bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional significance of coactivation of vagal and sympathetic cardiac nerves.
Authors: Koizumi K , Terui N , Kollai M , Brooks C M
Published: May 2006

Simultaneous recording of activity in the vagal and sympathetic supplies to the heart has revealed that in reflexly and centrally evoked activity these two "antagonists" do not necessarily change action reciprocally. Coactivation occurs in chemoreceptor reflexes and related reactions, upon stretching of the sinoatrial nodal region of the right atrium and when certain hypothalamic regions are stimulated. The objective of the present work was to assay the physiological importance of coactivation of the two potentially antagonistic cardiac nerves in anesthetized dogs. Output from the heart was monitored by recording volume flow in the thoracic aorta just below the aortic arch; cardiac contractility was measured as left ventricular dp/dt. Tape recordings of vagus and sympathetic nerve activity during chemoreceptor and baroreceptor reflexes, during reciprocal and nonreciprocal changes produced by hypothalamic stimulation, and during hypoxia and hypercapnia were used to trigger stimulators feeding a stimulus per action potential to cardiac vagus and sympathetic nerves after central connections were cut. The vagus stimulation alone produced a decrease in aortic blood flow; stimulation of the sympathetic nerve alone resulted in increased aortic blood flow. Simultaneous stimulation of vagus and sympathetic, however, produced an even greater cardiac output (measured by aortic blood flow). Intermediate degrees of heart rate and strength of myocardial contraction were maintained in coactivation. Obviously, an association of increased vagus and sympathetic actions, which can be effected reflexly or by action of higher centers, is of physiological benefit. In control reactions that relate cardiac function to body need, both reciprocal and synergistic actions (coactivation) of cardiac nerves are used.
Acid phosphatase polypeptides in Saccharomyces cerevisiae are encoded by a differentially regulated multigene family.
Authors: Rogers D T , Lemire J M , Bostian K A
Published: May 2006

Two clones from a lambda phage collection containing yeast genes regulated by inorganic phosphate were shown by low-stringency hybridization to select three mRNAs that direct the in vitro synthesis of repressible acid phosphatase (EC 3.1.3.2) polypeptides p60, p58, and p56. By higher stringency hybridization one yeast fragment [8 kilobases (kb)] selects p60 mRNA and the other (5 kb) selects p56 mRNA. These EcoRI digestion fragments were subcloned in yeast transformation vectors and hybridization selection assignments were confirmed by measuring enzyme and mRNA levels in transformants. Enzyme and mRNA levels in (8-kb) high copy number transformants grown in high inorganic phosphate medium revealed a hitherto undetected acid phosphatase protein, P57, which is believed to correspond to the constitutive enzyme encoded by PHO3. The identify of the 8-kb fragment purported to contain the PHO5/PHO3 genes was confirmed by genetic mapping of an integrated copy of this fragment. The site of integration of the 5-kb fragment was demonstrated to be unlinked to the PHO5/PHO3 genes.
Surface receptors for pancreatic hormones in dog and rat hepatocytes: qualitative and quantitative differences in hormone-target cell interactions.
Authors: Bonnevie-Nielsen V , Polonsky K S , Jaspan J J , Rubenstein A H , Schwartz T W , Tager H S
Published: May 2006

In order to evaluate potential differences in the kinetics of peptide hormone-receptor interactions in hepatocytes of different species, we developed a simple procedure for the isolation of canine hepatocytes. Cells (obtained by collagenase perfusion of an extirpated dog liver lobe) were isolated with uniform high viability and yield. In addition, isolated dog hepatocytes tolerated incubation for at least 4 hr in defined medium with only a slight decrease in viability and with no change in the kinetics of [125I]iodoinsulin or [125I]iodoglucagon binding to cell-surface receptors. Comparisons of peptide hormone interactions with isolated dog and rat hepatocytes showed that (i) [125I]iodoglucagon associated with specific membrane receptors more rapidly than did [125I]iodoinsulin, for both rat and dog hepatocytes and at both 30 degrees C and 37 degrees C; (ii) the steady-state binding of [125I]iodoglucagon at 30 degrees C was greater than that of [125I]iodoinsulin in dog hepatocytes, but the reverse relationship held in rat hepatocytes; (iii) the rate of dissociation of [125I]iodoinsulin from hepatocytes of both species was enhanced by the presence of the unlabeled hormone, whereas the rate of dissociation of receptor-bound [125I]iodoglucagon was enhanced by the presence of unlabeled glucagon only in hepatocytes derived from the dog; and (iv) [125I]iodopancreatic polypeptide bound to neither rat nor dog hepatocytes, although the [125I]iodotyrosylated peptide bound to rat hepatocytes with an unusually high apparent dissociation constant. While confirming essential findings of pancreatic hormone binding to isolated hepatocytes, this comparison suggests that both qualitative and quantitative aspects of hormone-target cell interactions can show interspecies variability.
Human interferons protect plants from virus infection.
Authors: Orchansky P , Rubinstein M , Sela I
Published: May 2006

Unfractionated human leukocyte interferon, as well as highly purified subspecies of this interferon, and a purified recombinant of human leukocyte interferon produced in bacteria are active in suppressing multiplicability of tobacco mosaic virus in tobacco leaf discs. Human fibroblast interferon exhibits diverse levels of antiviral activity against tobacco mosaic virus but becomes as active as human leukocyte interferon upon incubation with glycosidases. The effect of interferon is reversible; normal multiplication of tobacco mosaic virus resumes upon removal of interferon.
Long-term human cytolytic T-cell lines allospecific for HLA-DR6 antigen are OKT4+.
Authors: Krensky A M , Reiss C S , Mier J W , Strominger J L , Burakoff S J
Published: May 2006

Human allospecific cytotoxic T lymphocyte lines were established by weekly stimulation of peripheral blood lymphocytes with allogeneic Epstein-Barr virus-transformed lymphoblastoid cell lines in the presence of interleukin 2. The cytotoxic T lymphocyte lines were stimulated by either JY (which expresses HLA-A,B and -DR) or Daudi (which expresses HLA-DR but not -A,B antigens). Specificity of the effector cell lines was determined by antibody blocking and by patterns of cytolysis against a panel of target cell lines. The phenotype of the effector cells was identified by both negative (antibody and complement lysis) and positive (fluorescence-activated cell sorter) selection. The anti-JY lines only recognized targets bearing HLA-A2, B7 antigens and were composed exclusively of OKT4-8+ effector cells. The anti-Daudi lines, on the other hand, specifically recognized targets bearing DR6 and eventually contained only OKT4+8- cells. Thus, stimulation by Daudi cells can result in the generation of OKT4+ CTL lines that are allospecific for DR6 antigen.
Adenoviral protein-primed initiation of DNA chains in vitro.
Authors: Ikeda J E , Enomoto T , Hurwitz J
Published: May 2006

The initiation of DNA chains by the 80-kilodalton form of the adenovirus terminal protein has been studied. This protein, which can be covalently linked to dCMP, is isolated complexed to a 140-kilodalton protein possessing DNA polymerase activity. In the presence of adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex requires the addition of ATP and nuclear extract from uninfected cells in addition to Mg2+ and dCTP. When single-stranded DNA is used in place of the adenovirus DNA-protein, the formation of the 80-kilodalton protein-dCMP complex occurs in the absence of ATP and nuclear extract. In the presence of the four dNTPs, the complex yields DNA chains of various sizes between 100 and 300 nucleotides. The products formed with bacteriophage phi X174 single-stranded circular DNA as the template are site specific, predominantly derived from the sequences between nucleotides 2363 and 2977 and between nucleotides 3760 and 4206. These small dNA chains are blocked at their 5' ends with the 80-kilodalton protein but possess free 3'-OH ends that are susceptible to degradation by exonuclease III and can be elongated to replicative form II products with DNA polymerase I of Escherichia coli or eukaryotic DNA polymerase beta preparations. A protein priming model explaining the different requirements for initiation with adenovirus DNA-protein and with phi X174 DNA is presented.
A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos.
Authors: Conaway R C , Lehman I R
Published: May 2006

Preparations of DNA polymerase alpha from early embryos of Drosophila melanogaster catalyze the ATP-dependent synthesis of DNA with single-stranded M13 DNA or poly(dT) templates. In the case of M13 DNA, GTP, but not UTP or CTP, can replace ATP. The reaction is completely dependent on added template and is not inhibited by alpha-amanitin. Alkaline hydrolysis of the product synthesized in the presence of [alpha-32P]dATP and poly(dT) generates 32P-labeled 3'(2') adenylate, showing that a covalent ribo-deoxynucleotide linkage is formed. Furthermore, incorporation of ribonucleotides occurs at the 5' end of the newly synthesized polynucleotide chain. These findings are consistent with the hypothesis that a ribo-oligonucleotide primer is synthesized by primase action and subsequently elongated by DNA polymerase. Under the appropriate conditions, DNA polymerase I from Escherichia coli can elongate primers formed by primase in the presence of ATP and poly(dT). Primase activity copurifies with DNA polymerase alpha and may be part of the multisubunit polymerase molecule.
Site of premature termination of late transcription of simian virus 40 DNA: enhancement by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole.
Authors: Skolnik-David H , Hay N , Aloni Y
Published: May 2006

Sedimentation analysis of pulse-labeled RNA synthesized in nuclei isolated from simian virus 40-infected cells revealed an abundance of short cellular and viral RNAs. The relative amount of the short chains is increased in nuclei isolated from cells treated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). The short viral RNAs were purified by hybridization to and elution from simian virus 40 DNA on filters, and their sizes were determined by gel electrophoresis. A major band of 93- to 95-nucleotide-long RNA was observed along with additional minor bands. Identical bands were revealed when the viral RNA was purified from nuclei of cells pretreated with DRB. The major band was identified as an aborted transcript of a RNA that initiated at the major initiation site (nucleotide 243). We have found that the DNA region where the RNA stops is A+T rich and is immediately preceded by a G+C-rich region that exhibits dyad symmetry, resembling the termination signal in prokaryotes. These observations show that RNA polymerase II responds to the same termination signal as the prokaryotic enzyme and suggest that a mechanism of attenuation regulates simian virus 40 late transcription.
Purification of two calcium/calmodulin-dependent forms of cyclic nucleotide phosphodiesterase by using conformation-specific monoclonal antibody chromatography
Authors: Hansen R. Scott , Beavo J. A.
Published: May 2006

A procedure for nondenaturing immunopurification of bovine calmodulin-dependent 3′,5′-cyclic-nucleotide phosphodiesterase (3′,5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) is described that utilizes chromatography on a conformation-specific monoclonal antibody column. Hybridomas derived from spleen cells of mice immunized with Ca
2+
/calmodulin/phosphodiesterase were screened for antiphosphodiesterase antibody production. A stable cell line was established that secretes a monoclonal antibody that binds to the Ca
2+
/calmodulin/enzyme complex with an approximate
K
d
of 10
-9
M. The dissociation constant was increased by two orders of magnitude when calmodulin interaction with the enzyme was inhibited by Ca
2+
chelation. This differential reactivity was utilized for affinity chromatography of heart and brain phosphodiesterases on monoclonal antibody columns. Highly purified phosphodiesterases were eluted in good yield with buffer containing EGTA. The immunopurified enzymes from heart and brain exhibited specific activities of ≈300 units/mg when assayed at millimolar concentrations of cGMP or cAMP. Calmodulin stimulated both enzymes 10- to 15-fold over basal activity under these conditions. However, analysis of the two preparations by NaDodSO
4
/polyacrylamide gel electrophoresis revealed an apparent subunit of
M
r
61,000 for the brain enzyme, in contrast to the
M
r
59,000 cardiac subunit. The observed difference was not an artifact of tissue homogenization because both forms were detected after purification from mixed-tissue homogenates. These results suggest that mild, biospecific elution from a conformation-specific monoclonal antibody column may be a general technique applicable to the rapid isolation of proteins whose antigenic determinants can be altered with specific ligands.
Primary structure of a cytochrome P-450: coding nucleotide sequence of phenobarbital-inducible cytochrome P-450 cDNA from rat liver.
Authors: Fujii-Kuriyama Y , Mizukami Y , Kawajiri K , Sogawa K , Muramatsu M
Published: May 2006

We determined the coding nucleotide sequence of phenobarbital-inducible cytochrome P-450 mRNA of the rat by analysis of three cloned cDNAs and by primer extension methods. The deduced amino acid sequence of the cytochrome is composed of 491 amino acids, and its predicted molecular weight and amino acid composition concur with those determined with the purified protein. The sequence of one of the three cloned cDNAs is not identical with that of the other two. In their 922 overlapping nucleotides, 14 nucleotide substitutions occur, and 7 of them result in 6 amino acid changes, therefore indicating the presence of at least two similar but distinct mRNAs for phenobarbital-inducible cytochrome P-450. These substitutions occur in a limited portion of the sequence, apparently forming some sort of a "variable region."
Agrobacterium
Ti plasmid indoleacetic acid gene is required for crown gall oncogenesis
Authors: Liu S.-T. , Perry K. L. , Schardl C. L. , Kado C. I.
Published: May 2006

A gene (
iaaP
) necessary for virulence and indoleacetic acid (IAA) production has been located on a nopaline Ti plasmid of
Agrobacterium tumefaciens
C58.
iaaP
function was established by using transformation to insert nopaline or octopine Ti plasmids into an avirulent, Ti plasmid-free mutant 1D1293-3 that was defective in IAA synthesis (
iaaC
-
). The resulting transformants produced increased levels of IAA and virulence was restored. When these transformants were cured of their Ti plasmid, virulence and high IAA production levels were concomitantly lost. A Tn5 mutagenized TiC58 plasmid, deficient in the ability to direct increased synthesis of IAA, was inserted by transformation into mutant 1D1293-3. The resulting transformants 1D1293-3 (TiC58::Tn5) remained avirulent and
iaaP
-
. Restriction analysis of the TiC58::Tn5 plasmid DNA identified the
iaaP
gene at 20.9 kilobases to the left of the T-DNA. A major aromatic-amino-acid aminotransferase is coded by the
iaaC
gene, but not by the
iaaP
gene. The possible reasons for the
iaaP
locus to be situated outside the T-DNA region are discussed.
Functional conservation near the 3' end of eukaryotic small subunit RNA: photochemical crosslinking of P site-bound acetylvalyl-tRNA to 18S RNA of yeast ribosomes.
Authors: Ofengand J , Gornicki P , Chakraburtty K , Nurse K
Published: May 2006

Escherichia coli acetylvalyl (AcVal)-tRNA1Val became crosslinked to both yeast and spinach chloroplast ribosomes upon irradiation (300 nm) in the presence of poly(U2,G). Yields were 25-30% and 33%, respectively, compared to 45% for E. coli. Crosslinking occurred to the P site, only to the 40S subunit, and 90% of that was to the 18S rRNA. The crosslink could be photolyzed at 254 nm with the same first-order kinetics as for the E. coli ribosome complex previously studied. The AcVal-tRNA that split off could be crosslinked again when irradiated at 300 nm, showing that the crosslink was photoreversible. There was a strong codon specificity for crosslinking. With pG-U-U, 85% crosslinking was obtained after 20 min of irradiation; with G-U-A, only 3% crosslinking occurred. All of these properties are the same as those previously reported for the E. coli ribosome crosslink that occurs via cyclobutane dimer formation between the 5' anticodon base 5'-carboxymethoxyuridine-34 and cytidine-1400 of the 16S RNA. Cytidine-1400 is in the center of a 17-mer that has been almost totally conserved among the small subunit rRNAs of all species so far examined, including yeast. Crosslinking of tRNA in the same way to both yeast and E. coli ribosomes shows that there has been a functional conservation as well in this region of the small subunit rRNA. This region may be involved in some essential aspect of the decoding process that is common to both prokaryotic and eukaryotic protein synthesis systems.
Phorbol ester induction of leukemic cell differentiation is a membrane-mediated process.
Authors: Cooper R A , Braunwald A D , Kuo A L
Published: May 2006

Phorbol esters are potent inducers of macrophage-like differentiation in the HL-60 promyelocytic leukemia cell line. The sequence of events by which they bring about this transition is poorly understood. However, it is known that phorbol esters bind to the surface membrane of HL-60 cells and to various other cells as well. Our studies were directed toward determining the biologic importance of this membrane association. [3H]Phorbol dibutyrate (PBu2) was specifically bound by HL-60 cells with a Kd of 23 nM and with 1.9 X 10(5) binding sites for [3H]PBu2 per cell. There was no internalization of bound [3H]PBu2. Specific binding was fully reversible upon washing in fresh medium, and [3H]PBu2 added thereafter bound normally to its receptor. Within 10 min of binding, PBu2 stimulated [14C]choline incorporation into phosphatidylcholine, with a rapid return to normal upon removal of the PBu2. Membrane-bound PBu2 progressively inhibited DNA synthesis, with 70% inhibition by 8 hr. This process was interrupted if the PBu2 was removed, and little recovery of DNA synthesis occurred in previously inhibited cells. Between 8 and 16 hr, PBu2 induced adherence of cells to plastic, but only in those cells in which phosphatidylcholine synthesis was stimulated, and this process was also interrupted if PBu2 was removed prior to 16 hr. Similarly, nonspecific esterase, which develops after 72 hr of incubation, was induced in cells exposed to PBu2 for the initial 16 hr but not in cells exposed for 5 hr. These studies demonstrate that phorbol esters exert their effects while retained at the cell surface. Inhibition of cell growth and the acquisition of surface and enzymatic properties that characterize macrophages are separable events, each of which proceeds through a receptor-mediated, transmembrane process. The stimulation of phosphatidylcholine synthesis appears to be a part of that process.
Novel free radicals in synthetic and natural pheomelanins: distinction between dopa melanins and cysteinyldopa melanins by ESR spectroscopy.
Authors: Sealy R C , Hyde J S , Felix C C , Menon I A , Prota G , Swartz H M , Persad S , Haberman H F
Published: May 2006

Synthetic pheomelanins from enzymic oxidation of the 3,4-dihydroxyphenylalanine (dopa) derivative 5-S-cysteinyldopa have been examined by ESR spectroscopy. These alkalisoluble polymers contain a novel kind of free radical that is spectroscopically distinct from that found in eumelanins. Delocalization of the unpaired electron onto a nitrogen atom and the ability of the radical to chelate complexing metal ions strongly suggest an o-semiquinonimine structure. The synthetic pheomelanin was compared with natural red pigments extracted from human red hair and from red chicken feathers. Spectroscopically, the chicken feather pheomelanin is almost identical to synthetic cysteinyldopa pheomelanin. In contrast, the pigment from red hair has a major spectral component very similar to that found in dopa melanin, with a smaller component corresponding to that found in cysteinyldopa melanin.
Conformational analysis of the calcium--A23187 complex at a lipid--water interface.
Authors: Brasseur R , Deleers M , Malaisse W J , Ruysschaert J M
Published: May 2006

A possible conformation of the complex formed by one calcium ion and two molecules of the ionophore A23187 at a simulated lipid--water interface was predicted by a variant method for conformational analysis. This method takes into account, in addition to the Van der Waals energy, electrostatic interaction, and torsional potential, the alteration of electrostatic forces attributable to changes in dielectric constant at the interface and the transfer energy for each part of the complex as it moves through the lipid-water interface. The most probable conformer was characterized by a two-fold axial symmetry that was maintained during transition to the hydrophobic bulk conformation. Minor changes in the interfacial structure were sufficient to achieve the configuration characteristic of the hydrophobic bulk phase.
Stimulation of de novo synthesis of cytochrome P-450 by phenobarbital in primary nonproliferating cultures of adult rat hepatocytes.
Authors: Newman S , Guzelian P S
Published: May 2006

Primary monolayer cultures of nonproliferating parenchymal cells prepared from adult rat liver and maintained in serum-free medium responded to additions of phenobarbital with concentration-dependent increases in synthesis and accumulation of a cytochrome P-450 protein immunochemically and catalytically indistinguishable from that found in the livers of adult rats treated with phenobarbital. Maximal stimulation of the rate of synthesis of this cytochrome protein by phenobarbital, as much as 20-fold higher than in control cultures (1.01% of the rate of synthesis of total cellular protein), could be achieved when the drug was first added to cultures no older than 24 hr and then was maintained in the medium for 96 hr. In addition to phenobarbital, chemicals classified as "phenobarbital-like" inducers in vivo (mephenytoin, mirex, 2,2',4,4',5,5'-hexabromobiphenyl) induced synthesis in culture of this same immunoreactive protein. Supplementation of the medium with 0.1 microM H2SeO3 plus phenobarbital produced an average 2-fold enhancement in the rate of synthesis of this inducible cytochrome protein as compared to that in cultures receiving phenobarbital alone. Inasmuch as there was a decline in selenium content and in the activity of the seleno-enzyme glutathione peroxidase in hepatocyte cultures maintained in standard culture medium for more than 24 hr, the added selenium appears to correct a spontaneously acquired cellular deficiency in selenium. Contrary to the concept that liver cells placed in culture promptly dedifferentiate with general loss of specialized functions such as cytochrome P-450, our data demonstrate that expression of the phenobarbital-inducible form of cytochrome P-450 is not extinguished in culture, but rather it is masked transiently and is attenuated as the cells adapt to the imperfect conditions of the culture environment.
Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.
Authors: Nielsen P J , Thomas G , Maller J L
Published: May 2006

A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor.
Gene amplification in Rhynchosciara salivary gland chromosomes.
Authors: Glover D M , Zaha A , Stocker A J , Santelli R V , Pueyo M T , De Toledo S M , Lara F J
Published: May 2006

Late in the fourth larval instar, several regions of the Rhynchosciara americana salivary gland chromosomes undergo "DNA puffing." We have constructed a library of cloned cDNAs synthesized from poly(A)+RNA isolated from salivary glands during the period of development when the DNA puffs are active. From this library we have studied clones representative of three genes active during this period but not active at earlier developmental periods of the gland. One of these genes is not amplified during the developmental process and encodes a 0.6-kilobase RNA molecule. The other two genes are located within the DNA-puff sites C3 and C8 and encode 1.25-kilobase and 1.95-kilobase RNA molecules, respectively. We estimate from the quantitation of transfer hybridization experiments that each of these genes undergoes 16-fold amplification during DNA puffing.
Meiosis in haploid yeast
Authors: Wagstaff Joseph E. , Klapholz Sue , Esposito Rochelle Easton
Published: May 2006

Haploid yeast cells normally contain either the
MAT
a or
MAT
α mating-type allele and cannot undergo meiosis and spore formation. If both mating-type alleles are present as a consequence of chromosome III disomy (
MAT
a/
MAT
α), haploids initiate meiosis but do not successfully form spores, probably because the haploid chromosome complement is irregularly partitioned during meiotic nuclear division. We have demonstrated that the ochre-suppressible mutation
spo13-1
enables haploid yeast cells disomic for chromosome III and heterozygous at the mating-type locus to complete meiosis and spore formation, yielding two haploid spores. Previous studies have shown that the absence of the wild-type
SPO13
gene function permits diploid cells to bypass homologous chromosome segregation at meiosis I and proceed directly to meiosis II. During
spo13-1
haploid meiosis, cells enter prophase of meiosis I. Genetic recombination, monitored on the chromosome III disome, occurs at levels similar to those seen in diploids, indicating that the level of exchange between homologs is an autonomous property of individual chromosomes and not dependent on exchange elsewhere in the genome. Exchange is then followed by a single meiosis II equational chromosome division. Recombination in
spo13-1
haploids is blocked by the
spo11-1
mutation, which also eliminates recombination between homologous chromosomes during conventional diploid meiosis. We conclude that Spo
+
haploids expressing both a and α mating-type information attempt a
SPO13
-dependent meiosis I division, and that this division, in the absence of paired homologous chromosomes, is responsible for the failure of such haploids to complete normal gametogenesis. Our observations support the conclusion that initiation and completion of meiosis II and spore formation are not dependent on either completion of meiosis I or the presence of a diploid chromosome complement.
Internal triplication in the structure of human ceruloplasmin.
Authors: Takahashi N , Bauman R A , Ortel T L , Dwulet F E , Wang C C , Putnam F W
Published: May 2006

Amino acid sequence analysis of the 67,000-dalton (67-kDal) fragment that is the amino-terminal half of human ceruloplasmin has revealed internal triplication in the primary structure of the entire molecule. This is illustrated by comparison of 620 residues representing homologous domains of the 67-kDal fragment and of the 50-kDal and 19-kDal fragments that together comprise the carboxyl-terminal half of the molecule. The polypeptide chain is divided into three covalently linked homologous segments, each of about 340 residues. All three homology units have about 30% identity in sequence, and each pair exhibits at least 40% identity. The statistical significance of the 3-fold internal duplication was established by computerized analysis of the sequence. These results and studies of the sites of limited proteolytic cleavage support a model for the ceruloplasmin molecule consisting of an alternating structure of six domains of two different kinds (or possibly nine domains of three kinds). The 3-fold internal homology suggests that the ceruloplasmin molecule evolved by tandem triplication of ancestral genes.
Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes.
Authors: Deutsch P J , Wan C F , Rosen O M , Rubin C S
Published: May 2006

Cell surface and cryptic insulin receptors were solubilized from the particulate fraction of murine 3T3-L1 adipocytes with buffer containing 1% Triton X-100. Solubilized receptors were affinity crosslinked with 125I-labeled insulin and disuccinimidyl suberate and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography after specific immunoprecipitation. Two insulin-binding polypeptides were identified: the more abundant protein had a Mr of 130,000, corresponding to the size of the hormone-binding subunit of insulin receptors on the surface of target cells; the second polypeptide exhibited a Mr of 200,000 and appears to be a component of the latent pool because it was unaffected when 3T3-L1 adipocytes were exposed to trypsin under conditions that result in a 95% reduction in cell surface insulin-binding activity and the loss of the Mr 130,000 polypeptide in crosslinking experiments. Unexpectedly, the population of Mr 200,000 molecules in intact cells was accessible for limited cleavage by chymotrypsin, yielding a Mr 195,000 insulin-binding polypeptide. When 3T3-L1 adipocytes received a 15-min pulse of [35S]methionine, the predominant immunoprecipitated polypeptide had a Mr of 180,000. During a 1.5-hr chase, radioactivity in the Mr 180,000 species rapidly declined while the latent Mr 200,000 polypeptide became intensely labeled. After a 5-hr chase period, broad protein bands with Mrs of 130,000 and 90,000 were visualized as the major immunoprecipitated radioactive polypeptides. Thus, the Mr 180,000 species may be a very early biosynthetic precursor that may be subsequently processed to a Mr 200,000 form and one or both of the smaller receptor subunits at the cell surface.
Modulation of adenovirus transformation by thyroid hormone.
Authors: Fisher P B , Guernsey D L , Weinstein I B , Edelman I S
Published: May 2006

We have examined the effect of triiodothyronine (T3) on de novo transformation of a cloned population of Fischer rat embryo fibroblasts (CREF) by a temperature-sensitive mutant (H5ts125) of type 5 adenovirus and on the expression of the transformed phenotype in these cells. When CREF cells were grown in medium lacking T3 before, during, and after infection with H5ts125, the yield of transformed foci was half that in the cultures supplemented with 1 nM T3. Selective addition or removal of T3 during various phases of the transformation process indicated that the hormone exerted its maximal effect within 72 hr after viral infection. T3 was also required for optimal growth in agar of two clones of CREF cells previously transformed by type 5 adenovirus, wt-3A and ts-7E. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate could substitute for T3 in enhancing growth in agar of wt-3A but not of ts-7E, suggesting that the promoter and T3 modify anchorage-independent growth by different mechanisms. Normal CREF cells and both of the transformed CREF clones grew equally well in monolayer culture in medium containing or lacking T3. Both of the transformed CREF clones contained a lower number of nuclear T3 receptors than did CREF cells and they bound somewhat lower levels of phorbol dibutyrate. These results indicate that thyroid hormone modulates an early stage involved in adenovirus transformation and that it also enhances the expression of the transformed state in previously transformed cells.
Induction of an antiviral response by interferon requires thymidine kinase.
Authors: Lewis J A , Mengheri E , Esteban M
Published: May 2006

Mouse fibroblastoid cells (Ltk-) that lack thymidine kinase (tk) activity are unable to respond to murine beta-interferon by establishing antiviral activity or inducing the double-stranded RNA-dependent enzymes, oligo[(2'-5')A] polymerase and Mr 68,000 protein kinase. In contrast, the parental L-929 cell line or clonal derivatives of Ltk- cells into which the herpes virus tk gene was introduced by DNA-mediated gene transfer respond normally to interferon in developing resistance to viral infection and in inducing double-stranded RNA-dependent enzymes. Further evidence for a role of tk in the response to interferon was obtained by isolating revertants of tk+ clones that lost the herpes virus tk gene during growth in BrdUrd-containing medium. In such revertant sublines both tk enzyme activity and viral tk genes were undetectable and treatment with interferon failed to produce an antiviral effect or induce synthesis of the double-stranded RNA-dependent enzymes. Our results indicate that the ability of mouse L cells to respond to beta-interferon is dependent upon the presence of a functional tk gene. We propose that the induction of antiviral responses by interferon stringently requires a metabolite, the level of which is determined by tk activity. The system described may provide a means for elucidating the mechanisms by which responses to interferon are induced.
Electrophysiological and biochemical studies on enhancement of desensitization by phenothiazine neuroleptics.
Authors: Carp J S , Aronstam R S , Witkop B , Albuquerque E X
Published: May 2006

The actions of the phenothiazines chlorpromazine, prochlorperazine, and trifluoperazine were studied on the acetylcholine receptor-ionic channel complex of frog and rat skeletal muscle and of Torpedo californica to determine their role in pharmacological desensitization and their interactions with different states of the receptor-ionic channel complex. The phenothiazines depressed the peak amplitude of spontaneous and evoked endplate currents while having negligible effect on the decay time constants. Mean channel lifetime and single channel conductance were not altered by these drugs. They also produced a frequency-dependent depression of the peak amplitude of endplate potentials evoked by repetitive microiontophoresis at the extrajunctional region. In addition, these drugs enhanced the ability of carbamoylcholine to displace 125I-labeled alpha-bungarotoxin from receptor-rich membrane preparations of T. californica when used in concentrations that had no effect on 125I-labeled alpha-bungarotoxin binding alone (10 microM). Similarly, the phenothiazines inhibited the binding of tritiated ionic channel ligands, such as phencyclidine and perhydrohistrionicotoxin, a process also enhanced by the presence of carbamoylcholine. These data suggest that the phenothiazines augment agonist-induced desensitization primarily by interacting with the receptor-ionic channel complex prior to channel opening.
Isolation of genomic DNA clones spanning the entire fibronectin gene.
Authors: Hirano H , Yamada Y , Sullivan M , de Crombrugghe B , Pastan I , Yamada K M
Published: May 2006

Overlapping recombinant clones that appear to encompass the entire fibronectin gene have been isolated by step-wise screening of a library of chicken genomic DNA fragments. The first genomic clone was isolated by using a cloned fibronectin cDNA hybridization probe. The remaining clones were obtained by using defined fragments of this and successive genomic clones as probes. Their relationships and overlaps were determined by electron microscopy, restriction mapping, and heteroduplex analysis. Based on electron microscopic analysis of hybrids between these clones and fibronectin mRNA, the gene is approximately 48 kilobases long, more than 5 times larger than the corresponding mRNA. This large gene contains at least 48 exons interrupted by introns of highly variable size. The total exon size as estimated by R-loop analysis is 8 kilobases, similar to the mRNA for fibronectin. With the exception of the 3'- and 5'-terminal exons, the exons are small and roughly similar in size. The average exon size is 147 +/- 37 base pairs, corresponding to a protein unit of 50 amino acids. The nucleotide sequence of one of these exons was determined. The deduced amino acid sequence has marked homologies with one type of repetitive protein sequence unit known to exist in bovine fibronectin. These results suggest that the gene for fibronectin may have arisen by multiple gene duplications of a primordial gene or genes approximately equal to 150 base pairs long.
H2A.F: an extremely variant histone H2A sequence expressed in the chicken embryo.
Authors: Harvey R P , Whiting J A , Coles L S , Krieg P A , Wells J R
Published: May 2006

A cDNA clone bank has been constructed from chicken embryonic RNA. Clones hybridizing poorly to embryonic histone gene probes were selected as possible variant gene transcripts. The DNA sequence of one cDNA predicts an extremely variant H2A protein (H2A.F), which is 40% divergent from the most abundant H2A protein in chicken erythrocyte chromatin. The H2A.F gene is not highly conserved across large species barriers, but in the chicken there may be a family of linked genes. The H2A.F mRNA is approximately equal to 820 base pairs in length and, unlike most other histone mRNAs, is polyadenylylated. Significantly, the H2A.F transcript shows a limited tissue distribution in the chicken embryo.
Monoclonal antibody to chicken oviduct progesterone receptor.
Authors: Radanyi C , Joab I , Renoir J M , Richard-Foy H , Baulieu E E
Published: May 2006

Antibodies to molybdate-stabilized chicken oviduct progesterone receptor were raised in a Wistar rat and detected by interaction with homogeneous radioiodinated progesterone receptor. Spleen cells of this rat were then fused with mouse Sp2/0-Ag14 myeloma cells and the antibodies produced by the hybrid cells were detected by double immunoprecipitation using the 125I-labeled receptor. Cells of one of the positive cultures were then cloned by limiting dilution and one hybridoma cell line was studied. The monoclonal antibody produced was an IgG2b, and it reacted with the molybdate-stabilized "8-9S" form of the chicken oviduct progesterone receptor, labeled with either [3H]progesterone or [3H]ORG 2058 (a high-affinity synthetic progestin). Kd for the 8-9S progesterone receptor was approximately equal to 1 nM. Progesterone receptor-monoclonal antibody complexes were labeled with radioactive progesterone, suggesting that antibody does not prevent hormone binding. By using a [35S]methionine-labeled antibody, we were able to detect the progesterone receptor independently of its characteristic function of binding radioactive hormone. No crossreaction with human progesterone receptor was detected.
Sexuality in piroplasms as revealed by electron microscopy in Babesia microti.
Authors: Rudzinska M A , Spielman A , Lewengrub S , Trager W , Piesman J
Published: May 2006

Protozoa of the closely related genera Babesia and Theileria are intraerythrocytic parasites of vertebrates. They have a complex life cycle that includes development in an intermediate vector host, a tick. Whether sexual stages occur in the tick has been a subject of great controversy. The small size of the organism and the complexity of developmental stages in the gut of the tick have prevented a definitive solution of this problem. By means of a simple and straightforward although time-consuming method, it became possible to demonstrate gametes and their sexual fusion in Babesia microti developing in the gut of larvae of the tick Ixodes dammini. Tick larvae fed on hamsters infected with a human strain of B. microti were fixed and processed for electron microscopy. It was found that some of the parasites formed a unique structure shaped like an arrowhead. Because it was suspected that these forms might represent gametes, a search was made for pairs of parasites that were fusing and with each member of the pair emerging from a different erythrocyte. Such a fusing pair could not possibly represent a parasite undergoing division. By study of serial sections such pairs were indeed found. In every case one member of the pair of gametes had an arrowhead structure. This proves sexuality of B. microti and makes highly likely its existence in all members of the genera Babesia and Theileria.
Mouse sn-glycerol-3-phosphate dehydrogenase: molecular cloning and genetic mapping of a cDNA sequence.
Authors: Kozak L P , Birkenmeier E H
Published: May 2006

The isozymes of glycerol-3-phosphate dehydrogenase (GPDH; sn-glycerol-3-phosphate:NAD+2-oxidoreductase, EC 1.1.1.8) in tissues of the mouse are coded for by two structural genes, Gdc-1 and Gdc-2, located on chromosomes 15 and 9, respectively. In order to investigate the expression of these genes, we isolated a GPDH cDNA clone from a mRNA preparation isolated from brown adipose tissue. The GPDH cDNA clone was identified by colony hybridization and hybrid selection of a mRNA that was translated in vitro to produce immunoprecipitable GPDH protein. In blot analysis, the GPDH cDNA hybridized to a single mRNA species that migrated at the position of 23S ribosomal RNA. This GPDH cDNA clone was mapped to the Gdc-1 locus by identification of a restriction enzyme polymorphism present in genomic DNA isolated from Gdc-1 congeneic lines of mice.
Complete nucleotide sequence of the 26S rRNA gene of Physarum polycephalum: its significance in gene evolution.
Authors: Otsuka T , Nomiyama H , Yoshida H , Kukita T , Kuhara S , Sakaki Y
Published: May 2006

The complete nucleotide sequences of the 5.8S and 26S rRNA genes of Physarum polycephalum and the transcribed spacer between them were determined. Comparison of the sequences with those of the Escherichia coli 23S rRNA and yeast 26S rRNA genes showed that there are 16 highly homologous regions in the sequences of Physarum and E. coli and that eukaryotes have some eukaryote-specific extra sequences. The sequence immediately following the 5.8S-like region of E. coli 23S rRNA was found to be highly homologous to the 5' terminus of Physarum 26S rRNA, indicating that the eukaryote-specific 5.8S rRNA gene is derived from the 5'-terminal region of the prokaryote large rRNA gene.
Evidence for metabolic and functional discrimination of sterols by
Phytophthora cactorum
Authors: Nes W. David , Stafford Allen E.
Published: May 2006

When fed 10 ppm of one of the following sterols: cholesterol (cholest-5-en-3β-ol), wingsterol (21-isopentylcholesterol), desmosterol [cholesta-5,24(25)-dien-3β-ol], 24-methylenecholesterol [ergosta-5,24(28)-dien-3β-ol], or fucosterol [stigmasta-5,24(28)-dien-3β-ol], the pathogenic fungus
Phytophthora cactorum
, which is naturally unable to epoxidize squalene, accumulated each of the test compounds to similar levels. Fucosterol, the only sterol metabolized, was reduced to yield 24-ethylcholesterol. All the sterols tested induced the formation of sex structures. Fertilization and subsequent maturation of oospores capable of germination occurred only with the naturally occurring sterols. Wingsterol treatments resulted in aborted oospores. None of the sterols tested was inhibitory to growth, measured as changes in the 21-day mycelial dry weight. The results are consistent with the view that the accumulated sterol functions to regulate the life cycle of
P. cactorum
. However, the metabolism and kinds of recognition of the sterol molecule, in terms of uptake and effects on growth and induction of the various sexual events, contrast sharply with what is known for other oomycetous fungi such as
Achlya
and
Saprolegnia
. This implies that the evolutionary histories of the Oomycetes may be different.
Regulatory regions of ColE1 that are involved in determination of plasmid copy number.
Authors: Som T , Tomizawa J
Published: May 2006

The copy number of plasmid ColE1 has been known to increase when the Hae II-C segment downstream from the replication origin is deleted. The presence of the 306-base-pair (bp) Hpa II region in the segment is sufficient for reduction of the copy number. Plasmids harboring the region express a trans-acting function that is responsible for the copy number reduction and synthesize a unique protein. A protein specified by the region is purified to near homogeneity and identified as the 63-amino-acid protein encoded by the Hpa II segment. The region, which includes segments 19-25 bp and 53-311 bp downstream from the start site of the primer RNA, is involved in determination of sensitivity to the inhibitory function. In vivo transcription of the galK gene, which is directed by the primer promoter in a segment inserted in front of the structural gene, is inhibited by a plasmid carrying the Hpa II region. The inhibition is strong when the promoter segment contains up to 135 bp downstream from the primer RNA start site, whereas it is weak when only the region up to 52 bp downstream is present.
Structural and dynamic differences between supercoiled and linear DNA from proton NMR.
Authors: Bendel P , James T L
Published: May 2006

Proton NMR experiments on an intact plasmid supercoiled DNA molecule are reported. Spectra of the hydrogen-bonded exchangeable imino protons and nonexchangeable aromatic protons of pIns36 (7,200 base pairs; Mr 5 x 10(6)) were examined at 240 and 360 MHz. In contrast to linear DNA of comparable size in which the signals from these protons are broadened almost beyond detection, the supercoiled DNA exhibits a remarkably well-resolved spectrum in the range 12-15 ppm, with linewidths characteristic of linear DNA molecules with molecular weight nearly two orders of magnitude smaller. These results imply an extraordinary degree of mobility in accord with our earlier discovery of unusually long 31P spin-spin relaxation times in the supercoiled molecule. Alterations in the chemical shift of the imino proton resonance positions in supercoiled DNA compared to linear DNA imply that the structure is modified in the A . T base pairs with at least two distinct structures apparent from the spectrum. However, little or no alteration is evident in the resonances due to the G . C base pairs.
Genetic mapping in the major histocompatibility complex by restriction enzyme site polymorphisms: most mouse class I genes map to the Tla complex.
Authors: Winoto A , Steinmetz M , Hood L
Published: May 2006

From a genomic library constructed from sperm DNA of the inbred BALB/c mouse, we previously isolated 54 cosmid clones that contain 36 class I genes and can be divided by restriction map analyses into 13 gene clusters. We have isolated single- and low-copy DNA probes from each of these clusters to visualize restriction enzyme site polymorphisms in the DNAs from various congeneic and recombinant congeneic mice. These polymorphisms permit us to map each of the 13 cosmid clusters to a precise location in the major histocompatibility complex of the mouse. Thirty-one of 36 class I genes map into the Tla complex of the major histocompatibility complex whereas the remaining 5 genes map to the H-2 complex. Thus, all 36 class I genes are located in the major histocompatibility complex. Analysis of the number of restriction enzyme fragments visualized by the single- and low-copy DNA probes suggests that the class I genes in different inbred strains of mice probably undergo gene duplications and deletions, presumably by homologous but unequal crossing-over.