bluuebunny/crossref_metadata_2025 huggingface dataset:
Antibody-Mediated Suppression of Lymphoma: Participation of Platelets, Lymphocytes, and Nonphagocytic Macrophages
Authors: Shin Hyun S. , Hayden Michael L. , Gately Celia L.
Published: May 2006

Humoral antibody directed against a grafted murine lymphoma can suppress the growth of the tumor in mice of the inbred strain native to the tumor. Antibody, however, cannot suppress the tumor growth in mice given 500 R of whole-body irradiation. When the tumor-antibody inoculum is admixed with lymphocytes or macrophages obtained from peritoneal exudate, macrophages experimentally rendered nonphagocytic, or with platelets isolated from peripheral blood, the tumor growth is suppressed in irradiated mice. These results indicate that mechanisms other than phagocytosis may play an important role in antibody-mediated suppression of tumor growth
in vivo
.
Activation of Transcription of
hut
DNA by Glutamine Synthetase
Authors: Tyler Bonnie , Deleo Albert B. , Magasanik Boris
Published: May 2006

The correct transcription of the
hut
(histidine utilization) operon DNA of
Salmonella typhimurium
requires activation either by 3′:5′-cyclic adenosine monophosphate and catabolite-activating protein or by nonadenylylated glutamine synthetase (EC 6.3.1.2.). Thus, glutamine synthetase plays a regulatory role distinct from its enzymatic function.
Mechanism of Inhibition of Eukaryotic Protein Synthesis by Trichothecene Fungal Toxins
Authors: Cundliffe Eric , Cannon Michael , Davies Julian
Published: May 2006

The 12,13-epoxytrichothecenes, a group of sesquiterpenoid fungal antibiotics, inhibit protein synthesis in eukaryotic cells but do not share a common mode of action. Trichodermin stabilizes polyribosomes, prevents their disaggregation by puromycin, and also prevents the release of nascent peptides from ribosomes by puromycin. Nivalenol, T-2 toxin, and verrucarin A cause rapid and almost quantitative breakdown of polyribosomes in H-HeLa cells, a process which is inhibited by anisomycin, cycloheximide, or trichodermin. Similar effects of trichodermin, nivalenol, and verrucarin A are also observed in yeast spheroplasts. We conclude that nivalenol, T-2 toxin, and verrucarin A are potent and highly selective inhibitors of polypeptide chain initiation in eukaryotes, whereas trichodermin inhibits chain elongation and (or) termination. We have compared the structural formulae of various trichothecenes and suggest that the presence of substituents on carbon-15 of the common trichothecene ring may be important in determining the precise modes of action of this group of compounds.
Cyclic AMP-Mediated Induction of the Cyclic AMP Phosphodiesterase of C-6 Glioma Cells
Authors: Schwartz Joan P. , Passonneau Janet V.
Published: May 2006

Long-term regulation of the cyclic nucleotide phosphodiesterase of the C-6 rat glioma cell line has been studied. Both the low
K
m
and high
K
m
activities can be induced by elevation of intracellular cyclic AMP levels following either dibutyryl cyclic AMP or norepinephrine treatment of the cells. The enzymes are maximally induced by 3-4 hr. The presence of either cycloheximide or actinomycin D prevents induction by either dibutyryl cyclic AMP or norepinephrine. Evidence is presented that the norepinephrine effect is mediated by the β-catecholamine receptor. The increased phosphodiesterase activity causes a partial refractoriness to a second challenge with norepinephrine, which can be overcome by blockade of the induction with cycloheximide. The results suggest that just as short-term regulation of cyclic AMP levels occurs via changes in the rates of synthesis or degradation, long-term alterations of the system may also involve either the adenylate cyclase or the phosphodiesterase.
Equilibrium Sedimentation of Turnip Yellow Mosaic Virus
Authors: McGee J. H. , Kupke D. W. , Godschalk W. , Beams J. W.
Published: May 2006

Sedimentation equilibrium was achieved with turnip yellow mosaic virus at low speeds (600 rpm) in a magnetic ultracentrifuge. The experiments were carried out in the newly installed constant-speed rotor, equipped with automatic control and an electromagnetic drive. A particle mass of 5.55 × 10
6
daltons was calculated for the virus at vanishing concentrations, in essential agreement with the earlier results using the more tedious procedure with a freely coasting rotor. In order to interpret the observed departure from ideal behavior (nonassociating conditions), preferential interaction experiments were carried out by magnetic densimetry. These showed that a strong Donnan effect exists at pH 7, which contributed > 10
3
times more to the nonideality than did the excluded volume effect. A net negative charge of about 3.5 × 10
3
per particle was estimated at this pH.
Receptors from Glucocorticoid-Sensitive Lymphoma Cells and Two Classes of Insensitive Clones: Physical and DNA-Binding Properties
Authors: Yamamoto Keith R. , Stampfer Martha R. , Tomkins Gordon M.
Published: May 2006

Mouse lymphoma tissue culture cells (S49.1A) are normally killed by dexamethasone, a synthetic glucocorticoid hormone. Dexamethasone-resistant clones have been selected from this line, some of which retain the ability to specifically bind dexamethasone. Addition of [
3
H]dexamethasone to cultures, followed by cell fractionation, reveals that the nuclear transfer of hormone-receptor complexes in some of these variant clones is deficient (nt
-
), while others show increased nuclear transfer (nt
i
) relative to the parental line. Two independently selected members of each class have been studied here, in an effort to elucidate the molecular determinants involved in the receptor-nucleus interaction
in vivo
. The labeled receptors in cell-free extracts bind to DNA-cellulose, but only after previous incubation of the extract at 20°, similar to the treatment required for cell-free interaction of receptors with nuclei. More importantly, the apparent DNA-binding affinity of the nt
-
receptors is lower than the wild type, whereas the nt
i
receptors bind DNA with an affinity higher than the parental molecules. The parallelism of nuclear and DNA binding, together with the observations that the receptors from the variants have sedimentation properties different from the wild-type cells, lead us to conclude that (
i
) these variants may contain altered receptor molecules and (
ii
) DNA is probably the primary nuclear binding site for steroid receptors
in vivo
.
The Molecular Weight of the Major Glycoprotein from the Human Erythrocyte Membrane
Authors: Grefrath Stuart P. , Reynolds Jacqueline A.
Published: May 2006

The molecular weight of the major glycoprotein from the human erythrocyte membrane is 29,000, of which 55% is carbohydrate and 45% is protein. The binding of sodium dodecyl sulfate to this glycoprotein is anomalous when compared to water soluble proteins and leads to migration rates in sodium dodecyl sulfate-polyacrylamide gels that cannot be interpreted in terms of molecular weight. Anomalous sodium dodecyl sulfate binding may be a general characteristic of many intrinsic membrane proteins even if they are not glycoproteins, and such proteins are likely to have mobilities in sodium dodecyl sulfate-gel electrophoresis that do not correspond to the mobilities of water soluble proteins of identical molecular weight.
Nuclear Triiodothyronine-Binding Protein: Partial Characterization and Binding to Chromatin
Authors: Degroot Leslie J. , Refetoff Samuel , Strausser John , Barsano Charles
Published: May 2006

Nuclei were prepared by sucrose sedimentation of liver homogenates from rats given
125
I-labeled triiodothyronine
in vivo
. The nuclear extract obtained by treatment of the nuclear pellet with 0.4 M KCl contains the [
125
I]triiodothyronine that had been injected
in vivo
bound to protein(s). The triiodothyronine bound to nuclear protein(s)
in vivo
does not readily exchange with triiodothyronine added to the extract
in vitro
. This triiodothyronine·nuclear extract complex retains triiodothyronine during dialysis or exposure to anion exchange resin and migrates as a broad band on agarose-gel electrophoresis. It is rapidly destroyed by Pronase, by 8 M urea, and by
p
-chloromercuribenzoic acid, but not by RNase or by DNase. It is also susceptible to thermal inactivation at 37°, possibly through changes in the affinity of triiodothyronine to the nuclear binding protein(s), since the bound triiodothyronine becomes more readily dialyzable, is absorbed by an anion exchange resin, but retains its characteristic mobility on electrophoresis. The triiodothyronine·nuclear extract complex formed
in vivo
binds to crude liver chromatin
in vitro
at low salt concentration, but can be completely extracted again at KCl concentrations greater than 0.2 M.
Stability of Neuronal Microtubules to High Pressure
In Vivo
and
In Vitro
Authors: O'Connor Thomas M. , Houston L. L. , Samson Fred
Published: May 2006

Neuronal microtubules in a variety of nerve cell types are unaffected by high hydrostatic pressures over a range of 1400-10,000 pounds/inch
2
and periods of 10-45 min. Similarly, purified tubulin polymerized to form microtubules
in vitro
were not depolymerized by the same range of pressures. The depolymerization of microtubules in several types of non-neuronal cells, which has been reported, may have been over-generalized with regard to the direct action of pressure on microtubule stability.
Cholera Toxin and Cell Growth: Role of Membrane Gangliosides
Authors: Hollenberg Morley D. , Fishman Peter H. , Bennett Vann , Cuatrecasas Pedro
Published: May 2006

The binding of cholera toxin to three transformed mouse cell lines derived from the same parent strain, and the effects of the toxin on DNA synthesis and adenylate cyclase activity, vary in parallel with the ganglioside composition of the cells. TAL/N cells of early passage, which contain large quantities of gangliosides G
M3
, G
M2
, G
M1
, and G
Dla
, as well as the glycosyltransferases necessary for the synthesis of these gangliosides, bind the most cholera toxin and are the most sensitive to its action. TAL/N cells of later passage, which lack chemically detectable G
M1
and G
Dla
and which have no UDP-Gal:G
M2
galactosyltransferase activity, are intermediate in binding and response to the toxin. SVS AL/N cells, which lack G
M2
in addition to G
M1
and G
Dla
and which have little detectable UDP-GalNAc:G
M3
N
-acetylgalactosaminyltransferase activity, bind the least amount of toxin. The SVS AL/N cells are the least responsive to inhibition of DNA synthesis and stimulation of adenylate cyclase activity by cholera toxin. Gangliosides (especially G
M1
), which appear to be the natural membrane receptors for cholera toxin, may normally have important roles in the regulation of cell growth and cAMP-mediated responses.
Reversal of Pactamycin Inhibition of Methionyl-Puromycin Synthesis and 80S Initiation Complex Formation by a Ribosomal Joining Factor
Authors: Suzuki Hideo , Goldberg Irving H.
Published: May 2006

A crude mixture of polypeptide chain initiation factors (0.5 M KCl ribosomal wash) from reticulocyte ribosomes was fractionated by DEAE-cellulose column chromatography. Among several initiation factors obtained from the column, one factor eluting at 0.22-0.25 M KCl showed a remarkable ability to overcome the inhibition of Met-puromycin and 80S initiation complex formation caused by the antibiotic, pactamycin. Earlier experiments had shown that pactamycin does not prevent the binding of Met-tRNA
f
to the small ribosomal subunit but does interfere with the joining of the 60S ribosomal subunit to form the 80S initiation complex. A Lineweaver-Burk plot of initial rates of Met-puromycin formation showed that the interaction of the factor and pactamycin was of a competitive type.

In the absence of the factor, [
35
S]Met-puromycin was not synthesized and [
35
S]Met-tRNA
f
bound only to the small ribosomal subunit. The amount of [
35
S]Met-tRNA
f
bound to 80S ribosomes bearing endogenous mRNA and the amount of [
35
S]Met-puromycin formed were directly related to the amount of factor added. Thus, this factor can be termed a “joining factor,” and a simple assay of its activity can be devised based on its ability to overcome the pactamycin inhibition of the puromycin reaction.
lsomeric Phenylalanyl-tRNAs. Position of the Aminoacyl Moiety During Protein Biosynthesis
Authors: Hecht Sidney M. , Kozarich John W. , Schmidt Francis J.
Published: May 2006

The preparation of phenylalanyl-tRNA terminating in 3′-deoxyadenosine has been achieved by incubation of abbreviated tRNA (tRNA-CpC
OH
) with 3′-deoxyadenosine 5′-diphosphate and polynucleotide phosphorylase (EC 2.7.7.8), followed by aminoacylation. The isomeric phenylalanyl-tRNA terminating in 2′-deoxyadenosine was constructed by incubation of tRNA-CpC
OH
with 2′-deoxy-3′-
O
-phenylalanyladenosine 5′-diphosphate and polynucleotide phosphorylase. While tRNA is aminoacylated at the 2′-position, only the 3′-aminoacyl-tRNA is active as a peptide acceptor in the peptidyltransferase reaction. Both modified tRNAs were bound to the A-site as efficiently as unmodified tRNA, but neither was so efficient at P-site binding or as an acceptor in the peptidyltransferase reaction. Neither of the modified tRNAs acted as a donor in the peptidyltransferase reaction.
Characterization of Binding Components for Progesterone and 5α-Pregnane-3,20-dione in the Hamster Uterus
Authors: Leavitt Wendell W. , Grossman Charles J.
Published: May 2006

In the hamster uterus, a specific progesterone (pregn-4-ene-3,20-dione) receptor has been identified in the cytosol fraction. In the present study, we examined hamster uterine cytosol for the possible existence of specific macromolecules that bind the progesterone metabolite, 5α-pregnane-3,20-dione. When cytosol was analyzed by density-gradient centrifugation with sucrose-glycerol gradients and by Scatchard plot analysis of [
3
H]5α-pregnane-3,20-dione binding data, there was no evidence of specific binding components for this metabolite.
In vivo
treatment of proestrous hamsters with unlabeled progesterone, 5α-pregnane-3,20-dione, or cortisol for 1 hr revealed that only progesterone caused the depletion of progesterone-receptor sites from the uterine cytosol fraction. Incubation of uterine strips which had been preloaded with two different concentrations of [
3
H]-progesterone demonstrated that progesterone was metabolized to 5α-pregnane-3,20-dione and to a greater extent to 3α-hydroxy-5α-pregnan-20-one. The accumulation of 5α-pregnane-3,20-dione during progesterone metabolism appeared to be related to the availability of nonspecifically bound hormone. These studies (
i
) strongly suggest there is no specific receptor system for 5α-pregnane-3,20-dione in the uterine cytosol fraction, (
ii
) confirm the existence of a specific progesterone receptor in uterine cytosol, and (
iii
) provide evidence that progesterone itself mediates the uterine progestational response via interaction with a specific receptor system.
Serendibite, a Complicated, New, Inorganic Crystal Structure
Authors: Buerger Martin J. , Venkatakrishnan V.
Published: May 2006

Serendibite with very similar analyses is known from Ceylon and New York. The triclinic cell of symmetry
P
[unk] and volume 670.9 Å
3
contains 2Ca
1.64
Mg
2.64
Fe
0.27
II
Al
4.64
B
1.66
Si
3
O
20
, with 14 metal atoms and 20 oxygen atoms in the asymmetric unit. It was solved by the “direct” method of transforming the ordinary three-dimensional Patterson function into an approximation of the electron density by using conjugate peaks and minimum functions, followed by successive Fourier syntheses and least-squares refinement to
R
= 7.1%. This new structure is composed of interrupted brucite-type layers which form an octahedral framework, and winged single chains of tetrahedra. The structure has units of similar, but not identical, geometry to those of the minerals sapphirine and aenigmatite, and these similar units are assembled in a different way.
Muscle-Like Contractile Proteins and Tubulin in Synaptosomes
Authors: Blitz A. L. , Fine R. E.
Published: May 2006

Material in major bands with molecular weights corresponding to those of actin, brain tropomyosin, and myosin is present in purified rat synaptosomes dissolved in sodium dodecyl sulfate and subjected to electrophoresis on dodecyl sulfate-acrylamide gels. A band corresponding to tubulin appears to be the major constituent of synaptosomes, confirming the work of Feit and his coworkers. We have demonstrated by peptide mapping that the proteins in these bands have strong chemical similarities to actin, brain tropomyosin, myosin, and tubulin. We have prepared synaptic membrane, vesicle, and soluble fractions from synaptosomes. The polypeptide composition of synaptic membranes, as determined by dodecyl sulfate-acrylamide gel electrophoresis, is similar to that of synaptosomes, with tubulin, actin, and tropomyosin being major constituents. Synaptic vesicles have as their major polypeptide an unidentified protein with a molecular weight of 50,000; they also have many bands in common with synaptosomes. The soluble fraction predominantly contains actin and tubulin. The possibility that the muscle-like contractile proteins and tubulin are membrane-associated in various cell types is discussed, as is their possible role in neurotransmitter release.
Idiotypic Specificity of Surface Immunoglobulin and the Maturation of Leukemic Bone-marrow-derived Lymphocytes
Authors: Fu S. M. , Winchester R. J. , Feizi T. , Walzer P. D. , Kunkel H. G.
Published: May 2006

Certain cases of chronic lymphocytic leukemia are associated with monoclonal immunoglobulins in the serum. In the present study it was possible to demonstrate that the surface immunoglobulin of the leukemic lymphocytes was idiotypically identical to the serum monoclonal immunoglobulin of the same individual. This was done through the use of fluorescent antibodies that were prepared against the isolated immunoglobulin M kappa serum protein from patient Ei. Such antibodies, after absorption, failed to react with the lymphocytes of other individuals. Capping experiments indicated that both the immunoglobulin M and the immunoglobulin D on the leukemic cells possessed the same idiotypic specificity.

A population of plasma cells was also identified in the peripheral blood of patient Ei that showed strong intracellular staining with the idiotypic antiserum and was the apparent source of the specific serum immunoglobulin. The accumulated evidence indicates that the plasma cells were derived from the leukemic lymphocytes by a process of differentiation. Surface immunoglobulin D disappeared and surface immunoglobulin M decreased markedly in this process.
Structure and Molecular Weight of the 60-70S RNA and the 30-40S RNA of the Rous Sarcoma Virus
Authors: Mangel Walter F. , Delius Hajo , Duesberg Peter H.
Published: May 2006

The structure and molecular weight of the 60-70S RNA complex and the 30-40S RNA species of Rous sarcoma virus were analyzed in an electron microscope after treatment of the RNAs with the bacteriophage T4 gene-32 protein to stretch out the RNA strands. Although all RNA preparations treated with gene-32 protein showed considerable heterogeneity in length, a significant fraction of the RNA retained its original sedimentation coefficient after treatment to allow the following conclusions to be made: The 30-40S RNA was confirmed to be a linear polynucleotide with a molecular weight of about 3 × 10
6
. The 60-70S RNA exhibited a network structure with a molecular weight predominantly of about 6 × 10
6
. Therefore, the subunit hypothesis for the 60-70S RNA is confirmed. A model for the structure and molecular weight of the 60-70S RNA postulates that the complex consists of two 30-40S RNA subunits held together at many points. This model elucidates the biological observation that the infectivity of RNA tumor viruses is proportional to the amount of 30-40S RNA in a virus preparation and not to the amount of 60-70S RNA.
Regulation of Hemoglobin Synthesis in a Murine Erythroblastic Leukemic Cell: The Requirement for Replication to Induce Hemoglobin Synthesis
Authors: McClintock Patrick R. , Papaconstantinou John
Published: May 2006

The induction of hemoglobin synthesis by dimethylsulfoxide in cells infected with Friend leukemia virus has been shown to depend on the number of cell divisions after the addition of the inducing agent. In asynchronous cultures the cells have a generation time of 24 hr, and hemoglobin synthesis occurs after 48 hr in the presence of dimethylsulfoxide. Generation times were extended by lowering the serum content of the medium. In 7.5% and 2.5% fetal calf serum, the generation time was increased to 36 hr and 48 hr, respectively, and hemoglobin synthesis began at 72 hr and 96 hr, respectively. In the presence of
N
6
,
O
2
′-dibutyryladenosine 3′:5′-cyclic monophosphate the generation time was extended to 38 hr, and hemoglobin synthesis began after 72 hr in dimethylsulfoxide.

The number of cell doublings, under conditions where the generation time was 24 hr, was regulated by the size of the initial cell concentration. When a low inoculum (1.5 × 10
5
cells per cm
3
) was used, the culture was still in log phase after two doublings and hemoglobin appeared in log phase; a moderate initial inoculum (4.2 × 10
5
cells per cm
3
) took the culture into stationary phase after two doublings, and hemoglobin synthesis was induced in stationary-phase cells. Heavy inocula (3.7 × 10
6
cells per cm
3
) maintained the cells in stationary phase, and under these conditions no induction of hemoglobin synthesis occured. We conclude from these studies that the initiation of hemoglobin synthesis required two rounds of mitosis after treatment with dimethylsulfoxide.

In studies with synchronized cultures of cells infected with Friend leukemia virus, two mitoses were required for the initiation of hemoglobin synthesis. We conclude that two rounds of DNA synthesis and/or mitosis are also required for the dimethylsulfoxide-mediated induction of erythroid differentiation and hemoglobin synthesis in these cells.
A New Method for Congressional Apportionment
Authors: Balinski M. L. , Young H. P.
Published: May 2006

The problem of Congressional apportionment is explained together with a brief history of the methods used or considered for its solution. Reasons are given for rejecting the presently used method of equal proportions and for accepting a new method, the quota method, which is the unique method satisfying three essential axioms.
Translational Properties of Rabbit Globin mRNA after Specific Removal of Poly(A) with Ribonuclease H
Authors: Sippel A. E. , Stavrianopoulos J. G. , Schutz G. , Feigelson P.
Published: May 2006

Highly purified RNase H (RNA·DNA hybrid ribonucleotidohydrolase, EC 3.1.4.34) from calf thymus was used to specifically remove the poly(A) sequences of purified rabbit globin mRNA after its hybridization with poly(dT). The deadenylylated globin mRNA was repurified by a one-step procedure including a nitrocellulose column. The poly(A) size and the content of unmodified mRNA were determined by hybridization with [
3
H]-poly(U), and it could be shown that the RNase H digestion method effectively removes this terminal poly(A) sequence. No difference in activity was found between mRNAs with and without poly(A) to initiate, elongate, terminate, and release newly synthesized globin chains in exogenous-mRNA-dependent, cell-free, protein-synthesizing systems from wheat embryo, ascites Krebs II cells, and rat liver. Furthermore, poly(A)-free globin mRNA competed with the same efficiency as authentic globin mRNA against chick ovalbumin mRNA when translated under total mRNA saturation conditions. It is apparent that the 3′-terminal poly(A) sequence is not necessary to maintain the translationally active secondary and tertiary configuration of the globin mRNA molecule. Preincubation of intact and deadenylylated globin mRNA in the Krebs II ascites translational system indicates that the presence of the poly(A) sequence may stabilize the translationally active mRNA molecule.
Fluorescence Enhancement of Laccase Induced by Reduction of Cu(II) Sites
Authors: Goldberg Michel , Pecht Israel
Published: May 2006

The intrinsic fluorescence of laccase (
p
-diphenol:O
2
oxidoreductase, EC 1.10.3.2), emitted by its tyrosinyl and tryptophanyl residues, underwent significant enhancement upon reduction of the enzyme redox sites. The increase in quantum yield reached its maximum after the addition of approximately four reduction equivalents and depended on the wavelength of excitation:

54% increase for λ
ex
= 250 nm and 76% for λ
ex
= 305 nm.

A linear correlation between this enhancement and the reduction of the type 1 Cu(II) was observed both by direct measurement and by calculation on the basis of added reductant.

The implications of the fluorescence enhancement and its correlation with the reduction of the type 1 copper are discussed in terms of the possible quenching mechanisms. The possibility of a redox-induced structural transition in the protein is suggested.
Localization of
Escherichia coli
Ribosomal Proteins S4 and S14 by Electron Microscopy of Antibody-Labeled Subunits
Authors: Lake James A. , Pendergast Maryanne , Kahan Lawrence , Nomura Masayasu
Published: May 2006

Binding sites for antibodies specific for proteins S4 and S14 of the small subunit of
E. coli
ribosomes have been mapped on the surface of the subunit by electron microscopy. Antibody binding to reconstituted subunits was shown to depend specifically on the presence of
E. coli
S4 and S14. Anti-S14 IgG was found to bind to a limited region of the ribosome surface. In contrast anti-S4 IgG was found to bind to three separated regions of the ribosome surface, suggesting S4 has an elongated conformation
in situ
.
On the Mechanism of Pyridoxine Responsive Homocystinuria. II. Properties of Normal and Mutant Cystathionine β-Synthase from Cultured Fibroblasts
Authors: Kim Young Jin , Rosenberg Leon E.
Published: May 2006

Cystathionine β-synthase [L-serine hydrolyase (adding homocysteine), EC 4.2.1.22] was studied in cultured skin fibroblasts from two control subjects and three patients with pyridoxine-responsive homocystinuria. In crude cell sonicates, cystathionine synthase activity detected in each mutant line was less than 5% of control values. After differential centrifugation, ammonium sulfate fractionation, and calcium phosphate gel treatment, the specific activity of synthase from control lines increased 5- to 7-fold with 70-79% yield. These same steps led to only 2- to 3-fold purification of mutant synthase and a reduced yield (26-44%). Michaelis-Menten analyses with the partially purified enzyme revealed that each mutant synthase had a marked reduction in affinity for its coenzyme, pyridoxal 5′-phosphate, as well as reduced affinity and maximum velocity for both co-substrates, L-homocysteine and L-serine. Even at saturating concentrations of coenzyme, mutant synthase activity was less than 3% of control. Mutant synthase was also far more thermolabile than control enzyme. In the absence of added coenzyme, heating for 10 min at 55° led to complete loss of mutant activity whereas control activity was reduced by 60%. Significantly, addition of saturating concentration of coenzyme prior to heating increased thermostability of both control and mutant synthase, the fractional increase being considerably greater in the mutants. We conclude that these patients suffer from a mutation of the synthase apoenzyme which impairs coenzyme binding, and that this primary abnormality results in reduced total enzyme activity in two ways: by reducing holoenzyme formation; and by accelerating apoenzyme degradation. We propose that pharmacologic amounts of pyridoxine increase holoenzyme formation modestly, thereby enhancing catalytic activity and slowing apoenzyme turnover.
Density Gradient Separation of Lymphoid Cells Adhering to Protein-A-Containing Staphylococci
Authors: Ghetie V. , Nilsson K. , Sjöquist J.
Published: May 2006

A cell separation technique was designed based on the interaction between cell-surface-bound IgG and protein A of
Staphylococcus aureus
. The density of lymphoid cells coated with IgG antibodies against one of the surface markers was increased by adherence of staphylococci. Cells with adhering bacteria were separated from cells without bacteria by density gradient centrifugation in 11.5% sodium metrizoate. Bacteria were removed from the lymphoid cells by lysostaphin digestion.

The purity of separated cells was approximately 95% even when the proportion of a specific cell population was below 10% in the initial mixture. The viability and the ability of cells to multiply
in vitro
were not significantly impaired by the fractionating procedure.

The technique can generally be applied for cell separation, provided antibodies of the IgG class against specific surface markers are available.
The Polypeptide Subunit Structure of the DNA-dependent RNA Polymerase of
Zea mays
Chloroplasts
Authors: Smith H. J. , Bogorad L.
Published: May 2006

Analysis of the maize chloroplast DNA-dependent RNA polymerase by electrophoresis on polyacrylamide gels that contain sodium dodecyl sulfate shows that the enzyme is multimeric. It contains at least two polypeptides of 180,000 and 140,000 daltons. Polypeptides of lower molecular mass that are associated with the enzyme at several stages of purification may also be subunits of the enzyme.

Electrophoresis of a mixture of purified maize nuclear DNA-dependent RNA polymerase IIA and chloroplast polymerase on sodium dodecyl sulfate-polyacrylamide gels resolves the 160,000 dalton polypeptide of IIA from the 140,000 dalton polypeptide of the chloroplast enzyme, but does not resolve the 180,000 dalton components found in both enzymes. The polypeptides of less than 40,000 daltons in the highly purified maize nuclear IIA preparations are absent from preparations of purified maize chloroplast RNA polymerase.
Termination Point of Replication of Colicin E1 Plasmid DNA in Cell Extracts
Authors: Sakakibara Yoshimasa , Tomizawa Jun-Ichi
Published: May 2006

Closed-circular monomeric molecules were one of the major products of replication of colicin E1 plasmid DNA in cell extracts. However, when the plasmid DNA synthesized in the reaction mixture was labeled for 3 min after 27 min of incubation, most of the label was found in open-circular molecules. The open-circular molecules were converted to closed-circular molecules upon further incubation. The newly replicated open-circular molecules had a nick or small gap in their newly synthesized strand. The interruption was located at approximately 20% of the molecular length from the single site of cleavage by restriction endonuclease
Eco
R1 and at or very close to the origin of replication. The addition of 10 mM nicotinamide mononucleotide instead of nicotinamide adenine dinucleotide to extracts did not significantly affect the kinetics of the colicin E1 plasmid DNA synthesis. However, in the presence of nicotinamide mononucleotide the formation of completely replicated closed-circular molecules was suppressed and, instead, open-circular molecules accumulated with an interruption in the newly synthesized strand at the termination point of replication, which was located at or very close to the origin of replication.
An Altered Rate of Uridine Transport in Membrane Vesicles Isolated from Growing and Quiescent Mouse 3T3 Fibroblast Cells
Authors: Quinlan Dennis C. , Hochstadt Joy
Published: May 2006

Balb/c 3T3 and simian virus-40-transformed Balb/c 3T3 (SV-3T3) cells were examined for their ability to transport nucleosides. Confluent (quiescent) 3T3 cells transported uridine at a rate 3-4 fold lower than did subconfluent cells. Adenosine uptake was independent of cell population density. Both adenosine and uridine were transported at the same rate by confluent and sub-confluent SV-3T3 cells. A membrane vesicle population (plasma membrane and endoplasmic reticulum) was isolated from 3T3 and SV-3T3 cells by nitrogen cavitation. With membrane vesicles derived from SV-3T3 cells it was determined that uptake occurred by a mediated process. Uptake of adenosine and uridine by membrane vesicles isolated from 3T3 and SV-3T3 cells demonstrated the same pattern as found in whole cells; that is, membrane vesicles from confluent 3T3 cells transported uridine at a rate 3-fold lower than did membrane vesicles from subconfluent 3T3 cells. Much of the adenosine taken up was converted to inosine, hypoxanthine, and ribose 1-phosphate, whereas uridine transport resulted only in the accumulation of uridine. Results obtained with membrane vesicles indicate that the lowered rate of uridine transport by confluent 3T3 cells seems due to an alteration in the membrane itself or a component(s) thereof, rather than to changes in subsequent cellular metabolic processes.
Long-Range, pH-Dependent Effects on the Carbon-13 Nuclear Magnetic Resonance Spectra of Oxytocin
Authors: Deslauriers Roxanne , Walter Roderich , Smith Ian C. P.
Published: May 2006

The effects of hydrogen ion concentrations on the carbon-13 nuclear magnetic resonance spectra of oxytocin were investigated. The starting pD of 3.0 was increased stepwise to 8.4. A change of the state of protonation of the N-terminal amino group of oxytocin is accompanied by changes in chemical shifts of carbon-13 nuclei of amino-acid residues located in the 20-membered ring of the hormone. The resonance positions of the acyclic peptide portion, Pro-Leu-Gly-NH
2
, remain constant. The pD-induced chemical-shift changes of carbons up to five bonds removed from the site of protonation are interpreted in terms of “through-bond” and “through-space” mechanisms. Chemical-shift changes of carbons more than five bonds removed are proposed to have a conformational origin. It is suggested that a change in the charge density of the amino group perturbs the dihedral angle of the —CH
2
—S—S—CH
2
— moiety of oxytocin, which in turn significantly affects the overall conformation of the 20-membered ring of the hormone.
The Site of Force Generation in Muscle Contraction as Deduced from Fluorescence Polarization Studies
Authors: Nihei T. , Mendelson Robert A. , Botts Jean
Published: May 2006

The fluorescent dye,
N
(-iodoacetylamino)-l-naphthylanine-5-sulfonic acid, labeled exclusively the myosin cross-bridges in rabbit glycerinated psoas muscle fibers, without impairing their function. Fluorescence polarization was used to study cross-bridge orientation in rigor, relaxation, and contraction, as a function of sarcomere length. At a length where no overlap between thick and thin filaments occurs, rigor-inducing, relaxation-inducing, and contraction-inducing solutions all induced the relaxation attitude. At lengths where overlap does exist, the slowly-hydrolyzing ATP analog, “α,β-methylene ATP,” induced the relaxation attitude. The data were consistent with the A. F. Huxley-Simmons model of force generation. Combined with our earlier results, the data indicated that torque was generated at the actin-myosin interface.
Ribosyl-
trans
-Zeatin, A Major Cytokinin Produced by Crown Gall Tumor Tissue
Authors: Miller Carlos O.
Published: May 2006

A cell division factor in crystalline form has been isolated from ethanolic extracts of cultured
Vinca rosea
L. crown gall tumor tissue. The crystalline material shows the melting point, mass spectrum, ultraviolet-light absorbancy spectra, solubilities, chromatographic migration values, ion-exchange behavior, and growth activities expected of ribosyl-
trans
-zeatin, that is, 6-(4-hydroxyl-3-methyl-
trans
-2-butenylamino)-9-β-D- ribofuranosylpurine. This compound, which appears to be a major cell-division factor in the extracts, has also been crystallized from liquid media in which the tumor tissue had been growing and apparently was the only active compound in such media.
Formation
In Vitro
of Infective Joint Molecules of λ DNA by T4 Gene-32 Protein
Authors: Wackernagel Wilfried , Radding Charles M.
Published: May 2006

Half molecules of λ DNA that had been partially digested by λ exonuclease to expose homologous single strands were rejoined by the action of T4 gene-32 protein at 37° in the presence of Mg
++
. Measurements of infectivity in
su
-
spheroplasts and sedimentation in sucrose demonstrated the formation of
sus
+
joint molecules from two preparations of sheared λ DNA that carried
sus
mutations at opposite ends of the genome. The biological activity of joint molecules made by annealing at 75° was diminished by the addition of the gene-32 protein in the absence of Mg
++
, and largely restored by the subsequent addition of Mg
++
. The specific infectivity of joint molecules made by gene-32 protein at 37° was similar to that of joint molecules made by annealing at 75°. The experimental system described provides a possible model for simulating early steps in genetic recombination.
The N-Terminal Amino-Acid Sequence of Bovine Proparathyroid Hormone
Authors: Hamilton James W. , Niall Hugh D. , Jacobs John W. , Keutmann Henry T. , Potts John T. , Cohn David V.
Published: May 2006

Proparathyroid hormone (calcemic fraction-A) is a biosynthetic precursor of parathyroid hormone in bovine glands. Limited amounts of the isolated prohormone have been obtained for the purpose of initial structural studies. The results of automated sequence analysis on two separate preparations indicate that the N-terminal region of the prohormone consists of the sequence Lys-Ser-Val-Lys-Lys-Arg followed by a sequence exactly corresponding to residues 1 to 34 of bovine parathyroid hormone. Also observed was a minor sequence (about 12% of the total) in which the N-terminal lysine was absent. These data suggest that more than one species of prohormone may exist. Due to the small quantities of sample available, the analyses were restricted to the N-terminal portion of the prohormone only. However, because the amino-acid composition of the prohormone indicates it to be a molecule containing more than 100 amino acids, the possibility remains that additional residues occur at the C-terminus. Thus, the prohormone structure based on these data is believed to consist of the hexapeptide sequence above, followed by the known sequence of the 84 residues in parathyroid hormone, possibly followed by an additional sequence of 10-15 residues.
Intracellular Cyclic AMP Concentration Responds Specifically to Growth Regulation by Serum
Authors: Oey Jan , Vogel Arthur , Pollack Robert
Published: May 2006

Intracellular concentrations of cyclic AMP increase when cells are deprived of serum. Studies with the mouse fibroblast line 3T3, an SV40-transformed subline of 3T3, and six different revertant lines derived from this clone show that a marked increase in cyclic AMP occurs only when the serum concentration is reduced below the minimum necessary for growth of a given line. Conversely, density-dependent inhibition of growth is not accompanied by an increase in cyclic AMP concentration in any line.
Fractionation of DNA on Hydroxyapatite with a Base-Specific Complexing Agent
Authors: Pakroppa Werner , Müller Werner
Published: May 2006

We describe a chromatographic technique for separating mixtures of DNA of varying G + C content. The method employs a specially prepared high-capacity hydroxyapatite and a G·C-specific DNA ligand (2-methyl-3-amino-7-dimethylamino-5-phenyl-phenazinium cation, abbreviated PNR). DNA molecules rich in G·C pairs bind larger amounts of this dye and are eluted earlier from the hydroxyapatite column than are molecules rich in A·T pairs. The dye can easily be removed from DNA by dialysis or solvent extraction after the chromatographic separation. The resolution of the method approaches that of CsCl density gradient separation, and the capacity of the column is much larger than that of a typical density gradient experiment. Elution of the DNA is not dependent on molecular weight, so samples of different molecular weight can be separated on the basis of G + C content. The technique should be especially useful for separating G·C-rich minor components from DNA obtained form eukaryotic cells, as demonstrated by a fraction of DNA from calf thymus.
Parathyroid Hormone Receptors of Renal Cortex: Specific Binding of Biologically Active,
125
I-Labeled Hormone and Relationship to Adenylate Cyclase Activation
Authors: Di Bella Francis P. , Douša Thomas P. , Miller Sam S. , Arnaud Claude D.
Published: May 2006

Biologically active
125
I-labeled bovine parathyroid hormone (prepared by electrolytic iodination) and its synthetic NH
2
-terminal (1-34) biologically active fragment bound rapidly and specifically to a purified plasma membrane preparation from bovine renal cortex. Binding of labeled intact hormone or labeled NH
2
-terminal (1-34) peptide was inhibited competitively by unlabeled (1-34) peptide in the same range of concentrations that activated renal cortical 3′:5′-adenylate cyclase (EC 4.6.1.1) in these membranes. The concentrations of synthetic (1-34) peptide for half-maximal inhibition of binding of labeled hormone as well as half-maximal activation of the enzyme were about 0.6 μM (2.5 μg/ml). Therefore it is likely that the binding activity studied represents a physiologically important renal receptor for parathyroid hormone.

Biologically inactive (oxidized) forms of parathyroid hormone and (1-34) NH
2
-terminal peptide as well as calcitonin, glucagon, insulin, and epinephrine failed to competitively inhibit the binding of labeled (1-34) parathyroid hormone or activate adenylate cyclase in the renal cortical membrane preparation.

Observations with the NH
2
-terminal (1-34) biologically active fragment of parathyroid hormone suggest that the COOH-terminal region of the molecule is not required for receptor binding.
Control of Translation in Cultured Cells: Continued Synthesis and Accumulation of Messenger RNA in Nondividing Cultures
Authors: Rudland Philip S.
Published: May 2006

Nontransformed animal fibroblasts in tissue culture regulate total protein and ribosomal RNA synthesis coordinately with changes in the cellular growth state. Here it is shown that the amounts and rates of synthesis of cytoplasmic poly(A)-containing RNA, presumed to be mRNA, do not appreciably change with alterations in growth state. In nongrowing (resting) cultures of BALB/c 3T3 cells presumptive mRNA molecules predominantly accumulate as cytoplasmic ribonucleoprotein particles; the RNA from which can be chased into the polyribosomal structure upon activation of the resting cultures with animal sera. It is suggested that the decreased protein synthetic rate in resting as compared with growing cells is in part due to a failure of preexisting mRNAs to attach to ribosomes and that addition of serum to the tissue culture medium can partially overcome this translational lesion.
Globoid Cell Leukodystrophy: Deficiency of Lactosyl Ceramide Beta-Galactosidase
Authors: Wenger David A. , Sattler Martha , Hiatt William
Published: May 2006

Activity of lactosyl ceramide β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23) was found to be extremely low in enzyme preparations from liver, brain, and cultured skin fibroblasts from patients with Krabbe's disease. Leukocytes from one set of parents had enzyme levels approximately half those measured in control leukocytes. The low activity observed for this galactolipid hydrolase is the fourth enzymatic deficiency noted for this genetic disease. Beta-galactosidase activity toward galactocerebroside, psychosine, and monogalactosyl diglyceride is also low in patients with Krabbe's disease. Other lysosomal enzymes measured were found to be in the normal range. This enzymatic defect may provide a better explanation for the pathological and chemical findings previously reported for this syndrome.
The Membrane Junctions in Communicating and Noncommunicating Cells, Their Hybrids, and Segregants
Authors: Azarnia R. , Larsen W. J. , Loewenstein W. R.
Published: May 2006

Human Lesch-Nyhan cells, which are coupling and have gap junctions, were fused with mouse cl-lD cells, which are noncoupling and lack gap junctions. The resulting hybrid cells were coupling and had gap junctions while they contained the nearly complete complement of parent chromosomes. As the hybrid cells lost human chromosomes, clones appeared among the segregants, which had reverted to the noncoupling and junction-deficient trait of the mouse parent cell. The human cell appears to contribute a genetic factor to the hybrids that corrects the junctional deficiency of the mouse cell.
Solution by Recursion of the
N
-Body Electrostatic Schrödinger Equation
Authors: Knirk Dwayne L.
Published: May 2006

A generalized partial wave expansion of atomic wavefunctions has been shown to allow exact solution of the many-electron Schrödinger equation. This solution is constructed here by an algebraic recursion method, and several analytical properties of such wave-functions are easily obtained. Eigenstates of the atom correspond to such solutions which satisfy certain boundary conditions for infinite extension of the system. A method of locating these states is presented.
Localization of Acetylcholine Receptor by
125
I-Labeled α-Bungarotoxin Binding at Mouse Motor Endplates
Authors: Fertuck Helen C. , Salpeter Miriam M.
Published: May 2006

Exposed sternomastoid muscles of anaesthetized mice were bathed in
125
I-labeled α-bungarotoxin until all neurally evoked muscle contractions were eliminated. The distribution of label was then determined by electron microscope autoradiography. It was found that the label was localized at the top of the junctional folds, i.e., at the postjunctional membrane nearest the axon. Since the α-bungarotoxin had fully eliminated the physiological muscle response, these results indicate that the active acetylcholine receptor occupies a limited area of the junctional folds and is not distributed uniformly throughout this membrane. Specialized membrane densities seem to coincide with the labeled regions.
Models for Positional Information and Positional Differentiation
Authors: Babloyantz A. , Hiernaux J.
Published: May 2006

It is commonly thought that the formation of patterns in developing organisms is due to the existence of a gradient of a morphogen that determines the fate of cells as a function of position in the organism. A model is presented based on a molecular mechanism where the gradient is established by the active transport of a morphogen between source and sink. The cellular differentiation and the subsequent spatial pattern formation results from the interaction of this morphogen with the genetic regulatory mechanisms of cells. Some properties of the model are given and discussed in relation to grafting experiments in hydra.
A Soluble Super-Active Form of Insulin
Authors: Oka Takami , Topper Yale J.
Published: May 2006

Insulin-like material can be extracted from insulin-Sepharose. This soluble material elicits super-insulin responses from mammary epithelial cells. It stimulates the accumulation of α-aminoisobutyric acid by virgin mouse mammary cells, which are unresponsive to insulin itself. It exerts a greater stimulatory effect on mammary cells from pregnant mice than insulin. It also is more efficacious than insulin as a stimulant of DNA synthesis and the combined activities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and phosphogluconate dehydrogenase (EC 1.1.1.43). Its specific biological activity on these cells is more than five times greater than that of insulin.