bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear Magnetic Resonance Spectroscopy. Carbon-13 Spin-Lattice Relaxation Time Measurements of Amino Acids
Authors: Armitage Ian M. , Huber H. , Pearson H. , Roberts John D.
Published: May 2006

The
13
C spin-lattice relaxation times,
T
1
's, of several amino acids have been measured as a function of pD and concentration. A strong dependence of the carboxyl carbon
T
1
was observed for both pD and concentration and is believed to be due to intermolecular associations. For the carboxyl carbon, spin rotation is proposed as the predominant relaxation mechanism, whereas the other carbons are relaxed mainly by the dipole-dipole mechanism, and their relaxation times are relatively independent of changes in concentration and pD.
Effect of Coenzyme Q on Serum Levels of Creatine Phosphokinase in Preclinical Muscular Dystrophy
Authors: Folkers Karl , Nakamura Ryo , Littarru Gian Paolo , Zellweger Hans , Brunkhorst John B. , Williams Coyle W. , Langston John H.
Published: May 2006

Coenzyme Q
10
(CoQ
10
) exists in human tissue, and is indispensable to mitochondrial enzymes of respiration. CoQ was administered to children with preclinical muscular dystrophy, CoQ enzymology was emphasized, and serum creatine phosphokinase, CPK, (ATP:creatine
N
-phosphotransferase, EC 2.7.3.2) was repeatedly monitored.

A 40-week treatment of an infant, 1-2 years of age, reduced serum CPK (
P
< 0.001; total CPK assays, 76). A 40-week treatment of a boy, 3-5 years of age, reduced serum CPK (
P
< 0.01); treatment through 80 weeks reduced CPK (
P
< 0.001; total CPK assays, 118).

This response of preclinical dystrophy to CoQ implies a deficiency of CoQ in skeletal muscle that was actually found previously by assay of the activity of the succinate dehydrogenase:coenzyme Q
10
reductase of the rectus abdominis. The relationships among a CoQ deficiency in muscle, serum CPK, and use of CPK in muscle are uncertain; however, restoration of CoQ enzyme activity in muscle by oral administration of CoQ could lead to increased use of CPK in muscle to form phosphocreatine from creatine and ATP, with a corresponding decrease in serum levels of CPK. The great excess of CPK in serum comes from deteriorating muscle in which CPK is below normal.
Transplantation of Autoimmune Potential. I. Development of Antinuclear Antibodies in H-2 Histocompatible Recipients of Bone Marrow from New Zealand Black Mice
Authors: Morton Jane I. , Siegel Benjamin V.
Published: May 2006

Antinuclear autoantibodies (ANA) appeared in the plasma of lethally irradiated H-2d histocompatible DBA/2 and BALB/c mice several weeks after intravenous transplantation of 2 to 4 × 10
6
bone marrow cells from 3-week-old animals of the autoimmune New Zealand Black (NZB) strain. Little or no ANA development was observed in DBA/2 or BALB/c strains when syngeneic or nonautoimmune allogeneic marrow was grafted, or when NZB marrow was injected into untreated DBA mice or mice receiving 200 rads of x-irradiation. Transfer of 5 × 10
6
spleen cells from 8-day-old NZB mice into lethally irradiated BALB/c mice effected substantial ANA formation by the ninth day after transfer, compared to a 20-day latency following transfer of the same number of bone marrow cells. This earlier conversion with splenocytes may have been due to the presence of immunocompetent T and B cells, since stem cell numbers of the two tissues were similar. Transplantation of NZB marrow to lethally irradiated H-2 incompatible SJL/J (H-2s) and C57B1/6 (H-2b) strains brought about less ANA conversion than the transfer of compatible (SJL × NZB)F1 and (C57B1 × NZB)F1 marrow cells to the respective nonautoimmune SJL or C57B1 parental strain. Graft-versus-host reactions thus did not appear to play a requisite or determining role in the autoimmune development observed following grafting of NZB hemopoietic tissues. Reconstitution of lethally irradiated NZB mice with BALB/c or SJL/J bone marrow depressed the recurrence of ANA for 30 days, compared to rapid ANA recovery following NZB marrow injection. The characteristics that ultimately provoke or permit spontaneous auto-reactivity are inherent in the hemopoietic stem cell population of the NZB strain.
The Homopolyadenylate and Adjacent Nucleotides at the 3′-Terminus of 30-40S RNA Subunits in the Genome of Murine Sarcoma-Leukemia Virus
Authors: Rho Hyune Mo , Green Maurice
Published: May 2006

Adenosine is the major 3′OH-terminal nucleoside of the 60-70S RNA genome of the murine sarcoma-leukemia virus, its 30-40S RNA subunits, and the poly(A) segments derived by RNase treatment of both RNA species, as determined by periodate oxidation-[
3
H]-borohydride reduction. The binding 30-40S RNA to oligo(dT)-cellulose suggests that most viral RNA subunits contain poly(A). The molecular weight of poly(A) derived from viral RNA by digestion with RNase and purified by affinity chromatography is 64,000-68,000, as determined by gel electrophoresis. From the size of poly(A) and the poly(A) content of viral RNA (1.6%), it is estimated that there is about one poly(A) segment for each viral 30-40S RNA subunit. The results of 3′-termini labeling with [
3
H]borohydride,
in vivo
labeling with [
3
H]adenosine, and base composition of [
32
P]poly(A) indicate that a homopoly(A) segment is located at the 3′-end of a 30-40S RNA subunit. The homogeneous poly(A) segments isolated from RNase T1 digests of 60-70S [
32
P]RNA consist of one cytidylate, one uridylate, and about 190 adenylate residues, while those isolated from RNase A digests consist exclusively of adenylate residues. These results indicate that -G(C,U)A
190
A
OH
is the 3′-terminal nucleotide sequence of the viral 30-40S RNA subunits.
Short-Term Binding and Hydrolysis of Cyclic 3′:5′-Adenosine Monophosphate by Aggregating
Dictyostelium
Cells
Authors: Malchow D. , Gerisch G.
Published: May 2006

Transient binding of cyclic AMP to aggregating cells of
Dictyostelium discoideum
was measured under cyclic GMP excess in order to inhibit cyclic AMP hydrolysis by cell-bound phosphodiesterase. Cyclic GMP extended the period of half-maximal cyclic AMP binding from less than 5 sec to 1-2 min. With the same time course as bound cyclic AMP was released from the cells, labeled 5′-AMP appeared in the medium. Specificity, kinetics, and developmental regulation suggest that the cyclic AMP-binding sites exposed in living cells are identical with receptor sites for the chemotactic response. The functioning of the cyclic AMP-receptor/phosphodiesterase system and its formal similarity with the synaptic acetylcholine-receptor/esterase system are discussed.
In Vitro
Genetic Recombination of Bacteriophage λ
Authors: Syvanen Michael
Published: May 2006

DNA of bacteriophage λ recombines in a cell-free extract prepared from an induced
Escherichia coli
lysogen of bacteriophage λ. The assay for recombination
in vitro
takes advantage of the ability of such an extract to package λ DNA and to assemble complete phage particles. For example, when λ DNA that has been extracted from phage with the immunity of 434 is added to an extract, infectious λ
imm
434 particles are produced. The precursor DNA molecule in this packaging reaction is a multichromosomal polymer; circular monomers, for example, are not packaged.

Nevertheless, when 434 circular DNA monomers are added to an extract, some phage that contain the
imm
434 marker are produced. In this case, the circular DNA had recombined with λ DNA in the extract and thereby had become part of a polymeric structure, which by the normal packaging process could give rise to infectious particles with the
imm
434 marker. Genetic recombination is demonstrated when
imm
434 circular monomer DNA carries amber mutations in genes
A
and
B
; then most of the 434 plaque formers produced
in vitro
are
A
+
B
+
, the genotype of the endogenous λ DNA. Genetic crossing-over occurs through a region that contains the prophage attachment site, suggesting that recombination is carried out by the λ Int functions. The 434 recombinant plaque formers are particles physically identical to wild-type 434 particles, as judged by their buoyant density in a CsCl equilibrium gradient.
The Mechanism of Viral Replication. Structure of Replication Complexes of Encephalomyocarditis Virus
Authors: Thach Sigrid S. , Dobbertin Darrell , Lawrence Charles , Golini Fred , Thach Robert E.
Published: May 2006

The structure of the purified replicative intermediate of encephalomyocarditis virus was determined by electron microscopy. Approximately 80% of the replicative intermediate complexes were characterized by a filament of double-stranded RNA of widely variable length, which had a “bush” of single-stranded RNA at one end. In many examples one or more additional single-stranded bushes were appended internally to the double-stranded RNA filament. These results support the view that before deproteinization, replicative intermediate contains little if any double-stranded RNA.
Vitamin K Dependent Modifications of Glutamic Acid Residues in Prothrombin
Authors: Stenflo Johan , Fernlund Per , Egan William , Roepstorff Peter
Published: May 2006

A tetrapeptide, residues 6 to 9 in normal prothrombin, was isolated from the NH
2
-terminal, Ca
2+
-binding part of normal prothrombin. The electrophoretic mobility of the peptide was too high to be explained entirely by its amino-acid composition. According to
1
H nuclear magnetic resonance spectroscopy and mass spectrometry, the peptide contained two residues of modified glutamic acid, γ-carboxyglutamic acid (3-amino-1,1,3-propanetricarboxylic acid), a hitherto unidentified amino acid. This amino acid gives normal prothrombin the Ca
2+
-binding ability that is necessary for its activation. Observations indicate that abnormal prothrombin, induced by the vitamin K antagonist, dicoumarol, lacks these modified glutamic acid residues and that this is the reason why abnormal prothrombin does not bind Ca
2+
and is nonfunctioning in blood coagulation.
Some New Asymptotic Fixed Point Theorems
Authors: Browder Felix E.
Published: May 2006

For a continuous self mapping
f
of a locally convex topological vector space which is locally compact (i.e.,
f
maps a neighborhood of each point into a relatively compact set), it is shown that a sufficient condition for the existence of a fixed point is the existence of a compact attractor
K
0
such that each orbit under
f
has a point of
K
0
in its closure. The proof is based upon the circle of ideas of the Lefschetz fixed point theorem.
Cell-free Synthesis of Adenovirus 2 Proteins Programmed by Fractionated Messenger RNA: A Comparison of Polypeptide Products and Messenger RNA Lengths
Authors: Anderson C. W. , Lewis J. B. , Atkins J. F. , Gesteland R. F.
Published: May 2006

Cytoplasmic RNA extracted from human tissue culture cells infected with adenovirus type 2 was used to program protein synthesis in a cell-free system derived from mammalian cells. Analysis of the protein product by polyacrylamide gel electrophoresis revealed ten adenovirus-specific polypeptides. Five of these were further identified by analysis of tryptic peptides. Translation of RNA fractionated by sedimentation through sucrose gradients containing formamide demonstrated seven size classes of RNA, each of which programmed the synthesis of only one or two virus-specific polypeptides. Six of the virus-specific polypeptides were translated from RNAs much larger than expected for the size of the polypeptide.
On Some Principles Governing Molecular Evolution
Authors: Kimura Motoo , Ohta Tomoko
Published: May 2006

The following five principles were deduced from the accumulated evidence on molecular evolution and theoretical considerations of the population dynamics of mutant substitutions: (
i
) for each protein, the rate of evolution in terms of amino acid substitutions is approximately constant/site per year for various lines, as long as the function and tertiary structure of the molecule remain essentially unaltered. (
ii
) Functionally less important molecules or parts of a molecule evolve (in terms of mutant substitutions) faster than more important ones. (
iii
) Those mutant substitutions that disrupt less the existing structure and function of a molecule (conservative substitutions) occur more frequently in evolution than more disruptive ones. (
iv
) Gene duplication must always precede the emergence of a gene having a new function. (
v
) Selective elimination of definitely deleterious mutants and random fixation of selectively neutral or very slightly deleterious mutants occur far more frequently in evolution than positive Darwinian selection of definitely advantageous mutants.
Glutathione Turnover in the Kidney; Considerations Relating to the γ-Glutamyl Cycle and the Transport of Amino Acids
Authors: Sekura Ronald , Meister Alton
Published: May 2006

The overall turnover of glutathione in kidney and liver was determined in studies in which L-[
14
C]glutamate was administered to mice. Turnover was much more rapid (about 5 times greater) in kidney than in liver. Studies were also carried out in which 5-oxo-L-[
14
C]proline was administered; the first order rate constants for glutathione synthesis from 5-oxoproline in liver and kidney were not far from those found for synthesis of glutathione from glutamate in these tissues. The findings are in accord with the fact that the activities of the enzymes of the γ-glutamyl cycle are much higher in kidney than in liver. The findings of high turnover of glutathione in kidney and the rapid utilization of 5-oxoproline by this organ for glutathione synthesis are consistent with the function of the γ-glutamyl cycle
in vivo
and the proposed role of γ-glutamyl derivatives in amino-acid transport.
Translation
In Vitro
of Total Nuclear RNA from HeLa Cell Nuclei Infected with Adenovirus 2
Authors: Chatterjee Nando K. , Weissbach Herbert
Published: May 2006

Total heterogeneous nuclear RNA from HeLa cells infected with adenovirus for 18-20 hr stimulates amino acid incorporation into protein in a cell-free system from Ehrlich ascites tumors. This stimulation of protein synthesis by the nuclear RNA requires intact nuclei. The role of nuclei in this system is unknown, but evidence is presented that the nuclei are involved in the conversion of high-molecular-weight RNA to low-molecular-weight species. Some of the newly synthesized polypeptides appear to resemble the virion polypeptides.
Conservation of 70S Ribosomal RNA Genes in the Chloroplast DNAs of Higher Plants
Authors: Thomas J. R. , Tewari K. K.
Published: May 2006

Chloroplast DNAs of higher plants have been found to contain two chloroplast ribosomal RNA genes. The base sequences of these chloroplast ribosomal RNA genes in chloroplast DNAs have been studied by molecular hybridization. The results have shown that these genes have undergone little divergence in the evolution of either monocotyledonous or dicotyledonous plants.
Isolation of a Major Cell Surface Glycoprotein from Fibroblasts
Authors: Yamada Kenneth M. , Weston James A.
Published: May 2006

A cell surface component has been isolated in partially purified form from cultured chick embryo and chick heart fibroblasts. This glycoprotein is similar to a protein recently reported to be present at the surface of normal cells, and missing after neoplastic transformation. It is a major cell surface glycoprotein that is synthesized by cultured fibroblasts, but is not collagen. It is shown to be markedly trypsin-sensitive, and its recovery from the cell surface is dependent on cell density. It is excluded from Sephadex G-200, but is not rapidly sedimented by ultracentrifugation, and has an apparent molecular weight of 220,000. The isolation of this cell surface glycoprotein may now provide a means of determining its function.
Intracellular Restraint: A New Basis for the Limitation in Response to Oxidative Stress in Human Erythrocytes Containing Low-Activity Variants of Glucose-6-Phosphate Dehydrogenase
Authors: Gaetani Gianfranco D. , Parker John C. , Kirkman Henry N.
Published: May 2006

Several mechanisms recently proposed for regulation of the hexose monophosphate shunt require the concentration of NADP to be low or that of NADPH to be high. The present study indicates that the first enzyme of the hexose monophosphate shunt of human erythrocytes is under severe restraint even when these conditions do not exist. In human erythrocytes containing low-activity variants of this enzyme, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP
+
1-oxidoreductase; EC 1.1.1.49), measurements of the rate of oxidation of C-1 labeled glucose show that the enzyme is operating at a rate much closer to its maximum than in normal cells. This requires that the ratio of inhibitory NADPH to NADP be much lower in the variant cells than in normal cells. A small increase in oxidative rate, induced by naphthol, then causes a disappearance in reduced glutathione in the variant cells, presumably because a significant further decrease in NADPH occurs in these cells, whereas the same oxidative stress in normal cells would not lower the NADPH level appreciably. A low NADPH/NADP ratio in unstressed cells deficient in glucose-6-phosphate dehydrogenase is confirmed by direct measurement. The maximum activity of the variant enzyme in the cell, as measured with methylene blue to keep most of the NADP in the oxidized form, is only about 1/60 of that found in hemolysates, thus accounting for the failure to compensate for a relatively small oxidative stress
in vivo
in spite of an apparent sufficiency of enzyme. The reason for the limitation on maximum intracellular activity is unknown. A similar limitation is seen with normal cells incubated with methylene blue, where the maximum intracellular rate is also only about 1/60 of that found in hemolysates.
Collagenase Production by Endotoxin-Activated Macrophages
Authors: Wahl Larry M. , Wahl Sharon M. , Mergenhagen Stephan E. , Martin George R.
Published: May 2006

Peritoneal exudate macrophages, when exposed to bacterial lipopolysaccharide in culture, were found to produce collagenase (EC 3.4.24.3). This enzyme was not detected in extracts of the macrophages or in media from nonstimulated macrophage cultures. Lipidcontaining fractions of the lipopolysaccharide, including a glycolipid from the rough mutant of
Salmonella minnesota
(R595) and lipid A, were potent stimulators of collagenase production. The lipid-free polysaccharide fraction had no effect. Cycloheximide prevented the production of collagenase by endotoxin-treated macrophages, suggesting that it was newly synthesized.
The Use of Cytochalasin B to Distinguish Myoblasts from Fibroblasts in Cultures of Developing Chick Striated Muscle
Authors: Sanger J. W.
Published: May 2006

Cytochalasin B (5 μg/ml) elicits a differential effect on myoblasts and fibroblasts in culture. After 1 day in culture in the presence of the drug, two types of cells were observed, round cells and cells with elongated arms, designated “arborized” cells. Both cell types were examined in the electron microscope. The round cells contained aggregates of thin and thick filaments as well as a few intact sarcomeres. Within the arms of the arborized cells were bundles of intermediate sized filaments (100 Å in diameter). The round cells could be shaken off the culture dish, washed free of cytochalasin B, and recultured to form myotubes. The remaining arborized cells lost their stellate shape when the drug was removed. The progeny of these cells gave rise to normal fibroblasts. Cytochalasin B, thus could be used to identify and isolate myoblasts prior to their fusion into developing muscle. It is suggested that this differential effect of the drug can be used to prepare pure cultures of fusible muscle cells uncontaminated by fibroblasts.
Direction of Transcription of the Regulatory Gene
araC
in
Escherichia coli
B/r
Authors: Wilcox Gary , Boulter James , Lee Nancy
Published: May 2006

The protein product of the regulatory gene
araC
can be synthesized in a cell-free, protein-synthesizing system programmed with a λ

paraC
+
B

DNA template. Hybrid, renatured phage DNA molecules prepared with DNA from phages λ

paraC
+
B

and λ
paraC3B
(
araC3
is a nonsense mutation) were used to program the cell-free synthesis of the
araC
protein. The findings observed lead to the conclusion that the codogenic strand of the
araC
gene is on the light strand of the phage DNA. The
araB
gene is on the heavy strand, as determined by DNA·RNA hybridization. Thus, with regard to the standard
E. coli
map,
araC
is transcribed in a clockwise direction, whereas transcription of the
araBAD
operon has a counterclockwise orientation. The technique described should allow one to determine the direction of transcription of any gene that can be incorporated into the genome of a specialized transducing phage.
An
N
-Glycosidase from
Escherichia coli
That Releases Free Uracil from DNA Containing Deaminated Cytosine Residues
Authors: Lindahl Tomas
Published: May 2006

An enzyme that liberates uracil from single-stranded and double-stranded DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers in the absence of Mg
++
has been purified 30-fold from cell extracts of
E. coli
. The enzyme does not release uracil from deoxyuridine, dUMP, uridine, or RNA, nor does it liberate the normally occurring pyrimidine bases, cytosine and thymine, from DNA. The enzymatic cleavage of
N
-glycosidic bonds in DNA occurs without concomitant cleavage of phosphodiester bonds, resulting in the formation of free uracil and DNA strands of unaltered chain length that contain apyrimidinic sites as reaction products. The enzyme may be active in DNA repair, converting deaminated dCMP residues to an easily repairable form.
Quantitative Estimation of
Agrobacterium tumefaciens
DNA in Crown Gall Tumor Cells
Authors: Drlica Karl A. , Kado C. I.
Published: May 2006

Several reports suggest that
Agrobacterium tumefaciens
nucleic acids can induce transformation of the cells of susceptible host plants and that bacteria-free tissue cultures of transformed cells contain
A. tumefaciens
DNA, RNA, antigens, or bacteriophages. We assayed
Vinca rosea
tumor DNA for base sequence homologies with
A. tumefaciens
DNA by DNA·DNA solution enrichment and DNA·DNA filter saturation hybridization techniques. No homologies were found by either method. The filter saturation hybridization technique included model filters containing known percentages of bacterial DNA mixed with
V. rosea
leaf DNA. Using this sensitive technique, we found that no more than 0.02% of the crown gall tumor genome could be homologous to
A. tumefaciens
DNA. This upper estimate of homology corresponds to 0.2 bacterial genome equivalent per diploid tumor cell.
Mutations affecting sexual development in Phycomyces blakesleeanus.
Authors: Sutter R P
Published: May 2006

Although zygospore (mature zygote) formation in P. blakeslleeanus occurs in liquid glucoseglutamate medium, morphological observations are made more easily when cultures are grown on 1-mm-thick agar medium. Zygophores (sexually differentiated hyphae) develop prior to physical contact in crosses of (plus) and (minus) wild types. Zygophores interlock upon contact and then undergo six successive morphological changes to become a zygospore. Mutants with abnormal carotene synthesis exhibit aberrant sexual behavior. Some zygospores do form in crosses of carA mutants and wild types. Only paired zygophores form in crosses of wild type(plus) with car-42(minus), a beta-carotene-accumulating mutant. Zygophores form only on (minus) in crosses of wild type(plus) with carB(minus), carR(minus), carAcarR(minus), and carBcarR(minus) mutants, and only on (plus) in crosses of car-43(plus) with wild type(minus), car-42(minus), and carA(minus) mutants. Zygophores do not form in crosses of car-43(plus) with carB(minus), carR(minus), carAcarR(minus), and carBcarR(minus) mutants. These observations demonstrate that each mating type makes a chemical messenger that stimulates zygophore development in the opposite mating type.
Microbial production of ammonium ion from nitrogen.
Authors: Shanmugam K T , Valentine R C
Published: May 2006

Genetic manipulation of nitrogenase and key glutamate-forming enzymes can provide mutants that excrete fixed N2 as NH4+. A derepressed N2 fxation mutant (SK-24) has been isolated , which excretes up to 20.2 mumol of fixed N2 as NH4+ per mg of cell protein in 24 hr at room temperature. Biochemical analysis shows that this mutant, which requires glutamate for growth, releases fixed N2 as NH4+ into the environment because of (i) constitutive synthesis of nitrogenase and (ii) genetic blocks resulting in losses of glutamate synthase [L-glutamine:2-oxoglutarate aminotransferase (NADPH oxidizing), EC 2.6.1.53] and glutamate dehydrogenase [L-glutamate:NADP oxidoreductase (deaminating), EC 1.4.1.4] activities, enzymes essential for NH4+ assimilation into cell material. The parent strain (asm-1), missing only glutamate synthase activity, also actively excretes NH4+ during early phases of its growth but eventually reutilizes the NN4+. A miximum yield of 4.0 mumol of NH4+/ml per 24 hr has been noted for asm-1 only during the growth period. Biosynthesis of NH4+ PROCEEDS AT THE EXPENSE OF A Variety of fermentable sugars, such as sucrose or glucose, with a maximum energy conversion efficiency of about 5 glucose degraded per NH4+ formed. The use of microbes for production of NH4+ fertilizer is discussed.
Differences in the surface proteins of mouse B and T cells.
Authors: Trowbridge I S , Ralph P , Bevan M J
Published: May 2006

We have selectively labeled the surface of mouse thymus-dependent (T) and thymus-independent (B) lymphocytes by means of lactoperoxidase-catalyzed iodination. Examination of the labeled proteins by electrophoresis in the presence of sodium dodecylsulfate followed by autoradiography revealed differences in the cell surface proteins of B and T cells. Proteins from labeled thymocytes, T cell lymphomas, and both normal and activated peripheral T cells give a broad band of radioactivity corresponding to at least two protein components with apparent molecular weights of 170,000 to 190,000. This band is absent from autoradiographs of iodinated B cell proteins, which instead have another band corresponding to a protein with an apparent molecular weight of about 220,000. We have shown that these T and B cell marker proteins are synthesized by the cell and are not serum components selectively bound to the cell surface. We have also established that these proteins are the major iodinated species precipitated from Nonidet P-40 cell lysates by rabbit anti-mouse lymphocyte serum, suggesting that they are the major antigens recognized by rabbit antibodies to mouse lymphocytes.
Biosynthesis of polypeptides of cytochrome c oxidase by isolated mitochondria.
Authors: Poyton R O , Groot G S
Published: May 2006

Yeast mitochondria, incubated with radioactive amino acids in a "protein-synthesizing mixture" containing an oxidizable substrate and an ATP regenerating system, have been shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to incorporate label into polypeptides equivalent in molecular weight and relative amount ot those made in vivo in the presence of cycloheximide. The ability of these isolated mitochondria to synthesize "native" polypeptides was assessed by examining the incorporation of label into subunits of cytochrome c oxidase (EC 1.9.3.1). An analysis of immunoprecipitates formed by incubating cholate extracts of labeled mitochondria with an antiserum against holocytochrome c oxidase revealed that label was incorporated into three polypeptides of sizes equivalent to those of cytochrome c oxidase subunits I, II, and III, shown from earlier studies in vivo to be translated on mitochondrial ribosomes. Further evidence that these polypeptides made in vitro are "native" and identical to subunits I, II, and III was provided by the observation that labeled polypeptides equivalent in size to subunits I-III- ARE ALSO IMMUNO-PRECIPITATED BY ANTISERUM AGAINST SUBUNITS V plus VII, an antiserum that can precipitate subunits I, II, and III only when they are complexed to the cytoplasmically synthesized subunits, V and VII, of the enzyme. These results suggest that isolated mitochondria are capable of synthesizing three subunits of cytochrome c oxidase and assembling them into a holoenzyme.
A nonidentifiability aspect of the problem of competing risks.
Authors: Tsiatis A
Published: May 2006

For an experimental animal exposed to k greater than 1 possible risks of death R1, R2, ..., Rk, the term i-th potential survival time designates a random variable Yi supposed to represent the age at death of the animal in hypothetical conditions in which Ri is the only possible risk. The probability that Yi will exceed a preassigned t is called the i-th net survival probability. The results of a survival experiment are represented by k "crude" survival functions, empirical counterparts of the probabilities Qi(t) that an animal will survive at least up to the age t and eventually die from Ri. The analysis of a survival experiment aims at estimating the k net survival probabilities using the empirical data on those termed crude. Therorems 1 and 2 establish the relationship between the net and the crude probabilities of survival. In particular, Theorem 2 shows that, without the not directly verifiable assumption that in their joint distribution the variables Y1, Y2, ..., Yk are mutually independent, a given set of crude survival probabilities Qi(t) does not identify the corresponding net probabilities. An example shows that the results of a customary method of analysis, based on the assumption that Y1, Y2, ..., Yk are independent, may have no resemblance to reality.
Translation and identification of the mRNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.
Authors: Both G W , Moyer S A , Banerjee A K
Published: May 2006

Vesicular stomatitis virus messenger RNA has been transcribed in vitro from the viral genome by the virion-associated RNA polymerase in quantities suitable for translation. Wheat germ cell-free extracts programmed with the isolated in vitro 12-18S RNA fraction synthesize polypeptides similar to the viral N, NS, and M proteins, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping of the in vitro products and the viral marker polypeptides. In addition, the RNA synthesized in vitro also codes for a protein of molecular weight 63,000 which may be a nonglycosylated form of the viral glycoprotein G. The 12-18S RNA has been partially separated into individual messenger species and these have been identified by the proteins for which they code. There are four monocistronic messenger species in the in vitro 12-18S RNA and the coding capacity of three of these molecules agrees with the estimated molecular weight of the polypeptide assigned to it.
Methylated nucleotides block 5'-terminus of vaccinia virus messenger RNA.
Authors: Wei C M , Moss B
Published: May 2006

Studies on the nature and location of the methylated nucleotides in mRNA synthesized in vitro by vaccinia virus particles revealed an unusual 5'-terminal structure. Evidence that the pyrophosphate group is blocked by 7-methylguanosine and that both 2'-O-methyl-adenosine and 2'-O-methylguanosine occupy penultimate positions was presented. According to this model, the 5'-termini of vaccinia virus mRNAs are: 7MeG-5'ppp-5'GMepNp and 7MeG-5'AMepNp.
Site-directed mutagenesis: effect of an extracistronic mutation on the in vitro propagation of bacteriophage Qbeta RNA.
Authors: Flavell R A , Sabo D L , Bandle E F , Weissmann C
Published: May 2006

It has been proposed that the nucleotide sequences of the 3' terminal extracistronic regions of phage RNA plus and minus strands have been strictly conserved during evolution because they are stringently required for recognition by the viral replicase. We have devised a method to generate point mutations at selected sites in the genome of phage Qbeta. The in vitro synthesis of Qbeta RAN with G leads to A transition in the 16th position from the 3' end, i.e., in the terminal extracistronic region of the genome, is outlined. When a mixture of about 60% wild-type and 40% mutant RNA was repeatedly replicated, the mutant RNA was enriched to 80%, showing that at least this point mutation in the terminal sequence of Qbeta RNA does not impair its in vitro replication, but in fact slightly accelerates it.
Crystal structure of human erythrocyte carbonic anhydrase B. Three-dimensional structure at a nominal 2.2-A resolution.
Authors: Kannan K K , Notstrand B , Fridborg K , Lövgren S , Ohlsson A , Petef M
Published: May 2006

The three-dimensional structure of carbonic anhydrase B (EC 4,2,1,1; carbonate hydro-lyase) from human erythrocytes has been determined to high resolution. Parallel and antiparallel pleated sheet makes up the predominant secondary structure of the enzyme. The tertiary structure is unique for its folding and is very similar to the structure is unique for its folding and is very similar to the structure of the isoenzyme, human erythrocyte carbonic anhydrase C. The essential metal ion, zinc, is firmly bound to the enzyme through three histidyl ligands and located at the bottom of a 12-A deep conical cavity. The zinc ligands are involved in a number of hydrogen bond formations with residues in the immediate vicinity of the active site cavity. Some of the similarities and differences in the sidechain orientation and active site topography of the two isoenzymes are also discussed.
Matrix method for fluctuations and noise in kinetic systems.
Authors: Chen Y D
Published: May 2006

In a series of papers we were concerned with the question of how to calculate the concentration noise power spectra of an ensemble of multi-state linear kinetic systems when the rate constants of the systems are assumed to be known. We have used a standard eigenvalue-eigenfunction method to solve the differential equations which govern the regression of the means and derived the noise power spectrum as a function of the eigenvalues and eigenfunctions of the relaxation matrix of the system. In this paper, we have obtained an equation which relates the noise spectrum matrix of the fluctuations directly to the relaxation matrix of the means. As a result, the noise power spectrum can be calculated through matrix operations without the necessity of an eigenvalue-eigenfunction calculation. The present formalism is particularly useful in the evaluation of kinetic rate constants when the noise spectrum data of concentration fluctuations are given. Possible applications to biochemical systems are briefly discussed.
DNA replication in vitro starting with an intact phiX174 phage.
Authors: Jazwinski S M , Kornberg A
Published: May 2006

Conversion of the single-stranded DNA in the intact phiX174 phage particle to the duplex replicative form (RF) has been demonstrated in lysates form phage-sensitive cells. The conversion is resistant to rifampicin and requires participation of both a "membrane" fraction of the lysate and a multienzyme replicative system. The lipopolysaccharide phage receptor, while essential, does not replace the membrane fraction. Clear, nonsedimentable extract fractions prepared with a certain nonionic detergent can replace the membrane fraction. Purification of the activity in these extracts by adsorption to polypropylene film yields a fraction with a 5-fold increase in activity relative to lipopolysaccharide and 50-fold increase relative to protein. The low buoyant density (1.03 g/cm3) suggests a high phospholipid or detergent content in this fraction.
Neutron scattering measurements of separation and shape of proteins in 30S ribosomal subunit of Escherichia coli: S2-S5, S5-S8, S3-S7.
Authors: Engelman D M , Moore P B , Schoenborn B P
Published: May 2006

Neutron scattering measurements done on E. coli 30S ribosomal subunit specimens in which specific pairs of proteins were deuterated have enabled us to estimate the distances between the labeled proteins. The distances between centers of gravity of three protein pairs have been determined: S2-S5 (105 A), S3-S7 (115 A), and S5-S8 (35 A). A method for extracting shape information about these proteins from the neutron scattering profiles is demonstrated. The method shows that S5 and S8 are compact and S2 is extended.
Assembly of microtubules onto kinetochores of isolated mitotic chromosomes of HeLa cells.
Authors: Telzer B R , Moses M J , Rosenbaum J L
Published: May 2006

The kinetochores of isolated HeLa cell chromosomes attached to an electron microscope specimen grid, fixed in formaldehyde, and stained with alcoholic phosphotungstic acid are visible as dark, preferentially stained structures distinct from the chromatin with which they are associated. When unfixed chromosomes are immobilized by attachment to grids and incubated with chick brain tubulin, microtubules are observed to assemble onto the kinetochores. This demonstrates the competence of kinetochores in isolated chromosomes to act in vitro as microtubule assembly sites and suggests that they also possess this capacity in vivo. In addition, the results provide a possible means for isolating and characterizing kinetochores.
Increased cellular levels of spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A.
Authors: Fillingame R H , Jorstad C M , Morris D R
Published: May 2006

There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) blocks synthesis of these polyamines by inhibiting S-adenosylmethionine decarboxylase. Previous results showed that when cells are activated in the presence of MGBG the synthesis and processing of RNA, as well as protein synthesis, proceed as in the absence of the drug. In contrast, the incorporation of [methyl-3H]thymidine into DNA and the rate of entry of the cells into mitosis are inhibited by 60% in the presence of MGBG. Several experiments suggest that MGBG inhibits cell proliferation by directly blocking polyamine synthesis and not by an unrelated pharmacological effect: (1) the inhibitory action of MGBG is reversed by exogenously added spermidine or spermine; (2) inhibition of DNA synthesis by MGBG shows the same dose-response curve as does inhibition of spermidine and spermine synthesis; and (3) if MGBG is added to cells which have been allowed to accumulate their maximum complement of polyamines, there is no inhibition of thymidine incorporation. MGBG-treated and control cultures initiate DNA synthesis at the same time and show the same percentage of labeled cells by autoradiography. Therefore, it appears that in the absence of increased cellular levels of polyamines, lymphocytes progress normally from G0 through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication.
Linkage disequilibrium over space and time in natural populations of Drosophila montana.
Authors: Baker W K
Published: May 2006

The previously described allelic frequencies and linkage disequilibrium among the active and null alleles of four tightly linked loci coding for the alpha-esterases were found to be maintained by one population for 5 years, and were found to be present in two other populations which were shown to be genetically distinct from the first. It appears that enzyme variants coded by these highly polymorphic loci are being maintained in the populations by selective forces.
Collisional energy transfer and quenching of electronic excitation
Authors: Lin S. H. , Eyring H.
Published: May 2006

The purpose of this paper has been to explore in a preliminary way the nature and mechanism of collisional energy transfer and quenching of electronic excitation. For this purpose, the Born approximation has been used, and the triplet-triplet and singlet-singlet transfer, and the triplet-triplet and singlet-singlet quenching have been studied. It has been shown theoretically that (
i
) the singlet-singlet transfer constants (or cross sections) are always larger than the triplet-triplet transfer constants (or cross sections) for the same system of donor and acceptor; (
ii
) for the singlet-singlet transfer, the observed cross section varies linearly with respect to the spectral overlap between the donor emission and the acceptor absorption; (
iii
) the reason that the quenching constants (or cross sections) are always smaller than the energy transfer constants (or cross sections) is due to the fact that for the quenching the vibration of the acceptor hardly participates in accepting the electronic excitation and for the energy transfer only part of the excited electron energy of the donor is converted into the energy of nuclear motion; and (
iv
) the polar acceptor molecules are better quenchers than nonpolar acceptor molecules.
On complexifications of real manifolds
Authors: Kulkarni R. S.
Published: May 2006

This paper studies the problem of obtaining complexifications of a differentiable manifold which have desirable analytic or algebraic properties and which are minimal in the sense described below. It is seen that there is a significant difference between analytic and algebraic complexifications.
Nucleotide sequence of microvariant RNA: another small replicating molecule.
Authors: Mills D R , Kramer F R , Dobkin C , Nishihara T , Speigelman S
Published: May 2006

Microvariant RNA, a small self-replicating molecule (114 nucleotides long), has been isolated from Qbeta replicase reactions incubated in the absence of exogenous template. Its complete nucleotide sequence has been determined. Comparison with MDV-1 RNA, a somewhat larger endogenous Qbeta replicase product (220 nucleotides long) that had previously been characterized, revealed no significant sequence similarity. Since Qbeta replicase can mediate the synthesis of both of these disparate RNA molecules, primary sequence cannot be the sole determining factor in the processes of enzyme recognition and replication. This implies that the key is to be found in the secondary or tertiary structures. The availability of two different replicating molecules of defined sequence should aid in identifying these critical structural features.
Purification of folate binding factor in normal umbilical cord serum.
Authors: Kamen B A , Caston J D
Published: May 2006

Human umbilical cord serum was found to contain both free folate and folate complexed to a high-molecular weight factor. The complexed folate was bound to a very high affinity binder and was present in concentrations equivalent to as much as 60 ng of 5-methyltetrahydrofolic acid per ml of serum. Acidification of the serum caused disassociation of the folate-binder complex. Released folates were separated from binder by Sephadex gel filtration, zonal centrifugation through sucrose gradients, or adsorption onto activated charcoal. The separated binding factor, either saturated or unsaturated with folate, had a molecular weight of about 40,000 on Sephadex G-200 chromatography. Binding of [3H]pteroylglutamic acid was rapid and, as in the original endogenous folate-binder complex, was essentially irreversible at neutral pH. The affinity and specificity of the binder were examined by competition experiments using [3H]pteroylglutamic acid and nonradioactive folate derivatives. Oxidized folates were bound in preference to reduced derivatives, but only three to four times more unlabeled 5-methyltetrahydrofolic acid than pteroylglutamic acid was required to produce an equal level of competition. The strong affinity for 5-methyltetrahydrofolic acid, the main serum folate, suggests that the binder could be part of the mechanism by which the fetus concentrates maternally supplied folate for its growth and development.
Requirement for cellular protein synthesis in reversal of ethidium-bormide-induced inhibition of cell transformation by murine sarcoma virus.
Authors: Roa R C , Bose S K
Published: May 2006

Cultures of mouse Balb 3T3 fibroblasts exposed to a noncytotoxic dose of ethidium bromide for 16-18 hr are unable to produce foci after infection with murine sarcoma virus. Such cultures regain susceptibility to infection when incubated for 6-8 hr in drug-free growth medium. Pretreated but not untreated cultures exhibit sensitivity toward brief (6 hr) exposure to cycloheximide, chloramphenicol, and actinomycin D before infection. Pretreatment with cordy-cepin inhibits the ability of cultures to produce foci after infection. The recovery of ethidium-bromide-treated cultures requires the synthesis of cellular proteins which may have some important role in the establishment of RNA tumor virus infection.
Intramolecular arsanilazotyrosine-248-Zn complex of carboxypeptidase A: a monitor of multiple conformational states in solution.
Authors: Harrison L W , Auld D S , Vallee B L
Published: May 2006

The red azoTyr-248-Zn complex of arnilazocarboxypeptidase, previously used to demonstrate differences in conformation of the enzyme in crystals and in solution, has now provided means to detect multiple conformations of the enzyme in solution by stopped-flow pH and temperature jump experiments. These studies identify two distinct processes. Er + H+ in equilibrium Ey (I), is the extremely rapid, Kfast about 10(5) sec-1, pH dependent dissociation of the metal complex. Ey in equilibrium Ey' (II), is much slower, Kslow about 5 sec-1, pH independent interconversion of two distinct populations of protein molecules, Ey and Ey', in which the yellow azo-Tyr-248 is different conformations. These two conformations can be differentiated readily by stopped-flow pH-jump experiments, since I is three to four orders of magnitude faster than II. Mathematical expressions derived from this mechanism accurately predict all observations over the pH range from 6.0 to 8.5. In a previous stopped-flow pH-jump experiment, Lipcomb and coworkers [Quiocho, F. A., McMurray, C. H. & Lipcomb, W. H. (1972), Proc. Nat. Acad. Sci. USA 69, 2850-2854] recognized only a single process with a rate constant of about 6 sec-1, but not the major, very rapid rate observed here. The failure to detect this fast process led to the postulation of a number of explanations intended to account for the detection of only a single, slow rate. The present observations show that the premise for those conjectures is not valid. The azoprobe reveals the existence of rapidly interconvertible substructures of carboxypeptidase A, and the results support the view that in solution, enzymes can adopt multiple, readily interconvertible and related conformations which could then either facilitate or impede catalysis. In crystals, rearrangement of molecular structure could be severely impaired or restricted, and crystallization might single out either active or inactive conformations. In the latter case, such crystals would have greatly reduced activities and markedly altered catalytic behavior, as is observed for carboxypeptidase A. In combination with detailed kinetic analysis of crystals, conformational analysis in solution should be a valuable guide to discern enzyme mechanisms and select crystals for x-ray structure analysis.