bluuebunny/crossref_metadata_2025 huggingface dataset:
MRNA-directed synthesis of catalytically active mouse beta-glucuronidase in Xenopus oocytes.
Authors: Labarca C , Paigen K
Published: May 2006

Catalytically active mouse beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) is formed when Xenopus oocytes are injected with mouse RNA enriched for poly(A)-containing mRNA sequences. With the RNA from androgen-induced kidneys, the efficiency of translation is comparable to that of endogenous Xenopus messenger, and the fidelity of translation is high. Detection of glucuronidase messenger by formation of a catalytically active product is several orders of magnitude more sensitive than detection by incorporation of isotopically labeled amino acids. As well as providing a sensitive technique for examining the regulation of gene expression, the system makes available an opportunity to study the regulation of post-translational polypeptide processing of a lysosomal enzyme.
Hormonal effects on intracellular and secreted casein in cultures of mouse mammary epithelial cells on floating collagen membranes.
Authors: Emerman J T , Enami J , Pitelka D R , Nandi S
Published: May 2006

Cultured on floating collagen membranes in the presence of lactogenic hormones, dissociated normal mammary epithelial cells from prelactating mice acquire the ultrastructural and biochemical characteristics of differentiated mammary secretory cells in vivo. The cells on floating collagen membranes in medium containing insulin alone have sparse secretory organelles, and a small amount of casein can be detected in these cells with a sensitive radioimmunoassay. These cells resemble counterpart cells in early-pregnant mice. When the cells are exposed to insulin, cortisol, and prolactin, the secretory apparatus is elaborated and significant increases in intracellular and extracellular casein are observed. In this environment, the intracellular casein content is generally four to eight times greater than in freshly dissociated cells or cells cultured in insulin alone. The amount of casein secreted into the medium by floating-collagen-membrane cultures in the three hormones is from 25 to 200 times greater than that secreted by cultures in insulin alone. Cells cultured on plastic substrates in either hormone combination fail to show any increase in intracellular or extracellular casein. On floating collagen membranes, the cells differentiate in response to hormones as they do in vivo and in organ culture. This cell-culture system provides an opportunity to study direct effects of environmental factors on mammary differentiation at the cellular level.
Ligand-independent cap formation: redistribution of surface receptors on mouse lymphocytes and thymocytes in hypertonic medium.
Authors: Yahara I , Kakimoto-Sameshima F
Published: May 2006

Most of the mobile receptors on mouse lymphocytes and thymocytes, including immunoglobulins, H-2 antigens, Thy-1.2 antigens, some concanavalin A receptors, and some antigenic determinants detected by anti-thymocyte serum, were redistributed into caps when the cells were incubated in hypertonic medium (about 600 mOsM) in the absence of ligands. The caps reverted to the original distributions if the cells were transferred again to isotonic medium. The viability of the cells was not decreased after incubation in the hypertonic medium. Ligand-independent cap formation appeared to depend upon cellular metabolism. Different species of receptors appeared to move with different mobilities during the process of ligand-independent cap formation. Most microvilli on cells showing caps in hypertonic medium were associated with the regions of the caps. These results suggest that free receptors can be induced to form caps if the receptors are allowed to interact with the machinery of cap formation under special conditions.
Evolution of primate oncornaviruses: An endogenous virus from langurs (
Presbytis
spp.) with related virogene sequences in other Old World monkeys
Authors: Benveniste Raoul E. , Todaro George J.
Published: May 2006

Gene sequences related to a retrovirus (oncornavirus type D) isolated from a lung cell culture from spectacled langur (
Presbytis obscurus
) are found in multiple copies (20-40 per haploid genome) in langur cellular DNA; partially homologous virogene sequences are present in the DNA of related Old World monkey species. Primates thus contain gene sequences for at least two distinct classes of genetically transmitted oncornaviruses, the type C class (isolated from baboons) and the type D class described here. The langur virus is partially related to Mason-Pfizer monkey virus, a type D retrovirus isolated from rhesus monkeys. Nucleic acid hybridization studies suggest that Mason-Pfizer monkey virus, now infectious among primates, was derived from an endogenous virus of langurs or from another member of the primate sub-family Colobinae.
Differential lateral mobility of IgM and IgG receptors in mouse B lymphocyte membranes
Authors: Krolick Keith A. , Wisnieski Bernadine J. , Sercarz Eli E.
Published: May 2006

Anti-Ig induced redistribution of different Ig subclasses was studied as a function of temperature and correlated with membrane phase transitions as revealed by electron spin resonance spectroscopy. Fluorescein isothiocyanate-coupled anti-IgG2 and anti-IgM antibodies induced patching and capping that proceeded with increasing rates from 2° to 40° (measured at 2° intervals). Characteristic temperatures marked the onset of discontinuities in such rate changes. IgG2-bearing lymphocytes displayed discontinuities at 14°, 22°, 28°, and 36°, whereas IgM-bearing lymphocytes displayed discontinuities at 18°, 24°, 32°, and 38°. Electron spin resonance spectroscopy studies using the spin label 2,2-dimethyl-4-butyl-4-penty-
N
-oxyloxazolidine, a nitroxide-substituted decane, indicated that these temperatures are a function of hydrocarbon phase separations in the B lymphocyte membrane. With a glucosamine-derivative [2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl glucosamide] as a probe restricted to the outer monolayer of the plasma membrane, the temperatures 14° and 28° denoted the onset and end, respectively, of a fluidizing process in the outer monolayers of IgG2-bearing lymphocytes. Temperatures of 18° and 32° denoted these boundaries in IgM-bearing lymphocytes. Inner monolayer transitions are associated with the remaining temperatures. We conclude that membranes of IgM-bearing lymphocytes are less fluid than those of IgG2-bearing lymphocytes.
beta-Endorphin: pituitary and adrenal glands modulate its action.
Authors: Holaday J W , Law P Y , Tseng L F , Loh H H , Li C H
Published: May 2006

Hypophysectomized rats are supersensitive to the hypothermic effects of morphine and beta-endorphin injected intraventricularly as early as 1 week after surgery. At 2 weeks after surgery, there is a significant increase in the antinociceptive potency for these opiates. The route of opiate injection must be considered in interpretations of these results. The enhanced opiate potency following subcutaneous morphine injection in hypophysectomized rats may be partially explained by adrenal dysfunction, as demonstrated by a similar sensitization to subcutaneous morphine following adrenalectomy. By contrast, no enhancement of opiate potency was observed upon direct intraventricular injection of morphine or beta-endorphin in adrenalectomized rats. Furthermore, the potency of intravenous beta-endorphin is profoundly enhanced in adrenalectomized mice; five out of nine animals died when injected with a dose of the peptide that produced only mild analgesia in sham controls. The complete reversal of this intravenous beta-endorphin supersensitivity by dexamethasone implies a possible physiological interplay between the peptide and adrenal function.
Theory of connectivity for formally symmetric operators
Authors: Herrera Ismael
Published: May 2006

A previous paper introduced the notion of complete connectivity conditions and developed variational principles for diffraction problems subjected to such restrictions. Here, an abstract definition of formally symmetric operators is given and it is shown that the problem of connecting solutions of equations associated with this kind of operators leads to complete connectivity conditions. The variational principles previously developed as well as a present more general one are thus applicable. The problem of connecting solutions defined in different regions is basic for finite element formulations. Formally symmetric operators occur in many branches of science and engineering. Applications are given here to potential theory, wave propagation, elasticity, and a general class of boundary integral equations.
Discrete spiral modes in disk galaxies: Some numerical examples based on density wave theory
Authors: Bertin G. , Lau Y. Y. , Lin C. C. , Mark James W.-K. , Sugiyama L.
Published: May 2006

Discrete growing spiral modes are calculated, on the basis of principles of stellar dynamics, according to the recently developed asymptotic theory of density waves for spiral structure in galaxies. From the growth rates obtained, it appears likely that several spiral modes coexist in a given disk galaxy. General properties of these modes are described for two galaxy models, and some of their important observational implications are discussed.
Dinucleoside pyrophosphate are substrates for T4-induced RNA ligase.
Authors: England T E , Gumport R I , Uhlenbeck O C
Published: May 2006

RNA ligase isolated from bacteriophage T4-infected Escherichia coli will utilize a number of different compounds with the general structure Ado-5'PP-X as substrates in an ATP-independent reaction. The P-X portions of these molecules are transferred to the 3'-hydroxyl of an oligoribonucleotide to form a phosphodiester bond, and the Ado-5'P (AMP) portion is released. AMP, CMP, GMP, UMP, dTMP, NMN, alphaNMN, reduced NMN, FMN, Rib-5P, phosphopantetheine, and cyanoethylphosphate all have been added to [Cyd-3H](Ap)3C from their corresponding AMP adducts. Contrary to the relative lack of specificity of RNA ligase for the P-X -group added, the failure of NADP+, deamino-NAD+, epsilonNCD+, epsilon NAD and CoA to react indicates that the enzyme shows a high degree of selectivity for the AMP portion of the substrate. The diversity of chemical groups that can be efficiently added suggests that this reaction of RNA ligase will prove useful for the modification of the 3' ends of RNA molecules.
Activation of hormone-sensitive lipase and phosphorylase kinase by purified cyclic GMP-dependent protein kinase
Authors: Khoo John C. , Sperry Pamela J. , Gill Gordon N. , Steinberg Daniel
Published: May 2006

Cyclic GMP-dependent protein kinase, purified to homogeneity from bovine lung, was shown to activate hormone-sensitive lipase partially purified from chicken adipose tissue. The degree of activation was the same as that effected by cyclic AMP-dependent protein kinase although higher concentrations of the cyclic GMP-dependent enzyme were required (relative activities expressed in terms of histone H2b phosphorylation units). Activation by cyclic AMP-dependent protein kinase was completely blocked by the heat-stable protein kinase inhibitor protein from skeletal muscle but activation by the cyclic GMP enzyme was not inhibited. Lipase fully activated by cyclic AMP-dependent protein kinase showed no further change in activity when treated with cyclic GMP-dependent protein kinase. Lipase activated by cyclic GMP-dependent protein kinase was reversibly deactivated by purified phosphorylase phosphatase (from bovine heart); full activity was restored by reincubation with cyclic GMP and cyclic GMP-dependent protein kinase. Cholesterol esterase activity in the chicken adipose tissue fraction, previously shown to be activated along with the triglyceride lipase by cyclic AMP-dependent protein kinase, was also activated by cyclic GMP-dependent protein kinase. Crude preparations of hormone-sensitive triglyceride lipase from human or rat adipose tissue and cholesterol esterase from rat adrenal were also activated by cyclic GMP-dependent protein kinase. Purified phosphorylase kinase (rabbit skeletal muscle) was also shown to be activated by cyclic GMP-dependent protein kinase. The present results, together with those of other workers on histone phosphorylation, suggest that the substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinase may be similar. This is discussed in the light of a model recently proposed with regard to the relationship between the subunit structures of the two kinases. The physiologic significance of the findings remains to be established.
Estradiol receptor of calf uterus: interactions with heparin-agarose and purification.
Authors: Molinari A M , Medici N , Moncharmont B , Puca G A
Published: May 2006

Heparin attached covalently to agarose beads binds the "native" form of the estradiol receptor with very high affinity. Chondroitin sulfate does not bind to the receptor. When the receptor is complexed with hormone, the affinity is at least 10 times higher. Only the "native" and not the "nuclear" or the "derived" (i.e., after activation by a calcium-dependent enzyme) forms of the estradiol receptor interact with heparin. The "native" estradiol-receptor complex is purified to homogeneity after chromatography on columns of heparin-agarose, Sephadex G-200, and DEAE-cellulose, followed by two more Sephadex G-200 columns. The purified molecule is a single polypeptide of molecular weight 69,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. The sedimentation coefficient on sucrose gradients is 4.3 S, the Stokes radius from gel filtration is 36.5 A, and the isoelectric point is 6.4. The purified [3H]estradiol-receptor complex exchanges the radioactive hormone with estradiol or other estrogenic steroids, but not with testosterone, 5alpha-dihydrotestosterone, or progesterone.
Prostaglandin regulation of macrophage collagenase production.
Authors: Wahl L M , Olsen C E , Sandberg A L , Mergenhagen S E
Published: May 2006

The production of collagenase (EC 3.4.24.3) by endotoxin-stimulated macrophages was significantly inhibited by indomethacin, indicating that prostaglandins (PGs) mediate this effect. Inhibitions of collagenase production by indomethacin was overcome by addition of exogenous PGE2 at 10 nM whereas the addition of 0.1 and 1.0 micrometer PGE2 increased the enzyme production to 3 times that achieved by endotoxin. Although the addition of prostaglandin alone to macrophage cultures did not stimulate collagenase production, the simultaneous addition of PGE1 or PGE2 and endotoxin enhanced collagenase activity 2- to 10-fold. This increase was detectable at PGE concentrations of 10 nM and was optimal at 0.1-1.0 micrometer. PGF2alpha had little effect on either the enhancement of collagenase production by endotoxin-stimulated macrophages or its restoration in cultures inhibited by indomethacin. Radioimmunoassay of prostaglandins in the culture media revealed that macrophages exposed to endotoxin secreted 40-fold more PGE2 than did unstimulated cells. The increase in PGE2 was detected 4 hr after exposure to endotoxin and was maximal at 14 hr. The peak PGE2 concentrations in the culture media were similar to those of exogenous PGE2 (about 10 nM) needed to restore collagenase production in indomethacin-treated cultures. These findings demonstrate the involvement of PGE in the endotoxin-activation of macrophages with resultant production of collagenase.
Contacts between the lac repressor and the thymines in the lac operator.
Authors: Ogata R , Gilbert W
Published: May 2006

We have identified important points of contact between the lac repressor and the lac operator by crosslinking the repressor to bromouracil-substituted operator. We substituted bromouracils for thymines in a 55-base-long restriction fragment containing the lac operator and labeled one or the other 5' end with 32P. Ultraviolet irradiation of this fragment produced single-strand breakds at the bromouracils. We examined breakage at each bromouracil in the sequence by denaturing the DNA and displaying the UV-generated fragments on a polyacrylamide gel. In the presence of lac repressor, UV radiation failed to break at specific sites. We attribute this to a competing reaction in which the DNA crosslinks to the repressor rather than breaking. These crosslinkable sites thus define positions at which the lac repressor protein lies close to the methyl group of a thymine in the major groove of DNA.
Complexes of inorganic pyrophosphate, orthophosphate, and calcium as stimulants of 3T3 cell multiplication
Authors: Rubin H. , Sanui H.
Published: May 2006

Addition of 0.1-0.5 mM sodium PP
i
for 17 hr to confluent cultures of BALB/c 3T3 cells in low serum concentrations stimulated the incorporation of [
3
H]thymidine into DNA to an extent equal to that produced by high serum concentration. PP
i
prevented much but not all of the cell detachment that accompanies decreasing the serum concentration of confluent cultures and it increased the saturation density of cultures in high serum concentrations. The stimulation had a sharp concentration dependence and was associated with the appearance in the medium of a flocculent precipitate. Stimulation and precipitate formation were dependent on Ca
2+
and inorganic orthophosphate (HPO
4
2-
) and were inhibited by Mg
2+
. More than half the Ca
2+
requirement could be met with Sr
2+
. In the absence of PP
i
, supranormal concentrations of either Ca
2+
or HPO
4
2-
caused graded increases in [
3
H]thymidine incorporation and total cell yield. The effect of supranormal [Ca
2+
] depended on [HPO
4
2-
] and vice versa, and the Ca
2+
requirement could be partially met by Sr
2+
. The stimulation was associated with increasing turbidity of the medium. Various other complexing agents of Ca
2+
, including the divalent cation ionophore A 23187, failed to produce stimulation of 3T3 cells. We conclude that water insoluble complexes of PP
i
, HPO
4
2-
, and Ca
2+
or, at much higher concentrations, the latter two together, stimulate 3T3 cells and we speculate that this is brought about by the association of these complexes with the cell membrane.
Polypeptide hormone binding sites in vivo: initial localization of 125I-labeled insulin to hepatocyte plasmalemma as visualized by electron microscope radioautography.
Authors: Bergeron J J , Levine G , Sikstrom R , O'Shaughnessy D , Kopriwa B , Nadler N J , Posner B I
Published: May 2006

The rationale of the specific-binding assay was applied to the detection of the liver insulin receptor in vivo. Quantitative electron microscope radioautography indicated that, 3 min after an intraportal injection, 125I-insulin was exclusively located to the hepatocyte plasmalemma.
Selective loss of wheat germ agglutinin binding to agglutinin-resistant mutants of Chinese hamster ovary cells.
Authors: Stanley P , Carver J P
Published: May 2006

The binding of 125I-labeled wheat germ agglutinin (WGA) to parental and three distinct WGA-resistant Chinese hamster ovary cell lines possessing modified cell surface carbohydrate structures has been examined over a 10(6)-fold range of WGA concentrations. The Scatchard plot for WGA binding to parental cells was complex and exhibited positively cooperative binding at the high affinity sites. One of the WGA-resistant mutants (WgaRIII) was apparently not altered in its WGA-binding ability compared with parental cells. However, two of the WGA-resistant lines (WgaRI and WgaRII) had distinct alterations in their WGA-binding properties specific to certain regions of the binding curve. Neither appeared to be affected in either the highest or lowest affinity regions of the binding curve. Thus, lectin-resistant cell mutants altered in specific lectin-binding sites at the cell surface provide a direct approach to analysis of the complex binding parameters that characterize the interaction of WGA with the plasma membrane.
Error estimate for the diffusion approximation of the Wright--Fisher model.
Authors: Ethier S N , Norman M F
Published: May 2006

The Wright--Fisher model and its approximating diffusion model are compared in terms of the expected value of a smooth but arbitrary function of nth-generation gene frequency. In the absence of selection, this expectation is shown to differ in the two models by at most a linear combination (with coefficients depending only on the derivatives of the smooth function involved) of the maximum mutation rate and the reciprocal of the population size.
Proposal for the biologically active conformation of opiates and enkephalin.
Authors: Gorin F A , Marshall G R
Published: May 2006

A model for the opiate receptor has been defined by using a computer-based molecular display and x-ray crystallographic input data. The model can explain the stereochemical fashion in which the morphine, morphinan, and oripavine classes of compounds interact with the receptor. The minimal structural unit of the enkephalins demonstrated to be pharmacologically active. Tyr-Gly-Gly-Phe, was also fitted to this model by using a systematic search of conformational space.
Structure of transition-metal cluster compounds: Use of an additional orbital resulting from the
f, g
character of
spd
bond orbitals
Authors: Pauling Linus
Published: May 2006

A general theory of the structure of complexes of the transition metals is developed on the basis of the enneacovalence of the metals and the requirements of the electroneutrality principle. An extra orbital may be provided through the small but not negligible amount of
f
and
g
character of
spd
bond orbitals, and an extra electron or electron pair may be accepted in this orbital for a single metal or a cluster to neutralize the positive electric charge resulting from the partial ionic character of the bonds with ligands, such as the carbonyl group. Examples of cluster compounds of cobalt, ruthenium, rhodium, osmium, and gold are discussed.
Transition state structures of a dipeptide related to the mode of action of beta-lactam antibiotics.
Authors: Boyd D B
Published: May 2006

The tetrahedral adducts formed during nucleophilic attack by a hydroxyl ion on the carbonyl carbon of a model dipeptide, glycylglycine, were studied by modified-intermediate-neglect-of-differential-overlap molecular orbital calculations. This dipeptide is taken to represent the D-alanyl-D-alanine terminus of the polypeptides involved in the cross-linking transpeptidation reaction of peptidoglycan in bacterial cell walls. It was found that nucleophilic attack on one face of the carbonyl carbon leads to a transition intermediate species structurally similar to that afforded by the bicyclic nucleus of penicillins and cephalosporin antibiotics. The results support the concept that the beta-lactam antibiotics, which are known to inhibit various bacterial cell wall enzymes, may act as transition state analogs. Also, the structure formed from nucleophilic attack on the so-called alpha face of the dipeptide is more similar to the antibiotic structures than is that from attack on the opposite face. In agreement with other types of experiments, the results suggest that the alpha face may be the one approached by a nucleophile in the receptor site(s) of the appropriate cell wall enzymes.
Identification of the major adducts formed by reaction of benzo(a)pyrene diol epoxide with DNA in vitro.
Authors: Straub K M , Meehan T , Burlingame A L , Calvin M
Published: May 2006

Covalent binding of the benzo[a]pyrene metabolite (+/-)7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to calf thymus DNA was investigated. Enzymatic hydrolysis of the carcinogen-modified DNA and subsequent separation via reversed-phase high-pressure liquid chromatography resulted in the detection and isolation of seven distinct products. High-resolution mass spectrometry indicates that these products are covalent adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine. The deoxyguanosine and deoxyadenosine adducts involve binding between the activated hydrocarbon (benzo[a]pyrene diol epoxide) and exocyclic amino groups of the respective purines.
Spatial requirement for coupling of iodotyrosine residues to form thyroid hormones.
Authors: Cahnmann H J , Pommier J , Nunez J
Published: May 2006

A linear random copolymer of tyrosine and lysine and two synthetic oligopeptides containing two tyrosine residues in addition to lysine residues give thyroid hormone (thyroxine and triodothyronine) residues in good yield upon enzymatic iodination with thyroid peroxidase. These synthetic peptides may serve as simple models for thyroglobulin, the protein in which biosynthesis of the thyroid hormone takes place. For the formation of significant amounts of hormone, such model compounds must contain at least two properly spaced tyrosine residues.
Developmental control of messenger RNA for hepatic tryptophan 2,3-dioxygenase
Authors: Killewich Lois A. , Feigelson Philip
Published: May 2006

The enzyme tryptophan 2,3-dioxygenase [EC 1.13.11.11; L-tryptophan:oxygen 2,3-oxidoreductase (decyclizing)] first appears in the livers of young rats around the 15th postnatal day, and increases to the adult level by the 22nd day. Studies have shown that the appearance and subsequent development of the enzyme activity result from an increase in the rate of its synthesis and thus in the amount present in the liver. In this study, we have attempted to ascertain whether the appearance and development of tryptophan 2,3-dioxygenase mRNA coincided with, and thus led to, the development of enzyme activity, or whether the biosynthesis of this enzyme was due to a developmental event enabling translation of a preexisting, sequestered, reservoir of its mRNA. Using a cell-free protein-synthesizing system based on a wheat germ S30 supernatant, we measured the level of tryptophan 2,3-dioxygenase mRNA in the livers of rats between 0 and 22 days of age. We found that functional tryptophan 2,3-dioxygenase mRNA is not detectable in rat liver until the 15th postnatal day. It increases to the adult level by the 22nd postnatal day, in parallel with the enzyme. The appearance and development of tryptophan 2,3-dioxygenase are the direct consequence of the parallel appearance and development of its mRNA.

It has been shown that glucocorticoids, which induce tryptophan 2,3-dioxygenase activity in adult rats, are capable of inducing the appearance of this enzyme precociously in 8- and 10-day-old rats. We have found that it is also possible to induce tryptophan 2,3-dioxygenase catalytic activity with hydrocortisone precociously in 4-day-old rats. Moreover, precocious induction of enzyme activity and the induction that occurs during the enzyme's normal developmental rise to the adult level between 15 and 22 days, are mediated through parallel increases in the level of tryptophan 2,3-dioxygenase mRNA.

The present findings indicate that glucocorticoids are developmental hormones that act upon the postnatal hepatocyte to evoke elevated levels of the mRNA species coding for tryptophan 2,3-dioxygenase; the findings are compatible with the hypothesis that such hormones act by initiating and accelerating transcription of the structural genes coding for the α and β protomers of this enzyme.
Distribution of rat liver albumin mRNA membrane-bound and free in polyribosomes as determined by molecular hybridization
Authors: Yap S. H. , Strair Roger K. , Shafritz David A.
Published: May 2006

Recently, we purified rat liver albumin mRNA and prepared albumin [
3
H]cDNA. Using albumin [
3
H]cDNA in molecular hybridization experiments, we have now determined the distribution of albumin mRNA sequences in membrane-bound and free liver polyribosomes prepared by techniques in which there is high recovery of polyribosomes without evidence of degradation. By using molecular hybridization to measure specific mRNA sequence content, numerous problems could be avoided in interpretation of results as obtained by cell-free protein synthesis or immunological methods. Under these conditions, 98% of albumin mRNA sequences in polyribosomes are found in the membrane-bound fraction.
Sites of transcription initiation
in vivo
on
Xenopus laevis
ribosomal DNA
Authors: Reeder Ronald H. , Sollner-Webb Barbara , Wahn Harvey L.
Published: May 2006

We report the results of a novel method for locating sites of transcription initiation using a complex of capping enzymes from vaccinia virions that catalyze the reaction pppG +
S
-adenosylmethionine + (p)ppXpYpZp..... →
7m
GpppXpYpZp..... [Ensinger, M. J., Martin, S. A., Paoletti, E. and Moss, B. (1975)
Proc. Natl. Acad. Sci. USA
72, 2525-2529]. This enzyme complex will cap di- or triphosphate termini but will not cap monophosphate or hydroxyl termini.
Xenopus laevis
40S precursor rRNA from oocytes is capped by these enzymes, and we conclude that it has 5′-polyphosphate termini. Therefore, 40S RNA must represent the primary transcript of amplified
X. laevis
ribosomal DNA. The majority of 40S molecules with polyphosphate termini begin with the sequence (p)ppAAG. There is evidence, however, that the 5′ terminus may be heterogeneous. The majority of all detectable initiation events were localized close to the region coding for the 5′ end of the 40S RNA. No initiation sites were detected in the nontranscribed spacer, but an apparent initiation site in the middle of the transcribed region was also observed.
The bicistronic nature of lens alpha-crystallin 14S mRNA.
Authors: Chen J H , Spector A
Published: May 2006

The A2 and B2 polypeptide chains of calf lens alpha-crystallin are synthesized on a 14S, 1500-nucleotide mRNA and a 10S, 735-nucleotide mRNA, respectively. The 10S mRNA is theoretically compatible with the size of the B2 chain, but the 14S mRNA contains approximately twice the required number of nucleotides necessary for A2 chain synthesis. This fact raises the question of the function of the additional nucleotide sequence in the 14S mRNA. The following observations on 14S mRNA suggest that it may contain an additional cistron. (i) Under a number of denaturing conditions, 14S mRNA continues to retain its initial size characteristics. (ii) In addition to synthesis of the A2 chain, 14S mRNA directs the synthesis of another polypeptide with the same electrophoretic mobility as that of the B2 chain. (iii) Molecular hybridization of the 14S mRNA with the cDNA produced from the 10S mRNA suggests that 2 mol of the cDNA bind to 1 mol of the 14S mRNA. (iv) Examination of the nucleotide sequences of the 10S and 14S mRNAs by two-dimensional maps of RNase A and T1 digests indicates marked similarity. The overall data suggest that the additional cistronic component may carry coding information for an alpha-crystallin polypeptide or a closely related polypeptide species.
DNA sequencing with chain-terminating inhibitors
Authors: Sanger F. , Nicklen S. , Coulson A. R.
Published: May 2006

A new method for determining nucleotide sequences in DNA is described. It is similar to the “plus and minus” method [Sanger, F. & Coulson, A. R. (1975)
J. Mol. Biol.
94, 441-448] but makes use of the 2′,3′-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage ϕX174 and is more rapid and more accurate than either the plus or the minus method.
Initiation of polypeptide synthesis with various NH2-blocked aminoacyl-tRNAs under the direction of alfalfa mosaic virus RNA 4.
Authors: Castel A , Kraal B , Kerklaan P R , Klok J , Bosch L
Published: May 2006

Initiation of polypeptide synthesis in a cell-free system of Escherichia coli directed by alfalfa mosaic virus RNA 4 was studied by using either fMet-tRNA or Ac-Phe-tRNA as initiator tRNA. Initiation with fMet-tRNA yielded a product that was identical to the authentic viral coat protein except that the NH2-terminal serine was preceded by fMet instead of being acetylated. When Ac-Phe-tRNA was used as initiator, the biosynthetic product was 10-12 amino acid residues longer, the extra amino acids being located at the NH2-terminus. fMet-tRNA and Ac-Phe-tRNA did not compete for ribosomes during initiation of protein synthesis, as became evident from incorporation studies using both initiator tRNAs simultaneously. It is concluded that E. coli ribosomes recognize two sites on the 5' end of alfalfa mosaic virus RNA 4 that are separated by a region of about 30 nucleotides. The results are in complete agreement with the 5'-terminal nucleotide sequence of this RNA [Koper-Zwarthoff, E. C., Lockhard, R. E., RajBhandary, U. L., Alzner-deWeerd, B. & Bol, J. F. (1977) Proc. Natl. Acad. Sci. USA 74, 5504-5508].
Electron microscopy of fibers and discs of hemoglobin S having sixfold symmetry
Authors: Ohtsuki M. , White S. L. , Zeitler E. , Wellems T. E. , Fuller S. D. , Zwick M. , Makinen M. W. , Sigler P. B.
Published: May 2006

Aggregated forms of deoxyhemoglobin S were examined with a field emission transmission electron microscope. Images of isolated helical fibers were obtained from sickled cell lysates stained directly on the electron microscope grid. Optical and digital analyses of the electron micrographs showed that the fibers are similar to those characterized by J. T. Finch, M. F. Perutz, J. F. Bertles, and J. Döbler [(1973)
Proc. Natl. Acad. Sci. USA
70, 718-722] in that they consist of stacked discs each composed of six hemoglobin molecules. The fibers exhibit an outer diameter of 160-170 Å and an inner diameter of about 60 Å with an axial spacing of 58 Å per disc. The fiber can be described as a helix consisting of 56 discs per helical turn. We observed discs of six hemoglobin molecules, which may be stable substructural components of the fibers. They were observed in preparations of hemoglobin fibers and exhibited 6-fold symmetry by power spectrum analysis. A reconstructed image of a disc digitally filtered for 6-fold symmetry has a maximum external diameter of ∼170 Å and a central hole of 60 Å diameter and is similar to the axial projection of a single disc from a low-resolution, three-dimensional reconstructed model of a fiber.
Nerve growth factor prevents vinblastine destructive effects on sympathetic ganglia in newborn mice.
Authors: Chen M G , Chen J S , Calissano P , Levi-Montalcini R
Published: May 2006

Vinblastine injections in newborn mice produce severe atrophy of sympathetic ganglia; after a 7-day treatment the ganglia are 80% reduced in volume. Histological examinations show that this effect is due to a marked decrease in the neuronal cell population. The most precocious ultra-structural alterations are localized in the nuclear compartment, followed by axonal swelling and microtubule disappearance. Simultaneous injections of nerve growth factor entirely prevent the noxious effects of the vinca alkaloid, and result in partial appearance of the growth effects of nerve growth factor. Such protective action is not due to inhibition of vinblastine uptake which is the same in control mice and in mice pretreated with nerve growth factor. It is suggested that nerve growth factor prevents the vinca alkaloid action by favoring the assembly or organization, or both, of microtubules, which, from in vitro studies, have been proved to be inaccessible to vinblastine.
Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts.
Authors: Ash J F , Louvard D , Singer S J
Published: May 2006

The surface distributions of three different membrane integral proteins, beta2-microglobulin (part of the histocompatibility antigen complex), aminopeptidase (alpha-aminoacyl-peptide hydrolase; EC 3.4.11.2), and the Na+,K+-ATPase (ATP phosphohydrolase; EC 3.6.1.3) on human fibroblasts grown in monolayer culture have been studied with their specific antibodies by immunofluorescence. On the same cells, the distribution of intracellular actin was observed by a spectrally distinct fluorescent staining procedure. If each of the antibody reagents was permitted to cluster its specific protein in the plane of the membrane, these clusters apparently became linked, through the membrane, to actin- and myosin-containing filaments (stress fibers) underneath the membrane, and were thereby immobilized. From these and other experiments, it appears that most, if not all, integral proteins can, upon clustering, form such transmembrane linkages to actin and myosin. A molecular mechanism for the formation of these linkages is proposed which postulates that actin is associated with the cytoplasmic surface of plasma membranes by peripheral attachment to a ubiquitous integral protein X in the membrane; when other integral proteins are induced to form clusters, they become bound to X and hence to actin (and myosin). The possible physiological role of these transmembrane linkages is briefly discussed.
Molecular evolution of human and rabbit beta-globin mRNAs.
Authors: Kafatos F C , Efstratiadis A , Forget B G , Weissman S M
Published: May 2006

The primary structures of human and rabbit beta-globin mRNAs are compared. Using as a standard the extent of nucleotide substitutions inferred from the hypervariable amino acid residues of fibrinopeptides A and B, which are thought to change largely by neutral evolution, we show that not all silent mutations in globin mRNA are neutral. The divergence of the sequences is limited in part by the selective usage of synonymous codons. The divergent nucleotides tend to be distributed nonrandomly: in the coding region silent substitutions are most rare in segments that are also deficient in substitutions leading to replacements.
Assignment of the human gene for galactose-1-phosphate uridyltransferase to chromosome 9: studies with Chinese hamster-human somatic cell hybrids.
Authors: Mohandas T , Sparkes R S , Sparkes M C , Shulkin J D
Published: May 2006

Chinese hamster-human somatic cell hybrids were analyzed for the expression of human galactose-1-phosphate uridyltransferase (GALT; UDPglucose:alpha-D-galactose-1-phosphate uridyltransferase, EC 2.7.7.12) by electrophoresis and for the presence of human chromosomes cytogenetically with the aid of Q-banding. Three of the 10 randomly chosen independently derived primary hybrid lines showed the presence of human GALT. Human chromosome 9 was consistently present in the hybrid lines expressing human GALT and consistently absent in the lines not expressing it. Biochemical analysis alone of 11 independently derived hybrid lines showed human GALT to be syntenic with known chromosome 9 markers (soluble aconitase, adenylate kinase 1, and adenylate kinase 3). Previous studies on chromosome assignment of this locus, utilizing somatic cell hybrids, have yielded inconsistent results; one group assigned GALT to chromosome 2, and another assigned it to chromosome 3. However, we believe that, based on our results and other published evidence, the correct assignment of the human GALT locus is to chromosome 9.
Isolation and characterization of a lambdapolA transducing phage.
Authors: Kelley W S , Chalmers K , Murray N E
Published: May 2006

A plaque-forming lambdapolA phage was isolated from a population of transducing phage made in vitro from Escherichia coli DNA and a phage vector digested with restriction endonuclease HindIII. Amber mutations, in genes whose products are necessary for late protein synthesis (Q) and cell lysis (S), were crossed into the lambdapolA phage. Infection of either polA+ or polA- bacteria with this phage, under conditions permitting DNA replication but preventing phage production and lysis, elevated the levels of DNA polymerase I to between 75- and 100-fold that detected in a wild-type strain. The kinetics of enzyme production suggest that the polA gene is transcribed from its own promoter rather than from any of the well-characterized phage promoters. The fragment of E. coli DNA within the lambdapolA phage comprises approximately 5000 base pairs, sufficient to accommodate the polA gene and one, or two, coding sequences for smaller proteins.
Increased dexamethasone resistance of cystic fibrosis fibroblasts.
Authors: Epstein J , Breslow J L , Davidson R L
Published: May 2006

We demonstrate previously that fibroblasts from cystic fibrosis (CF) patients are significantly more resistant to the toxic effects of ouabain than normal human fibroblasts are. Ouabain is generally assumed to act primarily at the level of the cell membrane and to cause killing of cells by inhibiting ion transport. We have therefore examined the ability of normal and CF cells to survive exposure to ethacrynic acid, another inhibitor of ion transport, and to survive colchicine and aminopterin, resistance to which has been associated with membrane alterations in other cells. The effect of dexamethasone, which has a sterol nucleus similar to that of ouabain but is thought to have a different site of cellular action, was also tested. Exposure of the cells to ethacrynic acid, colchicine, or aminopterin did not reveal any differences in survival between normal and CF fibroblasts. However, CF cells survived exposure to dexamethasone significantly better than normal cells did. These results suggest that normal and CF cells do not differ in terms of a generalized resistance to ion transport inhibitors or to drugs that must pass through the membrane to be active. Instead, the results raise the possibility that CF cells have an enhanced resistance to drugs that have the sterol nucleus found in ouabain and dexamethasone.
Variable and constant parts of the immunoglobulin light chain gene of a mouse myeloma cell are 1250 nontranslated bases apart.
Authors: Brack C , Tonegawa S
Published: May 2006

A DNA fragment carrying an immunoglobulin gene coding for both variable (V) and constant (C) regions of a mouse lambda light chain was enriched about 15-fold from a total endonuclease EcoRI digest of a plasmacytoma (HOPC 2020) DNA by preparative agarose gel electrophoresis. The DNA fraction was used for cloning of a lambda chain gene in the phage lambdagtWES vector. After screening [Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182] of about 70,000 plaques, each arising from an independent transfection event, we isolated one clone (Ig 303) that contained both a Vlambda and a Clambda DNA sequence. Electron microscopy of R-loops formed between the cloned DNA and purified lambda chain mRNA (HOPC 2020) revealed that the Vlambda and Clambda DNA sequences are separated by a 1250-base DNA fragment.
Physiological regulation of antigen binding to T cells: role of a soluble macrophage factor and of interferon.
Authors: Lonai P , Steinman L
Published: May 2006

A soluble product of macrophages (MF) and mouse viral interferon (IF) increase both major histocompatibility antigenic determinants and the number of antigen-binding cells in nonstimulated T cell-enriched mouse lymphocyte cultures. MF increases Ia and not H-2 antigens; IF increases H-2 but not Ia antigens. The increased antigen binding due to MF can be inhibited by anti-Ia but not by anti-H-2 sera, whereas IF-induced binding is sensitive to anti-H-2 but not to anti-Ia sera. The specificity of IF- or MF-induced binding of branched synthetic polypeptides by T cells is different from that of B cells and similar to the specificity of the Ir gene regulation. MF increases antigen binding only in Ir high-responder animals. The IF-induced antigen binding is not dependent on the Ir genotype. MF-reactive cells express the Ly-1 marker, and the IF-reactive antigen binders express the Ly-2 phenotype. It is suggested that MF and IF are physiological mediators of antigen binding by T cells.
Interspecies radioimmunoassay for the major structural proteins of primate type-D retroviruses.
Authors: Colcher D , Teramoto Y A , Schlom J
Published: May 2006

A competition radioimmunoassay has been developed in which type-D retroviruses from three primate species compete. The assay utilizes the major structural protein (36,000 daltons) of the endogenous squirrel monkey retrovirus and antisera directed against the major structural protein (27,000 daltons) of the Mason-Pfizer monkey virus isolated from rhesus monkeys. Purified preparations of both viruses grown in heterologous cells, as well as extracts of heterologous cells infected with squirrel monkey retrovirus or Mason-Pfizer monkey virus, compete completely in the assay. Addition of an endogenous virus of the langur monkey also results in complete blocking. No blocking in the assay is observed with type-C baboon viruses, woolly monkey virus, and gibbon virus. Various other type-C and type-B viruses also showed no reactivity. An interspecies assay has thus been developed that recognizes the type-D retroviruses from both Old World monkey (rhesus and langur) and New World monkey (squirrel) species.
Chemiluminescent reactions and electrophilic oxygen donating ability of 4a-hydroperoxyflavins: general synthetic method for the preparation of N5-alkyl-1,5-dihydroflavins.
Authors: Kemal C , Chan T W , Bruice T C
Published: May 2006

The synthesis of N5-methyl- and N5-ethyl-1,5-dihydroflavin mononucleotides is reported. These compounds show no bioluminescence activity with bacterial luciferase. This feature is interpreted in terms of steric hindrance between the N5-alkyl group and a hydrogen bonding group at the active site of the luciferase. The chemiluminescence observed on reaction of N5-alkyl-1,5-dihydroflavins with oxygen and aldehydes has been shown to occur via formation of a mixed peroxide of flavin and aldehyde and to be associated with a primary deuterium isotope effect when [1-2H]aldehyde is substituted for aldehyde. The time course for light emission has been compared for aldehyde and ketone substrates. The suggestion is entertained that the peroxide bond of 4a-hydroperoxyflavin is sufficiently polarized to allow this species to act as the oxidant per se at the active site of mixed function oxidases. The second-order rate constants for reaction of hydroperoxides with thioxane and I- are compared. 4a-Hydroperoxy-3,5-dimethyllumiflavin is shown to convert thioxane to its sulfoxide 1.8 X 10(5) times faster than t-butyl hydroperoxide.
Lipid and protein segregation in Escherichia coli membrane: morphological and structural study of different cytoplasmic membrane fractions.
Authors: Letellier L , Moudden H , Shechter E
Published: May 2006

Lipid and protein segregations can be induced in E. coli cytoplasmic membranes by conformational transitions of their lipid hydrocarbon chains from a disordered to an ordered state. For E. coli strain K 1059 (an unsaturated fatty acid auxotroph) supplemented with linolenic acid, the segregation leads to large areas of membrane surfaces having distinctly different morphological characteristics (smooth compared with strongly particulated fracture faces, as visualized by freeze fracture electron microscopy). The different regions are physically separated by osmotic lysis of spheroplasts at temperatures below those of the order-disorder transition of the lipid hydrocarbon chains. The analysis of the different cytoplasmic membrane fractions provides a direct demonstration and allows a direct analysis of the segregation. As compared to the nonfractionated membranes, the membrane regions corresponding to the smooth fracture surfaces are poor in proteins, rich in lipids, and enriched in saturated fatty acids, while the membrane regions corresponding to the strongly particulated fracture surfaces are rich in proteins, poor in lipids, and enriched in unsaturated fatty acids. Quantitative information about the extent of these segregations is obtained from high-angle x-ray diffraction of the different membrane fractions and of the corresponding total lipid extracts.