bluuebunny/crossref_metadata_2025 huggingface dataset:
Excitation-contraction coupling: role of K-activation within the transverse tubular system.
Authors: Reuben J P , Katz G M , Brandt P W , Suarez-Kurtz G , Dekin M S
Published: May 2006

Long-duration Ca action potentials induced in crustacean muscle fibers after prolonged exposure to quaternary ammonium ions are accompanied by attenuated tensions with unique time courses. The tensions have three phases. The initial phase, correlated with the upstroke of the spike, is a rapid increase in tension followed by relaxation to or near to resting level (on-tension). In the second phase, tension rises slowly as the spike plateau declines. The final phase is another rapid increase and decay in tension that is correlated with termination of the action potential (off-tension). To observe these tensions, fibers must be exposed to 50-100 mM tetrabutylammonium ion for about 1 hr or to lower concentrations for longer periods (e.g., 5 mM for 20-30 hr). To obtain a similar response in fibers treated with tetraethylammonium ion, higher concentrations or longer soaking periods, or both, are required. Because neither caffeine-induced tensions in intact fibers nor contractile protein and sarcoplasmic reticulum function in skinned fibers were modified by quaternary ammonium ions, their site of action appears to be limited to surface or transverse tubular system membranes, or both. The unique tensions can be explained by considering the mode by which quaternary ammonium ions block K channels in conjunction with a scheme in which activation of K channels within the transverse tubular system controls the driving force for influx of Ca ions.
Protein binding sites are conserved in U1 small nuclear RNA from insects and mammals.
Authors: Wieben E D , Madore S J , Pederson T
Published: May 2006

To gain insight into the ribonucleoprotein (RNP) structure of small nuclear RNAs, HeLa cell poly(A)+ mRNA was translated in a reticulocyte lysate, and the in vitro binding of 35S-labeled proteins to individual small nuclear RNA species was examined by using human autoimmune antibodies. A Mr 32,000 protein binds to U1 RNA but not to U2, U4, U5, or U6. The resulting U1 RNP complex is recognized both by Sm and RNP antibodies. U2 RNA also forms a complex with protein, which is recognized by Sm antibody. Thus, the lack of binding of the Mr 32,000 protein to U2 RNA is not due to a failure of U2 to bind specific proteins in the in vitro system. Similar translation-assembly experiments with Drosophila poly(A)+ mRNA reveal that a Mr 26,000 protein identified previously in Drosophila U1 RNP [Wieben, E. D. & Pederson, T. (1982) Mol. Cell. Biol. 2, 914-920] also binds to U1 RNA in vitro. When the translation products of HeLa or Drosophila mRNA are presented with U1 RNA of the other species, the Mrs 32,000 and 26,000 proteins recognize binding sites on the heterologous U1 and, in both cases, form complexes recognized by RNP antibody. These results establish that a Mr 32,000 protein is unique to U1 RNA in human cells and that the U1 RNA binding sites for this and a Mr 26,000 homologue have been highly conserved in evolution. These sites may be the identical 13 nucleotides at the 5' ends of human and Drosophila U1 RNA or a highly conserved aspect of U1 secondary structure.
Headgroup interactions in mixed phospholipid bilayers
Authors: Sixl Frank , Watts Anthony
Published: May 2006

2
H NMR methods have been used to study how bilayer-forming phospholipids interact with each other at the membrane surface. Aqueous dispersions of dimyristoyl-
sn
-phosphatidylcholine (Myr
2
-PtdCho), dimyristoyl-
sn
-phosphatidylethanolamine (Myr
2
-PtdEtn), and dimyristoyl-
sn
-phosphatidyl-3-glycerol, specifically deuterated at different positions in their headgroups, give well-resolved
2
H NMR powder spectra. These spectra are characteristic of a lipid bilayer with quadrupole splittings that are sensitive to the amplitude of headgroup motion. In binary mixed bilayers of deuterated lipids with an unlabeled component, all parts of the deuterated headgroup monitor the presence of the second lipid from changes in the measured quadrupole splittings. The headgroups of the charged lipids, dimyristoyl-
sn
-phosphatidylserine and dimyristoyl-
sn
-phosphatidyl-3-glycerol, interact to the largest extent with the choline moiety of Myr
2
-PtdCho and the ethanolamine moiety of Myr
2
-PtdEtn, whereas a somewhat smaller but still marked alteration in headgroup motion was observed in Myr
2
-PtdCho/Myr
2
-PtdEtn mixtures. The large changes in the deuterium quadrupole splittings for the zwitterionic lipids after addition of a charged lipid suggest that either a strong perturbation in the hydrogen bonding occurs or changes take place in the water structure at the membrane surface, or possibly both.
In vitro translation and characterization of a unique histidine-rich protein mRNA in the avian malaria parasite Plasmodium lophurae.
Authors: Wallach M , Boeke J D
Published: May 2006

The histidine-rich protein (HRP) of the avian malaria parasite Plasmodium lophurae contains 70% histidine. It is found in dense cytoplasmic granules and during the erythrocytic cycle it accumulates to represent 10% of the dry weight of the parasite. In the present work the HRP mRNA was studied by in vitro translation and by the use of a polyhistidine oligonucleotide probe. The HRP mRNA contains 2,000-2,100 nucleotides encoding a protein with an apparent molecular weight of 50,000. In addition a HRP of molecular weight 35,000-40,000 is also produced in vitro, probably as a result of proteolytic cleavage of the molecular weight 50,000 polypeptide which corresponds to in vivo labeled and purified HRP. The HRP represents a much larger proportion of the in vitro products synthesized in the homologous cell-free system compared to the rabbit reticulocyte system, and it reflects more closely the pattern of protein synthesis seen in vivo. In addition, HRP mRNA is more abundant in polysomes isolated from young parasites than in polysomes from mature schizonts. These results indicate that the HRP accumulates as a result of amplified translation of its mRNA at certain stages of its erythrocytic cycle.
Critical experimental test of the possibility of "paternal leakage" of mitochondrial DNA.
Authors: Lansman R A , Avise J C , Huettel M D
Published: May 2006

Most previous data suggesting maternal inheritance of mtDNA have come from single-generation mating experiments, and most of the analytical techniques utilized would not have detected paternal mtDNA molecules in progeny at levels less than about 5%. Long-term mating experiments, in which a fertile female lineage derived from hybridization of two species with distinguishable mtDNAs is backcrossed recurrently to the male parental species, provide an ideal opportunity to assess possible low-level paternal leakage. We have analyzed the 45- and 91-generation backcross progeny of such matings between two species of lepidopteran insects [Heliothis (Noctuidae)], using autoradiographic techniques that can detect rare mtDNA molecules in less than 1 part per 500. The analysis failed to detect any paternal mtDNA and sets an upper limit of paternal leakage at about 1 molecule per 25,000 per generation in this system.
Long-wavelength-absorbing forms of bacteriochlorophyll
a
in solutions of Triton X-100
Authors: Gottstein J. , Scheer H.
Published: May 2006

At least three forms of Triton X-100-solubilized bacteriochlorophyll
a
(BChl
a
) have been characterized by UV/visible/near-IR absorption and CD spectra. One, absorbing at 770 nm, is similar to a monomeric solution in methanol. The two others have strongly red-shifted absorption peaks (860 nm and 930, 835 nm) and intense and complex CD bands in this region, indicative of strong interaction of at least two and three molecules of BChl
a
, respectively.
pH and the recycling of transferrin during receptor-mediated endocytosis.
Authors: Dautry-Varsat A , Ciechanover A , Lodish H F
Published: May 2006

At pH 5.4 apotransferrin (iron-free transferrin) binds to cell-surface transferrin receptors to the same extent and with the same affinity as does diferric transferrin at pH 7.0. Apotransferrin is quickly dissociated from its receptor when the pH is raised to 7.0. These and other results strongly support a simple model that explains the cycling of transferrin during a single cycle of receptor-mediated endocytosis. Diferric transferrin binds to cell-surface receptors, and the transferrin-receptor complex is endocytosed. The pH of the endocytic vesicle is lowered to 5.5 or below; this causes dissociation of iron from the transferrin-receptor complex, but apotransferrin remains bound to its receptor. The iron remains within the cell, and the apotransferrin-receptor complex is recycled to the cell surface. Upon encountering the neutral pH of the medium, apotransferrin is dissociated from the cell.
Recombination involving transposable elements: role of target molecule replication in Tn1 delta Ap-mediated replicon fusion.
Authors: Muster C J , Shapiro J A , MacHattie L A
Published: May 2006

Donor DNA molecules carrying Tn1 or Tn3 deletion mutants do not need to replicate in order to participate in replicon fusion recombination events during which the Tn1/Tn3 element is duplicated. We have assayed Tn1 delta Ap-mediated replicon fusion events involving plasmid R388 and the bacteriophage lambda-derived plasmid p lambda CM, and we find that the role of the recipient molecule is distinct. When p lambda CM carries Tn1 delta Ap, replicon fusion occurs in more than 1% of all cells assayed, whether or not p lambda CM::Tn1 delta Ap can replicate. In contrast, when R388 carries Tn1 delta Ap, replicon fusion occurs only when the p lambda CM target can replicate. Blocks to p lambda CM replication by prophage repressor or amber mutations of the O and P cistrons reduce replicon fusion so that it occurs in less than 1 out of 10(5) cells assayed.
Autologous and allogeneic typing of human leukemia cells: definition of surface antigens restricted to lymphocytic leukemia cells.
Authors: Naito K , Yamaguchi H , Horibe K , Shiku H , Takahashi T , Suzuki S , Yamada K
Published: May 2006

Serum from a patient (CO) with acute lymphoblastic leukemia was reactive in immunoadherence assays with autologous leukemia cells but not with autologous blood lymphocytes or bone marrow cells during complete remission. Extensive absorption tests with an array of leukemia cells and normal cells were performed in order to define the specificity of the reaction. The autologous leukemia reactivity was either completely or partially absorbed with acute lymphoblastic leukemia cells obtained from 10 of 14 patients but not with acute nonlymphoblastic leukemia cells or normal hematopoietic cells. Some cultured lymphocytic leukemia lines also showed expression of the antigen. The absence of alloantibodies in CO's serum detectable in our assay allowed direct testing of CO's serum on allogeneic target cells. The results of these tests correlated with the results of absorption tests, indicating that small populations of antigen-positive cells were not missed with absorption tests. In addition, absorption tests with CO-positive allogeneic leukemia target cells showed the same pattern of antigen distribution as tests with autologous targets. CO reactivity was not removed by absorption with cell lines expressing human T-cell leukemia virus, nor did CO's serum react with adult T-cell leukemia cells from three patients.
Degree of mapping for nonlinear mappings of monotone type: Strongly nonlinear mapping
Authors: Browder Felix E.
Published: May 2006

The classical degree function developed earlier for pseudomonotone operators from a reflexive Banach space to its dual is extended for the special case of Sobolev spaces to the sum of a pseudomonotone mapping (representing a nonlinear elliptic operator) and a strongly nonlinear lower order term.
Topological and percolation properties of potential mapping with glueing
Authors: Zeldovich Ya. B.
Published: May 2006

The potential movement of particles forms a continuous mapping of the Lagrangian space on the space of Eulerian coordinates of particles. The force-free inertial movement with random smooth initial velocity field is considered. The condition of particles on caustics glued together to form singularities leads to a peculiar structure with localized regions of rarefied matter surrounded by glued surfaces with higher-order singularities on them.
Regulation of three key enzymes in cholesterol metabolism by phosphorylation/dephosphorylation.
Authors: Scallen T J , Sanghvi A
Published: May 2006

Our laboratories have investigated the role of phosphorylation/dephosphorylation in the regulation of three key enzymes in cholesterol metabolism. 3-Hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase (EC 1.1.1.34), the major regulatory enzyme in cholesterol biosynthesis, is inhibited by phosphorylation. Acyl-CoA:cholesterol O-acyltransferase (ACATase; EC 2.3.1.26) and cholesterol 7 alpha-hydroxylase (EC 1.14.13.7), key regulatory enzymes in the utilization of cholesterol, are activated by phosphorylation. In view of these results, we propose that short-term regulation of the concentration of intracellular unesterified cholesterol is achieved by a coordinate phosphorylation/dephosphorylation of these three enzymes. For example, if cholesterol enters the liver cell, HMG-CoA reductase would be inhibited by phosphorylation and biosynthesis of cholesterol would be reduced; however, reactions utilizing cholesterol would be activated, due to the phosphorylation of ACATase and cholesterol 7 alpha-hydroxylase. Thus, the phosphorylation/dephosphorylation of these three enzymes provides an elegant short-term mechanism for the homeostasis of intracellular unesterified cholesterol.
Role of carbohydrate in multimeric structure of factor VIII/von Willebrand factor protein.
Authors: Gralnick H R , Williams S B , Rick M E
Published: May 2006

The carbohydrate moiety of the factor VIII/von Willebrand (vW) factor protein is important in the expression of vW factor activity and the intravascular survival of the protein. Studies of normal human factor VIII/vW factor protein indicate that there is a requirement of a full complement of penultimate galactose for the maintenance of a normal multimeric structure. Release of penultimate galactose by beta-galactosidase or modification by galactose oxidase results in loss of the largest molecular weight multimers and increased numbers of intermediate and smaller multimers. In contrast, terminal galactose on the factor VIII/vW factor protein does not appear to play a significant role in the maintenance of the multimer organization. The abnormalities in multimeric structure and molecular size were demonstrated by NaDodSO4/polyacrylamide/agarose gel electrophoresis, NaDodSO4/glyoxyl-agarose electrophoresis, and sucrose density ultracentrifugation. These studies indicate that the penultimate galactose plays a role in the maintenance of the largest multimers of the factor VIII/vW factor protein. This may explain why, in some patients with variant forms of vW disease, a carbohydrate abnormality also may affect the multimeric structure of the plasma factor VIII/vW factor protein.
Pepsin digestibility of proteins in sorghum and other major cereals
Authors: Mertz Edwin T. , Hassen Mohamed M. , Cairns-Whittern Carleen , Kirleis Allen W. , Tu Lichuan , Axtell John D.
Published: May 2006

We have shown previously that sorghum is highly digestible in the rat. However, other workers have shown that sorghum is much less digestible than wheat, maize, and rice in young children. Because the rat does not show these digestibility differences, we developed an empirical pepsin digestion method, first reported in 1981, which simulates the digestion values found in children. In this report the method has been improved and used to analyze wheat, maize, rice, millet, and sorghum and certain processed samples of millet and sorghum. The pepsin digestion values parallel those found in children for wheat, maize, rice, and sorghum. In addition, a processed sorghum product that gave a high digestion value in children also gave a high value with the
in vitro
pepsin method.
Identification of a labile protein involved in the G1-to-S transition in Saccharomyces cerevisiae.
Authors: Popolo L , Alberghina L
Published: May 2006

The biochemical nature of the start process that commits budding yeast to DNA synthesis is not known. Kinetic evidence has suggested recently that short-lived protein(s) may have to accumulate to a critical level before the cell cycle may progress towards DNA synthesis and cell division. We investigated by high-resolution two-dimensional electrophoresis whether, in a cdc25-1 mutant strain of Saccharomyces cerevisiae that had been blocked at the regulatory step called "start" by growth at a restrictive temperature, short-lived proteins are synthesized during the recovery of growth at a permissive temperature. Of the approximately equal to 500 proteins resolved by the two-dimensional electrophoresis, 6 were short-lived. Only one of them (Mr = 100,000, pI approximately equal to 4.8-5) appears to be specifically made during the G1-to-S transition at start. A regulatory role for cell cycle progression in yeast is suggested for this protein, p100.
Labile nature of the visual recovery promoted by reverse occlusion in monocularly deprived kittens.
Authors: Mitchell D E , Murphy K M , Kaye M G
Published: May 2006

Kittens were monocularly deprived by closing one eye at the time of natural eye opening for periods that ranged from 4 to 14 weeks. This eye was then opened, and the other eye was closed for an approximately equal period of time. During this period of reverse occlusion, the vision of the initially deprived eye improved from apparent blindness to a level of good visual acuity. Surprisingly, however, this recovery was largely eliminated in only 2 weeks once the initially nondeprived eye was opened to restore visual input to both eyes. This finding has important implications for the nature of the mechanism(s) responsible for the dramatic physiological effects of monocular occlusion on the visual cortex. It may also help to elucidate recent observations on patching therapy in human amblyopia.
Identification of Epstein-Barr nuclear antigen polypeptide in mouse and monkey cells after gene transfer with a cloned 2.9-kilobase-pair subfragment of the genome.
Authors: Fischer D K , Robert M F , Shedd D , Summers W P , Robinson J E , Wolak J , Stefano J E , Miller G
Published: May 2006

A new antigenic polypeptide was identified in mouse L cells and in monkey COS-1 cells in which Epstein-Barr nuclear antigen (EBNA) was expressed as the result of gene transfer with cloned fragments of Epstein-Barr virus (EBV) DNA. The same-size protein (Mr, approximately equal to 78,000) was seen in stably transformed mouse cells harboring only the BamHI K fragment [approximately equal to 5.2 kilobase pairs (kbp)] or its BamHI/HindIII subfragment, I1f (approximately equal to 2.9 kbp). Thus, the latter DNA fragment is sufficient to code for the protein. In transfected COS cells, a deletion mutant of the I1f fragment (approximately equal to 2.3 kbp) gave rise to a truncated protein (Mr, approximately equal to 52,000), whereas the BamHI K fragment yielded a full-sized Mr 78,000 species. This finding indicates that EBNA is encoded by viral genes. In Burkitt lymphoma lines or in immortalized lymphocytes, variation in the size of the I1f fragment correlated with the apparent molecular weight of the EBNA polypeptide. EBNA is truncated in two Burkitt lymphoma lines, Raji (Mr, 67,000) and P3JHR-1 (Mr, 70,000), which have deletion mutant I1f genes. EBNA in human lymphoid cells bearing a complete I1f fragment as part of the entire EBV genome is the same size (Mr, 78,000) as EBNA found after gene transfer of I1f alone into mouse or monkey cells. Therefore, these expression systems make an authentic EBNA after transfer of the appropriate EBV genes.
Template requirements for initiation of phage phi 29 DNA replication in vitro.
Authors: García J A , Peñalva M A , Blanco L , Salas M
Published: May 2006

The template requirements for the formation of the phi 29 protein p3-dAMP initiation complex in vitro have been studied. The initiation reaction requires the parental protein p3 but not an intact DNA molecule. Protein p3-containing fragments from the left- or right-hand DNA ends were active as template for formation of the initiation complex provided they had a minimal size: a 26-base-pair-long fragment was active whereas a 10-base-pair-long one was essentially inactive. However, the activity of the latter was restored by ligation of an unspecific DNA sequence. phi 29 DNA internal fragments, as well as denatured phi 29 DNA, were inactive as template for the initiation reaction. The terminal protein-DNA complex isolated from Bacillus phage phi 15 was active in formation of the phi 29 p3-dAMP complex, whereas the protein-DNA complex isolated from Bacillus phage GA-1 or from the pneumococcal phage Cp-1, both with a morphology similar to that of phage phi 29, as well as that obtained from adenovirus, were inactive.
Different central nervous system cell types display distinct and nonrandom arrangements of satellite DNA sequences.
Authors: Manuelidis L
Published: May 2006

Paraformaldehyde-fixed tissue from mouse cerebellum was hybridized with biotin-labeled satellite DNA for identification of centromeres. By using avidin-peroxidase conjugates, it was possible to define the nuclear position of centromeres at the ultrastructural level. Three-dimensional analysis of well-resolved centromere arrays were aided by computer reconstruction of serial sections. Different cell types displayed distinct, nonrandom centromere locations. In Purkinje neurons, the majority of detected sequences were clustered together around the central nucleolus, whereas in granule neurons, more numerous, dispersed centromere clusters were associated with the nuclear membrane. In Purkinje cells, peroxidase-labeled regions corresponded to dense heterochromatic aggregates were detected in Purkinje cells of several different species. These observations suggest that in these highly differentiated cells, the nuclear position of centromeres is maintained in evolution despite species differences in centromeric DNA sequence. Such defined ordering of centromeres may be integral to specific functional capacities.
A human autosomal phosphoglycerate kinase locus maps near the HLA cluster.
Authors: Szabo P , Grzeschik K H , Siniscalco M
Published: May 2006

The human cDNA probe pPGK824 , of Singer-Sam et al. [Singer-Sam, J., Simmer , R. L., Keith, D. H., Shively , L., Teplitz , M., Itakura , K., Gartler , S. M. & Riggs, A. D. (1983) Proc. Natl. Acad. Sci. USA 80, 802-806] was used to isolate a genomic clone lambda PGK-1 containing a portion of an autosomal locus for phosphoglycerate kinase (PGK). A unique sequence subclone (pGK-1) of lambda PGK-1 was then used to map this locus to the region p23-q12 of human autosome 6--i.e., to the interval that contains the major human histocompatibility locus (HLA). Since an autosomal gene coding for testis-specific PGK in the mouse has been shown to be closely linked to the H2 locus and to the T/t-complex locus [ Eicher , E. M., Cherry, M. & Flaherty , L. (1978) Mol. Gen. Genet. 158, 225-228], it is suggested that the lambda PGK-1 recombinant clone contains part of the human gene for the testis-specific isozyme of PGK. In addition, the subcloned pGK-1 detects an EcoRI restriction fragment length variant and may therefore prove useful for further genetical analysis of the HLA region and specifically for testing the hypothesis that spina bifida and anencephaly may be the human equivalent of the murine defects due to the T/t-complex locus. Our findings support the generally held hypothesis that a large number of structural loci clustered around the histocompatibility genes have been conserved in a close linkage association throughout a large evolutionary interval.
Selective expression of a plasmid cat gene at a late stage of Bacillus subtilis sporulation.
Authors: Mongkolsuk S , Lovett P S
Published: May 2006

The cat-86 gene in plasmid pPL603 specifies chloramphenicol acetyltransferase (CAT) and is selectively expressed in Bacillus subtilis at a stage in sporulation in which internal spores are first observed (approximately T8). The gene is unexpressed in vegetatively growing cells. cat-86 expression and spore formation are both blocked when cells are grown in excess glucose. cat-86 expression at T8 is due to selective transcription of the gene, since cat-86 mRNA is undetectable in vegetatively growing cells but is readily demonstrated in sporulating cells. The transcription start site for cat-86 mRNA from sporulating cells is within a 203-base-pair restriction fragment designated P1, which is located upstream from the cat coding region on pPL603 . Deletion of P1 from pPL603 eliminates the sporulation -associated expression of cat-86. Host sporulation genes, whose function is absolutely required for cat-86 expression at T8, include six early sporulation, spo0 , genes and spoIIE . Therefore, pPL603 provides a novel system in which the in vivo expression of a known, plasmid-linked gene is dependent on sporulation-specific changes in B. subtilis.
Recombination of transfected DNAs in vertebrate cells in culture.
Authors: Bandyopadhyay P K , Watanabe S , Temin H M
Published: May 2006

We studied the frequency of homologous and illegitimate recombinations between transfected noninfectious retroviral DNA molecules in chicken embryo fibroblasts. The frequency of recombination was determined by the formation of infectious virus and varied with the extent of homology between the DNA molecules at the site of recombination, but only when there were regions of homology surrounding this region. While homologous recombination led to the formation of wild-type virus, illegitimate recombination resulted in formation of infectious virus with alterations at the site of recombination. Apparent homologous recombination was also observed between transfected and chromosomal DNAs in D17 dog cells.
Genetic control of major histocompatibility complex-linked immune responses to synthetic polypeptides in man: poly(L-phenylalanine, L-glutamic acid)-poly (DL-alanine)--poly(L-lysine) and L-glutamic acid, L-alanine, L-tyrosine (60:30:10).
Authors: Chan M M , Bias W B , Hsu S H , Meyers D A
Published: May 2006

Vigorous lymphoproliferative responses to synthetic polypeptides poly(L-phenylalanine, L-glutamic acid)-poly(DL-alanine)--poly(L-lysine) [( Phe,G)-A--L], and L-glutamic acid, L-alanine, L-tyrosine (60:30:10) (GAT) were observed in cells from 92 unrelated subjects. Thirty-three percent responded to (Phe,G)-A--L and 77% to GAT. No HLA association was observed with responses to these two antigens. Family studies indicated that two complementary immune response (Ir) genes are required for response to each antigen. Eleven matings were informative for linkage analysis between HLA and these Ir genes. Families in which the complementary genes are in coupling gave maximal lod scores (log of the odds) of 4.50 for (Phe,G)-A--L and 7.57 for GAT for 0 = 0. In a HLA-B/D recombinant family, the Ir- PheGAL genes are mapped towards the HLA-D region. The localization of Ir-GAT genes close to HLA-B was provided by a HLA-A/B recombinant.
Extrapancreatic insulin gene expression in the fetal rat.
Authors: Muglia L , Locker J
Published: May 2006

Analysis of gene expression in rat yolk sac, a primitive endodermal structure, revealed a low level of developmentally regulated insulin production. At 14 days of gestation, a 2.4-kilobase (kb) RNA species hybridized to cloned insulin gene probes. This species increased throughout gestation. At 16 days, a second transcript of 0.72 kb became visible and, by 18 days, the 0.72-kb transcript predominated. In the pancreas, the fully processed insulin mRNA is 0.55 kb long. Over the same time period in the fetal liver (also a tissue of endodermal origin, as is the pancreas), only the 2.4-kb transcript was detected; no hybridizing transcripts were detected in adult liver RNA. Gel filtration studies and insulin radioimmunoassay of acid/ethanol-soluble peptides showed approximately equal amounts of proinsulin and insulin in 18-day yolk sac, a result suggesting that the transcripts in this tissue are translated. On the other hand, a lower level of insulin and the lack of proinsulin in fetal liver were compatible with a pancreatic origin of hepatocyte insulin by receptor binding rather than intrahepatic insulin synthesis.
Recognition of the muscarinic receptor by its endogenous neurotransmitter: binding of [3H]acetylcholine and its modulation by transition metal ions and guanine nucleotides.
Authors: Gurwitz D , Kloog Y , Sokolovsky M
Published: May 2006

Agonist binding to the muscarinic receptor in rat cerebral cortex membranes was studied by using the neurotransmitter itself, [3H]acetylcholine [( 3H]AcCho). By using 10 microM atropine or oxotremorine to define specific binding, it was possible to demonstrate specific binding of [3H]AcCho that was sensitive to muscarinic but not to nicotinic ligands. Equilibrium binding experiments with 5-240 nM [3H]AcCho indicated specific binding of the ligand to a saturable population of muscarinic receptors (361 +/- 29 fmol/mg of protein; Kd = 76 +/- 17 nM). This value represented 25% of the available binding sites for a labeled antagonist in the same preparation and corresponds to the proportion of high-affinity agonist binding sites observed previously in competition experiments with labeled antagonists. Inclusion of transition metal ions (e.g., 2 mM Ni2+) in the assay increased the equilibrium binding of [3H]AcCho (628 +/- 38 fmol/mg of protein, Kd = 86 +/- 21 nM) but did not affect equilibrium binding of 3H-labeled antagonists, indicating conversion of low- into high-affinity muscarinic agonist binding sites. The increase developed slowly over 30 min of incubation at 25 degrees C but could be reversed rapidly (approximately equal to 2 min) by the chelating agent EDTA or by guanine nucleotides. These data directly reveal a slow though quickly reversible interconversion of low- into high-affinity muscarinic agonist binding sites.
Partial deglycosylation of chloroplast coupling factor 1 (CF1) prevents the reconstitution of photophosphorylation.
Authors: Maione T E , Jagendorf A T
Published: May 2006

Treatment of spinach chloroplast coupling factor 1 (CF1) with a mixture of glycosidases resulted in the removal of approximately 75% of the carbohydrate associated with the enzyme. The ATPase of CF1 was not activated by this treatment nor were its heat-activated or methanol-dependent ATPase activities inhibited. The deglycosylated enzyme, however, was unable to catalyze photophosphorylation when recoupled with CF1-depleted thylakoids. The glycosidase-treated protein competed with native CF1 for specific binding sites on the depleted membranes and was able to reconstitute proton uptake in uncoupled thylakoids. The inhibition of photophosphorylation could not be overcome with added delta subunit. We conclude that deglycosylated CF1 was unaffected in its ability to bind to the membrane sector of the chloroplast proton-pumping ATPase (CF0) but was altered in some property essential for photophosphorylation but not ATPase activity.
Intragenic regions required for LamB export.
Authors: Benson S A , Bremer E , Silhavy T J
Published: May 2006

Escherichia coli strains containing a series of lamB-lacZ fusions have been isolated and characterized. Each of these fusions specifies a hybrid protein with LamB sequences at the NH2 terminus and a large functional COOH-terminal fragment of beta-galactosidase. The amount of LamB present in the various hybrid proteins ranges from as few as 4 amino acids to a complete signal sequence (25 amino acids) plus 49 amino acids of the mature protein. With respect to hybrid protein export these fusions fall into three classes. Hybrid proteins with an incomplete LamB signal sequence or those that have a complete signal sequence plus 27 or fewer amino acids of the mature LamB protein are not exported and remain in the cytoplasm. In contrast, fusion strains attempt to export hybrid proteins that contain a complete signal sequence plus 39 or 43 amino acids of mature LamB. However, these proteins are not localized to the outer membrane. Finally, a hybrid protein that is slightly larger, containing 49 amino acids of mature LamB, is found in the outer membrane in appreciable amounts. These fusions, together with previously described lamB-lacZ fusions, have enabled us to define more precisely the minimal amount of lamB required to initiate the process of protein export. Moreover, they genetically locate a signal that appears to guide LamB to the outer membrane. This signal is within a region of amino acid homology shared by other major outer membrane proteins [ Nikaido , H. & Wu, H. C. P. (1984) Proc. Natl. Acad. Sci. USA 81, 1048-1052].
Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork.
Authors: Ottiger H P , Hübscher U
Published: May 2006

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.
Altered mRNA splicing in monkey cells abortively infected with human adenovirus may be responsible for inefficient synthesis of the virion fiber polypeptide.
Authors: Anderson K P , Klessig D F
Published: May 2006

Messenger RNA encoding the fiber protein of the human adenovirus serotype 2 (Ad2) capsid is inefficiently translated in abortively infected African green monkey kidney cells. The amount of fiber mRNA present in the cytoplasm of abortively infected monkey cells is less than that in productively infected cells by a factor of 5-10 but synthesis of the fiber polypeptide is reduced by a factor of more than 100. Evidence from a variety of experiments indicates that the defect does not lie in the translational apparatus of the monkey cell but may best be explained by differences in the fiber messages made in abortively versus productively infected cells. Here we report that fiber mRNA isolated from abortively infected monkey cells is processed differently than that made in productively infected cells. Primer extension analysis of the 5' ends of fiber messages from several different productive and abortive infections shows a direct correlation between synthesis of the fiber polypeptide in vivo and the presence of the "x" and/or "y" ancillary leaders on messages encoding the fiber polypeptide. Of all the mRNAs encoded by the major late transcriptional unit of Ad2 only the fiber message can contain the x and y leaders, and the fiber protein is the only late Ad2 protein reported to be glycosylated. We speculate that these leader sequences play a role in the synthesis of this glycoprotein, as well as that of the Ad2 19-kilodalton glycoprotein encoded by early region 3, whose mRNA also contains the x and y leaders.
Accurate initiation of human epsilon-globin RNA synthesis by Escherichia coli RNA polymerase in isolated nuclei of K562 erythroleukemia cells.
Authors: Gilmour R S , Allan M , Paul J
Published: May 2006

The human epsilon-globin gene was transcribed in vitro in isolated K562 cell nuclei by using exogenous Escherichia coli RNA polymerase (EC 2.7.7.6). Newly formed RNA transcripts were distinguished from nuclear RNA molecules by (i) incorporating mercurated UTP into RNA under conditions in which the endogenous polymerase II is inactive and (ii) subsequently isolating the mercurated RNA by affinity chromatography on thiolated Sepharose. A specific 5'-end-labeled probe spanning the epsilon-globin gene cap site was used in nuclease S1 mapping studies to examine the in vitro initiation site of the isolated transcripts. It was found that transcription occurred from the coding strand only and originated almost entirely from a point that was identical to that of the major cap site for epsilon-globin mRNA in vivo.
Expression of a nuclear and a cytoplasmic Epstein-Barr virus early antigen after DNA transfer: cooperation of two distant parts of the genome for expression of the cytoplasmic antigen.
Authors: Takaki K , Polack A , Bornkamm G W
Published: May 2006

Expression of Epstein-Barr virus (EBV) antigens was studied after transfection of cloned EBV DNA fragments into baby hamster kidney (BHK) cells. A set of seven widely overlapping clones covering the whole genome of the non-defective Epstein-Barr virus strain M-ABA was used for transfection. Transfer of the cosmid clones into BHK cells resulted in expression of two distinct antigens, as revealed by indirect immunofluorescence using human anti-EBV sera. Staining with human sera of different reactivity against EBV-associated antigens revealed that both types of antigens were related to the early antigen complex. The first type of antigen was detected only in the nuclei of BHK cells that had received DNA of a clone containing HindIII-G, -H, -E, -I2, -O, -I1, and -P. The second type of antigen was found in the cytoplasm of cells cotransfected with clones containing Sal-A and HindIII-I2, -O, -I1, -P, and -C, whereas transfection of both individual clones failed to induce the antigen. Further analysis with subclones identified HindIII-G (5 kilobases) and HindIII-I2 (3 kilobases) plus the rightmost 3 kilobases of Sal-A as the sequences responsible for expression of the nuclear and the cytoplasmic antigen, respectively. The fact that two distant regions of the viral genome are required for expression of a viral antigen provides evidence for intergenomic regulation that can be studied in vitro.
Maintenance of high disequilibrium in the presence of partial selfing.
Authors: Hastings A
Published: May 2006

The minimum strength of selection necessary to maintain high linkage disequilibrium in two-locus, two-allele, deterministic discrete time genetic models with high rates of partial selfing is determined. The relationship between linkage and partial selfing is shown to be complex. The major effect of high selfing rates is to decrease the strength of selection necessary to maintain disequilibrium, but only for large recombination rates. With partial selfing, stable equilibria of these models require strong selection to maintain high values of disequilibrium.
Identification and primary sequence of an unspliced human urokinase poly(A)+ RNA.
Authors: Verde P , Stoppelli M P , Galeffi P , Di Nocera P , Blasi F
Published: May 2006

Human urokinase cDNA clones have been identified from a cDNA library prepared from total RNA of human fibroblasts transformed by simian virus 40 [Okayama, H. & Berg, P. (1983) Mol. Cell. Biol. 3, 280-289]. Synthetic oligonucleotides, corresponding to urokinase protein sequence, were used as probes. The cloned cDNA covers most of the coding sequence and the entire 3' untranslated region. The nucleotide sequence of one of the clones identifies this as a copy of a partially spliced polyadenylylated precursor to urokinase mRNA. The introns separate functionally different domains of the enzyme. Human urokinase mRNA has been identified by RNA blot and its size was estimated at 2500 nucleotides.
Different evolutionary behavior of structurally related, repetitive sequences occurring in the same Balbiani ring gene in
Chironomus tentans
Authors: Höög Christer , Wieslander Lars
Published: May 2006

The Balbiani ring 2 (BR 2) gene in
Chironomus tentans
is highly internally repeated. Two types of related repeat units—the α and β types—are tandemly arranged in separate blocks, which together are likely to form the major part of the gene. Every repeat unit has one constant region and one subrepeat region. Here we analyze the length and sequence of a number of repeat units of both types and compare the units within and between the blocks. The ≈100 α repeat units are essentially invariant regarding length and sequence. In contrast, when the ≈70 β repeat units are compared, six length variants are found, four of which have been sequenced. The length variations reside in the subrepeat regions and are due to different numbers of whole or half subrepeats. Furthermore, the subrepeat regions differ by several base-pair substitutions, many of which change the amino acid sequence. On the other hand, all β-type constant regions are of equal length and are virtually homogeneous in sequence. The observed length distributions in combination with analysis of the basepair substitutions in the α-and β-type constant and subrepeat regions suggest that the α and β blocks are of different age, that seemingly homologous repeated regions may play different functional roles at the protein level, and that sequence correction mechanisms are likely to operate to different extents on the constant and subrepeat regions within the β block.
Transfer of nuclei from a parasite to its host
Authors: Goff Lynda J. , Coleman Annette W.
Published: May 2006

During the normal course of infection, nuclei are transferred via secondary pit connections from the parasitic marine red alga
Choreocolax
to its red algal host
Polysiphonia
. These “planetic” nuclei are transmitted by being cut off into specialized cells (conjunctor cells) that fuse with an adjacent host cell, thereby delivering parasite nuclei and other cytoplasmic organelles into host cell cytoplasm. Within the foreign cytoplasm, planetic nuclei survive for several weeks and may be active in directing the host cellular responses to infection, since these responses are seen only in host cells containing planetic nuclei. The transfer and long-term survival of a nucleus from one genus into the cytoplasm of another through mechanisms that have evolved in nature challenge our understanding of nuclear-cytoplasmic interactions and our concept of “individual.”
Processing of complex heteroduplexes in Escherichia coli and Cos-1 monkey cells.
Authors: Abastado J P , Cami B , Dinh T H , Igolen J , Kourilsky P
Published: May 2006

Upon transformation into Escherichia coli or Cos-1 monkey cells, heteroduplex DNA made of two sequences containing many nucleotide mismatches yields a wide array of different molecules, some with a patchwork structure. Thus, complex heteroduplexes can be processed to generate many genetic variants.
Risk-efficient estimation of the mean exponential survival time under random censoring.
Authors: Gardiner J C , Susarla V
Published: May 2006

The paper proposes a sequential estimator theta of the parameter theta of an exponential distribution when the data are censored. It is shown that theta is asymptotically risk efficient when the loss is measured by the squared error of estimation of theta plus a linear function of the number of observations and that theta is asymptotically normal as the cost per observation goes to zero.
Use of short DNA oligonucleotides for determination of DNA sequence modifications induced by benzo[a]pyrene diol epoxide.
Authors: Wei S J , Desai S M , Harvey R G , Weiss S B
Published: May 2006

Various organic agents that alkylate DNA are known to induce mutations in bacterial and animal cells. The precise nature and location of modified DNA sequences in such mutants are often difficult to ascertain. In this report, a 10-base-pair oligomer (BamHI linker) is treated with (+/-)-trans-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide and inserted into replicative form DNA of phage M13 by ligation at a specific restriction site. Escherichia coli are transfected with the recombinant DNA containing the alkylated target, progeny viral plaques are selected, and their DNAs are subjected to DNA sequence analysis at the region of oligomer insertion. For the alkylated inserts used in this study, the DNA sequence analysis of progeny viral DNA showed that nucleotide deletions were present in every clone examined. These deletions occurred primarily, but not exclusively, at G-C cluster regions, varied from 1 to 24 base pairs in length, and included both target and nontarget nucleotides. A second type of repair, which restores most of the original nucleotide bases in the alkylated insert, is also implied by the DNA sequence data obtained.
Differential sleep-promoting effects of five sleep substances nocturnally infused in unrestrained rats.
Authors: Inoué S , Honda K , Komoda Y , Uchizono K , Ueno R , Hayaishi O
Published: May 2006

Sleep-inducing and sleep-maintaining effects of five different putative sleep substances were compared by the same nocturnal 10-hr intracerebroventricular infusion technique in otherwise saline-infused, freely moving male rats. Delta-sleep-inducing peptide (2.5 nmol), which induces electroencephalogram delta (slow)-wave patterns, was rapidly effective in increasing both slow-wave sleep and paradoxical sleep but the effects were not long-lasting. Muramyl dipeptide (2 nmol) induced excessive slow-wave sleep in the middle of the infusion period, accompanying a simultaneous elevation of brain temperature. However, paradoxical sleep was not affected. Component B of sleep-promoting substance (2 brainstem equivalents), a partially purified extract from rats deprived of sleep for 24-hr, was markedly effective in inducing and maintaining both kinds of sleep. Prostaglandin D2 (0.36 nmol) was more effective in enhancing sleep at the later period of the infusion period. Uridine (10 pmol) caused a mild but long-lasting increase in sleep, especially in paradoxical sleep. Thus, each substance exhibited compound-specific sleep-modulating properties.
Monoclonal antibodies distinguish subtypes of retinal horizontal cells.
Authors: Young L H , Dowling J E
Published: May 2006

Sixteen hybridomas have been identified that secrete antibodies specific to horizontal cells in the carp retina. The hybridomas have been classified into three groups based on their antibody staining patterns: group I, staining associated with all horizontal cells; group II, staining associated with the most abundant subtype of horizontal cell (CH1); and group III, staining associated with other subtypes of horizontal cells. Most of the hybridomas fall in group II; some of these antibodies stain the entire horizontal cell, but others are specific only to the cell perikarya and do not stain axonal processes. Our results suggest that there are surface molecules specific (i) to all retinal horizontal cells, (ii) to individual subtypes of horizontal cells, and (iii) to portions of horizontal cells. Furthermore, a group II antibody, which recognizes a 48- to 50-kDa membrane protein, has been found to provide a substrate selective for horizontal cell growth. Horizontal cells plated on coverslips coated with this antibody remain healthy in culture and extend long and elaborate processes for at least 3 weeks.