bluuebunny/crossref_metadata_2025 huggingface dataset:
Parathyroid hormone stimulates bone formation and resorption in organ culture: evidence for a coupling mechanism.
Authors: Howard G A , Bottemiller B L , Turner R T , Rader J I , Baylink D J
Published: May 2006

We have developed an in vitro system, using embryonic chicken tibiae grown in a serum-free medium, which exhibits simultaneous bone formation and resorption. Tibiae from 8-day embryos increased in mean (+/- SD) length (4.0 +/- 0.4 to 11.0 +/- 0.3 mm) and dry weight (0.30 +/- 0.04 to 0.84 +/- 0.04 mg) during 12 days in vitro. There was increased incorporation of [3H]proline into hydroxyproline (120 +/- 20 to 340 +/- 20 cpm/mg of bone per 24 hr) as a measure of collagen synthesis, as well as a 62 +/- 5% increase in total calcium and 45Ca taken up as an indication of active mineralization. A physiologic concentration (1 pM) of parathyroid hormone was found to stimulate bone resorption over control levels in this system. Parathyroid hormone stimulated the release of [3H]hydroxyproline from the bone shafts but not from the cartilage ends, indicating the specificity of the response. With 1 pM parathyroid hormone we observed an acute inhibition of bone formation, followed (after 12-16 hr) by a chronic stimulation of bone formation during the 12-day incubation. Both mineral uptake and matrix formation were enhanced at approximately the same rate during the 12-day incubation. The chronic enhancement of formation required parathyroid hormone only for the initial 8-10 hr of incubation. These results could be explained by the production or release of a factor from bone to stimulate formation in response to the acute increase in resorption--a "coupling factor." Indeed, dialyzed culture medium conditioned by actively resorbing bones stimulated bone formation over controls when added to organ cultures at a 1:20 dilution. The factor is larger than 12,000 daltons as determined by dialysis. The factor is specific for the bone shaft and did not affect the cartilage ends.
Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes.
Authors: Cawley D B , Simpson D L , Herschman H R
Published: May 2006

We have constructed a toxic hybrid protein that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibiting protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. We tested a series of glycoproteins for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, two glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose-terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialogalactoorsomucoid) had no appreciable effect on the activity of the conjugate. We tested the ASF-DTA conjugate on six cell types; except for primary rat hepatocytes, none of them were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is cell-type specific, and its action is mediated by a well-characterized receptor, whose mechanism of receptor-ligand internalization has been extensively investigated.
Distribution of UV light-induced damage in a defined sequence of human DNA: detection of alkaline-sensitive lesions at pyrimidine nucleoside-cytidine sequences.
Authors: Lippke J A , Gordon L K , Brash D E , Haseltine W A
Published: May 2006

The distribution of UV light-induced damage to the highly reiterated alpha sequence of human DNA was investigated. The results show that the distribution of UV light-induced cyclobutane dimers within a defined sequence is similar whether the DNA is exposed to UV light as part of the chromosome of intact cells or as naked DNA. However, the cellular environment shields the nuclear DNA, resulting in about 50% decrease in apparent dose. A new type of UV photodamage was detected. Treatment of UV light-irradiated DNA with hot alkali results in strand breaks at positions of cytidine located 3' to pyrimidine nucleosides. The chemical nature and biological significance of the pyrimidine nucleoside-cytidine lesion is discussed.
Bacteriorhodopsin: partial sequence of mRNA provides amino acid sequence in the precursor region.
Authors: Chang S H , Majumdar A , Dunn R , Makabe O , RajBhandary U L , Khorana H G , Ohtsuka E , Tanaka T , Taniyama Y O , Ikehara M
Published: May 2006

mRNA for bacteriorhodopsin from Halobacterium halobium has been partially purified. By using this mRNA as template in the presence of reverse transcriptase RNA-dependent DNA nucleotidyltransferase and a 5'-[32P] synthetic oligodeoxyribonucleotide corresponding to amino acids 9-12 of bacteriorhodopsin as primer, we have isolated the major 5'-[32P]cDNA product, approximately 80 nucleotides long, and determined its sequence. Based on the cDNA sequence, the 5'-proximal sequence of bacteriorhodopsin mRNA is G-C-A-U-G-U-U-G-G-A-G-U-U-A-U-U-G-C-C-A-A-C-A-G-C-A-G-U-G-G-A-G-G-G-G-G-U-A-U-C -G-C-A-G-G-C-C-C-A-G-A-U-C-A-C-C-G-G-A-C-G-U-C-C-G. This includes the expected sequence for amino acids 1-8 and shows that bacteriorhodopsin is synthesized as a precursor that is at least 13 amino acids longer (Met-Leu-Glu-Leu-Leu-Pro-Thr-Ala-Val-Glu-Gly-Val-Ser) at the NH2 terminus. Agarose/urea gel electrophoresis of the partially purified mRNA showed several bands; of these, a major one hybridized with 5'-[32P]cDNA. These results suggest that the bacteriorhodopsin mRNA in the partially purified preparation is homogeneous in size and that it constitutes a substantial portion of the RNA preparation subjected to electrophoresis.
Amino acid sequence of Escherichia coli alkaline phosphatase.
Authors: Bradshaw R A , Cancedda F , Ericsson L H , Neumann P A , Piccoli S P , Schlesinger M J , Shriefer K , Walsh K A
Published: May 2006

The complete amino acid sequence of the Escherichia coli alkaline phosphatase subunit [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1, isozyme 3] has been determined. The monomer contains 449 amino acid residues in a single unglycosylated polypeptide chain having a calculated Mr of 47,029. Isozyme 1 has an additional arginine residue at the NH2 terminus that presumably results from variability in processing of precursor molecules. Sequence data were obtained from both manual and automatic Edman degradation of the tryptic and cyanogen bromide peptides, as well as other peptides derived therefrom. The two disulfide bonds were determined from analyses of the appropriate peptic peptides. This structure confirms earlier reports of the sequence surrounding the active-site serine and both the NH2- and COOH-terminal cyanogen bromide fragments. A secondary structure prediction places nearly half the residues in alpha-helical segments that have 13% and 16%, respectively, in beta-strand and beta-turn orientations.
Differential effects of transforming avian RNA tumor viruses on avian macrophages.
Authors: Durban E M , Boettiger D
Published: May 2006

Functionally differentiated chicken macrophages were derived by in vitro differentiation of embryonic yolk sac cells and were characterized by several macrophage-specific cell markers. Uniform, infected, virus-producing cultures were obtained after exposure of these macrophages to avian myoblastosis virus (AMV), avian myelocytomatosis virus (MC29), myeloblastosis-associated virus (MAV-2), and Prague strain of Rous sarcoma virus (PR-B RSV). Both AMV and MC29 induced morphological transformation typical of the in vivo leukemias induced by these virus strains. Analysis of the expression of macrophage-specific markers in these two transformed cell types demonstrated that different markers of the mature macrophage were suppressed by each virus, even though the parental cell immediately preceding the transformation event was a mature macrophage in both cases. Cells infected with PR-B RSV and MAV-2 showed no observable difference from uninfected macrophages in terms of morphological characteristics, growth rate, or expression of the differentiated functions of macrophages. Ths system provides demonstrations of a cell type that produces infectious, transforming RSV but fails to respond by functional alterations induced by the transforming gene, src.
Self-renewal of hemopoietic stem cells during mixed colony formation in vitro.
Authors: Humphries R K , Eaves A C , Eaves C J
Published: May 2006

Replating experiments have shown that the self-renewal of pluripotent hemopoietic stem cells can be studied in vitro by clonal analysis techniques. The number of daughter stem cells detectable in individual primary clones produced in vitro varies markedly from one clone to another. These findings are consistent with a general model of stem cell differentiation in which the choice to self-replicate or not is ultimately determined at the single-cell level by a mechanism involving a random-event component that is intrinsic to the stem cell itself. Hemopoietic stem cells were identified by their ability to generate macroscopic-sized colonies having a visible erythroid component (i.e., gross red color) in standard methylcellulose assays containing medium conditioned by pokeweed mitogen-treated spleen cells and erythropoietin. In assays of replated primary or secondary colonies, inclusion of irradiated marrow-cell feeders was found to be an additional requirement. The mixed erythroid-megakaryocyte-granulocyte nature of colonies identified simply as macroscopic and erythroid was confirmed by cytochemical stains for lineage-specific markers. Marked variation in self-renewal was a feature of marrow stem cells both before and after maintenance in flask culture, although the overall self-renewal capacity exhibited by flask-cultured cells was approximately 5-fold higher. Variation in self-renewal was not correlated with primary colony size, which also varied over a wide range (0.2-9 X 10(5) nucleated cells per colony). Variation in stem cell self-renewal has been previously associated with hemopoietic stem cell proliferation in vivo. Its persistence in vitro in assays of dilute single-cell suspensions casts doubt on the significance of microenvironmental influences in directing stem cell differentiation.
Differentiation of fetal liver cells in vitro.
Authors: Freeman A E , Engvall E , Hirata K , Yoshida Y , Kottel R H , Hilborn V , Ruoslahti E
Published: May 2006

Fetal mouse liver hepatocytes proliferate on a substrate of irradiated pigskin epidermis scored with scalpel blade slits to permit cell access to the basement membrane. At the time the cells are explanted, fetal genes, such as those responsible for production of alpha-fetoprotein (AFP) and gamma-glutamyltransferase (GGTase), are strongly expressed. The levels of GGTase decrease rapidly and become undetectable within 2 weeks. The levels of AFP decrease more gradually but become undetectable after 3-5 weeks in culture. As the AFP levels decrease, there is a concomitant increase in albumin production. Hydrocortisone prolongs production of AFP (for up to 8 weeks) but not of GGTase, and it decreases albumin production for up to 8 weeks. Once cells lose AFP expression, addition of hydrocortisone does not restart it. Based on these data, fetal mouse liver hepatocytes, cultured on pigskin, seem to be an excellent in vitro model for liver cell maturation.
Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia.
Authors: Heldin C H , Westermark B , Wasteson A
Published: May 2006

A cellular receptor for platelet-derived growth factor (PDGF) was demonstrated by incubation of 125I-labeled PDGF with human foreskin fibroblast cultures followed by liberation of cell-bound radioactivity with Triton X-100. The cellular binding of labeled PDGF in the presence of increasing amounts of unlabeled PDGF showed saturation; Scatchard analysis of binding data indicated a single class of receptors having kd = 1 X 10(-9) M. The number of PDGF binding sites was approximately 3 X 10(5)/cell. Labeled PDGF binding reached an apparent equilibrium after 3 hr at 4 degrees C. At 37 degrees C, it passed a maximum after 30 min and then decreased with time due to degradation of the tracer. A large excess of unlabeled PDGF reduced labeled PDGF binding by more than 90% whereas similar doses of epidermal growth factor, fibroblast growth factor, or insulin had no effect. It was concluded that PDGF did not share receptors with these factors. PDGF receptors were found on skin fibroblasts, normal and malignant glial cells, smooth muscle cells, and 3T3 cells but not on epithelial-derived cells, neuroblastoma cells, endothelial cells, or peripheral lymphocytes.l As only the receptor-positive cells--i.e., the connective tissue- and glia-derived cells--are responsive to stimulation with PDGF, these findings imply a functional significance of the PDGF receptor.
Embryonic proteins in somatic embryos of carrot
Authors: Sung Z. R. , Okimoto R.
Published: May 2006

The translational profile of cultured carrot cells (callus) was compared with that of the somatic embryos derived from them. The two tissues synthesize almost the same number and kinds of polypeptides except for two “embryonic” proteins. These were found in the somatic embryos but were nearly undetectable in the callus. Both embryo development and the production of embryonic proteins were induced by the same trigger (transfer of the callus to fresh medium) and were suppressed by the same factor (2,4-dichlorophenoxyacetic acid). But the appearance and disappearance of the proteins occurred several days prior to embryo formation and to the conversion of embryo to callus, respectively. Carrot cell lines incapable of embryogenesis could not synthesize the embryonic proteins. These findings indicate that the embryonic proteins play a key role in the process of embryo development. The function of these proteins is presently unknown; however, they can serve as early developmental markers for studying the mechanisms underlying somatic embryogeny in plants.
Models of speciation by sexual selection on polygenic traits
Authors: Lande Russell
Published: May 2006

The joint evolution of female mating preferences and secondary sexual characters of males is modeled for polygamous species in which males provide only genetic material to the next generation and females have many potential mates to choose among. Despite stabilizing natural selection on males, various types of mating preferences may create a runaway process in which the outcome of phenotypic evolution depends critically on the genetic variation parameters and initial conditions of a population. Even in the absence of genetic instability, rapid evolution can result from an interaction of natural and sexual selection with random genetic drift along lines of equilibria. The models elucidate genetic mechanisms that can initiate or contribute to rapid speciation by sexual isolation and divergence of secondary sexual characters.
Syntheses of all singly labeled [
15
N]adenines: Mass spectral fragmentation of adenine
Authors: Barrio Maria Del Carmen G. , Scopes David I. C. , Holtwick Joseph B. , Leonard Nelson J.
Published: May 2006

Syntheses of all five of the singly labeled [
15
N]adenines are now provided. The presence or absence of two-bond
15
N-
1
H spin couplings in their
1
H NMR spectra confirm the location of the isotope in each case. The fragmentation patterns in their mass spectra are indicative of the sequential losses of HCN units and of CH
2
N
2
from adenine upon electron impact.
All binomial identities are verifiable
Authors: Zeilberger Doron
Published: May 2006

Sister Celine Fasenmyer's technique for obtaining pure recurrence relations for hypergeometric polynomials is formalized and used to show that every identity involving sums of products of binomial coefficients can be verified by checking a finite number of its special cases.
Discrete-length repeated sequences in eukaryotic genomes.
Authors: Pearson W R , Morrow J F
Published: May 2006

Two of the four repeated DNA sequences near the 5' end of the silk fibroin gene hybridize with discrete-length families of repeated DNA. These two families comprise 0.5% of the animal's genome. A repeated sequence with a conserved length has also been found in the short class of moderately repeated sequences in the sea urchin. The discrete length, interspersion, and sequence fidelity of these moderately repeated sequences suggests that each has been multiplied as a discrete unit. Thus, transposition mechanisms may be responsible for the multiplication and dispersion of a large class of repeated sequences in phylogenetically diverse eukaryotic genomes. The repeat we have studied in most detail differs from previously described eukaryotic transposable elements: it is much shorter (1300 base pairs) and does not have terminal repetitions detectable by DNA hybridization. A simple technique for identifying such discrete-length repeated sequences is described.
Stoichiometry and composition of an aminoacyl-tRNA synthetase complex from rat liver.
Authors: Johnson D L , Yang D C
Published: May 2006

The particulate aminoacyl-tRNA synthetases of rat liver were copurified about 1000-fold with more than 20% yields for individual synthetase activities. Measurements of aminoacylation activities showed that lysyl-, arginyl-, leucyl-, isoleucyl-, and methionyl-tRNA synthetases in the purified complex cosedimented at 18 S. The molecular weight of the synthetase complex is about one million, as estimated by gel filtration. The stoichiometry of the synthetase in the complex was determined by active site titration with aminoacyl adenylates. Results indicate that the 18S synthetase complex contains one subunit of methionyl-tRNA synthetase and two subunits of lysyl-tRNA synthetase. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the 18S synthetase complex contains eight major protein bands. Proteins with subunit molecular weights of 104,000, 92,000, 69,000, and 67,000 are present in molar ratios of 1:1:2:2, while proteins with subunit molecular weights of amounts. These results suggest that the particulate aminoacyl-tRNA synthetases exist as a heterotypic multienzyme complex with defined structure.
Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins: requirement for T7 RNA polymerase.
Authors: Romano L J , Tamanoi F , Richardson C C
Published: May 2006

The primary origin of bacteriophage T7 DNA replication is located 15% of the distance from the left end of the T7 DNA molecule. This intergenic segment is A + T-rich, contains a single gene 4 protein recognition site, and is preceded by two tandem promoters for T7 RNA polymerase [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. Analysis by electron microscopy shows that T7 DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] and gene 4 protein initiate DNA synthesis at randomly located nicks on duplex DNA to produce branched molecules. However, upon the addition of T7 RNA polymerase and ribonucleoside triphosphates 14% of the product molecules have replication bubbles, all of which are located near the primary origin observed in vivo; no such initiation occurs on T7 deletion mutant LG37 DNA, which lacks the primary origin. We have also studied initiation by using plasmids into which fragments of T7 DNA have been inserted. DNA synthesis on these templates is also dependent on the presence of T7 RNA polymerase and ribonucleoside triphosphates. DNA synthesis is specific for plasmids containing the primary origin, provided they are first converted to linear forms.
Comparison of the proteins of two immunologically distinct intermediate-sized filaments by amino acid sequence analysis: desmin and vimentin.
Authors: Geisler N , Weber K
Published: May 2006

Although all intermediate-size filaments (10-nm filaments) seem to show similar morphology and share a number of biochemical properties, different cell- and tissue-specific subclasses have been distinguished by immunological experiments and by differences in apparent molecular weights and isoelectric points of the major constituent proteins. In order to understand the degree of possible homology between these proteins, we have begun amino acid sequence analysis of the polypeptides. Here we characterize a large fragment of chicken gizzard and pig stomach desmin as well as the corresponding fragment from porcine eye lens vimentin. The fragments are situated at the carboxyl end and consist of 138-140 amino acid residues--i.e., some 28% of the corresponding polypeptide chains. The results show that the two immunologically distinct porcine proteins are different gene products. They show a related amino acid sequence but differ in 36% of the residues present in the carboxy-terminal region. Thus tissue specificity overrides species divergence. These results are discussed in the light of previous immunological experiments. They lend further support to the hypothesis that intermediate filaments belong to a multigene family, which is expressed in line with certain rules of differentiation during embryogenesis.
Removal of beta subunit of the eukaryotic polypeptide chain initiation factor 2 by limited proteolysis.
Authors: Mitsui K , Datta A , Ochoa S
Published: May 2006

It is generally considered that the eukaryotic polypeptide chain initiation factor 2 (eIF-2) from rabbit reticulocytes consists of three nonidentical subunits termed alpha, beta, and gamma, in order of increasing molecular weight. However, a recent report [Stringer, E. A., Chaudhuri, A., Valenzuela, D. & Maitra, U. (1980) Proc. Natl. Acad. Sci. USA 77, 3356-3359] suggested that this factor is made up of only two subunits. In this paper we show that limited proteolysis of rabbit reticulocyte eIF-2 leads to loss of the beta subunit. This modified eIF-2 has the same activity as the native factor in promoting ternary (eIF-2.GTP.Met-tRNAi) and 40S (eIF-2.GTP.Met-tRNAi.40S ribosome) initiation complex formation. Like native eIF-2, the protease-treated factor can restore translation in heme-deficient lysates. On the other hand, the treated factor is less stable than the native protein.
Identification of an electron transfer locus in plastocyanin by chromium(II) affinity labeling.
Authors: Farver O , Pecht I
Published: May 2006

Cu(II)--plastocyanin from French beans (Phaseolus vulgaris) is reduced quantitatively by Cr(II)aq ions to give a substitution-inert Cr(III) adduct of Cu(I)--plastocyanin. Enzymatic proteolysis of this derivative by thermolysin led to the identification of the Cr(III) binding peptide. This contains four potential ligands for the metal ion: aspartate-42 and -44 and glutamate-43 and -45. In the three-dimensional fold of plastocyanin, this stretch is very close to tyrosine-83. The emission intensity and its pH dependence observed for the tyrosines in this tryptophan-devoid protein differ markedly in the Cr(III) adduct. That difference is interpreted as reflecting proximity and interaction between the latter metal ion and tyrosine-83. The distance between the copper center and the suggested Cr(III) binding site is approximately 12 A. The intervening region contains an array of highly invariant aromatic residues. These are proposed to be involved in the electron transfer process. A mechanism for that process is presented that involves interaction between the d electrons of the metal ions with d pi-pi* delocalization through a weakly coupled pi* system. The rationale of this electron transfer pathway for the reactivity of plastocyanin with inorganic redox agents is discussed.
Mechanism of action of the lexA gene product.
Authors: Brent R , Ptashne M
Published: May 2006

Genetic experiments have suggested that the lexA gene product of Escherichia coli represses a number of genes involved in the response to DNA damage, including recA and lexA. We purified the lexA gene product from bacterial strains that bear plasmids that direct the synthesis of large amounts of the protein. Purified lexA protein bound to two symmetrical DNA sequences in front of lexA and one in front of recA, protecting them from digestion with DNase I and blocking methylation of purines in the major groove. lexA protein repressed transcription of both genes in vitro. lexA protein binds to the two sites in front of the lexA gene with approximately the same affinity and with greater affinity to the single site in front of the recA gene. The affinity of lexA protein for its operator sites was measured under conditions that mimic conditions in vivo. Differences in the affinity with which lexA protein binds to the operators of genes it represses may account for the differences in the timing and extent of their induction after DNA damage.
Rapid purification of a high-affinity plasminogen activator from human blood plasma by specific adsorption on fibrin/Celite.
Authors: Husain S S , Lipinski B , Greuwich V
Published: May 2006

A preparation of fibrin precipitated over a solid Celite (diatomaceous earth) matrix that selectively binds 50-70% of the plasminogen activator present in human blood plasma is described. Affinity chromatography of plasma on fibrin/Celite followed by gel filtration led to a 29,000-fold purification of the plasminogen activator. The activator, referred to as the high-affinity plasminogen activator, is characterized by its ability to be strongly adsorbed by fibrin. Smaller amounts of other plasminogen activators and essentially all plasminogen were not bound to fibrin. The high-affinity plasminogen activator is a single-chain unstable protease with a molecular weight of 65,000-70,000. The high-affinity plasminogen activator has a low specific activity (500 CTA units/mg) compared to tissue or urine plasminogen activators (100,000-200,000 CTA units/mg) (CTA, Committee on Thrombolytic Agents).
Glia maturation factor promotes contact inhibition in cancer cells.
Authors: Lim R , Nakagawa S , Arnason B G , Turriff D E
Published: May 2006

The effect of bovine glia maturation factor on the growth pattern of cancer cells was investigated in the rat glioma cell line 354A. When the cells were grown in the serum-free defined medium N2 in the absence of the factor, the cells proliferated with a doubling time of 24 hr without showing contact inhibition. After reaching confluency, the cell layer formed numerous foci from which heaps of cell colonies arose. The addition of glia maturation factor to the culture stimulated cell division in the logarithmic phase but prevented overgrowth once the cells arrived at confluency. The ability of glia maturation factor to restore contact inhibition suggests a regulatory role in normal and neoplastic cells.
Temperature-sensitive RNA polymerase mutants of a picornavirus.
Authors: Lowe P A , King A M , McCahon D , Brown F , Newman J W
Published: May 2006

Temperature-sensitive (ts) RNA polymerase mutants of a picornavirus are reported. Two foot-and-mouth disease virus (FMDV) mutants designated ts 22 and ts 115 have been characterized. As judged by isoelectric focusing, both have charge alterations in P56a, the FMDV RNA polymerase protein. Virus RNA synthesis in cells infected with the mutants is severely impaired at the nonpermissive temperature. RNA polymerase purified from baby hamster kidney cells infected with these mutants exhibits a marked ts transcribing activity in vitro. Spontaneous revertants of both mutants have P56a polypeptides that are indistinguishable from the parental proteins on the basis of charge. The revertants regain the ability to synthesize virus RNA in vivo at the nonpermissive temperature. RNA polymerase purified from the revertants remains transcriptionally active at the nonpermissive temperature.
Deletion mapping a eukaryotic promoter.
Authors: Struhl K
Published: May 2006

The phenotypes of 24 mutants that successively delete DNA sequences adjacent to the 5' end of the Saccharomyces cerevisiae (yeast) his3 structural gene are described. Deletions retaining greater than 155 base pairs before the mRNA coding sequences are phenotypically indistinguishable from the wild-type his3 allele. Deletions having end points between 113 and 65 base pairs before the transcription initiation site express his3 at reduced levels. Mutations retaining less than 45 base pairs are indistinguishable from null alleles of the his3 locus. These results indicate (i) that a sequence(s) located 113--155 base pairs upstream from the transcribed region is necessary for wild-type expression and (ii) that the T-A-T-A box (a sequence in front of most eukaryotic genes) is not sufficient for wild-type promoter function. Thus, the yeast his3 promoter region appears large when compared with prokaryotic promoters, suggesting that it may be more complex than a simple site of interaction between RNA polymerase and DNA.
Expression and processing of bacterial beta-lactamase in the yeast Saccharomyces cerevisiae.
Authors: Roggenkamp R , Kustermann-Kuhn B , Hollenberg C P
Published: May 2006

The mode of expression in Saccharomyces cerevisiae of the bacterial antibiotic resistance gene coding for beta-lactamase (EC 3.5.2.6) is described. Yeast transformants, containing hybrid plasmid pMP78-1 consisting of pBR325 in a 2-micrometers DNA vector, synthesize an active beta-lactamase protein. The enzyme was purified about 100-fold over crude extracts. With regard to activity, molecular weight, and binding to specific antibodies the yeast beta-lactamase was indistinguishable from the purified enzyme from Escherichia coli. Because the bacterial enzyme is synthesized as a preprotein with subsequent maturation, the results suggest that S. cerevisiae is able to convert the preprotein to the mature beta-lactamase. This was confirmed by in vitro experiments showing that the bacterial preprotein can be processed by crude extracts of S. cerevisiae.
Specific antibody-dependent interactions between macrophages and lipid haptens in planar lipid monolayers.
Authors: Hafeman D G , von Tscharner V , McConnell H M
Published: May 2006

The binding of guinea pig peritoneal macrophages to planar lipid monolayers on alkylated glass is shown to be highly specific, requiring lipid hapten in the monolayer and specific anti-hapten IgG antibody. This is true for both "fluid" and "solid" monolayer membranes, in which the lateral diffusion coefficients of fluorescent lipid probes and bound fluorescent antibodies differ by at least two orders of magnitude. The region of the (macrophage membrane)--(monolayer membrane) contact is readily observed by using an epifluorescence microscope and fluoresceinated IgG antibodies or fluorescent lipids. The fluorescence intensity of IgG antibodies bound to lipid haptens in fluid monolayer membranes in the region of the (macrophage membrane)--(monolayer membrane) contact was significantly enhanced in the early stages of binding (first 10 min at 24 degrees C), due to a diffusive flux of the fluorescent antibodies into the region of membrane--membrane contact. Cellular activation takes place immediately during a 10-min warm-up to 37 degrees C and can be recognized by rapid symmetrical cell spreading, the formation of "black holes" around the cells (probably due to superoxide-facilitated photochemical bleaching of the fluorophore), and the release of the lysozomal enzyme cathepsin B. Specific antibody-dependent [1-14C]-glucose oxidation by these macrophages on fluid and solid monolayers is quite similar to that reported previously for fluid and solid bilayer vesicle target membranes. These results are significant for understanding the molecular interactions between membranes that are necessary for a macrophage cytotoxic response.
Monoclonal antibodies that demonstrate specificity for several types of human lung cancer.
Authors: Cuttitta F , Rosen S , Gazdar A F , Minna J D
Published: May 2006

Monoclonal antibodies with selectivity for human lung cancer were produced by immunizing BALB/c mice with an established line of human small cell lung cancer (NCI-H69) and fusing the mouse spleen cells to mouse myeloma line X63-Ag8.653. The resulting hybrid cells were initially screened by immunoautoradiography for production of antibodies that would react with NCI-H69 and another small cell lung cancer line (NCI-H128) but not its autologous B-lymphoblastoid line (NCI-H128BL). Stable monoclonal antibody-producing lines were isolated by repeated cloning. Three independently derived monoclonal antibodies, designated 525A5, 534F8, and 538F12, were found to react with three of the major types of human lung cancer (small cell, adenocarcinoma, and squamous carcinoma). They did not react with bronchioloalveolar and large cell lung cancers, myeloma, lymphomas, leukemias, osteogeneic sarcoma, mesothelioma, hypernephroma, malignant melanoma, simian virus 40-transformed human fetal lung cells, skin fibroblast lines, human B-lymphoblastoid lines, human erythrocytes, and rodent cells. Interestingly, these antibodies also bound to three out of three human neuroblastomas and two out of three breast cancers but failed to react with mouse neuroblastoma and rat pheochromocytoma. The monoclonal antibodies reacted with human small cell lung cancer tumors obtained at autopsy, but had insignificant reactions with normal human lung, liver, spleen, and skeletal muscle. We conclude that monoclonal antibodies have been generated that react with common antigenic determinants expressed on several human lung cancer types, neuroblastoma, and some breast cancers, but are not detectable by our current assays on a variety of other human tumors or normal adult human tissues. Such antibodies are of potential clinical and biological importance.
Mutant mouse tottering: selective increase of locus ceruleus axons in a defined single-locus mutation.
Authors: Levitt P , Noebels J L
Published: May 2006

The central catecholamine neuron system in the mutant mouse tottering was examined by fluorescence histochemistry and biochemical analysis of catecholamine content. This single-locus neurological mutation expresses a reproducible alteration in central nervous system physiology characterized by spontaneous spike-wave and focal motor seizures in the absence of any previously recognized disturbance of cellular organization or brain size. Histochemical analysis showed a significant increase in the number of noradrenergic axons in terminal fields innervated by the nucleus locus ceruleus when compared with the wild type. A concomitant 100-200% rise in norepinephrine levels is found in the same areas, including hippocampus, cerebellum, and dorsal lateral geniculate. Catecholamine fibers and transmitter content in areas innervated by a second major noradrenergic system arising from the brainstem lateral tegmental neurons are unaltered. The terminal axons and transmitter content were both unchanged in nuclei receiving a dense dopaminergic innervation. Despite the hypertrophy of the locus ceruleus axonal plexus, the number and size of locus ceruleus cell somata were identical in both wild-type and tottering mice. These findings are consistent with a specific gene-linked alteration of developmental events controlling the number of axons produced by a single neuronal population in the mammalian brain.
Induction of hepatic synthesis of serum amyloid A protein and actin.
Authors: Morrow J F , Stearman R S , Peltzman C G , Potter D A
Published: May 2006

Major changes in the mRNA population of murine liver occur after administration of bacterial lipopolysaccharide, an agent that causes increases in the concentrations of acute-phase serum proteins. The mRNA for one of these, serum amyloid A, is increased at least 500-fold compared to the normal level. It becomes one of the most abundant hepatic mRNAs, and serum amyloid A synthesis comprises about 2.5% of total hepatic protein synthesis in the acute-phase response. Its synthesis is tissue-specific in that amyloid A mRNA was not detected in the kidney, an important site of amyloid fibril accumulation. The protein synthesized in largest amount by acute-phase liver tissue in culture is cytoplasmic actin. Its relative rate of synthesis is increased about 5-fold compared to the normal tissue; that of serum albumin is decreased to about one-third of its normal rate. The concentration of mRNA for serum albumin is decreased by a similar amount. Starting with induced liver RNA, we have constructed a recombinant plasmid containing most of the DNA sequence encoding the serum amyloid A polypeptide.
Accumulation of newly synthesized mRNAs in response to human fibroblast (beta) interferon.
Authors: Colonno R J
Published: May 2006

Treatment of human fibroblast cells with human fibroblast (beta)interferon for up to 8 hr resulted in the accumulation of at least four mRNAs. The mRNAs were isolated from cellular polysomes and characterized by stimulation of translation in a wheat germ cell-free protein synthesis system. The mRNAs appear as early as 2 hr after exposure to interferon and can be translated in vitro into proteins having molecular weights of 61,000, 62,000, 64,000, and 68,000. The response is not elicited by mouse interferon or insulin and does not occur in the presence of actinomycin D. Chase experiments indicated that the induced mRNAs remain ribosome-associated for at least 3 hr after their synthesis. The appearance of the induced mRNAs correlated directly with the onset of an antiviral state. Velocity sedimentation of the induced mRNAs on sucrose gradients demonstrated that each of the four induced proteins are encoded by different-sized mRNAs.
Interconverting mu and delta forms of the opiate receptor in rat striatal patches.
Authors: Bowen W D , Gentleman S , Herkenham M , Pert C B
Published: May 2006

The binding of a radiolabeled "mu receptor" prototype opiate, dihydromorphine (H2morphine), and the binding of a "delta receptor" prototype, [D-Ala2,D-Leu5]enkephalin (D-Enk), to slide-mounted rat caudate slices were simultaneously compared quantitatively and visualized by autoradiography. Generally, D-Enk bound to opiate receptors distributed evenly throughout the entire striatum (type 2 pattern), whereas H2morphine labeled discrete islands or patches of receptors (type 1 pattern). In the presence of Mn2+ (3 mM) or other divalent cations, however, Na+ and GTP at 25 degrees C caused an increase in D-Enk binding at the expense of H2morphine binding at striatal opiate receptor patches. Thus, these conditions shifted D-Enk binding from an even pattern to one that included both an even and patchy distribution. These incubation conditions not only promoted D-Enk binding to striatal patches but also enabled the opiate receptor to regulate adenylate cyclase with the same (P less than 0.01) ligand selectivity pattern as that obtained by the displacement of D-Enk binding. The relative affinity of opiate receptors in striatal patches for opiate peptides, naloxone, and morphine appears to be a function of incubation conditions and coupling to adenylate cyclase and is not indicative of distinctly different opiate receptors. We postulate a three-state allosteric model consisting of mu agonist-, mu antagonists-, and adenylate cyclase-coupled delta-agonist-preferring states, whose equilibrium may be regulated by a sulfhydryl group mechanism.
Hydration of single crystals of dipalmitoylphosphatidylcholine.
Authors: Urabe T , Asher S A , Pershan P S
Published: May 2006

Optical birefringence and x-ray measurements are reported for single crystals (0.5 mm X 2 mm X 5 micrometers) of dipalmitoylphosphatidylcholine as a function of temperature and relative humidity. Large single crystals grown in CHCl3/acetone at a low water concentration undergo an irreversible phase transformation at 20 degrees C and 70% relative humidity or at 60 degrees C and 65% relative humidity. After this transformation occurs, the crystals can be made to undergo a reversible transformation into and out of the L alpha phase. The x-ray-determined mosaic spread in the hydrated crystal is less than approximately 0.04 degrees both above and below the main transition.
Periodic synthesis of microtubular proteins in the cell cycle of Physarum.
Authors: Laffler T G , Chang M T , Dove W F
Published: May 2006

Periodic polypeptide labeling over the naturally synchronous nuclear replication cycle of Physarum polycephalum was analyzed by fluorography of two-dimensional electropherograms. Two sets of polypeptides, denoted as P and Q, showed strong periodicity; they were maximally labeled just prior to mitosis. This periodicity was shown to reflect synthesis rather than turnover or recovery. Both P and Q copolymerized with porcine microtubular proteins and displayed electrophoretic properties similar to those of porcine tubulins. The significance of the periodic synthesis of these microtubular proteins is discussed as a possible component within the chain of events that establishes the high mitotic synchrony of Physarum syncytia.
Synergistic interaction between 1-beta-D-arabinofuranosylcytosine, thymidine, and hydroxyurea against human B cells and leukemic blasts in vitro.
Authors: Streifel J A , Howell S B
Published: May 2006

Isobologram analysis was used to examine the interaction between 1-beta-D-arabinofuranosylcytosine (Ara-C), thymidine (dThd), and hydroxyurea. All three pairs of drugs, as well as the triple combination, were synergistic against a human B cell line in vitro across a broad range of concentrations. Synergy was associated with an increase in the Ara-C nucleotide pool and Ara-C triphosphate concentration. dThd increased, and hydroxyurea decreased, the incorporation of Ara-C into trichloroacetic acid-insoluble macromolecules per unit time. Hydroxyurea was more effective than dThd at equimolar concentrations in increasing the acid-soluble Ara-C pool. Maximal stimulation of Ara-C triphosphate formation by dThd occurred at 1 mM and was associated with reduction of the deoxycytidine triphosphate pool to 31% of control. At the same concentration, hydroxyurea increased Ara-C triphosphate formation to a greater extent but increased deoxycytidine triphosphate to 116% of control. When tested at clinically achievable concentrations on blasts from patients with acute leukemia, hydroxyurea increased the Ara-C nucleotide pool in all six cases studied, whereas dThd decreased the Ara-C nucleotide pool. These results indicate that in SB cells dThd and hydroxyurea work by different mechanisms to augment the Ara-C nucleotide pool and that hydroxyurea may be more effective than dThd as a modulator of Ara-C activity in patients with acute leukemia.
Potassium current suppression by quinidine reveals additional calcium currents in neuroblastoma cells.
Authors: Fishman M C , Spector I
Published: May 2006

Quinine and quinidine have been evaluated with regard to their effects on the electrical activity of neuroblastoma cells. Under voltage-clamp conditions, we have found that quinine and quinidine block both the voltage-dependent and Ca2+-dependent K+ conductances. Blockage of the voltage-dependent K+ channel is manifest as an increase in the amplitude and in the duration of the action potential. Blockage of the Ca2+-dependent K+ channel in Na+-free (replaced by Tris) solutions containing 6.8 mM Ca2+ and tetraethylammonium ion or 4-aminopyridine (to block the voltage-dependent K+ current) is seen as a further prolongation of the Ca2+ action potential and diminution of the after-hyperpolarization. A critical role of the Ca2+-dependent K+ conductance in modulation of the rate and duration of trains of Ca2+ action potentials is shown by the use of low concentrations (5-40 microM) of quinine or quinidine, which diminish the Ca2+-dependent K+ conductance in a graded manner. After complete blockade of K+ currents, the peak Ca2+ currents are enhanced at all voltages, especially at values more positive than -30 mV, where a steady-state inward current appears as well. In this same voltage range, the decay of the Ca2+ current exhibits two time constants--that of the transient inward current, which is about 20 msec, and a much slower (approximately 2000 msec) component. It is suggested that neuroblastoma cells have two types of calcium channels--one which generates the Ca2+ action potential and a second, distinguished by activation at more depolarized levels and by a slow rate of inactivation, which underlies the calcium entry necessary to activate the Ca2+-dependent K+ conductance.
Fourier-transform analysis of normal photoelectron diffraction data for surface-structure determination
Authors: Hussain Z. , Shirley D. A. , Li C. H. , Tong S. Y.
Published: May 2006

A direct method for surface-structure determination from normal emission photoelectron diffraction (NPD) data is presented. Fourier transforms of the calculated NPD intensities yield peaks at adsorbate—substrate normal interlayer distances. Applications are demonstrated, using theoretical NPD curves for the Se/Ni system calculated by dynamical theory. These results show that interplanar spacings between the overlayer and as many as four substrate layers can be determined with an accuracy of better than 3%.
Repetitive DNA sequences cotranscribed with developmentally regulated Dictyostelium discoideum mRNAs.
Authors: Zuker C , Lodish H F
Published: May 2006

We have isolated a clone of Dictyostelium discoideum genomic DNA, pB41-6, that contains sequences that are reiterated and interspersed in the genome. The expression of these reiterated sequences is developmentally regulated, and they appear to be linked only to developmentally regulated mRNAs. We have used clone pB41-6 to isolate a set of complementary cDNA clones. Analysis of two selected clones indicates that they encode developmentally regulated mRNA species and, by means of the repetitive elements they contain, hybridize to many developmentally regulated mRNAs. In addition, we have defined two families of mRNA species carrying these repetitive sequences: one is induced at 5.5 hr of development and the other is induced between 5.5 and 15 hr of development. Since most of the mRNA species that have sequences complementary to clone pB41-6 accumulate in a coordinated fashion during development, these sequences may play an important regulatory role in the developmental program of Dictyostelium.
Role for membrane potential in the secretion of protein into the periplasm of Escherichia coli.
Authors: Daniels C J , Bole D G , Quay S C , Oxender D L
Published: May 2006

The leucine-specific binding protein of Escherichia coli is a periplasmic protein that is synthesized as a precursor and subsequently is processed during its secretion into the periplasmic space. The processing of both the leucine-specific binding protein and a plasmid-coded beta-lactamase is inhibited by phenethyl alcohol and by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP). The levels of CCCP that inhibit processing also produce significant decreases in the membrane potential. Valinomycin, a potassium ionophore, also inhibits processing of the leucine-specific binding protein in spheroplasts. Processing can be restored in CCCP-treated cells and in valinomycin-treated spheroplasts by dilution of the treated cells in fresh medium. These results suggest a role for membrane potential in the secretion of periplasmic proteins. A model is presented which suggests that membrane potential plays a primary role in the proper orientation of the precursor signal sequence within the membrane, thus promoting processing and secretion.