bluuebunny/crossref_metadata_2025 huggingface dataset:
Fetal membrane collagens: identification of two new collagen alpha chains.
Authors: Burgeson R E , El Adli F A , Kaitila I I , Hollister D W
Published: May 2006

Human fetal membranes contain two new genetically distinct collagen polypeptide chains which are subunits of one (or two) new molecular species of collagen. These new polypeptide chains, which we have tentatively named alphaA and alphaB, have been directly compared with the polypeptide chain subunits of Types I, II, and III human collagen and Type IV collagen from bovine lens capsule. Both alphaA and alphaB exhibit characteristic profiles on carboxymethyl-cellulose chromatography and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The distribution of methionine residues along both new chains is different from known collagen chains as manifest by distinctly different cyanogen bromide peptide profiles on carboxymethyl-cellulose chromatography and/or sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both alphaA and alphaB exhibit contents of amino acids and glycine typical of collagens, and comparison with the observed and reported compositions of collagen chains of Types I-IV collagens reveals notable differences, particularly in the content of alanine, leucine, isoleucine, and the basic amino acids, lysine, hydroxylysine, and arginine. The new collagen species containing both alphaA and alphaB may be separated in the native (triple-helical) state from other native collagen species by differential salt precipitation. The observations that both chains coprecipitate in the same narrow NaCl range, and that the ratio of alphaA:alphaB is constant, suggest the possibility of a single new species of collagen with a subunit structure alphaA [alphaB]2.
Handedness of crossover connections in beta sheets.
Authors: Richardson J S
Published: May 2006

In a crossover connection, the polypeptide chain leaves one end of a beta sheet, forms a loop of any length and any conformation, and reenters the same beta sheet from the opposite end. Of the 85 examples of crossover connections which occur in the known protein structures, 83 are righthanded and only two are lefthanded. It is proposed that consistent handedness, even in long irregular loops, could be produced by the preferred twist direction of extended chain and the righthandedness of alpha-helices, provided certain conditions hold during the protein folding process.
beta-endorphin is a potent analgesic agent.
Authors: Loh H H , Tseng L F , Wei E , Li C H
Published: May 2006

beta-Endorphin, an opiate-like peptide, has potent antinociceptive properties when it is administered directly into the brain and assayed in the the tail-flick, hot-plate, and writhing tests in mice and in the wet shake test in rats. On a molar basis, beta-endorphin is 18 to 33 times more potent than morphine and its actions are blocked by the specific opiate antagonist, naloxone hydrochloride. The activity of beta-endorphin in vivo is also compared to other peptides that show opiate-like activity in assays in vitro.
Initiation of translation directed by 42S and 26S RNAs from Semliki Forest virus in vitro.
Authors: Glanville N , Ranki M , Morser J , Kääriäinen L , Smith A E
Published: May 2006

The proteins synthesized in vitro in response to 42S and 26S RNAs from Semliki Forest virus were labeled with formyl-[35S]methionine from initiator tRNA. One protein which comigrated with viral capsid protein was labeled under the direction of 26S RNA, and only one labeled peptide was detected after digestion with trypsin. Further digestion with pronase gave rise to the dipeptide fMet-AsN. Several labeled polypeptides were found in the 42S RNA directed product and these had molecular weights of up to 150,000. However, tryptic digestion of the product yielded only one formylmethionyl-labeled peptide, which had a different mobility from that directed by the 26S RNA. Further digestion with pronase gave a single dipeptide, fMet-Ala. This indicates that nonstructural proteins as large as 150,000 daltons are probably synthesized from one initiation site on the 42S RNA. Translation starting from the internal initiation site on the 42S RNA, which is equivalent to that on the 26S RNA, could not be detected under the conditions used. Internal initiation sites which are similarly inactive have also been detected in other viral RNAs (e.g., brome mosaic virus, tobacco mosaic virus, and polyoma 19S RNA) and this suggests that, although eukaryotic mRNAs can contain more than one initiation site for protein synthesis, only the site nearer the 5' terminus is active in vitro.
Beta-endorphin: stimulation of growth hormone release in vivo.
Authors: Dupont A , Cusan L , Garon M , Labrie F , Li C H
Published: May 2006

Two micrograms of beta-endorphin (beta-lipotropin61-91) injected intraventricularly in rats that had been treated with antiserum against somatostatin led to a 6- and 10-fold stimulation of the concentration of plasma growth hormone (somatotropin) measured 10 and 20 min after injection of the peptide, whereas 400 mug of methionine-enkephalin led to a 4- to 6-fold increase of levels of plasma growth hormone at 10 min with a rapid return to basal levels at later time intervals. At doses of 5 and 25 mug, beta-endorphin led to a 20- to 30-fold stimulation of levels of plasma growth hormone, the maximal effect being measured between 20 and 30 min after injection. These data suggest the possible role of the endogenous opiate-like peptides in the control of growth hormone secretion.
Microscopic theory of irreversible processes
Authors: Prigogine I. , Mayné F. , George C. , De Haan M.
Published: May 2006

The microscopic theory of irreversible processes that we developed is summarized and illustrated, using as a simple example the Friedrichs model. Our approach combines the Poincaré's point of view (dynamical interpretation of irreversibility) with the Gibbs-Einstein ensemble point of view. It essentially consists in a nonunitary transformation theory based on the symmetry properties of the Liouville equation and dealing with continuous spectrum. The second law acquires a microscopic content in terms of a Liapounov function which is a quadratic functional of the density operator. In our new representation of dynamics, which is defined for a restricted set of observables and states, this functional takes a universal form. We obtain, in this way, a semi-group description, the generator of which contains a part directly related to the microscopic entropy production. The Friedrichs model gives us a simple field theoretical example for which the entropy production can be evaluated. The thermodynamical meaning of life-times is explicitly displayed. The transition from pure states to mixtures, as well as the occurrence of long tails in thermodynamic systems, are also briefly discussed.
Erythropoietin: isolation by affinity chromatography with lectin-agarose derivatives.
Authors: Spivak J L , Small D , Hollenberg M D
Published: May 2006

Affinity chromatography using agarose-bound lectins was used to isolate erythropoietin from crude preparations of sheep plasma and human urinary erythropoietin. On the basis of previous estimates of the sugar content of the hormone, six lectins (wheat germ agglutinin, phytohemagglutinin, Ricinus communis 120, soybean agglutinin, concanavalin A, and limulin) were chosen for study. Only wheat germ agglutinin-agarose and phytohemagglutinin-agarose derivatives had significant affinity for erythropoietin. By use of wheat germ aggutinin-agarose columns, erythropoietin could be separated from over 95% of the initial starting protein, resulting in an 8-to 100-fold purification and a recovery of at least 40% depending on the source of the hormone. Affinity chromatography with agarose-bound lectins provides a simple rapid method for isolating erythropoietin from crude preparations of the hormone.
Nalidixic acid resistance: A second genetic character involved in DNA gyrase activity
Authors: Gellert Martin , Mizuuchi Kiyoshi , O'Dea Mary H. , Itoh Tateo , Tomizawa Jun-Ichi
Published: May 2006

ATP-dependent DNA supercoiling catalyzed by
Escherichia coli
DNA gyrase was inhibited by oxolinic acid, a compound similar to but more potent than nalidixic acid and a known inhibitor of DNA replication in
E. coli
. The supercoiling activity of DNA gyrase purified from nalidixic acid-resistant mutant (
nalA
R
) bacteria was resistant to oxolinic acid. Thus, the
nalA
locus is responsible for a second component needed for DNA gyrase activity in addition to the component determined by the previously described locus for resistance to novobiocin and coumermycin (
cou
). Supercoiling of λ DNA in
E. coli
cells was likewise inhibited by oxolinic acid, but was resistant in the
nalA
R
mutant. The inhibition by oxolinic acid of colicin E1 plasmid DNA synthesis in a cell-free system was largely relieved by adding resistant DNA gyrase.

In the absence of ATP, DNA gyrase preparations relaxed supercoiled DNA; this activity was also inhibited by oxolinic acid, but not by novobiocin. It appears that the oxolinic acid-sensitive component of DNA gyrase is involved in the nicking-closing activity required in the supercoiling reaction. In the presence of oxolinic acid, DNA gyrase forms a complex with DNA, which can be activated by later treatment with sodium dodecyl sulfate and a protease to produce double-strand breaks in the DNA. This process has some similarities to the known properties of relaxation complexes.
Alterations in insulin binding accompanying differentiation of 3T3-L1 preadipocytes.
Authors: Reed B C , Kaufmann S H , Mackall J C , Student A K , Lane M D
Published: May 2006

Expression of the adipocyte phenotype by differentiating 3T3-L1 preadipocytes occurs upon exposure of the cells to insulin. Differentiation-linked changes in 125I-labeled insulin binding to 3T3-L1 cells were monitored and compared with those in nondifferentiating 3T3-C2 controls treated similarly. Without chronic insulin treatment, 3T3-L1 cells failed to express the adipocyte phenotype but maintained a level of 25,000-35,000 insulin-binding sites per cell. Treatment of 3T3-L1 cells with insulin resulted in an initial suppression of insulin binding followed by a 12-fold increase that paralleled the appearance of differentiated cells. A maximum of 170,000 insulin-binding sites per cell was attained for a population in which greater than 75% of the cells had differentiated. The increase of insulin receptor level appears to be differentiation-dependent and is not a general response of cells to the culture conditions. 3T3-C2 cells maintained in the presence of insulin for 30 days exhibited the undifferentiated phenotype and suppressed levels of insulin binding (35,000 sites per cell). The binding capacity of 3T3-L1 cells for epidermal growth factor remained unchanged between 25,000 and 40;000 sites per cell and was independent of the state of differentiation. Thus, induction by insulin in receptor-specific changes. Insulin receptors increase in number but epidermal growth factor receptors remain constant.
beta-Lipotropin: localization of cells and axons in rat brain by immunocytochemistry.
Authors: Watson S J , Barchas J D , Li C H
Published: May 2006

Using specific antisera to human beta-lipotropin, we have visualized cells and axons with beta-lipotropin-like immunoreactivity in rat brain and pituitary. The beta-lipotropin so localized is well delineated and contained in the cytoplasm of cells and in beaded axons. Areas of greatest beta-lipotropin content are hypothalamus (with cell bodies in the medial basal hypothalamus and arcuate regions), periventricular nucleus of the thalamus, ansa lenticularis, zona compacta of the substantia nigra, medial amygdaloid nucleus, zona incerta, periaqueductal central gray area, locus ceruleus, and a few fibers in the reticular formation. The question of the exact relationship of beta-lipotropin and methionine-enkephalin remains open, because some brain areas contain both substances and some areas contain only one or the other.
How to label nerve cells so that they can interconnect in an ordered fashion.
Authors: von der Malsburg C , Willshaw D J
Published: May 2006

We present a method for setting up topographically ordered mappings between two sheets of nerve cells. A set of chemical markers that express the neighborhood relationships within the presynaptic sheet is induced by the fibers into the postsynaptic sheet. The markers are used to guide the fibers to their terminal sites. A case for which this idea may be relevant is the retinotectal projection; our model exhibits types of plasticity found experimentally. The fact that the postsynaptic markers remain after removal of the projecting fibers suggests an important difference between development and regeneration. This paper concentrates on explaining the basic idea, and in addition presents a set of preliminary computer simulations.
Histone H3 disulfide dimers and nucleosome structure.
Authors: Camerini-Otero R D , Felsenfeld G
Published: May 2006

The arginine-rich histone, H3, isolated from avian erythrocytes, can dimerize by forming a disulfide linkage between the single cysteine sulfhydryl residues at position 110 of the H3 polypeptide chain. The H3 dimer can be substituted for undimerized H3 in experiments in which the nucleosome is reconstituted from DNA and mixtures of the four "core" histones, H2A, H2B, H3, and H4. We report here that reconstituted nucleosomes containing H3 dimer are indistinguishable, by a number of criteria, either from native nucleosomes or from reconstitutes containing H3 monomer. The criteria include the pattern of susceptibility of the complex to nucleases, the amount of DNA supercoiling induced by histone binding, and the hydrodynamic properties of reconstituted nucleosome "core" preparations. The results suggest that the residues in the neighborhood of position 110 on each H3 molecule are in close contact in the nucleosome. If, as has been proposed, the nucleosome has a dyad axis, then the disulfide bridge between H3 molecules must lie on this axis.
Microsurgically produced homozygous-diploid uniparental mice
Authors: Hoppe Peter C. , Illmensee Karl
Published: May 2006

Shortly after fertilization, either the male or the female pronucleus was microsurgically removed from 202 F
1
hybrid eggs derived from crosses of two inbred strains. Subsequent incubation of these haploid eggs in medium containing cytochalasin B, which inhibits cytokinesis but not nuclear division, enabled the remaining pronucleus to become diploid. After nuclear diploidization and transfer to regular culture medium, cleavage commenced normally, and a total of 135 successfully manipulated eggs continued in development and yielded 93 morulae and blastocysts. These embryos were surgically transferred to the uteri of pseudopregnant foster mothers who gave birth to seven live female offspring. Five of the females were derived from the maternal genome (gynogenesis) and the remaining two mice inherited only the paternal genes (androgenesis), depending on whether the female or male pronucleus had been retained in the egg, respectively. Homozygosity for a number of genetic loci positioned on different chromosomes and effecting the coat color phenotype and strain-specific allelic variants of several enzymes, urinary and plasma proteins, and hemoglobins could be demonstrated unequivocally in all instances. Chromosomal analysis revealed a normal diploid karyotype including two X chromosomes. Thus far, six of the seven homozygous-diploid (isogeneic) females have proved to be fertile and have given birth to progeny corresponding only to the pronuclear genotype of the mother.
Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis.
Authors: Ehrlich S D
Published: May 2006

One S. aureus plasmid coding for tetracycline resistance, pT127, and four plasmids (pC194, pC221, pC223, and pUB112) coding for chloramphenicol resistance have been introduced by transformation into B, subtilis. The plasmids replicate in--and confer antibiotic resistance upon--their new host. These experiments show that the potential for genetic exchange between diverse bacterial species is greater than has been commonly assumed.
Elimination of naturally occurring crosslinks in vaccinia virus DNA after viral penetration into cells.
Authors: Pogo B G
Published: May 2006

Vaccinia virus DNA, extracted from purified virus or from the cytoplasmic fraction of virus-infected cells very shortly after infection, was analyzed by sedimentation in alkaline and neutral sucrose gradients. The sedimentation properties of vaccinia DNA under denaturing conditions changed, immediately after penetration into the cell, from the characteristic circular viral DNA (crosslinked double-stranded linear DNA) to nicked circular DNA or to single-stranded molecules. This transition occurred at the time of uncoating of the virus and with a slight change in the DNA size, as judged by sedimentation in neutral sucrose. These results indicate that the crosslinks, that held the complementary strands of the genome together, are removed after penetration. When vaccinia DNA was incubated with the supernatant fraction of virus-infected cells, a similar change in the sedimentation properties of the DNA under denaturing conditions was observed. It is concluded that the endonuclease present in the supernatant of infected cells eliminated the crosslinks in the DNA, and that this enzymatic hydrolysis may be the mechanism by which crosslinks are removed prior to DNA replication.
Calabi's conjecture and some new results in algebraic geometry
Authors: Yau Shing-Tung
Published: May 2006

We announce a proof of Calabi's conjectures on the Ricci curvature of a compact Kähler manifold and then apply it to prove some new results in algebraic geometry and differential geometry. For example, we prove that the only Kähler structure on a complex projective space is the standard one.
A DNA nicking-closing enzyme encapsidated in vaccinia virus: partial purification and properties.
Authors: Bauer W R , Ressner E C , Kates J , Patzke J V
Published: May 2006

Vaccinia virus cores contain an activity which is able to relax both left-and right-handed superhelical DNA. This virus-specific nicking closing enzyme has been highly purified and differs from the corresponding host enzyme in salt optimum, in sedimentation coefficient, and in polypeptide composition as determined on sodium dodecyl sulfate/polyacrylamide gels. The enzyme is probably newly synthesized after the cessation of host protein synthesis which follows virus infection. The most highly purified preparation contains two polypeptides, one of molecular weight 24,000 and the other 35,000. The former polypeptide is a major constituent of the virus (7% of total protein by weight), whereas the latter is present in a much smaller amount (0.2%). Chromatography with denatured DNA-cellulose reveals that the activity is predominately associated with those fractions enriched in the polypeptide of greater molecular weight.
Origin of replication of colicin E1 plasmid DNA.
Authors: Tomizawa J I , Ohmori H , Bird R E
Published: May 2006

Cleavage maps of colicin E1 plasmid DNA and its smaller derivative, pNT1 DNA, were constructed by using restriction endonucleases. The nucleotide sequence of a region that contains the orgin of replication was determined. The site of the nucleotide from which DNA replication is initiated was determined with 6S L-fragments, the DNA fragment first made on colicin E1 plasmid DNA. The fragments were labeled with [gamma-32P]ATP and polynucleotide 5'-hydroxyl-kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) at the 5'-OH groups which were uncovered by alkali treatment. The site is one of three consecutive nucleotides, dA, dA, and dC, located at a unique position. One or a few rA residues were found to be attached to some of the DNA molecules. The transition from the primer RNA to DNA occurs in a region consisting of a segment of five A residues. Both sides of this segment are rich in G and C.
Light collection and harvesting processes in bacterial photosynthesis investigated on a picosecond time scale
Authors: Campillo A. J. , Hyer R. C. , Monger T. G. , Parson W. W. , Shapiro S. L.
Published: May 2006

Fluorescence lifetimes have been determined for four strains of
Rhodopseudomonas sphaeroides
. Chromatophore samples were excited with a single picosecond flash, and the fluorescence was detected with a streak camera. The decay times are 100 psec in strains 2.4.1 and Ga, and 300 psec in the carotenoidless strain R-26. These times are related to the transfer of energy from the light-harvesting antenna pigment molecules to the photochemical reaction center. In strain PM-8 dpl, which lacks reaction centers, the lifetime is 1.1 nsec. In addition, we have obtained curves relating the quantum yield of fluorescence to the photon density of the excitation pulse. These curves can be fit with a simple model that relates excitonic processes to properties of the photosynthetic unit and that qualitatively describes differences between the mutant strains.
Bioassay determinations of thymopoietin and thymic hormone levels in human plasma.
Authors: Twomey J J , Goldstein G , Lewis V M , Bealmear P M , Good R A
Published: May 2006

Thymopoietin is a thymic hormone that induces differentiation of thymocytes from precursor cells which arise in hemopoietic tissues. This paper describes a sensitive in vitro assay for the induction of Thy 1.2 antigen on null lymphocytes from germ-free athymic (nu/nu) mice. The sensitivity and specificity of the bioassay were increased by adding high concentrations of ubiquitin (a nonspecific inducer) to the induction incubations. The bioassay was sufficiently sensitive to detect thymopoietin at less than 0.25 ng/ml. A dose-response relationship was shown between thymopoietin concentration and the percentage of cells induced to express Thy 1.2 antigen. When normal human plasma was assayed, induction was registered with activity corresponding to thymopoietin at greater than 1 ng/ml in plasma from infants or young adults. Activities in the thymopoietin range of 0.25 ng/ml were registered with plasma from healthy subjects over 50 years of age. Thymectomy was followed by loss of this inductive activity from the plasma. This bioassay permits clinical studies on T (thymus-derived) cell inducers released by the human thymus into the circulation.
Structural adaptations of lactate dehydrogenase isozymes.
Authors: Eventoff W , Rossmann M G , Taylor S S , Torff H J , Meyer H , Keil W , Kiltz H H
Published: May 2006

The three-dimensional structures of dogfish M4 (muscle) and pig H4 (heart) lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) have been determined and correlated with the amino acid sequences of the dogfish M4, pig M4, pig H4, chicken M4, and chicken H4 lactate dehydrogenase isozymes. These results have been related to the known differences of physicochemical properties between the M and H lactate dehydrogenase isozymes. By far the largest structural alterations occur in the transition between the "apo" and "ternary complex" conformational states of the enzyme rather than between species or isozymes. The major catalytic difference can be explained by a replacement of alanine (in the M chain) with a glutamine (in the H chain) in the vicinity of the binding site of the coenzyme phosphates. The known immunological differentiation of the M and H isozymes is consistent with the differences in their amino acid sequences.
Cleavage of C2 by C1̄s̄ into the antigenically distinct fragments C2a and C2b: Demonstration of binding of C2b to C4b
Authors: Nagasawa Shigeharu , Stroud Robert M.
Published: May 2006

The activation of complement component C2 by C1̄s̄ is a major reaction step leading to the assembly of two related macromolecular enzymes in the classical complement pathway C3 convertase and C5 convertase. The present studies clearly document the smaller fragment, C2b, that results when human C2 reacts with C1̄s̄. We have identified and characterized C2b (34,000 daltons) as a single protein on disc electrophoresis and immunoelectrophoresis. C2a (73,000 daltons), the larger fragment from this reaction, has a more acidic nature and C2b is more basic. These fragments can also be detected by their different antigenic determinants. When the C2-C4b complex is activated in the fluid phase by C1̄s̄ and allowed to decay, it dissociates into C2a and the C2b-C4b complex. Furthermore, when C2 is bound to C4b-Sepharose and then reacted with C1̄s̄, only the C2a fragment is released from the solid phase C2-C4b-Sepharose into the fluid phase, and the C2b fragment remains noncovalently bound to C4b-Sepharose. These results suggest that the C2b portion of C2 contains a stable binding site for C4b and, after the decay release of C2a from this C3 convertase, the C2b fragment remains bound. Thus, the decay release of C2a may represent a temperature-dependent dissociation from C2b.
Oxygen "pulsed" cytochrome c oxidase: functional properties and catalytic relevance.
Authors: Antonini E , Brunori M , Colosimo A , Greenwood C , Wilson M T
Published: May 2006

The kinetics of the reaction of cytochrome c with solubilized mammalian cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) has been studied by a stopped-flow technique under two different experimental situations: (i) the completely oxidized enzyme (resting oxidase as obtained from the preparation) was mixed with reduced cytochrome c, and (ii) the completely reduced enzyme in the presence of reduced cytochrome c was exposed to a "pulse" of O2 (pulsed oxidase). Both sets of experiments were performed with either "limiting" or "excess" O2 (relative to oxidase), in the presence or absence of CO. Both the pre-steady-state events and the steady-state kinetics of cytochrome oxidase are found to be different in the two cases. This shows that the product of the reaction of fully reduced oxidase with O2 (pulsed oxidase) is functionally different from the oxidase as prepared (resting oxidase). These differences are interpreted with the assumption of a different rate of intramolecular electron transfer in the pulsed and resting oxidases. Implications of these experimental findings are discussed in the general framework of a tentative model for the catalytic cycle of the oxidase.
Synthesis of human interferon by Xenopus laevis oocytes: two structural genes for interferons in human cells.
Authors: Cavalieri R L , Havell E A , Vilcek J , Pestka S
Published: May 2006

Human fibroblasts and leukocytes produce interferons which may be distinguished by their antigenic and species specificity as well as by their molecular weight distributions. To elucidate the basis for these differences, we isolated mRNA from induced human fibroblasts and lymphoblastoid (Namalva) cells and studied the products of translation in Xenopus laevis oocytes. The mRNA from the respective cells yielded translation products, in oocytes, that were characteristic of the cells from which the mRNA was derived. We conclude that human cells contain at least two structural genes for interferon, coding for polypeptides differing in primary sequence. Fibroblasts synthesize a single species of interferon; lymphoblastoid cells synthesize two species, the fibroblast and leukocyte types.
On the translational error theory of aging
Authors: Edelmann Patricia , Gallant Jonathan
Published: May 2006

Theoretical treatments of error feedback in translation have revealed that two different modes of behavior are possible, depending on the values of certain parameters. In mode I, the error frequency will rise steadily toward randomness, inevitably reaching whatever value is catastrophic for cell survival; the “error catastrophe” theory of aging implicitly assumes this mode of behavior. In mode II, the error frequency will converge to a stable value, which may or may not have toxic consequences. We have performed an experimental test of the behavior of the translation system in
Escherichia coli
cells: we altered the system's intrinsic fidelity by means of the error-promoting drug streptomycin, and monitored the kinetics of change in error frequency by means of a specific assay of one kind of mistranslation (incorporation of cysteine into flagellin). We find that the system behaves according to mode II. Moreover,
E. coli
cells in which the error frequency has stabilized at a value as high as 50 times greater than normal continue to proliferate, albeit abnormally slowly, and their viability is not detectably reduced. Earlier results by Gorini and his associates point in the same direction. These observations diminish the plausibility of the error catastrophe theory of aging.
Fibroblast heterogeneity and prostaglandin regulation of subpopulations
Authors: Ko S. David , Page Roy C. , Narayanan A. S.
Published: May 2006

The effects of prostaglandin E
2
(PGE
2
) upon the synthesis of protein and DNA, and membrane transport of proline and thymidine, by human diploid fibroblasts were studied. At a concentration range of 1-10 μM, PGE
2
inhibited protein synthesis and membrane transport by 45-50%. Serum-activated DNA synthesis and thymidine transport were also inhibited by approximately 50% in cells made quiescent and synchronous by serum deprivation. To determine whether prostaglandin inhibits some of the cells completely or all of the cells partially, radioautographic and cell-counting experiments were done. In cultures pulse-labeled with [
3
H]thymidine 12-33 hr after serum activation, prostaglandin exposure reduced the number of labeled nuclei by 42%. Sixty-five hours after serum activation, the total cell numbers present in the PGE
2
-exposed cultures were reduced by 25%. Furthermore, in the fibroblast cultures derived from cells previously maintained in 10 μM PGE
2
for 14 days, PGE
2
had no effect on DNA synthesis, indicating that the PGE
2
-sensitive cells had disappeared from the cultures. Thus, PGE
2
appears to inhibit growth and synthesis of a subpopulation of cells while not affecting the remaining insensitive cells. Prostaglandins may play an important role in connective-tissue differentiation and in some pathologic alterations by regulating fibroblast subpopulations.
Intracellular distributions of mechanochemical proteins in cultured fibroblasts
Authors: Heggeness Michael H. , Wang Kuan , Singer S. J.
Published: May 2006

We have used methods that have allowed simultaneous fluorescent staining of intracellular actin together with either myosin, filamin, or tubulin in normal rat kidney fibroblasts in monolayer culture. In the main portions of the cell body, the actin, myosin, and filamin are all present in two structures: in one, the three proteins are present in the same fiber bundles (stress fibers); in the other, there is a diffuse distribution of the three proteins. On portions of the cell periphery however—in the basal regions of microspikes, in ruffles, and in regions of cell-cell contact—actin and filamin are present, but myosin is severely depleted or absent. Microtubules are present in the cell body in a distribution independent of the stress fibers and are mostly absent from the cell periphery. Microspikes and ruffles are highly dynamic structures on the cell surface, and regions of cell-cell contact generally result from the association of ruffles on the two contacting cells. Therefore, the presence of filamin and actin but not myosin in these specialized regions on the cell surface, together with the recent demonstration [Wang, K. & Singer, S. J. (1977)
Proc. Natl. Acad. Sci. USA
74, 2021-2025)] that pure filamin interacts with individual F-actin filaments in solution to form fiber bundles and sheet-like structures, suggest that
in vivo
filamin-actin interactions play an important role in the control of actin filament structure, in cell motility, and in the stabilization of cell-cell contacts.
Rapid speciation and chromosomal evolution in mammals.
Authors: Bush G L , Case S M , Wilson A C , Patton J L
Published: May 2006

To test the hypothesis that population subdivision into small demes promotes both rapid speciation and evolutionary changes in gene arrangement by inbreeding and drift, we estimated rates of speciation and rates of chromosomal evolution in 225 genera of vertebrates. Rates of speciation were estimated by considering the number of living species in each genus and the fossil record of each genus as well as information about extinction rates. Speciation rate was strongly correlated with rate of chromosomal evolution and average rates of speciation in lower vertebrate genera were one-fifth those in mammalian genera. Genera with high karyotypic diversity and rapid speciation rates may generally have small effective population size (Ne), whereas large Ne values may be associated with karyotypically uniform genera and slow rates of speciation. Speciation and chromosomal evolution seem fastest in those genera with species organized into clans or harems (e.g., some primates and horses) or with limited adult vagility and juvenile dispersal, patchy distribution, and strong individual territoriality (e.g., some rodents). This is consistent with the above hypothesis regarding the evolutionary importance of demes.
pppA2'p5'A2'p5'A: an inhibitor of protein synthesis synthesized with an enzyme fraction from interferon-treated cells.
Authors: Kerr I M , Brown R E
Published: May 2006

A low molecular weight inhibitor of cell-free protein synthesis effective at subnanomolar concentrations is formed on incubation of cytoplasmic extracts from interferon-treated cells with double-stranded RNA and ATP. It can be conveniently synthesized by incubating a poly(I).poly(C)-Sepharose-bound enzyme fraction from such cells with [3H]- or [alpha- or gamma-32P]ATP. The radioactive inhibitor has been characterized by its behavior on DEAE-Sephadex in the presence of urea and on the basis of the products obtained on enzymic, alkaline, and sequential degradation by periodate oxidation and beta elimination. Its structure appears to be pppA2'p5'A2'p5'A. We have found no evidence for any modification or abnormality other than the 2'-5' linkage. On occasion the inhibitor preparations have included what seems to be the corresponding dimer (pppA2'p5'A), tetramer [ppp(A2'p)3A], pentamer [ppp(A2'p)4A], and higher oligomers in decreasing amounts. The trimer, tetramer, and pentamer are similar in activity, but the dimer is less potent if active at all.
Escherichia coli recA gene product inactivates phage lambda repressor.
Authors: Roberts J W , Roberts C W , Craig N L
Published: May 2006

Phage lambda repressor is inactivated and cleaved into two detectable fragments during incubation with purified Escherichia coli recA gene protein in vitro, in a reaction that requires ATP. This reaction reproduces the recA-dependent inactivation of repressor that occurs in vivo during induction of the SOS functions. The proteolytic activity may reside in the recA protein itself and may be a fundamental activity of it.
Acid catalysis of the formation of the slow-folding species of RNase A: Evidence that the reaction is proline isomerization
Authors: Schmid Franz X. , Baldwin Robert L.
Published: May 2006

Unfolded RNase A is known to contain an equilibrium mixture of two forms, a slow-folding form (U
1
) and a fast-folding form (U
2
). If U
1
is produced after unfolding by the slow
cis-trans
isomerization of proline residues about X-Pro imide bonds, then the formation of U
1
should be catalyzed by strong acids. Therefore, the rate of formation of U
1
has been measured at different HClO
4
concentrations. After rapid unfolding of the native protein in concentrated HClO
4
at 0°, the slow formation of U
1
was measured by use of refolding assays. Catalysis of its formation was found at HClO
4
concentrations above 5 M. The uncatalyzed reaction follows apparent first-order kinetics but, in the acid-catalyzed range, two reactions are found. The faster reaction produces two-thirds of the slow-folding species and shows acid catalysis above 5 M HClO
4
. Catalysis of the slower reaction begins at 8 M HClO
4
. The faster reaction shows a 100-fold increase in rate at 10.6 M HClO
4
over the rate of the uncatalyzed reaction of 5 M. The activation enthalpy of the uncatalyzed reaction has been measured in two sets of unfolding conditions: ΔH
‡
is 21.5 kcal/mol (1 kcal = 4.2 × 10
3
J) in 3.3 M HClO
4
and 21.0 kcal/mol in 5 M guanidine HCl, pH 2.5.

Both acid catalysis of the formation of U
1
and its high activation enthalpy are consistent with the rate-limiting step being
cis-trans
isomerization either of X-Pro imide bonds or of peptide bond. The rate of the uncatalyzed reaction is in the range expected for proline isomerization and is 0.1% of that of peptide bond isomerization; thus, the simplest explanation for the formation of U
1
is proline isomerization. Earlier data, showing that the kinetic properties of the U
1
⇄ U
2
reaction in refolding conditions differ from those of proline isomerization, can be explained if there is kinetic coupling between early steps in the folding of U
1
and its conversion to U
2
.

The existence of two acid-catalyzed reactions that are distinguished by the HClO
4
concentration at which catalysis begins suggests that at least two essential proline residues produce slow-folding species of RNase A by isomerization after unfolding. Because protonation of imide bonds is responsible for acid catalysis of proline isomerization, the slower reaction probably involves an imide bond with a low pK. It may be the bond connecting Lys-41 and Pro-42, because the positive charge on Lys-41 could make this bond more difficult to protonate.
Enzymatic conversion of 5'-phosphate-terminated RNA to 5'-di- and triphosphate-terminated RNA.
Authors: Spencer E , Loring D , Hurwitz J , Monroy G
Published: May 2006

We have isolated from vaccinia virus cores an enzyme, 5'-phosphate-polyribonucleotide kinase, that in the presence of ATP and Mg2+ catalyzes the conversion of 5'-phosphate and 5'-diphosphate termini of RNA to the 5'-triphosphate species. With the exception of dATP, other nucleoside triphosphates were inactive as phosphate donors; activity with dATP was 10% of that observed with ATP. The purified enzyme did not phosphorylate 5'-hydroxyl- or 5'-monophosphate-terminated polydeoxyribonucleotides, although a variety of 5'- monophosphate-terminated RNA chains were active as phosphate acceptors. By using a coupled system of 5'-phosphate-polyribonucleotide kinase and guanylyltransferase in the presence of ATP, GTP, Mg2+, and S-adenosylmethionine, capping of 5'-P-, 5'-PP-, and 5'-PPP-RNA was demonstrated; in the absence of 5'-phosphate-polyribonucleotide kinase only 5'-PPP-RNA was capped by guanylyltransferase.
Energy coupling in DNA gyrase and the mechanism of action of novobiocin
Authors: Sugino Akio , Higgins N. Patrick , Brown Patrick O. , Peebles Craig L. , Cozzarelli Nicholas R.
Published: May 2006

Escherichia coli
DNA gyrase catalyzes negative supercoiling of closed duplex DNA at the expense of ATP. Two additional activities of the enzyme that have illuminated the energy coupling component of the supercoiling reaction are the DNA-dependent hydrolysis of ATP to ADP and P
i
and the alteration by ATP of the DNA site specificity of the gyrase cleavage reaction. This cleavage of both DNA strands results from treatment with sodium dodecyl sulfate of the stable gyrase-DNA complex that is trapped by the inhibitor oxolinic acid. Either ATP or a nonhydrolyzable analogue, adenyl-5′-yl-imidodiphosphate (App[NH]p), shifts the primary cleavage site on ColE1 DNA. The prevention by novobiocin and coumermycin A
1
of this cleavage rearrangement places the site of action of the antibiotics at a reaction step prior to ATP hydrolysis. The step blocked is the binding of ATP because coumermycin A
1
and novobiocin interact competitively with ATP in the ATPase and supercoiling assays; the
K
i
values are more than four orders of magnitude less than the
K
m
for ATP. This simple mechanism accounts for all effects of the drugs on DNA gyrase. Studies with App[NH]p, another potent competitive inhibitor of reactions catalyzed by gyrase, show that cleavage of a high energy bond is not required for driving DNA into the higher energy supercoiled form. With substrate levels of gyrase, App[NH]p induces supercoiling that is proportional to the amount of enzyme; a -0.3 superhelical turn was introduced per gyrase protomer A. We postulate that ATP and App[NH]p are allosteric effectors of a conformational change of gyrase that leads to one round of supercoiling. Nucleotide dissociation favored by hydrolysis of ATP returns gyrase to its original conformation and thereby permits enzyme turnover. Such cyclic conformational changes accompanying alteration in nucleotide affinity also seem to be a common feature of energy transduction in other diverse processes including muscle contraction, protein synthesis, and oxidative phosphorylation.
O4-(5'-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus.
Authors: Rothberg P G , Harris T J , Nomoto A , Wimmer E
Published: May 2006

Virion RNA of poliovirus type 1 has been analyzed for the linkage between genome-protein VPg and the polyribonucleotide chain. Hydrolysis of the linkage with acid or alkali and enzymatic degradation lead to the conclusion that the bond is neither a phosphodiester such as nucleotidyl-(P-O)-serine (or threonine) nor a phosphoramidate such as nucleotidyl-(P-N)-amino acid. VPg-RNA can be iodinated by the Bolton and Hunter reagent [iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester] but not by the chloramine-T or lactoperoxidase procedures, an observation suggesting that VPg does not contain accessible tyrosine. However, VPg can be labeled with [3H]tyrosine in vivo. Hydrolysis of VPg-[32P]pUp with 5.6 M HCl at 110 degrees yielded 32P-labeled O4-(3'-phospho-5'-uridylyl)tyrosine that could be cleaved with micrococcal nuclease to O4-[32P]phosphotyrosine and uridine 3'-[32P]phosphate. These data establish that VPg is linked to the poliovirus genome by a bond between the O4 of tyrosine and the 5'-P atom of the terminal uridylic acid residue. The 5' end of polio genome RNA can now be described as VPg(Tyr-O)-pU-U-A-A-A-A-C-A-G.
Quantitative determination of the lateral diffusion coefficients of the hormone-receptor complexes of insulin and epidermal growth factor on the plasma membrane of cultured fibroblasts.
Authors: Schlessinger J , Shechter Y , Cuatrecasas P , Willingham M C , Pastan I
Published: May 2006

Fluorescent derivatives of insulin and epidermal growth factor bound to 3T3 mouse fibroblasts are mobile on the cell surface, with similar diffusion coefficients, D approximately (3--5) x 10(-10) cm2/sec at 23 degrees C. Increasing the temperature to 37 degrees C results in rapid receptor immobilization. The immobilization is attributed to aggregation of hormone-receptor complexes, their internalization, or a combination of both processes.
Transcription termination: Nucleotide sequence at 3′ end of tryptophan operon in
Escherichia coli
Authors: Wu Anna M. , Platt Terry
Published: May 2006

We have determined the RNA and DNA sequences in the region specifying termination of transcription at the end of the tryptophan (
trp
) operon of
Escherichia coli
. A 3′-terminal mRNA fragment of about 150 nucleotides yielded oligonucleotide products that could be assigned to the end of
trpA
(the last structural gene in the operon) by correlation with the amino acid sequence of the protein product. Analysis of the DNA corresponding to this region served to align the few noncoding RNA oligonucleotide sequences and demonstrated that termination of
trp
transcription occurs
in vivo
at a site 36 nucleotides after
trpA
, with greater than 95% efficiency. In two different strains partially defective in the transcription termination factor rho, the purified transcript is much longer and more complex, suggesting that a significant amount of read-through occurs in these strains. This is consistent with evidence [Guarente, L. P., Mitchell, D. H. & Beckwith, J. (1977)
J. Mol. Biol.
112, 423-436] that efficient termination
in vivo
at the end of the
trp
operon is a rho-dependent event. The
trp
terminator (
trp t
) shares several features with other known sites of transcription termination, including (
i
) a 3′-terminal RNA sequence of several uridine residues, C-A-U-U-U-U
OH
, (
ii
) a G·C-rich region in the DNA immediately preceding the site of termination, followed by an A·T-rich region, and (
iii
) a region of dyad symmetry in the DNA which, in the transcript, is capable of forming a stable hairpin containing seven G·C base pairs and one A·U base pair in its stem.
Sequence organization and transcription at two heat shock loci in Drosophila.
Authors: Livak K J , Freund R , Schweber M , Wensink P C , Meselson M
Published: May 2006

The heat shock loci of Drosophila melanogaster chromosome subdivisions 87A and 87C have been studied by using cloned DNA. Both sites contain a number of copies of a 2,4-kilobase (kb) region homologous to mRNA for the 70,000-dalton heat shock protein. In situ hybridization to chromosomal RNA shows that transcripts of this sequence accumulate at both sites after temperature elevation. At 87C there is a 1.5-kb repeated sequence homologous to another heat shock RNA. One cloned segment includes two to three tandem copies of this sequence located 0.8 kb from the beginning of a 2.4-kb message region. RNA complementary to the 1.5-kb repeat acccumulates at 87C after temperature elevation, but does not code for any known heat shock protein. In the sibling species D. simulans, there are sequences located and transcribed at 87A and 87C that are homologous to the melanogaster 2.4-kb message sequence. The 1.5-kb repeat, however, is absent from 87C in simulans and no heat shock RNA homologous to it can be detected.
K
+
-resistant mutants and “adaptation” in
Paramecium
Authors: Shusterman Cheryl Laffer , Thiede Eric W. , Kung Ching
Published: May 2006

A class of mutants of
Paramecium tetraurelia
has been isolated by adding a critical amount of K
+
to mutagenized, autogamized cultures. Adding 35 mM K
+
to the medium kills the wild type. The mutants can grow at 35 mM K
+
and some survive an addition of up to 80 mM K
+
. Thirty-three lines of such mutants have been studied. These mutants have few or no behavioral abnormalities in culture medium.

Paramecia can “adapt.” Hildebrand and Dryl showed that
P. caudatum
incubated in 15 mM K
+
for over 15 min fail to give avoiding reactions when challenged with Ba
2+
or a thermal gradient [Hildebrand, E. & Dryl, S. (1976)
Bioelectrochem. Bioenerg
. 3, 543-544]. We have observed a similar phenomenon in wild-type
P. tetraurelia
. However, the K
+
-resistant mutants show little or no such “adaptation,” i.e., they perform avoiding reactions when challenged with Ba
2+
after prolonged incubation in 15 mM K
+
. The K
+
resistance and the lack of “adaptation” are strictly correlated. Because
all
33 lines of mutants show this correlation, “adaptation” and killing by K
+
must share a mechanism. This mechanism may be studied by finding the basis of the K
+
resistance in the mutants. The possible ionic bases of “adaptation” are discussed.
Identification of insulin in rat brain.
Authors: Havrankova J , Schmechel D , Roth J , Brownstein M
Published: May 2006

Insulin concentrations in acid/ethanol extracts of the whole rat brain were on the average 25 times higher than plasma insulin levels. Brain insulin was indistinguishable from authentic pancreatic insulin, based on its behavior in radioimmunoassay, radioreceptor assay, and bioassay and its chromatographic pattern on Sephadex G-50 column chromatography. Insulin was found in all regions of the brain examined, but distribution was uneven. Some regions had insulin concentrations as much as 100 times higher than in plasma; levels at least 10 times higher were found in other regions. The role of insulin in the central nervous system is not clear at present but, because both insulin and insulin receptors are abundant in the central nervous system, an extensive physiological regulation of the central nervous system by insulin is proposed.