Yeh M Y , Hellström I , Brown J P , Warner G A , Hansen J A , Hellström K E
Published: May 2006

Mouse NS-1 myeloma cells were fused with spleen cells from mice that had been immunized with cells from a human melanoma, M1804. Hybrid cells were grown in selective medium and tested for production of antibody to surface antigens of M1804 cells. Three hybrids that produced antibodies that bound to the melanoma cells but not to autologous skin fibroblasts were cloned. Antibodies produced by two of the clones were cytotoxic to M1804 cells in the presence of rabbit complement. Extensive specificity tests showed that the antibodies produced by the clones bound strongly only to M1804 cells; significant, although weaker, binding occurred with 2 of 11 allogeneic melanomas. Apart from weak binding of the antibody produced by one of the clones to a breast carcinoma, binding assays of five carcinomas, one sarcoma, and fibroblasts from 17 individuals were negative, as were cytotoxic tests of 10 lymphoblastoid cell lines and peripheral blood lymphocytes from 68 normal donors and 12 chronic lymphocytic leukemia patients. This suggests that we have identified one or more determinants of a melanoma-associated antigen(s), whose expression is limited to a small proportion of melanomas.
Mitrochondrial NADH dehydrogenase in cystic fibrosis.
Authors: Shapiro B L , Feigal R J , Lam L F
Published: May 2006

We have shown that skin fibroblast from patients with cystic fibrosis (CF) and from carriers for CF [heterozygotes (HZ)] consume more O2 than do their controls. When the mitochondrial electron transport inhibitor rotenone was added to the cells, the relative inhibition of O2 consumption was CF greater than HZ greater than controls (P less than 0.005 in both comparisons). Because rotenone specifically inhibits NADH dehydrogenase, [NADH: (acceptor) oxidoreductase, EC 1.6.99.3], which is the enzyme of energy-conserving site 1 of the mitochondrial electron transport system, activity and kinetics of this enzyme system were studied in fibroblast homogenates. NADH dehydrogenase activity was equal in cells from the three genotypes. At pH 8.0, affinity of the enzyme for its substrate was CF greater than HZ = controls; at pH 8.6, affinity was CF greater than HZ = controls (P less than 0.005 for the differences). pH optima for the genotypes were without exception 8.6 (CF), 8.3 (HZ), and 8.0 (control). HZ and control lines were distinguished unequivocally in a blind test on the basis of differences in pH optima. Purified mitochondrial preparations revealed pH optima identical to those found in whole cell homogenates. These data suggest that the mutant gene responsible for CF is expressed in the complex mitochondrial NADH dehydrogenase system.
Density matrix method for orbital localization
Authors: Caldwell Dennis , Redington Patrick , Eyring Henry
Published: May 2006

A method is presented for localizing molecular orbitals, based on diagonalizing subunits of the density matrix. First, nonbonding orbitals are found by diagonalizing the monatomic subunits; then, diatomic σ or π bonding and antibonding orbitals are obtained from the diatomic subunits for all bonded pairs of atoms; finally, the delocalized π-orbitals for particular chromophores are found by projecting the first set out of the self-consistent field (SCF) Hamiltonian. The results show good general agreement with other localization methods, with advantages in the ability to display group orbitals in complex molecules which most closely resemble the SCF orbitals for simple prototypes.
Polyadenylylation of proteins in reovirions.
Authors: Carter C A
Published: May 2006

The reovirus oligoadenylates exist in two states within the virion: free and bound to viral proteins. The latter class of oligonucleotides, after digestion with Penicillium (P1) nuclease, yields adenylic acid and an adenosine-containing compound that is positively charged at pH 1.7, 3.5, or 6.5. In a mixture of [35S]methionine- and [3H]adenosine-labeled reovirus disrupted by sodium dodecyl sulfate/urea, approximately 4% of the radioactivity in [35S]methionine-labeled proteins coelutes with [3H]adenosine-labeled material at a net charge of -1.5 when analyzed by ion-exchange chromatography on DEAE-cellulose. This material migrates in sodium dodecyl sulfate/polyacrylamide gels with mu polypeptides and with a small protein, viii. Radioactivity is not released when the complex is boiled in buffer containing sodium dodecyl sulfate and urea or boiled in 80% dimethyl sulfoxide or when viral RNA is extracted with phenol. Digestion with Pronase converts the [3H]adenosine-labeled compound to oligomers of net charge -8 to -12 which contain nuclease P1- and alkaline phosphatase-sensitive adenylic acid residues as well as adenosine in a P1- and phosphatase-resistant linkage. These data indicate that reovirus contains structural proteins that are covalently bound to an oligoadenylate moiety.
Kinetics of the induction of three translation-regulatory enzymes by interferon.
Authors: Kimchi A , Shulman L , Schmidt A , Chernajovsky Y , Fradin A , Revel M
Published: May 2006

Three enzymes that cause inhibition of mRNA translation, eukaryotic initiation factor 2 protein kinase PK-i, oligoisoadenylate synthetase E, and phosphodiesterase 2'-PDi, have been recently isolated from interferon-treated cells. We show that the rise in these three enzyme activities may be used to study the response of uninfected cells to interferon. For each enzyme, a specific microassay that can be carried out on extracts from 2-5 x 10(4) monolayer cells from mouse, monkey, or man was developed. With these assays, the kinetics of induction of the three enzymes in mouse L cells are compared. The dose dependence for protein kinase PK-i induction is shown to be similar to that for the development of the antiviral state. Actinomycin D and anti-interferon serum block enzyme induction if added to the cells early after interferon treatment. The quantitative measurements of the intracellular level of these enzymes provide a new and convenient model to study the cell's response to interferon.
Hydrostatic pressure in small phospholipid vesicles
Authors: Tanford Charles
Published: May 2006

The internal solvent-filled cavity of a singlewalled spherical phospholipid vesicle must be at essentially the same pressure as the aqueous medium outside the vesicle. Whether or not the bilayer itself is under elevated pressure cannot at present be determined.
Structure of cytochrome a3-Cua3 couple in cytochrome c oxidase as revealed by nitric oxide binding studies.
Authors: Stevens T H , Brudvig G W , Bocian D F , Chan S I
Published: May 2006

The addition of NO to oxidized cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) causes the appearance of a high-spin heme electron paramagnetic resonance (EPR) signal due to cytochrome a3. This suggests that NO coordinates to Cu+2a3 and breaks the antiferromagnetic couple by forming a cytochrome a+33-Cu+2a3-NO complex. The intensity of the high-spin cytochrome a3 signal depends on the method of preparation of the enzyme and maximally accounts for 58% of one heme. The effect of N-3 on the cytochrome a+33-Cu+2a3-NO complex is to reduce cytochrome a3 to the ferrous state, and this is followed by formation of a new complex that exhibits EPR signals characteristic of a triplet species. On the basis of optical and EPR results, a NO bridge between cytochrome a+23 and Cu+2a3 is proposed--i.e., cytochrome a+23-NO-Cu+2a3. The half-field transition observed at g = 4.34 in the EPR spectrum of this triplet species exhibits resolved copper hyperfine splittings with [A+2] = 0.020 cm-1, indicating that the Cu+2a3 in the cytochrome a+23-NO-Cu+2a3 complex is similar to a type 2 copper site.
Decrease in epidermal growth factor receptor levels and production of material enhancing epidermal growth factor binding accompany the temperature-dependent changes from normal to transformed phenotype.
Authors: Guinivan P , Ladda R L
Published: May 2006

Normal rat kidney (NRK) cells infected with a temperature-sensitive mutant of Kirsten sarcoma virus (Ts cells) exhibited normal monolayer morphology identical to that observed for uninfected cells (NRK cells) at the nonpermissive temperature, 39 degrees C, but grew as multilayered foci resembling NRK cells transformed by the wild-type virus (KNRK cells) at 32 degrees C, the permissive temperature. NRK cell division was stimulated by epidermal growth factor (EGF), and these cells showed high levels of EGF receptors, as determined by 125I-labeled EGF binding. KNRK cells were unresponsive to EGF and no EGF receptors were detectable. Ts cells also were unresponsive to EGF at both temperatures, but exhibited just detectable EGF binding at 32 degrees C and 10-15% of NRK cell binding at 39 degrees C. Use of EGF added to the culture medium by these cells paralleled the receptor levels. Crossfeeding experiments among NRK, KNRK, and Ts cultures indicated that Ts cells at the permissive temperature and KNRK cells at both temperatures produced a heat-stable substance(s) which stimulated DNA synthesis in NRK cells independent of the presence of serum or of EGF. Conditioned medium from the transformed cultures also significantly enhanced EGF binding to NRK cells. These studies demonstrated a correlation between the transformed phenotype and the receptor levels of a potent cell mitogen, EGF, which was readily reversible in the Ts cultures. In addition, cultures expressing the transformed phenotype produced material that did not compete for the EGF receptor but did enhance EGF binding, in contrast to other reports involving sarcoma virus-transformed cells.
Regulation of ribosomal protein synthesis in Escherichia coli by selective mRNA inactivation.
Authors: Fallon A M , Jinks C S , Strycharz G D , Nomura M
Published: May 2006

In an Escherichia coli strain lysogenic for lambda spc2 transducing phage, an extra copy of ribosomal protein (r-protein) genes in the spc and alpha operons are carried on the phage chromosome. Expression of genes in the spc operon in this merodiploid strain was compared with that in a control "haploid" strain carrying lambda trkA phage. It was found that the synthesis rate of spc mRNA, relative to other reference mRNA in the merodiploid strain, is about 2-fold higher than that in the control strain; yet, no dosage effect was observed in the synthesis rate of r-proteins in the spc or alpha operon. The spc mRNA was found to be more rapidly degraded in the merodiploid strain than in the control strain, and its steady-state amount, relative to reference mRNA, was only slightly higher in the merodiploid strain than in the control strain. Thus, E. coli cells have the ability to regulate the rate of r-protein synthesis regardless of the rate of transcription of r-protein genes, presumably by inactivation of the mRNA followed by its degradation. A model is proposed which involves selective inactivation of r-protein mRNA by a feedback mechanism. The model can explain coordinated synthesis of r-proteins and other observations related to selective expression of certain alleles in diploid strains.
Healing of
mat
mutations and control of mating type interconversion by the mating type locus in
Saccharomyces cerevisiae
Authors: Strathern Jeffrey N. , Blair Lindley C. , Herskowitz Ira
Published: May 2006

Homothallic yeasts switch cell types (mating types a and α) at high frequency by changing the alleles of the mating type locus,
MAT
a and
MAT
α. We have proposed in the cassette model that yeast cells contain silent
MAT
a and
MAT
α blocs (“cassettes”), copies of which can be substituted at the mating type locus for the resident information. The existence of silent cassettes was originally proposed to explain efficient switching of a defective
MAT
α locus (
mat
α) to a functional
MAT
α locus. We report here that this “healing” of
mat
mutations is a general property of the mating type interconversion system and is not specific to the class of
mat
α mutations studied earlier: a defective
MAT
a (
mat
a
1
) switches readily to
MAT
a and various
mat
α loci switch readily to
MAT
α. These observations satisfy the prediction of the cassette model that all mutations within
MAT
a and
MAT
α be healed. These studies also identify
MAT
functions that control the switching process: the same functions known to promote sporulation and prevent mating in a/α cells also inhibit the switching system in a/α cells. Finally, we present additional characterization of a natural variant of
MAT
α,
MAT
α-inc [Takano, I., Kusumi, T. & Oshima, Y. (1973)
Mol. Gen. Genet
. 126, 19-28] that is insensitive to switching. Our observation that
MAT
α-inc acts in
cis
suggests that it may be altered in a site concerned with excision of
MAT
α-inc or its replacement by another cassette.
Testosterone and its precursors and metabolites enhance guanylate cyclase activity.
Authors: Vesely D L
Published: May 2006

Both testosterone and cyclic GMP stimulate DNA synthesis. Because cyclic GMP and testosterone seem to have similar actions, the objective of this investigation was to determine if testosterone and its precursors might have part of their mechanism of action through stimulation of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2], the enzyme that catalyzes the formation of cyclic GMP from GTP. The precursors--namely, progesterone, pregnenolone, 17 alpha-progesterone, 17 alpha-hydroxypregnenolone, androstenedione, and dehydroepiandrosterone--caused a 2- to 3 1/2-fold enhancement of guanylate cyclase activity in rat liver, kidney, skeletal muscle, and ventral prostate at a concentration of 1 microM. These precursors are generated from cholesterol, which had no effect itself on guanylate cyclase activity. Testosterone, 19-nortestosterone, 17-methyltestosterone, and 5 alpha-dihydrotestosterone enhanced guanylate cyclase activity 2- to 5-fold in the same tissues at 1 microM. Etiocholanolone, androsterone, and epiandrosterone, metabolites of testosterone metabolism, enhanced guanylate cyclase activity 1 1/2- to 2-fold at this same concentration. Dose-response relationships revealed that testosterone and its precursors and metabolites had their maximal effect at 1 microM but still had some effect at 0.001 microM. The data in this investigation suggest that the guanylate cyclase-cyclic GMP system plays a role in the mechanism of action of testosterone and its precursors.
Localization of horseradish peroxidase-alpha-bungarotoxin binding in crustacean axonal membrane vesicles and intact axons.
Authors: Chester J , Lentz T L , Marquis J K , Mautner H G
Published: May 2006

A conjugate of alpha-bungarotoxin with horseradish peroxidase was used to visualize alpha-bungarotoxin binding sites at the fine structural level in isolated axonal membrane vesicles from lobster walking leg nerve. These plasma membrane vesicles have previously been shown to exhibit saturable binding of [3H]nicotine and [3H]acetylcholine. Binding of the toxin was identified in the axon plasma membrane and could be blocked by pretreatment with excess free alpha-bungaratoxin or d-tubocurarine. Binding sites for alpha-bungarotoxin were identified by the same technique in sections of intact nerve fibers from both lobster and spider crab and were found to be localized primarily in the axolemma rather than in the Schwann cell membrane.
Occurrence and role of tightly bound adenine nucleotides in sarcoplasmic reticulum of rabbit skeletal muscle.
Authors: Aderem A A , McIntosh D B , Berman M C
Published: May 2006

Freshly isolated sarcoplasmic reticulum vesicles contain 0.05 mol of tightly bound ADP and 0.03 mol of tightly bound ATP per mol of Ca2+, Mg2+-ATPase (ATP phosphohydrolase, EC 3.6.1.3). These values were increased to 0.1-0.2 mol ADP and 0.2-0.3 mol ATP per mol of ATPase after incubation of vesicles in the presence of MgATP and Ca2+ at 25 degrees C and pH 7.0. Half-maximal enrichment of tightly bound nucleotides was obtained with 2.5 mM ATP and 0.32 microM free Ca2+. Uncoupling of calcium transport from ATPase activity by mild acidic conditions or with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at pH 7.0 decreased the ability of the membranes to be enriched with tightly bound nucleotides and also decreased the content of tightly bound nucleotides of previously enriched membranes. Tightly bound [3H]nucleotides could only be partially displaced by reincubation under enrichment conditions. Tightly bound nucleotides are associated with energized calcium translocation but do not appear to be directly involved in the catalytic cycle.